Protein Kinase-mediated Decisions Between Life and Death (Advances in Experimental Medicine and Biology, 1275) 3030498433, 9783030498436

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Advances in Experimental Medicine and Biology 1275

Ayse Basak Engin Atilla Engin  Editors

Protein Kinase-mediated Decisions Between Life and Death

Advances in Experimental Medicine and Biology Volume 1275 Series Editors Wim E. CrusW, Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, CNRS and University of Bordeaux, Pessac Cedex, France Haidong Dong Radeke, Departments of Urology and Immunology, Mayo Clini, Rochester, MN, USA Heinfried H. Radeke, Institute of Pharmacology & Toxicology, Clinic of the Goethe University Frankfurt Main, Frankfurt am Main, Hessen, Germany Nima Rezaei, Research Center for Immunodeficiencies, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of Cardiovascular Science, School of Life Science, Shanghai University, Shanghai, China

Advances in Experimental Medicine and Biology provides a platform for scientific contributions in the main disciplines of the biomedicine and the life sciences. This series publishes thematic volumes on contemporary research in the areas of microbiology, immunology, neurosciences, biochemistry, biomedical engineering, genetics, physiology, and cancer research. Covering emerging topics and techniques in basic and clinical science, it brings together clinicians and researchers from various fields. Advances in Experimental Medicine and Biology has been publishing exceptional works in the field for over 40 years, and is indexed in SCOPUS, Medline (PubMed), Journal Citation Reports/Science Edition, Science Citation Index Expanded (SciSearch, Web of Science), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical Abstracts Service (CAS), and Pathway Studio. 2018 Impact Factor: 2.126. More information about this series at http://www.springer.com/series/5584

Ayse Basak Engin  •  Atilla Engin Editors

Protein Kinase-mediated Decisions Between Life and Death

Editors Ayse Basak Engin Department of Toxicology Faculty of Pharmacy Gazi University Ankara, Turkey

Atilla Engin Department of General Surgery Faculty of Medicine Gazi University Ankara, Turkey

ISSN 0065-2598     ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-3-030-49843-6    ISBN 978-3-030-49844-3 (eBook) https://doi.org/10.1007/978-3-030-49844-3 © Springer Nature Switzerland AG 2021, Corrected Publication 2021 Chapter 5 is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). For further details see license information in the chapter. This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Contents

1 Protein Kinase-Mediated Decision Between the Life and Death����������������������������������������������������������������������������   1 Atilla Engin 2 Aging and Protein Kinases���������������������������������������������������������������� 35 Ayse Basak Engin and Atilla Engin 3 The Connection Between Cell Fate and Telomere������������������������  71 Ayse Basak Engin and Atilla Engin 4 Dark-Side of Exosomes�������������������������������������������������������������������� 101 Atilla Engin 5 Signal Transduction in Immune Cells and Protein Kinases�������� 133 Monica Neagu and Carolina Constantin 6 Role of Protein Kinase C in Immune Cell Activation and Its Implication Chemical-Induced Immunotoxicity�������������� 151 Emanuela Corsini, Erica Buoso, Valentina Galbiati, and Marco Racchi 7 Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles Effect������������������������������������ 165 Ayse Basak Engin 8 Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes ������������������������������������������������������������������������ 195 Ayse Basak Engin and Atilla Engin 9 Bile Acid Toxicity and Protein Kinases������������������������������������������ 229 Atilla Engin 10 N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia������������������������������������������������������ 259 Atilla Engin and Ayse Basak Engin 11 Alzheimer’s Disease and Protein Kinases�������������������������������������� 285 Ayse Basak Engin and Atilla Engin 12 Bacterial Protein Kinases���������������������������������������������������������������� 323 Evren Doruk Engin

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13 Indoleamine 2,3-Dioxygenase Activity-Induced Acceleration of Tumor Growth, and Protein Kinases-Related Novel Therapeutics Regimens ������������������������������������������������������������������ 339 Ayse Basak Engin and Atilla Engin 14 A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases Can Be A Potential Therapeutic Target for Anti-cancer Therapy�������������������������������� 357 Basak Celtikci 15 Protein Kinases in Hematological Disorders �������������������������������� 383 Mufide Okay and Ibrahim C. Haznedaroglu 16 Metabolic Stress and Immunity: Nutrient-Sensing Kinases and Tryptophan Metabolism���������������������������������������������������������� 395 Johanna M. Gostner, Dietmar Fuchs, and Katharina Kurz Correction to: Signal Transduction in Immune Cells and Protein Kinases���������������������������������������������������������������������   C1 Index���������������������������������������������������������������������������������������������������������� 407

Contents

Contributors

Erica Buoso  Department of Drug Sciences, Università di Pavia, Pavia, Italy Basak Celtikci  Hacettepe University, Faculty of Medicine, Department of Medical Biochemistry, Ankara, Turkey Carolina  Constantin Immunology Department, Victor Babes National Institute, Bucharest, Romania Colentina University Clinical Hospital, Bucharest, Romania Emanuela Corsini  Laboratory of Toxicology, Department of Environmental Science and Policy, Università degli Studi di Milano, Milan, Italy Atilla  Engin Department of General Surgery, Faculty of Medicine, Gazi University, Ankara, Turkey Ayse  Basak  Engin  Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey Evren Doruk Engin  Ankara University, Biotechnology Institute, Gümüşdere Campus, Keçiören, Ankara, Turkey Dietmar  Fuchs Institute of Biological Chemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria Valentina Galbiati  Laboratory of Toxicology, Department of Environmental Science and Policy, Università degli Studi di Milano, Milan, Italy Johanna M. Gostner  Institute of Medical Biochemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria Ibrahim  C.  Haznedaroglu Hacettepe University, Medical School, Department of Hematology, Ankara, Turkey Katharina Kurz  Department of Internal Medicine II, Infectious Diseases, Pneumology, Rheumatology, Medical University of Innsbruck, Innsbruck, Austria Monica  Neagu  Immunology Department, Victor Babes National Institute, Bucharest, Romania Faculty of Biology, University of Bucharest, Bucharest, Romania Colentina University Clinical Hospital, Bucharest, Romania

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Mufide  Okay Hacettepe University, Medical School, Department of Hematology, Ankara, Turkey Marco  Racchi Department of Drug Sciences, Università di Pavia, Pavia, Italy

Contributors

1

Protein Kinase-Mediated Decision Between the Life and Death Atilla Engin

Abstract

Protein kinases are intracellular signaling enzymes that catalyze the phosphorylation of specific residues in their target substrate proteins. They play important role for regulation of life and death decisions. The complexity of the relationship between death receptors and protein kinases’ cell death decision-making mechanisms create many difficulties in the treatment of various diseases. The most of fifteen different cell death pathways, which are reported by Nomenclature Committee on Cell Death (NCCD) are protein kinase signal transduction-­ mediated negative or positive selections. Tumor necrosis factor (TNF) as a main player of death pathways is a dual-­ functioning molecule in that it can promote both cell survival or cell death. All apoptotic and necrotic signal transductions are conveyed through death domain-containing death receptors, which are expressed on the surface of nearly all human cells. In humans, eight members of the death receptor family have been identified. While the interaction of TNF with TNF Receptor 1 (TNFR1) activates various

A. Engin (*) Department of General Surgery, Faculty of Medicine, Gazi University, Besevler, Ankara, Turkey

signal transduction pathways, different death receptors activate three main signal transduction pathways: nuclear factor kappa B (NF-ĸB)-mediated differentiation or pro-­ inflammatory cytokine synthesis, mitogen-­ activated protein kinase (MAPK)-mediated stress response and caspase-mediated apoptosis. The link between the NF-ĸB and the c-Jun NH2-terminal kinase (JNK) pathways comprise another check-point to regulate cell death. TNF-α also promotes the “receptor-­ interacting serine/threonine protein kinase 1” (RIPK1)/RIPK3/ mixed lineage kinase domain-like pseudokinase (MLKL)dependent necrosis. Thus, necrosome is mainly comprised of MLKL, RIPK3 and, in some cases, RIPK1. In fact, RIPK1 is at the crossroad between life and death, downstream of various receptors as a regulator of endoplasmic reticulum stress-induced death. TNFR1 signaling complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of transforming growth factor β-activated kinase 1 (TAK1), inhibitor of nuclear transcription factor κB (IκB) kinase (IKK) α/IKKβ, IκBα, and NF-κB. IKKs affect cell-survival pathways in NF-κB-independent manner. Toll-like receptor (TLR) stimulation triggers various signaling pathways dependent on myeloid differentiation factor-88 (MyD88), Interleukin-1 receptor (IL-1R)-associated

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_1

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kinase (IRAK1), IRAK2 and IRAK4, lead to post-translational activation of nucleotide and oligomerization domain (NLRP3). Thereby, cell fate decisions following TLR signaling is parallel with death receptor signaling. Inhibition of IKKα/IKKβ or its upstream activators sensitize cells to death by inducing RIPK1-dependent apoptosis or necroptosis. During apoptosis, several kinases of the NF-κB pathway, including IKK1 and NF-κB essential modulator (NEMO), are cleaved by cellular caspases. This event can terminate the NF-κB-derived survival signals. In both canonical and non-canonical pathways, IKK is key to NF-κB activation. Whereas, the activation process of IKK, the functions of NEMO ubiquitination, IKK-­ related non-canonical pathway and the nuclear transportation of NEMO and functions of IKKα are still debated in cell death. In addition, cluster of differentiation 95 (CD95)-mediated non-apoptotic signaling and CD95- death-inducing signaling complex (DISC) interactions are waiting for clarification. Keywords

Nuclear factor kappa B essential modulator (NEMO) · Tumor necrosis factor receptor-­ associated factor 2 (TRAF) · mitogen-­ activated protein (MAP) kinase mitogen-activated protein kinase (MAPK) · cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) · Fas-associated death domain (FADD) · Tumor necrosis factor-related apoptosis-­ inducing ligand (TRAIL) · Tumor necrosis factor receptor associated-protein with death domain (TRADD) · receptor-interacting serine/threonine protein kinase (RIPK) · E3 ligase linear ubiquitin chain assembly complex (LUBAC) · Shank-associated RH domain-interacting protein (SHARPIN)

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Introduction

Protein kinases are intracellular signaling enzymes that catalyze the phosphorylation of specific residues in their target substrate proteins. Despite the basic regulatory roles played by protein phosphorylation, it is not precisely known how phosphorylation directly modifies protein functions (Zeke et al. 2016). Nevertheless, it has been known for a long time that, protein kinases play an important role for regulation of life and death decision made in response to various stress signals. Indeed, these stresses are closely linked to physiological mechanisms in the control of cell fate that is implicated in the pathophysiology of human diseases (Matsuzawa et al. 2002). Since their identification more than 20  years ago, the death receptors have been intensively studied with respect to their cell death-triggering activities via protein kinases (Siegmund et  al. 2017). However, the complexity of relationship between the death receptors and protein kinases’ cell death decision-making efficacy has been maintained. In this chapter, the balance between the cell death and cell death-relevant activities of these receptors-linked metabolic pathways and related protein kinases will be debated at the molecular level.

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 ross-Talk Between Cell-­ C Death Morphologies and Protein Kinases

The Nomenclature Committee on Cell Death (NCCD) periodically proposes unified criteria for the definition of cell death and of its different morphologies (Kroemer et al. 2009) . In this context, NCCD reported a new guideline for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. According to this, irreversible degeneration of vital cellular functions has been reviewed, and fifteen different cell death pathways are classified (Galluzzi et al. 2018). Most of them are protein kinase signal transduction-­ mediated negative or positive selections, which are defined as regulated cell death (RCD). Of

1  Protein Kinase-Mediated Decision Between the Life and Death

these, intrinsic apoptosis is initiated by a variety of cellular disturbances including DNA damage, endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) overload, replication stress, microtubular alterations or mitotic defects (Pihán et  al. 2017; Vitale et  al. 2017). In this type of death, ER stress results with a complex and dynamic signaling network termed the unfolded protein response (UPR). Indeed, the UPR involves a complex signaling transduction pathway initiated by at least three type of stress sensors or transducers localized to the ER membrane including inositol-requiring enzyme 1 (IRE1) α, Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) α (Hetz et  al. 2015; Wang and Kaufman 2016). Upon activation, PERK signaling blocks protein synthesis by phosphorylating the translation eukaryotic initiation factor 2 (eIF2) α, reduces the overload of misfolded proteins inside the ER. The mammalian eIF2 kinases PERK and general control nonderepressible 2 (GCN2) repress translation of most mRNAs but selectively increase translation of ATF4. This process results in the induction of the downstream gene, transcription factor C/EBP homologous protein (CHOP) (Harding et  al. 2000) . GCN2 may play pivotal roles in multiple protein translation checkpoints in both the nucleolus and cytosol. After induction of GCN2/eIF2α phosphorylation, ATF4 expression overrides PERK/ Akt (Protein kinase B, PKB)-mediated adaptation and induces apoptosis through ATF4dependent expression of pro-­ apoptotic factors (Nakamura and Kimura 2017; Shin et al. 2015). Under chronic ER stress, ATF4 contributes to apoptosis by upregulating the CHOP. Therefore, CHOP has a role in the induction of cell death under conditions associated with malfunction of the ER (Zinszner et al. 1998). The CHOP protein undergoes stress-inducible phosphorylation by stress-inducible members of the p38-mitogenactivated protein (MAP) kinase (MAPK) family. Phosphorylation is associated with enhanced transcriptional activation by CHOP.  CHOP upregulates proapoptotic factor antiapoptotic B-cell leukemia/lymphoma 2 (Bcl-­2) homology (BH)-3 only or Bcl-2 interacting mediator of cell

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death (BIM) and downregulates the Bcl-2. IRE1Bcl-2-associated X protein (BAX)/ Bcl-2 homologous antagonist/killer (BAK) complex and Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) triggers the activation of the apoptosis signal-regulating kinase 1 (ASK1) (Chiribau et  al. 2010; Puthalakath et  al. 2007; Wang and Ron 1996). Activated BIM binds the BAX/BAK complex to increase the influx of calcium into the mitochondria and triggers the release of cytochrome c. The critical step for intrinsic apoptosis is irreversible and widespread mitochondrial outer membrane permeabilization (MOMP), which causes apoptotic cell death with the activation of the caspase proteases (Chiribau et al. 2010; Tait and Green 2010). In response to apoptotic stimuli, interactions of two key proapoptotic BCL-2 proteins mediate MOMP.  Thereby, the interaction between BAX and BAK, either preserves survival or initiates MOMP (Luna-Vargas and Chipuk 2016). BAX retro-translocation depends on pro-survival Bcl-2 family proteins, whereas inhibition of retrotranslocation correlates with BAX accumulation on the mitochondrial membrane. Upon induction of apoptosis, BAX retro-translocation ceases as the mitochondrial pools of BAX and BAK undergoes activation by pro-apoptotic BH3-only proteins. BAX activator molecules cleave to residues of BH3 interacting domain death agonist (BID) protein and the BIM BH3 peptide. Cytochrome c release are synergistically induced by these proteins (Edlich et al. 2011; Kuwana et al. 2005). In fact, the BH3-only protein BIM, exists as three splice variants BIM-S (short), BIM-L (long), and BIM-EL (extra long) with different pro-apoptotic potency. In response to activation of the extracellular signal–regulated kinase (ERK) 1/2 pathway, only BIM-EL is phosphorylated on at least three sites (Ley et  al. 2005). BH3-only proteins are essential to induce ER stress-mediated cell death. Therefore, many forms of apoptosis require direct activation of BAX and BAK at the mitochondria by a member of the BID, BIM, or p53 upregulated modulator of apoptosis (PUMA) family of pro-death proteins. Death signals initiated at ER induce BIM and PUMA to activate mitochondrial apoptosis (Kim et al. 2009a; Ren

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A. Engin

Fig. 1.1  The molecular mechanisms of Fas/FasL-induced cell death. Fas receptor oligomerization leads to the formation of a death-inducing signaling complex. Fas activates two independent pathways to induce cell death: one of these, Daxx-JNK pathway that involves JNK activation and is blocked by Bcl-2, and the second pathway is FADD-caspase-8 that is Bcl-2: insensitive. Fas-induced MCP-1 and IL-8 serve as “find-me” signals for apoptotic cells. (Abbreviations: AA-Bcl-2: Antiapoptotic Bcl-2, ASK1: apoptosis signal-regulating kinase 1, ATF4: activating-­ factor 4, BAD: sensitizers inactivating the prosurival Bcl-2 proteins, BAK: BCL-2 antagonist killer, BAX: BCL-2 associated X apoptosis regulator, Bcl-2: antiapoptotic B-cell leukemia/lymphoma 2 protein, BID: BH3‐interacting domain death agonist, BH3-domain-only proapoptotic factor, BIM: Bcl-2 Interacting mediator of cell death, Casp: caspase, CD95: death receptor, APO-1, Fas receptor, CD95L: CD95 with its natural ligand (CD178), cFLIP: cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein, cFLIPL: cFLIPLong, cFLIPR: cFLIPRaji, cFLIPS: cFLIPShort,

CHOP: transcription factor C/EBP homologous protein or CCAAT-­binding homologous protein, Cytc: cytochrome c, Daxx: Fas death domain–associated protein, DD: Death domain, DED: death effector domain, DISC: death-inducing signaling complex, ER: endoplasmic reticulum, FAAD: Fas (The receptor CD95)-associated death domain, IRE1α: inositol-­ requiring enzyme 1 α, IL-8: Interleukin-8, JNK: c-JunNH2-­terminal kinase, MOMP: mitochondrial outer membrane permeabilization, Mt: mitochondria, p phosphorylated, GCN2: general control nonderepressible 2, p38MAPK: p38 mitogen-activated protein kinase, MEK: mitogen-activated protein kinase (MAPK) kinase, MCP-1: Monocyte chemoattractant protein-1 or CCL2, chemokine (C-C motif) ligand 2, PA-Bcl-2: Proapoptotic Bcl-2, PERK: Protein kinase R-like endoplasmic reticulum kinase, PLAD: N‐terminal preligand assembly domain, PUMA: p53 upregulated modulator of apoptosis, TRAF2: TNF receptor-associated factor 2, TRAIL: tumor necrosis factor-­related apoptosis inducing ligand, tBID: truncated (t) Bid, UPR: unfoldedprotein response)

et  al. 2010) (Fig.  1.1). The small guanosine triphosphate effector p21-activated kinase 1 (PAK1) plays an important role in apoptosis. Thereby, PAK1 silencing induces apoptosis by stimulating the expression of PUMA and p21 (Wang et  al. 2018, p.  1). The MAPK regulates cell death and survival following cellular stress or cytokine signaling (Kyriakis and Avruch 2012). Specific variant of intrinsic apoptosis, anoikis is protein kinase-related programmed cell death, which is induced upon cell detachment from extracellular matrix (Paoli et al. 2013). Anoikis is

important regarding the tumor progression. Aggregation-induced stabilization of epidermal growth factor receptor (EGFR) and consequent MAPK1/ERK2 survival signaling inhibits anoikis. Therefore, disruption of aggregation in Erb-­ B2 receptor tyrosine kinase 2 (ErbB2)-positive cells is enough to induce anoikis. As mentioned above, neoplastic cells can evade anoikis upon activation of MAPK1/ERK2 caused by cellular aggregation, and EGFR stabilization mediated by ErbB2 (Buchheit et  al. 2014; Rayavarapu et  al. 2015). In healthy cells, BIM molecules are bound

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to LC8 cytoplasmic dynein light chain and thereby sequestered to the microtubule-­associated dynein motor complex. Actually, the dynein complex is comprised of two functional subcomplexes. First is the motor domain, which is responsible for adenosine triphosphate (ATP) hydrolysis and movement along microtubules, second is the cargo attachment subcomplex. Once activated, ERK2 supports anoikis resistance by promoting the cytosolic sequestration of BIM-EL.  Freed BIM translocate together with LC8 to Bcl-2 and neutralize its anti-apoptotic activity. Apoptotic stimuli disrupt the interaction between LC8 and the dynein motor complex (Nyarko and Barbar 2011; Puthalakath et  al. 1999). Mechanistically, ERK/MAPK survival signaling protects the cells from anoikis through ErbB2 and EGFR by facilitating the formation of a protein complex containing BIM-EL, Beclin-1, and LC8. This process, functions to sequester BIM-EL from the mitochondria and thereby blocks anoikis (Buchheit et  al. 2015). ERK1/2-­ dependent dissociation of BIM-EL from pro-­ survival proteins is the first step in destruction of BIM variant (Ewings et al. 2007) . On the other hand, mitochondrial dysfunction causes the release of cytochrome c, translocation of BAX, and activation of caspase cascade. In any case, cleavage of poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP), which is a specific substrate for caspase-3, may lead to apoptosis (Pal et al. 2010). Three major pathways have been defined for PARP-mediated necroptotic cell death due to uncontrolled PARP activity; first, cellular energy deficiency due to depletion of nicotinamide adenine dinucleotide (NAD+) as the substrate of PARPs, second, poly(ADP-ribose) (PAR)-mediated translocation of apoptosis inducing factor (AIF) from ­mitochondria to nucleus, the third is the crossrelation between PAR-mediated cell death or survival kinases and phosphatases (Virág et al. 2013; Ying 2008). The PAR/AIF pathway of cell death is termed parthanatos to distinguish it from caspase-­dependent apoptosis, necrosis and other cell death pathways. Mitochondrial AIF release and its translocation to the nucleus is the triggering point for parthanatos (Fatokun et  al. 2014;

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Wang et al. 2009). Furthermore, the exposure to advanced oxidative protein products causes the loss of mitochondrial membrane potential, and intracellular ROS generation increases. Proapoptotic proteins, such as BAX, caspase 9/ caspase 3, and PARP-1 are activated, whereas anti-apoptotic Bcl-2 protein is downregulated. In addition, increases in reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)4, ERK1/2 and p38 MAPK expressions induce apoptosis (Sun et al. 2016). Indeed, MAPK signaling is important for determining cell fate decisions in a diverse number of organisms and cell types (Alberola-Ila and Hernández-Hoyos 2003). Furthermore, MAPK phosphatase (MKP) activity controls cell fate by regulating the threshold of T-cell receptor (TCR) signaling that is able to induce positive selection (Bettini and Kersh 2007). The regulatory dephosphorylation of ERK1/2 is mediated by protein-­ tyrosine specific phosphatases, protein-serine/ threonine phosphatases, and dual specificity phosphatases. However, the combination of kinases and phosphatases make the overall process reversible (Roskoski 2012). c-Jun NH2-­ terminal kinase (JNK), p38 and ERK5 are MAPKs essential for negative selection, but do not influence positive selection. The phosphorylation of ERK1/2 is important for positive selection but not for negative selection. The MAP kinase kinase (MEK)-ERK pathway is also involved in negative selection. Although, Ras/ raf-1/MEK pathway exhibits a partially blocked positive selection, it does not play a role in negative selection (Alberola-Ila et  al. 1996; Bommhardt et al. 2000; O’Shea et al. 1996; Wu et al. 2018). Wyrsch et al. claimed that cell death is activated by downstream of ERK1/2 and AKT, whereas cell survival correlates with the phosphorylation of p38, stress-activated protein kinase (SAPK)/JNK, and cyclic adenosine monophosphate (AMP) response element-binding protein (CREB) (Wyrsch et  al. 2012). Thus, the induction of ERK leads to positive selection, while the induction of p38 and JNK results in negative selection (Mariathasan et al. 2001). But there is a cellular threshold for active ERK1/2 levels, which affects the cell fate between death

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and growth arrest. Therefore, ERK1/2 mediates death signaling dependently of its kinase activity. Indeed, ERK1/2 is a focal point of Raf/MEK/ ERK pathway-mediated growth arrest and death signaling (Hong et  al. 2018; Wu et  al. 2015) . After stimulation, ERK1/2 are phosphorylated by MEK1/2 and detach from the anchoring proteins. This detachment exposes ERK1/2 to the additional phosphorylation during the nuclear translocation signal of the kinases. This event results in the translocation of ERK1/2 to the nucleus (Berti and Seger 2017; Caelles et  al. 2017). In contrast, cytosolic retention of ERK1/2 blocks the access to the transcription factor substrates that are responsible for the mitogenic response. Hereby, cytosolic ERK1/2, besides inhibiting survival and proliferative signals in the nucleus, potentiates the catalytic activity of some proapoptotic proteins such as death-associated protein kinases (DAPKs) in the cytoplasm (Mebratu and Tesfaigzi 2009). DAPKs regulate many signaling events of the cell death pathways, including apoptosis, autophagy and membrane blebbing (Bovellan et al. 2010). In fact, activation of different death receptors joins on the activation of three main signal transduction pathways: nuclear factor kappa B (NF-ĸB)-mediated differentiation or inflammation, MAPK-mediated stress response and caspase-mediated apoptosis (Sessler et al. 2013). In this context, eight members of the death receptor family have been identified in humans, which can be divided into four structurally homologous groups (Kavuri et  al. 2011; Sessler et al. 2013). When, extracellular stress signals are sensed by specific transmembrane receptors, extrinsic apoptosis is initiated by the binding of lethal ligands to various death receptors (Wajant 2002). Two types of receptors are activated in extrinsic apoptosis. First, death receptors, whose activation occurs after binding to cognate ligands. Second, dependence receptors, whose activation occurs when the levels of their specific ligand drop below a specific threshold. Dependence receptors induce two opposite intracellular signals depending on the availability of their ligand. While in the presence of ligands, they mediate classical signal transduction of survival, without ligands they mediate cell death (Gibert and

A. Engin

Mehlen 2015; Mehlen and Thibert 2004; Thibert and Fombonne 2010). Extrinsic apoptosis is initiated by the formation of the death-inducing signaling complex (DISC). Caspase-8 binding via Fas (The receptor CD95)-associated death domain (FADD) protein to the death receptor is an indispensable initiating step in DISC formation and NF-ĸB activation. Whereas, caspase-10 as a negative regulator of cell death, shifts the apoptotic cell death response following DISC formation to the activation of NF-κB and cell survival. Caspase-8 and its regulator cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) controls death signaling by binding to the death-receptor, which is bound to FADD (Horn et  al. 2017; Irmler et  al. 1997) (Fig.  1.1). Protein kinase C (PKC) activation blocks FADD recruitment and caspase-8 activation and thus DISC formation. In contrast, inhibition of PKCs enhances the activity of the Fas pathway by rapidly increasing FADD recruitment, caspase-8 activation, and DISC formation (Gómez-Angelats and Cidlowski 2001). As mentioned above, caspase-dependent or independent “intrinsic apoptosis” is triggered by intracellular stress conditions of mitochondrial pathways, including DNA damage, oxidative stress, cytosolic calcium overload, excitotoxicity and ER stress (Galluzzi et  al. 2012). Whereas, caspase inhibition induces a shift from an apoptotic to mixed cell death morphology or even to features of necrosis or autophagic cell death (Golstein and Kroemer 2005). Autophagic cell death happens with a lysosome-dependent degradation pathway that promotes cell homeostasis in response to stress such as nutrient deprivation, oxidative stress or DNA damage. Autophagy regulates cell fate following DNA damage, and has a pivotal role in the maintenance of nuclear and mitochondrial genomic integrity. This pathway is modulated by several protein kinases (Vessoni et  al. 2013). In contrast, necrotic cell death or necrosis is morphologically characterized by a gain in cell volume, swelling of organelles, plasma membrane rupture and subsequent loss of intracellular contents (Kroemer et  al. 2009). Necrotic cell death is the consequence of extensive crosstalk between molecular events at different cellular levels, and may be finely reg-

1  Protein Kinase-Mediated Decision Between the Life and Death

ulated by a set of signal transduction pathways (Festjens et  al. 2006; Golstein and Kroemer 2007).

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activation. Even a small amount of change in the relative activities of NF-κB can have dramatic effects on the ability of TNF-α to induce cell survival or death (Chang et al. 2006) (Fig. 1.2). In fact, FADD is an adaptor protein that is a critical 3 Determination of Cell Life via component of the death receptors’ apoptotic signaling pathway. FADD and its apoptotic partner, Tumor Necrosis Factor-αcaspase-8, is effective in necroptosis (Zhang Protein Kinase Cross-Talk et  al. 1998). Caspase-8 belongs to the cysteine In many cases, firstly, DAPK1 activity is signifi- protease family, which plays a crucial role in cantly induced by both TNF-α and interferon- programmed cell death. Death receptor stimulagamma (IFN-γ) signals. Subsequently, DAPK1 tion of caspase-8 triggers sufficient effector casmediates the pro-­apoptotic activity of TNF-α and pase activation to commit the cell to apoptotic IFN-γ via the NF-ĸB signaling pathways (Yoo death (Varfolomeev et  al. 2005). On the one et al. 2012). Whereas, Receptor-interacting pro- hand, caspase-­8 prevents the receptor-interacting tein (RIP) is required for necrotic cell death, ­ serine/threonine protein kinase 3 (RIPK3)which is induced by TNF and TNF-related dependent necrosis without inducing apoptosis apoptosis-­inducing ligand (TRAIL). In contrast by functioning in a proteolytically active comto its role in NF-ĸB activation, RIP also requires plex with cFLIPL. On the other hand, recruitment its own kinase activity for death signaling (Holler of cFLIPL to this complex leads to partial caset  al. 2000). TRAIL can induce several distinct pase-8 activation, which results in cell survival signaling outcomes ranging from cell death, via (Oberst et  al. 2011). As mentioned above, apoptosis or necroptosis, to gene activation and caspase-­mediated cleavage may also functionally cytokine production (Lafont et  al. 2017). RIP1 inactivate kinases, thereby terminating survival containing a C-terminal death domain (DD) signals. Similarly, it is thought that caspases can interacts with the adaptor molecule FADD pro- also inhibit survival signaling by inactivating the tein. FADD promotes participation of the initia- NF-κB pathway. During apoptosis, several tor caspase-8 by interacting with death effector kinases of the NF-κB pathway, including inhibitor domains (DEDs). This signaling platform initi- of NF-κB kinase 1 (IKK1) and NF-κB essential ates the cell death induction, which is tightly modulator (NEMO), are cleaved by cellular casregulated by antiapoptotic protein, pases. This event can thereby terminate the cFLIP.  Thereby, cFLIP isoforms facilitates the NF-κB-derived survival signals (Frelin et  al. cell death through necroptosis (Feoktistova et al. 2008; Levkau et al. 1999) (Fig. 1.2). In addition, 2011). c-FLIP contains three different isoforms, the enzymatic activity of the FADD-caspase-8-­ which are Long (L), Short (S) and Raji (R). All cFLIPL complex blocks RIPK3-dependent sigthree isoforms possess two DEDs and thereby naling including necrosis. cFLIPL also blocks bind to the CD95-DISC. Of these, cFLIPL has a RIPK3-independent apoptosis promoted by the proapoptotic role only upon moderate expres- FADD-caspase-8 complex alone (Dillon et  al. sion, either in combination with strong receptor 2012). Thus, Fas, TRAIL and TNF receptors can stimulation or in the presence of high amount of initiate cell death by two alternative pathways, one one of the other short cFLIP isoforms (Fricker relying on caspase-8 and the other dependent on the et  al. 2010). By inducing cFLIPL synthesis, RIPK (Holler et  al. 2000). Caspase activity can NF-kB activation prevents caspase-8 activation. be adjusted through caspase-associated recruitAt this period, the TNF Receptor 1 (TNFR1)- ment domain (CARD) inhibitor of apoptosis proassociated complex I dissociates and the FADD-­ teins (cIAP) and posttranslational modifications containing complex II appears. The amount of such as, nitrosylation, phosphorylation or ubiquicFLIPL in the cell determines the extent of pro-­ tination. Both the caspase activation process and caspase-­8 recruitment to FADD and its rate of intrinsic enzymatic activity are under the control

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Fig. 1.2 TNFα-induced cell death dependent JNK activation and c-FLIPL degradation. RIPK1, TRADD, and TRAF2 dissociate from TNFR1 and form complex I. FADD and caspase-8 are recruited on complex 1 to form complex II. In the absence of NF-ĸB activity from complex I, complex II can initiate caspase-8 activation and cell death. NF-ĸB inhibits cell death through upregulation of antiapoptotic genes such as c-FLIP, which directly inhibits caspase activation in complex II or through suppression of JNK activity. If c-FLIPL levels are low (low NF-kB, high JNK1), caspase-8 is recruited to FADD and activated, leading to apoptotic cell death. (Abbreviations: cIAP: E3 ubiquitin (Ub) ligases cellular inhibitor of apoptosis protein, ER: endoplasmic reticulum, FADD: Fas (The receptor CD95)-associated death domain, HOIL: heme-­oxidized iron regulatory protein 2 (IRP2) ubiquitin ligase-1, HOIP: HOIL1-interacting protein, IKK: inhibitor of kappa B (IκB) kinase, JNK: c-JunNH2-terminal

kinase, LUBAC: E3 ligase linear ubiquitin chain assembly complex, MAPK: mitogen-activated protein kinase, MEK: mitogen-activated protein kinase (MAPK) kinase, MLKL: mixed lineage kinase domain-like pseudokinase, MyD88: myeloid differentiation factor-88, NEMO: NF-κB essential modulator or IKKɣ, NF-ĸB: nuclear factor kappa-­ light-­ chain-enhancer of activated B cells, RIPK1: receptor-interacting protein kinases 1, RIPK3: receptor-­interacting protein kinases 3, ROS: reactive oxygen species, SHARPIN: Shank-associated RH domaininteracting protein, TAK1: transforming growth factor β-activated kinase 1, TIRAP: TIR domain-containing adapter protein, TNF: Tumor necrosis factor, TNFR1: TNF Receptor 1, TRADD: TNF: receptor associated-protein with death domain, TRAF2: TNFR-associated factor 2, TRIF: Toll/IL-1 receptor (TIR) domain-containing adapter inducing IFN-β)

of modified kinases and phosphatases. Mutually, caspase-mediated activation of kinases promotes apoptosis, kinases and phosphatases are activated by caspase cleavage to enhance the cell death process (Kurokawa and Kornbluth 2009). In most cell types, the RIPK1 and RIPK3 are key signaling molecules in necrosis and are regulated by caspases and ubiquitination. Moreover, TNF stimulation induces the formation of a necrosome in which RIP3 is activated. Later, the necrosome induces mitochondrial complex I-mediated production of ROS and cytotoxicity (Vandenabeele et al. 2010). The cleavage of aspartic acid at site

324 fragment of RIPK containing DD accelerates apoptosis by enhancing interaction between TNF receptor associated-­ protein with death domain (TRADD) and FADD.  RIPK is selectively cleaved and causes TNF-induced apoptosis as early as 1  hour following the stimulation. Interestingly, RIPK cleavage by caspase-8 blocks NF-κB pathway, whereas also activates TNFinduced proapoptotic signaling pathway. This means that RIPK cleavage has an important role in shifting cells from life to death in response to TNF (Lin et al. 1999). RIPK1 is a master regulator of cell fate decisions. It was identified as a

1  Protein Kinase-Mediated Decision Between the Life and Death

direct substrate of MAP kinase-­activated protein (MAPKAP) kinase-2 (MK2) in the TNFR1 signaling pathway. In this respect, MK2-mediated phosphorylation of RIPK1 limits cytosolic activation of RIPK1. The subsequent assembly of the death complex is a crucial checkpoint for cell fate (Dondelinger et  al. 2017; Menon et  al. 2017). Recent studies have been suggested that the MK2 acts as a key player in determining whether cells live or die in response to TNF and toll-like receptor (TLR) signaling (Oberst 2017). MK2 primarily binds to p38α MAPK, and later is activated by p38α via multiple proline-directed phosphorylation in a stress-dependent manner (Ronkina et al. 2008). In fact, MK2 is one of the DNA damage checkpoint kinases that functions in parallel with the other downstream effector kinases to regulate the DNA damage and cell cycle arrest in mammalian cells (Manke et  al. 2005). In the p38 SPAKs pathway, MK2 serves both as an effector of p38 by phosphorylating substrates and as a determinant of cellular localization of p38. Following phosphorylation of MK2  in nucleus, nuclear p38-MK2 complex is transferred to the cytoplasm (Ben-­Levy et  al. 1998). Eventually, the transforming growth factor-β (TGF-β)activated kinase 1 (TAK1)-p38-MK2 kinase cascade directly limits the lethal potential of cytosolic and complex-I-­ associated RIPK1, thereby TNF-induced activation of MK2 selectively protects cells from RIPK1 kinase-dependent death (Jaco et  al. 2017). In the absence of p53, cells depend on a third cell-cycle checkpoint pathway involving p38MAPK/MK2 for cellcycle arrest and survival after DNA damage (Reinhardt et  al. 2007). Essentially, p38MAPK and MK2 are components of a general stress kinase pathway (Kyriakis and Avruch 2001). On the other hand, previously it was considered that p53 promotes cell death or permanently inhibits cell proliferation, however recently it has become clear that p53 can also contribute to cell survival as well (Kruiswijk et  al. 2015). Thus, the p38/ MK2/Apoptosis-­ antagonizing transcription factor (AATF) signaling pathway is a critical repressor of p53-driven apoptosis. Thus, AATF impacts on the cellular outcomes of the p53 response. Upon genotoxic stress, cytoplasmic pools of

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myosin regulatory light chain (MRLC)-bound AATF are phosphorylated through the p38MAPK/ MK2 checkpoint kinase complex. Phosphorylation of AATF results in the disruption of cytoplasmic MRLC-­ AATF complexes. Following nuclear localization, AATF represses the pro-apoptotic p53 target gene expression (Höpker et al. 2012). As mentioned above, while TNF has the capability to induce cell death, this response is suppressed unless some cell death checkpoints are disrupted (Ting and Bertrand 2016). Even so, TNF as a main player of death pathways is a dual-functioning molecule in that it can promote both cell survival or cell death. In this context, the balance between these two opposing responses is achieved by generating the appropriate protein kinase activation (Legarda-Addison et al. 2009). If this balance deteriorates towards cell death; following upon binding of TNF-α to TNFRI, TRADD and RIP1 associate with FADD and caspase-8, forming a cytoplasmic complex (complex II). Thus, TNFR1-mediated-signal transduction includes a checkpoint, resulting in cell death via complex II.  In this case, NF-κB fails to be activated via complex I (Micheau and Tschopp 2003). The DD at the cytoplasmic end of TNFRI rapidly joins with TRADD. Indeed, the interaction of TNF with TNFR1 activates several signal transduction pathways. A common feature of each pathway is the TNF-induced formation of a multiprotein signaling complex at the cell membrane (Chen and Goeddel 2002; Muppidi et  al. 2004). Of them, TNFR1-associated complex (complex I) that contains RIP1, TRAF2, and cIAP1 activates the IKK/NF-ĸB pathway. Subsequently, TRADD, RIP1, and TRAF2 dissociate from TNFR1, moving to the cytosol. FADD and caspase-8 bind to this cytosolic complex and they constitute complex II (Varfolomeev and Ashkenazi 2004). It has been shown that FADD and caspase-8 are absolutely required for TNFR1-mediated cell death. TNFR1-mediated cell death occurs in a very short period. Thus, within a few minutes after binding of TNF to TNFR1, RIPK1, TRADD, the TRAF2, and cellular inhibitor of apoptosis proteins cIAP1 and cIAP2 are recruited to TNFR1 to form complex

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I. The membrane complex I is mainly composed of TNFR, TRADD, RIP, TRAF2, cIAPs and IKKs. A few hours later, RIPK1, TRADD, and TRAF2 dissociate from TNFR1 and recruit FADD and caspase-8 to form complex II, which can induce apoptosis (Muppidi et  al. 2004) (Fig. 1.2). In fact, cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I.  This suggests that complex-II originates from both RIPK1 in complex-­I and cytosolic RIPK1 (Jaco et al. 2017). Cytosolic complex II containing FADD and caspase-8 is referred to as Complex IIa. In physiological conditions, apoptosis as well as necroptosis is prevented by dimerization between caspase-8 and the cFLIPL.  Active caspase-8  in Complex IIa not only initiates the caspase cascade and the apoptotic program, but also cleaves and inactivates essential necroptosis mediators such as RIPK1 and RIPK3. In contrast, stabilization of RIPK1 and recruitment of RIPK3 convert complex IIa to complex IIb, which is referred as necrosome. Hence, inhibition of caspase-8 or its upstream adaptor FADD primes cells for necroptosis by preserving the integrity of RIPK1 and RIPK3 (Chan et al. 2015). In apoptotic program, upon ligand binding, the TNF receptor family member, Fas forms a complex with FADD through DD:DD interactions. FADD then recruits caspase-8 or caspase-10 through their respective death-effector domains (DEDs). This event results in the activation of caspase in FADD-­ Caspase-­8 complex (Ferrao and Wu 2012). The above-mentioned events occur in two steps; in the first step, TNFR1 recruits the TRADD protein, which in turn recruits RIP1 via the DD interaction at its C-terminal DD and TRAF2 via its N-terminal domain. Thereby, TRADD together with TNFR1, DD and TRAF2 constitute complex I as a membrane bound-complex. Complex I leads to the sequential activation of MAPK kinase kinase, MAPK, JNK, TAK1, IKK and NF-κB. In the next step, the TRADD-RIP1-DD-­ TRAF2 complex dissociates from TNFR1 and associates with the FADD and caspase-8 to form the cytoplasmic proapoptotic complex II (Blackwell et  al. 2009; Micheau and Tschopp 2003). The balance between these two steps is

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provided by the expression of cFLIP in response to NF-κB activation and TRAF2-mediated recruitment of cIAP (Kreuz et  al. 2001; Park et  al. 2000). Furthermore, the balance between NF-ĸB and JNK efficiency is one of the important factors, which determines (De Smaele et al. 2001) cell fate. While NF-ĸB stimulation limits the duration of TNF-induced JNK activity, NF-ĸB inhibition is associated with prolonged TNF-induced JNK activation and apoptosis (Varfolomeev and Ashkenazi 2004). Eventually, the link between the NF-ĸB and the JNK pathways downregulates JNK signaling induced by the TNFR, thereby NF-ĸB-dependent inhibition of the JNK pathway is another check-point to regulate cell death (De Smaele et  al. 2001). In this respect, NF-ĸB-mediated inhibition of cell death involves attenuating the TNF-induced activation of JNK. Transient activation of JNK upon TNF treatment is associated with cellular survival, whereas prolonged JNK activation contributes to cell death (Wullaert et  al. 2006). TNF-α-induced apoptosis requires the JNK1-­ dependent degradation of the caspase-8 inhibitor cFLIPL. JNK antagonizes NF-ĸB during TNF-α signaling by promoting the proteasomal elimination of cFLIPL (Chang et  al. 2006). cFLIPL is essential for suppression of TNF-α-induced prolonged JNK activation and ROS accumulation (Nakajima et  al. 2006). It should be noted that disruption of either the necroptotic pathway or the apoptotic pathway rebalances the system in favor of lethality by the other pathway (Dillon et al. 2014). FADD overexpression cannot induce JNK activation. Therefore, Fas activation of JNK is not secondary to FADD-related activation. Interestingly, Fas death domain–associated protein, Daxx overexpression activates JNK to a level similar to that of Fas. Indeed, Fas activates two independent pathways to induce cell death: one of these, Daxx-JNK pathway that involves JNK activation and is blocked by Bcl-2, and the second pathway is FADD-caspase-8 that is Bcl-2 insensitive. FADD and Daxx binds to distinct surfaces of the Fas DD (Yang et  al. 1997) (Fig. 1.1). The interaction of cFLIPL with Daxx inhibits JNK activation by preventing the normal interaction of Daxx and Fas (Kim et  al. 2003).

1  Protein Kinase-Mediated Decision Between the Life and Death

Fas-induced production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-­ 8) promote chemotaxis of phagocytes toward apoptotic cells, suggesting that these factors serve as “find-me” signals (Cullen et al. 2013). Taking into consideration the other cell-death mechanisms in that, necroptosis is a newly discovered pathway of regulated necrosis that requires RIPK3 and mixed lineage kinase domain-like pseudokinase (MLKL). Necroptosis can be triggered by FasR, TRAILR1/2 or death receptor 3 in different manners but the best characterized one is TNFR1-induced necroptotic type (Pasparakis and Vandenabeele 2015). In this context, RIPK1- and RIPK3-MLKL-mediated necroptosis is the most well-known form of regulated necrosis (Vanden Berghe et al. 2014). As far as it is known, MLKL is the only substrate for RIPK3 that is essential for necroptotic cell death (Khan et  al. 2014). The activation of MLKL occurs in a multimolecular complex, which is called necrosome. As mentioned above, necrosome is mainly comprised of MLKL, RIPK3 and, in some cases, RIPK1. The phosphorylation of MLKL is necessary for its translocation and accumulation in the plasma membrane. Subsequently, MLKL mediates necroptosis by inducing the plasma membrane rupture (Rodriguez et al. 2016). Thus, the MLKL domain acts as a latch to restrain the N-terminal four-­ helix bundle domain and that unleashing this domain results in formation of a high-molecular-­ weight, membrane-localized complex and subsequent cell death (Hildebrand et  al. 2014). Although the MLKL-tethered pseudo-kinase domain is itself catalytically inactive, its necroptotic signal is induced by RIPK3-mediated ­phosphorylation. In other words, inactive pseudokinase domain functions as a molecular switch and that RIPK3-mediated phosphorylation triggers this switch by inducing a conformational change in MLKL (Murphy et  al. 2013). In this context, phosphorylation of MLKL at Threonine (Thr)357/Serine (Ser)358 residues by human RIPK3, are critical for necrotic cell death, and stimulates the oligomerization of MLKL and its translocation to plasma membranes (Sun et  al. 2012a; Vanden Berghe et al. 2016). In this event,

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after the oligomerization of MLKL and its translocation to lipid rafts of plasma membrane, MLKL complex increases the sodium influx, which enhances osmotic pressure, eventually leading to membrane rupture (Chen et al. 2014). TNF-α promotes RIPK1/RIPK3/MLKL-­ dependent necrosis. TNF receptor signaling complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of TAK1, IKKα/IKKβ, IκBα, and NF-κB (Mohideen et al. 2017). Phosphorylation targets IκBα for polyubiquitination and subsequent degradation by the proteasome, thus releasing NF-κB (Chen et  al. 1995). As it is generally known, following dissociation of NF-κB from IκB, it is activated at the multiple serine residues, before it undergoes nuclear translocation (Takada et al. 2004). In this regard, IKK/NF-κB-RIPK1 signaling pathway is a critical determinant of tissue homeostasis and inflammation. The regulation of cell death signaling plays an important role in the maintenance of tissue homeostasis (Kondylis et  al. 2017). Contrarily, in resting cells, NF-κB dimers are sequestered into the cytoplasm through interaction with IκB proteins. IKK complex has three catalytic components, which are composed of IKKα, IKKβ and IKKγ subunits. The third component of IKK regulatory complex, IKKγ is known as NEMO (Chen 2005; Pomerantz and Baltimore 2002). NF-κB activation is basically provided by two distinct pathways, which are known as the canonical and non-canonical pathways (Pomerantz and Baltimore 2002). The non-canonical pathway is characterized by a distinctive slower kinetics, requires neither IKKβ nor NEMO (Hu et  al. 2013). In the canonical pathway, which is the predominant for NF-κB signaling, cells are stimulated with TNF-α or interleukin-1β (IL-1β). Canonical signaling of IKK/NF-ĸB pathway largely depends on NEMO. In this case, NEMO functions as a regulatory non-enzymatic scaffold protein (Liu et  al. 2012). TNF-α and IL-1β induces NF-ĸB by activating the IKK complex, which phosphorylates IĸB proteins. Ubiquitination and degradation of IĸB through its phosphorylation causes the translocation of NF-ĸB to the nucleus (Pomerantz and Baltimore

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2002). As noted, the key function of the IKK required for efficient TNF-induced activation of complex is to phosphorylate IκBs and the NF-κB NF-ĸB and JNK, resulting in apoptosis inhibition precursors. Both phosphorylation and ubiquitina- (Haas et al. 2009). Deletion of TNFR1 decreases tion, whether to be destructive or not, are crucial apoptotic cell death and infiltration of immune post-translational events in these processes cells as well as production of pro-inflammatory (Scheidereit 2006). IκB kinase dependent but cytokines. These events are associated with NF-κB independent signaling events have been diminished JNK activation (Cubero et al. 2013). shown to influence various cell fate decision pro- The activation of the IKK complex is followed by cesses. IKKs not only affect cell proliferation, the phosphorylation and resultant degradation of but also cell survival pathways, in an NF-κB-­ IκB-α. Thus, free NF-κB enters the nucleus and independent manner. In this respect, different induces target gene expression (Oeckinghaus and types of polyubiquitin can be able to bind to Ghosh 2009). The activation of NF-κB signaling NEMO and may contribute to IKK activation not only directly prompts cell growth and prolif(Hinz and Scheidereit 2014). E3 ligase linear eration but also suppresses cell death by upreguubiquitin chain assembly complex (LUBAC) lating antiapoptotic molecules that inhibit the activates the canonical NF-ĸB pathway by bind- function of caspases. HOIP is cleaved by effector ing to NEMO.  In this context, LUBAC conju- caspases upon TNF stimulation. The balance gates linear polyubiquitin chains onto specific between the cell death and survival is regulated lysine residues of NEMO (Tokunaga et al. 2009). by survival signaling that participates in the supFurthermore, LUBAC forms part of the TRAIL-­ pression of the caspase cascade. In contrast, supR-­associated complex I and the secondary com- pression of the HOIP and the LUBAC functions plex II upon TRAIL stimulation and that it forms by the effector caspases in the NF-κB signaling, linear ubiquitin chains in both complexes. This attenuates the inhibitory role of NF-κB in death means that, LUBAC and M1-ubiquitin chains are signaling and further accelerates cell death or components of complex I and II of TRAIL sig- sensitizes cells to cell death (Joo et al. 2016). naling (Lafont et  al. 2017). Within both comA typical early event in NF-κB signaling is the plexes, LUBAC limits activation of caspase-8 receptor-mediated recruitment of adapter proand promotes IKK complex recruitment, thereby teins that contain protein-protein interaction restricting apoptosis and leading to pro-­ domains (Hinz and Scheidereit 2014). Adaptor inflammatory cytokine production. Consequently, protein, RIP homotypic interaction motifs LUBAC restricts TRAIL-induced necroptosis by (RHIM), play a key role in cell death. RHIM in limiting the formation of the necroptosis-­ Toll/IL-1 receptor (TIR) domain-containing mediating complex (Lafont et  al. 2017). adapter inducing IFN-β (TRIF) is essential for Moreover, LUBAC regulates the TNF signaling TRIF-induced apoptosis and simultaneously conpathway by linearly ubiquitylated NEMO leading tributes to TRIF-induced NF-ĸB activation. to the activation of IKK kinases. Co-expression of Substantially, TRIF-induced apoptosis is not the LUBAC subunits, non-catalytic dependent on its ability to activate either IFN ­Shank-­associated RH domain-interacting protein regulatory factor-3 (IRF3) or NF-ĸB but it is pri(SHARPIN), and heme-oxidized iron regulatory marily dependent on the presence of an intact protein 2 (IRP2) ubiquitin ligase-1 (HOIL1)- RHIM.  Because, an intact RHIM is required to interacting protein (HOIP) promotes linear drive assembly of this caspase-8 activating comubiquitination of NEMO and subsequent activa- plex (Kaiser and Offermann 2005). However, not tion of NF-κB signaling. Thus, the SHARPIN-­ all RHIM-mediated interactions lead to cell HOIP complex might act as an upstream regulator death. RIPK3 can bind to the RHIM for TRIF-­ of IKK activation (Gerlach et  al. 2011; Ikeda dependent necroptosis (Chan et al. 2015). In this et  al. 2011). LUBAC enhances NEMO interac- context, the RHIM is the core domain that regution with TNF-RSC and stabilizes this protein lates activation of the necrosome. To date, three complex. The formation of this complex is RHIM-containing proteins have been reported to

1  Protein Kinase-Mediated Decision Between the Life and Death

activate the kinase activity of RIPK3 within the necrosome: RIPK1, TRIF, and DNA-dependent activator of interferon regulatory factors (DAI) (Vanden Berghe et al. 2016). Of these, DAI regulates nucleotide and oligomerization domain (NOD), leucine-rich repeat-containing protein family, pyrin domain containing protein 3 (NLRP3) inflammasome activation as well as induction of apoptosis and necroptosis. In contrast, DAI deficiency reduces inflammatory responses, and epithelial damage (Kuriakose et  al. 2016). NLRP3 is a member of the NOD-­ like receptors (NLRs) family of cytoplasmic pattern recognition receptors, which plays a critical role in cell death (Ting et al. 2008). Although, the expression of NLRP3 is tightly controlled by the activity of multiple signaling receptors, its activation is dependent on priming activity by the signaling receptors in human cells. This is a critical checkpoint for NLRP3 activation. Thus, NLRP3 expression is controlled by signals culminating in the activation of NF-κB. Dual step mechanism in transcriptional activation of NLRP3 inflammasome prevents the accidental or uncontrolled NLRP3 activation (Bauernfeind et  al. 2009). NLRP3 is also activated by a two-step deubiquitination mechanism, which is initiated by the pattern recognition receptor, TLR4. Signaling by the TLR4 through myeloid differentiation primary response protein 88 (MyD88) can rapidly and non-transcriptionally activate NLRP3 inflammasome by stimulating its deubiquitination. This process is primarily dependent on mitochondrial reactive oxygen species production. Moreover, inhibition of NLRP3 deubiquitination completely blocks NLRP3 activation in human cells (Juliana et  al. 2012). Inflammasome sensor molecules connect to caspase-1 via N-terminal pyrin domain (PYD) of apoptosis-associated speck-­like protein containing a caspase recruitment domain (ASC) adaptor protein. Indeed, ASC adaptor consists of two death-fold domains: PYD and CARD. Because of the NLRP3/caspase-1 co-localization within mitochondria, ASC interacts with NLRP3 via PYD and recruit procaspase-1 via a homotypic caspase recruitment domain interaction (Vajjhala et  al. 2012). In fact, TLRs and NLRs are two major forms of innate immune sensors

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which provide immediate responses against tissue injury and stress conditions. In this context, RIPK1, RIPK3, FADD, FLIP and caspase-8 are incorporated into compatible TLR signaling modules. These modules have a high capacity to switch cell death (Blander 2014). In fact, TLRs activate distinct signaling pathways, which are mediated by the five different TIR-containing adaptor proteins known as MyD88, MyD88 adaptor-like protein (MAL), TRIF, TRIF-­related adaptor molecule (TRAM) and sterile alpha-and armadillo motif-containing protein (SARM). Furthermore, MAL recruits MyD88 to TLR2 and TLR4, and TRAM recruits TRIF to TLR4 to allow for the IRF3 activation (Fornarino et  al. 2011; O’Neill and Bowie 2007). In the last case, TLR4 activation leads to the translocation of IRF3 into the nucleus. In this manner, phosphorylation of IRF3 enhances IL-1β mRNA synthesis. Following degradation of IKK, IκBβ ensures the IRF3-regulated NF-κB activation and phosphorylation of p65. Phosphorylated NF-κB p65 is translocated into the nucleus and induces target gene expression (Bagchi et  al. 2013). Cell fate decisions following TLR signaling is parallel with death receptor signaling. In this case, RIP3-­ dependent programmed necrosis is suppressed by caspase-8, either initiated directly by TRIF-­ RIP3-­MLKL pathway or indirectly via TNF activation and the RIP1-RIP3-MLKL necroptosis pathway. In contrast, inhibition or elimination of caspase-8 during stimulation of TLRs results in RIPK3-dependent programmed necrosis that occurs through either TRIF or MyD88 signal transduction (Kaiser et al. 2013). As mentioned above, TLRs trigger innate immune responses through different signaling pathways, which is mediated by TIR domain-­ containing adaptors; such as MyD88, TIR domain-containing adapter protein (TIRAP) and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-­independent pathway from TLR3 and TLR4 (Yamamoto et al. 2003). MyD88-signaling pathway is essential for the induction of inflammatory cytokines triggered by all TLRs (Takeda and Akira 2004). In addition, TIRAP/MyD88 complex, as a second

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adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways. In this process, overexpression of TRIF activates the NF-ĸB-­dependent promoters (Yamamoto et  al. 2002). TRAF6 interacts with TRIF through the TRAF domain of TRAF6. In contrast, disruption of TRAF6-binding motifs of TRIF results in a reduction in the TRIF-induced activation of the NF-κB-dependent promoter. TRIF is mainly involved in TLR3-mediated signaling, thereby one of the roles of TRIF is to maintain the link between TLR3 and TRAF6 for NF-κB activation, independently of MyD88 and IL-1R-associated kinase (IRAK)1 (Sato et  al. 2003). IRAK1 is an essential signaling molecule required for early NLRP3-ASC oligomerization and activation, whereas IRAK4 is required for both early, and late phases of NLRP3 activation in case of prolonged TLR4 stimulation (Fernandes-Alnemri et  al. 2013). While the kinase activity of IRAK1 decreases within 1 h of TLR2 stimulation coincident with IRAK1 degradation, the kinase activity of IRAK2 is sustained and peaked at 8 h after stimulation. Thus, IRAK2 together with the IRAK4 is critical for the late phase of NLRP3 activation in prolonged TLR responses (Kawagoe et  al. 2008). Collectively, TLR stimulation triggers a rapid signaling pathway dependent on MyD88, IRAK1, IRAK2 and IRAK4, that leads to post-translational activation of NLRP3. The TIRs all contain a cytoplasmic TIR domain that makes complexes with the bifunctional adaptor protein MyD88. The DD of MyD88 recruits the kinases IRAK1 and IRAK4 into a signaling c­ omplex, via their DDs. In this case, autophosphorylation of IRAK4 leads to the activation of its kinase activity (Cushing et  al. 2014). In humans, DD containing homologues MyD88, IRAK4, and IRAK1/2 assemble into oligomeric signaling complexes to initiate a phosphorylation cascade that results in the degradation of IκB. Consequently, NF-ĸB translocates from the cytoplasm to the nucleus (Ferrao and Wu 2012). Fundamentally, TLR3 stimulation at intermediate or late periods can activate two pathways that promote NLRP3 activation. These pathways contain downstream of the TRIFRIPK1 complex depending on the nature of the

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signaling molecules. Although Caspase-8 scaffolding function is crucial in both pathways, it is required only at the late-phase of necroptotic pathway for activity of RIPK3/MLKL complex (Kang et al. 2015). Interestingly, loss of RIPK1 sensitizes primary cells to both TNFR1-FADDcaspase-8-mediated apoptosis induced by TNF, and RIPK3-mediated necroptosis induced by TLR ligation via TRIF. TRIF can recruit RIPK3 directly when TLR3 or TLR4 are stimulated and this results in the activation of TRIF-RIPK3MLKL pathway (Dillon et al. 2014). Caspase-8 inhibition leads to induction of a TRIF-RIPK1RIPK3-dependent late-phase pathway of NLRP3 activation in response to TLR3 signaling. RIPK3 and its kinase activity are not required for priming of NLRP3 by the intermediate-phase pathway. But RIPK3 and its kinase activity are needed for activation of NLRP3 by the late-phase pathway (Kang et al. 2015). In fact, MLKL is a functional substrate for RIP3K that serves as an adaptor protein for necrosis signal transduction. Contrarily, without MLKL, RIP3 fails to trigger phosphorylation on the mitochondrial enzymes that are required for necrosis downstream of the RIP1/3 kinases (Murphy et  al. 2013; Sun et  al. 2012b). In brief, MLKL is not critical for inflammasome activation in the early or intermediate pathways, but is particularly required during the late phase TRIF-RIPK1-RIPK3-dependent pathway (Kang et al. 2015). As mentioned above, Complex I leads to the sequential activation of MAPK kinase kinase3 (MAP3K3 or MEKK3). The MEKK3-dependent pathway, together with the IL-1-mediated TAK1-­ dependent one produces two parallel signaling for activation of NF-κB. The TAK1-independent but MEKK3-dependent pathway involves IKK-ɣ phosphorylation and IKK-α activation. This process results in NF-ĸB activation through IĸB-α phosphorylation and subsequent dissociation from NF-ĸB without IĸB-α degradation (Yao et al. 2007). The MAPKs are a family of serine/ threonine kinases, their activations are achieved through kinase cascades, MEKK 1–4. These cascades connect cell-surface receptors to specific transcription factors and other regulatory proteins (Su and Karin 1996). Overexpression of

1  Protein Kinase-Mediated Decision Between the Life and Death

MEKK3 increases NF-κB activity and enhances expression of cell survival molecules (Samanta et al. 2009). TLR8-mediated, MEKK3-dependent NEMO phosphorylation plays an important role in the activation of IKK complex. TLR8-mediated IĸBα phosphorylation and subsequent NF-κB activation occurs in a different manner from the classical MyD88-NF-κB pathway (Qin et  al. 2006). Both TAK1-dependent and MEKK3-­ dependent pathways are regulated at the level of IRAK.  The TAK1-dependent pathway causes IKKα/β phosphorylation and IKKβ activation, leading to classic NF-κB activation through IκBα phosphorylation and degradation. In contrast, TLR8-mediated NF-κB and JNK activation are TAK1-independent and MEKK3-dependent. This may be a regulatory mechanism at the level of receptor complexes that determines the usage of TAK1-dependent versus MEKK3-dependent pathways in TIR signaling (Fraczek et al. 2008). In this context, it is thought that IRAK in the TLR8 pathway probably utilizes MEKK3 to mediate NF-κB and JNK activation instead of TAK1 (Qin et al. 2006). In brief, the impact of the IKK/NF-κB signaling pathway in mammalian life is a continuously growing area, with the number of diseases. However, the activation process of IKK, the functions of NEMO ubiquitination, IKK-related non-canonical pathway and the nuclear transportation of NEMO and functions of IKK-α are still debated (Hinz and Scheidereit 2014). The recruitment of TAK1 and the IKK complex to the complex I leads to the activation of NF-κB, which drives the transcription of ­pro-­survival genes. In contrast, with the inhibition of NF-κB signaling or protein synthesis, the cell death-inducing complex II develops (Micheau and Tschopp 2003; Wang et al. 2008). In this context, TAK1 functions as a regulator of the TNFR1-mediated cell survival/death signaling through adjusting multiple cell death checkpoints. Thus, inactivation of NF-κB via inhibition of TAK1 is a cell death checkpoint for necroptosis. TAK1 can also regulate cell death through NF-κB-independent mechanisms involving the induction of two cell death complexes: the caspase-­8-activating complex, which consists of RIP1-FADD-caspase 8 and the necroptotic cell

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death complex, which consists of RIP1-RIP3-­ FADD (Guo et  al. 2016). The early phase of TNF-α-induced necroptosis is triggered by TAK1 inhibition, which is a NF-κB-independent death, but the late-onset necroptosis is NF-κB-­ dependent. The anti-necroptotic effect of NF-κB is related to the upregulation of FLIP and cIAPs (Guo et al. 2016). Contrarily, TAK1 prevents cell death complex formation by interacting and stabilizing RIP1  in the complex I.  In this respect, TAK1 functions as a molecular switch in TNFR-1 signaling by regulating the formation of both cell death complexes (Li et  al. 2014). Furthermore, activation of TAK1 blocks cell death complex formation and necroptotic cell death, in contrast, inhibition of TAK1 promotes the necroptotic signaling pathway (Guo et al. 2016). All apoptotic and necrotic signal transductions are conveyed through death domain-­ containing death receptors, which are expressed on the surface of nearly all human cells. In humans, eight members of the death receptor family have been identified. All can be divided into four structurally homologous groups; namely: (1) The p75 neurotrophin receptor (NTR) (consisting of ectodysplasin A receptor, death receptor 6 and p75 NTR); (2) The TNFR1 and death receptor 3, (3) The CD95 (CD95/FAS) and (4) The TRAILR: TRAILR1 and TRAILR2 (Kavuri et al. 2011; Sessler et al. 2013). TNFR1 is the prototypic member of a sub-family within the TNF receptor superfamily that contains an essential protein interaction domain called the DD. DD-containing death receptors include Fas/ CD95/ accumulation of photosystem one 1 (APO-1), TRAIL receptor 1 and 2, death receptor 3, death receptor 6 and ectodysplasin A receptor (EDAR) (Chan et  al. 2015). The DD adaptors FADD and TRADD control cellular outcomes that range from apoptosis to gene activation. Thus, the TNF-mediated TRADD-, FADD- and caspase-8-dependent apoptotic complex IIa inhibits NF-κB (Wilson et al. 2009). The stimuli perceived by these death receptor families are interpreted and transmitted to the target cells through three major signal transduction pathways, which contain multiple cell death checkpoints. Consequently, NF-κB-mediated

16

differentiation or pro-inflammatory cytokine synthesis, MAPK-mediated stress response and caspase-­ mediated apoptosis can be regulated through these receptors (Sessler et  al. 2013). Although TRAF2 or RIP can be independently recruited to the TNFR1 complex, neither of them alone is capable to transduce the TNF signal that leads to IKK activation. In response to TNF, IKK is recruited to the TNFR1 complex, and this recruitment is accomplished through TRAF2. The activation of IKK in the TNFR1 complex requires the presence of RIP (Devin et al. 2000). The major function of TNFR1, like that of death receptor 3 (TRAMP/APO-3), is to induce prosurvival and pro-inflammatory genes, in contrast to some other death receptor family members such as CD95 (FAS/APO-1), TRAILR1 (death receptor 4) and TRAILR2 (APO-2/TRICK/ death receptor 5/KILLER). Furthermore, cell-to-cellmediated death signals are induced by activation of these death receptor-ligand systems. Besides TNF itself and the CD95 (Fas/APO-1) ligand (FasL/Apo1L), the TRAIL/Apo2L belongs to the subfamily of ligands that is responsible for extrinsic induction of cell death (Falschlehner et al. 2009). CD95L is cleaved by metalloproteases and so exists in two different forms: a transmembrane form (m-CD95L) and a soluble ligand form (s-CD95L) (Le Gallo et al. 2017). Of these, transmembrane CD95L (m-CD95L) is a potent inducer of cell death (Siegmund et al. 2017). The simultaneous induction of apoptotic and nonapoptotic signaling pathways in cells exposed to cytotoxic CD95L is necessary for an efficient immune response. Fas/CD95-induced apoptosis is associated with the production of cytokines and chemokines. As mentioned above, Fasinduced production of MCP-1 and IL-8 from apoptotic cells serves as “find-me” signals for phagocytes. In addition to CD95L, RIPK1 and IAPs are required for optimal production of cytokines and chemokines in response to Fas receptor stimulation. In dying cells, production of cytokines in turn recruit professional phagocytes via a cellular inhibitor of apoptosis (Cullen et  al. 2013). Engulfing dying cells by phagocytes provides a molecular link between cell death and the

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efficient or impaired immune response (Le Gallo et al. 2017). Secondary signaling pathway in kinase pathway activation by TNF is Apo2L/TRAIL complex, which is not only a receptor-associated DISC, but also a secondary intracellular signaling complex (Varfolomeev et al. 2005). Binding of TRAIL to TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5 or KILLER) induces formation of the DISC, also termed as complex I, composed of FADD, caspase-8/10 and cFLIPL/S (Kischkel et  al. 2000). Ligation of death receptor 5 by Apo2L/TRAIL leads to rapid recruitment of FADD and caspase-8 to the receptor, forming a DISC. Caspase activation by the primary signaling complex of Apo2L/TRAIL promotes the formation of a secondary complex, which in turn leads to kinase pathway activation and apoptosis induction (Varfolomeev et  al. 2005). In brief, Apo2L/TRAIL assembles a primary complex that signals apoptosis and a secondary intracellular complex that stimulates kinase pathway activation. Activation of p38 and IKK by Apo2L/ TRAIL is closely depended on RIPK1, thereby RIPK1 depletion causes cell death that is triggered by Apo2L/TRAIL.  On the other hand, TRAF2 depletion sensitizes cells to death induction by Apo2L/TRAIL, FasL, as well as TNF (Varfolomeev et  al. 2005). In contrast to the apoptosis-initiating events triggered by binding of FasL and Apo2L/TRAIL to their corresponding death receptors, TNF binding to TNFR1 may lead to activation of the IKK/NF-κB/JNK/p38 MAPK signaling pathways. In this case, the TNF-induced formation of a multiprotein signaling complex at the cell membrane is the common feature in a wide spectrum of human diseases (Chen and Goeddel 2002). Apoptosis initiation by TNF relies on the formation of a secondary intracellular signaling complex, composed of TRADD, RIP1, and TRAF2, as well as FADD and caspase-8 (Micheau and Tschopp 2003). Death receptor activation drives the complex IIB signaling platform that includes RIP1, caspase-8, FADD, and cFLIP. This complex maintains control over caspase-8-dependent apoptosis as well as RIP3-dependent necroptosis (Kaiser et  al.

1  Protein Kinase-Mediated Decision Between the Life and Death

2013). In the NF-κB-dependent cell death checkpoint, the overexpression of TRAF1, TRAF2, cIAP-1, and c-IAP-2 may result in their increased recruitment to signaling complexes and in an augmented NF-κB response. This, in turn, upregulates caspase-­ 8 homologue cFLIP levels and thus indirectly leads to the anti-apoptotic response of the TRAF1, TRAF2, cIAP-1, and cIAP-2 proteins. As caspase-8 levels are quite stable, only small variations in cFLIP levels may decide whether a cell will respond by living or dying mediated by Fas or TNF (Micheau et  al. 2001). cFLIP, as an important NF-κB-dependent regulator of death receptor, induces apoptosis. Thereby, cFLIP has key role in the anti-apoptotic response of NF-ĸB activation (Kreuz et al. 2001). In contrast, stress signals, such as TNF-αmediated JNK activation accelerates turnover of the NF-ĸB-induced anti-­apoptotic protein cFLIP, which is an inhibitor of caspase-8. This process depends on JNK-­mediated phosphorylation and activation of the itchy (oncogene) E3 ubiquitin protein ligase, which specifically ubiquitinates cFLIP and induces its proteasomal degradation (Chang et  al. 2006). The ubiquitin-proteasome pathway plays a crucial role in both the canonical and non-­canonical pathways of NF-κB activation (Doherty et al. 2002). The canonical NF-κB pathway is activated by IKK, which phosphorylates IκB, leading to its degradation. Eventually, NF-κB transcription factors are released. The ubiquitin binding function of IKKγ was required by all the cFLIP variants. In the case of cFLIPL, the LUBAC is necessary, presumably to generate a ubiquitinated substrate to interact with IKKγ (Baratchian et al. 2016). RIPK1 is at the crossroad between life and death, downstream of various receptors as a regulator of ER stress-induced death. The pro-­ apoptotic role of RIPK1 is virtually independent of its kinase activity, is not regulated by its cIAP1/2-mediated ubiquitylation, and does not rely on the direct regulation of JNK or the transcription factor, CHOP (Estornes et al. 2014). In mammalian cells, the UPR emerges from three ER-anchored receptors: IRE1, PERK, and ATF6 (Lin et  al. 2007). Activated PERK and ATF6 have been shown to induce expression of

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the transcription factor CHOP, which promotes cell death (Tabas and Ron 2011). IRE1 may promote intrinsic apoptosis by activating JNK. Indeed, IRE1 interacts with TRAF2, allowing the downstream activation of the apoptosis signal-regulating kinase 1 (ASK1)/JNK pathway leading to apoptosis (Kim et  al. 2009b; Urano et al. 2000). In fact, the cytoplasmic part of IRE1 bound TRAF2 is an adaptor protein that couples plasma membrane receptors to JNK activation. Mechanistically, MAP kinase-JNK pathway is activated under ER stress condition and that IRE1 has a crucial role in this process by recruiting TRAF2 (Urano et al. 2000). This means that, RIP and IRE1α together with TNFR1 form a signaling complex in response to ER stress. This complex, in turn leads to JNK activation (Yang et al. 2006). Inhibition of NF-ĸB significantly decreases ER stress-induced cell death in a caspase-­ 8-dependent manner. Furthermore, blocking TNFR1 signaling also significantly inhibits ER stress-induced cell death. In contrast, ER stress induces down-regulation of TRAF2 expression, which impairs TNF-α-induced activation of NF-ĸB and JNK and initiates the apoptotic signaling through the membrane death receptors (Hu et al. 2006). However, cell death is not only dependent on death receptor signaling. Through a different pathway, cIAP1 and cIAP2 suppress TNF-α stimulated cell death by preventing formation of the TNFR1 pro-apoptotic signaling complex (Varfolomeev and Vucic 2008). In this respect, cellular IAP proteins interact directly with TRAF2, or via TRAF2, are recruited to TNF receptor-associated complexes, where they regulate apoptotic and NF-κB signaling (Rothe et al. 1995). The network of poly-ubiquitin chains tightly regulates the dynamic assembly of complex I and the subsequent activation of the NF-κB pathway. These ubiquitin chains are also required for the activation of the TAK1-IKK kinase cascade (Ting and Bertrand 2016). Eventually, inactive NF-κB is sequestered in the cytoplasm through its interaction with the IĸBs. In response to various stimuli, IĸBs are phosphorylated by IKK at serines 32 and 36 and are rapidly leads to proteasome-mediated degradation after conjuga-

18

tion with ubiquitin. The degradation of IĸBs results in the release of NF-κB in the cytoplasmic fraction and allows its translocation into the nucleus and the subsequent stimulation of its target genes (Baeuerle and Baltimore 1996). Indeed, ubiquitination is a critical checkpoint for necroptosis. The ubiquitins within Complex I functions to recruit the IKK complex and to promote survival through NF-κB dependent and independent pathways. The RIP intermediate domain binds to the signalosome, which is signaling organelle for the IkB phosphorylating complex. It is assembled into a stable macromolecular complex, containing equal amounts of three unique components, IKK1, IKK2, and the IKK regulatory subunit NEMO.  Binding of RIP to NEMO may initiate the activation of the IKK complex by TNF. NEMO together with RIP and the two other signalosome components IKK1 and IKK2, is recruited to the p55 TNF receptor upon its stimulation. Changes in the cellular levels of NEMO are directly proportional with the alteration of NF-κB activation. It may also dramatically enhance the phosphorylation of the transcription factor c-Jun. Regulation of the IKK signalosome by TNF involves interactions of NEMO with RIP within the p55-TNFR complex (Zhang et al. 2000). In brief, triggering of the p55 TNFR, induces binding of RIP to NEMO, a component of the IKK “signalosome” complex, as well as recruitment of RIP to the receptor together with the three major signalosome components, NEMO, IKK1 and IKK2 (Zhang et  al. 2000). The TNF-alpha-induced polyubiquitination of RIP1 at Lys-377 is required for the activation of IKK and NF-ĸB.  Polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, which is a regulatory subunit of the IKK complex (Ea et al. 2006). As mentioned above, almost all NF-ĸB activation pathways concentrate on IKK. Phosphorylation of IĸB with IKK results in polyubiquitination of IĸB and its degradation. Proteasomal degradation of IĸB proteins liberates IĸB-bound NF-κB transcription factors, which translocate to the nucleus. IKK activation is dependent on the attachment polyubiquitin

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chains to RIP.  IKK has two catalytic subunits, IKKα and IKKβ, and a regulatory subunit NEMO.  NEMO has critical role in IKK and NF-ĸB activation. Furthermore, RIP is destabilized in the absence of NEMO (Häcker and Karin 2006; Wu et al. 2006). Recruitment of NEMO to ubiquitinated RIP1 is a key step in the TNFR1 signaling pathway that determines whether RIP1 triggers a necrotic death response (O’Donnell et al. 2012). Indeed, the ubiquitination of RIP is required for the TNF-α-induced NF-κB activation through recruiting TAK1 to the TNFR1 complex (Li et al. 2006). RIPK1 activity and RIPK3 contributes to TNF-induced apoptosis in the conditions of cIAP1/2 depletion or TAK1 inhibition. This suggests that inhibition of RIPK1 activity or depletion of RIPK3 under cell death conditions is not always a prerequisite for the involvement of necroptosis. In this case, TAK1 inhibition induces assembly of the cytosolic RIPK1/Fas-associated protein with DD/caspase-8 apoptotic TNFR1 complex IIb. This means that TAK1 is a NF-κB-­ independent cell death checkpoint in the TNFR1 apoptotic pathway (Dondelinger et  al. 2013) (Fig. 1.2). Moreover, cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells. Constitutively ubiquitinated RIP1 associates with the pro-­ survival kinase, TAK1 (Bertrand et  al. 2008). Absence of polyubiquitination of RIP1 attenuates the upstream event that is necessary for NF-ĸB signaling. Indeed, cells missing both cIAP1 and 2 are sensitized to TNF-α-mediated apoptosis, and cytosolic cell death-inducing complex II, which is formed upon stimulation of TNFR1 (Mahoney et  al. 2008). Thus, loss of cIAPs promotes the spontaneous formation of an intracellular platform that activates either apoptosis or necroptosis. In this respect, triggering of the membrane-bound receptor is critically influenced by ripoptosome. Ripoptosome is a unique complex, which contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. As a member of this complex, cFLIP controls ripoptosome formation by blocking the association of FADD, RIP1, and caspase-10 with caspase-8. Although, ripoptosome is necessary, it is not sufficient for cell death (Feoktistova et al.

1  Protein Kinase-Mediated Decision Between the Life and Death

2011). Thus, cFLIP can regulate the formation of the death receptor-independent apoptotic platforms, through the ripoptosome. Additionally, cFLIP is also involved in a non-apoptotic cell death pathway known as programmed necrosis or necroptosis (Tsuchiya et al. 2015). The core components RIP1, FADD, and caspase-8 of ripoptosome also assemble in response to genotoxic stress-induced depletion of cIAP1 and cIAP2. It forms independently of TNF, CD95L/FASL, TRAIL, death-receptors. Furthermore, ripoptosome formation converts proinflammatory cytokines into pro-death signals. Eventually, ripoptosome can kill cells in a caspase-dependent (apoptosis) and caspase-independent (necroptosis) manner. IAP-mediated inactivation of RIP1 and ripoptosome occurs in an ubiquitylation-­ dependent manner (Tenev et al. 2011). Principally, c-IAP1 and c-IAP2 are required for activation of NF-κB and MAPK by members of the TNFR family. c-IAPs are also required for the recruitment of IKKβ, NEMO, and RBCK1/HOIP to TNFR signaling complexes. Moreover, TNFRs stimulate the NF-κB pathway by triggering the translocation of c-IAPs, TRAF2, and TRAF3 from the cytosol to membrane fractions. Later, IAP proteins and TRAF 2/3 are degraded by proteasomal and lysosomal processes (Varfolomeev et al. 2012). As mentioned above, RIPK1 as a kinase at the crossroad of life and death, has a central role in the signaling pathways, which activate downstream of several TNFR and TLR family members (Declercq et  al. 2009). The recruitment of caspase-8 to TLR3 requires RIP1. But it is negatively modulated by cIAP2-TRAF2- TRADD-E3 [cIAP2- TRAF2- TRADD-ubiquitin ligase complex (E3)] which regulates RIP1 ubiquitination. Although FADD and caspase-8 are required for TNFR1-mediated cell death, caspase-8 recruitment and activation within the TLR3 death-­ signaling complex is not dependent on FADD (Estornes et al. 2012). Nevertheless, RIP1-DD is shown to be important for binding to other death receptors, such as TNFR1, TRAIL-R1 and TRAIL-R2, and to DD-containing adaptor proteins such as TRADD and FADD (Meylan and Tschopp 2005; Stanger et al. 1995).

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As mentioned by Dondelinger et  al., RIPK1 activity and RIPK3 contribute to TNF-induced apoptosis during cIAP1/2 depletion or TAK1 inhibition (Dondelinger et al. 2013). In the same conditions, the loss of cIAPs leads to a dramatic sensitization for the CD95 ligand (CD95L)related killing process. This form of cell death can only be blocked by a combination of RIPK1 and caspase inhibitors. In these instances, cFLIPL protects cells from death by interfering with RIP1 recruitment to the DISC and complex II (Geserick et al. 2009). In fact, CD95-related pro-and anti-­ apoptotic signaling is regulated at the DISC, complex II and the mitochondria levels. Especially, procaspase-8 and c-FLIP are accepted as two DED proteins, which are essential for the initiation of the apoptotic and non-apoptotic signals at the level of CD95-DISC (Lavrik and Krammer 2012). In addition, control of the E3 ligase activity of cIAPs is critical for their antiapoptotic function. Thus the ubiquitin chains generated by cIAP1/2 allow further recruitment of LUBAC, which adds complexity to the ubiquitin network by conjugating RIPK1, NEMO, TRADD and TNFR1 with M1-linked poly-ubiquitin chains (Haas et  al. 2009; Tokunaga et  al. 2009). In fact, poly-­ ubiquitin (pUb) chains are produced by the formation of a peptide bond between the α-amino group of the N-terminal methionine (M) of one ubiquitin and the C-terminal glycine of another ubiquitin. This compound is termed M1-linked linear polyubiquitin (M1-pUb) chains (Kirisako et  al. 2006). LUBAC generates M1-pUb chain and specifically regulates the canonical NF-κB pathway. Specific deubiquitinases inhibit LUBAC-induced NF-κB activation by different molecular mechanisms. LUBAC and linear ubiquitination-­ regulating factors contribute to immune and inflammatory processes and apoptosis (Tokunaga 2013). Indeed, the interactions between deubiquitinases and the LUBAC ubiquitin ligase are involved in controlling the extent of TNF-α-induced NF-κB activation in cells by the generation of linear ubiquitin chains by LUBAC (Takiuchi et al. 2014). Selective binding of NEMO to linear ubiquitin chains is essential for NF-kB activation by TNF-α

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and other agonists. Since the IKKa and IKKb molecules are present in complex with the regulatory component NEMO, linear ubiquitin chains catalyzed by the E3 complex LUBAC affect the IKK complex via direct binding to the neighboring NEMO. Consequently, NEMO binding to linear ubiquitin chains plays an important role in the activation of the canonical NF-κB pathway (Rahighi et al. 2009). As indicated in cell death pathways, NF-ĸB activation involves three steps of ubiquitination, which are consisted of proteasomal degradation of IĸB, processing of NF-ĸB precursors, and activation of TAK1 and IKK complexes (Skaug et al. 2009). From these alternatives, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, all options result linear pUb of NEMO, which plays crucial roles in IKK activation (Fujita et al. 2014). As previously reported by Pomerantz et  al., NF-κB is inactive in resting cells as it resides in the cytoplasm bound to inhibitor proteins called inhibitors of NF-κBs. In the case of NF-ĸB activation, the ubiquitin mediated protein degradation system is involved in both the canonical and non-canonical pathways (Chiu et  al. 2009; Pomerantz and Baltimore 2002). The canonical pathway is activated by most NF-κB stimulatory ligands, including TNF-α and IL-1β (Chiu et al. 2009). Thus, in the same metabolic pathway, the IKK complex is also activated upon stimulation with inflammatory cytokines or TLR ligands (Iwai 2014) (Fig.  1.2). During the stimulation with TNF-α, in addition to adaptor molecules, ­proteasomal degradation of IĸB, processing of NF-ĸB precursors, and activation of the TAK1 and IKK complexes steps in NF-ĸB activation are directly subjected of ubiquitination (Skaug et al. 2009). TRAF1, TRAF2 and TRAF3 bind to the intracellular receptor through two different binding sites. However, TRAF1 and TRAF3 have opposite effects to TRAF2 on NF-κB activation via TNFR2 (Cabal-Hierro et  al. 2014). In fact, ubiquitin ligases are critical components of the ubiquitination process. Thus, cIAP1 and cIAP2 suppress TNF-α stimulated cell death by preventing formation of the TNFR1 pro-apoptotic signaling complex (Varfolomeev and Vucic 2008;

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Vaux and Silke 2005). The recruitment of Really Interesting New Gene (RING) domain-­containing c-IAP1 or c-IAP2 to the TNFR2 signaling complex requires a TRAF2-TRAF1 heterocomplex. TRAF1 and TRAF2 can associate with the cytoplasmic domain of TNFR2 as a heterodimeric complex in which only TRAF2 contacts the receptor directly (Bradley and Pober 2001; Varfolomeev and Vucic 2008; Vaux and Silke 2005). Because of the absence of an N-terminal ring domain, TRAF1 is not arranged into C-rich associated with RING and TRAF (CART) domains. This property makes TRAF1 unique amongst the TRAF family. Although, TRAF proteins acts as intracellular adaptors, their association with different intracellular proteins facilitate their recruitment to cell surface receptor complexes. Both homo- and heterodimers mediated by TRAF-RING domains have the capacity to synthesis ubiquitin chains (Bradley and Pober 2001; Middleton et al. 2017). Thus, control of the E3 ligase activity of cIAPs is critical for their antiapoptotic function. In this case, the RING domain mediates ubiquitination by facilitating the direct transfer of ubiquitin from ubiquitinconjugating enzymes (E2s) to lysine residues on the target substrate. RING-­type ubiquitin ligases (E3s) act together with E2s to mediate ubiquitination and are implicated in cellular processes (Metzger et  al. 2014). Furthermore, TRAF3 recruits a TRAF2-cIAP1-­cIAP2 E3 complex to NF-κB-inducing kinase (NIK), which is a key component of the noncanonical pathway of NF-κB activation. Induction of noncanonical NF-κB signaling by extracellular signals involves degradation of TRAF3 and the concomitant enhancement of NIK expression (Liao et  al. 2004; Vallabhapurapu et al. 2008). In fact, noncanonical NF-κB pathway is activated through non-death TNF receptor members. In this respect, ultimately activated two kinases NIK as well as IKKα decide for life or death of cells (Remouchamps and Dejardin 2015). The RING domain activity and the specific binding of the TRAF domain to NIK are two critical components of TRAF3 suppression of NIK protein levels (He et  al. 2007). As a central signaling component of the noncanonical NF-κB pathway,

1  Protein Kinase-Mediated Decision Between the Life and Death

NIK is a MAP3K member. While the level of NIK is extremely low in resting cells, in the stimulated cells NIK accumulation triggers the downstream signaling events in non-canonical NF-κB pathway. The NIK accumulation is a result of both its stabilization and de novo synthesis. NIKubiquitin ligase complex, TRAF2 directly interacts with cIAP1/2. TRAF3, connects to cIAP1/2 via dimerization with TRAF2, and serves as an adaptor to recruit TRAF-cIAP E3 complex to NIK (Sun 2011). Collectively, NIK activation is a slow process that comprises signal-induced TRAF3 degradation and NIK accumulation. This process is a result of NIK stabilization, and de novo synthesis. Induction of noncanonical NF-κB signaling is a consequence of the escape of NIK from TRAF3-mediated negative regulation (Liao et  al. 2004). In this context, following RING domain dimerization, increased RING-type ubiquitin E3 ligase activity of cIAP1 and cIAP2 plays important roles in NF-κB regulating pathways. The Ubiquitin-associated (UBA) domain enhances the activity of cIAP1 by binding to ubiquitin chains. Resultant high levels of IAPs inhibit caspase-­mediated apoptosis (Budhidarmo and Day 2014; Gyrd-Hansen and Meier 2010). Both TNFR1 and TNFR2 can activate canonical NF-κB and JNK-MAPK signaling, while TNFR2, alone can also activate non-canonical NF-κB signaling. RING protein is a key player in TNFR1and TNFR2-induced signaling. TRAF2 shows a multiple receptor-specific functions (Borghi et al. 2016). In other words, the integrity of the TRAF2RING domain plays a critical role in JNKdependent gene expression in response to TNF-α stimulation, whereas it has an important role in efficient suppression of the non-canonical NF-κB pathway in resting cells. Therefore, in addition to NF-κB-dependent expression of anti-­ apoptotic proteins such as cIAP1/2 and cFLIP, TRAF2 RING domain-dependent retention of cIAP1  in the TNFR1 complex might be required for the inhibition of TNF-α-induced cell death (Zhang et al. 2010). In both canonical and non-canonical pathways, IKK is key to NF-κB activation. Supporting evidences show that ubiquitination and deubiquitination play a central role in IKK regulation by diverse NF-κB signaling pathways

21

(Chen 2005; Krappmann and Scheidereit 2005). These signaling complexes regulate apoptotic and NF-κB signaling. The activation of NF-ĸB blocks the activation of caspase-­8. These proteins are required to fully suppress TNF-induced apoptosis, when NF-ĸB is inactive (Rothe et al. 1995; Varfolomeev and Vucic 2008; Vaux and Silke 2005; Wang et al. 1998). In the non-canonical NF-κB pathway, cIAP proteins are negative regulators that ubiquitinate NIK, causing its proteasomal degradation and inhibition of signaling (Varfolomeev et al. 2007). Through TRAF2 interactions, cIAP1 and cIAP2 are recruited to TNFR1- and 2-associated complexes where they regulate receptor-mediated apoptosis. TRAF1, TRAF2, and cIAP1, cIAP2 are targets of NF-κB transcriptional activity. (Wang et al. 1998). Within minutes after stimulation, TNFR1 assembles complex-I by recruiting the adaptors TRADD and TRAF2, the kinase RIPK1, and the E3 ubiquitin ligases cIAP1 and cIAP2 (Silke 2011; Ting and Bertrand 2016). In this case, cIAP1 and cIAP2 proteins are recruited to TNFR1-associated signaling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity (Dynek et al. 2010). RING domain contains cellular IAP1 and IAP2 (c-IAP1 and c-IAP2) (Vaux and Silke 2005). LUBAC, composed of SHARPIN in postsynaptic density, HOIL-1L and HOIP.  SHARPIN-­ containing complexes can linearly ubiquitinate NEMO and activate NF-κB.  Deletion of SHARPIN drastically reduces the amount of LUBAC, which resulted in attenuated TNF-α-­ mediated activation of NF-κB (Tokunaga et  al. 2011). Indeed, SHARPIN deficiency leads to rapid cell death upon TNF-α stimulation via FADD- and caspase-8-dependent pathways. In contrast, SHARPIN activates NF-κB and inhibits apoptosis via distinct pathways (Ikeda et  al. 2011). In brief, LUBAC is a linear ubiquitination complex of the NEMO, which is required for NF-κB activation (Tokunaga et  al. 2009). Moreover, in the absence of LUBAC components, TNF fails to activate canonical NF-ĸB effectively, and consequently, cFLIP levels are insufficient to prevent caspase-8-mediated cell

22

death. Under normal conditions, cFLIPL suppresses TNF-induced cell death by heterodimerizing with caspase-8. This inhibits formation of complex-II and the necrosome by cleaving RIPK1, RIPK3. Eventually, NF-κB signaling is attenuated, apoptosis and inflammation is induced (Feng et  al. 2007; Oberst et  al. 2011; Tokunaga 2013). As mentioned above, the harmful effects of TNF are initiated when TNF binds to its receptors on the surface of target cells. TNFR1 is ubiquitously expressed. TNF binds to TNFR1 and composes the TNF-RSC (Walczak 2011). LUBAC attaches linear chains to NEMO and possibly other TNF-RSC components, thereby stabilizing the entire complex and increasing the retention times of NEMO and other signaling components. LUBAC, in addition to promoting retention of NEMO in the TNF-­ RSC, helps retain cIAP1, TAK1, RIP1, and TRAF2 recruitment to the TNF-RSC.  LUBAC recruitment is diminished in the absence of TRAF2 and almost undetectable in the absence of cIAP1 and 2. cIAPs are required to recruit LUBAC, and LUBAC activity in turn helps retain cIAPs within the complex, thereby ultimately increasing the activation of NF-κB and JNK (Haas et  al. 2009). During TNF-α -mediated NF-κB activation, RIP1 is modified with linear ubiquitin in the TNF receptor-signaling complex. By enabling linear ubiquitination in the TNF receptor signaling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation (Gerlach et al. 2011). Following TNF stimulation, the adaptor molecule RIP1 recruits to the TNF receptor, becomes poly-ubiquitinated by K63-linked chains, and recruits the IKK signaling complex in NEMO-­ dependent manner. Simultaneous recruitment of another kinase complex, the TAK1/TAB complex allows phosphorylation and activation of the IKK kinase subunits by TAK1. Alternatively, pUb NEMO may directly recruit the TAK1/TAB complex (Laplantine et al. 2009). Nuclear IKK complex have been found to act directly at the chromatin level of induced genes and to mediate responses to DNA damage (Kovalenko and Wallach 2006; Scheidereit 2006). IKKβ and NEMO are essential for rapid NF-ĸB activation

A. Engin

by proinflammatory signaling cascades, that are triggered by TNF-α (Häcker and Karin 2006). The amount of RIPK1 in the NEMO complex can be regarded as an indirect measure of RIPK1 ubiquitination. Polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO (Ea et al. 2006). RIP is also destabilized in the absence of NEMO binding and undergoes proteasomal degradation. This indicates the presence of critical role of NEMO in IKK activation, and a link between cytokine-­ receptor proximal signaling and IKK and NF-ĸB activation (Wu et al. 2006). In fact, IKKε, TRAF associated NF-ĸB activator (TANK)-binding kinase (TBK1), NF-ĸB-activated kinase (NAK), or TRAF2-associated kinase exhibit structural similarity to IKKα and IKKβ. These protein kinases mediate phosphorylation of IĸB proteins and represent a convergence point for most signal transduction pathways leading to NF-ĸB activation (Häcker and Karin 2006). In contrast, in the absence of ubiquitination, RIP1 serves as a pro-­ apoptotic signaling molecule by engaging caspase-­ 8. Therefore, RIP1 is a dual-function molecule that can be either pro-survival or pro-­ death depending on its ubiquitination state, and this serves as an NF-ĸB-independent cell-death switch early in TNF signaling (O’Donnell et al. 2007). Even there is no defect in TNF-mediated RIPK1 ubiquitylation or NF-ĸB/MAPK activation in MK2-deficient conditions, loss of NF-ĸB signaling can sensitize cells to TNF-induced death. MK2 not only phosphorylates RIPK1  in complex-I but also modifies a substantial pool of RIPK1 outside of this complex (Jaco et al. 2017). RIPK1, a master regulator of cell fate decisions, is identified as a direct substrate of MK2 in the TNFR1 signaling pathway. Hence, MK2 phosphorylation of RIPK1 is a crucial checkpoint for cell fate in inflammation and infection (Menon et al. 2017). Thus, phosphorylation of RIPK1 by MK2 limits cytosolic activation of RIPK1 and the subsequent assembly of the death complex that drives RIPK1 kinase-dependent apoptosis and necroptosis (Dondelinger et al. 2017). Inhibition of IKKα/IKKβ or its upstream activators sensitizes cells to death by inducing RIPK1 kinase-­ dependent apoptosis or necroptosis. Furthermore,

1  Protein Kinase-Mediated Decision Between the Life and Death

the IKK complex phosphorylates RIPK1 at TNFR1 complex I and protects cells from RIPK1 kinase-dependent death in NF-ĸB-independent fashion (Dondelinger et al. 2015).

4

Conclusion

Although targeting RIPK1 seems to be the best therapeutic strategy, combination with inhibitors of other cell death pathways provide superior advantage. Interaction between NF-ĸB and JNK determines the outcome of TNF-α signaling by inducing cFLIPL.  Thus, even subtle changes in the relative activities of NF-κB and JNK1 can have rather dramatic effects on the ability of TNF-α to induce cell survival versus cell death. Moreover, TNF is a bifunctional molecule that can increase both cell survival and cell death. In cell death, the balance between these two opposing responses is achieved by the generation of appropriate protein kinase activation. In fact, NF-κB/IKK pathway is an attractive target, however its therapeutic interventions in human disease are still very limited. Classical cell death inducers, caspase-8 and RIPK3 cause inflammasome activation. RIPK3-mediated phosphorylation triggers this switch by inducing a conformational change in MLKL.  Although RIPK3-MLKL-mediated necroptosis is the well-­ known form of regulated necrosis, the precise mechanisms of this interaction is waiting for clarification. On the other hand, in both canonical and non-­ canonical pathways, IKK is key to NF-κB activation. Proteasomal degradation of cIAP1 and cIAP2, via IAP antagonists trigger activation of the canonical and noncanonical NF-κB pathways. Noncanonical NF-κB signaling is largely controlled by NIK. Canonical signaling of IKK/ NF-ĸB pathway largely depends on NEMO.  In the absence of LUBAC components, TNF fails to activate canonical NF-ĸB effectively. Interestingly, signals from the same receptor discriminates to activate the canonical pathway from noncanonical pathway, and also connects NIK and IKK signals to activate the noncanonical pathway with a different kinetics of cell death.

23

Interaction between different modalities of cell death in  vivo is an attractive topic for further research and will generate further insight into the therapeutic targeting of protein kinases and their role for regulation of life and death decision.

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Aging and Protein Kinases Ayse Basak Engin and Atilla Engin

Abstract

Keywords

Recently, aging has been tried to be explained with large numbers of theories, but none of them can elucidate the changes occurring in the aging process alone. A unified theory encompassing the mechanisms of genetic factors and repair systems in aging is becoming increasingly required. Almost 37 protein kinases contribute to all processes of aging and senescence. Furthermore, these kinases not only regulate the large number of metabolic pathways related to aging processes, but also control these pathways through 12 checkpoints. Thus, in this chapter, the metabolic targets of protein kinases signal transduction pathways were discussed in terms of the aging perspective under five headings, which are the indispensable stages of the aging process. Although the most popular classical aging theories have been stated as DNA damage theory, mitochondrial theory, free radical theory, and telomere theory, it was concluded that the aging process is controlled by protein kinases regardless of the different theories.

DNA damage · DNA damage response (DDR) · Base excision repair (BER) · Nucleotide excision repair (NER) · Ataxia-­ telangiectasia mutated (ATM) · Ataxia- and Rad3-related (ATR) · DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) · Polo-like kinase-1 (PLK1) · Mammalian target of rapamycin complex 1 (mTORC1) · Insulin-like growth factor signaling (IIS)

A. B. Engin (*) Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey A. Engin Department of General Surgery, Faculty of Medicine, Gazi University, Ankara, Turkey

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Introduction

The causal relationship between the biological changes that occur with time and aging is not fully understood. Numerous theories of aging have been proposed, but none of these fully explained all aspects of aging (Maynard et  al. 2015). According to program hypotheses, although the imbalance on chemical processes caused by differential gene expression and hormonal changes may contribute to aging, there is no pre-determined timeline for aging (da Costa et al. 2016). Nevertheless, it is thought that aging can be clarified by four different theories such as, evolutionary theory, DNA damage/repair theory, the mitochondrial and free radical theory, telomere theory (Maynard et al. 2015).

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_2

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The evolutionary theory of aging claims that aging is the result of a decline in the force of natural selection. Epigenetic and posttranslational processes together with environmentally influenced genetic pathways play key-roles in aging and strongly affect gene expressions through different ways such as the posttranslational modifications of proteins, the production of harmful peptides due to proteolysis, the age-related uncoupling in elastin receptors (Robert et  al. 2010; Tosato et al. 2007). Age-related increases in somatic mutations and other forms of DNA damage suggest that the capacity for DNA repair is an important determinant of the rate of aging at the cell and molecular levels (Promislow 1994). Thus, poly (adenosine diphosphate (ADP)-ribose) polymerase-1 (PARP-1) is a key player in the immediate cellular response to stress-induced DNA damage. DNA-base excision repair (BER), DNA-damage signaling, regulation of genomic stability, and regulation of transcription and proteasomal function is correlated with cellular poly (ADPribosyl)ation capacity of mammalian life. While PARP-1 overactivity can cause cell suicide by nicotinamide adenine dinucleotide (NAD+) depletion (Bürkle 2001), higher PARP-1 activity levels are associated with longer life spans. Thus, a higher poly (ADP-ribosyl)ation capacity in cells from long-­lived species showed that it might contribute to the efficient maintenance of genome integrity and stability over their longer life span (Grube and Bürkle 1992). On the other hand, in many human somatic tissues, decline in cellular division capacity with age is linked to the fact that the telomeres, which protect the ends of chromosomes, get progressively shorter as cells divide (Kim Sh et al. 2002). The loss of telomeric DNA is commonly attributed to the so-called “end replication” problem. Oxidative damage is repaired less well in telomeric DNA than elsewhere in the chromosome, and oxidative stress accelerates telomere loss. Oxidative stress has an even bigger effect on the rate of telomere loss as compared to other factors. (von Zglinicki 2002). According to “Mitochondrial Theory”, an important connection between molecular stress and

A. B. Engin and A. Engin

aging is ­suggested by the accumulation of mitochondrial DNA (mtDNA) mutations with age (Wallace 1999). mtDNA damage and mutations may accumulate with advancing age until they reach a threshold level where they influence the bioenergy capacity of the cell or tissue. So, in elderly subjects, there is very low activity of cytochrome c oxidase (COX), which is indicative for mtDNA defect. This means that, age-related increases in the frequency of COX-deficient cells are associated with mtDNA mutation (Brierley et  al. 1998; Kopsidas et  al. 1998, 2000). Consequently, when mtDNA mutation reaches a high level which is likely to suffer from impaired adenosine triphosphate (ATP) production, results in a decline in tissue energy production. Actively proliferating cells, which suffer from somatic mutations and telomere erosion because of the repeated requirement for DNA replication, are more vulnerable than postmitotic cells. If proliferating cells reduce their rate of division in the remaining of their lives, a corresponding shift develops in the balance between different mechanisms of molecular damage accumulation due to intrinsic molecular aging (Kirkwood 2005). Furthermore, dysfunctional mitochondria not cleared by autophagy in senescent cells produce reactive oxygen species (ROS), which cause cellular damage including DNA damage (Weichhart 2018). In fact, senescence provides a barrier against tumor development by preventing proliferation of cells with damaged DNA (Müllers et al. 2017). Mitochondrial oxidative stress is not considered to have a completely negative effect on age-­related parameters. Mitochondrial stress and ROS are assumed to be able to form protective mechanisms as well as damage. In this adaptive response, induction of the mitochondrial unfolded protein response causes an increased lifespan in the absence of overt mitochondrial dysfunction (Bennett and Kaeberlein 2014). This idea is referred as “mitohormesis” (Ristow and Zarse 2010). In this context, the free radical theory of aging postulates that aging changes are caused by free radical reactions. These are classified as follows: (1) the relationship of the average life spans and basal metabolic rates, (2) the effect

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of food restriction on endogenous free radical reactions and life span, (3) the increase in autoimmune disorders and aging-related changes in immunological functions of T cells, (4) the effect of ionizing radiation, (5) the effects of free radical in the pathogenesis of specific diseases (Effros 2005; Harman 1981, 1992, 1993). Of these, oxidative damage due to accumulation of reactive oxygen and nitrogen radicals, is the main inducer of aging and adversely affects the aging process (Liu and Xu 2011). ROS are thought to generate as many as 50,000 DNA lesions per human cell per day including base modifications, single-­ strand breaks (SSBs), double strand breaks (DSBs), and inter-strand cross-links (Lindahl 1993; Maynard et al. 2015). On the other hand, it has been proposed that telomere length may not be a strong biomarker of survival in older individuals, but it may be an informative biomarker for healthy aging (Babizhayev et  al. 2014). In fact, telomeres localize at the ends of chromosomes in humans, and repeat over 2000 times TTAGGG sequence. Telomere shortening, which is sensitive to oxidative stress, promotes leading to the cessation of cell division and this results in cellular senescence (Prasad et al. 2017). Although telomere lengths within cells are highly variable, cells accumulate short telomeres as they approach senescence (Martens et al. 2000). The accumulation of telomere attrition over time depends both on the number of cell divisions and on the number of base pairs lost per DNA replication (Boonekamp et  al. 2017). Since the aforementioned theories concern the various cellular and molecular levels, it is unexpectedly complicated to explain all aspects of aging in a single frame. Nevertheless, a unified theory encompassing the efficiency of genetic maintenance and repair systems is becoming increasingly required. Protein kinases are the common regulator of multiple metabolic pathways involved in the pathophysiology of aging and the age-related processes. Therefore, in this chapter the metabolic targets of protein kinases signal transduction pathways were discussed in terms of aging perspective. It is thought that this debate will reveal common aspects of the aging phenomenon.

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2

 NA Damage, DNA Repair D Pathways and Protein Kinases

Genomic DNA damage is a type of environmental stress, and when cells are exposed to gene-­ stress, cell cycle progression is delayed by a mechanism called the DNA damage checkpoint. If cells must proliferate, it is necessary to down-­ regulate the checkpoint response in order to tolerate the cellular DNA damage stresses (Wakida et  al. 2017). Among the pro-mitotic activities, human cyclin-dependent-kinases (CDKs) are the primary driving forces that order and coordinate cell cycle events. S phase and mitotic onset are brought about by the action of multiple different cyclin-CDK complexes (Swaffer et  al. 2016). The CDKs, cell division control protein 2 (cdc2)cdc13 complex can control the onset of S phase and, in addition, ensures that there is only one S phase per cell cycle (Stern and Nurse 1996). CDK activity persists at low levels after DNA damage. This low level of CDK activity allows both cell cycle progression and promotes cell cycle exit at a decision point in G2 phase. (Müllers et al. 2017). Cells evolve a highly organized and coordinated effort to ameliorate genotoxic stress called the DNA damage response (DDR). This response underlies the ability to sense and signal problems in its DNA, to arrest cell-cycle progression with cell-cycle checkpoints and activate appropriate DNA repair mechanisms, or to eliminate cells with unrepairable genomes (Maréchal and Zou 2013). The DDR is a molecular mechanism that cells have evolved to sense DNA damage, transduce these signals and promote their repair. Cells have DDR activity that continually monitor the integrity of the DNA and function, by this way, prevents the occurrence of deleterious mutations and rearrangements (Harper and Elledge 2007; Lovejoy and Cortez 2009). The DDR signaling pathway is activated by aberrant DNA structures induced by DNA damage or DNA replication stress. The sensors of this pathway are the proteins that directly recognize these aberrant DNA structures and activate the most upstream DDR

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kinases. The DDR signaling pathway consists of a protein kinase cascade as well as mediator proteins that facilitate the phosphorylation events within the DDR network (Maréchal and Zou 2013). Prominent DNA repair pathways in mammalian cells are BER that removes damaged bases, nucleotide excision repair (NER) that removes bulky DNA adducts, and mismatch repair (MMR) that recognizes base incorporation errors. BER excises mostly oxidative and alkylation DNA damage. NER removes bulky, helix-distorting lesions from DNA.  MMR reverses replication errors. Breaks in the DNA backbone are repaired via double-strand break repair (DSBR) pathways. DSBR is achieved by either error-prone rejoining of the broken DNA ends nonhomologous end joining (NHEJ) or accurately repairing the DSB using information on the undamaged sister chromatid, homologous recombination (HR) (Iyama and Wilson 2013; Maynard et al. 2015; Sirbu and Cortez 2013; Wyman and Kanaar 2006). The repair of more complex lesions involving multiple DNA processing steps is regulated by DDR (Lovejoy and Cortez 2009). Cell division is closely connected to DDR, which involves DNA repair pathways that reverse DNA lesions, as well as checkpoint pathways that inhibit cell cycle progression while repair occurs. CDKs are involved in the DDR, especially in DNA repair by HR and in activation of the checkpoint response (Trovesi et  al. 2013). In any case, the DDR signaling pathway is activated by aberrant DNA structures, which are induced by DNA damage or DNA replication stress. The sensors of this pathway are the proteins that directly recognize these aberrant DNA structures and activate the upstream DDR kinases (Maréchal and Zou 2013). During the S phase, since they are required to faithfully replicate their genomes, eukaryotic cells create multiple replication forks where DNA synthesis is carried out (Bell and Dutta 2002). Mannose receptor, C type 1 (Mrc1) is a structural component of the replication fork required for both DNA replication and checkpoint signaling. Thus, in response to DNA replication interference, the Mec1 kinase is recruited to the sites of replication blocks and phosphory-

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lates a component of the DNA ­replication complex (Osborn and Elledge 2003). Rad9 and Rad53, as well as other DNA damage checkpoint proteins such as Mec1, Mec3, checkpoint kinase (CHK)1 and Dun1, are required for complete DNA-damage-induced cell-cycle arrest after loss of telomerase function. Loss of telomere capping and loss of telomere sequences provokes telomeric senescence (Grandin et  al. 2005). The highly conserved effector kinases Rad53 and CHK1 are directly targeted by phosphatidylinositol-­ 3 kinase-related kinases (PIKK) and are responsible for the amplification of the checkpoint signal, as well as for the phosphorylation of key proteins that modulate different aspects of cellular physiology (Longhese et al. 2003). The DDR is regulated by the PIKKs and they phosphorylate hundreds of proteins that maintain genome integrity through regulation of cell cycle progression, DNA repair, apoptosis, and cellular senescence. Thereby, PIKKs play central roles in the control of cell growth, gene expression, and genome surveillance and repair in eukaryotic cells. In this respect, mammalian cells express mainly six different PIKKs: ataxia-­telangiectasia mutated (ATM), ataxia- and Rad3-­related (ATR), mammalian target of rapamycin (mTOR), DNAdependent protein kinase catalytic subunit (DNA)-PKcs, human suppressor of morphogenesis in genitalia (hSMG-1) and transformation/ transcription domain-associated protein (TRRAP) (Abraham 2004; Lempiäinen and Halazonetis 2009; Lovejoy and Cortez 2009). Upon senescence-inducing signals, ATM and ATR pathways initiate a cascade of phosphorylations that prevent the inhibition of the p53 and inhibit cell division cycle 25A protein (Cdc25A). These DDR reactions provide the activation of a DNA damage checkpoint, which arrests cell cycle progression as to allow for repair, and prevention of the transmission of damaged or incompletely replicated chromosomes. In this process, the resulting DNA repair mechanisms includes direct repair, BER, NER, DSBR, and cross-link repair (Sancar et al. 2004). The ATM kinase initiates a major signaling pathway that particularly responds to double-strand breaks, which are among the most severe genomic lesions. Virtually,

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a genome-wide proteomic screen identifies over 700 protein targets that are potentially phosphorylated by ATM (Matsuoka et al. 2007). Moreover, as the molecular markers of DNA damage kinases ATM and ATR are activated in senescent cells. These markers include nuclear foci of phosphorylated histone H2AX and their co-localization with DNA repair and DNA damage checkpoints. Senescent cells contain activated forms of the DNA damage checkpoint kinases, CHK1 and CHK2. Inactivation of these checkpoint kinases in senescent cells can restore cell-cycle progression into S phase (d’Adda di Fagagna et al. 2003). ATR kinase activity is necessary for the rapid exchange of ATR at DNA-damage-sites, which in turn promotes CHK1-phosphorylation. ATRkinase-dead (KD) traps a subset of ATR and replication protein A (RPA) on chromatin, where RPA is hyper-phosphorylated by ATM/DNA-­ dependent protein kinase, catalytic subunit (DNA-PKcs) and prevents downstream repair (Menolfi et  al. 2018). Mechanistically, DNA damage response is initiated by the activation of PIKKs (ATM, ATR, DNA-PKcs), phosphorylation of histone H2AX and is continued by the recruitment of the Mre11-Rad50-Nbs1 or the Rad9-Hus1-Rad1 complex to damage sites (Ciccia and Elledge 2010). Once it is recruited to DNA damage sites, the ATM kinase rapidly activates many downstream target proteins via phosphorylation, thereby causing checkpoint proteins to trigger cell cycle arrest, and DNA repair enzymes to fix the damaged DNA (Matsuoka et  al. 2007). If DNA damage exceeds a certain threshold, it is inevitable that the cells undergo apoptosis or aging (d’Adda di Fagagna 2008). The DNA dependent protein kinase (DNA-PK) is a trimeric nuclear complex consisting of a large protein kinase and the Ku heterodimer that regulates kinase activity (Jin et al. 1997). The localization of PIKKs to subcellular compartments promotes kinase activation by concentrating each kinase with an activating protein. ATM, ATR, and DNA-PK are activated through protein-protein interactions with Mre11, Topoisomerase ­IIβ-­ binding protein (TopBP1), and Ku70/80, respectively (Lovejoy and Cortez 2009). Persistent DNA double-strand breaks act as

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silencing initiation sites. TopBP1 is essential for localization to the X chromosome of silencing sensors, and effectors, including ATR, γH2AFX, and canonical repressive histone marks (ElInati et  al. 2017). TopBP1 directly stimulates ATR kinase activity via binding to the surface of ATRinteracting protein (ATRIP) within the ATRATRIP complex (Kumagai et al. 2006). However, TopBP1 is not required for the localization of ATR to sites of damage or for the basal kinase activity of ATR (Mordes et  al. 2008). TopBP1 contacts with a region on ATR called the PIKK regulatory domain (PRD) that lies between the kinase and FK506 binding protein12-rapamycin associated protein (FRAP)-ATM-TRRAP carboxy-terminal FRAP-ATM-TRRAP-C-terminal (FATC) domains (Mordes et  al. 2008). PIKKs contain an extreme C-terminus called the FATC domain. FATC domain contributes to phosphorylation of target proteins (Ogi et al. 2015). These interactions are essential for ATR activation and checkpoint signaling. TopBP1 functions in both replication initiation and DDR signaling checkpoint responses both within S phase and following DNA damage (Garcia et  al. 2005). PRD is important for DNA-­ PKcs regulation (Mordes et al. 2008). Ku70/80 combined with DNA ends induces DNA-PKcs kinase activity. Mutations in the DNA-PKcs PRD block autophosphorylation, thereby, Ku70/80 may contact the C-terminal region of DNA-PKcs containing the PRD (Lovejoy and Cortez 2009; Mordes et al. 2008). The signal, that activates ATM and DNA-PKcs is a DSB. While ATR responds to DNA replication blocks, it also leads to the formation of single stranded DNA (ssDNA) gaps. All three kinases are gathered to the DNA lesion site, which promotes kinase activation (Lovejoy and Cortez 2009; Shiloh 2003). Actually, DSB is a cytotoxic DNA lesion caused by oxygen radicals, ionizing radiation, and radiomimetic chemicals. Cells fight with DNA damage by activating the DDR, which leads to either the damage-repair and cellular survival or to the programmed cell death. However, approximately 40% of DSB-induced phosphorylations are ATM-­independent (Bensimon et al. 2010). As mentioned above, the sixth PIKK family member, TRRAP, unlike the

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other PIKKs, does not possess kinase activity. It functions as a cofactor of oncogenic transcription factor pathways (McMahon et al. 1998). Analysis of proteins, which are phosphorylated in response to DNA damage recognized by ATM and ATR has indicated the presence of more than 900 regulated phosphorylation sites encompassing over 700 proteins. In response to DNA damage, hundreds of proteins are phosphorylated at Ser/ThrGlu motifs and additional sites in an ATM- or ATR-­dependent manner, whereas kinase activity of DNA-PKcs affects a smaller number of targets (Matsuoka et al. 2007). By analyzing 2689 phosphorylation sites in wild-type and various kinase-­ null cells, 62 phosphorylation sites from 55 proteins have been found to be controlled by the DNA damage checkpoint (Smolka et  al. 2007). The Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and accumulates the ATM at the broken DNA molecules. ATM is activated by Mre11/Rad50, particularly in the presence of DNA (Lee and Paull 2005). TopBP1 functions as a general activator of ATR. In case of mutation of the ATR, PRD does not affect the basal kinase activity of ATR but prevents its activation. So, TopBP1-mediated ATR activation is required for checkpoint signaling and cellular viability (Mordes et  al. 2008). The ATR kinase and its regulatory partner ATRIP coordinate checkpoint responses to DNA damage and replication stress. Replication protein A-coated ssDNA is the critical structure at sites of DNA damage that accumulates the ATR-­ ATRIP complex and facilitates its recognition of substrates for phosphorylation. Subsequently, it initiates checkpoint signaling. (Cortez et  al. 2001; Zou and Elledge 2003). The mechanisms that regulate TopBP1-access to ATR–ATRIP are complex. They include post-translational modifications and accumulation of TopBP1 independently of ATR–ATRIP sites of replication stress or DNA damage. DNA replication stress triggers the activation of CHK1. This pathway simultaneously requires the chromatin loading of the ATRIP-ATR complex (Delacroix et al. 2007; Lee et al. 2007). When DNA is damaged, the checkpoint mechanism is activated to allow cells to have enough time for repair. When damage is

A. B. Engin and A. Engin

repaired, cells division continuously progress. This is called as checkpoint recovery. If damage is too severe to repair, this leads to apoptosis. If cells undergo checkpoint adaptation, damage is partly repaired. In all these cases, a serine-­ threonine kinase, Polo-like kinase-1 (PLK1) regulates many key factors within damage response (Hyun et  al. 2014). The checkpoint-mediated pathways protect the replication fork integrity. However, problem may occur when DNA replication is initiated. When the DNA replication forks can encounter damaged DNA, replication forks stall, a signaling cascade mediated by DNA damage checkpoint kinases are activated simultaneously (Jossen and Bermejo 2013). Since CDKs target the RAD9 subunit to modulate DNA damage checkpoint activation, CDK-PLK1-­ dependent phosphorylation of RAD9 suppresses checkpoint activation. Therefore, high DNA synthesis rates are maintained during DNA replication stress. CDK-mediated PLK1-dependent signaling response suppresses the ability of the DNA damage checkpoint to detect DNA damage (Wakida et  al. 2017). The expression of PLK1 begins to increase from S/G2 phase, and its activity peaks at mitosis. PLK1 also plays a role in overall DNA damage response including DNA checkpoint activation, checkpoint maintenance, damage recovery and DNA repair. Severe mitotic DNA damage causes continuous inactivation of PLK1 and other mitotic kinases, (Hyun et  al. 2014). In brief, the suppression of DNA damage checkpoint signaling may promote cell proliferation even under genotoxic stress conditions (Wakida et al. 2017). mTOR pathway, together with the insulin/ insulin-like growth factor 1 (IGF-1), adenosine monophosphate (AMP) kinase, and protein kinase A (PKA), is in a central point, which has been on nutrient-responsive signal transduction pathways (Keith and Schreiber 1995; Stanfel et  al. 2009). The importance of the mTOR complex 1 (mTORC1) in mammalian aging was first documented in studies performed by the National Institute on Aging’s Interventions Testing Program (ITP). In 2009, the ITP reported that treating mice with the mTORC1 inhibitor rapamycin beginning at 600 days of age extends

2  Aging and Protein Kinases

median and maximal lifespan of both male and female subjects (Harrison et  al. 2009; Miller et al. 2007). As regulator of aging, and cellular senescence mTOR belongs to the PIKK family, and it comprises at least two catalytic subunits, including mTORC1 and mTORC2. While mTORC1 promotes protein synthesis through the phosphorylation of two key effectors, p70S6 Kinase 1 (S6K1) and eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4EBP), S6K1 is directly phosphorylated by mTORC1 (Saxton and Sabatini 2017). mTORC2 is activated by phosphoinositide 3-kinase (PI3K), and it phosphorylates the AKT (protein kinase B) on serine 473. A main target of AKT is tuberous sclerosis 2 (TSC2). TSC2 forms a heterodimeric complex with TSC1 and eventually inhibits mTORC1 (Weichhart 2018). mRNA translation caused by mTORC1 inhibition slows aging by reducing the accumulation of oxidative stress (Saxton and Sabatini 2017; Selman et al. 2009). Whereas, deleting the mTORC1 substrate ribosomal S6K1 increases lifespan (Selman et  al. 2009). Consequently, the lifespan-enhancing effects of mTOR inhibitors is linked to mTORC1 (Lamming et al. 2012). The mTORC1 complex is a central regulator for longevity. Considering its role in aging, mTORC1 promotes messenger RNA (mRNA) translation, repress autophagy, and modulate mitochondrial metabolism (Johnson et  al. 2015). Autophagy declines with age resulting in the accumulation of damaged proteins. Thus, mTORC1 actively suppresses autophagy by phosphorylating Ser/Thr kinase Unc-51 like autophagy activating kinase (ULK1) and, accordingly, inhibition of mTORC1 induces autophagy. Although mTORC1 inhibition enhances overall life span, adverse effects should be taken into consideration (Weichhart 2018). Even so, mTOR activity promotes cellular growth and cell division in response to nutrient synthesis and growth factor through a complicated network of interactions in aging and health (Johnson et  al. 2015; Laplante and Sabatini 2012; Shimobayashi and Hall 2014). Senescence is characterized with increased cell size, senescence-associated β-galactosidase activity (SA-βgal), development of senescence-associated

41

secretory phenotype (SASP), activation of ROS and, DNA damage. In addition to induction of cell cycle inhibitors (p53/p21), PI3K/Akt/mTOR and 5′ adenosine monophosphate-­activated protein kinase (AMPK) pathways are activated (Kumar et al. 2019). The secretion of proinflammatory mediators by SASP cells contribute to aging. Senescent cells accumulate with age, and express SASP. mTORC1 induces the SASP. mTORC1 inhibition suppresses the secretion of some SASP components by interfering with the senescent cell surface bound Interleukin 1α (IL1A)- nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity in senescent cells feedback loop (Herranz et  al. 2015; Laberge et  al. 2015). Although they are in a state of permanent cell-­ cycle arrest, unlike apoptotic cells, senescent cells remain metabolically active and undergo widespread gene expression changes, of which the SASP is part (Campisi and d’Adda di Fagagna 2007). Thus, senescent cells can alter their microenvironment for as long as they persist. In fact, the genotoxic stress activates the DDR and p38-mitogen-activated protein kinases (MAPK) signaling pathways. p53 and NF-κB, have a major role in the regulation of cellular senescence and aging. The efficiency of the p53 signaling pathway declines during aging whereas that of NF-κB is enhanced through an unknown mechanism. NF-κB and CCAATenhancer-­binding proteins β (C/EBPβ) activate the transcription of SASP genes (Freund et  al. 2010; Salminen and Kaarniranta 2011). Reduction of the membrane-bound cytokine IL1A diminishes transcription of inflammatory genes regulated by the proinflammatory transcription factors NF-κB. mTOR controls the SASP by differentially regulating the translation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2 kinase, MAPKAPK2) through 4EBP1 (Herranz et  al. 2015; Laberge et al. 2015). Signaling via p38α MAPK, and the persistent DDR associated with senescence contribute to SASP induction. Initiation and maintenance of cytokine response requires the DDR proteins ATM and CHK2 (Di Micco et al. 2006; Freund et al. 2011; Rodier et al. 2009). Indeed,

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the ATM, ATR (ATM- and Rad3-Related), and DNA-PKCs (DNA-­dependent protein kinase C) kinases are the upstream DDR kinases in mammalian cells (Abraham 2004). Epidermal growth factor receptor (EGFR) or mitogen-activated protein kinase kinase (MEK)/ extracellular signal–regulated kinase (ERK) inhibitors delay DNA damage resolution. MEK/ERK inhibitors or mitogen-­sensitive kinase 1 (MSK1) depletion enhance cell death. Expression of nuclear PKCδ activates ERK and MSK1, that ERK activation is required for MSK1 activation, and that both ERK and MSK1 activation are required for apoptosis (Ohm et  al. 2019). MAPKAPK2 activity increases during senescence but declines upon mTOR inhibition. MAPKAPK2 is located downstream of p38α MAPK 34 pathway, a known SASP regulator. p38MAPK regulates the SASP independently of the canonical DDR. p38MAPK induces the SASP by increasing NF-κB transcriptional activity (Freund et al. 2011; Herranz et al. 2015). Block of autophagic degradation of the transcription factor GATA family of zinc finger transcription factor 4 (GATA4) as a senescence and SASP regulator results in NF-κB activation and SASP induction and facilitate senescence (Kang et  al. 2015). In contrast, degradation of GATA4 by selective autophagy contributes to growth arrest and accumulation of senescence markers. GATA4 indirectly regulate other senescent phenotypes, notably growth arrest, through the SASP (Kang et al. 2015). Collectively, SASP has at least two beneficial roles. First, certain key SASP factors act in an autocrine feedback loop to reinforce the senescence growth arrest. Second, the SASP might signal to the immune system to clear senescent cells (Freund et al. 2010).

3

Calorie Restriction and Aging

Three major signaling pathways involved in longevity regulation have been described as the insulin/IGF signaling (IIS), the target of rapamycin (TOR)/ortholog of the mammalian S6 kinase Sch9, and adenylate cyclase/PKA (AC/PKA). Reducing activity of these pathways is known to

promote health and lifespan extension (Fontana et  al. 2010). Deletion or inactivation of various components of the Ras-AC-PKA pathway delays both replicative and chronological aging (Fabrizio et  al. 2001). The stress resistance transcription factors multicopy suppressor of SNF1 mutation proteins 2 (Msn2) and Msn4 are required for the effect of reduced chronological lifespan extension (Fabrizio et  al. 2001). Lifespan can be extended by limiting glucose or by reducing the activity of the glucose-sensing cyclic-AMP-­ dependent kinase (PKA) (Lin et  al. 2002). Msn2p/4p are maintained in the cytoplasm by the activity of PKA. As cAMP levels fall, PKA activity decreases, and dephosphorylated Msn2p/4p relocalize to the nucleus (Görner et  al. 1998). Ribosomal DNA (rDNA) stabilization and lifespan extension by both calorie restriction and TOR signaling is due to the re-localization of the transcription factors Msn2p and Msn4p from the cytoplasm to the nucleus. TOR and sirtuins may be part of the same longevity pathway, and that they may promote genomic stability during aging (Medvedik et al. 2007). The other major growth and aging pathways are related on mTOR and SCH9. The deletion of both the Akt and SCH9 in combination with calorie restriction extends life span almost ten-fold. The Ras/cAMP/PKA/ Rim15/Msn2/4 and the Tor/SCH9/Rim15/Gis1 pathways largely affect the calorie restrictiondependent stress resistance and extension of life span (Wei et  al. 2008). TORC1 directly phosphorylates SCH9p kinase, which is a negative regulator of both chronological and replicative aging (Kaeberlein et  al. 2005). Additionally, SCH9p kinase is a key downstream effector of mtDNA-encoded oxidative phosphorylation (OXPHOS), ROS and chronological life span (CLS) in the mTOR-mitochondria pathway (Pan and Shadel 2009). Mitochondria also generate ROS as byproducts of the electron transport process, which is a major way they are thought to contribute to the aging process (mitochondrial theory of aging) (Balaban et al. 2005; Bonawitz and Shadel 2007). The extension of CLS by reduced TOR signaling involves an increase in the OXPHOS complexes that increases oxygen consumption, decreases ROS production, and

2  Aging and Protein Kinases

thereby limits damage to cellular components (Pan and Shadel 2009). Signaling through IIS/TOR pathways starts with the binding of ligands, such as insulin and IGF-1 and IGF-2 in the case of mammals, to specific receptors, which in turn activate the PI3K/ AKT/mTOR intracellular signaling cascade that regulates metabolism and stress resistance and consequently aging. Reduced signaling through the insulin/IGF-1/mTOR signaling pathway has been proposed as an essential mechanism by which a calorie restriction diet extends lifespan (Lamming 2014). mTORC1 is very important in response to decreased PI3K/AKT/mTOR signaling. Through regulation of key residues on AKT and serum- and glucocorticoid-induced protein kinase 1 (SGK), mTORC2 regulates the Forkhead box transcription factor O (FOXO) and p38 MAPK pathways (Lamming et al. 2014). Insulin sensitivity is not required for extended longevity. Although insulin resistance is undesirable, it is not sufficient to block extended longevity (Lamming 2014). However, signaling through IGF-1 receptor or hybrid insulin-IGF-1 receptor complexes activates PI3K/AKT/mTOR signaling and regulates aging (Lamming 2014; van Heemst 2010). In calorie restriction, reducing mTORC1 activity along the PI3K/AKT/mTOR pathway significantly increase life span (Lamming et  al. 2014). Circulating IGF-1 is almost always found bound to the IGF binding proteins (IGFBPs) which regulate its activity, bioavailability, and retention. But, the effect of IGFBP2 expression on lifespan is not known (Boisclair et al. 2001). The levels of adiponectin and leptin are significantly higher in old subjects compared to young, whereas those of IGF-1 are lower in old subjects (the European research network project “Myoage”) (Bucci et  al. 2013). Overexpression of adiponectin in mice leads to a significant increase in lifespan on both normal and high-fat diets (Otabe et al. 2007). Effects of adiponectin is meditated by the adiponectin receptors. While adiponectin receptor 1 (AdipoR1), activates the AMPK, AdipoR2 activates the Peroxisome proliferator-activated receptor α (PPARα) signaling pathway. Insulin sensitivity is mediated by the activation of AMPK.  In this context AMPK

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inhibits mTORC1, decreases the activation of its substrate, S6K1 and reduces the inhibitory effect of serine phosphorylation on IRS1. Activation of the liver kinase B1 (LKB1)/AMPK/TSC1/2 pathway reduces the p70 S6 kinase-mediated negative regulation of insulin signaling, via insulin sensitivity increasing effect of adiponectin (C. Wang et al. 2007a). Activated by mTORC1, S6K1 directly phosphorylates the insulin receptor substrate-1 (IRS1), and promotes IRS1 degradation (Um et  al. 2004). On the other hand, adiponectin prevents endothelial progenitor cells senescence by inhibiting the ROS/p38 MAPK/ p16INK4A signaling cascade independent of its insulin-sensitizing activity (Chang et  al. 2010). However, hyperglycemia accelerates the onset of endothelial progenitor cells’ senescence leading to the impairment of proliferative activity. It is claimed that, this effect of hyperglycemia might be related to the phosphorylation of p38 MAPK (Kuki et  al. 2006). Furthermore, the number of cells in the adipose-derived stromal/progenitor cell population with high adipogenic capacity is inversely proportional with the increase of senescent cells. Cellular senescence contributes to dysfunctions in adipose-derived stromal/progenitor cell replication, adipogenesis, triglyceride storage, and adipokine secretion (Mitterberger et al. 2014). Increased plasma leptin levels with aging diminish leptin action. This may be dependent on leptin resistance in elderly subjects, who have abdominal obesity and insulin resistance (Gabriely et al. 2002). Leptin induces the PI3K/ AKT/mTOR signaling pathway via Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3)-mediated phosphorylation of IRS, as well as via activation of the growth hormone (GH)/IGF-1 axis (Park and Ahima 2014). Calorie restriction in adults causes beneficial metabolic, hormonal, and functional changes, but the precise amount of calorie intake or body fat mass associated with optimal health and maximum longevity in humans is not known. In addition, it is possible that even moderate calorie restriction may be harmful in patients’ population, who have minimal amounts of body fat (Fontana and Klein 2007). The molecular signaling pathways mediating the anti-aging effect of

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calorie restriction include sirtuins, PPARγ coactivator-1α, AMPK, insulin/IGF-1, and TOR (Testa et al. 2014). Cytoplasmic Sirtuin 2 (SIRT2) and its cofactor NAD+ are upregulated in a variety of tissues during calorie restriction (Haigis and Sinclair 2010; F. Wang et al. 2007b). SIRT2 controls the mitotic checkpoint kinase gene budding uninhibited by benzimidazole-­ related 1 (BubR1) abundance. The age-related decline in BubR1 levels is mediated in part through a decline in NAD+ and SIRT2 activity. In contrast, rising in BubR1 activity due to increasing NAD+ levels or SIRT2 activity has beneficial effects on health span and lifespan in mammals (North et al. 2014). Genetic evidences have established that TOR complex activity requires the small guanosine triphosphate (GTP)-binding proteins (GTPase) Ras homolog enriched in brain (Rheb). The TSC1/ TSC2 functions as a Rheb GTPase activator and inhibits mTOR signaling. Ku70/80 activates DNA-PK in the presence of DNA ends (Gottlieb and Jackson 1993). Whereas, a small Rheb-GTP activates mTORC1. Rheb binds to a C-terminal portion of mTOR that includes the PRD (Inoki et al. 2003; Long et al. 2005). A major upstream regulator of mTORC1 is the tuberous sclerosis complex comprised of the Tsc1 (aka hamartin) and Tsc2 (aka tuberin) heterodimer. TSC1/TSC2 acts as a GTPase activating protein (GAP) for the Rheb small GTPase by converting active Rheb-­ GTP to inactive Rheb-guanosine diphosphate (GDP) (Aspuria and Tamanoi 2004; Inoki et al. 2003). Loss of function of either TSC1 or TSC2 leads to hyperactive Rheb and chronic mTORC1 activity, stimulating cellular growth and proliferation (Martin et  al. 2014). mTORC1 controls ribosomal biogenesis, protein translation and autophagy, mediated by substrates that include S6K1, 4E-BP1, ULK1 (Kennedy and Lamming 2016). Activation of mTORC1 requires the co-­ ­ localization of mTORC1 with GTP-bound Rheb at the lysosomal surface. In the absence of amino acids and insulin, neither mTORC1 nor Rheb localize to the lysosome (Cummings and Lamming 2017). Whereas, mTORC2 activity is stimulated by insulin, IGF-1 and leptin via the activation of PI3K (Park and Ahima 2014).

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mTORC2 localizes to the ribosome-rich mitochondria-­associated endoplasmic reticulum membrane (MAM). mTORC2 deficiency disrupts MAM, causing mitochondrial defects including increases in ATP production, and calcium uptake (Betz et al. 2013). The abundance of ribosomal subunits and activity of the ribosome is largely controlled by mTORC1, thus indirectly links mTORC2 to the stimulation of mTORC1 by amino acids, glucose, cellular energy, and oxygen (Kennedy and Lamming 2016). Inactivated inhibitor of NF-κB (IK) kinase α/β IKKα/β interacts with rictor and mTOR.  This results in a decrease in mTORC2 activity (Xu et  al. 2013). Insulin stimulates the AKT-­dependent phosphorylation of TSC2, stimulating the departure of TSC from the lysosome, and permitting RhebGTP to activate mTORC1. The spatial recruitment of mTORC1 and the TSC complex to Rheb at the lysosomal surface serve to integrate diverse growth signals (Menon et al. 2014). mTORC1 is also sensitive to the availability of glucose, like amino acids which induce localization of mTORC1 to the lysosome (Efeyan et al. 2013). Glucose may regulate mTORC1 through three separate mechanisms: AMPK, v-ATPase/ Ragulator/Rag interaction and localization, and direct inhibition of mTORC1 by hexokinase-II (Kennedy and Lamming 2016). As the PI3K– AKT pathway is the critical mediator of insulin action regulation of the TSC1–TSC2 complex by AKT, AKT-mediated phosphorylation of TSC2 and subsequent activation of mTORC1 play a role in downstream of insulin. Thus, high mTORC1 signaling has been implicated in the development of insulin resistance (Manning 2004). In addition to persistent mTORC1 signaling, increased mTORC2 signals promote insulin resistance due to mTORC2-­mediated degradation of IRS-1, which normally relay the signal from the insulin receptor to PI3K and Akt. Downstream of PI3K, the survival kinase, Akt, is completely refractory to activation by IRSdependent growth factor pathways. (Destefano and Jacinto 2013; Shah et al. 2004). mTORC2 is also a well-characterized activator of SGK and protein kinase C (PKC) (Gan et al. 2012; Li and Gao 2014). Adenosine A1 receptor-

2  Aging and Protein Kinases

mediated adenosine-induced t­ ranslocation of PKCε to mitochondria is mediated by a caveolin3-dependent mechanism and this process is agerelated (Kang et  al. 2017). Although rapamycin extends lifespan, and it also prevents the onset of age-related diseases, numerous negative side effects would seem to preclude its wide-­scale use as an anti-aging compound. In these cases, it is thought that specific inhibition of mTORC1 is sufficient to extend lifespan (Lamming et  al. 2013). In mammals, reduced dietary protein or essential amino acid intake can extend longevity, improve metabolic fitness and increase stress resistance. TOR inhibition reduces inhibitory phosphorylation of general control nonderepressible 2 (GCN2), thus promoting eIF2α phosphorylation. In case of calorie restriction, the mTORC1 pathway controls protein synthesis most directly by phosphorylating and inhibiting a repressor of cap-dependent mRNA translation, 4E-BP1. Phosphorylation of 4EBPs by mTORC1 results in their dissociation from eIF4E at the 5′ 7-methyl-GTP cap of mRNAs, facilitating cap-­ dependent translation. (Huang and Manning 2008). Therefore, mTORC1 activation promotes cell growth protein synthesis through activation of S6K and inhibition of 4E-BP (Fingar et  al. 2002). Three activating transcription factor 4 (ATF4) transcriptional targets contribute to reduced mTORC1 signaling through GCN2 activation. Firstly, crosstalk between Growth-arrest and DNA-damage-inducible protein 34 GADD34 and the mTOR signaling pathways contributes to the response of the protein synthetic machinery to environmental stress. Indeed, GADD34 is a phosphatase that removes an inhibitory phosphate on TSC2, thus repressing mTORC1, while at the same time turning down the GCN2 response by removing the phosphate on Ser51 of eIF2α (Watanabe et  al. 2007). Secondly, induction of 4EBP1 and the suppressor of the mRNA 5′ cap-­ binding protein eIF4E is involved in cell survival under Endoplasmic reticulum (ER) stress. Lastly, 4EBP1, and the TSC activator/mTORC1 repressor regulated in development and DNA damage responses 1 (REDD1) is up-­regulated at the tran-

45

scriptional level during ER stress. This mechanism involves activation of Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), phosphorylation of eIF2α, and increased ATF4 expression (Whitney et al. 2009; Yamaguchi et  al. 2008). REDD1 as a critical transducer of the cellular response to energy depletion, is induced by energy stress and play a role in AMPK-mediated TSC1–TSC2-dependent regulation of mTORC1 (Sofer et  al. 2005). REDD1 inhibits mTORC1 by reversing Akt-mediated inhibition of the TSC1–TSC2 complex (Huang and Manning 2008). GCN2 deletion alone has no effect on lifespan, indicating that the reduced protein synthesis in old cells that is mediated by eIF2α phosphorylation does not optimize the normal lifespan. The reduction in protein synthesis during aging is mediated via activation of the GCN2 kinase, which inhibits translational initiation through eIF2α, in combination with Ssd1-­ mediated delivery of mRNAs to aggregated P-bodies (Hu et al. 2018). When nutrients are abundant and cells are growing, TOR kinase is active, it phosphorylates and inactivates the 4EBP1 repressor of CAP-­ dependent translation and activates the S6 kinase 1 to promote ribosome biogenesis via phosphorylation of Rps6 (Blenis 2017). Mechanistically, elevated levels of the mRNA binding protein Ssd1  in old cells reduce protein synthesis by delivering mRNAs to aggregated P-bodies. The Ssd1-mediated reduction in protein synthesis during aging enables optimization of the normal lifespan, because Ssd1 loss shortens lifespan, while Ssd1 overexpression extends lifespan. In parallel, the phosphorylation of the GCN2 substrate eIF2α increases in old but not young cells, further reducing global protein synthesis. Overexpression of its downstream effector GCN4 extends lifespan in a manner dependent on autophagy (Hu et al. 2018). Calorie restriction induces dramatic changes that resemble those of younger individuals, in three key pathways of the skeletal muscle transcriptional profile. Long-term calorie restriction-­ responsive pathways associated with longevity in humans are IGF-1/insulin signaling, mitochondrial biogenesis, and inflammation (Mercken

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46

et al. 2013). Four pathways have been implicated in mediating the calorie restriction effect. These are the IGF-1/insulin signaling pathway, the sirtuin pathway, the AMPK pathway and the TOR pathway (Speakman and Mitchell 2011). Reactive species increase protein tyrosine kinases (PTKs) activation, which is counterbalanced by decreased protein tyrosine phosphatases (PTPs), leading to a shift in the PTK/PTP balance. The delicate balance between PTK and PTP is disturbed during aging and inflammation, both of which lead to NF-κB activation via NF-κB-inducing kinase (NIK)/IKK and MAPKs (Jung et  al. 2009). Senescence marker protein-30 (SMP30) deficient mice induces a shorter lifespan and redox changes. Overexpression of SMP30 prevents oxidative stress. The depletion of SMP30 increases redox-related PTK/PTP imbalance and protein phosphastase 1 (PP1)/PP2A inactivation. PP2A inactivation via PTK/PTP imbalance provoked by oxidative stress causes NF-κB activation, which contributes to the accumulation of oxidative stress in aging (Jin Jung et  al. 2013; Jung et al. 2015).

4

Telomeres and Aging

Cellular senescence is triggered by stresses. Senescent cells display a wide variety of characteristics: cell cycle arrest, morphological transformation, unscheduled oncogene activation, telomere dysfunction, DNA damage via ROS, induction of SA-β-GAL activity, increase in senescence-associated heterochromatic foci (SAHF), SASP, autophagy, the induction of replicative senescence by telomere attrition (Kuilman et al. 2010). The ends of chromosomes, called telomeres, contain telomeric DNA that forms a cap structure in cooperation with ­telomeric proteins to prevent the activation of the DDR, and chromosome fusion at this point of chromosome (Muraki and Murnane 2018). Telomeres progressively shorten in almost all dividing cells and most human cells do not express sufficient telomerase activity to fully maintain telomeres. In the absence of cell-cycle checkpoint pathways, cells bypass a cellular

growth arrest, and telomeres continue to shorten. Chromosome breakage fusion-bridge cycles lead to a high rate of apoptosis (Shay and Wright 2005). Although telomere sequence is identical in mice and humans and telomeres serve the same role, their telomeres are 5–10 times longer than in humans, but their lifespan is 30 times shorter. However, telomerase deficiency in both humans and mice accelerates telomere shortening (Calado and Dumitriu 2013). Considering telomere theory of aging, telomeric DNA repair efficiency is lower in cells from an aged donor in comparison to cells from a young donor. Therefore, it is thought that this decline in telomeric repair with aging is of functional significance to an age-­ related decline in genomic stability (Kruk et al. 1995). Telomere shortening or dysfunction triggers stress response that, through the induction of cell cycle inhibitory proteins, limits the replicative potential of cells (Kuilman et al. 2010). In this case, Cdk1 activity controls the timing of telomere elongation by regulating the single-­strand overhang at chromosome ends (Frank et al. 2006). When telomeres reach a critical minimal length, their protective structure is disrupted (Kuilman et  al. 2010). Deficiencies in telomere repair may result in accelerated senescence and aging as well as genetic instability that facilitates malignant transformation (Lansdorp 2000). Thereby, telomere integrity is essential for cell survival and genetic stability (Ferreira et al. 2004) (Fig. 2.1). Telomerase upregulation leads to telomere elongation with reprogramming. Mutations in telomerase significantly inhibit reprogramming. Short, dysfunctional telomeres cannot be efficiently elongated because the telomerase enzyme is inactivated or functionally impaired. Thereby, dysfunctional telomeres can initiate genomic instability, cellular senescence, and aging (Batista and Artandi 2013). In fact, telomeres are composed of tandem repeats of the TTAGGG sequence bound by a six proteins complex known as shelterin, which protects chromosome ends (de Lange 2005). In this structure, three telomeric proteins that directly bind telomeric DNA: protection of telomeres 1 (POT1), telomeric repeat binding factor 1 (TRF1), TRF2, and

2  Aging and Protein Kinases

47

Fig. 2.1  Obesity reduces life expectancy through the alteration of metabolic pathways, which are evidenced biochemically in the relationship between caloric restriction and longevity. Therefore, obesity results in epigenetic age acceleration of metabolically active tissues.

Senescence-associated features are characterized with lipotoxicity, senescence-associated β-galactosidase activity (SA-β-gal), development of senescence-associated secretory phenotype (SASP), increasing in reactive oxygen species (ROS) and DNA damage.

together with the three associated proteins: TERF1 (TRF1)-interacting nuclear factor 2 (TIN2), repressor and activator protein-1 (RAP1) and Tripeptidyl Peptidase 1 (TPP1), form shelterin (Palm and de Lange 2008). Although the shelterin complex serves to prevent telomeres ­ from being recognized as a DDR, paradoxically many DDR proteins localize to telomeres (Guo et al. 2007). Actually, shelterin has DNA remodeling activity that changes the structure of the telomeric DNA, thereby protecting chromosome ends (de Lange 2005; Palm and de Lange 2008). Shelterin represses a multitude of DDR pathways that threaten mammalian telomeres, and distinct shelterin subunits are related to different signaling and repair pathways (Doksani and de Lange 2016). Induction of telomere dysfunction through the removal of shelterin components also activates ATM or ATR-dependent signaling and cell cycle arrest (Palm and de Lange 2008). One of the best characterized shelterin members, TRF1 protects telomeres via binding to telomeric DNA. This process is regulated post-translationally (Bianchi et al. 1997; Boskovic et al. 2016). The TRF1 is regulated by the PI3K signaling pathway. There are three AKT phosphorylation sites on TRF1. Thus, inhibition of PI3K and of its downstream target AKT by small molecules results in decreased TRF1 levels and increased

telomeric DNA damage. Therefore, the PI3K/ AKT pathway plays a central role in the regulation of telomere protection (Méndez-Pertuz et al. 2017). The telomere length is maintained in most cases by the telomerase. The multifunctional shelterin complex associates with telomeres to coordinate multiple telomere alterations, including telomere elongation by telomerase (Kim et al. 2017). Within this complex, TRF2 plays an essential role by blocking the ATM signaling pathway at telomeres and preventing chromosome end fusion. TRF2 is phosphorylated on serine 323 by ERK1/2. Furthermore, the telomere stability is under direct control of one of the major pro-­oncogenic signaling pathways (RAS/ RAF/MEK/ERK) via TRF2 phosphorylation. (Picco et al. 2016). The presence of active types of ERK1/2 may prevent stable ERK1/2/TRF2 interactions to occur and thus favors proper Shelterin formation in normal situations. Indeed, ERK and PI3K pathways both promote cell cycle progression during G2/M, thereby, regulation of ERK1/2 activity is required all along the cell cycle for a physiological G1/S and G2/M transition (Roberts et al. 2002). Adaptive response to physiological stress increases TRF1 and TRF2 protein and mRNA levels, expression of DNArepair and its response genes, CHK2, Ku80 and protein content of phosphorylated p38 MAPK. In

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this process, TRF1 and TRF2 bind to the double stranded region of telomeric DNA (Donate and Blasco 2011; Ludlow et  al. 2017). In contrast, myocardial apoptosis in human heart failure is the results of defective expression of the TRF2, telomere shortening, and activation of the DNA damage checkpoint kinase, CHK2 (Oh et  al. 2003). In these cases, loss of TRF2 in telomeres leads to activation of the ATM kinase at the natural ends of human chromosomes, and arrest in the cell cycle (Karlseder et  al. 1999). Although ATM is not essential for elongation of the shortest telomeres, activation of ATM leads to telomere elongation. Otherwise ATR may compensate for loss of ATM (Feldser et al. 2006; Lee et al. 2015). However, the response to loss of POT1 is dependent on the ATR kinase but not on ATM. As the TRF2 is still present within the telomeres, ATM kinase remains repressed when POT1 is removed. The TRF2 and POT1 subunits are also efficient in blocking the two DNA repair pathways that could harm telomeres (de Lange 2009). In mammals, DNA DSBs are primarily repaired by NHEJ and error-free homologous recombination repair (HRR). In the presence of telomeric DSBs, repair would be the end-joining of broken telomeric fragments that favors correct repair telomere-­telomere fusions, potentially resulting from classical, DNA-PK-dependent c-NHEJ.  When c-NHEJ is inactivated, an alternative form of end-­joining (alt-NHEJ) removes DSBs. PARP1-­ dependent alt-NHEJ pathway shares a common DNA excision mechanism with HRR (Chiruvella et  al. 2013; Doksani and de Lange 2016; Iliakis et al. 2015; Konishi and de Lange 2008). c-NHEJ is the primary DSBR pathway in mammalian cells, and is involved in the repair of 95% of DSBs (Muraki and Murnane 2018). TRF2 may suppress NHEJ and prevents chromosome end-­to-­end fusions. Therefore, chromosome end fusions and senescence in human cells may be caused by loss by TRF2 from shortened ­telomeres. Deprotected telomeres become processed as sites of DNA damage, rapidly activating the DNA damage signaling cascade and leading to NHEJ-mediated repair reactions. The effect of cell cycle phase on telomere dysfunction indicates that telomere repair

A. B. Engin and A. Engin

by NHEJ occurs primarily in G1, being repressed in G2 by higher CDK activity (Konishi and de Lange 2008; van Steensel et  al. 1998). Furthermore, loss of the shelterin protein TRF2 activates the ATM kinase pathway, stimulates the formation of DNA damage induced foci, up-regulates p53, and arrests cells at the G1/S checkpoint. TRF2 and POT1 acts independently to repress these two DNA damage response pathways. TRF2 represses ATM, whereas POT1 prevents activation of ATR.  Either ATM or ATR signaling is required for efficient NHEJ of dysfunctional telomeres. Inactivation of DNA damage CHK1/2 in senescent cells restore cell-cycle progression into S phase that is activated with a direct contribution from dysfunctional telomeres (Celli and de Lange 2005; d’Adda di Fagagna et al. 2003; Denchi and de Lange 2007). CHK2 deletion improves telomerase-­ deficientassociated phenotypes, including lifespan and age-associated pathologies in the signaling of dysfunctional telomeres (García-Beccaria et  al. 2014). Phosphorylation of the TRF1 at an ATM/ ATR target site (S367) leads to loss of TRF1 from telomeres. The integrity of the telomerase complex reduces via ATM and ATR depletion (Tong et  al. 2015). TRF1-mediated telomere length regulation in human cells is achieved by ATM.  Inhibition of human ATM results in increased TRF1 at the telomere, and phosphorylation of TRF1 on serine 367 by ATM reduces the interaction of TRF1 with telomeres and abrogated its ability to inhibit telomere lengthening. S367 phosphorylation of TRF1 by ATM is important for the regulation of telomere length and stability (McKerlie et  al. 2012; Wu et  al. 2007, p.  11). One of the three associated proteins of shelterin, RAP1 plays a vital role in maintaining telomere homeostasis by regulating telomere length, inhibiting telomere excision, protecting telomere from telomere-telomere fusion, and telomere from unregulated activation of the DNA damage checkpoint (Wellinger and Zakian 2012). RAP1 has two domains, which include the N-terminal breast cancer 1, early onset (BRCA1) C terminus (BRCT) domain and the C-terminal protein–protein interaction Rap1 C-terminus (RCT) domain (Shore 1994). The telomeric pro-

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tective function of RAP1 is largely provided by its RCT domain, which recruits shelterin proteins RAP1-interacting factor (RIF)1/RIF2. While overexpression of either RIF1 or RIF2 decreases telomere length, co-overexpression of these proteins can reverse the telomere elongation (Hardy et al. 1992; Wotton and Shore 1997). Recruitment of humanRAP1 (hRAP1) to telomeres prevents chromosome fusions caused by the loss of TRF2/ hRAP1 from chromosome ends despite activation of a DNA damage response. In this case, hRAP1 inhibits NHEJ, interacts with DNAPK-C at mammalian telomeres and provides genome stability (Sarthy et  al. 2009). hRAP1 alters the affinity of hTRF2 and its binding preference on telomeric DNA.  Moreover, the TRF2-RAP1 complex has higher ability to re-model telomeric DNA than either component alone (Arat and Griffith 2012). The involvement of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) in telomere protection has also been linked to DNA-dependent protein kinase (DNA-PK) since hnRNP-A1 interacts with and could be the direct substrate of DNA-PK (Ting et  al. 2009). DNA-PK is composed of the DNA-binding Ku70/Ku80 subunit and the catalytic DNA-PKcs subunit, and is the critical regulator of the NHEJ pathway of double-­stranded break repair. NHEJ is a complex and versatile process that can repair DNA double stranded breaks (DSBs) (Davis et  al. 2014). Ku complex binds to DSBs. At a DSB, there are two DNA ends. Ku-DNA end complex serves as a node at which the nuclease, polymerases and ligase of NHEJ dock (Lieber 2008, 2010). NHEJ is a major pathway for repair of DSBs. Artemis (multifunctional nuclease) and DNA-dependent-PKcs together form a key nuclease for NHEJ in vertebrate organisms. The Artemis: DNA-PKcs complex is able to endonucleolytically cut a variety of types of damaged DNA overhangs. Thus, Artemis/DNA-PK carries out a limited and tightly coordinated removal of very short ­ oligonucleotides from both strands (Ma et al. 2005; Yannone et al. 2008). Although DNA-PKcs is necessary for the prevention of telomere fusions, it is not for the maintenance of telomere length (Gilley et al. 2001). TRF2/RAP1 and DNA–PKcs complexes sustain a dual protec-

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tion at telomeres against end-joining (EJ): on the one hand, through TRF2/RAP1 complex prevents c-NHEJ-­mediated end fusion at the DNA– PK end-binding step, on the other hand, through the DNA-PK complex counteracts classical ligase 4 (LIG4)-independent EJ mechanism (Bombarde et al. 2010). hnRNP-­A1 is phosphorylated by DNA-PKcs during the G2 and M phases and that DNA-PKc-dependent hnRNPA1 phosphorylation promotes the RPA-­to-­POT1. In addition, Ku70/Ku80 and DNA-­PKcs is linked to telomere protection (Sui et al. 2015). PI3-K/AKT pathway, one of the best characterized growth signaling cascades, regulates a variety of cellular function including cell proliferation, survival, metabolism, and DNA repair. In addition to Ku70/Ku80 and DNA-PKc, the AKT signaling pathway also plays an important role in telomere protection (Han et al. 2013). In vertebrate cells the telomeric duplex is packaged by a complex of six proteins termed shelterin. Three shelterin subunits, TRF1, TRF2, and POT1 directly recognize TTAGGG repeats. They are interconnected by three additional shelterin proteins, TIN2, TPP1, and Rap1, forming a complex that allows cells to distinguish telomeres from sites of DNA damage (de Lange 2005). Actually, one of the three associated proteins of shelterin, TPP1 are required to bridge the TRF1 and TRF2 subcomplexes. TPP1 and binding partner of POT1 form the sixth protein of the shelterin complex at telomeres. Telomere damage and reduced TPP1 dimerization as a result of AKT inhibition was also accompanied by diminished recruitment of TPP1 and POT1 to the telomeres (Finucane et  al. 2015; Han et  al. 2013). Mammalian cells have two distinct end-resection pathways that are regulated by DNA damage signaling, through RIF1-mediated inhibition. However, telomeres are protected from hyperresection through the repression of the ATM and ATR kinases by TRF2 and TPP1-bound POT1, respectively (Kibe et  al. 2016). Thus, shelterin can protect chromosome ends as a TRF2-bound TIN2/TPP1/POT1 complex. TIN2 is the only mechanism by which TPP1/POT1 heterodimers bind to shelterin and function in telomere protection (Frescas and de Lange 2014a, 2014b).

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Although TIN2 affects the TRF2-dependent repression of ATM kinase signaling, it does not affect TRF2-mediated inhibition of telomere fusions (Takai et al. 2011). RPA associates with telomeres during S phase of the cell cycle in humans and plays a key role in DNA replication and activation of the ATR checkpoint. In fact, RPA-coated telomeric two single-stranded DNA (ssDNA) is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex (Zou and Elledge 2003). The major role of TIN2 is to stabilize TPP1/ POT1 on the ssDNA (Takai et al. 2011). However, POT1 antagonizes RPA binding to ssDNA. Thereby, POT1 and TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. The telomeric repeat-containing RNA (TERRA) promotes POT1 binding to telomeric ssDNA by removing hnRNPA1. In this respect, hnRNPA1, TERRA and POT1 together displace RPA from telomeric ssDNA after DNA replication and preserve genomic integrity (Flynn et al. 2011). Damaged mammalian telomeres can activate both ATM and ATR and address the mechanism by which TRF2 and POT1 act independently to repress these two DDR pathways. TRF2 represses ATM, whereas POT1 prevents activation of ATR.  Either ATM or ATR signaling is required for efficient NHEJ of dysfunctional telomeres (Denchi and de Lange 2007). When DNA damage cannot be repaired, a choice between permanent arrest and checkpoint adaptation must be made. While permanent arrest endangers future lineages, continued proliferation is associated with the risk of genome instability (Klermund et al. 2014).

A. B. Engin and A. Engin

target interactions (Breving and Esquela-­ Kerscher 2010). In total, there are more than 45,000 miRNA target sites within human 3′ untranslated regions (3’UTRs), and more than 60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs. miRNAs regulate the stability and translation of mRNA by perfect or imperfect base pairing at the 3′ UTR of the mRNA. They bind to a target mRNAs at complementary sites within the 3′-UTR, triggering the suppression of the target gene (Bartel 2009; Friedman et  al. 2009). DDR is initiated by activation of the PI3K-like kinases (ATM, ATR, DNA-PKcs), phosphorylation of histone H2A histone family member X (H2AX) and recruitment of the Mre11-Rad50-­ Nbs1 or the Rad9-Hus1-Rad1 complex to damage sites (Ciccia and Elledge 2010). miR-421, suppresses ATM expression by targeting the 3′-untranslated region (3’UTR) of ATM mRNA transcripts leading to altered S phase cell cycle checkpoint (Hu et al. 2010). Several miRNAs are transcriptionally regulated by p53, that contributes to apoptosis and acute senescence (Braun et al. 2008; Yan et al. 2009, p. 17–92). miRNAs that have a putative p53-binding element in their genomic regions, indicate these miRNAs are transcriptionally activated by p53 (Wan et  al. 2011). As many as one fourth of miRNAs are significantly upregulated after the DNA damage in an ATM-dependent manner. The ATM kinase directly binds to and phosphorylates hnRNPK-­ homology (KH)-type splicing regulatory protein (KSRP), leading to increased KSRP activity in miRNA processing (Xinna Zhang et  al. 2011). The expression of the regulatory protein, KSRP or KHSRP is increased by PI3K-AKT activation (Ruggiero et  al. 2007). KSRP-­dependent miR5 MicroRNAs, Protein Kinases NAs are consisted of a class of miRNAs whose expression is induced in ATM-­dependent manner and Aging in the DDR (Xinna Zhang et al. 2011). KSRP is a MicroRNAs (miRNAs) are small non-coding key regulatory protein that accelerates the proRNAs generally act as post-transcriptional regu- duction of a subset of miRNAs that regulate cell lators of gene expression by binding to miRNA-­ activities in response to DNA damage. In miRNA recognition elements (MREs) in target transcripts. biogenesis, KSRP is activated via ATM (Wan miRNAs are extensively regulated at the levels of et al. 2011). miRNA promoter transcription, methylation, DDR decides the cell’s fate either to repair miRNA processing, RNA editing, and miRNA-­ DNA damage or to undergo apoptosis (Hu and

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Gatti 2011). Consequently, there is an extensive crosstalk between miRNAs and the canonical DDR signaling pathway (Arjumand et al. 2018). DNA damage can regulate miRNA expression at the transcriptional level. It is claimed that p53 plays a critical role in this regulation. However, the ATM/CHK2 (ATR/CHK1)-p53/Mouse double minute 2 homolog (MDM2)-p21 pathway is the major one that controls the DNA damageinduced G1/S checkpoint. Whereas, DNA damage-induced G2/M checkpoints are controlled by p53-dependent and p53-­independent pathways. DNA damage induced- miRNA-21 negatively regulates G1/S transition, while it also affects the DNA damage-induced G2/M checkpoint (Hu and Gatti 2011). However, miRNA response to DNA damage for p53- or KSRP-independent miRNAs are still unknown (Wan et al. 2011). Appearance of complex and strongly cell-type dependent patterns of altered miRNA expression after the exposure of cells to DNA damage indicates that alterations in miRNA form an essential regulatory component during the DDR (Hattori et  al. 2014). As small interfering RNAs (siRNAs) and miRNAs, two classes of short RNAs have been identified. These act through RNA-induced silencing complexes (RISCs). However, it is not known, how and why miRNA allocation between the RISC complexes changes with age for selected miRNAs (Grigoriev and Bonini 2014). It is thought that miRNA-­mediated mRNA destabilization might be a consequence of translational repression. In this respect, the miRNAs are incorporated into an RISC, which recognizes target mRNAs and results in translational inhibition or destabilization of the target mRNA (Garzon et al. 2009; Guo et al. 2010). Therefore, at least 60% of human protein-coding genes are under selective pressure to maintain pairing to miRNAs (Friedman et al. 2009). A miRNA can pair to an mRNA and thereby specify the post-transcriptional repression of that protein-coding message (Friedman et  al. 2009). miRNAs are potential sensors of aging and cellular senescence. Due to their capability to bind to the UTR of mRNA of specific genes, miRNAs can prevent the translation of these genes (Llave et al. 2002; Williams et  al. 2017). Mature miRNAs are loaded onto

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Argonaute (AGO) proteins and together form the core of the RISC.  The miRNA guide strand directs the RISC to the target mRNAs (Nishikura 2016). miRNAs regulate the stability and translation of mRNA by perfect or imperfect base pairing at the 3′ UTR of the mRNA.  In the human genome, approximately 30% of genes are estimated to be targeted by miRNAs (Bartel 2004). A site falling within a UTR with high overall conservation is far less likely to be conserved due to miRNA targeting than is one falling within a rapidly evolving UTR (Lewis et al. 2005). miR-376a represses phosphoribosyl pyrophosphate synthetase-1 (PRPS1) and threonine-tyrosine kinase (TTK) involved in DNA/RNA synthesis and mitosis checkpoint, respectively (Kawahara et al. 2007). Mesenchymal stem cells (MSCs) are as susceptible as other cells to molecular alterations that result from biologic aging (Yu et  al. 2011; Zhou et al. 2008). miRNAs play an integral role in the changes of various protein kinases associated with the aging process. Many upregulated miRNAs target to decrease the levels of both mRNA and functional protein, including the MAPK/ERK and NF-κB pathways, and CDK6. mRNA levels of cell-cycle regulatory molecules such as CDKs are downregulated, thereby cell proliferation is reduced. The miRNAs directed toward the MAPK/ERK system are expressed at higher levels in cells from aged donors. Elevated levels of NF-κB regulated by miRNA activity play a central role in the onset and progression of the aging process in MSCs (Pandey et al. 2011). Senescent cells are remarkably resistant to apoptosis. Causes of age-­related resistance to apoptosis are functional deficiency in p53 network, increased activity in the NF-κB- inhibitors of apoptosis (IAP)/c-Jun N-terminal kinase (JNK) axis, changes in molecular chaperones, miRNAs, and epigenetic regulations (Salminen et  al. 2011b). Calorie restriction, which is a dietary measure known to delay aging, causes excessive increase in circulating miRNAs linked to growth and insulin signaling pathway (Schneider et  al. 2017). miRNAs that regulate four main nutrient-sensing pathways involved in the pathogenesis of age-­ related diseases, namely insulin/IGF-1 (glucose-­

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sensing), mTOR (high amino acid level sensor), and low-energy state sensors AMPK and sirtuins pathways (Victoria et al. 2017). Indeed, miR-451 is a potent inhibitor of the AMPK signaling pathway by directly targeting calcium-binding ­ protein 39 (CAB39). CAB39 is a necessary coactivator of the serine/threonine kinase 11 (STK11, also known as LKB1), which subsequently phosphorylate AMP-bound AMPK (Godlewski et  al. 2010). miR-451 is negatively regulated through the phosphorylation and inactivation of its direct transcriptional activator organic cation transporter 1 (OCT1) by AMPK.  The capacity to transcriptionally regulate the CAB39/STK11/AMPK axis makes miR-­ 451 a major effector of glucose-regulated AMPK signaling (Ansari et al. 2015). NAD+-dependent deacetylase, SIRT1 is implicated in the control of metabolism and lifespan. SIRT1 mediates the beneficial metabolic effects of calorie restriction. Nuclear receptor Farnesoid X Receptor (FXR)/ Small Heterodimer Partner (SHP) cascade pathway controls the expression of miR-34a and its target SIRT1 (Lee and Kemper 2010). By activated FXR, SHP is recruited to the miR-34a promoter and suppresses its transcription by inhibiting the p53. Inhibition of miR-34a contributes to increased expression of SIRT1 (Chang et al. 2007). miR-34a is a posttranscriptional regulator of SIRT1 during the regulation of cell death and metabolism. miRNA-34a decreases SIRT1 levels by binding to the 3′-untranslated region of SIRT1 mRNA. miRNA-34 inhibition of SIRT1 leads to an increase in acetylated p53 and expression of p21 and p53-upregulated modulator of apoptosis (PUMA), transcriptional targets of p53 that regulate the cell cycle and apoptosis, respectively (Lee et  al. 2010; Yamakuchi et  al. 2008). As SIRT1 is a potent inhibitor of NF-κB signaling, SIRT1 is downregulated by NFκB-­ induced miR-34a. SIRT1 stimulates oxidative energy production via the activation of AMPK, PPARα and PPAR-γ coactivator 1α (PGC-1α) simultaneously. All these inhibit NF-κB signaling in aging. Whereas, NF-κB signaling downregulates SIRT1 activity through the expression of miR-34a and ROS (Kauppinen et  al. 2013). Likewise, miR-217 overexpression reduces

A. B. Engin and A. Engin

SIRT1 and FOXO1 activity and promotes senescence (Menghini et al. 2009). SIRT1, in addition to playing roles that are important for normal growth and lifespan, can contribute to oxidative damage in mammals by activating IRS-2/Ras/ ERK signaling downstream of insulin/IGF-1 receptors. Either the inhibition of SIRT1 or of Ras/ERK1/2 is associated with resistance to oxidative damage (Li et  al. 2008). miRNAs target genes, which are involved in the production of ROS and inflammatory cytokines, are a characteristic feature of senescent cells in aging. In this respect, miR-335 and miR-34a inhibits superoxide dismutase 2 (SOD2) and thioredoxin reductase 2 (Txnrd2) expression, through binding to the corresponding binding sites in the 3′-UTRs of SOD2 and Txnrd2 genes. These enzymes, which are located in the mitochondria, delay cellular aging by detoxifying ROS (Bai et  al. 2011; Bhaumik et al. 2009). In addition, miRNA, which is downregulated in senescence, increases nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4) activity and oxidative stress (Vasa-­Nicotera et al. 2011). FOXO family are indispensable for SIRT1-­ dependent cell survival against oxidative stress. SIRT1 not only regulates p53-dependent cell death, but also FOXOs-dependent cell survival signals under oxidative stress conditions (Hori et  al. 2013). AMPK negatively regulates p53 acetylation via phosphorylation of SIRT1 (Lau et al. 2014). SIRT1 binding to and deacetylating of the p53 protein inhibits p53-dependent apoptosis, leading to increase of lifespan. In this case, telomerase reverse transcriptase (TERT) is activated through the down regulation of the SIRT1 protein levels, inactivation of p53 deacetylation, decrease of the p53/Sp1 protein-protein complexes. Sp1 (specificity protein- amino acids 83–78) factors are essential for the cellular responses to p53 activation by genotoxic stress (Koutsodontis et  al. 2005; Vorovich and Ratovitski 2008). However, phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK targets, suggesting an important role of downstream kinases

2  Aging and Protein Kinases

in amplifying DDR signals (Beli et al. 2012). Sp1 is the transcription factor for both SIRT1 and microsomal glutathione transferase (Mgst1), whereas nuclear factor erythroid 2-related factor 2 (Nrf2) is the transcription factor of Mgst1 only. Up-regulation of both miR-34a and miR-93 constitutes an inescapable repression of two vital oxidative defense genes, by targeting not only the oxidative molecules, but also transcription factors controlling their activation (Li et al. 2011). Low GH-IGF-1 signaling is associated with extended longevity. However, this pathway is paradoxically linked with age-­ related diseases during normal aging in humans, which is supported by mutations. The accumulation of miRNA let-7  in aging tissues contributes to the systemic insulin resistance that accompanies aging. Indeed, Lin28a/b and let-7 modulates glucose metabolism through interaction with the insulin-PI3K-mTOR pathway. Loss of Lin28a or overexpression of let-7 results in insulin resistance and impaired glucose tolerance. These metabolic alterations occur through the let-7-mediated repression of multiple components of the insulin-­ PI3K-­mTOR pathway, including IGF1R, insulin receptor 2 (INSR), and IRS2 (Barzilai et al. 2012; Garinis et al. 2008; Schumacher et al. 2008; Zhu et al. 2011). Autophagy activation is tightly regulated in the cell based upon nutrient availability and cell stress. As the mTOR signaling pathway serves as a focal point for integration of metabolic information, growth factor signaling and stress, autophagy pathway activity is regulated by the mTORC1 complex (Han et  al. 2012). miRNA let-7 can suppress mTOR-induced anabolism and autophagic catabolism without turning off the insulin signaling pathway by targeting three members of the mTOR pathway, namely, Map4k3, RagC, and RagD. miRNA let-7 regulates the expression and activity of the genes that encode the components of the amino acid sensing pathway, thereby establishing let-7 as a key node in the mTORC1 regulatory circuit (Dubinsky et al. 2014). Subcellular redistribution of IRS-1 is regulated by the mTOR-dependent pathway. This facilitates proteasomal degradation of IRS-1, thereby down-regulating Akt. The redistribution of IRS-1 negatively regulates insulin-stimulated

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glucose transport. miRNAs appear to regulate insulin/IGF-1/mTORC1 and IRS-1/mTORC1/ S6K pathways crosstalk. miR-182, miR-223, and miR-142-3p mediate expression of IGF-1R and FOXO3A as well as activation of the insulin/ IGF-1 pathway signaling via phosphorylation of AKT and mTOR expression (Olivieri et al. 2014; Takano et al. 2001). miRNAs regulate the AKT/ mTOR signaling pathway via targeting of phosphatase and tensin homolog (PTEN). miR-19 is downregulated in four different cell types undergoing replicative senescence. The CDK inhibitor, p21/CDKN1A mRNA levels are negatively correlated with the miR-17-92. Furthermore, miR-­ 17, miR-19b, miR-20a, and miR-106 are novel biomarkers of cellular aging. Eleven miRNAs, such as mhsa-miR-23a, hsa-miR-23b, hsa-­ miR-­ 24, hsa-miR-27a, hsa-miR-29a, hsa-­ miR-­ 31, hsa-miR-100, hsa-miR-193a, hsa-miR-221, hsa-miR-222 and hsa-let-7i are consistently up-regulated in replicative senescent cells. Additionally, miR-21 over-expression reduces the replicative lifespan by reducing PTEN expression and concomitantly increasing AKT phosphorylation and mTORC1 activity. Of those miR-19 activates the AKT-mTOR pathway, thereby functionally antagonizing PTEN to promote cell survival (Dellago et al. 2013; Dey et al. 2011; Hackl et al. 2010; Olive et al. 2009). Novel miRNAs and all known miRNAs, identified by deep sequencing analysis demonstrated that the levels of many circulating miRNAs are increased with age, and that the increases can be antagonized by calorie restriction. However, the causal relationship of the miRNAs whose circulating levels are increased with age, and decreased by calorie restriction, still could not be clarified (Dhahbi et al. 2013).

6

Managing Energy Homeostasis and Resistance to Stress in Aging

Williams et al. stated that “All living beings are programmed to death due to aging and age-­ related processes” (Williams et  al. 2017). The correct regulation of energy homeostasis through

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molecular pathways regulated by AMPK enhances health and survival (Ruiz et al. 2016). Enhanced PGC-1α expression by SIRT3 via AMPK activation is responsible for increased hTERT expression and delayed endothelial senescence. In contrast, SIRT3 knockdown causes increased oxidative stress and premature senescence, by depleting hTERT expression (Karnewar et al. 2018). AMPK controls autophagy through mTOR and ULK1 signaling which augments the quality of cellular cleaning activity. Moreover, AMPK-induced stimulation of FOXO/ abnormal DAuer Formation (DAF-16), Nrf2/ SKN-1, and SIRT1 signaling pathways improves cellular stress resistance. The loss of sensitivity of AMPK activation to cellular stress with aging impairs metabolic regulation, increases oxidative stress and reduces autophagic clearance (Salminen and Kaarniranta 2012). Oxidative stress and DNA damage are among the factors that mediate senescence-associated miRNA expression changes. Senescence is induced by mitochondrial damage (Faraonio et  al. 2012). The Nrf2/Cap’n’ collar (CNC) family of transcription regulators are defined by the presence of the CNC domain and adjacent basic region. These are located within their DNA-binding domain. Kelch-like ECH-associated protein 1 (Keap1)-Nrf2-antioxidant response element (ARE) signaling plays a significant role in protecting cells from oxidative and electrophilic stresses. Upon recognition of chemical signals of oxidative molecules, Nrf2 is released from Keap1, escapes proteasomal degradation, and translocates to the nucleus. Nrf2 directs metabolic reprogramming during stress. The expression of cytoprotective genes, which are ­ transactivated by Nrf2, enhance cell survival (Hayes and Dinkova-Kostova 2014; Kensler et al. 2007; Uruno et  al. 2015). The mammalian Nrf transcription factor skinhead-1 (SKN-1)-DNA-­ binding region is localized to the CNC.  SKN-1 controls specific aspects of oxidative stress defense, such as redox balance, mitochondrial function and Unfolded protein response (UPR). SKN-1 is activated by stress signals, such as ER stress and ROS and senses the activity of multiple cellular processes, such as IIS and mTORC1/2

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(Blackwell et al. 2015). Under ER stress conditions, SKN-1 that is present in the nucleus might be captured to bind target promoters through cooperative interactions with X-box binding protein 1 (XBP-1) or other co-regulators. It has been reported that the PERK phosphorylates and activates Nrf2. Activation of PERK triggers the UPR which is both necessary and sufficient for dissociation of cytoplasmic Nrf2/Keap1 and subsequent Nrf2 nuclear import. The expression of Nrf2 enhances the cell survival following ER stress (Cullinan et  al. 2003; Cullinan and Diehl 2004). The regulation of cell survival occurs via two distinct pathways. The first involves the translation-dependent accumulation of ATF4, and the second is mediated by PERK-dependent phosphorylation of inactive Nrf2 (Cullinan et al. 2003). Unlike ATF4, Nrf2 is synthesized in unstressed cells, but is maintained in latent cytoplasmic complexes with Keap1 (Itoh et al. 1999). Through inhibiting miR-92a expression and regulating Nrf2-Keap1-ARE signal pathway, the oxidative stress reaction or inflammation can be suppressed (Liu et  al. 2017). Senescence-­ associated miRNAs are involved in the decline of Nrf2 expression, thus contribute to the repression of adaptive responses during cell senescence (Kuosmanen et al. 2018). Significant increase in SA-β-gal activity and protein expression, p53 and p16 protein expression, proliferation index (PI), malondialdehyde (MDA) concentration, SOD and glutathione peroxidase (GPX) activity, and significant decrease in telomerase activity develop in aging cells compared to young cells. A significant activation of PI3K/Akt/mTOR signaling occurs in aging cells. Therefore, the PI3K/ Akt/mTOR/S6K1 signal pathway is an important indicator in regulating the replicative senescence of human (Tan et  al. 2016). AMPK activation restores the NAD+ levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD+ synthesis. AMPK prevents oxidative stress-induced senescence by improving autophagic flux and NAD+ homeostasis (Han et al. 2016). In fact, unfolded proteins that accumulate in the ER stress, are sensed by the transmembrane proteins ATF-6, PERK, and

2  Aging and Protein Kinases

inositol-requiring protein 1α (IRE-1α), acting in conjunction with the ER chaperone HSP-4 (BiP) (Cullinan et  al. 2003; Hetz 2012). Under ER stress conditions, the transcription factor ATF-6 is released by processing in the Golgi, and the PERK inhibits translation by phosphorylating the initiation factor eIF-2α (Blackwell et al. 2015). SKN-1 is inhibited via phosphorylation of glycogen synthase kinase (GSK-3). Whereas, AKT phosphorylates and inhibits GSK-3, which mediates inhibition of Nrf2. GSK-3 is primarily inhibited by serine phosphorylation in response to insulin by either MAPKAPK-1 or p70 ribosomal S6 kinase (p70S6k) (Cross et  al. 1995). During aging the constitutive expression of many SKN-­ 1-­ regulated genes declines progressively (Ewald et al. 2015). Nrf2 and other Nrf/CNC proteins help drive lifespan extension in mammals (Blackwell et al. 2015). FOXO participates in cell cycle arrest, apoptosis, and metabolic processes via phosphorylation and acetylation besides its function in stress resistance and longevity (Accili and Arden 2004) (Fig. 2.2). The AMPK-FOXO pathway is one of the major contributors to contrasting regulation by calorie restriction initiated in different ages. Autophagy is a protective cellular response to nutrient deprivation. Aging is associated with a decline in autophagy, and enhancing autophagy promotes longevity (Madeo et al. 2015). On the other hand, FOXOs promote apoptosis signaling by either inducing the B-cell leukemia/lymphoma-­2 (Bcl2)-family of mitochondria-­targeting protein expression and stimulating expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or enhancing levels of various CDK inhibitors (CDKIs) (Xinbo Zhang et  al. 2011). FOXO/DAF-16 as a key transcription factor, could integrate different signals from these pathways to modulate aging, and longevity via shuttling from cytoplasm to nucleus. DAF-16/FOXO contains the DNA binding domain that recognizes a core consensus TTGTTTAC sequence known as the DBE (DAF-16 Binding Element) (Murphy et al. 2003). DAF-16/FOXO integrates with the signals of at least four different pathways, which are insulin and IGF-1 signaling

55

pathway, TOR pathway, AMPK pathway and JNK signaling pathway (Sun et al. 2017). AMPK activity is determined by cellular AMP/ATP ratios. Activation of AMPK is associated with increased longevity and improved health span (Bitto et  al. 2015; Hardie 2007). Furthermore, AMPK can inhibit ER and oxidative stresses which are involved in metabolic disorders and the aging process. The AMPK pathways involve in the inhibition of NF-κB signaling and suppression of inflammatory responses, which have a significant impact on both health span and lifespan (Salminen et al. 2011a). Proliferating mammalian cells have a cell-­ cycle checkpoint that responds to glucose availability. The glucose-dependent checkpoint occurs at the G1/S boundary and is regulated by AMPK. Cell-cycle arrests, despite continued amino acid availability and active mTOR. AMPK activation induces phosphorylation of p53. Phosphorylation of p53 is required to initiate AMPK-dependent cell-cycle arrest. AMPK-­ induced p53 activation promotes cellular survival in response to glucose deprivation, and cells that have undergone a p53-dependent metabolic arrest can rapidly reenter the cell cycle upon glucose restoration. However, persistent activation of AMPK leads to accelerated p53-dependent cellular senescence (Jones et al. 2005). Insulin and IIS pathway, which is a signal transduction cascades modulating aging and longevity, consists of four different steps: insulin-like peptides (ILPs), an insulin/IGF-1 receptor (DAF-2), a phosphoinositide 3-kinase (AGE-1/phosphoinositide kinase AdAPter subunit (AAP-1)/PI3K), serine/threonine kinases (PDK-1, AKT-1 and AKT-2) and the pivotal downstream FOXO transcription factor (DAF-16) (Sun et al. 2017). DAF-16/FOXO is phosphorylated from the direct upstream AKT kinases mediated signal transduction and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In the presence of survival factors, AKT phosphorylates FOXO-3A (FKHRL1), the FOXO family of transcription factor, and provides the FKHRL1’s retention in the cytoplasm. Contrarily, withdrawal of survival factors leads to FKHRL1 dephosphorylation and nuclear translocation (Brunet et  al. 1999).

56

A. B. Engin and A. Engin

Fig. 2.2  Main metabolic pathways that regulate mammalian longevity. CR and mTOR inhibition promote lifespan. NAD+ and SIRT1 levels are highly protective during aging. In CR, reducing mTORC1 activity along the PI3K/AKT/mTOR pathway significantly increase life span. mTORC1/S6K1 activity is increased in high calorie intake, which leads to the degradation of IRS1, and insulin resistance. The SASP activity is regulated by the mTOR pathway, and both are suppressed by rapamycin. These create a potential basis for developing rational strategies to evade from deleterious effects of genotoxic exposures, in normal aging processes. (Abbreviations: AdipoR1: adiponectin receptor 1, Akt: protein kinase B, AMPK: AMP-activated protein kinase, ATG13: mammalian autophagy-­related gene 13, ATM: Ataxia Telangiectasia Mutated, Bax: antiapoptotic B-cell leukemia/lymphoma 2 protein (BCL2)- associated X apoptosis regulator, C: high calorie intake, CR: caloric restriction, DSB: double strand breaks, 4E-BP: eukaryotic translation initiation factor 4E (eIF4E)-binding protein, FOXO: forkhead Box O1, GH: growth hormone, HMGB1: high mobility group box protein 1, IGF-1: insulin-like growth factor 1, IGF-1R: insulin-like growth factor 1 receptor, IKKα/β: IκB kinase α

and β, IL-1β: interleukin-1β, IR: insulin receptor, IRS-1: insulin receptor substrate-1, JAK: janus kinase, MAPK: p38-mitogen-activated protein kinases, MAPKAPK2: MAPK-activated protein kinase 2, MSK1/2: mitogenand stress-activated protein kinases 1 and 2, mTORC1: mammalian target of rapamycin complex 1, NAD+: nicotinamide dinucleotide, NEMO: NF-κB essential modifier, NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells, PDK-1: phosphoinositide-dependent kinase 1, PGC1α: peroxisome proliferator-­ activated receptor γ (PPAR-γ) coactivator 1α, PI3K: phosphatidylinositol 3-kinase, PIKK: phosphatidylinositol 3-kinase-related kinase, PIP3: phosphatidylinositol 3,4,5-trisphosphate, PKCζ: protein kinase Cζ, RhebGTP: Ras homolog enriched in brain (Rheb) GTPase, Rp: rapamycin, S6K: ribosomal protein S6 kinase, SA-β-gal: senescence-­ associated β-galactosidase activity, SASP: senescenceassociated secretory phenotype, SIRT1: silent information regulator T1, sirtuin 1, TAB: TAK1-­ binding protein, TAK1: TGFβ-activated kinase 1, TGFβ: transforming growth factor β, TNFα: tumor necrosis factor α, TRAF: TNF-receptor-associated factor, TSC: tuberous sclerosis, hamartin, ULK1: unc-51 like kinase 1)

The negative regulation of the IIS pathway with DAF-­18/PTEN and B56 regulatory ­subunit of the PP2A holoenzyme (PPTR-1)/PP2A ­ mutations exhibit short longevity, because DAF-18/PTEN antagonizes PI3Ks. PPTR-1/PP2A dephosphorylates the ATK kinases, which finally affect the intracellular distribution of DAF-16/FOXO (Padmanabhan et al. 2009; Solari et al. 2005; Sun et  al. 2017). IGF-1 promotes cell growth, pro-

liferation, and cell-cycle progression by inducing the IGF-1 receptor (IGF-1R). The IGF-1R activates the TOR pathway (Oldham and Hafen 2003), promotes cell survival via AKT (Song et  al. 2005). The IIS and TOR pathways each influence aging by regulating SKN-1 and DAF16. SKN-1/Nrf1/2/3 is critical for oxidative stress resistance and promotes longevity under reduced IIS. TORC1 opposes both SKN-1/Nrf and DAF-

2  Aging and Protein Kinases

16/FoxO, so that when TORC1 is inhibited these factors increase transcription of protective genes (Robida-Stubbs et  al. 2012). DAF-16 is necessary for AMPK function in oxidative stress resistance and longevity. If DAF-16 is inhibited, the increased longevity caused by overexpression of AMPK is reverted. Moreover, FOXO/DAF-16dependent transcription is regulated by AMPK (Greer et al. 2007). It is thought that there is a crosstalk between the IIS and AMPK pathways. On the other hand, the JNK signaling pathway interacts with the IRS-1 and the AKT protein kinase. Thus, JNK inhibits insulin signal transduction through the insulin-stimulated tyrosine phosphorylation of IRS-1 and activates AKT1 via the JNK-interacting protein (JIP1). However, JIP1 expression can exert effect independent of JNK activity (Aguirre et  al. 2000; Kim et  al. 2003). JNK signaling antagonizes IIS pathway. While JNK directly phosphorylates DAF-16/FOXO and promotes its nuclear localization, phosphorylated DAF-16/ FOXO by IIS pathway-AKT inactively retains in the cytoplasm (Sun et al. 2017). Insulin signaling is predominantly involved in nutrient regulation, whereas IGF-1 regulates growth in higher organisms (Bitto et al. 2015).

7

Conclusion

Although it has been recently suggested that there are four main theories in the etiology of aging, the main cause of aging is DNA damage and disruption of genome integrity due to many destroying mechanisms. Almost thirty-seven protein kinases are involved in all aspects of aging and senescence. These kinases not only regulate the large number of metabolic pathways related to aging processes, but also control these pathways through twelve checkpoints. Given that these protein kinases and their checkpoints have common function in the earlier theories of aging, it is thought that the different theories related to aging process might be joined in a single frame. In this chapter, aging-related protein kinases are discussed under five headings, including DNA damage and DNA repair pathways, calorie restriction

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and aging, telomeres and aging, miRNAs and aging, controlling of energy h­omeostasis and resistance to stress. As a fact that, each section of this chapter inherently contained relevant parts of classical aging theories such as DNA damage theory, mitochondrial theory, free radical theory, telomere theory.

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69 break ends by Artemis nuclease and DNA-dependent protein kinase. Nucleic Acids Res. 2008;36:3354–65. https://doi.org/10.1093/nar/gkn205. Yu JM, Wu X, Gimble JM, Guan X, Freitas MA, Bunnell BA.  Age-related changes in mesenchymal stem cells derived from rhesus macaque bone marrow. Aging Cell. 2011;10:66–79. https://doi. org/10.1111/j.1474-9726.2010.00646.x. Zhang Xinbo, Tang N, Hadden TJ, Rishi AK.  Akt, FoxO and regulation of apoptosis. Biochim Biophys Acta. 2011;1813:1978–86. https://doi.org/10.1016/j. bbamcr.2011.03.010. Zhang Xinna, Wan G, Berger FG, He X, Lu X. The ATM kinase induces microRNA biogenesis in the DNA damage response. Mol Cell. 2011;41:371–83. https:// doi.org/10.1016/j.molcel.2011.01.020. Zhou S, Greenberger JS, Epperly MW, Goff JP, Adler C, Leboff MS, Glowacki J.  Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts. Aging Cell. 2008;7:335–43. https://doi. org/10.1111/j.1474-9726.2008.00377.x. Zhu H, Shyh-Chang N, Segrè AV, Shinoda G, Shah SP, Einhorn WS, Takeuchi A, Engreitz JM, Hagan JP, Kharas MG, Urbach A, Thornton JE, Triboulet R, Gregory RI, DIAGRAM Consortium, MAGIC Investigators, Altshuler D, Daley GQ. The Lin28/let-7 axis regulates glucose metabolism. Cell. 2011;147:81– 94. https://doi.org/10.1016/j.cell.2011.08.033. Zou L, Elledge SJ.  Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science. 2003;300:1542–8. https://doi.org/10.1126/ science.1083430.

3

The Connection Between Cell Fate and Telomere Ayse Basak Engin and Atilla Engin

Abstract

Abolition of telomerase activity results in telomere shortening, a process that eventually destabilizes the ends of chromosomes, leading to genomic instability and cell growth arrest or death. Telomere shortening leads to the attainment of the “Hayflick limit”, and the transition of cells to state of senescence. If senescence is bypassed, cells undergo crisis through loss of checkpoints. This process causes massive cell death concomitant with further telomere shortening and spontaneous telomere fusions. In functional telomere of mammalian cells, DNA contains double-­stranded tandem repeats of TTAGGG. The Shelterin complex, which is composed of six different proteins, is required for the regulation of telomere length and stability in cells. Telomere protection by telomeric repeat binding protein 2 (TRF2) is dependent on DNA damage response (DDR) inhibition via formation of T-loop structures. Many protein kinases contribute to the DDR activated cell cycle A. B. Engin (*) Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey A. Engin Department of General Surgery, Faculty of Medicine, Gazi University, Ankara, Turkey

checkpoint pathways, and prevent DNA replication until damaged DNA is repaired. Thereby, the connection between cell fate and telomere length-associated telomerase activity is regulated by multiple protein kinase activities. Contrarily, inactivation of DNA damage checkpoint protein kinases in senescent cells can restore cell-cycle progression into S phase. Therefore, telomere-­ initiated senescence is a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres. In this review, in addition to the above mentioned, the choice of main repair pathways, which comprise non-homologous end joining and homologous recombination in telomere uncapping telomere dysfunctions, are discussed. Keywords

Telomere · Telomerase · Hayflick limit · Shelterin complex · telomeric repeat binding factor 2 (TRF2) · DNA damage response (DDR) · T-loop · Human telomeric reverse transcriptase (hTERT) · DNA double-strand breaks (DSBs) · Ataxia-telangiectasia mutated (ATM) · Ataxia- and Rad3-related (ATR) · Nonhomologous end joining (NHEJ) · Homologous recombination (HR) · Homology directed repair (HDR)

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_3

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1

Introduction

Organisms with linear chromosomes, while protecting the chromosomal ends from DNA breaks, prevent loss of genomic information through progressive chromosome shortening caused by the semi-conservative replication of DNA (Levy et  al. 1992). However, telomerase has not been detected in normal somatic cells, thereby these cells lose telomeres with age. Telomere attrition serves as a key checkpoint in the control of cell proliferation by triggering replicative senescence (MacNeil et al. 2016). Replicative senescence is induced by deprotected telomeres. Telomere deprotection is an epigenetic signal passed between cell generations to ensure that replication-associated telomere-dependent growth arrest before genome instability can occur (Cesare et al. 2013). If senescence is bypassed, cells undergo crisis through loss of checkpoints. This process causes massive cell death concomitant with further telomere shortening and spontaneous telomere fusions (Hayashi et al. 2015). Although the mechanism of cell death is precisely unknown, the cell death rate increases significantly with time as the mitotic arrest period increases. Therefore, prolonged mitosis is the main mechanism that limits cellular life span upon bypass of senescence (Hayashi et al. 2015). What is real is the fact that life span is governed by cell division, not time. The regular loss of telomeric DNA could serve as a mitotic clock in the senescence timetable, counting cell divisions. Abolition of telomerase activity results in telomere shortening, a process that eventually destabilizes the ends of chromosomes, leading to genomic instability and cell growth arrest or death (Counter 1996). Indeed, telomerase activity exhibits endogenous circadian rhythmicity in humans. Human telomeric reverse transcriptase (hTERT) mRNA expression oscillates with circadian rhythms and are under the control of circadian locomoter output cycles protein kaput (CLOCK)brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1 (BMAL1) heterodimers (Chen et  al. 2014). Regular work schedules have circadian oscillation of telomerase activity while night shift working disturbs the

circadian rhythms of telomerase activity. (Chen et  al. 2014). Furthermore, senescence decreases the ability of cells to transmit circadian signals to their clocks (Kunieda et  al. 2006). However, telomerase-­reconstituted cells reset the circadian oscillation of rhythmic gene expression. The circadian rhythm resetting involves in the activation of phosphorylated extracellular signal-regulated kinase (pERK)- cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB) and p38-CREB pathways (Qu et  al. 2008). The circadian clock plays an important role in determining the strengths of cellular responses to DNA damage including repair, checkpoints, and apoptosis. In mammalian cells, two checkpoint signaling pathways have been described as the ataxia-telangiectasia and Rad-­3-­ related (ATR)- checkpoint kinase (Chk) 1 pathway and ataxia-telangiectasia mutated (ATM)-Chk2 pathway. The latter is mainly activated by double strand breaks (Sancar et  al. 2010). The circadian clock affects the DNA damage checkpoint response, either by controlling the transcription of the checkpoint proteins or by direct participation of the clock proteins in the checkpoint response. (Unsal-­ Kaçmaz et  al. 2005). Thus, the DNA damage response (DDR) activates cell cycle checkpoint and survival pathways, and prevent DNA replication until damaged DNA is repaired. Many protein kinases contribute to this process. So, transforming growth factor-beta (TGF-β) activated kinase (TAK) 1 is a mediator of the alternative DNA damage response pathway. The p38 mitogenactivated protein kinase (MAPK)/MAPKactivated protein 2 (MAPKAP-2) kinases also mediate this response (Reinhardt et  al. 2007; Yang et al. 2011). On the other hand, the cyclin-­ dependent kinase (CDK) activity is imperative for repair of DNA damage in checkpoint-arrested cells. In contrast, checkpoint activation inhibits the mitotic inducer CDK1 by up-regulation of the kinase Wee1 and down-regulation of the phosphatase cell division cycle 25 (Cdc25), leading to increased Tyr15 phosphorylation (Wohlbold and Fisher 2009). Human tyrosine kinase, Wee1 is required for the G2 checkpoint through mediating inhibitory phosphorylation of

3  The Connection Between Cell Fate and Telomere

the Tyr-15 residue on CDK1 (Parker and Piwnica-­ Worms 1992). Therefore, Wee1 inhibition leads to abnormally high CDK1 activity, resulting in G2 checkpoint abrogation followed by mitotic catastrophe, which consists of the premature entry into mitosis, and unrepaired lethal DNA damage (De Witt Hamer et  al. 2011). ATM, which is a member of the phosphoinositide-3 kinase (PI3K)-like kinase (PIKK) family, takes part among the first proteins to respond to DNA double-strand breaks (DSBs) (Rodier et al. 2009). Vast number of kinases, like ATM, ATR, and the DNA dependent protein kinase (DNA-PKcs or DNA protein kinase catalytic subunit), phosphorylate various protein targets in order to repair the damage (Czornak et al. 2008). In this context, Polo-like kinase 1 (Plk1)-mediated phosphorylation is involved in both telomeric repeat binding protein 1 (TRF1) overexpression-­ induced apoptosis and its telomeric DNA binding ability (Wu et  al. 2008, p.  1). Contrarily, PI3K and AKT inhibitors reduce the telomeric foci of TRF1, as an essential component of the shelterin complex, and lead to increased telomeric DNA damage and fragility (Méndez-Pertuz et  al. 2017). As mentioned above, the connection between cell fate and telomere length-associated telomerase activity is regulated by multiple protein kinase activities. In this chapter, tasks of checkpoint protein kinases regarding telomeric integrity and telomere attrition during the cell survival or death are discussed.

2

“ Hayflick Limit” Setting Factors

Composed of repetitive nucleotide sequences (TTAGGG for mammals) associated proteins, telomeres protect important genetic information of linear chromosomes from deletion arising due to the “end replication” problem. So, cultured normal human cells have limited capacity to divide, after which they become senescent, which is known as the ‘Hayflick limit‘(de Lange 2002a; Hayflick 1998; Olovnikov 1996; Shay and Wright 2000). If clones expressing hTERT bypass the

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expected senescence point, they exceed their normal life span at least 20 population doublings. In replicative senescence restoration of telomerase activity can influence the mitotic clock that determines life span. This “telomerase reactivation concept” means the removal of the short telomere barrier in tumor progression (Nugent and Lundblad 1998). In fact, the process of DNA replication causes progressive shortening of linear chromosomes at the telomeres (de Lange 2002a; Shay and Wright 2000). Cells sense telomere length shortening and respond with cell cycle arrest at a certain size of telomeres referring to the “Hayflick limit.” In addition to regulating the cell replicative senescence, telomere biology plays a fundamental role in regulating the chronological post-mitotic cell aging (Liu et al. 2019). Thus, telomere shortening leads to the attainment of the Hayflick limit, and the transition of cells to state of senescence. The cells subsequently enter a state of crisis, accompanied by massive cell death (Rubtsova et al. 2012). Telomeres shorten DNA sequences in the division of normal cells, while telomerase keeps telomere length constant in immortal cell populations (Olovnikov 1996). Although, mouse telomere shortens approximately 100 times faster than that in humans, the rate of increase in the percentage of short telomeres, rather than the rate of telomere shortening per month is a significant predictor of lifespan (Vera et  al. 2012). In this regard, the telomeres of most laboratory animals are 5 to 10 times longer than in humans, but their lifespan is 30 times shorter (Calado and Dumitriu 2013). In mammalian cells, telomere DNA contains double-stranded tandem repeats of TTAGGG followed by terminal 3′ G-rich singlestranded overhangs. Telomere DNA contains the T-loop structure, where the telomere end folds back on itself and the 3′ G strand overhang invades into the double-stranded DNA, so it is called D-loop (Palm and de Lange 2008). Cellular mechanisms involved in genome maintenance include the stabilization of chromosome ends by telomeres and a system of DDR pathways that promptly detect double-stranded breaks (Nandakumar and Cech 2013). Each individual DNA replication event of human telomerase-neg-

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ative somatic cells leads to a loss of 100  base pairs of telomeric sequence, resulting in a progressive decline in telomere length with each cellular division. Telomere length and life span is uncoupled in a post-mitotic setting, thereby, separate pathways are valid for replication-dependent and replication-independent aging (Raices et  al. 2005). Actually, human somatic cells can only undergo approximately 50 to 80 cellular replication events before becoming senescent. Thus, telomere length is essential for normal cellular function and proliferation as well as chromosome stability. In the absence of proper telomere complex formation, the double-stranded break repair pathway can be initiated, and this results in apoptosis or senescence. Senescent cells contain activated forms of the DNA damage checkpoint kinases, CHK1 and CHK2. On the contrary, inactivation of DNA damage checkpoint kinases in senescent cells can restore cell-cycle progression into S phase. Thus, telomere-initiated senescence reflects a DNA damage checkpoint response that is activated with direct contribution from dysfunctional telomeres (d’Adda di Fagagna et al. 2003; Raices et al. 2005; Takai et al. 2003). ATM, ATR, and DNA-dependent protein kinase (DNA-PK), are PIKKs. These kinases function as the primary transducers of DNA damage signals (Shiloh 2003). DNA-PKcs is required to prevent endogenous DNA damage accumulation. ATR controls a DNA damage-­ induced G2/M checkpoint, independent of ATM and DNA-PKcs. However, DNA-PKcs is central to DNA repair in nonproliferating cells, and restricts DNA damage accumulation, whereas ATR controls damage-induced G2 checkpoint control and apoptosis in proliferating cells (Enriquez-Rios et al. 2017). Both ATM and ATR are recruited to telomeres during the late stages of telomere replication. Firstly, nuclease processing generates overhang, and then extended by telomerase activity (Verdun and Karlseder 2007). In fact, telomeres are the physical ends of eukaryotic linear chromosomes. Telomeres form special structures that cap chromosome ends to prevent degradation by nucleolytic attack and to distinguish chromosome termini from DSBs.

A. B. Engin and A. Engin

Telomeres are composed primarily of repetitive DNA-associated proteins. So, these interact specifically with double- or single-stranded telomeric DNA or with each other, forming highly ordered and dynamic complexes involved in length regulation. Telomerase is a unique ribonucleoprotein complex, comprised of a protein catalytic subunit TERT, and a telomeric RNA component (hTR or TERC) that serves as the template for telomere extension during de novo addition of TTAGGG repeats onto chromosome ends (Celeghin et al. 2016) (Fig. 3.1). To generate active telomerase, hTERT “must translocate to the nucleus” and assemble with the RNA component of telomerase. The molecular chaperones heat shock protein 90 (Hsp90) and p23 maintain hTERT in a conformation that enables nuclear translocation. Inhibition of nuclear transport of hTERT results in cytoplasmic accumulation, and cytoplasmic hTERT is rapidly degraded, thereby telomerase activity is abrogated (Jeong et  al. 2016; Lagadari et  al. 2016). Forkhead box O3 (FOXO3a) binds to the cellular Myelocytomatosis (c-Myc) promoter, and this interaction activates the transcription of the c-Myc gene. Higher levels of c-Myc recruited to the hTERT promoter activates hTERT gene expression (Yamashita et  al. 2014). Furthermore, efficient hTERT expression requires adenosine monophosphate (AMP)activated protein kinase (AMPK) activity (Jo et  al. 2018). Although, TERT expression and telomerase activity are often very low or undetectable in somatic cells (Blasco 2005), deletion of either hTR or TERT may lead to telomere shortening and genomic instability (Liu et  al. 2000b). TERT inhibition impairs cell cycle progression and enhances the pro-apoptotic effects of DNA-damaging agents in TERT-positive cells (Celeghin et al. 2016). Whereas, overexpression of TERT can dramatically increase the life span in the context of overexpressing tumor suppressor genes such as p53, p16, and p19 (Tomás-Loba et  al. 2008). Consequently, mammalian somatic cells that naturally lack of telomerase activity show telomere shortening with increasing age leading to cell cycle arrest and senescence (de Lange 2002b; Giardini et  al. 2014; Shay and Wright 2000). Indeed, telomerase activity is

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Fig. 3.1  The reverse transcriptase subunit of telomerase, hTERT, contains the catalytic activity of the enzyme, whereas the associated RNA component, hTR, serves as the template for synthesis of telomeric sequences. Both Hsp90 and p23 subunits are essential for restoring telomerase activity. The Hsp90-p23 chaperone complex with immunophilin FKBP52 interacts with hTERT and enables its nuclear translocation through microtubules. The shelterin complex is composed of TRF1, TRF2, TIN2, POT1, TPP1 and RAP1. All these proteins directly bind to the double-stranded telomeric repeats. RNAs assemble with four evolutionarily conserved scaffold proteins, which consist of the pseudouridine synthase dyskerin; RNA interacting components NHP2 and NOP10; and GAR1, upon maturation. NAF1 binds to dyskerin in the cytoplasm to stabilize the protein, allowing for the recruitment of the other components, NOP10 and NHP2. Furthermore, NHP2 can only associate with the pre-RNP tetramer in the presence of NOP10. Therefore, NOP10-NHP2 likely bind to dyskerin as a heterodimer. Then, NAF1 can act as a nucleolar shuttle, bringing an

inactive precursor complex to the nascent hTR to form the pre-RNP complex. HSP90-like chaperone regulates the free dyskerin levels, protecting the protein from degradation. Together, GAR1 and NAF1 create a heterodimer. Upon reducing NAF1 affinity for dyskerin, NAF1 separates from the complex and leaves the site for GAR1. (Abbreviations: AKT: Protein kinase B, ATM: Ataxia telangiectasia mutated, c-Abl TK: c-abl proto-­ oncogene, which encodes a cytoplasmic protein-tyrosine kinase, DNA-PK: DNA dependent protein kinase, FKBP52: FK506-binding protein of 52-kDa, GAR1: pseudouridylation-catalysis enhancer, HSP90: Heat Shock Protein 90, hTERT: human reverse transcriptase subunit of telomerase, hTR: RNA component human telomerase RNA, NAF1: Nuclear assembly factor 1, Plk1: Polo-like kinase 1, POT1: protection of telomeres 1, RAP1: transcriptional repressor/activator protein, RNP: ribonucleoprotein, TIN2: TRF1 interacting protein 2, TPP1: POT1 and TIN2 interacting protein1, TRF: Telomeric-repeat binding factor)

essential for maintaining telomere length throughout cellular lifespan. Early in human development, telomerase is constitutively active in cells but after birth it is active only in stem cells and germ cells (Liu et al. 2007). Besides the catalytic activity dependent telomere maintenance, catalytic activity-­independent effects of telomerase also be involved in the regulation of cell cycle. The telomere/telomerase system intervenes the proliferative activity of the cell by one of two ways. The first is inhibition of the telomere maintenance by inhibiting the telomerase

activity, and the second is activating the residual telomerase enzyme or inducing telomerase expression (Tárkányi and Aradi 2008). However, senescence is associated with hTERT expression, in addition to the telomere length and telomerase activity. The downregulation of hTERT by siRNA markedly decreases telomere length and telomerase activity, whereas the overexpression of hTERT increases telomere length and telomerase activity. To ensure that, activation of the PI3K/ protein kinase B (AKT) signaling pathway is mediated by hTERT in the senescence

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(Zhao et al. 2015). TERT belongs to the group of dual-targeted proteins. TERT prevents telomere erosion by the canonical function, thereby causes replicative senescence and genetic instability. Besides telomere extension, TERT also exhibits other non-telomeric activities such as cell cycle regulation, modulation of cellular signaling and gene expression, augmentation of proliferative lifespan as well as DDR (Ale-Agha et al. 2014). Replicative senescence is induced by partially deprotected telomeres, which activate a DDR without telomere fusions. Unlike genomic breaks, deprotected telomeres that are recognized as DNA damage but remain in the fusion-resistant intermediate state activate differential ATM signaling where CHK2 is not phosphorylated (Cesare et  al. 2013). Chromosome end-to-end fusions during crisis cause spontaneous mitotic arrest, amplifying telomere deprotection, which determines cellular fate. Exacerbation of mitotic telomere deprotection by disruption of telomeric repeat binding factor 2 (TRF2) increases the ratio of cells that die during mitotic arrest. If senescence is bypassed through loss of checkpoints, massive cell death occurs concomitant with further telomere shortening and spontaneous telomere fusions (Hayashi et  al. 2015). In contrast, TRF2 overexpression, which partially suppresses mitotic telomere deprotection, reduces cell death after mitotic arrest, suppresses telomere dysfunction-induced foci (TIF) and delays crisis. Since most cell death during pre-crisis is associated with mitotic arrest, it is proposed that prolonged mitosis is the main mechanism that limits cellular life span upon bypass of senescence (Hayashi et al. 2012, 2015) (Fig. 3.2). In fact, senescence is a stress response, and there are interconnections between the unfolded protein response (UPR) signaling and the different aspects of the cellular senescence programs. In this context, it is suggested that endoplasmic reticulum (ER) homeostasis restoration may be efficacious, to prevent the cell death (Pluquet et al. 2015). Inhibition of telomerase activity may enhance vulnerability to cell death caused by ER stress (Hosoi et al. 2016). Thus, over-expression of TERT diminishes ER stress-induced cell death. Indeed, ER stress induces TERT expression to withstand

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environmental stress. This mechanism is defined as the “ER stress-TERT axis“(Hosoi et al. 2014). In contrast, depletion of hTERT sensitizes cells to undergo apoptosis under ER stress. However increased hTERT expression reduces ER stress-­ induced cell death independent of DNA damage signaling (Zhou et  al. 2014). Telomeric DNA continually decreases in size in the absence of telomerase in normal cells, and this is followed by cellular senescence. Only abnormal or cancer cells are immortal, and these cells express the enzyme telomerase that prevents shortening (Gomez et al. 2016; Hayflick 1998). Nevertheless, ectopic expression of the enzyme telomerase, which is capable of elongating telomeres, counteracts telomere shortening driven by cell division and bypasses the senescence arrest (Bodnar et al. 1998). In cell-cycle arrest, senescent cells show dramatic changes in terms of gene expression, metabolism, epigenome. Senescent cells exhibit a distinct secretome profile, known as the “Senescence-Associated Secretory Phenotype (SASP)” (Coppé et al. 2008). SASP mediates the interactions between senescent and neighboring cells after DNA damage. Thus, the inflammasome and interleukin 1 (IL-1) signaling are activated in neighboring cells. IL-1α expression can reproduce SASP activation, resulting in senescence. Thereby, a “chronic” SASP effect induces senescence in adjacent young cells, contributing to tissue dysfunction (Acosta et  al. 2013; Caruso et  al. 2004). CHK1 signaling dependent protein kinase, ATM depletion reduces the secretion of SASP factors. While IL-6 secretion is decreasing 50-fold, IL-8 secretion 10-fold reduces (Rodier et  al. 2009). Pre-­ senescent levels of cytokines receptors are adequate to contribute to the rapid DDR growth arrest. Whereas, senescent cells activate a self-­ amplifying secretory network. In this period, overexpression of CXCR2-binding chemokines support growth arrest (Acosta et al. 2008). On the other hand, DDR involves the concerted action of protein complexes that take place at the DNA lesion. These complexes comprise phosphorylated nucleosomal histone variant (γ-H2AX), ATM, RAD-3 related kinase (ATR), p53 binding

3  The Connection Between Cell Fate and Telomere

Fig. 3.2 Uncapped telomeres activate DNA damage checkpoints. G2/M checkpoint prevents entry into mitosis. This is an essential mechanism for the maintenance of genome integrity. In response to telomere dysfunction, in addition to p53, phosphorylation of CHK1 and CHK2 in ATM/ATR-dependent manner are key event in preventing mitotic entry of uncapped telomeres during the G2/M transition. When telomeres are linearized by altered TRF2 function or mitotic arrest, ATM kinase is activated. Linearized DDR-positive telomeres do not fuse during mitotic arrest. This is consistent with the global NHEJ suppression during cell division, but also remain to be resistant to fusion and NHEJ, when passed into the subsequent G1 phase. Whereas, in protected telomere, TRF2 functions to both suppress ATM activity and modulate T-loop formation. ATM is suppressed at chromosome ends when telomeres are in a T-loop conformation. TRF2 is the only shelterin component that contributes to T-loop formation. (Abbreviations: AKT: Protein kinase B, AltNHEJ: Alternative non-homologous end-joining, ATM: Ataxia telangiectasia mutated, ATR: Ataxia telangiectasia

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and Rad3-related protein, CHK: Checkpoint kinase, CK2: Casein kinase 2, cNHEJ: Classical non-homologous end joining, D-loop: Displacement loop, DDR: DNA damage response, DNA-PK: DNA dependent protein kinase, DSB: Double-strand break, GAR1: Pseudouridylationcatalysis enhancer, HDR: Homology directed repair, HR: Homologous recombination, HSP90: Heat shock protein 90, hTERT: Human telomeric reverse transcriptase, hTR: Telomeric RNA component, MAD2L2: Mitotic arrest deficient 2-like 2 protein, P: Phosphorylation, PARP: poly(ADP-ribose) polymerase, PI3K: Phosphatidylinositol-­3-­kinase, Plk1: Polo-like kinase 1, POT1: Protection of telomeres 1, RAP1: Repressor activator protein 1, T-loop: telomere loop, TIN2: TRF1interacting nuclear factor 2, TCAB1: Telomere Cajal body protein 1, Tel2: Telomere maintenance 2 or TPP1 glutamate and leucine-rich, TPP1: Adrenocortical dysplasia protein homolog, TPP1S: POT1 and TIN2 interacting protein1 short isoform, TRF: Telomeric repeat binding protein, TTAGGG Telomere DNA double-stranded tandem repeats, TTI: Tel two-interacting protein)

protein-1 (53BP1), MRN (MRE11-RAD51-­ lated H2AX (γ-H2AX) promotes the spreading NBS1), and checkpoint kinase-1/2 (CHK1/2) of DNA damage factors along the damaged chro(Campisi and d’Adda di Fagagna 2007; Rodier matin and mediates the amplification of the DNA et  al. 2009; Rouse and Jackson 2002). When a damage signal. Thereby, H2AX phosphorylation DSB is formed, ATM acts near the lesion to dynamically link the DDR machinery to broken phosphorylate a carboxy-terminal serine of chromosomes (Stucki and Jackson 2006). H2AX, a histone variant, which is present Another member of DDR complex, MRN functhroughout the genome. Thus, ATM as the major tions as the DSB sensor in the ATM pathway kinase, is activated in the cellular response to (Paull and Lee 2005). MRE11 dimers, one of the DSBs (Burma et  al. 2001). In fact, phosphory- components of MRN, can bridge and align the

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two DNA ends (Williams et  al. 2008). MRN searches for free DNA, even on nucleosomecoated DNA.  Rad50 binds homoduplex DNA, whereas Mre11 is required for DNA end recognition and nuclease activities (Myler et al. 2017). In fact, MRN has been implicated in the generation of the telomeric overhang and the telomerase pathway. MRN is required for TRF1 phosphorylation by ATM kinase. Such phosphorylation results in the release of TRF1 from telomeres. Dissociation of TRF1 from telomerase facilitates the access of telomerase to the ends of telomeres (Chai et  al. 2006; Wu et  al. 2007). The MRN complex, and especially nijmegen breakage syndrome 1 (NBS1), is required for the alternative lengthening of telomeres (ALT) mechanism (Zhong et  al. 2007). Since MRN interacts with TRF2, its presence at telomeres is actively promoted by shelterin. (Dimitrova and de Lange 2009).

3

Shelterin Complex in Protection of Telomere

Central to the network of protein complexes at the telomeres take place six different proteins, which are collectively known as the telosome or “shelterin” (de Lange 2005; Liu et  al. 2004a). Indeed, the “Shelterin” complex is composed of six proteins including TRF1 and TRF2, the TRF1-interacting nuclear factor 2 (TIN2), repressor activator protein1 (RAP1), protection of telomeres 1 (POT1) and adrenocortical dysplasia protein homolog (TPP1). Of these, TRF1 and TRF2 bind to double-stranded telomeric sequences, whereas POT1 and TPP1 bind to the single-stranded 3′ overhang (Baumann and Cech 2001; Bianchi et  al. 1997; Bilaud et  al. 1997; Zhong et  al. 1992). Within this structure, telomere-binding proteins TRF1 and TRF2 interact with other telomere regulators including TIN2, TPP1, POT1, and RAP1 to ensure proper maintenance of telomeres. The TIN2 binds TRF2 directly, thereby bridging TRF2 to TRF1 (de Lange 2005; Liu et al. 2004a). Telomeres serve as protective molecules by shielding the loss of important genetic information as well as by

maintaining chromosome stability (Schumpert et  al. 2015). Nevertheless, shelterin proteins, TRF1 and TRF2 act as negative regulator for telomere length (Smogorzewska et al. 2000; van Steensel and de Lange 1997). In this context, TRF1 can function in the repression of replication problems at telomeres even when it is not connected to the rest of shelterin. However, the TRF1-TIN2 link is clearly important for the shelterin complex since it improves the expression level and telomeric accumulation of TRF1. Moreover, interaction of TIN2 with TRF1 is not only critical for the accumulation of TIN2 at telomeres but also increases the expression and telomeric accumulation of TRF1 (Frescas and de Lange 2014). As another member of shelterin complex, POT1 protects telomere ends from ATR-dependent DNA damage response, control 5′-end resection at telomere termini, and regulates telomerase-dependent telomere elongation. The first subunit of POT1, POT1a deletion results in chromosomal instability, while expression of POT1b provides the proper maintenance of chromosomal stability (He et al. 2006; Wu et al. 2006a). TPP1 interacts with both POT1 and TIN2. Thus, TPP1 binds to the carboxyl terminus of POT1 and recruits it to telomeres. In addition, TPP1 directly interacts with the telomerase for its recruitment to telomeres. On the one hand, TPP1-­ POT1 association enhances POT1 affinity for telomeric single-stranded DNA (ssDNA), on the other hand, inhibits telomerase access to the telomere. So, TPP1 and POT1 protect human telomeres, by both positively and negatively regulating telomerase access to telomere DNA (Liu et  al. 2004b; Wang et  al. 2007; Xin et  al. 2007). The central component of shelterin, TIN2 is critical for the function of POT1a and POT1b because it links TPP1/POT1 heterodimers to TRF1 and TRF2, simultaneously. This connection stabilizes TRF2 on the telomeres. POT1interacting protein 1 (PIP1) is connected to both POT1 and the TIN2. Furthermore, PIP1 could tether POT1 to the TRF1 complex (Ye et  al. 2004a, b). As described above, both TIN2 and TPP1 are key components mediating the six-protein complex assembly. Not only TIN2 but also TPP1 are required to bridge the TRF1 and TRF2

3  The Connection Between Cell Fate and Telomere

subcomplexes. Specifically, TPP1 helps to stabilize the TRF1-TIN2-TRF2 interaction and promote six-protein complex formation. POT1 is a specialized G-strand binding protein, whereas TPP1 interacts with both telomerase and other components of the shelterin complex (O’Connor et al. 2006). TRF1, TRF2 and POT1 bind directly to telomeric repeats and TIN2, TPP1 and RAP1 are interconnected to these proteins thus form a functional complex which caps telomeric end (Liu et al. 2004a). RAP1 forms a complex with TRF2 and affects the length and heterogeneity of human telomeres and thus play a role in telomere function and end protection (Li and de Lange 2003). Telomeric DNA end-binding proteins have generally been found to inhibit rather than stimulate the action of the chromosome end-­ replicating enzyme, telomerase. In fact, instead of inhibition of telomerase access to the telomere, POT1-TPP1 serves as transaction factor of shelterin complex for telomerase during the telomere extension (Wang et  al. 2007). As recently suggested that, TPP1-short (TPP1-S) and TPP1-long (TPP1-L) isoforms of TPP1 are distinct in somatic cells versus differentiated cells of the germline, regarding their ability to activate telomere synthesis. Telomerase recruited to the telomere by TPP1-L cannot efficiently extend chromosome ends in somatic cells, thereby TPP1-L is a telomerase-activation-incompatible isoform of TPP1 (Grill et al. 2019). Components of shelterin; TRF1, TIN2, RAP1, TRF2 act by regulating opening of the telomeric chromatin, whereas the POT1-TPP1 heterodimer directly affects telomerase activity (Churikov and Price 2008). The TRF2-interacting (RCT) domain of Rap1-TIN2 allele deficient in TRF1 binding fusion is fully functional in terms of chromosome end protection by TRF2, TPP1/POT1a, and TPP1/POT1b. When adequate TIN2 is loaded onto telomeres, its interaction with TRF1 is not required to mediate the function of TRF2 and the TPP1/POT1 heterodimers. Chromosome ends can be protected by TRF2-tethered TIN2/TPP1/ POT1 complex of shelterin (Frescas and de Lange 2014). The hTR contains the H/ACA-box motif. The H–ACA pre-RNP (ribonucleoprotein) complex involves dyskerin, NOP10, NHP2, and

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nuclear assembly factor 1 (NAF1). Two full RNP complexes bind to the hTR, one at each hairpin (Egan and Collins 2010). Dyskerin and NOP10 interact with hTR while GAR1 and NHP2 are recruited through protein–protein interactions to dyskerin and NOP10, respectively. Although NHP2 on its own bind RNA nonspecifically, this Dyskerin-NOP10-NHP2 core trimer specifically recognizes H/ACA RNAs (Egan and Collins 2012; Wang and Meier 2004). The core complex formation consists of H/ACA, Sm, and Heterogeneous nuclear ribonucleoproteins (hnRNP) proteins, is necessary for hTR stability and accumulation. Telomerase RNPs lacking interaction with Sm proteins or hnRNP C, leads to telomere elongation. Thus, H/ACA RNPs are essential for three fundamental cellular processes: protein synthesis, mRNA splicing, and maintenance of genome integrity (Fu and Collins 2007; Kiss et al. 2010). While Dyskerin-NOP10-­ NHP2 complex colocalizes at the site of hTR transcription, GAR1 does not. NAF1 and GAR1 bind to Dyskerin competitively. NOP10 associates with Dyskerin as a prerequisite for NHP2 binding (Darzacq et  al. 2006; Wang and Meier 2004). Therefore, GAR1 must join the complex later to form the mature RNP (MacNeil et  al. 2016). The complex preceding GAR1 association is known as the pre-RNP and includes the association factor NAF1. Human NAF1 cannot bind directly to the H/ACA domain of hTR, and requires the core trimer dyskerin-­NOP10-­NHP2 to be efficiently incorporated into the pre-RNP (Trahan and Dragon 2009). Furthermore, NHP2 can only associate with the pre-RNP tetramer in the presence of NOP10. Therefore, NOP10NHP2 likely bind to dyskerin as a heterodimer. Then, NAF1 can act as a nucleolar shuttle, bringing an inactive precursor complex to the nascent hTR to form the pre-RNP complex (MacNeil et al. 2016). The telomerase RNA subunit, hTR is shown to accumulate in Cajal bodies (CBs), subnuclear structures implicated in ribonucleoprotein maturation. NAF1 is also excluded from the CBs and nucleolus. Its subnuclear localization is an important regulatory mechanism for telomere length homeostasis in human cells (Cristofari et  al. 2007; Hoareau-Aveilla et  al. 2006). Once

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the pre-RNP is assembled, NAF1 must be substituted by GAR1 to form a mature complex (MacNeil et al. 2016). The exchange of NAF1p by GAR1p might be prompted by external factors that alter the oligomerisation state of NAF1p and GAR1p. The substitution of GAR1 for NAF1 is a key distinguishing characteristic between an active mature RNP complex and a pre-RNP complex (Leulliot et al. 2007). The p23 forms a complex with HSP90 and contributes to telomerase activity. Both hsp90 and p23 bind specifically to hTERT and influence its proper assembly with the template RNA, hTR (Forsythe et  al. 2001; Holt et al. 1999). Both chaperones associate stably with the N-terminus of hTERT and, interestingly, remain associated with active telomerase after proper folding of hTERT has taken place. Physical interaction between HSP90 and the hTERT promoter occurs in human immortalized and cancer cells, while absent from normal senescing cells which do not express telomerase. Inhibition of hsp90 results in decrease of the hTERT promoter activity, mRNA expression level and telomerase activity (Kim et  al. 2008). The activation and deactivation of telomerase which is mediated through transcriptional regulation of hTERT is key for maintaining control of cellular growth (MacNeil et  al. 2016). In this context, phosphorylation of hTEP1 and hTERT by protein kinase C-alpha PKCα and Akt/protein kinase B induces a marked increase in telomerase activity (Kang et al. 1999; Li et al. 1998). In contrast, c-Abl tyrosine kinase phosphorylates hTERT and inhibits hTERT activity (Kharbanda et  al. 2000). c-Abl associates with the DNAdependent protein kinase (DNA-PK) complex and ATM (Kharbanda et al. 2000; Shafman et al. 1997). The catalytic subunit of DNA-PK and ATM as the members of a family of phosphatidylinositol (PI) 3-kinase (PI3K)-like enzymes involve the control of telomere length (Kharbanda et al. 2000). Similarly, cytoplasmic C terminus of Hsc70-interacting protein (CHIP) physically associates with hTERT and regulates the cellular abundance of hTERT.  Overexpression of CHIP prevents nuclear translocation of hTERT and promotes hTERT degradation in the cytoplasm, thereby inhibiting telomerase activity (Lee et al.

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2010). Indeed, phosphorylation of hTERT is also important for nuclear localization. Phosphorylated hTERT and its nuclear import are correlated with telomerase activation (Liu et  al. 2001). Proteasomal degradation of ubiquitinated hTERT occurs in the cytosol, and as such, controlling nuclear import/export of the protein is key for regulating this degradation (MacNeil et al. 2016). Overexpression of Plk1 leads to an increase in hTERT protein levels without affecting the mRNA levels. Actually, Plk1 affects hTERT stability by inhibiting its ubiquitin-­mediated degradation. Thus, nuclear retention mediated through Plk1 prevents proteasomal degradation of hTERT and increases telomerase activity (Huang et  al. 2015). Protein Tyrosine Phosphatase 1B (PTP1B) undergoes phosphorylation during mitotic arrest and promotes mitotic cell death. Inhibition of Cdk1 or Plk1 during mitosis prevents PTP1B phosphorylation and inhibits PTP1B phosphatase activity (O’Donovan et  al. 2013). Increased telomerase recruitment is observed upon phosphorylation of the shelterin component TRF1 at an ATM/ATR target site. This phosphorylation leads to loss of TRF1 from telomeres. Phosphorylated (pS367) TRF1-­ containing foci may represent nuclear sites for TRF1 proteolysis. ATM and ATR depletion reduces assembly of the telomerase complex. However, ATM is required for the regulation of telomere length and stability in cells expressing POT1 that disrupts telomerelength homeostasis. Thus, human telomerase recruitment and telomere elongation are modulated by DNA-­ damage-­ transducing kinases (McKerlie et al. 2012; Tong et al. 2015). In fact, ATM and ATR regulate parallel damage response signaling pathways. ATM is activated by DSBs, whereas ATR is recruited to single-stranded regions of DNA. Although the two pathways are considered to function independently, ATRdependent phosphorylation of ATM activates ATM phosphorylation of Chk2, which has an overlapping function with Chk1  in regulating G2/M checkpoint arrest (Stiff et al. 2006). In fact, human telomeres contain two related TTAGGG repeat binding factors, TRF1 and TRF2 (Broccoli et  al. 1997). The DNA binding site of TRF1 is composed of two identical half sites which each

3  The Connection Between Cell Fate and Telomere

engage one Myb-type DNA binding domain in the TRF1 homodimer. There is no constraint on the distance between the two half sites, and the sites can be bound in direct or inverted orientation (Bianchi et al. 1997). Thus, a T-loop-based mechanism for telomere length regulation presents that both TRF1 and TRF2 are required for the length homeostasis of human telomeres. In this respect, TRF1 and TRF2 acts as a negative regulator of telomere length (Smogorzewska et al. 2000). Although most mammals have a single POT1 gene, rodents have two POT1 proteins that are functionally distinct. Thus, rodent telomeres contain two closely related POT1 proteins, POT1a and POT1b. Both POT1a and POT1b can repress ATR kinase signaling in G1 but repression of ATR in S/G2 requires POT1a (Gong and de Lange 2010; Palm et  al. 2009). In mammals, POT1 restricts telomere elongation by decreasing access to the DNA terminus. The DNA binding activity of POT1 is required for telomerase inhibition. But it is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding (Kelleher et  al. 2005; Lei et  al. 2005). In contrast, TPP1 promotes telomere elongation by recruiting telomerase, increasing telomerase processivity or both. In brief, TPP1 and POT1 form a complex with telomeric DNA that increases the activity and processivity of the human telomerase (Wang et  al. 2007; Xin et  al. 2007). TRF2 or POT1 deletion activates ATM- and ATR-dependent DNA damage checkpoints, respectively, through phosphorylation of checkpoint kinases CHK1 and CHK2 (Denchi and de Lange 2007; Guo et  al. 2007). In addition to the canonical ATM/ CHK2 and ATR/CHK1 signaling modules, the DDR network has connections with the diverse pathways involving PI3K/AKT, inhibitor of kappa B (IκB) kinase (IKK)/ nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and MAPKs. NF-κB essential modulator (NEMO) is the regulatory subunit of IKK. IKK phosphorylates the NF-κB inhibitor and associates with activated ATM after the induction of DSBs (Huang et  al. 2003; Wu et  al. 2006b). If telomeres are critically short or otherwise defec-

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tive, ATM-ATR signaling cannot be suppressed and a full DDR ensues. Whereas, inhibition of ATM expression or activity result in cell cycle reentry, indicating that stable arrest requires continuous signaling (d’Adda di Fagagna et al. 2003; Herbig et  al. 2004). If the extent of damage exceeds repair capacity, additional signaling cascades are activated to ensure elimination of these damaged cells. The DDR has traditionally been divided into two major kinase branches. The ATM/CHK2 module is activated after DNA DSBs, while the ATR/CHK1 pathway responds primarily to DNA single strand breaks or bulky lesions. Both pathways converge on CDC25, a positive regulator of cell cycle progression, which is inhibited by CHK1-mediated or CHK2mediated phosphorylation (Reinhardt and Yaffe 2009). As an oncogenic protein, CDC25 plays an important role in transitions between cell-cycle phases by dephosphorylating and activating CDKs. The activation and deactivation of CDC25 by kinases and phosphatases, respectively, maintain the level of CDK–cyclin activities and thus the genomic stability (Sur and Agrawal 2016). However, MEK1 destabilizes the CDC25B by phosphorylating the Ser249, leading to proteasomal degradation of CDC25B. Downregulation of CDC25B causes significant delay in entry into mitosis (Astuti et al. 2009). Under cellular stress, activated c-Jun N-terminal Kinase (JNK) and p38 phosphorylate Ser101 of CDC25B, resulting in the rapid degradation of CDC25B and cell cycle arrest. In this respect, JNK/p38-Cdc25B axis is a nongenotoxic stress-induced cell cycle checkpoint (Uchida et al. 2009). DDR can induce mitochondrial reactive oxygen species (ROS) and this is partially dependent on the activation of p53 and p21 pathways, besides the signals from ATM towards mammalian target of rapamycin complex 1 (mTORC1). Long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of ROS through serial signaling through growth arrest and DNA damage 45 (GADD45)-MAPK14 (p38MAPK)- growth factor receptor-­bound protein 2 (GRB2)- transforming growth factor beta receptor 2 (TGFBR2)-TGF-β. The ATM, Akt and mTORC1 phosphorylation cascade integrates

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DDR signaling towards the peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator-1β (PGC-1β)-dependent mitochondrial biogenesis. This event contributes to ROS-mediated activation of the DDR and cell cycle arrest. Thereby, a novel pathway has been identified involving ATM, Akt, mTORC1 and PGC-1β in the induction of senescence (Correia-­Melo et  al. 2016; Passos et  al. 2010). The locally increased ATM activity is important for phosphorylation of ATM substrates, which are CHK2 and the protein p53. ATM activates the CHK2 by direct phosphorylation on Thr-68 (Melchionna et al. 2000). In addition to p53, phosphorylation of CHK1 and CHK2 are key events in preventing mitotic entry of uncapped telomeres. Eliminating CHK1 and CHK2 functions in TRF2- or POT1-depleted cells results in progression into mitosis with a high frequency of damaged telomeres (Chang 2012). Inactivation of DNA damage checkpoint kinases in senescent cells can restore cell-cycle progression into S phase. Therefore, telomere-­ initiated senescence is a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres (d’Adda di Fagagna et  al. 2003). DDR results in activation of transcription factor p53 which is involved in DNA repair, cell-cycle arrest and apoptosis. p53 is a positive regulator of transcription of p21, a Cdk inhibitor, which is involved in the cell-cycle arrest observed during senescence (Victorelli and Passos 2017). In fact, damaged DNA is known to induce CHK1/CHK2-dependent phosphorylation of CDC25 phosphatases, which in turn prevents mitotic CDK activation and thus causes cell-­ cycle arrest (Bartek and Lukas 2003). Telomere shortening over successive cell divisions triggers a p53-dependent G2 checkpoint response that involves the ATM and ATR-­ dependent DNA damage signaling pathways. Short telomeres are more prone to persist in an uncapped state and to trigger the p53-dependent G2 checkpoint response. In such cells, the ATM/ ATR kinases are activated and p53 acts as an effector to delay mitotic entry. This process promotes the re-establishment of post-replicative telomere end-processing and protection (Jullien et al. 2013). Shelterin component, TRF2 prevents

ATM activation, while POT1 represses ATR signaling at telomeres. Telomere damage-­ activated ATR and ATM phosphorylate the p53, CHK1 and CHK2. Thus, activation of two independent pathways prevent progression into mitosis in uncapped telomeres. Surprisingly, telomere damage targets the Cell Division Cycle 25C (CDC25C) phosphatase for proteasome degradation in G2/M.  Therefore, CHK1/CHK2dependent phosphorylation of CDC25C at Ser 216 is required for CDC25C nuclear export and destruction. Demolition of CDC25C acts to sustain the G2/M arrest elicited by TRF2- or POT1depleted telomeres (Thanasoula et  al. 2012). If CDC25C is not phosphorylated, this results in both CDC25C stabilization and mitotic entry with uncapped telomeres. p53 further contributes to TRF2-specific G2/M arrest through downregulation of CDC25C (Chang 2012). If DNA breaks are not repaired, DDR is triggered immediately, then with cell cycle arrest, DSB accumulation leads to cell death (Gospodinov and Herceg 2013; Jeggo and Downs 2014).

4

Telomere Damage

The response to DNA breaks prevents the loss of genomic information through progressive chromosome shortening caused by the semi-­ conservative replication of DNA.  In humans, DNA ends, or telomeres are 5–15  kb of double stranded 50-TTAGGG-30/30CCCTAA-50 sequence repeats which terminate in a single stranded long 30  G-rich overhang of 130–210 bases in length. In this context, normal human chromosomes have long G-rich telomeric overhangs at one end. Overhang is four times greater than the amount of telomere shortening per division. A decrease of about 50 base pairs per generation suggest that a full deletion event is 100 to 200 base pairs. When cells get old after 80 doublings, about 4000 base pairs decrease in mean telomere length (Bohgaki et al. 2010; Levy et  al. 1992; Makarov et  al. 1997; Wright et  al. 1997). Most somatic cells lack the activity of the enzyme telomerase and experience, with cell division, a phenomenon called the “end-­

3  The Connection Between Cell Fate and Telomere

replication problem”. This occurs due to the intrinsic inability of DNA polymerases (Victorelli and Passos 2017). If the progressive loss of telomere repeats with cell division due to the “end-replication problem”, shelterin components may be displaced from telomere regions and subsequently destabilize the “T-loop” conformation. In fact, T loops are general mechanism for the protection and replication of telomeres (Griffith et  al. 1999; Victorelli and Passos 2017). This results in the exposure of the telomere end during the double-strand DNA break (Victorelli and Passos 2017). There are two major pathways by which a telomere can become uncapped or dysfunctional and lead to end-to-end chromosomal fusions. The first pathway involves progressive loss of telomeric sequences that occurs in the absence of telomerase due to the end-replication problem. The second major pathway for telomere dysfunction is the loss of a telomeric capping structure. In this case, telomeres are sufficiently long, and telomeric sequences are visible at the point of fusion between the misjoined chromosome ends, but the telomere end is unable to form the T-loop (de Lange 2002b; Espejel et al. 2002; Griffith et al. 1999). Telomeric DNA is present at the ends of eukaryotic chromosomes and is bound by telomere “capping” proteins, which are the Cdc13-Stn1-Ten1 (CST) complex and Ku (Yku70-Yku80). Inactivation of any of these complexes causes telomere “uncapping,” stimulating a DDR that frequently involves resection of telomeric DNA and stimulates cell cycle arrest (Dewar and Lydall 2012). CST function is correlated with POT1 levels at the telomere and CST depletion at the telomere and uncontrolled telomere elongation. POT1(CP) (homozygous S322L substitution in POT1) induces a proliferative arrest that could be bypassed by telomerase. POT1 is expressed at normal levels, binds TPP1 and telomeres, and blocks ATR signaling (Takai et al. 2016). In fact, telomeric integrity is efficient in aging, as telomere attrition serves as a key checkpoint in the control of cell proliferation by triggering replicative senescence. Two mechanisms are defined for telomere preservation in humans, as telomerase-

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mediated maintenance and alternative lengthening of telomeres (MacNeil et al. 2016; Shay et al. 2012). Approximately 80% of cancer cells are immortalized via activation of telomerase to maintain telomeres throughout rapid cellular proliferation (Shay et al. 2012). However, telomeres and subtelomeric regions are poor substrates for DNA replication; therefore, regions near telomeres are prone to replication fork stalling and chromosome breakage. DSBs near telomeres cannot be properly repaired. Thus, when DSBs occur near telomeres in normal cells, proliferation is stopped (Muraki and Murnane 2018). The telomeric DNA is shielded from DSB repair activities by specialized telomere proteins that bind along the duplex region of the telomere and to the overhang on the 3′ G-rich strand (Churikov and Price 2008). As mentioned above, the protection of chromosomal ends is recognized as DNA breaks. Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are crucial to prevent genomic instability throughout the cellular lifespan. Mitotic arrest deficient 2-like 2 protein (MAD2L2) accumulates at uncapped telomeres. This promotes Non-­ homologous end joining (NHEJ)-mediated fusion of deprotected chromosome ends. The activity of MAD2L2 depends on ATM kinase activity (Boersma et al. 2015). MAD2L2 is an interaction partner of nuclear receptor coactivator-3 (NCOA3). Overexpression of MAD2L2 suppresses the proliferation, migration, and clonogenicity of immortal cells by inducing the degradation of NCOA3. Whereas, NCOA3 promotes cancer cell proliferation through regulating the PI3K/AKT and Notch signaling pathway in RNA levels (Li et al. 2018). DSBs can arise in the case of telomere uncapping following erosion of telomeric repeats or loss of telomere-­binding proteins. Cells entering mitosis with uncapped telomeres generate chromatids that are joined in G1 to initiate bridge-fusion-breakage cycles, leading to the accumulation of deleterious chromosomal rearrangements (Chang 2012). Because the somatic cells cannot express telomerase, with each round of DNA replication results in progressive attrition of telomeric sequences. Eventually, this attrition culminates in

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the inability of chromosome ends, and at this crisis point, the resulting DDR halts further cell proliferation (Apte and Cooper 2017). DNA damage at non-telomeric regions is undetectable, thereby telomeric DNA damage shortens the average length of telomeres and leads to cell senescence (Sun et  al. 2015). Short telomeres become dysfunctional, signaling a p53-related DNA-damage response provokes senescence and apoptosis (Chin et al. 1999; d’Adda di Fagagna et  al. 2003). Short telomere patient–derived T cells also showed evidence of a DNA-damage DSB response and upregulated intrinsic apoptosis. Although short telomeres are acquired with aging, there is a threshold where this shortening is biologically and clinically relevant and sufficient to signal telomere dysfunction on a cellular level. Such a threshold may not be reached in most individuals with aging (Wagner et al. 2018). One or a few short telomeres are sufficient to induce cellular phenotypes, and the threshold of 400 base pairs has been suggested to be sufficient to induce a short telomere cellular response (Baird et al. 2003). The short, dysfunctional telomeres trigger a DDR that resembles the response elicited by DNA DSBs and loss of growth potential (d’Adda di Fagagna et  al. 2003; IJpma and Greider 2003). Telomerase does not act on every telomere in each cell cycle, but it exhibits an increasing preference for telomeres as their lengths decline (Hug and Lingner 2006). Senescent cells are identified by the expression of the cell cycle inhibitor p16INK4a and cell death by hairpin 1 and 2. Cell death markedly increases in cells expressing p16INK4a that have significant telomeric shortening. Although cell multiplication, mitotic index and telomerase increases, they do not compensate for cell death or prevent telomeric shortening (Chimenti et al. 2003). ATM kinase signal-transduction pathway is primarily responsible for the DNA damage-­induced phosphorylation of TRF2. The phosphorylated form of TRF2 at telomeres causes telomere-based crisis in telomerase-­independent, alternative lengthening of telomere pathway. The link between TRF2 phosphorylation and the DDR system represents direct crosstalk via a signaling pathway between the genomic stability, telomere mainte-

A. B. Engin and A. Engin

nance, and DNA repair (Tanaka et  al. 2005). Mutant template human telomerase RNAs induce DSBs-like damages in telomerase positive cells after phosphorylation of ATM and p53 via suppression of TRF2, which eventually leads to apoptosis (Mahalingam et al. 2011). In this case, ATM is a key mediator of the dysfunctional telomere response, and is the evidence of telomere fusions contributed cytotoxicity (Stohr and Blackburn 2008). On the other hand, a good correlation is found between telomerase activation and resistance to apoptotic cell death under hypoxic conditions. This result is dependent on the increase in telomerase activity via MAPK cascade signaling as a stress response against genotoxicity (Seimiya et  al. 1999). Increase in p53 and hypoxia-inducible factor 1-alpha (HIF-1α) in the hypoxic conditions indicates the induction of damage to genomic DNA by ROS that accelerates cell senescence through p53 activation (Cataldi et  al. 2009). Ectopic mTERT expression confers resistance to apoptosis induced by oxidative stress and other genotoxic insults. This resistance depends on the catalytic activity of mTERT. Although, the production of G-rich single stranded fragments during the telomere shortening triggers a p53 dependent cell cycle arrest, mTERT exerts its protective effect by antagonizing the p53 pathway (Lee et  al. 2005; Saretzki et al. 1999). In fact, hTERT maintains cell survival and proliferation via both telomerase enzymatic activity-dependent telomere lengthening and enzymatic activity-­ independent intermolecular interactions involving p53 and poly (adenosine diphosphate (ADP)-ribose) polymerase PARP (Cao et  al. 2002). TRF1 has a fundamental role in protecting telomeres from DNA repair activities. Thus, blocking replication-induced fragility prevents telomeric DNA damage. TRF1-depleted telomeres are prone to breakage. This leads to death which are associated with induction of telomere-instigated DNA damage, activation of the p53/p21 and p16 pathways, and cell cycle arrest (Martínez et  al. 2009; Sfeir et  al. 2009). TRF1 controls telomere length by inhibiting the action of telomerase at the ends of individual

3  The Connection Between Cell Fate and Telomere

telomeres. However, long-term overexpression of TRF1  in human cells results in progressive telomere shortening (van Steensel and de Lange 1997). PI3K/TRF1 inhibitor significantly decreases TRF1 foci of telomere, and induces telomeric DNA damage. Thereby, PI3K/AKT pathway plays a central role in regulation of telomere protection (Méndez-Pertuz et al. 2017). The AKT signaling pathway plays an important role in telomere protection. Telomere damage and reduced TPP1 dimerization as a result of Akt inhibition was also accompanied by diminished recruitment of TPP1 and POT1 to the telomeres (Han et  al. 2013). TPP1 and POT1 form a complex with telomeric DNA that increases the activity and processivity of the human telomerase. POT1-TPP1 switches from inhibiting telomerase access to the telomere, as a component of shelterin, to serving as a processivity factor for telomerase during telomere extension (Wang et al. 2007). On the other hand, caspase-dependent loss of the shelterin complex protein TRF2 from telomeres promotes a DDR that involves DNA-PK.  Prolonged mitotic stress is characterized by the sub-apoptotic activation of a classical caspase pathway, which promotes telomere deprotection, activates DNA damage signaling, and determines cell fate in response to a prolonged delay in mitosis (Hain et al. 2016). G-quadruplex-forming2 sequences occur naturally in the telomeres as well as promoter regions (Huppert and Balasubramanian 2005). When cells sense DNA damage or replication arrest, cell cycle checkpoints are activated that lead to cell cycle arrest. In addition to checkpoint activation DNA repair pathways are improved and, if the level of damage is severe, cell death occurs (Ciccia and Elledge 2010). Pu-27 is a Gly/ Cys-rich sequence that forms an intramolecular DNA quadruplex structure, which causes extensive DNA damage by inducing a brisk DNA damage response. Pu-27-mediated cell killing involves interference with c-Myc transcription (Islam et al. 2014). Its overexpression causes cell death, whereas inhibition of c-Myc expression of cells sensitizes to tumor necrosis factor (TNF)induced apoptosis. TNF-induced, cell death is dependent on Fas-associated protein with death

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domain (Liu et  al. 2000a). Although the exact mechanism by which Pu-27 causes dysfunction in the c-Myc promoter or the telomere shelterin complex is not known, c-Myc has some role in chromosomal rearrangement and remodeling through the telomere, and directly interacts with the catalytic subunit of telomerase. Subsequent to initial chromosomal breakages, new fusions follow, and the breakage-bridge-fusion cycles continue. In this process, c-Myc-dependent remodeling of the telomeres leads to chromosomal rearrangements (Flores et al. 2006; Louis et al. 2005). ATM deficient cells are more sensitive to Pu-27 and exhibit abundant telomere dysfunction-­induced foci. The shelterin complex does not properly protect the ATM deficient cells. This concept is defined as synthetic lethality. Cell death mediated by ligand-induced G-quadruplexes stabilization can be potentiated in cells deficient in DNA damage repair genes (Kaelin 2005; McLuckie et  al. 2013). On the other hand, the hnRNP-A1 is phosphorylated by DNA-PKcs during the G2 and M phases. DNAPK-dependent hnRNP-A1 phosphorylation promotes the replication protein A (RPA)-to-POT1 switch on telomeric single-stranded 3′ overhangs. In cells lacking hnRNP-A1 or DNAPKcs-­ dependent hnRNP-A1 phosphorylation, impairment of the RPA-to-POT1 switch results in DNA damage response at telomeres during mitosis as well as induction of fragile telomeres (Sui et al. 2015; Ting et al. 2009). ATM and ATR checkpoint kinases are critical for the full checkpoint response to DSBs (Brown and Baltimore 2003; Cortez et  al. 2001). Blunt DSB end and DNA end with short ssDNA tails are the substrates for ATM binding (Shiotani and Zou 2009), while ATR recruitment requires the formation of RPA-coated ssDNA arising from 5′–3′ nucleolytic degradation of the DSB end. The binding of ATR-interacting protein (ATRIP) to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA (Zou and Elledge 2003). ATM and ATR are activated by similar DNA structures at resected DSBs. While both ATM and ATR depend on the junctions of single- and double-stranded

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DNA for activation, they are oppositely regulated by the lengthening of single-stranded overhangs (SSOs). SSOs simultaneously attenuate ATM activation and potentiate ATR activation, thereby promoting an ATM-to-ATR switch during the process of DSB resection (Shiotani and Zou 2009). Indications of crosstalk at the level of the CHKs are largely due to interconversion of the DNA lesions. End resection converts DSBs into ssDNA structures that activate ATR while nucleases can cleave ssDNA to yield DSBs that activate ATM (Cimprich and Cortez 2008). Although ATM and ATR may collaborate in many ways, the phenotypes that result from the loss of ATM and ATR are dramatically distinct. The loss of ATR causes rapid lethality at the earliest embryonic stages. In brief, ATR is essential for cell viability (Brown and Baltimore 2000; de Klein et  al. 2000). In mammals, there are six PIKKs: ATM, ATR, DNA-PKcs, mTOR, transformation/ transcription domain-associated protein (TRRAP), and suppressor with morphological effect on genitalia 1 (SMG1). PIKKs typically function in the context of multi-protein complexes (Lovejoy and Cortez 2009). The chaperone proteins (TTT complex), comprising telomere maintenance 2 (Tel2), Tel2-interacting proteins 1 and 2 (TTI1 and TTI2) form a conserved trimeric complex called the “Triple T complex”, which stabilizes the PIKK enzymes. TTI1 and TTI2 protect cells from spontaneous DNA damage, and are required for the establishment of the intra-S and G2/M checkpoints. Moreover, Triple T complex constitutes multiple complexes with ATM. Deletion of TEL2 results in significantly reduced protein levels of ATM, ATR, and DNA-­ PKcs (Hurov et  al. 2010; Langouët et  al. 2013; Takai et  al. 2007). The ATM, ATR, and DNA-­PKcs PIKKs function in pathways involved in maintaining genomic integrity. ATM and ATR sense DNA DSBs. The PIKKs depend on Hsp90 and Tel2–TTI1–TTI2 (Triple T complex) for their maturation. It is obvious that the Tel2–TTI1–TTI2 complex is a PIKK-specific cochaperone for Hsp90 (Takai et al. 2010). The DNA-PKcs has a fundamental role in telomere length maintenance. DNA-PKcs is essential for both end-to-end fusions and apoptosis triggered

by critically short telomeres. In telomerase-­ deficient human somatic cells, DNA-PKcs abrogation leads to a faster rate of telomere degradation and cell cycle arrest (Espejel et  al. 2002). Casein kinase-2 (CK2) translocates to the cytoplasm. TTI1 and TEL2 are physiologically phosphorylated by CK2, thereby influencing the stability and function of PIKKs. Deletion of Tel2 results in significantly reduced protein levels for the PI3K-related kinases ATM and ATR, DNA-­ PKcs (Horejsí et  al. 2010; Takai et  al. 2007). These phosphorylations enable the TTT complex to bind and stabilize DNA-PKcs and ATM, later activates p53 (Takai et  al. 2007; Vousden and Prives 2009). In fact, DNA-PK acts as both a sensor and transmitter of DNA damage signals that directly impact cell fate. Phosphorylation of the ATM target site on p53 regulates p53 apoptotic function. The upstream effectors, DNA-PKcs and ATM selectively activate p53 to differentially regulate cell-cycle and apoptotic responses. ATM causes cell-cycle arrest but not apoptosis, whereas DNA-PKcs causes apoptosis but not cell-cycle arrest (Hill and Lee 2010; Sluss et al. 2004; Wang et al. 2000).

5

Non-homologous End-­ Joining Pathways at Telomeres

Homologous recombination (HR), and NHEJ are the principal pathways for DSB repair and the balance between them depends on the cell cycle stage, and type of DNA damage (Heyer et  al. 2010). There are two subtypes of NHEJ operate in many cells. These are referred to as classical NHEJ (cNHEJ) and alternative NHEJ (altNHEJ). In mammalian cells, NHEJ, which is the error-­ prone joining of DNA ends plays an important role in DSB repair, especially during the G1 phase of the cell cycle when no sister chromatid is available (Rodgers and McVey 2016). Second principles pathway, HR is required for accurate chromosome segregation and constitutes a key repair and tolerance pathway for complex DNA damage, including DNA DSBs, interstrand crosslinks, and DNA gaps (Heyer et  al. 2010).

3  The Connection Between Cell Fate and Telomere

The cNHEJ machinery recognizes breaks, however, indiscriminately joins them and is therefore potentially genotoxic (Lieber 2010). Whereas, HR is a key pathway to maintain genomic integrity between generations (Heyer et  al. 2010). Choice between HR and cNHEJ depends primarily on the cell cycle stage and the nature of the break. During G1 phase cNHEJ is dominant, but during S and G2 phases, both cNHEJ and HR compete (Arnoult et  al. 2017; Panier and Boulton 2014). Activation of cNHEJ leads to chromosome end-to-end fusions, while HR and altNHEJ remain inhibited by shelterin. In fact, these proteins are not a hallmark of a dysfunctional telomere. Contrarily, telomeres conceal ends of the linear chromosome from inappropriate repair, and provide a buffer to counteract replication-associated shortening (Smogorzewska et al. 2002; Verdun and Karlseder 2007). Fusions of deprotected telomeres, which occur through cNHEJ exclusively, are restricted to G1 phase. Thus, the processing of dysfunctional telomeres by NHEJ occurs primarily in G1, being repressed in S/G2  in a CDK-dependent manner (Celli and de Lange 2005; Konishi and de Lange 2008). In brief, the NHEJ is the major repair pathway in G1 cells when the sister chromatids for HR are lacking. DSB repair by NHEJ is a direct ligation of the two broken DNA ends (Pandita and Richardson 2009). Although telomere proteins shield the chromosome end from improper repair activities, a wide range of DDR proteins are also found at telomeres. Thus, the critical function of telomeres is their ability to distinguish the ends of linear chromosomes from DSBs, and avoid NHEJ (d’Adda di Fagagna et al. 2003; de Lange 2005; Riha et  al. 2006). Shelterin averts activation of three DNA damage response enzymes ATM, and ATR kinases and PARP1. Additionally, Shelterin blocks three DSB repair pathways, including cNHEJ, altNHEJ, and homology directed repair (HDR). Consequently, it prevents the hyper-­ resection at telomeres (de Lange 2018). The deleterious events at shelterin-free telomeres are the “end-protection problems” of six pathways related to six shelterin proteins. So, deletion of each individual shelterin proteins results in the

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end-protection problem. These events minimally involve the repression of ATM and ATR signaling as well as inhibition of DSB repair by NHEJ and HDR.  Shelterin protects the chromosome ends against classical NHEJ, and inadvertent activation of the ATM and ATR signaling (Sfeir and de Lange 2012). Fundamentally, POT1 is required to prevent the telomere from activating a catastrophic DDR (Churikov et al. 2006). Damage response primarily involves CHK1 signaling. CHK1 signaling depends on both ATM and ATR activation, but the damage response is thought to be mediated via ATR rather than ATM.  TRF2 represses ATM, while POT1 prevents activation of ATR. However, either ATM or ATR signaling is required for efficient NHEJ of dysfunctional telomeres (Churikov and Price 2008; Denchi and de Lange 2007). Telomeres are exposed to cNHEJ and ATM-dependent DNA damage signaling. TRF2 protects chromosome ends by maintaining the correct structure at telomere termini. A two-step mechanism is defined for TRF2-mediated end protection. At first step, the dimerization domain of TRF2 is required to inhibit ATM activation, which is the key initial step involved in the activation of a DDR.  At second step, TRF2 independently suppresses the propagation of DNA-damage signaling downstream of ATM activation. Functional telomeres exhibit a T-loop configuration. Thus, TRF2 is required for the formation and maintenance of T-loops. Consequently, within the shelterin complex, TRF2 uniquely serves to protect telomeres from two pathways that are initiated on free DNA ends. These pathways comprise cNHEJ pathway and ATM-dependent DNA damage signaling (Doksani et  al. 2013; Okamoto et  al. 2013; van Steensel et al. 1998). In TRF2 deprived telomeres ATM-dependent signaling is activated and telomeres fuse due to DNA Lig4-dependent NHEJ. Thereby, telomere fusions resulting from inhibition of the telomere-protective factor, TRF2 are generated by DNA ligase IV-dependent NHEJ (Karlseder et  al. 1999; Smogorzewska et  al. 2002; van Steensel et al. 1998). In fact, telomere protection by TRF2 consists of DDR inhibition via formation of T-loop structures, and inhibition

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of DDR response directly by TRF2 throughout the telomeric domain. The telomere-internal DSBs activate the ATM kinase-dependent signaling pathway and are repaired through a PARP1- and Lig3-dependent joining reaction as well as by HR. The ability of shelterin to repress ATM signaling, altNHEJ, and HR at chromosome ends is not functional at telomere-internal DSBs (Doksani and de Lange 2016). On the other hand, POT1 is one of the six factors in the shelterin complex, which protects telomeres from DNA damages. Overexpression of POT1 inhibits the protein stability of Lig3, which is the major regulator of altNHEJ, therefore suppressing the efficiency of altNHEJ (Yu et al. 2017). 53BP1 is a cNHEJ component and an ATM target that accumulates at DSBs and at uncapped telomeres. 53BP1 binds to the DNA-binding domain of p53 and enhances p53-mediated transcriptional activation (Fernandez-Capetillo et  al. 2002; Rappold et al. 2001; Takai et al. 2003; Wang et al. 2002). In addition, 53BP1 participates early in the DDR and is involved in cell cycle checkpoint. The NHEJ of DSBs is severely affected by 53BP1 deficiency. Depletion of 53BP1 results in cell cycle arrest in G2/M phase as well as genomic instability in human cells. The DDR protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by NHEJ as opposed to HR (Callen et  al. 2013; Ward et al. 2004). Cells from longer-lived species have an enhanced DDR. The presence of a greater number of 53BP1 foci is associated with decreased DNA fragmentation and a lower percentage of cells exhibiting micronuclei. Thereby, the number of 53BP1 foci that form in response to damage reflects the intrinsic capacity of cells to detect and respond to DNA harms (Croco et  al. 2017). Deletion of 53BP1  in cells with telomere uncapping as a result of TRF2 deficiency is established that NHEJ of dysfunctional telomeres is dependent on the binding of 53BP1 to damaged chromosome ends. So, depletion of TRF2 results in end-to-end chromosome fusions mediated by the cNHEJ pathway. In contrast, removal of TPP1-POT1 initiates chromosome fusions that are mediated by altNHEJ (Dimitrova et al. 2008; Rai et al. 2010). On the other hand,

A. B. Engin and A. Engin

deletion of 53BP1 in TRF1-deficient cells impairs the cNHEJ repair pathway and decreases the occurrence of chromosome-type end to end telomere fusions, while inducing a persistent DNA damage signal. By this process the ATR-­ dependent DDR and the HR repair pathway is activated. In particular, in the absence of 53BP1, dysfunctional telomeres accumulate higher levels of ssDNA and RPA indicative of increased resection. This, in turn, triggers a hyperactivation of the ATR–CHK1 pathway that further amplifies DDR signaling and that may lead to the G2 arrest (Martínez et  al. 2012). The persistent DNA damage foci (PDDF) contains γH2AX, activated (phosphorylated) ATM and ATM/ATR substrates (Rodier et  al. 2009). The phosphorylation of histone, H2AX facilitates the focal assembly of checkpoint and DNA repair factors, including 53BP1, mediator of DNA damage checkpoint protein 1 (MDC1)/nuclear factor with BRCT domain protein 1 (NFBD1) and NBS1 along the damaged chromatin and promotes the activation by phosphorylation of the transducer kinases, CHK1 and CHK2 (Smogorzewska and de Lange 2002). Indeed, nuclear factor with BRCA1 C terminus (BRCT) domain 1/ MDC1/NFBD1 is closely involved in DDR.  NFBD1 is phosphorylated by PLK1 and has an important role in G2/M transition. Both NFBD1 and PLK1 are co-expressed in cellular nuclei throughout G2/M transition (Ando et al. 2013). These signals are converged on p53/p21 and p16 pathways. Critically short human telomeres induce senescence either by activating p53 or by inducing the p16 pathways. Suppression of these pathways is required to suppress senescence of aged human cells (Smogorzewska and de Lange 2002). If DSBs cannot be repaired, they cause constitutive DDR signaling, prolonged p53-­ dependent growth arrest, and eventually an irreversible senescence arrest. Substantially, normal mammalian cells respond to dysfunctional telomeres by cellular senescence, which is permanent cell cycle arrest. The senescence response to telomere dysfunction is sustained primarily by p53. Additionally, p16 is a dominant second barrier to the unlimited growth of human cells (Beauséjour et  al. 2003; Rodier et  al. 2005).

3  The Connection Between Cell Fate and Telomere

These results strongly supported that telomere uncapping caused by TRF1 deletion delays mitotic entry in a p53–p21-dependent manner. Dysfunction of a telomere-binding protein is sufficient to produce severe telomeric damage even in the absence of telomere shortening (Martínez et al. 2009; Thanasoula et al. 2010). Depletion of TRF2 leads to ATM kinase activation. This response is mediated by p53 and the ATM kinase, consistent with activation of a DNA damage checkpoint. Telomeres lacking TRF2 directly signal apoptosis, because they resemble damaged DNA (Karlseder et al. 1999). Thereby, TRF2 plays an essential role by blocking the ATM signaling pathway at telomeres and preventing chromosome end fusion. The expression of non-phosphorylatable forms of TRF2 induces the DDR, leading to growth arrest. These indicate that the telomere stability is under direct control of Rat sarcoma (RAS)/rapidly accelerated fibrosarcoma ­(RAF)/mitogen-­activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways via TRF2 phosphorylation (Picco et  al. 2016). Within shelterin, TRF2 is unique in suppressing both ATM activation at chromosome ends and NHEJ-­ dependent telomere-telomere fusions. Thus, TRF2 represses DDR pathways (Denchi and de Lange 2007; Smogorzewska et  al. 2002). As mentioned above, TRF2 deletion results in accumulation of the DDR factors 53BP1 and γH2AX in addition to the activation of ATM kinase at telomeres. Consequently, the eventual covalent fusion of all chromosome ends takes place (Celli and de Lange 2005). These indicate that TRF2 inhibits NHEJ by preventing upstream ATM signaling (Van Ly et  al. 2018). Similarly, MRN functions as the DSB sensor in the ATM pathway, and the MRN/ATM pathway can protect telomeres from NHEJ, specifically after DNA replication (Dimitrova and de Lange 2009; Paull and Lee 2005). As mentioned above, DNA-PKcs is a member of the PIKK family of serine/threonine protein kinases that also ATM and ATR belong to. DNA-­ PKcs are recruited to DNA ends (Lempiäinen and Halazonetis 2009). The protein kinase

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activity of DNA-PKcs is essential for NHEJ.  When the major alternative DSB repair pathway, HR is suppressed, autophosphorylation of DNA-PKcs may play a role in regulating pathway choice as well as NHEJ progression (Cui et al. 2005; Dobbs et al. 2010). Moreover, activation of DNA-PKcs indicate the requirement of NHEJ pathway in the repair of telomerase inhibitor induced DNA damage. Indeed, the disruption of telomere length maintenance, induces cell cycle arrest and DSBs. DNA-PKcs activation occur in telomerase-inhibited cells as a response to DNA damage (Gurung et al. 2014). One hundred and fourteen proteins are described at TRF2-depleted telomeres. Amongst these proteins, 24 known DDR proteins of which 10 are associated with dysfunctional telomeres, and 15 interact with DNA damage sites. Eight of the selected proteins, Rad18, Rap80, Senp7, Ring1b, Pml, Fam50a, Fancd2 and Ppp2r5d localize at telomeres upon TRF2 depletion, and are tightly associated with dysfunctional telomeres (Bartocci et  al. 2014). Taking into consideration that TRF2-depleted cells undergo dramatic end-to-end chromosome fusions (Celli and de Lange 2005), of these proteins, RING Finger E3 ubiquitin ligase (Ring1b) expression shows a 50% reduction in the rate of NHEJ-­ mediated telomere fusions. The catalytic activity of Ring1b is dispensable for its role in promoting NHEJ at dysfunctional telomeres. Ring1b plays a critical role to maintain a compact chromatin status that is permissive for NHEJ at dysfunctional telomeres (Okamoto et al. 2013). Indeed, compact chromatin is more prone to undergo NHEJ-­ mediated DNA repair. Whereas, heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Heterochromatic DSBs are repaired by HR.  As participants in the DDR, human histone deacetylases, HDAC1 and HDAC2 recruit to DNA-damage sites. HDAC1/2 can promote NHEJ, which affect the ability of NHEJ factors to bind to DSB sites and function (Chiolo et  al. 2011; Miller et al. 2010).

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6

Conclusion

Cells sense telomere length shortening and respond with cell cycle arrest (Liu et al. 2019). Genomic instability, telomere attrition, epigenetic alterations and loss of proteostasis are the primary hallmarks that cause damage to cellular functions (Aunan et  al. 2016). Thus, telomere shortening is associated with aging, early senescence, and premature cell death (GonzalesEbsen et al. 2017). The increased telomere loss, chromosomal fusions, and telomere replication defects enhance the activation of the ATRdependent DDR (Wang et  al. 2013). The shelterin complex prevents activation of the MRN complex, which senses dysfunctional telomeres as DSBs to activate the ATM protein kinase (Deng et  al. 2009; Dimitrova and de Lange 2009). In fact, shelterin is required to prevent the aberrant repair of dysfunctional telomeres. Telomeres undergo end-to-end fusions via the classic NHEJ pathway in the absence of TRF2, while telomeres devoid of TPP1-POT1 are repaired by the altNHEJ pathway. Telomere fusions are due to the inhibition of the telomere-­ protective factor. Shelterin prevents the hyper-­ resection at telomeres by blocking cNHEJ, altNHEJ, and HDR pathways (Rai et al. 2010). Removal of shelterin components, or progressive telomere attrition results in telomere dysfunction, inactivation of DNA damage checkpoint kinases in senescent cells can restore cell-cycle course into S phase. ATM kinase activity at telomeres during S phase causes T-loop opening, however, this is reinstated during normal DNA replication (d’Adda di Fagagna et al. 2003; Herbig et al. 2004). While compact chromatin is more prone to undergo NHEJmediated DNA repair, heterochromatic DSBs expansion and relocalization of foci require checkpoint and are repaired by HR. On the other hand, uncontrolled telomere lengthening increases the risk of telomeric DNA abnormalities. Consequently, the more comprehensive studies are required regarding the cell death crisis due to telomere deprotection and prolonged mitosis that limits cellular life span upon bypass of senescence.

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4

Dark-Side of Exosomes Atilla Engin

Abstract

Exosomes are nanoscale extracellular vesicles that can transport cargos of proteins, lipids, DNA, various RNA species and microRNAs (miRNAs). Exosomes can enter cells and deliver their contents to recipient cell. Owing to their cargo exosomes can transfer different molecules to the target cells and change the phenotype of these cells. The fate of the contents of an exosome depends on its target destination. Various mechanisms for exosome uptake by target cells have been proposed, but the mechanisms responsible for exosomes internalization into cells are still debated. Exosomes exposed cells produce labeled protein kinases, which are expressed by other cells. This means that these kinases are internalized by exosomes, and transported into the cytoplasm of recipient cells. Many studies have confirmed that exosomes are not only secreted by living cells, but also internalized or accumulated by the other cells. The “next cell hypothesis” supports the notion that exosomes constitute communication vehicles between neighboring cells. By this mechaA. Engin (*) Department of General Surgery, Faculty of Medicine, Gazi University, Ankara, Turkey

nism, exosomes participate in the development of diabetes and its associated complications, critically contribute to the spreading of neuronal damage in Alzheimer’s disease, and non-proteolysed form of Fas ligand (mFasL)-bearing exosomes trigger the apoptosis of T lymphocytes. Furthermore, exosomes derived from human B lymphocytes induce antigen-specific major histocompatibility complex (MHC) class II-restricted T cell responses. Interestingly, exosomes secreted by cancer cells have been demonstrated to express tumor antigens, as well as immune suppressive molecules. This process is defined as “exosome-immune suppression” concept. The interplay via the exchange of exosomes between cancer cells and between cancer cells and the tumor stroma promote the transfer of oncogenes and onco-miRNAs from one cell to other. Circulating exosomes that are released from hypertrophic adipocytes are effective in obesity-related complications. On the other hand, the “inflammasome-induced” exosomes can activate inflammatory responses in recipient cells. In this chapter protein kinases-related checkpoints are emphasized considering the regulation of exosome biogenesis, secretory traffic, and their impacts on cell death, tumor growth, immune system, and obesity.

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_4

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A. Engin

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Keywords

Exosome · Extracellular vesicle · Immune synapse · Diacylglycerol kinase-α · Tumor-­ derived exosomes · Reactive oxygen species · NOD-like receptor NACHT, LRR and PYD domains-containing protein (NLRP3) · Phosphatase and tensin homolog (PTEN)

1

Introduction

As novel bio-carriers, exosomes are nanoscale extracellular vesicles that can transport cargos of proteins, lipids, DNA, and RNA.  Exosomes are of paramount importance in distant cell-cell communication because they can enter the circulation when secreted and pass through biological barriers. Therefore, exosomes can be utilized as “natural nanoparticles” to deliver drugs and genes (Jiang and Gao 2017). Furthermore, it is proposed that engineered exosome mimetics, which incorporate the natural exosome components into synthetic liposomes or nanoparticles may be used as pharmaceuticals (Barile and Vassalli 2017). Because of the rapid degradation of microRNAs (miRNAs) and their inefficient tissue specificity, it is recognized that exosomes can be promising tools for delivering small interfering RNAs (siRNAs) (Shahabipour et al. 2017). Exosomes, have been shown to contain various RNA species and to mediate their horizontal transfer to neighbouring- or distant recipient cell. It is suggested that these natural carriers of genetic material can be utilized to allow efficient systemic delivery of exogenous siRNA across biological barriers (El Andaloussi et  al. 2013; Kumar et  al. 2015). In recent years, exosomes have emerged as important players in intercellular communica­ tion. In addition to connexins, tunneling nanotubes and paracrine signaling, they represent an important mechanism for intercellular communication that involves intercellular transfer of extracellular vesicles (EVs) (Raposo and Stoorvogel 2013; Ridger et al. 2017; Rustom et al. 2004; Su and Lau 2014). Because the exosomes participate in many biological processes and diseases, inter-

est in the regulation of exosome biogenesis and secretory traffic has been increased. Although the molecular components that regulate these trafficking processes are managed by protein kinases-related checkpoints, this issue has not been emphasized any more. In this chapter protein kinases-regulated mechanisms have been discussed in the context of “next cell hypothesis”, “lethal exosomes concept”, uptake of the tumor-­ originated exosomes by cells of the immune system, and induction of autocrine or paracrine cell death during exosomes regulated immune function.

2

Structure and Functions of the Exosomes

Exosomes are spherical EVs with an outer lipid bilayer released from plasma membrane of almost all living cells and contain lipids, proteins and nucleic acids, such as DNA, protein coding, and non-coding RNAs. Owing to their composition, EVs can activate receptors at the target cell or transfer molecules to the target cells and thereby change the phenotype of these cells (Milbank et  al. 2016). Although the nomenclature of EVs is not entirely standardized, there are three types of EVs, including exosomes, microvesicles and apoptotic bodies, that are different in their size, formation, and release mechanisms. EVs serve a long-distance delivery of complex cellular messages. Thus, the molecules contained in EVs vary depending on micro-­ environmental conditions and transmit different messages to the recipient cells (Chistiakov et al. 2015; Hafiane and Daskalopoulou 2018; Todorova et  al. 2017). EVs are also classified according to their size and the pathway by which they are produced. These are grouped by size and origin, as exosomes (30–100 nm), microvesicles (100–1000  nm), and apoptotic bodies (1000– 5000  nm). Microvesicles and apoptotic bodies are formed directly via blebbing of the plasma membrane, whereas exosomes are produced via an endocytic pathway (Borges et  al. 2013; Gao et  al. 2017; Lakkaraju and Rodriguez-Boulan 2008). In this regard, exosomes are secretory

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products of endosomal origin. Whereas, multiveExosomal RNAs are not only stable and accessicular bodies (MVBs) or endosomal carrier ves- sible biomarkers, but they can be functionally icles are often larger than exosomes and transferred from parent cells to recipient cells. ‘irregularly’ shaped, depending on the number Exosomes contain both mRNA and miRNA, and morphology of the vesicles it contains which can be delivered to another cell, and can be (Ramachandran and Palanisamy 2012). The for- functional in this new location. These RNAs are mation of MVBs and their separation from the called “exosomal shuttle RNA” (Valadi et  al. ‘parent’ endosome appears to be regulated by 2007). At the first step of these nano-sized partihepatocyte growth factor-regulated tyrosine-­ cles formation, the RNAs are loaded into exokinase substrate (HRS) and annexin II, respec- somes, before the budding process, and RNA tively (Gruenberg and Stenmark 2004). Despite molecules bind to this raft-like regions retained at their nanoscale, the exosomes have a complex the membrane. The affinity of the RNA molecule structure consisting of a lipid bilayer containing to the raft-like regions is determined by binding membrane proteins surrounding their cargo in the motifs in the RNA sequence, and by the RNA lumen. Recently upon the increase in the number hydrophobic modifications (Janas et al. 2015). At and functions of exosomes, ExoCarta has been the second step, miRNAs transfer in exosomes organized as an exosome database that is classi- from T cells to antigen-presenting cells at immufied depending on the contents found in exo- nological synapses (ISs). Thus, these molecules somes, which consist of 41,860 proteins, 4946 can modulate gene expression in recipient cells. mRNAs and 2838 miRNAs (Burke et  al. 2014; During immune interactions miRNAs are transKeerthikumar et al. 2016). These nano-sized par- ferred in a unidirectional manner from the T-cell ticles that form as the internal vesicles of endo- to the antigen presenting cells (APC). This antisomal structures, after secretion, their lipid gen driven genetic communication is closely bilayer membranes protect their cargo from linked to the formation of the IS.  Indeed, the plasma and immune components and deliver the presence of specific antigen induces the formacargo to recipient cells (Gho and Lee 2017; tion of IS, and enhances this exosomal transition. Mugami et  al. 2019; Raposo and Stoorvogel Actually ISs are highly organized cell-cell con2013; Tkach and Théry 2016). This interchange tacts where, on antigen recognition, T-cell activaof signaling between cells is achieved by packag- tion is initiated and tuned. Transfer of miRNAs ing RNA species into exosomes endowed with by exosomes from T cells to antigen-presenting specific cell surface-targeting motifs. RNA cells at ISs indicates that gene expression in involved in transmitting information or mole- recipient cells has been able to be modulated via cules between cells is called exosomal RNA this transfer (Mittelbrunn et al. 2011). It is also (esRNA). The delivered RNA molecules are demonstrated that mRNAs, miRNAs, and cytofunctional, and mRNA can be translated into new kines carried by dendritic cell (DC)-derived exoproteins, while miRNAs target the host mRNAs somes interact with and influence immune cells in the recipient cell (Ramachandran and (Pitt et  al. 2014). Cellular RNAs may be selecPalanisamy 2012). Four potential modes for sort- tively sorted into exosomes (Janas et  al. 2015). ing of miRNAs into exosomes are proposed. The exosome sorting process depends on: (1) the These are consisted of the neural sphingomyelin- presence of a lipid-bilayer binding motif within ase 2 (nSMase2)-dependent pathway (Kosaka the RNA sequence; (2) RNA hydrophobic modiet  al. 2013), the miRNA motif and sumoylated fications; (3) RNA concentration in cytoplasm; heterogeneous nuclear ribonucleoproteins and (4) the presence of raft-like regions, enriched (hnRNPs)-dependent pathway (Villarroya-Beltri in specific lipids, in the MVB limiting membrane et al. 2013), the 3′-end of the miRNA sequence-­ (Janas et al. 2015). The fate of the contents of an dependent pathway (Koppers-Lalic et al. 2014), exosome depends on its target destination. The and the miRNA induced silencing complex sorting of various proteins to different cellular (miRISC)-related pathway (Frank et al. 2010). locations through exosomes is a meticulously

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regulated event. Complex “cargo-sorting” machinery for the packaging of protein, RNA, and other molecules contained in endosomes tightly regulates the exosome-mediated transfer of informational molecules (Ramachandran and Palanisamy 2012). As a consequence of their origin, exosomes from different cell types contain endosome-­ associated proteins, which are included Rab guanosine triphosphatases (GTPase), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), annexins, and flotillin (van Niel et al. 2006). Exosomes have a particular composition reflecting their origin in endosomes as intraluminal vesicles (Février and Raposo 2004). Tetraspanins play an important role in the formation of exosomes, cargo selection, target selection and their entry into recipient cells (Malla et al. 2018). Exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82. Interestingly, these molecules are concentrated on the internal membranes of multivesicular major histocompatibility complex (MHC) class II (Escola et al. 1998). In comparison to the plasma membrane, exosomal membrane has a similar content of the major phospholipids and cholesterol, and it has been organized as a lipid bilayer with a random distribution of phosphatidylethanolamines. In addition, there are tight lipid packing at neutral pH between the two leaflets of exosome membranes (Laulagnier et  al. 2004). Therefore lipids are important components of exosome membrane, and they play an important role in the biology of these vesicles. Thus, exosomes have been enriched with sphingolipids, cholesterol, and phosphatidylserine by two to three times more, compared with the donor cells (Skotland et  al. 2019). Phosphatidylinositol (PI)-phosphates (PIPs), are phosphorylated derivates of the membrane phospholipid, PI. PIP produced from PI(3) P by the action of the PIP kinase, phosphoinositide kinase, FYVE-type zinc finger containing (PIKfyve), is associated with MVBs (Ho et  al. 2012; McCartney et  al. 2014). Inhibition of PIKfyve increases exosome secretion and also induces secretory autophagy (Hessvik et  al.

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2016). Upon induction of autophagy, cytoplasmic cargo is trapped within double-membrane vesicles termed autophagosomes, which then fuse with MVBs to form amphisomes or directly with lysosomes for cargo degradation (Boya et  al. 2013; Skotland et  al. 2019). Inhibition of the class I PI(3)P kinase or Akt, as a downstream component in phosphoinositide 3-kinase (PI3K) signaling, increases the release of exosomal cargo (Ju et  al. 2014). Whereas, autophagyrelated gene, autophagy protein 5 (Atg5) depletion reduces exosome release (Guo et al. 2017). The presence of immunogenic molecules itself on the exosomal membrane for immune system activation makes them well suitable for interaction with their cell surface receptors thereby mediating signal transduction without the need for two cells to be in direct contact. Further, these vesicles can also fuse with the recipient cells leading to acquisition of novel molecules by the cells (Anand 2010; Valadi et al. 2007). Nevertheless, there are sufficient evidences that exosomes can enter cells and deliver their cargo. Various mechanisms for exosome uptake have been proposed, including clathrin-­ mediated endocytosis (CME), phagocytosis, macropinocytosis and plasma or endosomal membrane fusion. But, the mechanisms responsible for exosomes internalization into cells are still debated (Mulcahy et al. 2014). Firstly, exosomes released from DCs contain different miRNAs depending on the maturation of the DCs. Exosome-shuttle miRNAs delivered into the cells are protected from degradation by extracellular RNAses. Exosomes fuse with the target DCs, and release their content into the DC cytosol. So, exosome-­ shuttle miRNAs are functional, and they repress target mRNAs of acceptor DCs (Montecalvo et al. 2012). Endocytosed exosomes are sorted into the endocytic compartment of DCs for processing. Later, exosome-derived peptides in MHC-II molecules are loaded for presentation to CD4+ T cells. By this way, exosomes are internalized and processed by immature DCs for presentation to CD4+ T cells (Morelli et  al. 2004). Exosomes are also internalized by tumor cells via clathrin-dependent endocytosis. Specific glycoproteins enriched exosomes interact with

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other cells, and transfer their cargo to neighboring cells (Escrevente et  al. 2011). Interestingly, exosomes derived from Epstein-Barr virus-­ infected cells are internalized and transfer viral factors, including EBV-encoded latent membrane protein and miRNAs, to the recipient cells. Exosomes released from EBV-infected B cells are internalized via caveola-dependent endocytosis, which, in turn, contributes to phenotypic changes in the recipient cells (Nanbo et al. 2013). After binding to recipient cells, exosomes remain stably associated with the plasma membrane or dissociate, directly fuse with the plasma membrane, or be internalized through distinct endocytic pathways. When endocytosed, exosomes may subsequently fuse with the endosomal delimiting membrane or be targeted to lysosomes for degradation (Raposo and Stoorvogel 2013) (Fig. 4.1). In this process, the

cargo content of exosomes is received through two different pathways; clathrin-dependent endocytosis and clathrin-independent pathway. Second pathway consists of caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft-mediated internalization. In this context, there are three distinct mechanisms of interaction between exosomes and their recipient cells. Firstly, the exosomes fuse with target cell membranes and directly interact with the signaling receptors of these cells. Secondly, the exosomes fuse with the plasma membrane of recipient cells and deliver their content into the cytosol. Thirdly, the exosomes can be completely internalized into recipient cells (Koumangoye et al. 2011). However, it is likely that a heterogeneous population of exosomes may gain entry into a cell via more than one route. Nevertheless, the uptake mechanisms used by exosomes depend

Fig. 4.1  Exosome biogenesis in the parent cell and exosomal uptake by the recipient cell. miRNA genes are transcribed into primary miRNAs, and processed by the Drosha complex to form precursor miRNAs, which are exported from nucleus into the cytoplasm. miRNAs are transported by RBPs towards MVBs for exosome loading. During maturation, endosomes can be transported to the TGN, directed to lysosomes for degradation, or move along microtubules to fuse with plasma membrane. Thus, they release their ILVs as exosomes into the extracellular

space via parent cell membrane fusion. MLKL facilitates endosomal trafficking. Exosomes from the parent cell interact with the recipient cell. miRNAs activated in the recipient cell alter the phenotype of this cell. (Abbreviations. DGKα: Diacylglycerol kinase-alpha, ILVs: Intraluminal vesicles, miRNA: microRNA, MLKL: Mixed lineage kinase domain-like, MVB: Multivesicular bodie, PKD: Protein kinase D, Rabs: Small G-proteins, RBPs: RNA-­ binding proteins, TGN: Trans-Golgi network)

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on proteins and glycoproteins found on the surface of both the exosome and the target cell (Mulcahy et  al. 2014). Macropinocytosis is one of the efficient cellular uptake of exosomes. The active induction of macropinocytosis by stimulation of cancer cell-related receptors have shown that the activation of a tyrosine kinase receptor, epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes (Nakase et  al. 2015). As a clathrin-independent pathway, macropinocytosis is a distinguished endocytic uptake pathway. These vesicles carry the components sampled from the region around invaginated membrane ruffles (Doherty and McMahon 2009). Ruffled extensions of the plasma membrane protrude from the cell surface and encompass an area of extracellular fluid, subsequently this area of extracellular fluid is internalized entirely as a result of fusion of the membrane protrusions (Mulcahy et  al. 2014; Swanson 2008). This mechanism is similar to that of phagocytosis, but direct contact with the internalized material is not required. It is rac1-, actin- and cholesterol-dependent and requires Na+/H+ exchanger activity (Kerr and Teasdale 2009). In this case, cholesterol is prerequisite for the membrane localization of activated Rac1, actin reorganization, membrane ruffling and macropinocytosis (Grimmer et  al. 2002). Exosome release, which leads to net loss of cellular membrane and protein content, is negatively regulated by mechanistic target of rapamycin complex 1 (mTORC1). Exosome release, like autophagic flux, is also regulated by mTORC1, however, inhibition of mTORC1 does not significantly alter protein and miRNA profiles of exosomes (Zou et al. 2019). In fact, macropinocytosis can be stimulated in mammalian cells by ligands acting through either tyrosine receptor kinases (RTKs) or G-protein coupled receptors (GPCRs). Chemokine (C-X-C motif) ligand 12 (CXCL12)induced macropinocytosis contributes to the cytosolic AKT/ the tuberous sclerosis complex genes 2 (TSC2) pathway for activation of mTORC1. In addition to PI3K p110 isoforms, phospho-protein kinase B (pAKT) and phosphoTSC2 (pTSC2) require formation of ruffles and macropinocytic cups (Pacitto et  al. 2017).

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Stimulation of the endocytic pathways of macropinocytosis and phagocytosis by activated Rac1 is responsible for the increased internalization and subsequent degradation of extracellular proteins (Ahram et  al. 2000). Internalisation of some exosomes occur by a macropinocytotic mechanism without inducing a concomitant inflammatory response. Thus, oligodendrocytes secreted exosomes are internalised in microglia that do not have antigen-presenting capacity (Fitzner et al. 2011). The lipid raft-associated protein caveolin-1 (CAV1) negatively regulates the uptake of exosomes. Exosomes induce the phosphorylation of lipid rafts associated extracellular signal-­ regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Exosome uptake is dependent on ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. Ultimately, ERK1/2 activity is required for efficient exosome uptake (Svensson et al. 2013). In contrast to the others, Feng et  al. claimed that exosomes do not enter the internalization pathway involving caveolae, macropinocytosis and clathrin-coated vesicles. Rather, they suggest that cellular uptake of exosome is mediated by phagocytosis, and this pathway is important for exosome cell interactions. The cellular uptake of exosomes through phagocytosis has been thought to be reasonable for exosome-cell interactions and the exosome intracellular trafficking pathway (Feng et al. 2010). Whereas, phagocytosis is a receptor-mediated event that involves the progressive formation of invaginations surrounding the material destined for internalization, with or without the participation of enveloping membrane extensions as required for macropinocytosis (Doherty and McMahon 2009; Swanson 2008). In contrast, Heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived exosomes. Further, enzymatic depletion of cellsurface HSPG or inhibition of endogenous PG biosynthesis significantly attenuates exosome uptake and exosome-induced ERK1/2 signaling activation. Cancer cell-derived exosomes use HSPGs for their internalization and functional

4  Dark-Side of Exosomes

activity, as key receptors of macromolecular cargo (Christianson et  al. 2013). Some type of cancers release exosomes containing full-length, signaling-­ competent epidermal growth factor receptor (EGFR) ligands, which display increased invasiveness of recipient cancer cells 4 to 5-fold. EGFR ligand signaling via exosomes contribute to diverse cancer phenomena and priming of the metastatic niche (Higginbotham et al. 2011). Tyrosine receptor kinase B (TrkB)EGFR-sortilin (TES) complex in exosomes forms with the linkage of the two tyrosine kinase receptors with sortilin. Sortilin is a multi-functional receptor that interacts with a number of ligands including the apoptotic death receptor p75 neurotrophin (p75NTR, also known as NGFR) and the cell survival receptor, tropomyosin-related kinase (Trk) receptor. Trimeric sortilin–TrkB–EGFR complex in exosomes can be transferred to target cells (Wilson et al. 2014). In fact, the EGFR signaling pathway is connected to three major mitogen-­activated protein kinase (MAPK)-pathways and the PI3K/protein kinase B (AKT) survival pathway. The MAPK pathway consists of the ERK1/2 module, the p38 MAPK module, and the c-Jun N-terminal Kinase (JNK) module. TrkB activates PI3K/AKT and MAPK (MEK to ERK) signaling (Ho et al. 2002; Kim et al. 2004). Since exosomes is a novel mode of intercellular communication, ephrin (Eph) receptor tyrosine kinases and their membrane-tethered ephrin ligands have very important roles in exosomes-­ related biological processes (Gong et  al. 2016). Indeed, the Eph receptors are the largest family of tyrosine kinases, including nine EphA and five EphB receptors in the human genome (Barquilla and Pasquale 2015). They are transmembrane proteins and their extracellular region includes an N-terminal domain that binds the Eph ligands. The binding of an Eph receptor to an Eph on a neighboring cell leads to the formation of Eph receptor–Eph clusters that signal bidirectionally, generating “forward” signals in the Eph receptor–expressing cell and “reverse” signals in the Eph-expressing cell (Pasquale 2016). Once endocytosed EphB2 may be sorted to MVBs destined to generate exosomes. Eph receptor/Eph commu-

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nication occures at the exosome–cell interface (Gong et al. 2016).

3

The Next Cell Hypothesis

Exosomal nanoparticles expressed by tumoral cells interact with membrane lipid raft domains of sensitive tumoral target cells to disorganize the Notch-1 receptor complex. This complex implicates a disintegrin and metalloproteinase 17 (ADAM 17) and presenilin of γ-secretase partners regulating the Notch-1 survival pathway. The disorganization of the Notch-1 complex-1 firstly, activates pro-apoptotic phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and glucose synthase kinase-3β (GSK-3β). Secondly, leads to decreased expressions of hairy and enhancer-of-split homolog-1 (Hes-1) and cyclin D1, and finally, drives cells toward the mitochondria-dependent apoptosis (Ristorcelli et  al. 2009). PTEN gene products produced in one cell are able to enter recipient cells and contribute to PTEN functions. Purified PTEN-Long protein is able to enter tumor xenografts and downregulate PI3K signaling as well as cause tumor cell death (Hopkins and Parsons 2014). PI3K and AKT function in a signaling pathway by promoting growth and survival. Whereas, as a dual specificity protein phosphatase, PTEN suppresses these growth-promoting and survival signals by dephosphorylating the phospholipid products of PI3K, and negatively regulating the PI3K/AKT/PKB signaling axis (Myers et  al. 1998; Stambolic et  al. 1998). The “next cell hypothesis” is supported by the observation that PTEN-Long protein is elevated in histiocytes directly adjacent to tumors. Actually, PTEN-Long is a membrane-permeable lipid phosphatase that is secreted from cells and can enter other cells. These “next” cells may be responding to tumor initiation and may be acting to inhibit the aberrant proliferation of the neighboring cell (Hopkins et  al. 2013; Hopkins and Parsons 2014). PTEN activity plays critical tumor suppressive roles by acting both as a gatekeeper and landscaper tumor suppressor (Hopkins and Parsons 2014). Indeed, exosomes exposed cells

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produce labeled protein kinases, which are expressed by other cells. This means that these kinases are internalized by exosomes, and transported into the cytoplasm of recipient cells. Many studies have confirmed that exosomes are not only secreted by living cells, but also internalized or accumulated by the other cells. The “next cell hypothesis” supports the notion that exosomes constitute communication vehicles between neighboring cells. Ste20-related proline/alanine-­ rich kinase (SPAK), oxidative stress response 1 (OSR1), and Na-K-Cl cotransporter 1 (NKCC1) are packaged by cells in exosomes, that these microvesicles are released in the extracellular environment and internalized by neighboring cells. Functional transporters and kinases not only colocalize at the plasma membrane of donor cells, but are also found in exosomes (Koumangoye and Delpire 2016). SPAK and OSR1 anchor to their targets and phosphorylate downstream Ser/Thr residues. The major targets of SPAK and OSR1 are membrane proteins. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. SPAK and OSR1 mainly function as scaffolding proteins in exosomes (Piechotta et al. 2002, 2003). It is also showed that both SPAK and OSR1 kinases after entering cells through exosomes bind to their transporter target and are preferentially expressed at the plasma membrane. These kinases in exosomes are functional and maintain NKCC1  in a phosphorylated state (Koumangoye and Delpire 2016). Exosomes secreted by cancer-associated fibroblasts (CAFs) can reprogram the metabolic machinery following their uptake by cancer cells. CAF-derived exosomes inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells (Zhao et  al. 2016). Additionally, tumor-related exosomes educate macrophages into protumor phenotype. They also selectively induce effector T cell apoptosis via Fas/FasL interaction and enhance regulatory T cell proliferation and function by releasing transforming growth factor-beta (TGF-β) (Chen et  al. 2017).

Furthermore, TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, enhancing the growth of cancer cells, independently of angiogenesis (Guido et  al. 2012)

4

Exosomes and Cell Death

Exosomes have pleiotropic biological functions, including immune response, antigen presentation, intracellular communication and the transfer of RNA and proteins. Because the exosomes contain inactive forms of both mRNA and miRNA that can be transferred to another cell and be functional in that new environment, initiate many miRNA profiling. These exosomal miRNA profiles can be used as diagnostic biomarkers of diseases (Simpson et al. 2009). Exosomes participate in the development of diabetes and its associated complications (Castaño et  al. 2019). Individuals with diabetes had significantly higher levels of exosomes in their circulation than euglycemic control participants. Insulin resistance increases exosomes secretion. Furthermore, the levels of insulin signaling proteins are altered in exosomes from individuals with high levels of insulin resistance and β-cell dysfunction (Freeman et  al. 2018). miRNAs and long noncoding RNAs (lncRNAs)containing exosomes have been shown to regulate insulin signals in target tissues and, affect cell viability, and modulate inflammatory pancreatic cells in diabetes (Chang and Wang 2019). β-cells secrete miRNAs that can be transferred to neighboring β-cells. Exposure of donor cells to pathophysiological conditions commonly associated with diabetes modifies the release of miRNAs and affects the survival of recipient β-cells. Thereby, transfer of exosomal miRNAs constitutes a novel cell-to-cell communication mechanism regulating the activity of pancreatic β-cells (Guay et  al. 2015). In addition, T-lymphocyte exosomes trigger apoptosis, and the expression of genes involve in chemokine signaling, including [C-C motif] chemokine ligand 2 (Ccl2), Ccl7, and Cxcl10, in β-cells. The induction of these genes may promote the recruitment of immune

4  Dark-Side of Exosomes

cells and exacerbate β-cell death during the autoimmune attack. Exosomal-miRNA transfer is a communication mode between immune and insulin-­secreting cells, in type 1 diabetes (Guay et  al. 2019). Cytokine-treated pancreatic β-cell line secrete exosomes containing miRNAs that are transferred to neighboring β-cells, leading to apoptosis in the recipient cells by new communication mechanism based on cell-to-cell miRNA transfer. In this context, increase in the levels of miRNA-21, miRNA-29, miRNA-34a and miRNA-146a contribute to destruction of insulin-­ secreting pancreatic β-cells and then in the development of diabetes (Guay et al. 2011, 2012; Guay and Regazzi 2013). Thus, exosomes-derived miR-15a from pancreatic β-cells under high-­ glucose conditions leads to diabetes progression via their transfer to different cell types and results in oxidative stress by significant downregulation of AKT3. Consequently, overexpressed miR-15a leads to apoptotic cell death (Kamalden et  al. 2017). Similarly, miRNA-1 and miRNA-133a show a positive correlation with myocardial steatosis. Especially miRNA-133a has been shown to increase significantly in type 2 diabetic patients (de Gonzalo-Calvo et al. 2017). These miRNAs increase in the serum of patients who have suffered from an acute myocardial infarction. Circulating miR-133a originates mainly from the injured myocardium and localized inside exosomes. This can be used as a marker for cardiomyocyte death (Kuwabara et  al. 2011). During oxidative stress, cardiac progenitor cells (CPCs) release more miRNA-21, which targets the protein programmed cell death 4 (PDCD4), ultimately reducing the cleaved version of caspase-3. CPC-derived exosomal miR-21 has an inhibiting role in the apoptosis pathway through downregulating PDCD4 (Xiao et  al. 2016). High glucose and insulin increases the secretion of Sonic Hedgehog (Shh)-positive adipocyte-derived exosomes (AdipoDEs). Thereby, circulating Shh-­ positive exosomes are increased in type 2 diabetes patients. The insulin-resistant adipocytes release more Shh-positive exosomes. The AdipoDEs carrying Shh induce pro-inflammatory or M1 polarization of macrophages. Inhibitors of Ptch and PI3K block the M1 polarization induced by

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AdipoDEs, which also suggests that AdipoDEs carrying Shh mediate M1 macrophage polarization through the Ptch/PI3K signaling pathway. In brief, AdipoDEs carrying Shh stimulate the M1 macrophage polarization through patched (Ptch) and PI3K pathways, which lead to insulin resistance in adipocytes. AdipoDEs carrying Shh might be a potent effector of chronic inflammation which occurrs in insulin resistance (Song et al. 2018). Pancreatic tumor-derived exosomes trigger lipidosis and glucose intake inhibition. While insulin and PI3K/AKT signaling is inhibited, insulin-induced forkhead box O1 (FoxO1) nuclear exclusion is preserved and glucose transporter type 4 (GLUT4) trafficking is impaired. Thereby, exosomal miRNAs contribute to insulin resistance through the insulin and PI3K/AKT/ FoxO1 signaling pathways (Wang et al. 2017). In the proteomic characterization of the content of mesenchymal stem cells-derived exosomes, more than 730 proteins have been identified. Moreover, within exosomal cargo, a wide range of miRNAs were found, which can control functions related to neural remodeling as well as angiogenic and neurogenic processes (Reza-Zaldivar et al. 2018). In this respect, exosomes secreted from many cell types, contain pathogenic proteins including full-length amyloid precursor protein (flAPP) and amyloid precursor protein (APP) metabolites. However, the function of these exosomes in Alzheimer disease (AD) remains to be elucidated (Zheng et  al. 2017). These exosomes contain APP and are capable of efficiently transferring APP to normal primary neurons. Following the exosomes secretion, the spread of amyloid protein in the brain serves to amplify the neronal damage in AD (Zheng et  al. 2018b). Astrocytes surrounding amyloid plaques in brain of AD patients show caspase 3 activation and are apoptotic when co-­ expressing apoptosis response-4 (PAR-4) and ceramide. Ceramide generated by neutral sphingomyelinase 2 (nSMase2) is critical for amyloidinduced apoptosis. Amyloid peptide induces the secretion of PAR-4 and C18 ceramide-enriched exosomes. This means that neuronal apoptosis induction by PAR-4/ceramide-enriched exosomes critically contribute to AD (Wang et  al.

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2012). Since amyloid-β (Aβ) is well known to cause neuronal cell death, interestingly exosomes play essential role in the maintenance of the neuronal functions via the clearance of Aβ from the brain. Whereas, Aβ reduces exosome release from astrocytes by enhancing JNK phosphorylation, JNK inhibitors restore the decreased levels of exosome release induced by Aβ. Consequently, increased Aβ levels in the brain impair the exosome-mediated Aβ clearance pathway in AD (Abdullah et al. 2016; András and Toborek 2016). The neuron-derived exosomes, isolated from plasma of AD patients contain significantly higher levels of Aβ1-42, total tau, p-T181 tau and p-S396 tau, as compared to the controls. Determination of these exosomes provide a high predictability of disease development in the preclinical stage, 1 to 10  years before being diagnosed (Fiandaca et al. 2015). In diabetic patients, the level of the phosphorylated form of insulin receptor substrate 1 (IRS-1) in neural-derived exosomes also show a higher accuracy in predicting the development of AD. Exosomal levels of P-serine 312-IRS-1 and P-pan-tyrosine-IRS-1 and the ratio of P-serine 312-IRS-1 to P-pantyrosine-IRS-1 (insulin resistance factor, R) for AD and type 2 diabetes are found significantly high, up to 10  year prior to clinical onset (Kapogiannis et al. 2015; Mullins et al. 2017). In addition, the increased exosomal levels of lysosomal proteins, cathepsin D and lysosome-associated membrane protein 1 (LAMP1) and decreased levels of synaptic proteins (synaptophysin, synaptopodin, synaptotagmin-­2 and neurogranin) are also observed in the neuron-derived plasma exosomes of AD patients, reflect the pathology up to 10  years before clinical onset (Goetzl et  al. 2015, 2016a). Goetzl and colleagues found that the levels of cargo proteins in plasma exosomes derived from astrocytes are higher than those in neuron-derived plasma exosomes. Astrocyte-derived exosomes (ADEs) levels of β-site amyloid precursor protein-cleaving enzyme 1 (BACE-1), γ-secretase, soluble Aβ42, soluble amyloid precursor protein (sAPP)β, sAPPα, glial-derived neurotrophic factor (GDNF), P-T181-tau, and P-S396-tau are 3- to 20-fold higher than levels in neuron-derived exo-

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somes (NDEs) for patients and controls. BACE-1 levels are also a mean of 7-fold higher in ADEs than in NDEs. Levels of BACE-1 and sAPPβ are significantly higher and GDNF is significantly lower in ADEs of patients with AD (Goetzl et al. 2016b). Diacylglycerol kinase-α (DGKα) regulate the secretion of exosomes. The inhibition of DGKα kinase activity increase the secretion of exosomes bearing FasL (Alonso et  al. 2011). Exosomes participate in T lymphocyte-mediated cytotoxicity and activation-induced cell death (AICD). The secretion of the internal microvesicles loaded with FasL and apoptosis-inducing ligand (APO2L)/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation. DGKα is associated with the trans-­ Golgi network and late endosomal compartments. It is crucial for the control of cell activation and for the regulation of the secretion of lethal exosomes (Alonso et  al. 2005; Monleón et  al. 2001). In fact, FasL is a type II transmembrane protein and a member of the TNF family of proteins expressed by activated CD4+ and CD8+ cytotoxic T lymphocytes (CTL), natural killer (NK) cells. Both phosphorylation and ubiquitylation are necessary for the appearance of FasL within the MVBs. FasL is phosphorylated by the Src family tyrosine kinases (Fgr, Fyn and Lyn kinases) through binding to the proline-rich domain of FasL (Zuccato et al. 2007). As mentioned above, the secretory vesicle pathway involves several diacylglycerol (DAG)-controlled checkpoints at which DGKα may act; these include vesicle formation and fission at the transGolgi network (TGN), MVB maturation, as well as their transport, docking and fusion to the plasma membrane. Protein kinase D (PKD) is a cytosolic serine-threonine kinase that binds to the TGN and regulates the fission of transport carriers specifically destined to the cell surface. PKD is found to bind DAG, and this binding is necessary for its recruitment to the TGN.  Reducing cellular levels of DAG inhibits PKD recruitment and blocks protein transport from the TGN to the cell surface. Localized DAG accumulation is also

4  Dark-Side of Exosomes

required for cytotoxic T cell-mediated killing (Alonso et al. 2011; Quann et al. 2009). Peripheral golgi protein containing a PI-transfer domain, Nir2 is involved in maintaining a critical DAG pool in the golgi apparatus (Litvak et al. 2005). DGKα is essential for the negative control of DAG function in T lymphocytes. In fact, DGKs are members of a unique and conserved family of intracellular lipid kinases that phosphorylate DAG, catalysing its conversion into phosphatidic acid (PA). This reaction leads to attenuation of DAG levels in the cell membrane. DGKs provide a link between lipid metabolism and signaling (Mérida et  al. 2008). Apoptotic effect of the DGKα occurs via the regulation of the release of lethal exosomes by the exocytic pathway. Consequently, DGKα is crucial for the control of the lethal exosomes secretion. This in turn controls cell death (Alonso et al. 2005). The combination of positive signals triggered by receptor stimulation, and fine-tuning effect of a negative regulator indicates the kinase activity of DGKα control the secretory vesicle pathway that is responsible for the secretion of exosomes. The negative effect of DGKα kinase activity on traffic appears at the stage of formation of mature MVBs. In addition, for polarisation of MVBs and exosome secretion, a non-kinase function of DGKα is also necessary (Alonso et al. 2011). In T lymphocytes, stimulation of the T cell receptor induces the fusion of the exosomes with the plasma membrane. These “lethal exosomes” contain pro-apoptotic FasL, which are secreted as upon fusion with the plasma membrane. Thereby, non-proteolysed form of membrane-associated FasL (mFasL)-bearing exosomes trigger the apoptosis of T lymphocytes (Alonso et al. 2007, 2011). PKD is the major DAG effector that regulates MVB maturation and exosome secretion in T and B lymphocytes. DGKα-mediated DAG consumption limits PKD subcellular location and activation in T ymphocytes during the IS formation (Mazzeo et al. 2016). Dendritic cell (DC)-derived exosomes (DCexs) contain numerous plasma membrane and cytoplasmic DC components. Several plasma membrane molecules that are relevant for the immune functions of DCs are also expressed on

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the DCex bounding membrane. These include MHC class I and II molecules, intercellular adhesion molecule-1 (ICAM-1), integrins, and T cell co-stimulatory molecules such as CD40, CD80 and CD86 (Chaput et  al. 2006; Morelli et  al. 2004). These molecules are correctly oriented on the outer surface of the DCex membrane and are functional (Munich et  al. 2012). Furthermore, DCexs express on their surface TNF, FasL and TRAIL.  These exosomes can mediate essential innate immune functions triggering caspase activation and apoptosis in tumor cells (Chaput et al. 2006; Munich et al. 2012). Bone marrow cells are efficiently induced into DCs with typical DC characteristics. The anti-inflammatory cytokines TGF-β, FOXp3 and interleukin (IL)-10 are upregulated and IL-17 is downregulated. In contrast, exosomes produced by bone marrowderived dendritic cells (DEXs) could alleviate hepatic ischemia/reperfusion injury via modulating the balance between regulatory T cells (Tregs) and Th17 cells. DEXs transport heat shock protein 70 (HSP70) into naïve T cells and stimulate the PI3K/mTOR axis to modulate the balance between Tregs and Th17 cells and protect the liver from ischemia/reperfusion injury (Zheng et al. 2018a). However, proliferation of activated CD4+ T cells is dose-dependently suppressed, and CD4+ T-cell apoptosis is induced (Ren et al. 2011). Protein kinase C δ (PKCδ), a novel PKC isotype activated by DAG, regulates T cell receptor (TCR)-controlled MVB polarization toward IS and exosome secretion. Among several proapoptotic mechanisms, CTL kill Fas+ target cells by rapidly exposing intact, pre-formed FasL on the plasma membrane at the IS.  Newly synthesized FasL is stored in specialized secretory lysosomes in both CD4+ and CD8+ T cells and NK cells, and that polarized degranulation controls the delivery of FasL to the cell surface (Bossi and Griffiths 1999). In fact, Fas (CD95/Apo-1) ligand is a potent inducer of apoptosis and one of the major killing effector mechanisms of cytotoxic T cells. Thus, FasL activity has to be tightly regulated, involving various transcriptional and post-­ transcriptional processes. The activation of the Src-like kinases is absolutely required for Fas ligand degranulation (Kassahn et  al. 2009).

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Thereby, Fas and FasL are involved in down-­ regulation of immune reactions as well as in T cell-mediated cytotoxicity. FasL released from exosome induces cross-linking of the Fas death receptor on the target cell and leads to apoptosis (Nagata and Golstein 1995). In resting CTL, FasL is located at the limiting membrane of secretory MVBs (Bossi and Griffiths 1999). Because the membrane-associated FasL is released from T lymphocytes, via mFasL-bearing exosomes, mFasL in exosomes retains its activity in triggering Fas-dependent apoptosis, providing an alternative mechanism of cell death without cell-to-cell contact. The inhibition of DGKα increases the secretion of lethal exosomes bearing mFasL. Therefore, DGKα is crucial for the control of cell activation and also for the regulation of the secretion of lethal exosomes, which in turn controls cell death (Alonso et  al. 2005; Zuccato et  al. 2007). The IS serves as a focal point for exocytosis and endocytosis, directed by centrosomal docking at the plasma membrane. Upon TCR activation of CTL and the microtubule-­ organizing center (MTOC) in T-cells reorientation, lytic granules undergo fusion with the plasma membrane at the IS.  In fact, DAG is a lipid second messenger generated upon TCR activation and serves as a substrate for PKD, which also focuses in the center of the synapse, binding to DAG.  Blocking DAG production or impairing its localization prevents MTOC movement from the rear of the cell toward the synapse (Griffiths et  al. 2010; Quann et  al. 2009). As a consequence, the transport of pro-apoptotic FasL to the extracellular milieu occur with the relocalization of FasL to the cell surface and receptor-­ mediated target-cell recognition (Bossi and Griffiths 1999). The TCR has no intrinsic tyrosine kinase activity. Instead, the TCR is activated via cytoplasmic tyrosine kinases that localize to the TCR complex and initiate TCR-mediated signaling events (Clements et al. 1999). Therefore, FasL-containing lethal exosomes are secreted. CD3/TCR found in the exosomes come from the pool of complexes that have been activated. Therefore, proteins of the transduction machinery, tyrosine kinases of the Src family, and c-Cbl are also observed in the CD4+T cell-derived exo-

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somes (Alonso et al. 2005; Blanchard et al. 2002; Martínez-Lorenzo et al. 1999). The c-Cbl protein acts as an ubiquitin-protein ligase (E3) that can recognize tyrosine-­ phosphorylated substrates. Thus, c-Cbl, an adapter protein for receptor protein-tyrosine kinases (RPTKs), positively regulates RPTK ubiquitination in a manner dependent on its variant SRC homology 2 (SH2) and really interesting new gene (RING) finger domains (Joazeiro et al. 1999). Furthermore, exosomes derived from human B lymphocytes induce antigen-specific MHC class II-restricted T cell responses. These data indicate the role of exosomes in antigen presentation in  vivo (Raposo et  al. 1996). Epstein-Barr virus (EBV) infection of B cells induces the release of exosomes that harbor the viral latent membrane protein 1 (LMP1). LMP1 per se mimics CD40 signaling and induces proliferation of B lymphocytes and T cell-independent class-­ switch recombination. Thereby, exosomes harboring LMP1 enhance proliferation and drove B cell differentiation toward a plasmablast-like phenotype (Gutzeit et al. 2014). Moreover, within lymph nodes, exosomes associate with DCs, subcapsular sinus macrophages, B lymphocytes and stromal cells and regulate immune cells subsets (Hood 2017). There is an absolute dependence on the presence of CD4+ T cells, CD8+ T cells, and NK cells, where the loss of any one of these subsets lead to a complete loss of CTL response. Despite the potential role for B cells in the response to B cell-derived exosomal proteins, B cell depletion does not alter the exosome-induced CTL response (Saunderson and McLellan 2017). While exosomes are constitutively secreted by a variety of cell lineages and tumor cells, in T and B lymphocytes exosome secretion is triggered upon activation of cell surface receptors, which in turn regulates antigen-specific immune responses (Théry et  al. 2009). Exosomes are involved in important processes related to TCR-­ triggered immune responses, including T lymphocyte-­ mediated cytotoxicity, activation-­ induced cell death of CD4+ lymphocytes, antigen presentation, intercellular miRNA exchange. miRNAs are transferred during IS are able to modulate gene expression in recipient cells.

4  Dark-Side of Exosomes

These are efficient mechanisms for the rapid induction of autocrine or paracrine cell death during immune regulation (Mittelbrunn et  al. 2011; Monleón et al. 2001). Furthermore, PKCδ isotype has positive regulatory role in exosome secretion upon IS formation in T helper (Th) lymphocytes (Nascimento et  al. 2003). The molecular components that regulate some of these trafficking processes include PKD family members. PKD1 activity regulates fission of transport vesicles from the trans-Golgi network via direct interaction with the pre-existing DAG pool at this site (Baron and Malhotra 2002). PKD is the major DAG effector that regulates MVB maturation and exosome secretion in T and B lymphocytes. DGKα inhibition enhances exosome secretion in cells, this suggests that in addition to limiting PKD phosphorylation by PKC, DGKα controls direct PKD activation by DAG. PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic (Mazzeo et al. 2016). Because the exosomes have the ability to elicit potent cellular responses, patient-derived exosomes have been employed as a novel cancer immunotherapy in clinical trials, but this point lacks sufficient efficacy. However, cellular components shed from tumor cells or antigen presenting cells (APCs), such as DCs, macrophages and B cells, have been shown to be efficiently packaged in exosomes (Bell et  al. 2016; Tran et  al. 2015). As a mechanism to communicate with the microenvironment, tumour cells actively release large quantity of exosomes. These tumour-­ released exosomes play a critical role in promoting tumour growth and progression (Martins et  al. 2013; McAllister and Weinberg 2014). Tumour cell exosomes also deliver active Wnt proteins to regulate target cell β-catenin-­ dependent gene expression (Gross et  al. 2012). Pyruvate kinase type M2 (PKM2) plays a critical role in promoting the release of exosomes from the tumour cell. During exosome secretion, phosphorylated PKM2 forms a dimer structure with low pyruvate kinase activity but high protein kinase activity and then associates with

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synaptosome-­ associated protein 23 (SNAP-23) near cell’s membranes, leading to SNAP-23 phosphorylation at Ser95 and upregulation of tumour cell exosome release (Gao et  al. 2012; Wei et al. 2017). Curcumin [l,7-bis-(4-hydroxy-­ 3-methoxyphenyl)-l,6-heptadiene-3,5-dione], is a non-flavonoid polyphenol that inhibits tumor cells survival and increases reactive oxygen species’ (ROSs’) intracellular production and induces DNA damage. In curcumin-induced cell death, the phosphorylation of extracellularly regulated kinases (ERK)1/2 and p38 MAPK is stimulated, while the phosphorylation of p54 JNK and AKT is inhibited, c-Jun expression and phosphorylation is increased, and nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is prevented (Masuelli et al. 2017). When curcumin is incorporated into exosomes isolated from curcumin-­ treated pancreatic cancer cells, this compound is delivered to recipient pancreatic cancer cells via exosomes, promotes cytotoxicity and reduces cell viability via decreasing inhibitor of apoptosis (IAP) protein and mRNA expression. Furthermore, curcumin induces the expression of FOXO1 by inhibition of PI3K/AKT signaling, leading to cell cycle arrest and apoptosis, thereby reduces cell viability of recipient pancreatic cancer cells (Díaz Osterman et  al. 2016; Osterman et al. 2015; Zhao et al. 2015b). Interestingly, exosomes secreted by cancer cells have been demonstrated to express tumor antigens, as well as immune suppressive molecules such as programmed death-ligand 1 (PD-L1) and FasL. Concentrations of exosomes from plasma of cancer patients have been associated with spontaneous T cell apoptosis. This results in shortened survival of T-cells. This process is defined as “exosome-immune suppression” concept (Ichim et  al. 2008). T and B lymphocytes show inducible exosome secretion upon TCR and B-cell receptor (BCR) triggering. These vesicles affect the neighbouring recipient cells in various ways, from inducing intracellular signalling following binding to receptors to conferring new receptors, enzymes or even genetic material from the vesicles (de Saint Basile et  al. 2010; Théry et al. 2009). TRAIL is a promising member of the

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TNF family with selective effect on cancerous cells. Recent evidences showed that the membrane TRAIL-armed exosomes possess anti-­ tumor activity (Shamili et al. 2018). IS formation leads to polarization and membrane fusion of MVBs, which release intraluminal vesicles as FasL-containing exosomes that trigger Fas-­ dependent activation-induced cell death. FasL and APO2 ligand (APO2L)/TRAIL are stored inside cytoplasmic compartments that are approximately 500 nm in diameter, with characteristics of MVBs. The exosomes loaded with FasL and APO2L/TRAIL are secreted. This mechanism results in the rapid induction of autocrine or paracrine cell death during immune regulation. Thus the role of FasL and APO2L/TRAIL as effector mechanism in cell death is modified. In this case, mediators of activation-induced cell death (AICD) are CD95 FasL and APO2L/ TRAIL (Martínez-Lorenzo et al. 1999; Monleón et al. 2001). Indeed, FasL is a death mediator that binds to its receptor, Fas, and induces apoptosis. Life and death of T cells are determined by multiple factors. These are mainly, TCR triggering, co-­stimulation or cytokine signaling, caspase-8 (FLICE)-like inhibitory protein (FLIP) and haematopoietic progenitor kinase 1 (HPK1). The last molecule regulates the NF-κB pathway (Krammer et al. 2007; Nagata and Suda 1995). CTL degranulation leads to specific, TCR-controlled target cell apoptosis (de Saint Basile et al. 2010). MVB biogenesis and secretion are thus important for immune effector responses and T-cell homeostasis. The cytosolic serine/threonine kinases PKD1, PKD2 and PKD3 are expressed in many cells. PKD2 is the most abundant isotype in T lymphocytes (Irie et al. 2006; Matthews et al. 2010; Van Lint et  al. 2002). In fact, PKD has two DAG-­ binding domains (C1a and C1b) at the N t­ erminus, (Van Lint et  al. 2002), which mediate PKD recruitment to cell membranes. Protein kinase C (PKC) phosphorylation at the PKD activation loop further promotes PKD autophosphorylation and activation (Matthews et al. 1999). PKD1/2 is a major downstream effector of DGKα in the control of MVB traffic and exosome secretion in T and B cells. It is necessary for exosome secretion in T and some B cells, and thus contributes to

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crucial lymphocyte immune functions such as cytotoxic activity (Mazzeo et al. 2016). Mixed lineage kinase domain-like (MLKL) is a component of the “necrosome“. This multiprotein complex triggers TNF-induced cell death by necroptosis. Its essential nonenzymatic role in necroptotic signaling is induced by receptor-­ interacting serine-threonine kinase 3 (RIPK3)mediated phosphorylation (Murphy et al. 2013). The activated MLKL forms membrane-­ disrupting pores causing membrane leakage following necroptosis (Zhang et al. 2016). MLKL serves to facilitate endosomal function and generation of EVs. This function of MLKL is independent from RIPK1 or RIPK3. While its phosphorylation by RIPK3 enhancings cell survival via MBV and exosomes generation, on the other hand phospho-MLKL (pMLKL) contributes necroptotic death. Consequently, MLKL, besides mediating necroptotic death, also contributes to endosomal trafficking (Yoon et  al. 2017). Similarly, during hepatocyte lipotoxicity, activated MLK3 induces the release of the chemokine (C-X-C motif) ligand 10 (CXCL10)bearing vesicles from hepatocytes, which are chemotactic for macrophages. The missing link between hepatocyte lipotoxic damage and development of macrophage-associated inflammation indicates that proapoptotic lipotoxic signaling by MLK3 depends on release of proinflammatory exosomes from hepatocytes. Either genetic deletion or pharmacological inhibition of MLK3 prevents CXCL10 enrichment in exosomes (Ibrahim et al. 2016). The increase in CXCL10 correlates with the severity of liver fibrosis (Tacke et  al. 2011). MLK3 is a member of the mitogen-activated protein (MAP) kinase kinase kinase group. MLK3 is known to activate the ERK, JNK, and p38 MAPK pathways (Brancho et  al. 2005). Signal transducer and activator of transcription 1 (STAT1) is a p38 MAPK downstream target and p38 plays a key role in the serine phosphorylation of STAT1 (Goh et al. 1999). Consequently, in lipotoxic hepatocytes, MLK3 activates MAPK signaling cascade, resulting in the activating phosphorylation of STAT1, and CXCL10 transcriptional upregulation (Tomita et al. 2017).

4  Dark-Side of Exosomes

5

Exosomes and Tumor Growth

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step, they suppress DCs (Iero et  al. 2008; Liu et  al. 2006; Zhang et  al. 2012). Various malign tumors produce exosomes, which express FasL Cancer cells secrete excessive amounts of exo- and TRAIL on their surfaces (Iero et  al. 2008). somes compared to normal cells. The interplay Exosomes that carry active forms of FasL and via the exchange of exosomes between cancer TRAIL can target and induce apoptosis in acticells and between cancer cells and the tumor vated tumor-specific T cells. The induction of stroma may promote the transfer of oncogenes T-cell apoptosis by TDEs appears to be a novel and onco-miRNAs from one cell to another, lead- mechanism of tumor immune evasion (Abusamra ing to the reprogramming of the recipient cells et al. 2005). In this context, the TDEs inhibit NK (Kharaziha et al. 2012). Thus, exosomes derived cell cytotoxic activity and lead to reduction in the from motile hepatocellular carcinoma (HCC) cell percentages of these cells. Human tumor cell lines could significantly enhance the metastatic lines also produce exosomes that are capable of and invasive abilities of non-motile hepatocytes. inhibiting IL-2-stimulated NK cell proliferation The uptake of these shuttled molecules could (Liu et al. 2006). HCC-derived exosomes upregtrigger PI3K/AKT and MAPK signaling path- ulates the secretion of cytokines, such as IL-6, ways in non-motile hepatocytes with increased IL-10, IL-1β, and TNF-α in macrophages. secretion of active matrix metalloproteases-2 Whereas, melatonin treatment attenuates the (MMP-2) and MMP-9, and could facilitate the expression of these inflammatory cytokines and metastasis in liver parenchyma via mobilizing alters the immunosupressive status of macronormal hepatocyte (He et  al. 2015). Tumor-­ phages through STAT3 pathway (Cheng et  al. derived exosomes (TDEs) can actively suppress 2017). In fact, melatonin influences the phenotumor-specific immune responses. TDEs can type polarization of macrophages and affects the facilitate tumor immune evasion through differ- several cellular signaling pathways, such as ent mechanisms. They are internalized by NF-κB, STATs, and NOD-like receptor NACHT, CD11c+ cells and transported to the draining LRR and PYD domains-containing protein 3 lymph nodes. Circulating host-derived, MHC (NLRP3)/caspase-1 (Xia et  al. 2019). IL-6 and class II+ exosomes in tumor-bearing hosts are phosphorylated STAT3 (pSTAT3) are increased able to suppress the immune response specific to in an exosome dose-dependent manner when tumor antigens (Yang et  al. 2011, 2012). HCC-­ CD11b+ cells are stimulated with exosomes. derived exosomes have been shown to attenuate IL-6 and its stimulation of pSTAT3 represents an the cytotoxicity of T-cells and NK cells, and pro- oncogenic cytokine response. This effect in IL-6 mote the immuno-suppressive M2 macrophages, involves in blocking the maturation of CD11b+ N2 neutrophils, and the regulatory B cell subset. cells (Yu et al. 2007). Release of CD11b via exoThese exosomes harbor several immune-related somes induced by CpG-DNA (DNA containing non-coding RNAs and proteins that drive immunostimulatory cytosine-phosphate-guanine immune-escape and tumor progression (Han (CpG) motifs) is mediated by a Toll-like receptor, et al. 2019). Exosomes released from tumors are TLR9. This leads to powerful immunomodulacarried by the vascular system to distant cells, tory responses. The activation of p38 MAPK which take them up. Depending on the proteins, pathway and exosome formation are involved in mRNAs or miRNAs in the exosomes and the cell CpG-DNA-mediated CD11b release in macrotype, phagocytosis of exosomes may modulate phages (Hemmi et al. 2000; Kim et al. 2016). In the immune system. Uptake of the exosomes by contrast to TDEs, CD11b-expressing DC-derived cells of the immune system have three main exosomes also may activate NK cells, which effects: firstly they promote tumor growth by leads to tumor regression (André et  al. 2002; suppression of the number and activity of NK Viaud et al. 2009). Splenic macrophages and neucells. Secondly, they exert a broad array of detri- trophils are predominant phagocytes. Both macmental effects on the activity of T cells. At third rophages and DCs are required for augmentation

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of the CD4+ T-cell response in phagocytosis. After phagocytosis of antigens, exosomes containing antigens from macrophages are secreted partially in a ceramide-dependent manner and acted as transmitters to convey antigens to DCs, leading to an enhanced T-cell response. This novel pathway of cross-talk between macrophages and DCs, is characterized by increased rates of apoptosis (Xu et al. 2016). The number of neutrophils infiltrated in gastric cancer tissues is positively associated with lymph node metastasis. Furthermore, the supernatant from gastric cancer cells induces IL-1β and TNF-α expression in neutrophils. In this context, TDEs induce autophagy, and pro-tumor activation of neutrophils via HMGB1/TLR4/NF-κB signaling. Tumor-induced autophagy in neutrophils, leads to the release of factors that promotes the migration ability of gastric cancer cells (Zhang et  al. 2018, p. 2). On the other hand, TDEs differentiate monocytes into programmed cell death 1 (PD-­1) tumor-associated macrophages with M2 phenotypic and functional characteristics. These exosomes provide the production of a large number of IL-10, impair CD8+ T-cell function, and thereby promote cancer progression (Wang et al. 2018). MMPs also have been implicated in many processes involved in tumor progression (Drummond et al. 1999). The TNF-α converting enzyme, ADAM17 is a MMP domain-­disintegrin. It is responsible for the cleavage of transmembrane proteins. MMP inhibition increases the proportion of ADAM17 substrates, TNF and its receptors tumor necrosis factor receptor 1 (TNFR1) and TNFR2  in lipid rafts. Membrane cholesterol depletion increases the ADAM17-­ dependent alteration of these substrates. Thereby lipid rafts control this process (Tellier et  al. 2006). Exosome-mediated signaling increases stromal MMP1 secretion, and enhances tumor cell invasion. Gastrointestinal stromal tumor (GIST)-derived exosomes significantly increase MMP1 production, which in turn enhances GIST cell invasion. Indeed, GIST cells secrete a high number of oncogenic protein tyrosine kinase (KIT)-containing exosomes. Stromal cell uptake of GIST-derived exosomes induce tumor invasiveness (Atay et al. 2014). Additionally, transfer

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and uptake of these KIT-containing exosomes by surrounding stromal cells lead to enhanced AKT and MAPK signaling and phenotypic modulation of several cellular processes, including morphological changes, expression of tumor-associated markers, secretion of MMPs, and enhanced tumor cell invasion (Atay and Godwin 2014). On the other hand, a specific set of exosomal miRNAs modulate the tumor microenvironment (Rana et al. 2013). These miRNA carrying exosomes can be transferred among different cell lines through direct uptake. This concept also involves the oncogenic miRNAs, which can be transported through exosomes from different cell types facilitating cell to cell communication. These miRNAs influence the adjacent and distant normal cells which can lead to outcome in favor of tumor development and progression (Singh et al. 2014). The plasminogen activator urokinase receptor mRNA-containing exosomes are significantly increased in the plasma of gefitinib-resistant non-­ small-­cell lung cancer patients compared to that of gefitinib-sensitive patients. Plasminogen activator urokinase receptor induces geftinib-­ resistance through epidermal growth factor receptor (EGFR) tyrosine kinase/p-AKT/survivin signaling pathway, which plays an important role in tumor cell proliferation, migration and apoptosis. In addition, the exosomal mRNA of glycerol kinase 5 (GK5) in the plasma of patients with gefitinib-resistant adenocarcinoma is significantly higher. GK5 confers gefitinib resistance in lung cancer by inhibiting apoptosis and cell cycle arrest (Zhou et  al. 2018, 2019). In contrast, human pancreatic tumor nanoparticles, which are characterized by the proteomic analyses as exosome-­ like and rich in lipid rafts, decrease tumor cell proliferation. These nanoparticles increase B-cell leukemia/lymphoma-2 (Bcl-2) associated protein x (Bax) and decrease Bcl-2 expressions. The down-regulation of cyclin D1 and poly (adenosine diphosphate (ADP)-ribose) polymerase is triggered, so, the constitutively activated PI3K/AKT survival pathway is deactivated to drive tumor cells toward apoptosis (Ristorcelli et  al. 2008). As mentioned above, various signal transduction processes related to

4  Dark-Side of Exosomes

cell adhesion, migration, as well as to cell survival and proliferation, which play major roles in cancer development and progression, are dependent on lipid rafts. Despite lipid rafts contain mainly critical survival signaling pathways, including insulin-like growth factor 1 (IGF-1)/ PI3K/Akt signaling, these membrane domains also incorporate death receptor-mediated apoptotic signaling (Mollinedo and Gajate 2015). Actually, subtypes of lipid rafts can be distinguished according to their protein and lipid composition. These present a distinct signaling platform. Clustering raft-resident glycosylphosphatidylinositol-­anchored hemagglutinin at the outer monolayer can either enhance or inhibit specific signaling steps at the inner leaflet via the MEK/ERK pathway (Eisenberg et  al. 2006; Michel and Bakovic 2007). In fact, lipid rafts are cholesterol-enriched microdomains in cell membranes. Therefore, depletion of cholesterol inhibits both EGFR/Akt and EGFR/ERK signal transduction, and induces apoptotic cell death through down-regulation of Bcl-xL and up-­ regulation of caspase-3 (Oh et al. 2007). Exosomes induce PTEN and GSK-3β activation, while decreasing pyruvate dehydrogenase activity (Ristorcelli et  al. 2008). Expression of several endoplasmic reticulum (ER) stress markers; glucose-regulated protein 78, activating transcription factor 6 (ATF6), protein kinase R (PKR)-like ER kinase (PERK), and inositol-­ requiring enzyme 1α (IRE1α) are up-regulated in hepatocellular carcinoma tissues and negatively correlate with the overall survival. Expression of ER stress-related proteins positively correlate with CD68+ macrophage recruitment and PD-L1 expression in tumor tissues. ER-stressed tumor cells release the exosomes to up-regulate PD-L1 expression in macrophages, which subsequently inhibits T-cell function through an exosome miR-­ 23a-­ PTEN-AKT pathway, ultimately leads to immune evasion of tumor cells (Liu et al. 2019). The interaction of exosomal nanoparticles with pancreatic cancer cells leads to decreased expression of the intranuclear target of Notch-1 signaling pathway, and activates the apoptotic pathway. The expression level of Notch-1 pathway components is critical, because exosomal nanoparticles

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decrease the proliferation. Interactions of exosomal nanoparticles with target cells, at lipid rafts where Notch-1 pathway partners (Hes: hairy and enhancer-of-split homolog; ICN: intracytoplasmic Notch) are localized, prevent the functioning of the Notch-1 survival pathway. This process activates the apoptotic pathway, and determines the fate of tumoral cells (Ristorcelli et  al. 2009). Similarly, synthetic exosome-like nanoparticles (SELN) decrease cell survival due to activation of cell death with inhibition of Notch pathway, which interacts lipids between SELN and tumor cells ensues cell death (Beloribi et al. 2012).

6

Adipose Tissue and Exosomes

Adipose tissue, as an active endocrine organ, has been shown to release abundant exosomes to exert its auto/paracrine and endocrine functions in addition to well-described adipokines (Villarroya et al. 2013). Expansion of adipose tissue in obesity alters adipokine secretion of adipocytes, thereby increasing the risk of metabolic diseases. Five hundred and nine proteins have been identified from adipocytes, including 81 known adipokines. Approximately 78% of all the identified proteins have been categorized as exosome-­ associated proteins (Lee et al. 2015). Adipose tissue has recently been confirmed to constitute a major source of the exosomal miRNAs in circulation (Thomou et al. 2017), and therefore make a particular contribution to the circulating exosome level. Previous studies have reported that exosomes that are released from hypertrophic adipocytes impair endothelial cell functions. This indicates the effect of the exosomes on obesity-­ related atherosclerosis (Kranendonk et al. 2014a; Müller 2012). Thus, the formation of macrophage foam cells, which are characterized by the disruption of lipid homoeostasis in macrophages, is a vital feature of atherosclerosis (Chistiakov et  al. 2016). Consequently, exosomes participate in all the key processes of atherosclerosis from endothelial dysfunction, vascular wall inflammation to vascular remodeling (Yin et al. 2015). Neighboring

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endothelial cells transfer cav1-­ containing exosomes to adipocytes. Subsequently, adipocytes reciprocate by releasing exosomes to endothelial cells. Adipose tissue-derived exosomes contain proteins and lipids capable of modulating cellular signaling pathways. This mechanism facilitates transfer of plasma constituents from endothelial cells to the adipocyte (Crewe et  al. 2018). Fatty acid toxicity, in addition to chronic inflammation and oxidative stress changes all cellular functions, which are considered to play an essential role in the pathogenetic progress of obesity. Cells secrete exosomes containing proteins, lipids, nucleic acids, and membrane receptors, which mediate signal transduction and material transport to neighboring and distant cells. Thereby, exosome-­ associated miRNAs participate in multiple pathological processes including atherosclerosis, chronic inflammation, and insulin resistance (Yao et al. 2018). Compared to the lean subjects, obese have 44% lower capillary density and express 58% lower vascular endothelial growth factor (VEGF). The VEGF expression together with hypoxia-­inducible factor-1 (HIF-1) activity also requires PI3K- and TOR-mediated signaling. HIF-1α is an important signaling molecule in hypoxia to induce the inflammatory responses (Engin 2017). Therefore, the expression of VEGF and activation of the protein kinase A (PKA) signaling pathway in endothelial cells are significantly increased by hypoxia-exposed exosomes. Angiogenetic stimulatory activity of exosomes, which are released from hypoxic human adipose-­derived mesenchymal stem cell is significantly higher compared to that with exosomes from the normoxia group (Xue et al. 2018). Exosomes derived from mesenchymal stem cells stably overexpressing HIF-1α have an increased angiogenic capacity via an increase in the packaging of Jagged1. Jagged1 is the sole Notch ligand packaged into mesenchymal stem cell exosomes (Gonzalez-King et  al. 2017). In fact, hypoxia inhibits macrophage migration from the hypoxic adipose tissue, and also inhibits adipocyte differentiation from preadipocytes. Alterations in oxygen availability of adipose tissue directly affect the macrophage polarization

A. Engin

and are responsible from dysregulated adipocytokines production in obesity (Engin 2017). Hypoxic adipocyte-­ released exosomes have been enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). FASN-containing hypoxic adipocytes-derived exosomes increase the FASN levels in undifferentiated adipocytes. Additionally, exosomal proteins derived from hypoxic adipocytes affect lipogenic activity in neighboring preadipocytes and adipocytes (Sano et  al. 2014). Exosomes released from hypoxic adipocytes reduce insulin mediated phosphorylation of AKT, and impair insulin action in neighboring adipocytes (Mleczko et al. 2018). In fact, human adipose tissue-exosomes play a role in a reciprocal pro-­ inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic insulin resistance (Kranendonk et  al. 2014b; Sáez et  al. 2019). Furthermore, hypoxia along with higher concentrations of free fatty acids exacerbates macrophage-mediated inflammation in obesity. The metabolic status of adipocytes is a major determinant of macrophage inflammatory output. Macrophage/adipocyte fatty-acid-binding proteins act at the interface of metabolic and inflammatory pathways (Engin 2017a). Circulating exosomes are significantly increased with obesity. Sequential depletion of plasma exosomes modify plasma levels of macrophage migration inhibitory factor (MIF). Increase in exosome-associated MIF triggers ERK1/2 activation in macrophages (Amosse et al. 2018). Adipose tissue constitutes an important source of circulating exosomal miRNAs, which can regulate gene expression in distant tissues (Thomou et al. 2017). Indeed, development of lipid droplets, macrophage accumulation, macrophage polarization, TNFR-associated factor 6 activity, lipolysis, lipotoxicity and insulin resistance are effectively controlled by miRNAs in obesity (Engin 2017b). In this context, exosomal miRNAs have important roles in abdominal obesity, and cause metabolic alterations via posttranscriptional regulation of target genes. Ten exosomal

4  Dark-Side of Exosomes

differentially expressed (DE)-miRNAs are identified in subcutaneous adipose tissue (SAT) in patients with obesity in comparison to lean patients, whereas 58 DE-miRNAs are identified in visceral adipose tissue (VAT) of patients with obesity. The miRNA-4517 is a downregulated exosomal miRNA between SAT and VAT.  The other DE-miRNAs SAT-(hsa-miR-3156-5p and hsa-miR-4460) or VAT-(hsa-miR-582-5p, hsa-­ miR-­566 and miR-548) are specific (Yang et al. 2018). Exosomes in obesity and type 2 diabetes present altered miRNAs cargo and carry the molecules involved in inflammation, and immune efficiency. Adipocytes-released exosomes play an important role that may end in systemic insulin resistance in obesity (Pardo et  al. 2018). Thereby, adipose tissue-derived exosomes cause insulin resistance in hepatocytes via inhibiting insulin mediated AKT phosphorylation, with concomitant decrease in the expression of gluconeogenic genes (Kranendonk et  al. 2014c). To sum up, hypertrophic adipocyte produces more exosomes, and play an important role in obesity-­ related complications via intercellular communication that has emerged based on the release of exosomal cargo to recipient cells.

7

Exosomes and Inflammation

Inflammasomes are intracellular protein complexes of pattern recognition receptors (PRRs) and caspase-­1, with essential functions in regulating inflammatory responses of macrophages and DCs. The primary role of inflammasomes is to catalyze processing and secretion of ­pro-­inflammatory cytokines IL-1β and IL-18. The inflammasome allows the danger-exposed cells to release various proteins in order to alert and guide neighboring cells. The forming compartment, exosomes carries the local cargo proteins, including processed and unprocessed cytokines, or even entire inflammasome complexes (Cypryk et  al. 2018). Thereby, the “inflammasome-­induced” exosomes can activate inflammatory responses in recipient cells. In addition, inflammasome-­ derived exosomes directly activate NF-κB signaling pathway

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(Zhang et  al. 2017). This process consists two stages; firstly, signal 1 is provided by IL1R, TLRs, and the other plasma membrane receptors for extracellular nucleotides (PRRs). These receptors induce the transcription and expression of canonical NLRP3, as well as precursor forms of inflammatory cytokines IL-1β and IL-18, also lead to induction of expression of hundreds of other proteins (Cypryk et al. 2018; Pizzirani et al. 2007). Upon NLRP3 activation, both pro-form and mature IL-1β and IL-18 are observed in microvesicles released from cells. In this process, MLKL associates with endosomes and controls the transport of endocytosed proteins, thereby enhancing degradation of receptors and ligands, modulating their induced signaling and facilitating the generation of exosomes (Gulinelli et  al. 2012; Yoon et al. 2017). Secondly, signal 2 activates inflammasome which catalyzes processing of pro-IL-1β and gasdermin D.  Gasdermin D inserts its N-terminal domain into cell membrane and oligomerizes, forming a pore, which allows for direct secretion of small proteins, cytokines, and enhances ionic fluxes across the membrane (Cypryk et  al. 2018). The “inflammasome-­ induced” exosomes activate inflammatory responses in recipient cells (Zhang et al. 2017). Septic shock increases vascular permeability, leading to multiple organ failure including cardiac dysfunction, a major contributor to septic death. Septic exosomes contain higher levels of ROS than normal ones, which were effectively transported to endothelial cells leading to the generation of podosome (an actin-based dynamic membrane structure) clusters in target endothelial cells and thereby, causing zona occludens protein-1 (ZO-1) relocation, vascular leakage, and cardiac dysfunction (Mu et al. 2018). ROS-­ activated PKC, a small G protein Rho, Src tyrosine kinase, MLCK, ERK1/2 and p38 MAPK have been demonstrated to be crucial positive regulators of podosome assembly (Gu et al. 2007; Zhao et  al. 2015a). Furthermore, in endothelial monolayers, exosome-mediated transfer of cancer-­derived miR-105 efficiently destroys tight junctions and the integrity of these natural barriers against metastasis. Overexpression of miR-­ 105  in nonmetastatic cancer cells induces

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metastasis and vascular permeability in distant organs. Exosome-miR-105 levels in the blood and tumor are associated with ZO-1 expression and metastatic progression in early-stage breast cancer (Zhou et al. 2014). In sepsis, nitric oxide (NO) and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite (Gambim et al. 2007). Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of the receptor tyrosine kinase, Tie2 that is involved in the regulation of vascular integrity, and inflammation. Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/AKT/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Ang/Tie2, PI3K/ AKT/eNOS, and syndecan/syntenin signaling pathways play important roles in vascular growth and stabilization in inflammatory process (Ju et  al. 2014). Nevertheless, Ang1 and Tie2 form distinct signaling complexes at endothelial cellcell and cell-matrix contacts. The Ang1/Tie2 signal potentiates basal Notch signal controlling endothelial integrity and survival by up-­regulating Notch ligand delta-like 4 (Dll4). This process is achieved through PI3K/AKT pathway-mediated inhibition of GSK3β activation of β-catenin in the presence of endothelial cell-cell contacts. Blockade of Dll4/Notch signaling in tumor vasculature inhibits tumor growth by promoting non-productive angiogenesis associated with excessive sprouting from tumor vessels (Zhang et al. 2011). ROS contribute to tissue damage and remodelling mediated by the inflammatory response after injury (Hervera et  al. 2018). After platelet activation through ROS exposition, exosomes containing high concentrations of reduced nicotinamide adenine dinucleotide phosphate (NADPH) are released in heart blood vessels, those exosomes are internalized in endothelial cells, so lead to cell death and cardiac dysfunction (Monteiro et  al. 2017). On the other hand,

A. Engin

autophagy is one of the biological processes activated in response to a stressful condition. Exosomes that are released by human endothelial cells undergoing apoptosis and autophagy differ from classical apoptotic bodies because they do not contain nuclear components and are released independently of Rho-associated, coiled-coil containing protein kinase 1 activation. Exosomes naturally carry bio-macromolecules and play pivotal roles in both physiological intercellular crosstalk and disease pathogenesis (Momen-­ Heravi et  al. 2014; Pallet et  al. 2013). Lipids, which stimulate death receptor 5 (DR5), induce release of hepatocyte exosomes, which activate an inflammatory phenotype in macrophages (Hirsova et al. 2016). The inhibition of DGK-α in T lymphocytes increases the secretion of proapoptotic exosomes (Luo et al. 2004). Inhibition of DGK isoforms allows full activation of the DAG/Ras/ERK1/2 cascade (Los et  al. 2004), which represents a pathway related to important vascular signaling effectors. Most probably NO exposure promotes the release of exosomes from platelets by interfering in a similar pathway (Gambim et al. 2007). The production and release of FasL+ exosomes that co-express MHC class II molecules have the capacity to kill antigen-­ specific Th cells. Several lines of evidence indicate that FasL+ B cells and FasL+ MHCII+ exosomes have important roles in natural immune tolerance and have a great deal of therapeutic potential (Lundy et  al. 2015). Under conditions of primary necrosis, secondary necrosis and pyroptosis, THP-1-derived exosomes revealed that cells undergoing lytic forms of cell death generate a high number of exosomes compared with viable or apoptotic cells. These nanosized vesicules potentially act as mediators of cell-to-­ cell communication (Baxter et  al. 2019). Dying cells release damage-associated molecular patterns that-upon binding to evolutionary conserved PRRs-activate cells of the innate immune system to further stimulate inflammatory responses, hence initiate a massive cell death response establishing a highly feedforward cycle of inflammation and cell death (Brenner et  al. 2013). ER-stressed hepatocellular carcinoma cells release exosomes to up-regulate PD-L1

4  Dark-Side of Exosomes

expression in macrophages, which subsequently inhibits T-cell function through an exosome miR23a-PTEN-PI3K-AKT pathway. This indicates one of the tumor cells’ escape mechanisms from antitumor immunity (Liu et al. 2019). The accumulation of senescent cells and the low-­grade, systemic, inflammatory status that accompanies aging (inflammaging) are involved in the development of endothelial dysfunction. This phenomena are modulated by miRNA-containing exosomes. miRNAs can modulate all gene transcripts (Prattichizzo et  al. 2016). Inflammation activates molecular mechanisms contributing to muscle atrophy, including 5′ adenosine monophosphate-­ activated protein kinase (AMPK), p-38 MAPK and JNK, while inhibiting Akt-mediated myogenic signals. Exosomes released from inflamed myotubes induce myoblast inflammation and inhibit myogenic mechanisms while stimulating atrophic signals (Kim et al. 2018).

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5

Signal Transduction in Immune Cells and Protein Kinases Monica Neagu and Carolina Constantin

common to different TLRs, while some of them are specific for a certain type of TLR. Protein kinases involvement in innate immune cells are presented within the chapter emphasizing their coordination in many aspects of immune cell function and, as important players in immune regulation. In adaptive immunity T-cell receptor and B-cell receptor signaling are the main intracellular pathways involved in seminal immune specific cellular events. Activation through TCR and BCR can have common intracellular pathways while others can be specific for the type of receptor involved or for the specific function triggered. Various PKC isoforms involvement in TCR and BCR Intracellular signaling will be presented as positive and negative regulators of the immune response events triggered in adaptive immunity.

Abstract

Immune response relies upon several intracellular signaling events. Among the protein kinases involved in these pathways, members of the protein kinase C (PKC) family are prominent molecules because they have the capacity to acutely and reversibly modulate effector protein functions, controlling both spatial distribution and dynamic properties of the signals. Different PKC isoforms are involved in distinct signaling pathways, with selective functions in a cell-specific manner. In innate system, Toll-like receptor signaling is the main molecular event triggering effector functions. Various isoforms of PKC can be The original version of this chapter was revised. The correction to this chapter is available at https://doi.org/10.1007/978-3-030-49844-3_17

Keywords

M. Neagu Immunology Department, Victor Babes National Institute, Bucharest, Romania

Adaptive immune cells · Innate immune cells · Protein kinase · Intracellular signaling

Faculty of Biology, University of Bucharest, Bucharest, Romania Colentina University Clinical Hospital, Bucharest, Romania C. Constantin () Immunology Department, Victor Babes National Institute, Bucharest, Romania Colentina University Clinical Hospital, Bucharest, Romania

1

Introduction

Cells of the immune system whether appending to the innate or acquired arm undergo complex processes starting from their development

© The Author(s) 2021, Corrected Publication 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_5

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through their maturation and effective action displayed on the aggressor that triggered an immune response (Neagu 2012). Immune cellular responses activated by external and/or internal cues are governed by complex networks of signal transduction pathways. These intracellular pathways have as crucial regulators protein kinases. Isoforms of Protein kinase C (PKC) span the entire panel of immune cells and are involved in seminal immune processes. PKC families are enzymes involved in signaling pathways. Their action is to specifically phosphorylate substrates at serine/threonine aminoacids. Through this action PKCs regulate important cellular events like cell proliferation, differentiation, immune response and so on. PKCs consist of 10 kinase members. They have a highly catalytic kinase domain that is structurally conserved and a regulatory domain with a less conserved structure (Lim et al. 2015). In T lymphocyte regulation, there are different PKC isoforms with various physiological roles and non-redundant functions. Just to enumerate some of the PKCs, PKCθ and PKCα isoforms are involved in antigen-induced T cell activation, inhibition of T cell-mediated responses and in important pathological processes like allograft rejection and autoimmunity. Regulation of T cell proliferation and cell cycle progression have as key molecules distinct PKC isoforms that play positive roles during cell cycle progression, but there are also isoforms like PKCδ which serves as regulators. B cells are activated after antigen binding to B cell receptor (BCR) that needs for intracellular signaling CD79 molecules. In B cells pathways involving PKCβ are triggered. One common intracellular pathway with TCR signaling is the CARD11–BCL10–MALT1 (CBM) signalasome complex activating in the end the nuclear NFkB. Innate immune system detects pathogens through Toll-like receptors (TLRs), receptors that recognize specific molecular patterns expressed by the aggressors. Different TLRs would lead to various gene expressions, and would recognize different components of microorganisms. In the intracellular signaling events triggered upon TLR

activation various common or less common PKC isoforms are involved (Akira and Takeda 2004).

1.1

PKCs Traits

In the signal transduction pathway the extracellular signal that is considered the first messenger will be converted in another intracellular signal that will be the second messenger and this conversion is done at the plasma membrane. Further molecular conversions in the intracellular signaling events will take place in the cytoplasm, at organelle membranes like the nuclear envelope, and finally inside the nucleus. The main protein kinase (PK) in the immune system signaling is PKC because it can be activated by G proteins that are coupled to receptors and tyrosine kinase receptors, so that upon activation it can generate an array of intracellular signals. Activation of various isoforms of phospholipase C (PLC) generates diacylglycerol (DAG) and inositol phosphate 3 (IP3), both of them activating PKC (Nishizuka 1995). IP3 will induce the release of Ca2+ from intracellular stores that will activate certain PKC sub-families. The “immunological synapse” developed between specific T cell receptors (TCR) and the antigen presenting cell (APC) resides in the activation of PKC triggering the intracellular cascade as a response to a stimulus from the plasma membrane to the nucleus. Interestingly PKCs were found as transducer within the nucleus, pathway different from the cytoplasmic signalling event directly regulating gene expression (Lim et al. 2015). In the journey to develop the nucleus-driven intracellular signaling pathway PKC can trigger MAPK (mitogen-activated protein kinase) cascade by activating Raf (Rapidly Accelerated Fibrosarcoma). Also cytoplasmic PKC, when activated will phosphorylate the inhibitor of transcripiton factor nuclear factor (NF)kB (IkB). The phosphorylation will induce the proteolytic degradation of the inhibitor so that NFkB will translocate to the nucleus (Baeuerle and Henkel 1994). Without being exhaustive, this chapter will highlight some aspects regarding PKC isoforms

5  Signal Transduction in Immune Cells and Protein Kinases

involved in intra-cytoplasmatic signaling events that are involved in the development of the immune response whether triggered by innate or adaptive immune cells.

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in various disease from cancer to cardiovascular disease (Churchill et  al. 2008; Ali et  al. 2009; Palaniyandi et  al. 2009; Gonelli et  al. 2009; Bynagari-Settipalli et  al. 2010). TLRs are actually the family of receptors that will recognize PRRS and DAMPs to trigger an effective innate 2 Toll-Like Receptor Signaling immune response (Gay and Gangloff 2007; Kawai and Akira 2010). PKCs are involved in in the Innate System several intracellular signaling events induced by The innate immune system arm has numerous TLRs. Molecular events triggered from various first line defense functions, one of these functions TLRs differ, but the process is generally initiated engages the initial detection of microbes through after recruiting TIR (Toll/IL-1 Receptor pattern recognition receptors (PRRs). PRRs will homology) containing adaptor proteins [e.g. recognize the specific microbe molecular pattern, TIRAP (TIR Adaptor Protein) would recruit pathogen-associated molecular patterns MyD88 (myeloid differentiation factor 88), (PAMPs). PRRS can recognize also structures TRAM (TRIF-related adaptor molecule) would that result from damaged cells, damage associated recruit TRIF (TIR-domain-containing adapter-­ molecules patterns (DAMPs). When activated inducing interferon-β)]. Thereafter MyD88 PRRs will activate intracellular signaling would recruit IRAK 1,2,4 (IL-1R-associated pathways that will further induce complex kinase) and TRAF6 (tumor necrosis factor immune responses by activating the synthesis of receptor-associated factor-6). This event would inflammatory cytokines, e.g. type I interferon induce finally the pro-inflammatory genes (IFN), and an array of several other immune activation. TLR binding to TRIF would recruit mediators. This initial activation initiates TRAF6, βRIP1 (Receptor-interacting serine/ inflammation, and primes further antigen-specific threonine-protein kinase 1), and TAK1 (TGF-β-­ adaptive immune responses. These orchestrated activated kinase) and finally induce interferon-β responses has as final scope the clearance of (IFN) genes. The activation of TRIF pathway infecting microbes and the instruction of adaptive induces the pro-inflammatory cytokines secretion immune responses that is antigen-specific but to a lesser extent than MyD88 pathway. The (Janeway Jr and Medzhitov 2002). Amidst the involvement of various PKC isoforms in TLR signaling pathways, the family of PKCs signaling was proven when altering PKC enzyme appending to the larger family of protein serine / activity within innate immune cells, the process threonine kinases keeps the central role in of cytokine secretion was deregulated. Moreover, intracellular signal transduction. various TLR ligands activate different PKC Discovered more than 15  years ago PKCs isoforms in all innate immune cells, e.g. comprises isoforms that are divided in 3 monocytes, macrophages, dendritic cells and subfamilies: PKC-α, βI, -βII, and -γ; PKC-δ, -ε, neutrophils (Fronhofer et  al. 2006; Zhou et  al. -η, and -θ, and the atypical subfamily, PKCζ and 2006; Asehnoune et  al. 2005). These PKC λ/ι. These 3 sub-families were divided as such isoforms are expressed different in monocytes due to their characteristics, thus the first and macrophages, being involved also in mentioned sub-family requires calcium, DAG, monocyte differentiation. PKCα and β are and phosphatidylserine (PS); the second sub-­ activated during stimulation and monocyte-/ family requires DAG and PS in a calcium-­ macrophage-induced differentiation while PKCδ independent manner and the last sub-family expression decreases. Parihar et  al. have shown requires only PS (Newton 2003). As PKC with its that PKCδ links and phosphorylates caspase-3, various isoforms are involved in cell proliferation, inducing an increase in the apoptotic activity differentiation, apoptosis, and motility these (Parihar et  al. 2010). In neutrophils PKCδ enzymes have been discovered as being involved activation downstream of dectin-1 and Mac-1 is

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involved in the antifungal action but not the immunity responses (Ng Yan Hing et  al. 2004; function of macrophages (Li et  al. 2016). In Holm et  al. 2003). PPARγ regulates phorbol dendritic cells (DC) PKC-β expression and ester-induced NF-κB activation and TNF-α synsignaling is important in inducing DC thesis/secretion by hindering the activation of differentiation (Cejas et  al. 2005). It was also PKC-α (von Knethen et  al. 2007). More recent reported that as DCs are involved in induction of studies, have shown in human DCs that PKC-α chronic inflammatory diseases (Joffre et al. 2012) inhibition blocks also the IL-12p40 secretion that PKCs regulate caspase-3-dependent apoptosis was induced by the activation of TLR2/6, TLR (Alcain et al. 2017). 2/1, TLR5, but not through TLR3 (Langlet et al. In the signaling pathways of TLRs there are 2010). DCs from mice with the phenotype PKC-­ common molecules, but also specific ones. Thus α−/− need PKC-α activation for TLR2/1MyD88-dependent intracellular signaling event is activation of MAPK, NF-κB and AP-1; activation used by all TLRs except for TLR3. MyD88-­ that would lead to the secretion of TNFα, IL-6 dependent intracellular pathways activates and IL-10 (Langlet et  al. 2010). In an earlier MAPKK (mitogen-activated protein kinase study, Johnson et  al. found that a TLR3 ligand kinase) and IKK (IκB kinase) complex inducing can activate PKC-α. Hence, silencing PKC-α the nuclear translocation of AP-1 (Activator with siRNA, TLR3-stimulated IFN-β production Protein 1) and NF-κB (nuclear factor kappa-­light-­ was blocked in DCs. After silencing the PKCα chain-enhancer of activated B-cells). TRIF is the no hindering of IRF3 activation was recorded but principal adaptor protein in the MyD88-­ the IRF-3 transcriptional activity was inhibited independent events and can assembly with TRAF6 when induced by TRIF and TBK1 (TANKto activate AP-1 and NF-κB (Patel et al. 2012). binding kinase 1) over-expression. Probably this An overview of the major intracellular events effect is the consequence of the decreased IRF-3 where PKCs are involved in TLRs signaling is binding to CBP that needs PKC-α activation presented in Fig. 5.1 and some details regarding (Johnson et al. 2007). the involvement of PKCα, δ and ε in the TLR signaling events are further presented. 2.1.2 PKC-δ In innate immune cells, TLR-related cytokine secretion involves the activation of PKC-δ; this 2.1 PKC Isoforms Involved in TLR assertion was sustained by the fact that its inhibiSignaling tion decreases the activation of NF-κB, reduces the inflammatory cytokines secretion and the production of nitric oxide (Bhatt et  al. 2010; 2.1.1 PKC-α This is one of the conventional isoforms first dis- Ikewaki et al. 2007). PKC-δ interacts with TIRAP covered by using inhibitors that were inhibiting via the TIR domain during TLR signaling procytokine secretion in macrophages stimulated cess (Kubo-Murai et  al. 2007). If PKC-δ is via TLR (Loegering and Lennartz 2011; Foeyand depleted, the kinase activity in the complexed and Brennan 2004; Catley et  al. 2004). Several TIRAP is lost prooving that PKC-δ is the major other studies have shown the link between PKC-α kinase within TIRAP complex. In cecal ligation and TLR-related functions. Thus, earlier studies and puncture (CLP) animal model for sepsis, have shown that 264.7 RAW macrophages with TLR signaling was studied in  vivo. A PKC-δlow expression of PKC-α have also TNF-α, IL-1 inhibiting peptide was administered intra-tracheβ, iNOS, and NF-IL6 (CAAT/enhancer-binding ally in this model and the administration reduced protein β) low secretion upon LPS-activation the lung injury blocking sepsis-induced phos(Chano and Descoteaux 2002). Moreover the phorylation of this PKC-δ isoform. In this experilack of PKC-α is associated with deregulated mental model reduced levels of various phagocytosis, low killing capacity of pathogens, chemokines were obtained, reduced inflammaall these functions being non-TLR-related innate tory cells infiltration in the lungs and lower pul-

5  Signal Transduction in Immune Cells and Protein Kinases Fig. 5.1  TLR signaling main pathways. PKCs various isoforms can be found at various levels and appending to various TLR types. TLR2 after activation would associate with PKCα. PKCα is also involved in the TLR3 signaling, namely binding to the IRF3 – CBP complex (Interferon Regulatory Factor – CREB Binding Protein) and hence inducing the activation of IFN-β gene. PKCε is needed for TLR2/4 activation, as involved in TRAM phosphorylation. Activation of NFkB needs PKC-δ. PKC-ζ is also needed for TLR2/4 activation when it associates to TRAF6 and RhoA (Ras homolog gene family, member A) for transcription of p65 (nuclear factor NF-kappa-B p65 subunit). While both nuclear activation pathways induce Type I IFNγ, only CBP-p65 pathway induces IL-1, IL-6, TNFα

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monary edema. All these tissue effects were attributed to macrophages, endothelial cells, and epithelial cells (Kilpatrick et al. 2011). In activated macrophages, PKC-δ is positioned downstream of Sphingosine kinase 1 (SphK1) activation being required for NF-κB activation. In vivo inhibiting SphK1 reduced sepsis-­stimulated cytokine secretion and mortality in animal models. This isoform is a link in the activation chain TLR4-SphK1-PKC-δ -NF-κB-­ cytokine release (Snider et al. 2010).

Type I IFNg Cytokines

2.1.3 PKC-ε The first study showing PKCε involvement in innate immunity-related events was reported almost 20  years ago by Castrillo et  al. when PKC-ε−/− mice had spontaneous infections of the uterus and decreased survival rate upon experimental infection (Castrillo et  al. 2001). Further studies that have used PKC-ε specific inhibitors, have shown in the same PKC-ε−/− mouse model that PKC-ε is seminal for LPS-­ stimulated TNF-α and IL-12 secretion performed by DC and macrophages (Comalada et al. 2003;

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Koyanagi et  al. 2007). This experiment reinforced the involvement of PKC-ε in inflammation and host defense (Aksoy et  al. 2004). Actually PKC-ε is phosphorylated by all TLRs that need MyD88 (e.g. TLR1  – 9 except TLR3  in macrophages) (Faisal et  al. 2008). If PKC-ε can be phosphorylated also by PKC-α, the effects of PKC-α on cytokine secretion can be shared by PKC-ε (Saurin et al. 2008; Durgan et  al. 2008). Moreover, PKC-ε is involved in TLR4 activation but via TRAM pathway that induces IFN-β and RANTES (Regulated on Activation Normal T Cell Expressed and Secreted). Recombinant PKC-ε, phosphorylates TRAM, but this phosphorylation does not appear in PKC-ε−/− cells with reduced production of IFN-β. Thus, actually PKC-ε is a link in the flow of TLR4- PKC-ε  – TRAM- IFN-β pathway (McGettrick et al. 2006).

PKC-ζ is associated to RhoA in LPS-stimulated macrophages and by inhibiting RhoA or TRAF6, the PKC-ζ activation was blocked. Analogous experiments showed that inhibition of PKC-ζ blocked TAK1 phosphorylation (Huang et  al. 2009). Thus actually PKC-ζ is a link in the flow of TLR2/4-RhoA/TRAF6- PKCζ  – TAK1  – p65 – cytokine induction. Various forms of PKCs are involved in the large family of TLRs activation mediating important effector functions in innate immune cells.

3

 KCs in T-Cell Receptor P Signaling

In contrast to innate immunity, adaptive immunity discriminates minute differences between aggressors. The immune system encounters 2.1.4 PKC-ζ during its life span a myriad of antigens and As a more atypical isoform, component of IL-1R hence can specifically raise an immune response and TNFR intracellular signaling pathways PKC-­ towards all the encountered pathogens and ζ was reported more than 15 years ago (Hirai and antigens. Adaptive immunity would develop a Chida 2003; Duran et  al. 2003). PKC-ζ was slow but long-lasting specific immune response. shown to be involved in TLR, IRAK, RhoA, and T cells and B cells receptors are designed to NF-κB activation (Yang et al. 2007; Huang et al. recognize all potential antigens. 2009). Using PKC-ζ specific inhibitors, ERK The first stage of recognition in both cell types activation and TNF-α secretion was blocked is the specific linkage with the specific domains upon Mycobacterium tuberculosis (MTB) in the antigen followed by activation of a single stimulation (Yang et al. 2007). PKC-ζ was found specific cell type. This recognition would trigger associated with TLR2 but not TLR4. Thus, in cells to proliferate (clonal expansion). THP-1 cells (a cell line from an acute monocytic Recognition of specific domains on the antigen is leukemia) PKC-ζ does not associate with TLR2. done by a specific TCR structurally different IRAK phosphorylation is a molecular event in from that of BCR. Intracellular signaling the flow of TLR signaling and if it is degraded is triggered by the activation of TCR has both a negative control of the signaling pathway. common and different pathways with BCR as Using a set of protein kinase inhibitors, it was further described (Acuto and Cantrell 2000). suggested that IRAK is actually a PKC-ζ substrate. Several studies have highlighted a role for RhoA in TLR signaling (Chen et  al. 2009; 3.1 PKCs Families Shibolet et al. 2007; Manukyan et al. 2009; Lin et al. 2010). In monocytes it was found that the TCR as being the complex molecular structure activation of p65 through TLR2 ligands needs delivering the antigen specific recognition is a RhoA and PKC-ζ activation. PKC-ζ transitorily heterodimer of either αβ or γδ polypeptides that links to RhoA that is activated and such, PKCζ contain variable regions, responsible for the aids the observed effect of RhoA on gene binding to the complex of antigen peptide-major transcription. Huang et  al. have shown that histocompatibility complex (MHC). In order to

5  Signal Transduction in Immune Cells and Protein Kinases

efficiently deliver signals upon activation these chains associate with signal transducing components, CD3 complex, process needed for both cell expression and function accomplishment. The CD3 complex which has 3 parts, ζ part being intimately involved in the intracellular signaling pathway would develop the activation pathway (Favero and Lafont 1998). TCR will recognize the antigen presented by the antigenpresenting cells (APCs) and this “immunological synapse” will lead to T-cell differentiation and activation; a multifactorial process including PKC signaling. After TCR-MHC complex formation, PKC localizes to the complex and generates the activation of nuclear transcription factors for further induction of immune effector genes (Isakov and Altman 2002). Activated genes will generate both soluble molecules, like cytokines, chemokines, growth factors and transient cell surface molecules to induce T-cell proliferation and differentiation (Smith-Garvin et al. 2009; Fooksman et al. 2010; Yokosuka and Saito 2010). There are several PKC isoforms that are involved in the TCR intracellular signaling events.

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PKC to the oxidative state was proven in muscular disorders as well (Dobrowolny et al. 2018). In PKCθ-knockout mice, PKCθ involvement in T-cell signaling was reported more than 15 years ago (Pfeifhofer et al. 2003) and studies that related its dis-functionality in autoimmune diseases followed (Anderson et  al. 2006). In mouse experimental models of autoimmune encephalomyelitis it was shown that in PKCθ deficient mice type Th1 responses are deregulated. Th17 involvement in developing this experimental autoimmune encephalomyelitis needs PKCθ because it controls Th17 differentiation (Salek-­ Ardakani et al. 2005; Tan et al. 2006). PKCθ controls signal transducer and activator of transcription 3 (STAT3) after Th17 activation by association of activator protein 1 and NF-kB transcription factors to the STAT3 promoter (Kwon et al. 2012). PKC θ was reported to suppress Stat4, Tbet (T-box transcription factor) and IFNγ upon Th17 activation (Wachowicz et al. 2014). In immune memory control PKCθ is also involved. Thus in the antiviral responses performed by CD8+ T-cell, PKCθ is needed for the antigen immune recall upon re-infection with lymphocytic 3.1.1 PKCθ choriomeningitis virus and influenza virus The most predominant isoform used by T cell (Marsland and Kopf 2008) and for developing activation is PKCθ, while in different T cell memory T-cell per se (Teixeiro et al. 2009). subsets other forms can be identified as well In regulatory T cells (Treg) PKCθ is localized (Kong and Altman 2013). This PKCθ isoform is apart from the immunological synapse, process expressed in T cells within the haematopoietic that leads to a negative regulation of Tregs (Gupta cell population as shown in the early 90s (Baier et al. 2008; Zanin-Zhorov et al. 2010). The effect et al. 1993). Further studies have shown that after of negative regulation performed by PKCθ is an activation, PKCθ translocates to the plasma inhibited differentiation of Tregs via the AKT/ membrane associating with the co-stimulatory Foxo1/3a pathway (Ma et al. 2012). molecule CD28 through its V3 domain (Kong PKCθ has an important role in T-cell immune et  al. 2011; Yokosuka et  al. 2008). In CD28-­ response regulation and an outline of the main deficient mice it was shown that PKCθ needs the steps in TCR signaling involving PKCθ is presence of CD28 in activated T cells to develop depicted in Fig. 5.2. a mature immunological synapse. PKCθ is regulated also by the intracellular redox state, 3.1.2 PKC-α and in 2013 the first published study has shown DAG messengers are involved in TCR signaling, in naive T cells that the oxidized inactive PKCθ but more recently it was demonstrated that it can is recruited to the cell membrane and that this regulate the duration and amplitude of Ras/ERK process of PKC-θ activation is redox dependent signals. Thus, it was shown that PKCα is and needs de novo synthesis of glutathione (von transiently translocating, process mediated by Essen et al. 2013). Afterwards, the dependence of DAG generation in the contact area of the immune

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140 Fig. 5.2  TCR signaling pathway involving PKCθ. Upon TCR stimulation, activation of co-stimulatory CD8 induces the translocation of PKCθ; activation induces further the aggregation and phosphorylation of caspase-recruitment domain containing membrane-associated guanylate kinase protein-1 (CARMA1). Aggregated CARMA1 will complex with B cell lymphoma 10 (Bcl-10) and mucosal-associated lymphoid-tissue lymphoma-translocation gene 1 (Malt1). Malt1 in the CBM complex will induce the degradation of the IKK regulatory component, IkB, via activation of TRAF6 and transforming TAK1, the process leading to the activation and nuclear translocation of NF-kB

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synapse. It was demonstrated that Diacylglycerol kinase (DGK)ζ can negatively regulate PKCα translocation kinetics, while PKCα is self-­ limiting in the immune synapse. These two pillars DGKζ and PKCα would regulate the amplitude and duration of Ras/ERK activation after antigen recognition. In animal models characterized by deficient DGKζ it was demonstrated that increased DAG signaling induces enhanced PKCα-dependent L-selectin shedding. Thus Gharbi et al. depicted that the early activation of DAG-PKCα axis induces TCR biological responses (Gharbi et al. 2013). Markers that can detect pathologies associated to T cells were studied by immunohistochemistry

approaches, PKC isoforms included. Thus GADS (GRB2-related adaptor protein 2), DOK2 (Docking protein), SKAP55 (src family associated phosphoprotein), ITK (Interleukin-2 inducible T-cell kinase) markers were associated with lymphoid and myeloid precursor neoplasms. PKCα was found defective in angioimmunoblastic T-cell lymphoma and aberrantly expressed in classical Hodgkin lymphoma, Burkitt lymphoma and plasma cell myeloma. Probably future studies will develop PKCα as a new therapy target (Agostinelli et al. 2014). Further work on PKCα involvement in TCR signaling has shown that Ca2+ elevation is separate from the control of IκBα degradation,

5  Signal Transduction in Immune Cells and Protein Kinases

and that the regulatory action via Ca2+ is dependent on PKCα-mediated phosphorylation of p65. Therefore this recent study established a check point controller of Ca2+ signaling event as being PKCα (Liu et al. 2016). A study published in 2019 by Merida et al. has shown that in T lymphocytes, the interaction between DGKζ and Sorting Nexin 27 (SNX27) controls PKCα activation. When silencing SNX27  in T lymphocytes, PKCα activation is enhanced, finding that provides molecular basis for the DGKζ – PKCα deregulations in immune synapse impairment (Mérida et al. 2019).

3.1.3 PKCδ PKCδ isoform was first identified in a mouse model as a PKC family member that is required for the control of lytic granule exocytosis by CD8+ T cells. Thus PKCδ isoform, although it was shown to regulate this process, was found unessential for activation and cytokine production (Ma et  al. 2007). Other studies followed, showing PKCδ involvement in TCR activation. As shown in the previous section, TCR and costimulatory receptors activation have CARMA1 (caspase recruitment domain-containing membrane-­associated guanylate kinase 1) as a seminal protein. CARMA1 will recruit B-cell CLL/lymphoma 10 (Bcl10), mucosaassociated lymphoid tissue lymphoma translocation gene 1 (MALT1), and TRAF6 assembling a specific signalosome that would trigger NF-κB activation. In a cellular model with Jurkat T cells, PKCδ was shown to associate with CARMA1 and the over-­ expression of PKCδ inhibited NF-κB activation through CARMA1. Alternatively, when knocking down PKCδ, NF-κB activation and IL-2 secretion was noticed. All these results indicated PKCδ as a negative regulator in T cell activation (Liu et al. 2012). In 2019, it was shown that PKCδ regulates TCR-controlled multivesicular bodies polarization toward the immune synapse and exosome secretion. Moreover at the immune synapse the reorganization of F-actin is inhibited in T lymphocytes were PKCδ is obstructed. Therefore, PKCδ is recently proposed as actin

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reorganization regulator in the immune synapse (Herranz et al. 2019). In TCR signaling there are PKCs that up-regulate activation of adaptive immune functions, but also kinases that are negative regulators of this cell type.

4

B-Cell Receptor Signaling

Mature B lymphocytes are a critical component of the adaptive immunity participating to systemic defense by generating highly specific antibodies, regulatory cytokines, chemokines and presenting antigen to T cells to generate an immune response against tumors and pathogens (Harwood and Batista 2008). B cell activation is initiated upon specific antigen capturing by BCR, this event will trigger various intricate intracellular pathways mediated by Src-family kinases as well as Syk and Bruton’s tyrosine kinases (Btk)/Tec that cooperate to uptake the antigen-receptor complex. All this organized assemble of kinases and adaptor proteins form the so called signalosome that activates multiple signalling pathways (Packard and Cambier 2013; Lim et al. 2015). Besides antigen capturing, growth and proliferation of B cells are closely regulated through BCR signaling and other membrane receptors that would be activated by different cytokines (Jellusova and Rickert 2016; Taher et al. 2017). BCR is a heterodimeric transmembrane protein composed of heavy-chain and light chain immunoglobulins, CD79A/Igα and CD79B/Igβ and located on B cells outer surface (Stephenson and Singh 2017). The engulfed antigen via BCR is processed and presented on B cell surface as specific peptides associated with MHC-II molecules; this process results in the assignation of specific T helper lymphocytes that would provide a second signal for complete B cell activation. Further, activated B cells can differentiate to antibody-secreting plasma cells or enter into germinal centers (GCs) where cells are subjected to BCR affinity maturation and antibody class switch recombination (De Silva and Klein 2015). Subsequently B cells would

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leave the GC and differentiate into either high-­ affinity antibody secreting long-term plasma cells or dormant memory B cells that can reactivate as plasma cells upon re-encountering with the same antigen (Tsui et al. 2018; Kurosaki et al. 2010).

M. Neagu and C. Constantin

ease mechanisms and provide new therapeutic avenues for precision medicine (Taher et al. 2017). Another B cells subclass essential in immune homeostasis maintenance is represented by regulatory B lymphocytes (Bregs) that modulate immune responses mostly via anti-inflammatory cytokines (e.g. IL-10) enabling Tregs 4.1 PKCs Role in BCR Signaling commencement, and thus hindering anti-tumor Events responses. Aberrations in Breg numbers and function have been observed in immune-related Antigen binding to the BCR and further B cell pathologies, including cancer. However, despite a activation rely on PKC that is translocated from pivotal role for Bregs in upholding inflammation cytosol to the plasma membrane where spatially and carcinogenesis, the phenotypic diversity of there are regions comprising the tetraspanin the Bregs remains unclear (Mauri and Menon protein CD53 to sustain BCR-dependent PKC 2017; Sarvaria et al. 2017). The PKCβ is actively stimulation (Zuidscherwoude et al. 2017). involved in NF-kB signaling cascade upon BCR The PKC is a large family of Ser/Thr kinases engagement that initiate an influx of Ca2+ into the with pleiotropic expression and controlling many cytoplasm (Saijo et  al. 2002). Secondary cellular functions like development, mediators such as DAG are generated as part of differentiation, activation, lymphocytes survival the BCR activation cascade to initiate Ca2+ influx and immune responses development (Tan and and PKCs signaling events, leading to activation Parker 2003; Cooke et  al. 2017). From the of several transcription factors (Myc, NF-kB, previously section, the described isoforms of activator protein 1 etc.) essential for B-cell life PKC β, λ, and ζ variants prevail in B cells with program (Packard and Cambier 2013). PKCβ the most ubiquitously expressed and most The mandatory role of PKC isoforms and their important isoform in BCR signaling (Isakov involvement in NF-κB pathway modulation in 2018). lymphocytes is well known (Guo et al. 2004). The The role of PKCβ in regulating B-cell func- membrane localization of PKCβ is favorable for tion was initially revealed through PKCβ gene IκB kinase (IKK) complex activation that is knockout mice that exhibited decreased B-cell tightly regulated by Ca2+ levels (Guo et al. 2004; activation, no proliferation upon BCR activation Berry et al. 2018). The enzyme complex IKK is and altered T-cell-independent immune responses located in the upstream of the NF-κB signaling (Leitges et al. 1996). cascade playing an essential role for B cells In establishing the signalosome, the isoform β development and function (Kaisho et  al. 2001). of PKCs mediates phosphorylation of CARMA1 PKCβ has a significant role in IKK cascade as protein leading to a multipart complex comprising IKK becomes activated upon phosphorylation of CARMA1, Bcl10 and MALT1 linked to the adaptor protein CARMA1 by PKCβ; this will membrane lipid rafts (Shinohara et al. 2007). A fit together IKK and TAK1 allowing TAK1 to general outline of the intracellular signaling phosphorylate and activate IKK.  As a conseevents triggered by BCR activation regulated by quence, IkB is further phosphorylated by IKK PKCβ is presented in Fig.  5.3. However, leading to NF-kB activation, highlighting the role alterations in B cell responses can lead to of PKCβ in BCR-triggered NF-kB pathway pathological conditions such as autoimmune (Shinohara et al. 2005; Thome et al. 2010). diseases when high-affinity autoreactive B The central mechanism that regulates intracellymphocytes develop and produce auto-­lular Ca2+ levels in lymphocytes is represented by antibodies leading to tissue damaging. store-operated calcium entry (SOCE) through Outlining biomarkers that designate an abnor- calcium release-activated calcium (CRAC) commal B cell response can assist in elucidating dis- prising Stromal Interaction Molecule 1 and 2

5  Signal Transduction in Immune Cells and Protein Kinases Fig. 5.3  BCR signaling pathway involving PKCβ. B cells are activated after antigen binding to BCR that needs for intracellular signaling CD79 molecules. In B cells pathways involving PKCβ are triggered. One common intracellular pathway with TCR signaling is the CBM complex activating finally the nuclear NFkB. Secondary messengers (DAG) are generated upon PLCγ1 activation that due to generated calcium (Ca2+) would activate PKCβ. 3-phosphoinositide dependent protein kinase-1 (PDK1) activated through phosphoinositide 3-kinase (PI3K) would activate PKCβ. The signaling pathways lead to the activation of nuclear transcription factors: NFkB, activator protein 1 (AP1) and nuclear factor of activated T cells (NFAT) that will induce cytokines’ synthesis

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proteins (STIM) and Orai protein channels (Feske 2007). STIM and Orai channels discovery, almost a decade ago, has advanced knowledge regarding Ca2+ multiple actions such as gene expression activation program. Mostly, Ca2+ dynamics is induced by activation of antigen receptor that would trigger distinct patterns of gene expression responsible for a distinct transcriptionally fate in lymphocytes’ life. Interestingly, a clear connection between strength of antigen binding to BCR, pro-­ inflammatory transcription factors NF-κB and NFAT and Ca2+ levels effects was reported. Thus, a high affinity antigen interaction with BCR will cause STIM/ Orai-mediated Ca2+ entrance and activation of

both NF-κB and NFAT factors. An intermediary antigen affinity binding to BCR causes possibly Ca2+ fluctuations that preferentially activate NF-κB. Faint interactions in antigen-BCR complex will generate a transitory raise in cytosolic Ca2+ leading to the selective activation of NF-κB dependent gene expression. Thus Ca2+ levels dictated by bonding strength in antigen-receptor complex are able to regulate important checkpoints in canonical NF-κB signaling in both T and B lymphocytes. More accurately, in resting lymphocytes, homo- and heterodimers of NF-κB p50, p65, and c-Rel proteins are sequestered in the cytosol by IκB proteins including IκBα. BCR engagement activates a series of PTKs, β iso-

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forms in case of BCR, that subsequently activate PLCγ isoforms, cleaving PI(4,5)P2 to IP3 and DAG.  Further, IP3 binds to IP3 receptors channels from endoplasmic reticulum (ER) allowing release of Ca2+ in cytosol. The resulting collapse of Ca2+ level in ER induce STIM1 proteins oligomerization and stimulation of plasma membrane Orai1 channels enabling extracellular Ca2+ admission and a significant elevation in cytoplasmic Ca2+ level. STIM1 plays a dominant role in BCR-induced Orai1 activation while STIM2 regulates cytosolic Ca2+ levels in quiescent conditions (Roos et al. 2005; Liou et al. 2005; Berry et al. 2018). Additional roles for STIM/Orai in B cell life were revealed in Bregs where expression of immunosuppressive cytokines IL-10 and TGF-β are deficient in the absence of STIM/Orai mediated Ca2+ channels (Berry et al. 2018). Recent data designate a critical role of PKCβ in antigen polarization which is supposed to enable the effective antigen processed peptides linking to MHC-II molecules. Mice with PKCβ-­ deficient B cells would exhibit impaired antigen polarization and presentation that could hinder GC development, thus highlighting the significant role of PKCβ in B cells fate determination and metabolic program (Tsui et al. 2018). In the last years there were several studies revealing that PKC could intercede as a possible target in B cell diseases taking into account that deregulation in BCR signaling represents one of the mark for activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL); a number of BCR targeted therapies are current in progress, including kinase inhibitors against PKC (Stephenson and Singh 2017; Kuppers 2005). PKCβII is overexpressed in chronic lymphocytic leukemia (CLL) where it’s associated with poor prognosis and survival as well as with anticancer drug resistance development (Kabir et al. 2013; Kazi et  al. 2013). PKCβII is viewed as a potential therapeutic target for CLL treatment but inhibition of PKCβII with specific inhibitors (e.g., LY379196). This inhibitor had a minor effect on the cellular viability and failed to induce

M. Neagu and C. Constantin

a decrease of mRNA levels for PKCβII in CLL cells. Certain growth factors like VEGF seem to sustain PKCβII activity. In a milieu deprived of VEGF there is a clear inhibition of PKCβII by LY379196 (Abrams et  al. 2010). The role of PKCs in CLL cell survival is to inhibit pro-apoptotic signals and stimulate pro-survival ones (PKCβII) and activate anti-apoptotic effectors like NOTCH2 (PKCδ) (Hubmann et  al. 2010; Kang 2014). Furthermore, PKCδ regulates peripheral B-cell development and immune homeostasis (Salzer et  al. 2016) and mediates mitochondrial-dependent apoptosis in an epigenetic manner like phosphorylating histones at specific residues such as H3S10 or H3T45 known to be linked to apoptosis (Hurd et al. 2009; Park and Kim 2012). PKCδ is recognized to be involved in autoimmunity as recently, autosomal recessive mutations in gene encoding PKCδ have been identified in patients with systemic lupus erythematosus. Moreover Prkcd−/− knockout mice models revealed a distinctive role of PKCδ in controlling B-cell tolerance as immune complex-mediated glomerulonephritis, lymphadenopathy, splenomegaly, and multi-­organ infiltrations of B cells were observed in this animal model (Miyamoto et al. 2002). Therefore the mechanisms of B-cell tolerance could be elucidated studying PKCδ related deficiencies (Salzer et al. 2016) as well as its immune therapy targeting (Lim et al. 2015). Nevertheless, after almost three decades of cancer clinical trials with targeting PKC inhibitors, the results are still controversial for patient outcome. Increasing evidence from cancer genomic and proteomic outline provide a novel research line where PKCs isoenzymes largely act as tumor suppressors and therefore their activity should be restored instead of inhibited in antitumor approaches (Newton 2018). However the clear immunologic role of PKC in lymphocytes biology is still to be portrayed and progresses in this area are still to come (Harton 2019).

5  Signal Transduction in Immune Cells and Protein Kinases

5

Conclusion

PKCs in the immune system have become a check –point for seminal functions and control of intracellular signaling events. Nevertheless, after almost three decades of fundamental studies and cancer clinical trials targeting PKC with inhibitors, there is still information lacking. For example in the clinical trials results there are still controversies and sometimes even the clinical outcome of the patient subjected to this therapy worsed. Increasing evidence from cancer genomic and proteomic outline provide a novel approaches namely PKCs isoenzyme largely act as tumor suppressors and therefore their activity should be restored instead of inhibited in antitumor approaches (Newton 2018). However the clear immunologic role of PKC in lymphocytes biology is still to be portrayed. Apart from its potential role in antigen presentation and TCR/BCR triggered events immune cell activation/differentiation and transcriptomic pattern related events, still little is known about the contribution of PKC to cell’s immunobiology.

Acknowledgements This research was funded by UEFISCDI Projects PN-IIIP1-1.2-PCCDI-2017-0341 (acronym PATHDERM), PN-III-P1-1.2-PCCDI-2017-0782 (acronym REGMED) and PN.19.29.01.01/2019.

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6

Role of Protein Kinase C in Immune Cell Activation and Its Implication Chemical-Induced Immunotoxicity Emanuela Corsini, Erica Buoso, Valentina Galbiati, and Marco Racchi

chapter, current knowledge on the role of PKC in immune cell activation and possible implication in immunotoxicity will be described.

Abstract

Protein kinase C (PKCs) isoforms play a key regulatory role in a variety of cellular functions, including cell growth and differentiation, gene expression, hormone secretion, etc. Patterns of expression for each PKC isoform differ among tissues, and it is also clear that different PKCs are often not functionally redundant, for example specific PKCs mediate specific cellular signals required for activation, proliferation, differentiation and survival of immune cells. In the last 20 years, we have been studying the role of PKCs, mainly PKCβ and its anchoring protein RACK1 (Receptor for Activated C Kinase 1), in immune cell activation, and their implication in immunosenescence and immunotoxicity. We could demonstrate that PKCβ and RACK1 are central in dendritic cell maturation and activation by chemical allergens, and their expressions can be targeted by EDCs and anti-inflammatory drugs. In this E. Corsini (*) · V. Galbiati Laboratory of Toxicology, Department of Environmental Science and Policy, Università degli Studi di Milano, Milan, Italy e-mail: [email protected] E. Buoso · M. Racchi Department of Drug Sciences, Università di Pavia, Pavia, Italy

Keywords

Protein kinase C · Immunity · Hormones · Xenobiotics · Receptor for activated C kinase · Immunotoxicity

1

Introduction

Our interest in protein kinase C (PKC) and immunotoxicity started with the observation we made 20 years ago that aging was associated with a defective production of tumor necrosis factor-α (TNF-α) in rat alveolar macrophages in response to lipopolysaccharide (LPS), which correlated with a defective PKCβ membrane translocation, due to an age-dependent decrease in its anchoring protein RACK1 (Corsini et  al. 2002). This defective activation has consequence on the pathogenesis of those diseases in which the immune system and TNF-α plays a pivotal role (i.e. lung fibrosis). We demonstrated that aging was associated with reduced TNF-α release in the bronchoalveolar lavage fluid and reduced collagen deposition in rats exposed to silica (Corsini et  al. 2003). This result implies

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_6

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that extremes of age are not necessarily immunologically sensitive subpopulations, and that the understanding of the molecular mechanisms underlying toxicity/pathology is crucial to define the influence of age on the toxic responses and progression of diseases. More recently, we demonstrated that PKCβ was also central in dendritic cell activation following exposure to contact allergens, where the up-regulation of CD86, HLA-DR and interleukin (IL)-8 release were all PKCβ-dependent (Corsini et al. 2014a). The use of a RACK1 pseudosubstrate, which directly activates PKCβ, resulted in dose-related increase in CD86 expression and IL-8 release. The strong contact allergen dinitrochlorobenzene (DNCB) induced indeed a rapid (within 5 min) PKCβII translocation followed by reactive oxidative stress (ROS) production, which could be prevented blocking PKCβ activation, and subsequent surface marker up-regulation and cytokine production. Chemical allergens of different potency activated PKCβ with a different kinetic. This book chapter aims to portray the role of PKCs in immune cells activation and its implication in immunotoxicity.

2

 KC: From Discovery to Its P Role in Cell Homeostasis and Activation

At least 10 serine/threonine kinases constitute the PKC family: nine is the total number of genes in the PKC family identified in the human kinome, plus the splice variants PKCβI and βII, and a brain-specific variant that encodes only the catalytic domain of PKC ζ, end with 11 isoforms. PKC is conserved throughout evolution and it is present in all cell types, where it controls critical signal transduction pathways regulating activation, proliferation, differentiation, effector functions and death. PKC isoforms share a highly conserved catalytic kinase domain, and a less conserved regulatory domain responsible for binding to activators, i.e. diacylglycerol (DAG) and calcium (Ca2+), and to anchoring proteins, i.e. RACK1 (Lim et al. 2015).

The family of PKC enzymes was originally identified by Nishizuka and colleagues as serine/ threonine (Ser/Thr) protein kinases, and was named from Ca2+-phospholipid-dependent protein kinase or protein kinase C (Nishizuka 1995; Nakamura and Yamamura 2010). PKCs are classified into three subfamilies: classical or canonical, novel and atypical. Classical PKC includes α, βI, βII (alternate splicing variants of the PKCβ gene) and γ types, which require DAG, Ca2+ and phospholipids for activation. Novel PKC includes δ, ε, η, and θ isoforms, which require DAG but no Ca2+ for activation. Atypical PKC includes ζ and ι/λ (human/mouse) isoforms, which do not need DAG or Ca2+ for activation. Patterns of expression for each PKC isoform differ among tissues (Jaken and Parker 2000; Zeng et  al. 2012). The different PKCs are not functionally redundant, and specific PKCs mediate specific cellular signals required for activation, proliferation, differentiation and survival of immune cells (Leitges et al. 1996; Sun et  al. 2000; Miyamoto et  al. 2002; Pfeifhofer-­ Obermair et al. 2012). PKC isozymes have a similar structural architecture with functional domains that serve as binding partner for activator and co-factor molecules. There is an N-terminal domain that is called regulatory domain (and comprises a segment of approximately 35  kDa), connected via a hinge region to a catalytic or kinase domain at the C-terminal side of the molecule (approximately 45  kDa) (for a detailed review see Newton 2018). These structures contain functional subdomains that are essential for the activity of the kinase. One of the key element functioning as a molecular switch is the pseudosubstrate domain. It is represented by a stretch of amino acids reproducing the kinase site consensus with an Ala substituting the canonical Ser/Thr phosphorylation substrate. The binding of this domain with the catalytic site keeps each isoform in an inactive conformation, or a “folded conformation”. Interaction with specific activators reduce the affinity of the pseudosubstrate domain and allows the activation of the enzyme.

6  Role of Protein Kinase C in Immune Cell Activation and Its Implication Chemical-Induced…

The C1 domains are present in all PKC isozymes with null to high affinity to DAG.  Conventional PKC isoenzymes present low affinity binding C1 domains usually present in tandem as C1A and C1B where only one is the actual DAG binding site (Giorgione et  al. 2003). The same domains in novel PKC isoenzymes, bind DAG with higher affinity. These differences in binding affinity are functional to the fact that novel PKC enzymes should respond solely to agonist-evoked increases in DAG alone, whereas conventional PKC isozymes also need increases in intracellular Ca2+ (Giorgione et al. 2006). Increased membrane affinity of the C1 domain of protein kinase Cδ compensates for the lack of involvement of its C2 domain in membrane recruitment. It is worth to mention that the difference in affinity is due to a single amino acid variation and that this variation does not influence the binding of phorbol esters. Atypical PKC isozymes have just one C1 domain and it is not a DAG sensor and it is localized in close proximity to the pseudosubstrate segment and therefore participates structurally to the auto-inhibitory segment (Graybill et al. 2012). All conventional PKC isozymes have a Ca2+ sensing element which is denominated C2 domain. Novel PKC isozymes have a “novel” C2 domain that does not serve as a Ca2+ or plasma membrane sensor since it does not include those aminoacidic residues that are devoted to these functions. In addition, the C2 domains in novel PKCs have a peculiarly permutated β-strand fold compared to those in conventional C2 domains (Nalefski and Falke 1996). Atypical PKCs have a domain denominated PB1 domain which mediates protein scaffold binding. Partners engaging in this binding have the activation function of DAG binding to C1 domain of conventional and novel PKC isozymes which results in disengagement of the pseudosubstrate and kinase activation (Graybill et  al. 2012; Tsai et al. 2015; Tobias and Newton 2016). The kinase domain of PKC isozymes has the same overall structure of protein kinase A (PKA). The catalytic rate of PKC is lower than that of PKA but in general is comparable with that of

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other enzymes. The atypical isozymes are however much slower enzymes (Tobias et al. 2016). PKCs at variance with other kinases such as PKA and Akt (or protein kinase B), do not have a strong phosphorylation consensus sequence. It is important to have basic residues at the amino and/or carboxy terminal side of the consensus sequence and the presence of a hydrophobic residue at the P+1 position. This rather loose selectivity for specific residues translates into the difficulty to develop isoform specific inhibitors. The pseudosubstrate segment is a weak inhibitor and isoform specific sequences can be substrates for other kinases. Two specific bisindolylmaleimides are commonly used as PKC inhibitors: Gö6983, which inhibits conventional and novel PKC isozymes, and Gö6976, which inhibits conventional PKC isozymes (Wu-Zhang and Newton 2013). At the C-terminal PKCs have a domain that act as a regulator mostly of docking regulatory elements and to facilitate adenosine triphosphate (ATP) and substrate binding. Although not completely structured the mostly helical conformation suggest that this C terminal tail is a possible link with membrane during PKC maturation and activation (Yang and Igumenova 2013). In fact, the interaction of PKC with physiological stimulators results in a redistribution of the protein to subcellular compartments where it interacts with membranes and other proteins. The latter assumed a growing importance with the discovery of anchoring or scaffold proteins that support PKC activity and substrate targeting (Mochly-Rosen et  al. 1991). There are several proteins that can bind PKC in resting and activated conditions regulating the subcellular localization of the enzyme. One of the most interesting in relation to its role in immune cells is the so-called RACK1 (Ron and Mochly-Rosen 1995; Battaini et al. 1997; Buoso et al. 2017a, b). RACK1 is a protein that belongs to the tryptophan-aspartate 40 (WD40) motif repeat family, originally called also G-protein like because of its homology to the beta subunit of heterotrimeric G-proteins. WD repeat proteins share a common role as scaffolding protein complexes, they lack

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any enzymatic activity, but are modulated by post-translational modifications (Adams et  al. 2011; Gandin et al. 2013). RACK1 and its functions as PKC scaffold protein have been described by Mochly-Rosen’s group after cloning from a rat brain cDNA library screened for gene products that could bind activated PKCβII (Ron et  al. 1999). PKC/RACK1 interaction depends specifically on the presence of PKC activators (phospholipids, DAG and Ca2+) and suggest that RACK1 has a critical role in directing the relocation of PKC after its activation. The name RACK1 is derived from its interaction with PKC, however it is now known that RACK1 interacts with numerous proteins and plays a significant role in many fundamental physiological processes (Gandin et  al. 2013). Interestingly the peptide mimicking the site of interaction between PKC and RACK1 can be used to pharmacologically modulate in vitro and in  vivo specific PKC isozymes (Csukai and Mochly-Rosen 1999; Schechtman and Mochly-­ Rosen 2001). Another peculiar aspect of RACK1 is its transcriptional regulation. Human RACK1 is encoded by the gene rack1, localized on chromosome 5 and presenting 8 exons and 7 introns. Studies on its transcriptional regulation (Del Vecchio et al. 2009) led to the observation that the human gene has two alternative start sites of transcription (TSS) and several putative transcription factor binding sites among which Oct-1, Hand1/E47, which is implicated in the cardiomyocites differentiation, Elk-1 and Nkx2-­5, which are cardiac specific homeobox and myogenin/NF1 factor involved in muscle differentiation and growth. There are four binding sites for c-Rel, immune specific binding sites for Nuclear Factor-κB (NF-κB) transcription factor and a putative binding site for the glucocorticoid receptor (GR). We have characterized the role of cRel in the modulation of rack1 transcription (Buoso et al. 2013) and in more details we were able to demonstrate that the site identified on the rack1 gene promoter appears to be similar to the consensus for a negative glucocorticoid responsive element (GRE), at least in immune cells, suggesting a new role for corticosteroids in the regulation of inflammatory phenotype via

modulation of RACK1 expression and PKC activity (Buoso et al. 2011; Corsini et al. 2014b).

3

 KC Expression in Immune P Cells and Its Functions

PKC is central in many facets of the immune system. PKC isoforms participate in signaling cascades central to immune cell homeostasis and activation, including modulation of gene expression, proliferation, differentiation, migration and apoptosis as demonstrated using pharmacological inhibitors, ribonucleic acid (RNA) silencing and knockout mice (Altman and Kong 2016). Due to its central role in cell functions and the fine mechanisms that regulate its activation (e.g. correct spatiotemporal cellular localization, initiation and termination of PKC-­ regulated functions), PKC represents a potential target of immunotoxic compounds. In the immune system, PKC isoforms are important mediators of immune cell signaling through the immunological synapse, and directly regulating gene expression by acting as nuclear kinases (Lim et  al. 2015). Following expression and functions of PKC in the major immune cells, and some examples of immunotoxic compounds targeting PKC will be discussed. The different immune cells can express at different levels almost all PKC isotypes, i.e. all immune cells express PKCα, β, δ, ε, η, ζ, and ι/λ (Pfeifhofer-Obermair et al. 2012; Lim et al. 2015; Altman and Kong 2016), which may complicate to determine the specific cellular functions of the individual isotype, and suggests possible functional redundancy (Pfeifhofer-Obermair et al. 2012). The redundancy is also highlighted by the fact that genetic disruption of each PKC isoforms does not lead to a complete collapse of the immune system in rodents (Altman and Kong 2016). In Table 6.1 the expression of the different PKC isoforms in immune cells and their main role as showed in knockout mice. PKC isoforms are involved in the regulation of signal transduction pathways involved in both innate and adaptive immunity, leading to the expression of key genes and immune functions

6  Role of Protein Kinase C in Immune Cell Activation and Its Implication Chemical-Induced… Table 6.1  Expression of PKC isoforms in immune cells and defects in knockout mice PKC isoform PKCα

PKCβ

Expression in immune cells Ubiquitous, high in T cells, dendritic cells Ubiquitous, high in B cells and mast cells

PKCδ

Ubiquitous, high in B cells, mast cells, macrophages

PKCε

Ubiquitous

PKCη

Ubiquitous, high in T cells and macrophages

PKCθ

T cells, mast cells, monocytes, platelets

PKCζ

Ubiquitous

PKCι/λ

Ubiquitous

Knockout mouse phenotype Defects in T cell activation and T cell immunity Defects in B cell activation and survival; neutrophils and mast cells defects Defects in B cells homeostasis; proapoptotic, enhanced IL-2 production and proliferation Defects in macrophage activation and bacterial clearance Defects in T cells homeostasis and regulatory T cell functions, defective wound healing, increased skin tumor Defects in T cell activation, reduced IL-2 production, abrogated AP-1, NF-κB and NFAT transactivation, impaired Th2 immunity Defects in B cell receptor signalling, defects in Th2 responses Embryonic lethal

The table was adapted from Pfeifhofer-Obermair et  al. (2012), Lim et al. (2015), and Altman and Kong (2016). PKCγ is mainly expressed in the brain. Classical: α, β, γ. Novel: δ, θ, ε, η. Atypical: ζ, ι/λ (human/murine)

(Lim et al. 2015). Different isoforms are involved in distinct signaling pathways, with selective functions in a cell-specific manner, for example toll-like receptors (TLRs) signaling in the innate immunity and B cell receptor signaling in the adaptive immune response, both involve PKCβ activation, while, PKCθ is central in T cell immunological synapsis. While the mechanism of PKC signaling in the cytoplasm through the plasma membrane is overall well characterized, relatively less understood is the PKC signaling within the

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nucleus, despite the fact, that the ability of canonical PKC isoforms to phosphorylate histones was known since its discovery. PKC isoforms present in the nucleus are either translocated from the cytoplasm upon activation or exist constitutively within the nucleus. Within the immune system, PKC translocation to the nucleus seems to be the main mechanism in which PKC regulates immune cell differentiation upon stimulus with differentiation agonists, like macrophage colony stimulating factor, vitamin D3, trans-retinoic acid, phorbol 12-myristate 13-acetate, etc. (Lim et al. 2015). Although PKC isoforms have been detected in the nucleus of immune cells in the resting state, it is unclear how they are retained in the nucleus and whether they are structurally and functionally different from the translocated PKCs. It is postulated that PKC-binding proteins may play a role not only in the retention of PKC in the nucleus but also in the translocation of PKC into the nucleus. PKC has been shown to act as a kinase to phosphorylate histones, histone modifiers or transcription factors at specific serine and/or threonine residues leading to transcriptional activation or repression, e.g. both PKCα and PKCβ can phosphorylate histone H3 at threonine (T)6 and serine (S)10, while PKCε phosphorylates S10 only (Lim et al. 2015). In addition, PKC can also form transcriptional complexes on gene promoters. For example, PKCθ forms a transcriptional complex with RNA polymerase II (Pol II), with P300, with cRel on immune response gene promoters to lead to transcriptional activation; PKC-α, -δ, -ζ can phosphorylate p65; PKCβI is recruited to the estrogen receptor (ER) as part of a regulatory complex with Ran binding protein type and C3HC4-type zinc finger containing 1 (RBCK1) and ERα to regulate ERα promoter gene expression. Interestingly, PKC isoforms do not have canonical nuclear localization signals (NLS), however, they contain a nuclear targeting motif similar to the classical bipartite NLS, forming a putative NLS that is conserved across the different PKC isoforms, that allows nuclear translocation (reviewed in Lim et al. 2015).

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3.1

Innate Immunity

PKC inhibitors reduce LPS-stimulated cytokine secretion by monocytes/macrophages, linking The innate immune system is one of the two arms PKC activation to TLR signaling. PKCα, β, δ, ε, of the immune system found in vertebrates, the and ζ have been directly involved in multiple other being the acquired immune system. Its steps in TLR pathways (Loegering and Lennartz major functions are to recruit immune cells to 2011; Zhu et al. 2018). For example, PKCε has sites of infection or following tissue injury (sterile been shown to be an important player in both inflammation), to activate complement, to MyD88-dependent and -independent signaling phagocyte foreign materials and dead cells, to pathway during cell activation. In the MyD88-­ activate the adaptive immune system through dependent pathway, Toll–interleukin (IL)1 antigen processing and presentation. Central to receptor (TIR) domain containing adaptor the activation of innate immunity is the triggering (TIRAP) links MyD88 to the TLR4 receptor. of inflammation. Surveillance mechanisms PKCε is then recruited to TLR4 via MyD88 and involve pattern recognition receptors (PRRs) on phosphorylated on serine 346/368, which leads to the cell surface and in the cytoplasm. PRRs binding with 14-3-3β protein and the formation respond to pathogen-associated molecular of a complex with TLR4, TIRAP, MyD88 and patterns (PAMPs) or host-derived damage-­ TNF receptor-associated factor 6 (TRAF6) as associated molecular patterns (DAMPs) by well. In the MyD88-independent pathway, TRIF-­ triggering activation of NF-κB, AP-1 (activator related adaptor molecule (TRAM) is protein 1), CREB (cAMP response element-­ phosphorylated by PKCε allowing TLR4 to link binding protein), c/EBP (CCAAT/enhancer-­ with TRIF. TRIF then recruits kinases such as binding protein), and IRF transcription factors transforming growth factor activated kinase 1 (Newton and Dixit 2012). PAMPs include (TAK1) (through TRAF6), TANK binding kinase bacterial and viral nucleic acids, fungal β-glucan 1 (TBK1) and inhibitor of κB (IkB) kinase and α-mannan cell wall components, the bacterial epsilon (IKKε) for activation of NF-κB and protein flagellin, components of the peptidoglycan interferon regulatory factor (IRF) 3/7 signaling bacterial cell wall, and LPS from Gram-negative pathways to produce inflammatory cytokines bacteria. While DAMPs are endogenous (IL-1, IL-6, IL-8, TNFα) and Type I interferons molecules normally found in cells that get (Pfeifhofer-Obermair et al. 2012; Lim et al. 2015; released during necrosis and contribute to sterile Altman and Kong 2016). In neutrophils, PKCα, inflammation. DAMPs inform the body about and βhave been shown to regulate NF-κB danger, stimulate an inflammatory response, and dependent transcription both by enhancing finally promote the regeneration process. They nuclear translocation of NF-κB and also by include ATP, the allarmin cytokine IL1α, uric stimulating phosphorylation of the p65 subunit acid, the Ca2+-binding, cytoplasmic proteins (Asehnoune et al. 2005). S100A8 and S100A9, the DNA-binding, nuclear protein HMGB1, and hyaluronic acid degradation products to mention some (Vénéreau et al. 2015). 3.2 Acquired Immunity TLR4 and LPS signaling is the most studied. Following binding, it leads to the activation of Adaptive or acquired or specific immunity is two intracellular pathways: one MyD88 (myeloid defined by the presence of lymphocytes, either T differentiation primary response protein or B cells, and includes CD8+ cytotoxic T cells 88)-dependent and one MyD88-independent that are the effector cells that directly destroy (Lim et al. 2015). PKC isoforms are involved at tumor or target cells, CD4+ T helper cells (Th) many levels of the TLR signaling cascade during that regulate both CD T-cell and B-cell function, monocyte/macrophage/dendritic cell activation, and B cells that present antigen and produce also depending on the stimulus provided and the antibodies. Lymphocytes form the immunological cell type engaged. It has long been known that synapse with antigen presenting cells or upon

6  Role of Protein Kinase C in Immune Cell Activation and Its Implication Chemical-Induced…

antigen binding to the B cell receptor to activate signal transduction pathways that induce gene expression programs for lymphocyte functions.

3.2.1 T Cells Activation of T cells occurs when the T cell receptor (TCR) recognizes an antigen presented by the antigen presenting cells through MHC (major histocompatibility class) I or II. This leads to T cell differentiation in a process involving the activation of multiple pathways including PKC signaling (Lim et  al. 2015; Wieczorek et  al. 2017). In particular, a central role for PKCθ in the positive selection of thymocytes, and in the regulation of Th2-mediated immune response following TCR activation has been described (Lim et al. 2015). T cell activation occurs when the antigen presented by the antigen presenting cell on the MHC is complexed with the TCR together with the binding of costimulatory molecules CD28 and B7. This leads to the activation of Ca2+ signaling pathways and mitogen activated protein kinase (MAPK) pathways through phospholipase Cγ (PLCγ), GCK like kinase (GLK) activation and 3-phosphoinositide dependent protein kinase-1 (PDK1) activation through phosphoinositide 3 kinase (PI3K). DAG generated through PLCγ 1 activation binds to PKCθ, which is also phosphorylated by PDK1 and GLK.  Activation of PKCθ leads to the activation of NF-κB signaling pathways and gene expression programs. PKCθ appears to be particularly necessary for the development of robust immune responses that are controlled by both Th2 and Th17 cells. Besides to PKCθ, PKCα and PKCβ cooperate functionally to mediate de novo IL-2 transcriptional transactivation in response to antiCD3  in primary mouse T cells independently of the action of PKCθ, without affecting CD3-­induced transactivation of NF-κB, AP-1, NF-AT, or IL-2 mRNA stability or protein secretion (Lutz-Nicoladoni et  al. 2013). Regarding the other PKC isoforms expressed in T cells, PKCα has been also involved in IL 2 production and T cell proliferation, Th1 celldependent immune responses, IgG switch, autoimmunity and transplantation rejection; PKCβ has been involved in IL 2 production, T

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cell proliferation, and T cell migration; PKCε in preventing T cell apoptosis; PKCδ in promoting T cell apoptosis, and in contrasting IL 2 production and T cell proliferation, autoimmunity and transplant rejection; PKCζ in blocking T cell apoptosis and effector cell differentiation (Pfeifhofer-Obermair et al. 2012; Lim et al. 2015; Altman and Kong 2016).

3.2.2 B Cells B cells are activated upon antigen binding to the B cell receptor (BCR). Similar pathways to TCR are activated but the key PKC isoform involved is PKCβ. For both TCR and BCR signaling, the signaling pathways activated leads to the recruitment and activation of NF-κB, AP-1 and NF-AT (nuclear factor of activated T cells) to then produce cytokines (Lim et  al. 2015). Following the antigen binding to the BCR, a phosphorylation events by Src-family kinases as well as Syk and Bruton’s tyrosine kinase (Btk)/ Tec family kinases is initiated. This signals results in the organized assembly of kinases and adaptor proteins forming the signalosome, which in turn activates multiple signaling cascades. Among which central are secondary messengers like DAG and inositol-1,4,5-triphosphate to initiate Ca2+ and PKC downstream signaling pathways, leading to activation of transcription factors (Myc, NF-κB, AP-1). While B cells express multiple isoforms of PKC (α, β, δ, ε, η, ζ, and λ), PKCβ is the key PKC isoform that is important in BCR signaling (Lim et al. 2015).

4

 ole of PKC in Chemical-­ R Induced Immunotoxicity

4.1

Chemical Allergens

In 2005, Mizuashi et al. showed in dendritic cells (DC) that all sensitizers tested, namely nickel, formaldehyde, 2, 4-dinitrochlorobenzene, manganese chloride, and thimerosal, but none of the non-sensitizers, reduced the GSH/GSSG ratio [reduced glutathione (GSH)/oxidized (GSSG)], and induced p38 MAPK activation. The antioxidant N-acetyl-L-cysteine, which

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suppressed the reduction of the GSH/GSSG ratio, abrogated p38 MAPK activation and CD86 expression, a common marker of DC maturation induced by chemical sensitizers. Chemical-­ induced oxidation of the cell surface thiols appears to be one of the triggers of DC maturation, resulting in intracellular redox imbalance and ROS generation, mainly hydrogen peroxide or superoxide (Suzuki et  al. 2009; Kagatani et  al. 2010; Corsini et al. 2013, 2018a, b). Proteins that are reversibly modulated by ROS are of high interest. In this context, protein kinases and phosphatases have been described to be key elements in ROS-mediated signaling events. The major mechanism by which these proteins may be modified by oxidation involves the presence of key redox-sensitive cysteine residues. PKC isoforms have been shown to contain a unique structural feature that is susceptible to oxidative modification (Cosentino-­ Gomes et  al. 2012). In particular, two pairs of zinc fingers are found within the regulatory domain (sites of DAG and phorbol ester binding). Each zinc finger is composed of six cysteine residues and two zinc atoms. The high levels of cysteine residues render the regulatory domain susceptible to redox regulation (Giorgi et  al. 2010; Gopalakrishna and Jaken 2000). Oxidative stress destroys the zinc finger conformation, and the auto-inhibition is relieved, resulting in a PKC form that is catalytically active in the absence of Ca2+ or phospholipids. Currently, evidence supports the direct activation of different PKC isoforms by ROS generation (Gopalakrishna and Jaken 2000; Del Carlo and Loeser 2006). In the context of skin sensitization, PKC is of particular interest, as it has been demonstrated almost 20 years ago by Halliday and Lucas (1993) to be required for LC migration. Within the epidermis, PKCβ is exclusively expressed in LCs, and its downregulation impaired LC function with respect to contact hypersensitivity (Goodell et al. 1996). Furthermore, not only ROS can activate PKC, but PKCβ, PKCα, PKCε, and PKCζ have been involved in mitochondrial ROS production in different experimental models (Wang et al. 2006; Agudo-López et al. 2011). We recently demonstrated a central role of PKCβ and

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RACK1 in allergen-induced CD86 expression and IL-8 production in the human promyelocytic cell line THP1 as well as in primary human DC (Corsini et  al. 2014a). Dinitrochlorobenzene, p-phenylenediamine and diethylmaleate were used as contact allergens: the selective cell-­ permeable inhibitor of PKCβ and the broad PKC inhibitor GF109203X, completely prevented chemical allergen or LPS-induced CD86 expression and significantly modulated IL-8 release (50% reduction). On the contrary, the use of a RACK-1 pseudosubstrate, which directly activates PKCβ, resulted in dose-related increase in CD86 expression and IL-8 release, supporting the central role of PKCβ in the initiation of chemical allergen-induced DC activation. In addition, PKCβ is also a critical downstream signaling component of LPS-induced CD86 up-regulation, but the involvement of PKCε and possible other pathway in cytokine production was observed as well (Corsini et al. 2014a). To further investigate the role of PKCβ in DC activation, using the RACK1 pseudosubstrate we found that it induced rapid (5  min) and dose related PKCβ activation as assessed by its membrane translocation. Among the proteins phosphorylated we identified Hsp27 (Heat Shock Protein). The release of Hsp27 induced by RACK1 pseudosubstrate was also confirmed in peripheral blood mononuclear cells. Experiments of Hsp27 silencing and neutralization experiments with anti-Hsp27 antibody confirmed the central role of Hsp27  in RACK1 pseudosubstrate or LPS-induced cell activation, as assessed by IL-8 production and upregulation of CD86 (Corsini et al. 2016). Hsp27 has been associated in macrophages with upregulation and secretion of key pro- and anti-inflammatory cytokines (Salari et al. 2013). Hsp27 is a highly conserved 27  kDa stress protein, however, emerging data link Hsp27 to different signaling pathways regulating critical cellular functions, including cytokine production and immune responses (Liu et  al. 2010; Henderson and Kaiser 2013; Haslbeck and Vierling 2015). It is interesting to note that in both cases, the induction of CD86 appears more significantly affected compared to the reduction

6  Role of Protein Kinase C in Immune Cell Activation and Its Implication Chemical-Induced…

observed in IL-8 release. The induction of CD86 appears more dependent upon PKCβ activation compared to IL-8 production. Experimental evidence indicates that both TLR2 and TLR4, and NF-κB activation are involved in mediating the proinflammatory effect of extracellular HSP27 (Jin et al. 2014). The selective activation/inhibition of specific PKC isoforms may also provide novel therapeutic strategies to manipulate DC activation and contact allergy. Figure 6.1 captures the effects described.

4.2

Endocrine Disruptors

Industrialized countries are facing a significant increase in immune-mediated disorders, including allergy, autoimmunity and cancer, for

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which exposure to environmental factors, including endocrine disrupting chemicals, may provide a plausible explanation (Diamanti-­ Kandarakis et  al. 2009). Endocrine disruptor compounds (EDC) are chemicals able to interfere with the endocrine systems at different levels, including synthesis, secretion, transport, metabolism, receptor binding, or elimination of endogenous hormones. EDC are an emerging public health issue due to their potency, constant and universal human exposure. They have been associated with harmful effects in mammals, including developmental, reproductive, neurological, and immunological effects (Yang et al. 2015). We are exposed to EDCs directly or indirectly: directly through the presence of substances such as pharmaceuticals and phytoestrogens in plants; or indirectly from foods treated with EDCs like some pesticides.

Fig. 6.1  ROS and PKC and ROS in chemical allergen-­ advanced glycation end products, RACK1 receptor for induced DC activation. Abbreviations: GSH glutathione, activated C kinase 1 ARE anti-oxidant responsive element, RAGE receptor for

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Evidence is accumulating that EDC can affect the immune system, possibly resulting in adverse reactions, including immunosuppression, enhanced immunostimulation and autoimmunity (Corsini et al. 2018a). EDC can interfere with the immune system in humans and wildlife, and many have been shown to possess immunotoxic effects including immunosuppressive properties as well as the possibility to increase autoimmune reactions and enhance inflammation (Corsini et al. 2018a). The activation of PKC (assessed by CREB phosphorylation) by some EDCs [i.e. bisphenol A, endosulfan, nonylphenol, 17β-estradiol, diethylstilbestrol, 2,2-bis(p-hydroxphenyl)1,1,1-trechloroethane] has been observed (Li et  al. 2006). In addition, as mentioned above, nuclear PKC β1 has also been reported to modulate ERα transcriptional activity. RACK1 is a scaffolding protein involved in a variety of signaling pathways, controlling essential cellular processes and important biological events, including immune response, and cancer. Our published data supports the existence of a complex hormonal balance,

between glucocorticoids and androgens, in the control of RACK1 expression and immune cells activation, suggesting that RACK1 can be targeted by EDC.  Our results indicate that RACK1 could be a relevant target of EDC, responding in opposite ways to agonist or antagonist of androgen receptor (AR), representing a bridge between the endocrine system and the innate immune system (Buoso et  al. 2017a, b; Corsini et  al. 2018a). Very recently, investigating the regulation of RACK1 expression following exposure to selected estrogen active compounds, namely 17β-estradiol, diethylstilbestrol, and zearalenone, we also found that all compounds, with different kinetics, modulated RACK1 expression and, following a pretreatment with these compounds, the response to LPS was increased, confirming our working hypothesis. Interestingly, PKCmediated Hsp27 phosphorylation, mentioned above, may be also relevant in this contest, as Hsp27 plays an important role in mediating AR-mediated membrane-to-nuclear signal transduction (Li et al. 2018). In Fig. 6.2, results are summarized.

Fig. 6.2  The modulation of RACK1 could represent a key event that can bridge together steroid endocrine interference and immunotoxicity. AR androgen receptor,

DHEA dehydroepiandrosterone, DHT dihydrotestosterone, GR glucocorticoid receptor, GRE glucocorticoid responsive element

6  Role of Protein Kinase C in Immune Cell Activation and Its Implication Chemical-Induced…

5

Conclusions

PKC is a family of kinases centrally involved in intracellular signal transduction, implicated in a plethora of physiological and pathological conditions. It is not surprising that dysregulation of PKC leads to disease such as cancer, cardiovascular disease, psychiatric disorders and immune-mediated diseases. PKC isoforms can have different, and sometimes opposing effects, which complicated the understanding of their role and the possibility to use inhibitors/activators for therapeutic purposes. Specific inhibition of the interaction with substrates or anchoring proteins, and specific PKC isoforms, i.e. RACK1 and PKCβ, could pave the way for more targeted therapeutics. It is imperative to uncover how PKC function in its different roles to build on the knowledge and understanding for the development of further targeted therapeutics. This will also help in the understanding of chemical-induced immunotoxicity. PKC is central in many facets of the immune system, participating in signaling cascades involved in immune cell homeostasis and activation, including modulation of gene expression, proliferation, differentiation, migration and apoptosis, which make PKC a likely target of immunotoxic compounds with some examples presented in this chapter.

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7

Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles Effect Ayse Basak Engin

Abstract

Toxicity of metal nanoparticles (NPs) are closely associated with increasing intracellular reactive oxygen species (ROS) and the levels of pro-inflammatory mediators. However, NP interactions and surface complexation reactions alter the original toxicity of individual NPs. To date, toxicity studies on NPs have mostly been focused on individual NPs instead of the combination of several species. It is expected that the amount of industrial and highway-acquired NPs released into the environment will further increase in the near future. This raises the possibility that various types of NPs could be found in the same medium, thereby, the adverse effects of each NP either could be potentiated, inhibited or remain unaffected by the presence of the other NPs. After uptake of NPs into the human body from various routes, protein kinases pathways mediate their toxicities. In this context, family of mitogen-activated protein kinases (MAPKs) is mostly efficient. Despite each NP activates almost the same metabolic pathways, the toxicity induced by a single type of NP is A. B. Engin (*) Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey

different than the case of co-exposure to the combined NPs. The scantiness of ­toxicological data on NPs combinations displays difficulties to determine, if there is any risk associated with exposure to combined nanomaterials. Currently, in addition to mathematical analysis (Response surface methodology; RSM), the quantitative-structure-activity relationship (QSAR) is used to estimate the toxicity of various metal oxide NPs based on their physicochemical properties and levels applied. In this chapter, it is discussed whether the coexistence of multiple metal NPs alter the original toxicity of individual NP. Additionally, in the part of “Toxicity of diesel emission/exhaust particles (DEP)”, the known individual toxicity of metal NPs within the DEP is compared with the data regarding toxicity of total DEP mixture. Keywords

Metal nanoparticles (NPs) · Mitogen-­ activated protein kinase (MAPK) · Quantitative-structure-activity relationship (QSAR) · New technology diesel exhaust (NTDE) · Titanium dioxide (TiO2) · Zinc oxide (ZnO) · Silicon dioxide (SiO2) · Blood-brain barrier (BBB) · Diesel emission particles (DEP) · Cerium dioxide (CeO2)

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_7

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1

Introduction

Due to the unusual properties of nanoparticles (NPs), their potential applications in different areas are continuously expanded. Therefore, characterizing the toxicological effects of NPs have become extremely important (Dávila-Grana et  al. 2018). Moreover, because of the rapid development of this technology, it is expected that the amounts of NPs released into the environment will increase, and various types of NPs have been found simultaneously in the same medium. Analysis revealed that the combined toxicity types depend not only on their properties, but also on their exposure levels. Interactions of more than one NP and surface complexation reactions alter the original toxicity of individual NPs (Katsnelson et  al. 2015; Tong et  al. 2015). Recently, composition of NPs is grouped into five major categories by the “Nanotechnology Consumer Product Inventory (CPI)”. Of these, metal and metal oxide NPs comprise the largest nanomaterial composition group. The elemental composition of nanomaterials in the metal category is listed according to frequencies as silver (Ag), titanium (Ti), zinc (Zn), gold (Au), and other metals (magnesium (Mg), aluminum oxide (Al2O3), copper (Cu), platinum (Pt), iron (Fe) and iron oxides) (Vance et al. 2015). NPs are defined as having a size with at least one dimension 100 nanometers or less, modulate a wide range of biological reactions including immunomodulation, cytotoxicity and genotoxicity (Oberdörster 2010; Shi et al. 2013; Zolnik et al. 2010). Since nanomaterials can bind to a wide variety of substances, one of the distinctive features of industrial nanotoxicology is to carry out not only the individual ­toxicities of various metal oxide NPs, but also comparative assessments of their combined effects (Minigalieva et al. 2015; Oberdörster 2010). In this respect, one of the important results of industrial nanotechnology is the understanding of the harmful effects of the high amount of metal oxide NPs that emerge due to developments in automotive technology, and fuel composition (Gasser et  al. 2009). Preliminary toxicological data, that show marked differences in emissions and toxicity between new technology diesel

exhaust (NTDE) and traditional diesel exhaust (TDE). Compared to TDE, particulate matter (PM) levels have been reduced approximately 100-fold in NTDE, and similarly large reductions have also been achieved for numerous other diesel exhaust particles (DEPs) and gaseous species, including mutagens such as polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs. However, in NTDE elemental carbon decreases, while amount of the other metal elements increase (Hesterberg et al. 2011, 2012). The composition of NTDE is qualitatively different and the concentrations of particulate constituents are lower than TDE. However, because of the insufficient data, the evaluation of carcinogenic hazard of NTDE compared to TDE is recommended in future (McClellan et  al. 2012). DEP are composed of a carbonaceous core and adsorbed PAHs, metals, and other trace elements. DEP toxicity has been established as Group I carcinogen by The International Agency for Research on Cancer (IARC) (Bacchetta et al. 2012; Wichmann 2007). Structure/toxicity relationship analysis is conducted by using principal component analysis for a panel of NPs that included dry powders of metal oxides and DEP (Wang et  al. 2014). Metallic NP cytotoxicity consists of two determining effects as, the NP-derived effect due to its high mass-­specific surface, and the effect of the metal ions dissolved from NPs. However, both effects show ligand-dependent differences in cytotoxicity (Hahn et  al. 2012). Except for titanium dioxide (TiO2) NPs, all other metal NPs release their ions in cysteine solution. Cysteine binds to colloidal particles via thiol linkage, and it also acts as a heavy metal adsorber due to its ability to form metal ion complexes (Ding et al. 2009; Dişbudak et  al. 2002; Hahn et  al. 2012). Despite the efforts of the scientific community, the mechanisms of toxicity of NPs are still poorly understood. Nevertheless, quantitative-structureactivity relationship/quantitative-structure toxicity relationships (QSAR/QSTR) models are useful computational tools for the assessment of toxic effects of nanomaterials (Kleandrova et al. 2014a). Unified QSTR-perturbation model simultaneously evaluates ecotoxicity and cytotoxicity of NPs under different experimental

7  Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles-Effect

c­ onditions. In this context, diverse measures of toxicities, and multiple biological targets of NPs, in addition to compositions, sizes, shapes, exposure duration and coating agents of NPs are taken into consideration. This model has been created from 36,488 cases (NP-NP pairs) and obtained accuracies are higher than 98% (Kleandrova et al. 2014b). Considering the most highly used engineered nanomaterials (ENMs), nano-TiO2, and nano-zinc oxide (ZnO), the combined effects of ZnO dissolution and Zn adsorption on to nano-­ TiO2 control the concentration of dissolved zinc. Therefore, the fate and toxicity potential of soluble nano-ZnO, are influenced by the presence of nano-TiO2 (Tong et al. 2014). On the other hand, phenanthrene sorption by nano-TiO2 and nano-­ ZnO particles is enhanced significantly when coated by humic acid (HA). Thus, nano-TiO2 and nano-ZnO interact with phenolic OH, and COOH, respectively leading to different conformations of adsorbed HA. Interaction of HA phenolic OH with nano-TiO2 increases the polarizability of adsorbed HA, whereas interactions of COOH groups with nano-ZnO make the adsorbed HA be in a more condensed state with lower affinity (Yang and Xing 2009). Thereby, polarizability of the hydroxyl/metal complex, as reactivity of the particles and the cations determines cause of cytotoxic action by metal/metal oxide NPs. This classification model is used for virtual screening of toxic action of metal/metal oxide NPs (Shin et al. 2017). Although nanotechnology creates great opportunities for the progress of modern medicine, recent studies have shown evident toxicity of some NPs to living organisms. Comprehensive risk assessment is inefficient due to lack of available data and low adequacy of experimental protocols (Gajewicz et al. 2012). Thus, the QSAR method commonly used to predict the physicochemical properties of chemical compounds is applied to predict the toxicity of various metal oxide NPs (Puzyn et al. 2011). As a general evaluation form, toxicity studies on NPs have mostly been focused on individual NPs. There are very limited number of studies considering the interactions between metal oxide NP combinations and the effects of protein kinases-related pathways on their toxic-

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ity. In this chapter, whether the coexistence of multiple metal NP toxicity alter the original toxicity of individual NP is discussed.

2

Endothelial Transport of NPs

During the particle loading, endothelial cells is the first barrier of defense as come into direct contact with the particles. Adsorption of plasma proteins on the NP surface is the second important barrier of defense, which can lead to physical changes in NPs via activation of biochemical defense cascades (Karmali and Simberg 2011). When human endothelial cells were exposed to different amounts of the TiO2, silica (silicon dioxide; SiO2), Co and Ni NPs, all particle types are shown to be internalized except Ni, only Co particles possess cytotoxic effects. Furthermore, an impairment of the proliferative activity and pro-inflammatory stimulation of endothelial cells are induced by exposure to Co and, to a lesser extent, by SiO2 NPs (Peters et al. 2004). Following exposure to NPs, microvascular alterations result in disruption of the control of peripheral vascular response (Nurkiewicz et  al. 2006). The main impact of the exposure is alteration of functional complexes involved in cell adhesion, vesicular transport, and cytoskeletal structure. Those are the core cellular structures that are linked to the permeability and the integrity of the endothelial cells. In this case, endothelial cells react to preserve their semi-­permeable properties. Despite all, uptake of the extracellular proteins along with into the endothelial cells cause the NPs to escape from lysosomal degradation pathways (Kuruvilla et  al. 2019). Among three types of endothelial conformation as continuous, fenestrated, and sinusoidal, only the first two forms are selective cellular barriers to the passage of macromolecules between the circulating blood and the underlying extracellular matrix (Tuma and Hubbard 2003). In fact, an important function of the endothelium is to regulate the transport of NPs across the semi-permeable vascular endothelial barrier. Two cellular pathways have been identified controlling endothelial barrier function. These are p­aracellular pathway through

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inter-endothelial junctions, and the transcellular pathway via caveolae-­mediated vesicular transport. Src family protein tyrosine kinases contribute to the upstream signaling pathways that lead to endothelial hyperpermeability through both the paracellular and transcellular pathways (Hu et al. 2008; Komarova and Malik 2010). Different NP cargoes use different receptors that are localized to different entry sites in the plasma membrane. Consequently, at least one cargo molecule may use different entry ports such as caveolae versus clathrin-coated vesicles (Tuma and Hubbard 2003). Systemically applied NPs are quickly and simply taken up by phagocytic cells, mainly macrophages. Rest of the circulating particles is mostly internalized with clathrin-mediated endocytosis (dos Santos et  al. 2011). Micropinocytosis includes clathrin-coated vesicle endocytosis and small uncoated vesicles, whereas macropinosomes are efficient routes for non-selective endocytosis of solute macromolecules (Kirchhausen et  al. 2005; Swanson and Watts 1995). Transmembrane proteins are selectively accumulated in clathrin-coated pits at the plasma membrane and rapidly internalized in clathrin-coated vesicles (Sorkin 2004). ZnO NPs may impair endothelial cells and induce endothelial barrier dysfunction and are able to cross the endothelial barrier through paracellular and transcellular routes (Wu et  al. 2019). However, the protein corona affects the transcytosis of NPs, and their uptake by caveolae-mediated endocytosis does not necessarily lead to transcytosis (Ho et al. 2018). Gold nanoparticles (AuNPs) cause a pronounced reduction of protein kinase C, zeta (PKCζ)-dependent threonine phosphorylation of occludin and zonula occludens-1 (ZO-1), which results in the instability of endothelial tight junctions (TJs) and leads to proteasome-mediated degradation of TJ components. This impairment in the assembly of TJs between endothelial cells increase the permeability of the transendothelial paracellular passage and the blood–brain barrier (BBB). AuNPs increase endothelial paracellular permeability of BBB (Li et al. 2015). Similarly, SiO2 NPs could disturb BBB structure and function causing paracellular opening, and induce BBB inflammation, and these effects occur

A. B. Engin

through reactive oxygen species (ROS) and Rho-­ associated protein kinase (ROCK)-mediated pathways (Liu et al. 2017). As a special type of NP carriage, receptor-mediated transcytosis (RMT) facilitates the transport of nanomedicine across the BBB in protein-directed manner. Eleven proteins, including transthyretin (Ttr), and creatine kinase-muscle type (CKM), are identified as being capable of penetrating the endothelial cell layer by RMT.  Transcytosis of Ttr-quantum dot (QD) conjugates is a new putative transporter for nanomedicines across the BBB (Kim et al. 2015). Theaflavins (TFs) could enhance intestinal barrier function by increasing the expression of TJ-related proteins through the activation of 5′ adenosine monophosphate (AMP)-activated protein kinase (AMPK) (Park et al. 2015). AuNPs and an acid-cleavable linkage between the TF are designed to bind the NPs to transferrin receptors (TFRs). This NP core facilitates the NP RMT across the BBB. TF-TFR complex release the NP into the brain upon acidification during the transcytosis process (Clark and Davis 2015). In addition, if TF is conjugated with AuNPs to form a nanoconjugate AuNP-TFQ (quinone motif), its apoptotic ability increases significantly in comparison to the bare TF.  The presence of quinone motif in this complex induces ROS generation through the depolarization of mitochondria and results in the caspase-­ mediated apoptotic cell death (Maity et al. 2019). Once in contact with biological fluids, the NP surface is covered by a highly specific layer of proteins, forming the NP protein “corona”. This protein layer can impact on particle uptake and trafficking inside the cells (Lesniak et al. 2010). The corona confers biological identity to the NPs and interacts with the cellular machinery. Thereby it designates the NP destiny (Hellstrand et  al. 2009). Positively and negatively charged NPs bind with a wide range of proteins whereas neutral NP binds very little. The larger NPs bind fibrinogen with increasing affinity and a slower dissociation rate. Thereby, the changes in NP size can influence protein binding (Deng et  al. 2012, 2013). Thus, p38 mitogen-activated protein kinases (MAPKs) which are ­stress-­activated kinases are recently shown to be induced by pos-

7  Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles-Effect

itively charged mesoporous silica NPs, whereas negatively charged NPs induce ROS (T.-P.  Liu et  al. 2015b). There is a dynamic interaction between NPs and biomolecular species and other chemical and organic matter which ultimately results in biological corona formation. Conformational structure of the bio-corona, which is critically dependent on intrinsic NP properties, however, may also induce alterations in extracellular matrix (ECM) nature (Neagu et al. 2017). In addition, uptake of NP by endothelial cell, and NP-related endothelial damage is affected through multiple protein kinases activities. In fact, redox-active NPs can induce ROS production alone. When induction of oxidative stress of the purified and unpurified multi-walled carbon nanotubes are compared, unpurified multi-walled carbon nanotubes, which contains multiple metal oxides produce twice as high levels of ROS than the purified ones (Vitkina et al. 2016). Thus, oxidative DNA damage induced by multi-walled carbon nanotubes, and decreased mitochondrial function is thought to be dependent on the release of endogenous redox-active metal impurities such as iron NPs (Visalli et al. 2017). Nickel NPs (NiNPs), which are frequently present in multi-­ walled carbon nanotubes induce cyclooxygenase-­2 (COX-2) expression in macrophages via extracellular signal-related kinases 1/2 (ERK1/2) (Lee et al. 2012). NiCl2 amplifies the expression of pro-inflammatory mediators, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-­γ), interleukin 1beta (IL-1β), IL-6, IL-8 and IL-18, via activating the COX-2, and inducible nitric oxide synthase (iNOS), in addition to increasing mRNA expression levels of the transcription factor, the nuclear factor kappa-light-­chain-enhancer of activated B cells (NF-κB) (Deng et al. 2016). In this case, firstly, the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) pathway is activated by increasing eukaryotic initiation factor 2 alpha (eIF2α) and activating transcription factor 4 (ATF4) mRNA expression. Secondly, the inositol-­requiring protein 1 (IRE1) pathway is activated due to increase in IRE1 and X-box binding protein 1 (XBP1) mRNA expression. And thirdly, ATF6 mRNA expression increases due

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to ATF6 pathway activation. Thereby, endoplasmic reticulum (ER) stress is induced (Hongrui Guo et  al. 2016b). Actually, carbon black basically exhibits more potent toxicity by triggering ER stress in endothelial cells compared with DEP, which might be attributed to its less oxygen content (Wang et al. 2019). Similarly, SiO2 NPs could also induce dysfunction of endothelial cells through oxidative stress via c-Jun N-terminal kinase (JNK), p53 and NF-κB pathways, increase B-cell leukemia/lymphoma-2 (Bcl-2) associated protein x (Bax) expression and suppress Bcl-2 protein suggesting that exposure to SiO2 NPs may be a significant risk for the development of cardiovascular diseases such as atherosclerosis (Liu and Sun 2010). The downregulation of cellular adhesion molecule expression is involved in the disruption of endothelial cell homeostasis. The vascular endothelial growth factor (VEGF) receptor2 (VEGFR2)/Phosphoinositide3kinase (PI3K)/Protein kinase B (Akt)/Mammalian target of rapamycin (mTOR) and VEGFR2/MAPK/ ERK1/2/mTOR signaling pathways are involved in the cardiovascular toxicity triggered by SiO2 NPs (Duan et  al. 2014a). Thereby, inhibitory effect of SiO2 NPs on the PI3K/Akt/mTOR signaling pathway disturbs nitric oxide (NO)/ endothelial nitric oxide synthase (eNOS) system, induces inflammatory response, activates autophagy, and eventually leads to endothelial dysfunction (Duan et  al. 2014b). In a similar fashion, the exposure to TiO2 NPs increase cellular oxidative stress and DNA binding of NF-κB.  Phosphorylation of Akt, ERK1/2, JNK and p38 is increased in TiO2 exposed endothelial cells. Thus, TiO2 NPs induce endothelial inflammatory responses via redox-sensitive cellular signaling pathways (Han et al. 2013). Ultrafine cobalt (Co) NPs together with TiO2 NPs are very rapidly internalized, and significantly increase adhesion molecule (intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin) mRNA and protein levels and the release of monocyte chemoattractant protein-1 (MCP-1) and IL-8. These two NPs have significant differences between the extent of oxidative ­ stress-­ related enzyme and vascular adhesion molecule expres-

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sion, ROS production, and pro-­ inflammatory cytokine release despite the similar rate of NP internalization (Alinovi et al. 2015). The increase of intracellular ROS and catalase (CAT) activity is one common effect of NPs. When the endothelial cell layer is exposed to NPs including Au, Pt, SiO2, TiO2, ferric oxide (Fe2O3), oxidized multi-walled carbon nanotubes, with different surface chemistry and size, the NPs increase the level of intracellular ROS and the CAT activity to different degrees (Wen et  al. 2019). The Fe2O3, yttrium oxide (Y2O3), and ZnO NPs are internalized by human endothelial cells and are often found within intracellular vesicles, but Fe2O3 and cerium dioxide (ceria; CeO2) NPs do not provoke any inflammatory response at any of the concentrations (Kennedy et al. 2009). Thus, Gojova et al. found that, exposure of endothelial cells to Y2O3 or ZnO NPs significantly increase mRNA levels of the inflammatory markers IL-8, ICAM1, and MCP-­1, whereas Fe2O3 particles have no effect (Gojova et al. 2007). Furthermore, mRNA up-regulation is higher for ZnO than for Y2O3. Metal oxide NP types and composition influence the inflammatory response in endothelial cells. In addition, endothelial cell inflammation appears to correlate inversely with NP-specific surface area. Thus, Fe2O3 NPs, which have the largest specific surface area, fail to induce inflammation, whereas ZnO NPs have the smallest specific surface area and provoke the most pronounced inflammatory response (Gojova et  al. 2007). However, different findings have been obtained in this context. According to Zhu et al. iron oxide (Fe2O3 and Fe3O4) NPs induce cytoplasmic vacuolation, mitochondrial swelling and cell death in human endothelial cells. A simultaneous increase in NO production is evident due to coincidental elevation of endothelial eNOS activity. Moreover, phagocytosis and dissolution of NPs by monocytes provoke oxidative stress and mediate severe endothelial toxicity (Zhu et al. 2011). Contrarily, in chronic exposure to lead (Pb) the eNOS protein is unaffected, whereas the inducible iNOS protein is increased. Thus, increase in synthesis of NO and superoxide anions results in the uncoupling of eNOS and accumulation of reactive nitrogen species (RNS) (Gonick et  al.

1997; Vaziri et  al. 2001). Interestingly, a novel mechanism is described for the permissive role of fatty acid on iron intracellular translocation and subsequent oxidative injury. Although iron alone has little effect on endothelial cells, in combination with palmitic acid, iron-mediated toxicity is markedly potentiated, as reflected in mitochondrial dysfunction and apoptosis. So palmitic acid facilitates iron translocation into endothelial cells and causes intracellular iron overload and over-­ generation of ROS (Yao et al. 2005).

3

 rotein Kinase Mediated P Toxicity of NPs

After uptake of NPs into the human body from various routes, their toxicities occur largely through protein kinases pathways. As a family of serine/threonine-specific kinases MAPKs, including the ERK1/2, p38 MAPK, and JNK have important role in NPs toxicity. These are also called stress-activated protein kinases (SAPKs). SAPK transduces extracellular stimuli and are involved in the regulation of cell proliferation, gene expression, and apoptosis. Despite each NP activates the same metabolic pathways, the toxicity induced by a single type of NP can be different than the case of co-exposure to the combined NPs (Dávila-Grana et  al. 2018; Guyton et  al. 1996). However, previous work has indicated that several metals, including arsenic (As), chromium (Cr), Cu, Fe, Ni, vanadium (V), and Zn each can induce the phosphorylation of the MAPKs, such as ERK, JNK, and p38 (Samet et al. 1998). Thus, cellular response induced by different metal oxide NPs results in activation of MAPKs and NF-κB pathways. As mentioned above, ZnO NPs simultaneously activate p38 and SAPK/JNK. CeO2 and TiO2 NPs together activate ERK1/2 and p38, while Al2O3 NPs induce dose-­ dependently ERK1/2 activation only (Simón-­ Vázquez et  al. 2016) (Fig.  7.1). The toxicity induced by the ZnO NPs is mostly due to ion release. However, novel coating strategies reduce ZnO NP dissolution. In contrast, CeO2 NPs are taken up into intact caveolin-1 without inflammation or cytotoxicity. Consequently, CeO2 NPs

7  Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles-Effect

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Fig. 7.1  Coexistence of multiple metal NPs alters the original toxicity of individual NPs. The metal NPs-rich PM has stronger ability to generate ROS and to induce proinflammatory mediators release. The phosphorylation of p38 MAPK induced by metal NPs is shown to be critically important for the increases in inflammatory media. NPs, while increasing ROS production, significantly reduce the antioxidant capacity. The phosphorylation levels of ERK1/2, JNK and p38 MAPK are significantly altered after exposure to NPs. Thereby, cell death is triggered in human cells via oxidative stress-induced activation of pMAPK and PI3K/Akt/ CREB/Bcl-2 signaling pathways. The increase in Ca2+ influx activates NF-κB, ERK1/2 and p38 signaling pathway, cause the release of proinflammatory cytokines, and inflammation. Exposure to NPs leads to the simultaneous activation of TLR/MyD88, which triggers inflammation and cell death via IKK/NF-κB pathway. Furthermore, NPs increase ERK1/2 phosphorylation, and ROS generation via promoting NADPH oxidase activity. (Abbreviations: AhR: Aryl hydrocarbon receptor, Akt: Serine/threonine protein kinase B, ATF6: Activating transcription factor 6,

Bcl-2: B-cell lymphoma 2 protein, CREB: cAMP response element-binding protein, ER: Endoplasmic reticulum, ERK1/2: Extracellular signal-regulated kinase, HO-1: heme oxygenase 1, transcription of the antioxidative enzyme, IκB: Inhibitors of NF-kB, IκK: Inhibitor kappa B kinase, IL: Interleukin, IL-1β: Interleukin-1 beta, IL-8: Interleukin-8, IL-6: Interleukin-6, IRE1: Inositol-requiring protein 1, JNK: c-Jun N-terminal protein kinase, M1: Macrophage, MAPK: mitogen activated protein kinase, MyD88: Myeloid differentiation primary response gene 88, NFκB: Nuclear factor-­ kappa B, NO: Nitric oxide, NOX: Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NPs: nanoparticles, Nrf2: nuclear factor erythroid 2-related factor 2, ONOO: Peroxynitrite, p: Phosphorylated, p47phox: A key subunit of NADPH oxidase, PDGF-B: Platelet derived growth factor subunit B, PERK: Protein kinase RNA-like endoplasmic reticulum kinase, PI3K: Phosphatidylinositol 3-kinase, PM: Particulate matter, RNS: Reactive nitrogen species, ROS: Reactive oxygen species, TLR: Tolllike receptor, TNF-α: tumor necrosis factor alpha, UPR: Unfolded protein response)

suppress ROS production and increase cellular resistance to an exogenous source of oxidative stress (Buerki-Thurnherr et  al. 2013; Xia et  al. 2008). MAPK activation induced by Zn2+ ions involve the same pattern of p38 and SAPK/JNK phosphorylation at high concentrations. On the other hand, all metal oxide NPs can activate NF-κB, a pathway that is mainly related with inflammation and the immune response. Thus, the upregulation of IL-8 by Al2O3 and ZnO NPs is

due to activation of the NF-κB pathway (Simón-­ Vázquez et  al. 2016). In addition, metal oxide NPs (ZnO, CeO2, TiO2 and Al2O3) induce the changes in the expression levels of adhesion molecules and the C-X-C chemokine receptor type 4 (CXCR4) (Lozano-Fernández et  al. 2014). The TiO2 NPs significantly decrease Ca2+-ATPase, Ca2+/Mg2+-ATPase and Na+/K+-ATPase activities. The low-dose and long-term exposure to TiO2 NPs cause cardiac damage by increasing Ca2+/

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calmodulin-dependent protein kinase II (CAMKII) and α1/β1- adrenergic receptor (AR) expression and up-regulating protein kinase C epsilon type (PKCε) and phospho-ERK 1/2 (p-ERK1/2) in a dose-dependent manner (Yu et al. 2016). So, apoptotic and necrotic death due to TiO2 NPs are associated with oxidative stress and NF-κB pathway activation (Montiel-Dávalos et al. 2012). Whereas, SiNPs induce apoptosis in a different way. The surface of SiO2 contains a lot of hydroxyl radicals, which induce the ROS generation and oxidative damage in cells. Thus, SiO2 trigger apoptosis via PI3K/Akt/cAMP response element-binding protein (CREB)/Bcl-2 signaling pathway (Napierska et al. 2010; Zou et al. 2016). In fact, the classic signaling pathway of PI3K/ Akt regulates pro-survival proteins, NF-κB, CREB and Bcl-2  in addition to pro-­ apoptotic proteins. This requires phosphorylating activity of both the ERK and PI3K pathways. Both ERK and PI3K pathways simultaneously regulate p90 ribosomal S6 kinase (RSK). Subsequently, through RSK-induced transactivational activity of CREB, Bcl-2 transcription is regulated (Creson et al. 2009). Following environmental exposure, amorphous SiO2 (SiNPs)-induced oxidative stress results in endothelial injury. SiNPs-induced mitochondrial dysfunction is characterized by membrane potential collapse, and elevated Bax ultimately leads to apoptosis. While phosphorylated ERK, PI3K, Akt, and mTOR significantly decrease, phosphorylated JNK and p38 activated MAPK increase in SiNPs-exposed endothelial cells (Caixia Guo et al. 2016a). Moreover, SiO2-­ NPs can disturb the BBB structure, and function by inducing BBB inflammation. Thus, SiO2 NPs cause the BBB paracellular opening, oxidative stress and astrocyte activation in brains. These effects occur through ROS and ROCK-mediated pathways (Liu et  al. 2017). On the other hand, SiO2 NPs exposure activate ERK1/2 and JNK in the frontal cortex of mice. Increased tau phosphorylation, and neuroinflammation results in mood dysfunction and cognitive impairment in addition to synaptic changes via ERK activation (You et  al. 2018). The activation of NF-κB and MAPK/Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) pathway following NP exposure

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increase the expression of TNF-α, IL-1β, and IL-6, in the frontal cortex, corpus striatum, and hippocampus (Guo et  al. 2015; Parveen et  al. 2017). Like SiNPs, SiO NPs also lead to strong ER stress and unfolded protein response (UPR) induction, oxidative stress, activation of MAPK signaling and down-­regulation of p53 in human (Christen et  al. 2014). Whereas, NPs suspensions, which consist of C60 fullerene, TiO2 and SiO2 form agglomerates of different sizes for different types of NPs. The lack of a major toxic effect, although all NP suspensions induce concentration-dependent lysozyme release, extracellular oxyradical and NO production, the inflammatory effects of NPs shown to be dependent on rapid activation of the stress-activated p38 MAPK (Canesi et al. 2010). On the other hand, powder mixtures of tungsten carbide and metallic cobalt (WC-Co) are widely used in various products. Inhalation of WC-Co dust is known to cause “hard metal lung disease” and an increased risk of lung cancer. Exposure to WC-Co NPs induces angiogenic response by activating ROS, Akt, and ERK1/2 signaling pathways (Armstead and Li 2016; L.-Z. Liu et al. 2015a). Cadmium NPs (CdNPs) increase ROS production and the ROS-producing reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Cd translocates the p47(phox), a key subunit of NADPH oxidase, to the cell membrane. Cd induces matrix metallopeptidase 9 (MMP-9) expression via ROS-­ dependent epidermal growth factor receptor (EGFR)/Erk1/2, JNK1/2/Activator protein 1 (AP-1) and EGFR/Akt/NF-κB signaling pathways. Activation of these pathways subsequently facilitates the translocation of CdNPs in human endothelial cells (Lian et al. 2015). Despite there are not many studies, regarding the comparison of potential hazardous effects of three types of engineered metal NPs, Ag, CuO and ZnO NPs, they are applied as antibacterial agents against microorganisms. While Ag NPs are traditionally and most widely used as antimicrobial, CuO and ZnO NPs have been successfully used as biocides. Both NPs have negative surface charge, which results from oxygen atoms in CuO and ZnO (Bondarenko et  al. 2013; Xu et  al. 2012).

7  Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles-Effect

Although AgNPs do not initially contain oxygen, the surface of metallic AgNPs is oxidized under most environmental conditions and negatively charged hydroxo and oxo groups cause the negative surface charge of the particle (Levard et al. 2012). According to CPI, AgNPs are the most common metal in the metal and metal oxide category. AgNPs, with a smaller size and higher reactive activity, may induce much higher toxicity to endothelial cells compared with PM. AgNPs induce the injury and dysfunction of endothelial cells through the activation of NF-κB inhibitor (IκB) kinase (IKK)/NF-κB, which is associated with oxidative stress, suggesting that exposure to AgNPs may be a potential hazardous factor for early atherosclerosis (Shi et  al. 2014). Thus, AgNPs are taken up by vascular endothelial cells and induce intracellular ROS generation, which disrupts the integrity of endothelial layer (Hua Guo et al. 2016c). In this context, AgNPs induce a number of signature ER stress markers, including phosphorylation of RNA-dependent PERK and its downstream IeF2α, phosphorylation of IRE1, splicing of ER stress-specific X-box transcription factor-1, XBP1, cleavage of ATF6 and up-regulation of glucose-regulated protein-78 (GRP78) and CCAAT/enhancer-binding protein-­ homologous protein (CHOP/ growth arrest and DNA damage 153 (GADD153)) (Zhang et  al. 2012). Toxicologically, the most important joint property is that all the three NPs are soluble to some extent in aqueous media (Ivask et al. 2010). Three major phenomena driving the toxicity of these kinds of NPs consist of dissolution of NPs, cellular uptake of NPs and induction of oxidative stress and consequent cellular damages (Ivask et al. 2014). In addition, surface coating of metal oxide NPs modifies their toxicity. Nevertheless, the same coating molecules may enhance or reduce the toxic effects depending on their initial surface properties. The final toxicity reflects physicochemical modifications obtained upon coating such as changes in particle aggregation state, dissolution, zeta potential, and ion and free radical releasing to a solution (Stankic et  al. 2016). As mentioned above, NP interactions alter the original toxicity of individual NPs. ZnO NPs alone cause ER damage, neuroinflammation and

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vascular inflammation via protein kinases pathways. Thus, exposure of ZnO NPs alters the architecture of ER and mitochondria. ER-mitochondria encounter structure complex ERMES causes cellular toxicity due to lipid disequilibrium and proteostasis. In addition, exposure to these NPs activates the MAPKs of cell wall integrity (CWI), the MAPK of this pathway (Mpk1) and high-osmolarity glycerol (HOG) (the MAPK of the HOG-pathway; Hog1) pathways (Babele et al. 2018). ZnO NPs mediated neuroinflammation is induced via the Ca2+-dependent NF-κB, ERK and p38 activation pathways. In this context, ZnO NPs increase the number of autophagosomes and autophagy marker proteins such as microtubule-associated protein 1 light chain 3-isoform II (MAP-LC3-II) and Beclin 1 and lead to apoptotic and autophagic cell death. Phosphorylated Akt, PI3K and mTOR are significantly decreased during the ZnO NPs exposure (Liang et  al. 2018; Roy et  al. 2014). ZnO NPs treatment causes the activation of Ras-related C3 botulinum toxin substrate 1 (Rac1)/cell division control protein 42 homolog (Cdc42) and protein accumulation of mixed lineage kinase 3 (MLK3). The increase of ICAM-1 expression through Rac1/Cdc42-MLK3-JNK-c-Jun signaling pathway in ZnO NPs-treated endothelial cells result in vascular inflammation (Li et  al. 2012). However, exposure of human cells to ZnO NPs in combination with different concentrations of Al2O3, CeO2, TiO2 and Y2O3 NPs result in synergistic or antagonistic effects on the cell death in comparison with the exposure to ZnO NPs alone (Dávila-Grana et al. 2018). The inhibitory effect of ZnO NPs on bacterial ATP levels is reduced somewhat, depending on adsorption of Zn2+ by nano-TiO2. This means that, interaction of NPs, and surface complexation reactions alter the original toxicity of individual NPs. Thereby, comprehensive assessments of complex physicochemical interactions between toxicity of ENMs and various biological responses should be made (Tong et al. 2015). Thus, the combined ecological toxicity of TiO2 NPs and heavy metals is different when compared with their individual effects. The combined toxicity of nano-TiO2 and Cd2+ to microorganisms in absence of surfactant is antag-

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onistic because of the adsorption of Cd2+ to nanoTiO2 particles. However, in the presence of surfactant, such as sodium dodecyl benzene sulfonate the toxicity of Cd2+ is decreased or increased dose-­ dependently by TiO2 NPs (Li et  al. 2018). Additionally, when combined with Cd, TiO2 NPs reduce the metal-induced effects by significantly lowering ABC transport proteins, metallothioneins in abcb1 gene transcription, and DNA damage, whereas, enhanced effects are observed on NO production (Della Torre et  al. 2015). Both TiO2 NPs and Fe3O4/TiO2 NPs exposure induce apoptosis, inflammation, and carcinogenesis related signal protein alterations. When compared to TiO2 NPs alone, Fe3O4/TiO2 NPs induce higher signal protein expressions (Su et al. 2018). NiO NPs exposure alone causes both genotoxicity and ER stress-mediated apoptosis via protein kinases pathways. In this respect, NiO NPs exposure contribute to the increased protein contents of IRE-1α, PERK, eIF-2α, and their phosphorylated forms. These indicate that NiONPs can activate the pathways of ER stress-mediated apoptosis (Chang et al. 2017). In addition, NiO-­ NPs induce significant increase in chromosomal aberrations, micronuclei formation and, DNA damage. Thereby, NiO NPs induced toxicity is through cyto-genetic alterations, oxidative stress, apoptosis and liver toxicity. Mechanistically, NiO NPs toxicity is a result of the p­53/mitogen-­ activated protein kinase-activated protein kinase 2 (MAPKAPK-2) signaling via activation of caspases 8, 3, cyto c, pro-apoptotic bax and anti-­ apoptotic bcl-2 proteins (Saquib et al. 2017). It is well-known that, different forms of the same NP show different toxic effects. Despite differences in their uptake, NiNPs, NiO NPs and NiCl2 NPs exposures cause chromosomal damage. Of these, NiO NPs are most potent in causing DNA strand breaks and generating intracellular ROS.  However, the modulation of intracellular calcium by using inhibitors and chelators clearly prevents the chromosomal damage. Whereas, chelation of iron protects against NiO- and NiCl2induced toxicity only (Di Bucchianico et  al. 2018). Moreover, the comparative solubility of the Mn3O 4NPs and NiO NPs in a biological

medium is one of the important factors underlying the difference in their toxicokinetics and their degree of damage to neurons (Minigalieva et al. 2015).

4

 oxicity of Diesel Emission T Particles

Measurements of fractal dimensions of diesel emissions showed that about 60% of total number of particles are in the size range of 50–70 nm and 34% are in the size range of 75–120  nm (Xiong and Friedlander 2001). Although several studies have been conducted on the toxicity of metal oxide NPs, the aspect regarding their potential interactions with DEP, has still been poorly investigated. In vivo and in vitro studies report that DEP induces changes in markers of inflammation, including cytokines. Through interactions with membranous and cytosolic receptors, ion channels, and phosphorylation enzymes, molecules in the DEP trigger various cell signaling pathways, that may lead to the release of inflammatory markers directly or indirectly by causing cell death (Schwarze et  al. 2013). DEP, which is the major content of PAHs and metals, induce oxidative stress and inflammation. However, different DEPs induce different effects on the base of their PAHs or metals composition. Furthermore, expression of adhesion molecules by DEP results in endothelial cell activation, and the release of inflammatory markers from endothelial cells (Bengalli et al. 2019). Since in the real conditions the population is exposed to a mixture of different particles within DEP, it is thought that the co-exposure to these mixtures can lead to a different toxicity compared to the well-known biological responses induced by the single compounds (Zerboni et al. 2019). Cu2+ ions release is reduced by the presence of DEP. Thereby corresponding cytotoxicity of mixture decreases when compared with the exposure to CuO NPs alone (Zerboni et al. 2019). Ultrafine particles (UFPs) have an aerodynamic size of less than 100  nm. These are potentially most dangerous due to their small size, large surface area, deep penetration ability, and high con-

7  Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles-Effect

tent of redox cycling organic chemicals (Nel et al. 2006). These particles deposit in the alveolar region with much higher efficiency due to their strong diffusion capability (Heal et  al. 2012). Lipophilic fractions of DEP translocate through alveolar epithelial cells and trigger pro-­ inflammatory reactions in endothelial cells. Consequently, DEP contributes to endothelial dysfunction via oxidative stress through the generation of superoxide radicals and peroxynitrites that reduces the bioavailability of endothelium-­ derived NO (Brinchmann et  al. 2018; Miller et al. 2009). Furthermore, DEP increases apoptosis of endothelial progenitor cells (EPCs). The reduction in EPCs is associated with impaired neoangiogenesis and a marked increase in atherosclerotic lesion formation (Pöss et al. 2013). In fact, the toxic potential of various sizes of particles is correlated to their chemical composition and their capacity to induce oxidative stress. Organic DEP extracts induce oxidative stress response, leading to heme oxygenase 1 (HO-1) expression at normal reduced glutathione (GSH)/ oxidised glutathione (GSSG) ratios. At intermediary oxidative stress levels, increasing in Jun kinase activation, and IL-8 production results in cellular apoptosis in parallel with a sharp decline in GSH/GSSG ratios (Li et  al. 2002a, 2003). HO-1 is induced at low dose of DEP and with minimal decline in the GSH/GSSG ratios, whereas MAPK activation requires a higher dose of DEP and increasing levels of oxidative stress (Li et  al. 2002a; Xiao et  al. 2003). Although a strong correlation exists between the PM content of redox active chemicals and their capability to induce oxidative stress in macrophages and bronchial epithelial cells, the intracellular localization of the particles also play an important role in ROS production (Li et al. 2003; Xia et al. 2004). UFPs emitted from diesel engines stimulate cytosolic and mitochondrial superoxide production and increase the expression of oxidative response genes. UFP-induced generation of cellular ROS is mediated by JNK activation in endothelial cells (Li et al. 2009). The composition of UFPs from diesel emissions is highly heterogeneous and composed of primary and secondary particles. While primary particles are comprised of

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carbonaceous cores, with aromatic hydrocarbons, quinones and organometallic additives, the secondary component is formed in the atmosphere by condensing sulfuric acid and sulfates (Geller et al. 2005; Xiong and Friedlander 2001). However, the mortality caused by ischemic heart disease is significantly associated with elemental carbon and metal contents of UFPs (Kreyling et  al. 2002; Ostro et  al. 2015). In addition, the small size of UFPs allows to evade their clearance from the medium, leading to long-term particle accumulation. UFPs are significant contributors to the deposition of PAHs into the alveolar region of the lung. Actually, CD11c+ antigen presenting cells (APCs) derived from the alveolar space and lung parenchyma express differential co-stimulatory molecules and cytokines, upon stimulation with UFPs (Kawanaka et al. 2009; Kugathasan et al. 2008). Ambient particulate matter contains redox-active transition metals, redox-active quinones and polycyclic and halogenated PAHs which act synergistically to produce ROS.  Firstly, the cytotoxic and carcinogenic potential of PM is the result of redox cycling of persistent quinoid radicals, which generate large amounts of ROS. Secondly, the water-soluble fraction of PM elicits DNA damage via reactive transition metal-­ dependent formation of hydroxyl radicals, which play an important role for hydrogen peroxide production. These data indicate the importance of mechanisms involving redox cycling of quinones and Fenton-type reactions by transition metals in the generation of ROS (Valavanidis et  al. 2005). Thus, redox-active organic chemicals, PAHs and quinones are the major contributors to UFP-­generated ROS. Similar to ambient UFP, some engineered NPs are capable of abiotic ROS generation. Transition metals like Fe2+ on carbon nanotube or other metal NPs can generate superoxide via Fenton reaction. Thus, similar to ambient UFP, ROS generation by NPs lead to protein, lipids and membrane damage. The magnitude, localization and site of tissue injury depends on where the exposure to the NPs takes place (Jeng 2010; Li et  al. 2008; Lodovici and Bigagli 2011; Nel et  al. 2006). ROS accumulation as a result of either overproduction of super-

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oxides or inadequate antioxidant defense leads to oxidative stress (Cho and Kleeberger 2010). Nrf2, a transcription factor protects cells against the damaging effects of ROS via mediating the antioxidant and detoxification enzymes. Kelchlike ECH-­ associated protein 1 (Keap1) suppresses cellular Nrf2 in cytoplasm and drives its proteasomal degradation (Cho and Kleeberger 2010). UFPs enhance early atherosclerosis, partly due to their high content in redox cycling chemicals and their ability to synergize with known proatherogenic mediators in the promotion of tissue oxidative stress. Three subgroups of MAPKs are involved in both cell growth and cell death therefore, these protein kinases have paramount importance in determining cell fate. ROS activates ERKs, JNKs, and p38 MAPKs, but the mechanisms by which ROS can activate these kinases are unclear. However, it is known that GSH biosynthesis and particularly the regulation of transcription factor Nrf2 by GSH and downstream signaling during oxidative stress and inflammation in various pulmonary diseases are important (Araujo and Nel 2009; Biswas and Rahman 2009). As mentioned above, failure of cells to restore redox homeostasis induce airway inflammation. The epidermal growth factor receptor as well transition metal-­induced signaling is a common mediator for all NPs, whereas integrin-dependent Akt and ERK1/2 activation is particle-specific. Thus, particle-­induced intracellular oxidative stress is required for Akt and ERK1/2 activation (Weissenberg et  al. 2010). DEP extract demonstrates that quinones and PAHs are representative organic chemical groups that could contribute to the oxidant injury in the lung (Kumagai et al. 1997; Li et al. 2008). PAHs can be converted to quinones via biotransformation, through reactions involving cytochrome P450 1A1, epoxide hydrolase and dihydrodiol dehydrogenase (Penning et al. 1999). In addition to ROS, PM containing Fe, Ni, Cu, Co, and Cr metals are responsible for production and release of inflammatory mediators by the respiratory tract ­epithelium. Evidences indicate that these metals may contribute to the toxic effects of particulate air pollutants (Carter et  al. 1997). Of these, the exposure to nickel sulfate results in the

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induction of hypoxia-inducible genes and IL-8 production. The simultaneous exposure to iron in either ferric or ferrous form together with nickel, completely inhibit IL-8 production. In this condition, two different pathways involve in the induction of “hypoxia-like” stress and IL-8 production. Nickel can induce different signaling pathways with or without interference with iron metabolism (Salnikow et al. 2004). Among the transition metal ions, particularly iron, are present at low levels in biological systems. Whereas, this pathway, which is referred as the iron-catalyzed Haber-Weiss reaction generates the highly reactive hydroxyl radicals (Kehrer 2000). The DEP with the highest PAH and lowest metal content is more potent with respect to cytotoxicity and the expression and release of proinflammatory mediators, in comparison to the DEP-sample with lower PAH and higher metal content. Thus, the DEP-sample with the highest PAH and lowest metal content possesses a greater oxidative potential (Totlandsdal et al. 2015). Further analyses of the principal components indicated that while Fe and Si in the PM is correlated with IL-6 release, Cr is correlated with IL-8 increase (Becker et  al. 2005). Chemical species of the trace metals in PM exhibit significant differences, due to difference in sources of pollution. Traffic emissions and industrial activities are determined to be the two main sources of heavy metal pollution. However, higher potential health risks are posed by combined effects of Cu, Zn, Cd and Pb metals (Hou et al. 2019). Because of the metal contaminations, DEP pollutants show harmful effects on immunological pathways in macrophages that may mediate the development of pulmonary and systemic vascular effects (Bhetraratana et  al. 2019). Short-term exposure to highly concentrated ambient particles (CAPs) lead to significant increase of thiobarbituric acid reactive substances (TBARS) and oxidized proteins in rat lung indicating the presence of oxidative stress. This is accompanied by increased ­polymorphonuclear cells (PMN) in the broncho-­ ­ alveolar lavage fluid. Consequently, there are strong associations between increased TBARS accumulation and Al, Si, and Fe contents of CAP.  Same relationships are evident

7  Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles-Effect

between carbonyl content and Cr and Na concentrations, and between PMN count and Cr, Zn, and Na. These data demonstrate that oxidants are critical mediators of the inflammatory response elicited by PM inhalation (Rhoden et al. 2004). In fact, CAPs contain significantly larger amount of metals including Fe, Cu, Zn, Pb, Ni and V. Aqueous extracts from the metal-rich PM has stronger ability to generate ROS and to increase IL-8 and IL-6 release. The severity of inflammatory injury is directly proportional with the metal content, and the redox potential of CAP (Ghio 2004). Metals may synergize with organic PM components in this process leading to further increase in oxidative stress (Saldiva et al. 2002). A mechanism of biological effect common to many ambient air pollution particles is a disruption of iron homeostasis in cells and tissues. All surfaces of PM have some concentration of oxygen-­containing functional groups. As a result of its electropositivity, Fe3+ has a high affinity for oxygen-­ donor ligands and reacts with these groups at the particle surface, thus, iron is consumed (Ghio and Cohen 2005). In response to reduction in concentrations of requisite iron, a functional deficiency occurs intracellularly. Superoxide production by the cell exposed to a particle facilitates import of iron with the objective being the reversal of the metal deficiency. However, failure to resolve the functional iron deficiency following cell exposure to particles activates kinases and transcription factors resulting in a release of inflammatory mediators and inflammatory reaction (Ghio et  al. 2016). The phosphorylation of p38 MAPK induced by PMs is shown to be critically important for the increases in IL-1α and IL-1β expression. Moreover, PM markedly increases the mRNA and protein expression levels of matrix metalloproteinase 1 (MMP1) and COX-2 in human keratinocytes. Therefore, it is thought that, PMs exposure triggers skin ageing via p38 MAPK activation and interleukin secretion in epidermal keratinocytes (Kim et  al. 2019). Ni is a heavy metal found in PM. Ni ions increase extracellular matrix protein levels, including those of type I collagen and MMP9  in mouse lung tissues and cell lines. Moreover, Ni ions promote the phos-

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phorylation of Akt. Akt activation is a critical contributor to the Ni-exacerbated pulmonary fibrotic process (Yang et al. 2018). The greater concentrations of metals in DEP is associated with the JNK activation and increase in cytotoxicity (Seriani et al. 2015). DEP induces the generation of ROS, via superoxide anion and H2O2 generation in the endothelial cells. Transcription factor Nrf2 is translocated to the nucleus. Subsequently, it activates transcription of the antioxidative enzyme HO-1, and induces the release of vascular permeability factor VEGF-A.  Exposure to a low dose of DEP triggers actin cytoskeleton depolarization, reduces PI3K/Akt activity, and induces a p53/the ubiquitin ligase mouse double minute 2 homolog (Mdm2) feedback loop. Whereas, a high dose causes apoptosis by depleting Mdm2. ROS scavengers suppress DEP-induced oxidative stress (Tseng et al. 2017). DEPs stimulate the expression of mucin 4 (MUC4) via the 3 MAPKs p38/ CREB pathway in DEP-exposed airway epithelium (I.-H. Park et al. 2016a). In this case, UFPs translocate to the central nervous system and activate oxidative stress-related pathways. The transcription factor Nrf2 activation by ERK1/2 is a key regulator of cellular response to oxidative stress. DEP decreases the levels of antioxidant enzymes. The mitogen-activated protein kinase kinase (MEK)-ERK1/2 pathway is involved in regulating the antioxidant strategies to compensate the oxidative status induced by DEP (Farina et  al. 2016). Classically and alternative human macrophages increase the secretion of platelet-­ derived growth factor subunit B (PDGF-B) in response to DEP extract (DEPe). This occurrs via aryl hydrocarbon receptor (AhR)-activation. Human macrophages, whatever their polarization status, secrete PDGF-B in response to DEPe. Actually, PDGF-B is a target gene of AhR.  Therefore, induction of PDGF-B by DEP contributes to the deleterious effects towards human health triggered by such environmental contaminants (Jaguin et  al. 2015a). In M2 macrophages, DEPe exposure decreases the ­ expression of the CD200R, a typical membrane marker of M2, in addition to the reduction in the secretion capacity of chemokines, chemokine

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(C-C motif) ligand 17 (CCL17) (thymus and activation regulated chemokine; TARC), CCL18 (pulmonary and activation-regulated chemokine; PARC) and CCL22 (macrophage-derived chemokine; MDC) in T helper 2 (Th2) cells. The decreased chemokines secretion capacity of M2 polarizing macrophages exposed to DEPe is associated to a lower chemotaxis of CCR4+ cells. AhR and Nrf2 pathways are activated in both M1 and M2 type of macrophages after DEPe exposure. Furthermore, the DEPe-related decrease of IL-6 secretion is dependent on Nrf2, and AhR (Jaguin et  al. 2015b). The cytotoxicity of the DEPs is characterized by a time- and concentration-dependent increase in necrotic cells. DEPexposure exacerbated IL-6 and chemokine (C-X-C motif) ligand 8 (CXCL8) responses in toll like receptor 3 (TLR3)-primed cells, while TLR3-induced CCL5 is suppressed by DEP.  TLR3-priming and DEP-exposure results in possible additive effects on p38 phosphorylation and IκB-degradation, while DEP rather suppresses ERK and JNK-­ activation (Bach et  al. 2014). Previous studies have shown that the involvement of MAPKs play a crucial role in DEP-induced up-regulation of inflammatory mediator genes (Boland et  al. 2000; Li et  al. 2002b; Ma et  al. 2004). Exposure of primary human bronchial epithelial cells to DEP significantly increases IL-8 and IL-1β protein expression. Antioxidant glutathione S-transferase M1 (GSTM1) deficiency aggravates D ­ EP-­ induced IL-8 and IL-1β protein expression. DEP stimulation induces the phosphorylation of ERK1/2 and Akt, the downstream kinase PI3K, in GSTM1+ bronchial epithelial cells. In contrast, inhibition of ERK and PI3K activities block DEP-induced IL-8 and IL-1β expression (Wu et  al. 2012). Exposure to DEP with varying organic content differentially induces IL-8 expression in human airway epithelial cells. Thus, low organic-containing DEP stimulated IL-8 expression is predominantly NF-kB-­ dependent, whereas high organic-containing DEP induces IL-8 expression independently of NF-kB, rather requires AP-1 activity (Tal et  al. 2010). In fact, expression of IL-8 can be induced in lung epithelial cells through an NF-κB-­independent mechanism. The

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cell type-specific pattern of IL-8 gene expression is associated with the differential activation and promoter binding of the redox regulated transcription factors AP-1 and NF-κB (Roebuck 1999). DEP induces NF-κB activity by two- to three-fold via transforming growth factor-beta (TGF-β) activated kinase 1 (TAK1), and NF-κBinducing kinase (NIK) in lung epithelial cells. TAK1 and NIK are important mediators in DEPinduced NF-κB activation (Yun et  al. 2005). Activated TAK1 phosphorylates NIK, which stimulates IKK-α activity. TAK1 links tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) to the NIK-­IKK cascade in the IL-1 signaling pathway (Ninomiya-Tsuji et  al. 1999). Exposure to DEP at a non-cytotoxic concentration significantly increases the transactivation of NF-κB, but not AP-1. Furthermore, DEP promotes phosphorylation of Akt on Ser-473 and Thr-308  in a PI3K-­ dependent manner, and enhances phosphorylation of down-stream p70/ p85 S6 kinases (p70/p85S6K) as well as glycogen synthase kinase-­3beta (GSK3β). DEP has little effect on the phosphorylation of ERKs and p38 MAPK. Thus, DEP-induced transactivation of NF-κB is mediated by PI3K/Akt signaling pathway (Ma et al. 2004). DEP exposure induces signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation through a process that includes the generation of ROS and requires EGFR and Src activities in human endothelial cells (Cao et al. 2007). On the other hand, DEP exposure results in a decrease of total and EGFR-directed protein tyrosine phosphatases (PTPase) activity in human airway epithelial cells, independent of the organic content of these particles. Exposure to DEP also induces phosphorylation of the receptor tyrosine kinase EGFR in a manner that requires EGFR kinase activity. Because the PTPase activity normally functions to dephosphorylate EGFR, DEP-induced EGFR phosphorylation is the result of a loss of PTPase activity (Tal et  al. 2008). In this context, DEP-­ ­ associated quinones and reactive PAH metabolites can generate ROS and RNS that reversibly inactivate PTPases. In fact, organic DEP extracts induce oxidative stress response at

7  Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles-Effect

three stages. Firstly, leading to HO-1 expression at normal GSH/GSSG ratios. At second step, intermediary oxidative stress levels result in Jun kinase activation and IL-8 production. Finally, cellular apoptosis emerges in parallel with a sharp decline in GSH/GSSG ratios. One consequence of oxidative stimulation of NO generation is S-nitrosylation and inhibition of PTPs critical in cellular signal transduction pathways. Further, thiol oxidation leads to inactivation of PTPs and to superactivation of protein tyrosine kinases (PTKs) (Barrett et al. 2005; Chiarugi and Buricchi 2007; Li et al. 2002a). Transcription factor activation induced by DEP-associated ROS production has been linked to the MAPK pathways. Thus, DEP induces IL-8 and regulated upon activation, normal T cell expressed and presumably secreted (RANTES) production in addition to threonine and tyrosine phosphorylation of p38 MAP kinase in human bronchial epithelial cells (Fahy et al. 2000; Hashimoto et al. 2000). Long-­ term exposure to a high concentration of DEP increases microRNA-21 (miR-21) expression and then activates the phosphatase and tensin homolog (PTEN)/PI3K/AKT pathway in human bronchial epithelial cells. This pathway may lead to bronchial carcinogenesis (Zhou et al. 2015). Similar to PM, MMP-1, which is a collagenase involved in alveolar wall degradation, is induced by DEP. In this process DEP simultaneously increases ERK1/2 phosphorylation, and ROS generation via promoting NADPH oxidase (NOX4) activity (Amara et al. 2007). In contrast, human Thioredoxin-1 (hTrx1) suppresses DEP-­ induced ROS generation. Decreasing Akt phosphorylation by DEP results in apoptosis. However, apoptotic activity is prevented by hTrx1. Overall, hTrx1 exerts antioxidant effect and this protects against DEP-induced lung damage by regulating Akt-mediated antiapoptotic signaling (Kaimul Ahsan et al. 2005). mRNA levels of HO-1, glutamylcysteine ligase, glutathione peroxidase 2, TRX1, and thioredoxin reductase 1 (TRXRD1) are enhanced by co-treatment of Cr and As, whereas As alone reduces the mRNA expression level of TRX2. As increases Cr-induced pulmonary injury and that this effect may be exerted through a disruption in the bal-

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ance among several antioxidants (Tajima et  al. 2010). DEP markedly upregulates Fos leucine zipper transcription factor (fra-1) expression. Fra-1/DNA-binding activity is predominantly associated with the Jun-B and Jun-D in the AP-1 complex. Interestingly, DEP increases fra-1 but does not alter Jun-B and Jun-D protein levels, instead stimulates phosphorylation of ERK1/2-­ JNK1-­p38 MAPKs in alveolar epithelial cells. In addition, fra-1 has the opposite effect on MMP-9 promoter activity, and binds to the functional AP-1 site of the MMP-9 promoter after DEP stimulation. Collectively, fra-1 induction by DEP selectively regulates gene expression involved in alveolar epithelial cell injury and repair (Zhang et al. 2004). The different response towards the DEP/ZnO mixture with respect to the individual NPs of ZnO is attributed to the different mechanisms of interaction and endocytosis of DEP/ZnO NPs aggregates, compared to the ZnO NPs alone. The interaction of DEP with ZnO and CuO NPs may interact and change the surface reactivity between environmental UFP and engineered NPs. This interaction is largely dependent on the kind of NPs. DEP/Zn2+ mixture is more toxic than DEP or ZnO NPs alone. DEP/CuO NPs mixture reduces the bioavailability of Cu2+. Consequently, DEP/CuO NPs mixture has lower toxic effects compared to single CuO NPs. However, the co-­ exposure to metal oxide NPs together with DEP highly increases the toxicity of carbon-based UFP (Zerboni et  al. 2019). CuO NPs markedly increase inflammatory responses and collagen deposition, accompanied by the elevation of TGF-β1 and collagen I expression in lung tissue. CuO NPs induce fibrotic responses in the respiratory tract. This toxic effect is closely related to TGF-β1/mothers against decapentaplegic homolog 3 (Smad3) signaling, which is associated with the ERK, JNK and p38 phosphorylation (Ko et al. 2018; J.-W. Park et al. 2016b). The cellular toxicity of ZnO NPs dependent on the Zn2+ ions released from ZnO NPs in the cellular medium (Deng et al. 2009). Zn2+ ions induce inflammatory responses and increase oxidative stress, in addition to increasing intracellular [Ca2+] and decreasing mitochondrial membrane potential, and IL-8

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production (Vandebriel and De Jong 2012). The amount of Zn2+ ions released by the ZnO NPs and DEP/ZnO mixture and Cu2+ ions released by the CuO NPs alone and in DEP/CuO NPs mixture are similar. These findings support the ions dissolution hypothesis in the predicted toxicity of DEP/metal oxide mixture. Thus, pH-­dependent dissolution of ZnO NP inside of phagosomes is the main cause of ZnO NP-induced diverse progressive severe lung injury (Cho et al. 2011). The most frequently considered interactions between cellular responses and DEP are the induction of pulmonary oxidative stress and inflammation. The activation of bronchial epithelial TLRs results in increases in Th2 and Th17 cytokines (Muñoz et  al. 2019). Environmental ambient PM and DEP activate lung dendritic cells (DCs) in vivo and provoke a Th2 response (Bezemer et al. 2011). The road edge soils were moderately to highly polluted with Cd, Cu, Pb, and Zn. The safest distance to minimize metal pollution for agricultural production is proposed to be greater than 10 m away from the road edge (Krailertrattanachai et al. 2019). The highest levels of Pb, Cd, and Cr are present in sites with heavy traffic. The significant differences in the concentrations of Cu, Pb, Cd, and Zn among the different categories of roads are indicated as the contribution of traffic intensity to environmental contamination (Wang and Zhang 2018). T-cell apoptosis by Cd, more in CD4+ than in CD8+ cells appear to be related to higher depletion of intracellular glutathione. Th1 cells of CD4+ sub-­ population are more responsive to Cd than Th2 cells (Pathak and Khandelwal 2008). Vehicle emitted particles (VEP), which are collected from two different types of cars (diesel and gasoline) and locomotive increase percentage of CD4+ T cells, whereas cause a sharp decrease in basophil counts. These results indicate that VEP decreases the inhibitory effect of CD16+ monocytes on the proliferation of CD4+ T cell and suppresses polarization into a Th2 phenotype (Zakharenko et al. 2017). Recently, there have been many findings that exposure to DEP leads to neurodegenerative diseases. When taking into consideration of occupational hazard, it is observed that the con-

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centration of Pb in the urine and blood samples of traffic policemen have exceeded the highest limit of occupationally exposed outdoor workers (Liu et al. 2019). Moreover, in occupational exposure, significantly increased blood and urinary Pb levels are associated with the increased urinary neopterin, biopterin and delta-aminolevulinic acid, while blood dihydropteridine reductase activities are unchanged. Chronic Pb exposure permanently alters pteridine metabolism and may cause dopaminergic neurotransmission disorders via biopterin (Engin et al. 2006). Pb-mediated upregulation of ryanodine receptors leads to neurodegeneration via high levels of free calcium release. In this case, decreasing in the calcium-­dependent CaMKIIα/CREB signaling pathway, and activation of the calcium-dependent ERK/Bcl-2 apoptotic signaling pathway play an important role (Zhou et  al. 2020). Thus, the activation of JNK, PI3K, or Akt and MAPK signaling pathways are important for Pb-induced oxidative stress and the biomolecular consequences (Singh et  al. 2018). Chemical evaluation by Principal Component Analysis (PCA) revealed that road environments have been severely contaminated with trafficrelated elements. Thus, concentration of Cu in all road-environment samples is even higher, exceeds even up to 15 times its average concentrations established for the surrounding soils (Adamiec 2017). Chronic Cu exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway. Thus, copper induces the expression of the pro-apoptotic target molecule CHOP of ATF-4 and neuronal apoptosis. In addition, increased tau phosphorylation due to copper exposure is closely correlated with aberrant cdk5/p25 activation (Kitazawa et al. 2009; Q. Ma et al. 2015b). Since the brain regions such as hippocampus and cortex are deficient in antioxidant enzymes and rich in transition metals such as iron and copper, the degree of oxidative damage is more severe for these regions (Arya et al. 2016). On the other hand, cerium compounds have been used as a fuel-borne catalyst to lower the generation of DEPs and its toxicity. In this context, Envirox is a scientifically and commercially

7  Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles-Effect

proven diesel fuel combustion catalyst based on nanoparticulate CeO2 and has been demonstrated to reduce fuel consumption, greenhouse gas emissions, and particulate emissions. It is claimed that exposure to nano-size CeO2 as a result of the addition of Envirox to diesel fuel hinders the pulmonary oxidative stress and inflammation, decreases the number of particles in exhaust, and reduces atherosclerotic burden associated with exposure to standard diesel fuel (Cassee et al. 2012; Park et al. 2008; Zhang et al. 2016). The combustion process induces significant changes in the size and morphology of the particles; approximately 15 nm aggregates consisting of 5–7  nm faceted crystals in the fuel additive become 50–300 nm in the exhaust. The surfactant coating present on the CeO2 particles in the additive is lost during combustion, and by 30% of the observed particles in the exhaust, with the formed new surface coating, is approximately 2–5 nm thick (Dale et al. 2017). In fact, both CeO2 and DEP alone cause severe lung injury, separately. They exhibit different effects on pulmonary cellular responses. DEPs inhibit alveolar macrophage phagocytosis and weaken the innate immunity by inhibiting alveolar macrophage secretion of IL-1 and TNF-α. DEPs may also suppress cell-mediated immunity by inhibiting secretion of IL-12, which is a key cytokine for the initiation of Th1 cell development, from ­alveolar macrophages (Yin et  al. 2002). CeO2 NPs induce sustained inflammatory responses, which lead to lung fibrosis. DEP causes a switch of the pulmonary immune response from Th1 to Th2, while CeO2 induces inflammatory response in the lungs. CeO2 changes alveolar macrophage function from the M1 inflammatory classical subset to the fibrotic subset of M2 (Ma et  al. 2011, 2012; Park et al. 2007). Thus, CeO2 NPs strongly induce lung collagen formation, excessive accumulation of lung surfactants, and cellular mediators involving in the lung tissue remodeling process. But all these processes are not affected by the DEP. However, the addition of CeO2 NPs to DEP does not affect the previously developed DEP-­induced lung granuloma. It is claimed that inflammatory responses, which is observed in the lung due to CeO2 or DEP par-

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ticles exposures are hindered in the case of combined exposure to DEP/CeO2 (Ma et  al. 2014). Contrarily, cerium accumulation in the lungs results in histological changes, an increased Bax to Bcl-2 ratio, elevated cleaved caspase-3 protein levels, increased phosphorylation of p38 MAPK, and diminished phosphorylation of ERK-1/2MAPK (Rice et al. 2015). The stress-responsive transcription factor, AP-1, is significantly decreased in the cortical and subcortical fraction of the brain after DEP exposure. The addition of nanoceria to the diesel fuel reverses this effect via c-Jun activation. Whereas, the activation of another stress-related transcription factor, NF-κB cannot be inhibited (Lung et  al. 2014). However, tissue analysis indicates a concentration- and time-dependent accumulation of lung and liver CeO2 due to delayed clearance. DEP/ CeO2 exposure-induced edema increases the alveolar septa thickness. Consequently, DEP/ CeO2 induces more pulmonary adverse effects on a mass basis than DEP (Snow et  al. 2014). Cerium compounds as diesel fuel additives, increase fuel combustion efficiency, while decreasing diesel soot emissions. However, CeO2 NPs, which have been detected in the exhaust may raise a health concern (J. Ma et al. 2015a). Thus, high-dose respiratory exposure to CeO2 NPs cause lung inflammation, activation of MAPKs signaling, and cellular apoptosis (Rice et  al. 2015). CeO2 NPs alone significantly increase the production of ROS and malondialdehyde (MDA), and significantly reduce the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and CAT.  The phosphorylation levels of ERK1/2, JNK and p38 MAPK are significantly elevated after exposure to CeO2 NPs. Thereby, CeO2 NPs induce damage and apoptosis in human cells via oxidative stress and the activation of MAPK signaling pathways (Cheng et al. 2013). As mentioned above, oxidative stress and programmed cell death in the brain have been assumed as two mechanisms related to neurotoxicity of Pb. CeO2 and Y2O3 NPs have recently shown to have antioxidant effects, particularly when used together, through scavenging the amount of ROS required for cell apoptosis. The combinations of these NPs pres-

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ent neuroprotective effects against acute Pb-induced neurotoxicity (Hosseini et al. 2015). Co-administration of CeO2 and Y2O3 NPs recover Pb-caused oxidative stress markers and apoptosis indexes. This combination may potentially be beneficial for protection against Pb-caused acute neurotoxicity (Hosseini et al. 2015). Since, both NPs increase the total thiol concentration and Bcl-2 protein expression, combined exposure to CeO2 and Y2O3 NPs decrease ROS production, lipid peroxidation (LPO), Bax and caspase-3 proteins expression (Ghaznavi et  al. 2015). Accordingly, co-application of CeO2 and/or Y2O3 NPs reduces the oxidative stress and apoptosis and increases the β-cell viability, glucoseinduced ATP production and glucose-stimulated insulin secretion (Hosseini et al. 2013). Although CeO2 exposure alone increases the phosphorylation levels of ERK1/2, JNK and p38MAPK the effects of co-exposure to amorphous (a) SiO2/ CeO2 NPs on the protein kinases is not known. Subsequent to aSiO2-coated CeO2 NPs exposure, aSiO2 dissolves off the CeO2 core, and some of the CeO2 NPs transform to CePO4 and only Si is detected in tissues. aSiO2 coating reduces CeO2induced inflammation, phospholipidosis and fibrosis (J.  Ma et  al. 2015a). Polyethylene glycol-coated 3  nm CeO2 NPs (PEG-CNPs) promote neurogenesis via AMPK-mediated phosphorylation of PKCζ and activating CREB binding protein (CBP) (Arya et al. 2016). Brake wear contribute with significant amount of metals to PM, particularly within areas with high traffic density and braking frequency. In this context, the most abundant metals are Fe, Cu, Zn, Sn, and Sb. Brake wear can contribute up to 55% by mass to total non-exhaust traffic-related PM10 emissions and up to 21% by mass to total traffic-­ related PM10 emissions, while in freeways, this contribution decreases due to lower braking frequency (Grigoratos and Martini 2015; Kukutschová et al. 2011; Kwak et al. 2013). Fe content can reach up to 60  wt.% and varies according to the type of lining (Figi et al. 2010; Kukutschová et al. 2011). After Fe, Cu is the second element which is more representative of NPs

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emitted by brake wear. On the one hand the metals on brake wear particles damage the endothelial tight junctions with a mechanism involving oxidative stress, on the other hand increase pro-­ inflammatory responses (Gasser et  al. 2009; Iijima et  al. 2007). CuO NPs and TiO2 NPs exhibit the teratogenic potential. ZnO NPs cause the most severe lesions to the intestinal barrier, allowing NPs to reach the underlying tissues (Bacchetta et  al. 2012). Pb content has been reported in high amount, while other metals such as barium (Ba), Mg, Mn, Ni, tin (Sn), Cd, Cr, Ti, potassium (K) and antimony (Sb) have been found in lower concentrations (Kukutschová et al. 2011; Thorpe and Harrison 2008). Whereas, zinc-based NPs (Zn-NPs) mainly derive from tire wear. The frequency of metal emissions from brake linings/tire tread rubber in 2005 were detected as Cd, Cu, Pb, Sb, and Zn. The calculated Cu and Zn emissions from brake linings were unchanged in 2005 compared to 1998, indicating that brake linings still remain one of the main emission sources for these metals. In contrast, Pb and Cd emissions have decreased to one tenth compared to 1998. In addition, the results also show that tires are still one of the main sources of Zn and Cd emissions in the city (Harrison et al. 2012; Hjortenkrans et al. 2007). Sb associated with poorly crystalline Fe oxides decreases with distance from the road, and the content of Sb bounded to well-crystalline Fe oxides, and the residual Sb fraction are relatively constant around the road. Although roadside soils appear to inhibit brake lining-related Sb contamination, with significant but rather low ecotoxicological potential for input into groundwater (Földi et al. 2018). Sb trioxide (Sb2O3) causes a dose-dependent cytotoxicity. Sb2O3-induced excessive ROS is closely correlated with increased Nrf2 expression, and cell apoptosis. Furthermore, elevated Gadd45b promoter expression activates the phosphorylation of MAPKs upon Sb2O3 exposure. ROS and the SEK1 (an upstream regulator of JNK)/JNK pathway mediates the cytotoxicity of Sb2O3 exposure (Jiang et al. 2016).

7  Combined Toxicity of Metal Nanoparticles: Comparison of Individual and Mixture Particles-Effect

5

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greenhouse gas emissions, and particulate emissions. Contrarily, CeO2 NPs induce damage and Although each NP activates dose-dependently apoptosis in human cells via oxidative stress and more or less the same metabolic pathways, the the activation of MAPK signaling pathways. toxicity induced by a single NP type is different Although pulmonary adverse effects caused by from co-exposure to the combined NPs. Several DEP/CeO2 are controversial, co-administration metal NPs, including As, Cr, Cu, Fe, Ni, V, and of CeO2 and Y2O3 NPs prevent Pb-induced Zn can induce the phosphorylation of the neurotoxicity. MAPKs, such as ERK, JNK, and p38, however, Highly concentrated ambient particles contain they do not equally alter the activity of these pro- significantly larger amount of metals including tein kinases. ZnO NPs simultaneously activate Fe, Cu, Zn, Pb, Ni and Cr. All these metal NPs p38 and SAPK/JNK. CeO2 and TiO2 NPs together contribute to the toxic effects of particulate air activate ERK1/2 and p38, while Al2O3 NPs pollutants. The metal-rich PM has stronger abilinduce dose-dependently ERK1/2 activation ity to generate ROS and to increase IL-8 and only. However, all metal oxide NPs can activate IL-6 release. The phosphorylation of p38 MAPK NF-κB pathway, which is mainly related with induced by PMs is shown to be critically important inflammation and the immune response. for the increases in IL-1α and IL-1β expression. Transition metals in NPs mixture can generate NPs either alone or combined with other NPs superoxide via Fenton reaction. and DEP, while increasing ROS production, sigThe concentrations of metals in DEP is pro- nificantly reduce the antioxidant capacity. The portional with the JNK activation and increased phosphorylation levels of ERK1/2, JNK and p38 cytotoxicity, however, it is claimed that the DEP-­ MAPK are significantly altered after exposure to sample with the highest PAH and lowest metal NPs or DEP.  Thereby, cell death is triggered in content possesses a greater oxidative potential. human cells via oxidative stress-induced activaThe interaction of DEP with ZnO and CuO NPs tion of MAPK and PI3K/Akt/CREB/Bcl-2 sigmay interact and change the surface reactivity naling pathways. within environmental UFP and engineered NPs. This interaction is largely dependent on the kind of NPs. Thus, DEP/ZnO2 mixture is more toxic References than DEP or ZnO NPs alone. DEP/CuO NPs mixture reduces the bioavailability of Cu2+ ions. Adamiec E.  Road Environments: Impact of Metals on Human Health in Heavily Congested Cities of Poland. Therefore, DEP/CuO NPs mixture has lower Int J Environ Res Public Health. 2017:14. https://doi. toxic effects compared to single CuO NPs. The org/10.3390/ijerph14070697. co-exposure to metal oxide NPs together with Alinovi R, Goldoni M, Pinelli S, Campanini M, Aliatis I, Bersani D, Lottici PP, Iavicoli S, Petyx M, DEP highly increases the toxicity of carbon-­based Mozzoni P, Mutti A. Oxidative and pro-inflammatory UFP.  Whereas DEP-associated ROS production effects of cobalt and titanium oxide nanoparticles has been linked to MAPK pathways. DEP-exposure on aortic and venous endothelial cells. Toxicol In Vitro. 2015;29:426–37. https://doi.org/10.1016/j. results in additive effects on p38 phosphorylation tiv.2014.12.007. and IκB-degradation, while DEP rather suppresses Amara N, Bachoual R, Desmard M, Golda S, Guichard ERK and JNK-activation. However, MAPKs play C, Lanone S, Aubier M, Ogier-Denis E, Boczkowski a crucial role in DEP-­induced up-regulation of J. Diesel exhaust particles induce matrix metalloprotease-­1 in human lung epithelial cells via a NADP(H) inflammatory mediator genes. DEP induces the oxidase/NOX4 redox-dependent mechanism. Am J phosphorylation of ERK1/2 and Akt. On the other Physiol Lung Cell Mol Physiol. 2007;293:L170–81. hand, DEP induces NF-κB activity by two- to https://doi.org/10.1152/ajplung.00445.2006. three-fold via TAK1, and NIK. Araujo JA, Nel AE.  Particulate matter and atherosclerosis: role of particle size, composition and oxidaBoth CeO2 NPs and DEP alone cause severe tive stress. Part Fibre Toxicol. 2009;6:24. https://doi. lung injury. It was demonstrated that addition org/10.1186/1743-8977-6-24. of CeO2 NPs to DEP reduces fuel consumption,

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8

Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes Ayse Basak Engin and Atilla Engin

tance, and the reprogramming of β-cell for differentiation or dedifferentiation in T2D.  There is much debate about selecting proposed therapeutic strategies to maintain or enhance optimal β-cell viability for adequate insulin secretion in T2D. However, in order to achieve an effective solution in the treatment of T2D, more intensive clinical trials are required on newer therapeutic options based on protein kinases signaling pathways.

Abstract

Type 2 diabetes (T2D) is a worldwide serious public health problem. Insulin resistance and β-cell failure are the two major components of T2D pathology. In addition to defective endoplasmic reticulum (ER) stress signaling due to glucolipotoxicity, β-cell dysfunction or β-cell death initiates the deleterious vicious cycle observed in T2D. Although the primary cause is still unknown, overnutrition that contributes to the induction of the state of low-grade inflammation, and the activation of various protein kinases-related metabolic pathways are main factors leading to T2D. In this chapter following subjects, which have critical checkpoints regarding β-cell fate and protein kinases pathways are discussed; hyperglycemia-­induced β-cell failure, chronic accumulation of unfolded protein in β-cells, the effect of intracellular reactive oxygen species (ROS) signaling to insulin secretion, excessive saturated free fatty acid-induced β-cell apoptosis, mitophagy dysfunction, proinflammatory responses and insulin resisA. B. Engin (*) Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey A. Engin Department of General Surgery, Faculty of Medicine, Gazi University, Ankara, Turkey

Keywords

Type 2 diabetes (T2D) · β-Cell dysfunction · β-Cell death · Saturated free fatty acids (SFFA) · Glucose-stimulated insulin secretion (GSIS) · Reactive oxygen species (ROS) · Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase · Human islet amyloid polypeptide (hIAPP) · Fas receptors (FasR) · ER (endoplasmic reticulum) stress

1

Introduction

Type 2 diabetes (T2D) is a serious public health problem in the world. Approximately 150 million people worldwide had T2D in the year 2000. Zimmet et al. estimated that this number could be doubled by 2025 (Zimmet et  al. 2001). Beyond

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_8

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the predictions, the world prevalence of diabetes among adults, aged 20–79 years is 6.4% in 2010. This means that T2D affects 285 million adults. Furthermore, it is expected that this prevalence will increase to 7.7% by 2030, and reaches to 439 million adults. As a future projection between 2010 and 2030, it is predicted that the numbers of adults with diabetes will increase by 69% in developing countries, while this increase will be 20% in developed countries (Shaw et al. 2010). The total number of excess deaths attributable to diabetes worldwide is estimated to be 3.96 million in the age group 20–79  years, 6.8% of all-­ age global mortality. On the other hand, in developed countries, the mortality rate related to diabetes is 2.6 times higher than in developing countries (Roglic and Unwin 2010). According to data of 2013 of “International Diabetes Federation”, diabetes alone affects nearly 400 million people worldwide and accounts for about five million deaths annually (Wang et al. 2015). Insulin resistance and β-cell dysfunction are the two major components of T2D.  Binding of insulin to its receptor activates the insulin receptor subunit (InsRb) and insulin receptor substrate 1 (IRS-1). Approximately, the expression of 2637 genes are significantly changed via activity of insulin. In this context, phosphorylation of the 15 signaling proteins increase by 1.4- to over tenfold. The expression levels of 158 “insulin-­ responsive proteins” show significant changes. The phosphorylation levels of at least seven protein kinases show an average of threefold or more increase: such as protein kinase B (Akt), glycogen synthase kinase-3β (GSK3β), extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), p70 ribosomal protein S6 kinase (p70S6K), and nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) inhibitor (IκB) kinaseβ (IKKβ) (Wang et  al. 2015). Upon activation, kinases regulate cellular functions through T2D-susceptibility genes. However, some of the kinase-gene relationships are known, others are yet to be determined (Wang et al. 2015). One of the major factors in T2D, β-cell deficiency is attributed to an imbalance between the

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self-renewal and proliferation rate of β-cell, and the loss of β-cell via apoptosis (Butler et al. 2003, p., 2007; Rieck and Kaestner 2010). The loss of functional β-cell mass is not as much due to β-cell death but rather to de-differentiation of β-cells when these cells are exposed to metabolic stressors (Wang et al. 2017). When a pancreatic β-cell is exposed to high glucose, it responds with a compensatory increase in insulin secretion, and with β-cell proliferation and adaptive increase in β-cell mass. However, prolonged increases in glucose levels will paradoxically result in impaired β-cell function (Ferrannini 2010). Indeed, chronic glucotoxicity leads to T2D in five stages. First stage is a compensation phase, in which insulin secretion increases to maintain normoglycemia in the face of rising insulin resistance and decreasing β-cell mass. In this stage, glucose-stimulated insulin secretion (GSIS) is maintained permanently. The activation of glucokinase in β-cells controls the rate of glycolysis. In stage two glucose levels reach to approximately 5.0–6.5  mmol/l. This is a stable state of β-cell adaptation with loss of β-cell mass and disruption of function. Within this period glutathione disulfide (GSSG) gradually decreases. Stage three is a transient unstable period of early decompensation in which glucose levels rise relatively rapidly to the levels of frank diabetes of stage four. A stable decompensation with more severe β-cell dedifferentiation occurs. Final stage is characterized by severe decompensation representing a profound reduction in β-cell mass with progression to severe metabolic problems of T2D (Jansson et al. 1995; Meier et al. 2013; Weir et al. 2013; Weir and Bonner-Weir 2004). If prediabetic hyperglycemia is left unchecked, excessive plasma glucose, which is associated with β-cell failure can progress to T2D and leads to life-­ threatening secondary complications at final stage (Rourke et al. 2018). Although the primary cause is still unknown, inverse correlation between adiponectin and pro-­ inflammatory cytokines, defective GSIS from pancreatic β-cells, and insulin resistance are significantly associated with T2D.  During the uncontrolled hyperglycemic conditions, the GSIS can dramatically enhance reactive oxygen spe-

8  Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes

cies (ROS) production and manifestations of oxidative stress and, causes β-cell apoptosis (Fridlyand and Philipson 2004; Guo et al. 2018; Liu et al. 2016; Tong et al. 2017). Furthermore, in T2D, overnutrition is one of the major causative factors that contributes to induce the state of low-­ grade inflammation and the activation of various transcriptional mediated molecular and metabolic pathways, which increase the oxidative stress. In this context, it is thought that the raised level of tumor necrosis factor (TNF)-α induces insulin resistance by impairing the insulin signaling leading to the development of T2D (Akash et  al. 2018; Rehman and Akash 2016). In this process, JNK, ERK, p38, p70S6kinase, and NF-κB are activated by TNF-α in β-cells, whereas silencing mitogen-activated protein kinase kinase kinase kinase isoform-4 (MAP4K4) protects β-cells against TNF-α. Thereby, TNF-α-­induced decrease of IRS-2, and inhibition of glucosestimulated insulin secretion are recovered (Bouzakri et al. 2009). Although insulin-­induced signal transduction through protein kinases varies greatly among individuals, the differences in the phosphorylation of protein kinases are not due to differences in expression levels (Wang et al. 2015). There are very limited studies related to prevent the endoplasmic reticulum (ER) stressmediated pancreatic β-cell destruction via regulation of the protein kinases signaling pathways (Jung et  al. 2012; Simon-Szabó et  al. 2014). In this chapter, protein kinase signaling pathways and their potential checkpoints, which may play a role in the development of T2D as a result of β-cell damage, will be discussed in detail.

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enhancing glucose uptake in the liver. In fact, glucose phosphorylation by GK in the β-cells is the rate-limiting step that controls glucose-­ stimulated insulin secretion (Matschinsky 2009; Xu et  al. 2017). Therefore, GK has been an attractive target for anti-diabetic therapy over the past two decades. Thus, several GK activators (GKA) have been shown to reduce blood glucose levels in diabetic subjects (Grewal et al. 2014; Xu et al. 2017). Insulin is synthesized as preproinsulin and processed to proinsulin. Preproinsulin turns into proinsulin in the rough ER (rER) lumen. The folded proinsulin is transported from the ER to the Golgi apparatus, and then proinsulin along with the islet amyloid polypeptide (IAPP) precursor enters secretary vesicles. Proinsulin cleaves into insulin and C-peptide in these vesicles. Insulin and C-peptide are then stored in these secretory granules together with human islet amyloid polypeptide (hIAPP or amylin) (Fu et  al. 2013; Nishi et  al. 1990). The first phase entails the preparation of insulin granules for release. The second phase is insulin release from β-cells in response to glucose through the adenosine triphosphate (ATP)-sensitive K+[K(ATP)] channel-dependent pathway. The messengers controlling the second phase of insulin release are the same as for the first phase, which consists of ATP/adenosine diphosphate (ADP) ratio, membrane potential, [Ca2+]i, and the Ca2+ sensors. These steps are potentiated by additional signals, which are induced via glucose. These include citrate and malonyl coenzyme A (CoA), long-chain acyl-CoAs, diacyl glycerol, protein kinase C (PKC) isoforms, β-cell protein kinase A, phospholipases, and phosphoinositides (Bratanova-Tochkova et  al. 2002). An elevation 2 Glucose Homeostasis of intracellular Ca2+ is induced by increased Ca2+ and β-Cells in T2D influx through the voltage-dependent Ca2+ chanUnder physiological conditions, increased levels nels (VDCC). This is a primary driver of the of glucose induce the release of insulin from GSIS mechanism. However, further increases in secretory granules in the β-cell. Glucokinase intracellular Ca2+ stimulates mitochondrial gen(GK) serves as a glucose sensor of the insulin-­ eration of ROS, while Ca2+ enhances reduced producing pancreatic β-cells, and regulates nicotinamide adenine dinucleotide phosphate hepatic glucose production. Firstly, GK phos- (NADPH) oxidase (NOX)-dependent generation phorylates glucose to glucose-6-phosphate for of ROS in pancreatic β-cells via PKC activation lowering blood glucose concentrations by (Newsholme et al. 2007). Because of the β-cells’

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limited antioxidant capacity, upon Ca2+ stimulation of mitochondrial and NOX1 systems, ROS concentrations may increase rapidly and so easily reach damaging levels. On the other hand, excessive generation of ROS through increased NADPH oxidase activity indirectly damages β-cells by activating NF-kB/p38 MAPK JNK/ stress-activated kinases (SAPK) pathway. In this case, antioxidant capacity is rapidly consumed (Morgan et  al. 2007; Newsholme et  al. 2007). Suppression of NOX1 activity seems to be a new therapeutic strategy to preserve β-cell function in T2D (Weaver et  al. 2015). In contrast, NOXdependent ROS-­mediated signaling is necessary for endocrine cell differentiation, which provides a potential strategy for generation of β-cell (Liang et al. 2016). In addition, highly oxidative conditions induced by saturated free fatty acids (SFFAs) trigger aberrant ER Ca2+ release and thereby deplete ER Ca2+ stores. The resulting ER Ca2+ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca2+ release, cytosolic and mitochondrial matrix Ca2+ concentrations increase. Mitochondrial Ca2+ overload causes superoxide production and functional impairment, resulting in apoptosis (Ly et  al. 2017). Despite the K(ATP) channel-dependent mechanism of GSIS has been broadly accepted, it has become increasingly apparent that it does not fully describe the effects of glucose on insulin secretion (Jensen et  al. 2008). Dysregulation of intracellular Ca2+ homeostasis due to defects in the function of the mitochondrial ER membranes are also involved in the pathogenesis of insulin insensitivity and T2D.  Indeed, mitochondria-­ associated membranes have been shown to be critical for the transfer of stress signals from the ER to mitochondria (van Vliet and Agostinis 2018; Wang and Wei 2017). As another contributing factor, the formation of advanced glycation end products (AGEs) due to hyperglycemia augments ROS generation, and consequently, endogenous antioxidants are consumed increasingly

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(Nowotny et al. 2015). Excessive ROS formation chemically changes almost all cellular components (Rehman and Akash 2017). In this respect, pancreatic β-cells are extremely susceptible to oxidative stress damage, which is generated by chronic exposure to high levels of glucose and SFFAs. In this condition, β-cell dysfunction results in T2D (Sakai et  al. 2003). Meanwhile, oxidative stress leads to the activation of various transcriptional protein kinases, that consist of p38 MAPK, JNK, ERK1/2 and IKKβ. Of these, IKKβ induces the activation of NF-κB, while inhibition of ROS-induced MAPK phosphatases causes activation of JNK, which contributes to β-cell death (Hou et al. 2008; Rehman and Akash 2016). It has long been recognized that T2D develops as a result of the combined effects of insulin resistance and loss of pancreatic islet β-cells (Taylor 2017). Thus, histological studies of the pancreas in T2D patients consistently showed an approximately 50% reduction in number of β-cells compared with normal subjects, and that the mechanism underlying this is increased β-cell apoptosis (Butler et  al. 2003). β-cell loss appears to increase as duration of diabetes increases. While pancreatic insulin concentration is 30% lower in patients with T2D, the average β-cell mass is about 39% lower compared with matched controls (Rahier et al. 2008). Despite the fasting plasma glucose levels are at the upper limit of normal, the ability of the pancreas to mount a normal, brisk insulin response to an increasing plasma glucose level is lost 2 years before the detection of diabetes (Taylor 2013). In prediabetic individuals, while plasma insulin levels increase to maintain normal plasma glucose levels, hyperinsulinemia contributes to hypertriglyceridemia by stimulating de novo lipogenesis. This concept is defined as the “twin cycle hypothesis” of the T2D etiology (Schwarz et al. 2003; Taylor 2013). In advanced phase of T2D, suppression of IRS-1 by serine phosphorylation is one of the mechanisms leading to a decrease in IRS-1 tyrosine phosphorylation, phosphatidylinositol 3-kinase (PI3K) activity and glucose transport. In this context, lipotoxicity and ER stress, tumor necrosis factor-alpha (TNF-α)- suppressed IRS-1 activity and hyperinsulinemia

8  Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes

increase the serine phosphorylation of IRS-1. This process depends on p38 MAPK, JNK and IKKβ activations (Hou et al. 2008; Le MarchandBrustel et  al. 2003; Rehman and Akash 2016). Thus, individuals develop T2D when they exceed their personal fat threshold for safe storage of fat, whether body mass index is high or not (Taylor 2016). Meanwhile the accumulation of intracellular fatty acids causes an impairment of signaling through the IRS/PI3K and a decrease in the recruitment of glucose transporter type 4 (GLUT4) to the cell membrane of peripheral cells (Saini 2010). Eventually, the combination of excessive levels of fatty acids and glucose leads to decreased insulin secretion, and β-cell death by apoptosis. The ERK1/2 pathway, the metabolic sensor Per-Arnt-Sim kinase (PASK), and the activating transcription factor 6 (ATF6) branch of the unfolded protein response inhibit insulin gene expression through the glucolipotoxicity. It is therefore that glucolipotoxicity contributes to β-cell failure in T2D (Fontés et  al. 2009; Poitout et al. 2010; Schwarz et al. 2003). Consequently, in addition to glucolipotoxicity, decreased insulin output due to β-cell failure or death initiates the deleterious vicious cycle observed in T2D (Keane et al. 2015). For normal insulin signaling, the insulin receptor tyrosine kinase phosphorylates the tyrosine residues on IRS proteins, and induces activation of the PI3K-Akt pathway. Whereas, downregulation of the insulin receptor substrates due to constitutive activation of the Ras homologue enriched in brain (Rheb)/the mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K) pathway limits signal ­transmission from the insulin receptor to PI3K and accompanies insulin resistance (Saini 2010; Shah et al. 2004). Collectively, in advanced stage of T2D, insulin, IRS-2, Akt-β, and the forkhead transcription factor (FOXO1a) dysfunctions also result in insulin resistance (Schinner et al. 2005). In fact, dysfunction or death of β-cells are connected to various levels of ER stress. Because hyperglycemic state requires a greater amount of insulin, the main systemic event in T2D is the ensuring of more insulin levels by β-cells and the emergence of insulin resistance. Thereby, β-cells

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respond to such increased demand by increasing their cell mass and insulin secretion. In the preclinical period of T2D, hyperinsulinemia and β-cell hyperplasia develop to compensate for insulin resistance. Relative insulin deficiency in addition to insulin resistance should be detected for the diagnosis of the development of clinically manifest T2D (Fonseca et  al. 2011; Quan et  al. 2013). In brief, central to the process of glucose homeostasis are pancreatic β-cells, which sense elevations in plasma glucose. β-cells respond to these stimuli by releasing the appropriate quantity of insulin, and ensure the arrest of hepatic glucose output, and glucose uptake of peripheral tissues. Since the β-cell failure is associated with the transition from prediabetes to diabetes, functional capacity of β-cells has fundamental role in preventing T2D (Fig. 8.1). Recent data have shown that both insulin secretion and β-cell mass dynamics are regulated by the tumor suppressor liver kinase B1 (LKB1)5′ adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway and related kinases of the AMPK family (Rourke et al. 2018). It is demonstrated that a total of 12 human kinases related to AMPK are phosphorylated by LKB1 (Lizcano et al. 2004). LKB1 is an important regulator of pancreatic β-cell biology. This molecule is a master kinase, controlling the phosphorylation of at least 14 downstream kinases of the AMPK family. LKB1-dependent phosphorylation of distinct AMPK family members determines proper β-cell polarity and restricts β-cell size, total β-cell mass, and GSIS. Although LKB1 is essential for mitochondrial homeostasis in β-cells, it diminishes insulin secretion via the classic triggering pathway. In contrast to AMPK deficiency, LKB1 deficiency in β-cells provides an improvement of GSIS and glucose tolerance. In addition, mTOR/ The eukaryotic translation initiation factor 4E (eIF4)-binding protein-1/S6K signaling is also activated (Sun et al. 2010; Swisa et  al. 2015). LKB1 acts to restrict the size of β-cells via its effect on mTOR signaling and is important for the generation and maintenance of normal polarity of β-cells. Therefore, the modulation of β-cell size is regulated via LKB1AMPK-mTOR-rpS6 axis. In the absence of

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Fig. 8.1 Mechanism of the β-cell death in Type 2 Diabetes (T2D). The cellular toxicity induced by hIAPP, TNF-α, SFFAs and IL-1β in T2D has critical importance for the loss of β-cells. hIAPP causes release of proinflammatory cytokine and chemokine by macrophages. hIAPP formation induces JNK activation, which upregulates both the intrinsic and the extrinsic (Fas, FADD) pathways, resulting in β-cell apoptosis. Because the β-cells have limited antioxidant capacity, ROS concentrations may increase rapidly and so easily reach damaging levels. hIAPP is secreted from β-cell secretory vesicles together with insulin. Insulin resistance results in a higher amount of insulin biosynthesis, and thus of excessive hIAPP secretion. (Abbreviations: ASK1: apoptosis signal-­ regulating kinase 1; Akt: protein kinase B; ATF6: activating transcription factor 6; Bax: B-cell leukemia/ lymphoma-2 (Bcl-2) associated protein x (pro-apoptotic effector); Bim: BH3 domain-only member; CCL2: C-C motif chemokine ligand 2; CHOP: CCAAT-enhancerbinding protein C/EBP; Cp: Caspase; CXCL1: C-X-C motif chemokine ligand 1 that signals through the G-protein coupled receptor, CXC receptor 2; DD: CD95 encompasses an 87-amino-acid-long stretch designated the death domain; DED: death effector domain; DISC: death-­ inducing signaling complex; ER: endoplasmic reticulum; ERK: extracellular signal-regulated kinase; elF2α: eukaryotic translation initiation factor 2α; FADD:

Fas (The receptor CD95)-associated death domain; FAS: Fas cell surface death receptor; FAS-L: a homotrimeric membrane molecule on the regulator cells that binds its receptor Fas on responsive cells; FLIP: FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein; GLUT2: Glucose transporter 2; IKKβ: IκB kinaseβ; IR: Insulin resistance; iNOS: inducible nitric oxide synthase; hIAPP: human islet amyloid polypeptide; IL-1β: interleukin 1β; IL-1βR: interleukin 1β receptor; IRE1α: inositolrequiring protein 1α; JNK: c-Jun N-terminal kinase; KATP ch: ATP-sensitive K+channel; M1: M1 macrophage; MAPK: mitogen-activated protein kinase; MyD88: myeloid differentiation factor-88; mTORC1: mammalian transducers of regulated CREB Activity; Mt.: Mitochondria; NADPH-oxidase: (NOX: Reduced nicotinamide adenine dinucleotide phosphate oxidase) the flavin adenine dinucleotide (FAD)-binding C-terminal transmembrane domain; NF-κB: nuclear factor κB; PERK: protein kinase RNA (PKR)-like ER kinase; PKC: protein kinase C; proCs: pro caspase; ROS: reactive oxygen species; RNS: reactive nitrogen species; SAPK: stress-activated protein kinase; p70S6K: p70 ribosomal S6 kinase; SFFA: saturated free fatty acid; TLR4: Toll-like receptor 4; TNF-α: Tumor necrosis factor α; TNFR: TNF Receptor; TRADD: TNF receptor associated-protein with death domain; TRAF2: TNFR-associated factor 2; VDCC: voltage-dependent Ca2+ channels)

LKB1 or inactivity of its targets, increased transcriptional activity of cyclic AMP (cAMP) response element binding (CREB) protein could positively affect β-cell function. These results suggest that enhanced insulin secretion in LKB1 deficiency is independent of increased β-cell size and hyperactivation of the mTOR pathway. This

means that in β-cells lacking LKB1 or AMPK activity, the biosynthetic machinery remains active and maintains higher levels of intracellular insulin. However, LKB1 can restrict acute insulin secretion in  vivo (Granot et  al. 2009). Dephosphorylation of Ser-275 is essential for

8  Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes

both glucose and cAMP-mediated activation of CREB and transducers of regulated CREB activity (TORC2) in β-cells. In fact, glucose and cAMP promote nuclear relocalization of TORC2. Eventually, nuclear TORC2 can promote CREB activity even in the absence of stimuli. Thus, the phosphorylation status of Ser-275 on TORC2 is modulated by extracellular glucose in islet cells. CREB pathway receives stimuli from glucose via TORC2, which is a molecular link between glucose and the transcription factors, related to β-cell proliferation and survival. In this metabolic pathway microtubule affinity-­regulating kinase 2 (MARK2), a member of the AMPK family of Ser/Thr kinases, blocks TORC2-dependent CREB activity. Indeed, TORC2 represents the sole target of MARK2. Although Ser-171 of TORC2 is a critical site for negative regulation of TORC2  in β-cells, it is surprisingly found that neither Ser-171 nor Ser-275 is regulated by AMPK in β-cells (Jansson et  al. 2008). AMPK activation potentiates β-cell apoptosis induced by chronic high glucose. In this case, oxidative stress suppresses GK (Kim et  al. 2007). Stimulation of autophagy rescues β-cells from high-glucose-induced cell death (Han et  al. 2010). Chronic high glucose-stimulated autophagy is enhanced by inhibition of AMPK, whereas hIAPP significantly promotes autophagy through ROS-mediated AMPK signaling pathway in β-cell. The amyloid deposition is associated with both loss of β-cell mass and deterioration of insulin secretion in T2D, thereby inhibition of hIAPP-­ induced autophagy renders the β-cells more susceptible to hIAPP-induced oxidative stress and toxicity (Han et  al. 2010; Hull et  al. 2004; Xia et al. 2018). Since hIAPP leads to increase in AMPK phosphorylation at Thr172, AMPK signaling pathway is activated by hIAPP in β-cells (Xia et al. 2018). AMPK initiates the autophagy in cells other than β-cell through activating tuberous sclerosis complex (TSC1)1/2, which causes the inhibition of mTOR pathway. In contrast, glucolipotoxicity and hIAPP accumulation inhibits autophagy, via activation of mammalian target of rapamycin complex 1 (mTORC1) in β-cells. AMPK is an inhibitor of mTORC1 signaling. Deletion of well-known AMPK activator LKB1

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leads to activation of mTORC1 signaling in β-cells (Bachar-Wikstrom et al. 2013; Feng et al. 2005; Fu et al. 2009). The mTORC1 is a major regulator of β-cell cycle progression by modulation of cyclin D2, D3, and cyclin-­ dependent kinase 4 (Cdk4) activity (Balcazar et al. 2009). In addition, Akt induces β-cell proliferation in a Cdk4-dependent manner by the regulation of cyclin D1, cyclin D2, and p21 levels (Fatrai et al. 2006). Thus, induction of Akt results in progressive improvement in glucose tolerance and hyperinsulinemia as a result of increased β-cell mass and proliferation (Balcazar et al. 2009). As mentioned above, Akt activation is one of the important components linking growth signals to the regulation of β-cell proliferation. However, mTORC1 activity is modulated by multiple upstream signals, which include growth factors, amino acids, cytokines, energy and cellular stressors. mTORC1 is negatively regulated by TSC1 and TSC2 and positively regulated by the small G protein RHEB.  Phosphorylation and activation of TSC2 by AMPK and GSK3β controls cell growth by inhibiting mTOR signaling (Blandino-Rosano et al. 2012; Inoki et al. 2006).

3

 uman Islet Amyloid H Polypeptide in T2D

The main component of amyloid in β-cell is hIAPP, which contributes to β-cell dysfunction and death (Costes et al. 2013; Westermark et al. 2011). Increased insulin resistance in combination with reduced autophagy increases the toxic potential of hIAPP and enhances β-cell dysfunction and progression of T2DM (Shigihara et  al. 2014). Glucolipotoxicity, and its related hIAPP accumulation in the islet cells are the major factors activating β-cell apoptosis in T2D (Wali et al. 2013). Amylin or hIAPP is a peptide, which is formed of 37 amino acids produced together with insulin in the pancreatic β-cells. Following secretion from intracellular vesicles in T2D patients, cytotoxicity of extracellular hIAPP on β-cells is dependent on hIAPP-induced channel formation, or damage of the β-cells membrane (Mirzabekov et  al. 1996). The cellular toxicity

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induced by hIAPP is mainly mediated by its small pre-fibrillar oligomers, which are found in the human pancreas with T2D and have critical importance for the loss of β-cells (Westermark et al. 2011; Zraika et al. 2010). Initially, hIAPP is inserted in or adsorbed on the membrane, either as monomer or as oligomer. Because monomeric hIAPP has a strong tendency to insert in phospholipid monolayers, at initial step the monomeric hIAPP interacts with membranes much more. While negatively charged lipid interfaces play an important role as the first step of IAPP-­ induced membrane damage, insertion of hIAPP into phospholipid monolayers contributes to the progress in hIAPP-induced membrane damage in T2D (Engel et al. 2006; Lopes et al. 2007). In the next step, interactions of membrane-located hIAPP species with each other or with hIAPP species within the liquid phase lead to growth of fibrils at the membrane. The membrane itself promotes hIAPP fibril growth by increasing the local concentration of membrane-bound hIAPP.  The mechanism of membrane damage entails growth of a rigid hIAPP fibril on a flexible phospholipid bilayer, which results in a forced change in membrane curvature. This change leads to temporal membrane disruption (Engel et  al. 2008). In vivo, hIAPP is secreted from β-cell secretory vesicles together with insulin, approximately in a ratio of 1:10. At this ratio, insulin commonly inhibits hIAPP-induced membrane damage. Thereby, insulin plays a role as a natural inhibitor of hIAPP-induced membrane damage (Engel et al. 2008; Jaikaran et al. 2004). hIAPP fibrils accumulate either freely in the intracellular medium or the development site of the peptide’s oligomers are in secretory granules of the β-cells (Paulsson et al. 2006). An increased production of incompletely processed pro-insulin is a characteristic of patients with T2D. In T2D, any changes in storage or processing of insulin in the islet β-cells could result in destabilization of hIAPP.  In this context, the ratio of insulin to IAPP is critical for intermolecular interaction. Fibril formation is inhibited only when insulin is in a molar excess to IAPP. The proportion of the two peptides in the granules is critical for stabilization. More importantly, insulin resistance

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results in a higher biosynthesis of insulin and thus of hIAPP (Caillon et al. 2016; Jaikaran et al. 2004). Glucolipotoxicity, hIAPP accumulation and increased inflammation impair proinsulin folding, exceed the folding capacity of the ER, and lead to accumulation of misfolded proinsulin, with ER stress. Autophagy cannot be stimulated as an adaptive mechanism to eliminate misfolded proteins, while stimulation of mTORC1 represses autophagy (Bachar-­ Wikstrom et al. 2013). Although the mechanisms of the apoptotic behavior of β-cells could yet to be fully elucidated, there have been some hypotheses concerning the different pathways and triggers that induce cell death (Caillon et al. 2016). The extrinsic pathway involves hIAPP, which could activate the FAS receptor, present on the surface of β-cells. The activation of this “death receptor” results in apoptosis by in turn activating specific proteins caspase-8 and 3 (Law et al. 2010; Park et  al. 2014). The other pathway is linked to intracellular factors. Besides ER stress and unfolded protein response (UPR), other factors disturbing the main function of ER are enhanced hIAPP oligomerization and cell death. Among those, mitochondrial dysfunction, ROS generation, inhibition of autophagy can also be mentioned. Inhibition of lysosomal degradation increases vulnerability of β-cells to hIAPP-­ induced toxicity and, conversely, stimulation of autophagy protects β-cells from hIAPP-induced apoptosis (Rivera et  al. 2011). β-cell toxicity is induced by an inflammatory response linked to hIAPP. Indeed, the insulin resistance and production of hIAPP initiate an increase in the concentration of proinflammatory cytokines such as interleukin 1β (IL-1β). Fibrillogenic amylin can evoke a JNK1-mediated apoptotic pathway, which is partially dependent and partially independent of caspase-8, and in which caspase-3 acts as a common downstream effector (Park et al. 2012; Zhang et al. 2003). In this case, IL-1β plays a dual role by mediating amyloid-induced Fas receptors (FasR) upregulation and by inducing impaired proIAPP processing thereby potentiating amyloid formation (Park et  al. 2017). Actually, normal human pancreatic β-cells that do not constitutively express FasR, become

8  Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes

strongly Fas positive after IL-1β exposure, and are then susceptible to Fas-mediated apoptosis (Stassi et al. 1997). hIAPP causes release of proinflammatory cytokine and chemokine by macrophages (Stassi et  al. 1997). hIAPP-induced TNF-α release is almost entirely dependent on myeloid differentiation factor 88 (MyD88). This effect is regulated via IL-1α and/or IL-1β (Westwell-­Roper et  al. 2011). IL-1β mediates FasR-induced β-cell apoptosis via hIAPP (Park et  al. 2017). Prevention of hIAPP formation markedly reduces IL-1β production, β-cell Fas expression and apoptosis (Park et al. 2017; Park et al. 2012). IL-1 receptor antagonist (IL-1Ra) is a natural inhibitor of IL-1β and is secreted from β-cells. Impairing the balance between IL-1β and IL-1Ra by increasing IL-1β production and reducing IL-1Ra levels promotes the β-cell dysfunction and death (Hui et  al. 2017; Maedler et al. 2004). In human islets, the mechanism underlying glucose-induced β-cell apoptosis and impaired proliferation of β-cell is the up-regulation of FasRs, which interact with the constitutively expressed Fas-ligand (FasL) on neighboring β-cells. In T2D, glucotoxicity induces FasR expression, which can interact with the constitutively expressed FasL on neighboring β-cells expressed FasL (Maedler et  al. 2001). FasR-to-­ FasL interaction then leads to cleavage of procaspase-­ 8 to caspase-8. Activated caspase-8 promotes caspase-3 activation and DNA fragmentation in the Fas apoptotic pathway. However, despite abundant FasR surface expression induced by glucose, only a subpopulation of β-cells undergoes apoptosis. In this case, in apoptosis-­resistant Fas-positive β-cells, cellular caspase-8/FADD-like IL-1β–converting enzyme (FLICE)-inhibitory protein (FLIP) as a protective factor interferes with activation of Fas-induced caspases (Irmler et al. 1997; Maedler et al. 2002). Extrinsic apoptosis is initiated by the formation of the death-inducing signaling complex (DISC) in β-cell. Caspase-8 binding via Fas (the receptor CD95)-associated death domain (FADD) protein to the death receptor is an indispensable initiating step in DISC formation and NF-ĸB activation. So, caspase-8 and its regulator FLICE inhibitory

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protein cellular FLICE-like inhibitory protein (cFLIP) controls death signaling by binding to the death-receptor, which is bound to FADD. FLIP is basely expressed in human pancreatic β-cells. Fas activation, which recruits FLIP together with caspase-8, leads to a secondary degradation of the FLIP–caspase-8 complex. When FLIP expression is decreased, FasR activation promotes apoptosis leading to a loss of β-cells (Horn et al. 2017; Irmler et al. 1997; Maedler et al. 2002). In T2D, long-term exposure to elevated glucose concentrations result in a marked inhibition of the proliferative capacity of β-cells in parallel with the decreased cFLIP expression. Simultaneously hIAPP-induced caspase-­8 activation is associated with FasR activation and FLIP suppression. Consequently, in addition to impaired proliferation, β-cell apoptosis associates with decreased FLIP expression and induction of FasR and caspase-8  in T2D.  Therefore, intact FLIP protects human β-cells from glucoseinduced apoptosis and restores β-cell proliferation (Maedler et  al. 2002; Park et  al. 2014). hIAPP aggregates increase IL-1β levels in human islets that correlate with β-cell Fas upregulation, caspase-8-FLICE activation and apoptosis. All of these are reduced by IL-1Ra production. Indeed, IL-1β plays a dual role by mediating amyloidinduced Fas upregulation and β-cell apoptosis. Thus, blocking IL-1β preserves β-cells during the islet amyloid formation (Park et  al. 2017). Deletion of FasR protects islet β-cells from the cytotoxic effects of endogenously secreted hIAPP (Park et al. 2012). In contrast, β-cell FasR upregulation by endogenously produced hIAPP aggregates promotes caspase-8 activation, resulting in β-cell apoptosis (Park et al. 2014). Glucose-induced β-cell apoptosis is mediated by the intrinsic apoptosis pathway, which includes one B-cell leukemia/lymphoma-2 (Bcl-­ 2) homology domain (BH3-only proteins member, Bcl-2 interacting mediator of cell death (Bim) and pro-apoptotic effector, Bcl-2 associated protein x (Bax)). Thus, cellular stress activates the pro-apoptotic Bcl-2 family members (McKenzie et al. 2010). BH3-only protein BH3 interacting domain death agonist (Bid) is essential for death receptor-induced apoptosis of

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β-cell. Bid deficiency prevents FasL–induced β-cell survival and proliferation in T2D (Zhang β-cell apoptosis (McKenzie et al. 2008). In con- et al. 2018). As shown in these studies, prefibriltrast, Bcl-2 overexpression, loss of the BH3-only lar hIAPP aggregates promote islet dysfunction proteins Bim or p53 upregulated modulator of not only via direct toxicity to β-cells, but also by apoptosis (Puma), or loss of the multi-BH domain triggering a localized inflammatory response. protein Bax markedly protects the β-cell from Thus, strategies aimed at reducing hIAPP expresglucotoxicity. However, loss of Bax affords con- sion and aggregation may not only protect β-cells siderably less protection than combined loss of from apoptosis, but also ameliorate deficits in Bim and Puma in T2D (McKenzie et al. 2010). insulin secretion associated with proinflammahIAPP formation induces JNK activation, which tory cytokine release (Westwell-Roper et  al. upregulates both the intrinsic and the extrinsic 2011). (Fas, FADD) pathways, resulting in β-cell apoptosis (Subramanian et  al. 2012). In this regard, TNF receptor 1 (TNFR1), TNFR1-associated 4 β-Cell, ER Stress and Protein death domain protein (TRADD), FADD, and Kinases FLICE are expressed in the pancreatic β-cell. Thereby, TNF-α can cause apoptosis in pancre- It is fact that, intermittent hyperglycemia induces atic β-cells through TNFR1-linked apoptotic fac- a higher degree of apoptosis, thereby decreases tors, TRADD, FADD, and FLICE (Ishizuka et al. the insulin secretory capacity more in pancreatic 1999). Although, TNF-activated pathways capa- β-cells than chronic hyperglycemia (Kim et  al. ble of inducing apoptotic cell death are present in 2010). Constant demand from the body for insuβ-cells, caspase activation is the dominant path- lin biosynthesis and secretion, in response to way of TNF-induced cell death and is an impor- fluctuations in blood glucose levels, has made the tant mechanism of β-cell damage in β-cells dependent on UPR. The resulting insulin insulin-dependent T2D (Stephens et  al. 1999). resistance leads to excess insulin production in Furthermore, the genes encoding receptor-­ the β-cell. This increase in insulin biosynthesis interacting protein kinase 1 (RIPK1), RIP-­ overwhelms the folding capacity of the ER, leadassociated Ich-1/Ced-3 homologous protein with ing to chronic accumulation of unfolded protein a death domain (RAIDD), TNFR1, TRADD, in β-cells cause β-cell failure and T2D.  This BAX, BCL1/2, calcium activated neutral prote- chronic hyperactivation of ER stress signaling ase (CAPN) 1 and CAPN2 are much more highly invokes oxidative stress, and leads to β-cell dysexpressed in T2D β-cells than the healthy ones. function and eventually β-cell death (Fonseca Thereby the death receptor, TNFR1-mediated et  al. 2009; Scheuner and Kaufman 2008). pathway, mitochondrial BAX-related pathway, as Indeed, when the folding capacity of the ER is well as the CAPN1- and CAPN2-dependent exceeded, misfolded or unfolded proteins accupathway may be crucial in T2D (Ma and Zheng mulate in the ER lumen, resulting in ER stress. 2018). UPR is a consequence of the cytoprotective hIAPP formation impairs the balance between response to ER stress. Thus, paradoxically, UPR IL-1β and IL-1Ra and promotes β-cell dysfunc- signaling activation leads to opposite cell fates, tion and death. Restoring the IL-1β/IL-1Ra ratio that is adaptation to either survival or death may provide an effective strategy to protect (Rabhi et al. 2014). Thus, the UPR functions as a β-cells from hIAPP toxicity in T2D (Hui et  al. dual switch between life and death in β-cells. 2017). In this context, hIAPP formation leads to This dual switch mechanism is regulated by the reduced Akt phosphorylation in β-cells which is three main transmembrane protein sensors of the associated with elevated islet IL-1β levels. UPR, which are inositol-requiring protein 1α Inhibitors of amyloid or amyloid-induced IL-1β (IRE1α), protein kinase RNA-like endoplasmic production may provide a new approach to reticulum kinase (PERK), and ATF6 (Fonseca restore phospho-Akt levels thereby enhance et al. 2011; Ron and Walter 2007). Following ER

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stress increasing the protein-kinase activity, mediator of TRAF2-induced JNK activation PERK phosphorylates the eukaryotic translation (Nishitoh et  al. 1998; Urano et  al. 2000). initiation factor 2α (eIF2α) and inhibits transla- Activation of JNK alone is sufficient to induce tion of messenger RNA into protein. In this insulin resistance in pancreatic β-cells by inhibitrespect, PERK signaling pathway attenuates pro- ing insulin signaling in these cells, but it is not tein translation in response to ER stress (Harding enough to elicit β-cell death (Lanuza-Masdeu et  al. 1999). The two major signaling pathways et al. 2013). Chronically elevated concentrations that regulate protein synthesis during ER stress of leptin and glucose induce β-cell apoptosis are the PERK-mediated phosphorylation of the α through activation of the JNK pathway in human subunit of the translation initiation factor eIF2 at islets. In contrast, JNK inhibition protects β-cells serine 51 (eIF2α-P) and the network of mTORC1-­ from the deleterious effects of high glucose and targeted Akt phosphorylation (Appenzeller-­ leptin in T2D (Maedler et  al. 2008). ER stress-­ Herzog and Hall 2012; Pavitt and Ron 2012). The mediated pancreatic β-cell destruction can be master regulator of UPR is the PERK/eIF2α/ protected via regulation of the AMPK-PI3 kinase-­ ATF4 signaling pathway. In this pathway, eIF2α JNK pathway (Jung et al. 2012). is phosphorylated (eIF2α-P) by the kinase PERK, On the other hand, stimulation of the IRE1αwhile ATF4 induces selected amino acid trans- receptor for activated C kinase 1 (RACK1) interporters and aminoacyl-tRNA synthetases. action accompanies glucose-induced IRE1α Paradoxically, ATF4 activity leads cells to apop- phosphorylation and represents a specific tosis during chronic ER stress. The PERK/ glucose-­ sensing event in pancreatic β-cells. eIF2α-P/ATF4 signaling acts as a brake in the Under ER stress due to excessive glucose stimudecline of protein synthesis during chronic ER lation, RACK1, which is an adaptor protein for stress by positively regulating signaling down- activated protein kinase C, mediates IRE1α, stream of the mTORC1 activity. mTORC1 activ- RACK1, and protein phosphatase 2 (PP2A) comity is in parallel to the eIF2α-P mechanism and it plex formation. This complex promotes IRE1α independently causes the decline of protein syn- dephosphorylation by PP2A, thereby inhibits thesis during chronic ER stress (Guan et  al. IRE1α activation and attenuates IRE1α-­ 2014). In ATF4-mediated transcription program, dependent increase in insulin production. regulation of amino acid transport in β-cells dur- Defective signaling through the RACK1-PP2A ing ER stress involves responses leading to regulatory loop in the control of the IRE1α sigincreased protein synthesis, which can be protec- naling pathway results in ER stress-related gradtive during acute stress but can lead to apoptosis ual deterioration of β-cell functions and a during chronic stress (Krokowski et al. 2013). In reduction in pancreatic β-cells mass. This reprethis context, prolonged activation of the UPR in sents a potential pathophysiological mechanism β-cells leads to apoptosis, which limits c­ irculating in T2D (Qiu et  al. 2010; Rabhi et  al. 2014; insulin levels and leads to T2D (Oslowski and Scheuner and Kaufman 2008). Human islets Urano 2011). Thus, Krokowski et  al. have sug- from type 2 diabetic donors are reportedly 80% gested that increased expression of system amino deficient in the p21 (cell division control protein acid transporters in β-cells promotes develop- 42 (Cdc42)/ a subfamily of the Rho family of ment of T2D (Krokowski et al. 2013). GTPases (Rac))-activated kinase1, PAK1. PAK1 IRE1 as a central regulator of the UPR, is a is implicated in β-cell function and maintenance type I ER transmembrane kinase. High levels of of β-cell mass. PAK-inhibition leads to defective chronic stress lead to the recruitment of TNF-­ ERK and Akt activation, resulting in reduced proreceptor-­associated factor 2 (TRAF2) by IRE1. liferation and increased apoptosis. Eventually, ER stress activates apoptosis-signaling-kinase PAK1 deficiency potentially contributes to (ASK) 1 through formation of an IRE1-TRAF2-­ increased susceptibility to T2D (Ahn et al. 2016). ASK1 complex. Activated ASK1 promotes JNK Deficiency of p21 sensitizes pancreatic β-cells to and leads to apoptosis. Thus, ASK1 acts as a glucotoxicity. p21 acts as an inhibitor of

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ER-stress-associated tissue damage and that stimulation of p21 activity can be beneficial for the management of T2D (Mihailidou et al. 2015). β-cell is a highly specialized secretory cell which responds to elevated postprandial glycaemia by increasing mRNA proinsulin translation and insulin secretion (Itoh and Okamoto 1980). In this respect, β-cells adapt their secretory capacity to compensate the elevated glycaemia and the peripheral insulin resistance. In fact, the correct UPR and antioxidant response controlled by PERK-elF2α signaling are required for β-cell adaptation and survival. Increased proinsulin synthesis causes oxidative damage in β-cell and this reflects the β-cell failure associated with insulin resistance in T2D.  Mitochondrial swelling occurs in the eIF2α phosphorylation-deficient β-cells, suggesting altered mitochondrial function may play a role in ROS production (Back et al. 2009). In fact, chronic exposure to abnormally high blood glucose has detrimental effects on insulin synthesis/secretion, cell survival and insulin sensitivity through multiple mechanisms. Hyperglycemia increases the metabolic flux into the mitochondria and induces excessive generation of ROS which leads to chronic oxidative stress. Finally, oxidative stress activates stress-­ induced pathways that damage the β-cell (Robertson et al. 2003, 2004). Nox2 is necessary in the generation of ROS under high glucose concentrations. Rac1, is an integral member of the Nox2 holoenzyme. The Rac1-Nox2 signaling plays novel regulatory roles in high glucose-­ induced p38MAPK activation, loss in GSIS and the Nox2-p38MAPK signaling axis in the β-cell (Sidarala et al. 2015). Indeed, oxidative stress is an important risk factor for β-cell dysfunction in T2D. Endogenous antioxidant enzymes that can be induced in response to exposure to oxidative stress have the potential to inhibit glucose-­ triggered ROS signal, in addition to repressing the GSIS of β-cells (Pi et al. 2007). As a cellular adaptive response to oxidative stress challenge, nuclear factor erythroid 2-related factor 2 (Nrf2)mediated antioxidant induction plays paradoxical roles in pancreatic β-cell function. On the one hand, induction of antioxidant enzymes protects β-cells from oxidative damage and possible cell

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death, thus minimizing oxidative damage-related impairment of insulin secretion. On the other hand, the induction of antioxidant enzymes by Nrf2 activation blunts glucose-triggered ROS signaling, thus resulting in reduced GSIS. These two steps are relevant to impairment of β-cells occurring in the late and early stage of T2D, respectively (Pi et al. 2010). Together with hyperglycemia, hyperlipidemia results in insulin resistance, thereby both contributes to ROS production in β-cells. Oxidative stress and consequent activation of the JNK pathway are involved in progression of β-cell dysfunction found in T2D.  These findings suggest that antioxidants may improve outcomes of standard therapy of T2DM in humans (Kajimoto and Kaneto 2004; Robertson et  al. 2007). In this respect, specific antioxidants and ROS donors may need to be considered in future therapeutic approaches in T2D (Pi et al. 2010). However, even the successful antioxidant supplements do not easily penetrate the cell membrane. For T2D patients, taking antioxidant supplements may even exacerbate their diseased conditions because of the further dampening of ROS signaling by exogenous antioxidants. Thus, the Nrf2-mediated antioxidant response interacting with the antioxidant response element (ARE) represents a critically important cellular defense mechanism that serves to maintain intracellular redox homeostasis and limit oxidative damage. Thereby, Nrf2 activation contributes to the maintenance of intracellular glutathione levels, which in turn functions as a buffer for the accumulation of ROS during the UPR.  Furthermore, Nrf2-mediated antioxidant response plays a paradoxical role in insulin secretion. On the one hand, it protects β-cells from oxidative damage via minimizing oxidative damage-­related impairment of insulin secretion. On the other hand, situations leading to chronic induction of endogenous antioxidants due to oxidative stress blunts endogenous ROS signaling, resulting in reduced GSIS (Cullinan and Diehl 2004; Nguyen et  al. 2003; Pi et  al. 2010). To avoid interference with intracellular ROS signaling for insulin secretion, which is mostly required postprandially glucagon-like peptide-1 (GLP-1) receptor agonist (Exendin-4) promotes at least

8  Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes

three Nrf2-related adaptive responses; Nrf2 translocation, its binding activity to ARE, and increasing stabilization of Nrf2 by inhibition of ubiquitination. Thereby Exendin-4 contributes to the increase of antioxidant capacity and consequently can suppress β-cell apoptosis, improve β-cell function and protect against oxidative damage (Kim et al. 2017). GLP-1 enhances cellular levels of glutathione and the activity of its related enzymes, glutathione-peroxidase (GPx) and -reductase (GR) in β-cells through the translocation of the Nrf2. However, inhibition of protein kinase A (PKA) abolishes the GLP-1-derived protective effect on β-cells (Fernández-Millán et al. 2016). Under hyperglycemic conditions, increased intracellular methylglyoxal (MGO), which is mainly generated as a byproduct of glycolysis, induces cytotoxicity by activating oxidative stress, triggering mitochondrial apoptotic pathway, and ER stress-mediated IRE1α-JNK pathway. To counteract these effects of MGO, organism has an enzymatic glyoxalase defense system that catalyzes the conversion of MGO to d-lactate. Glyoxalase 1 (GLO1) is the key enzyme in this defense against glycation (Liu et al. 2017; Schalkwijk 2015). IRE1α plays a central role in β-cell adaptation to ER stress. On the one hand, hyperphosphorylation of IRE1α due to chronic high glucose exposure is a β-cell-protective mechanism which reduces insulin secretion in patients with T2D (Rabhi et  al. 2014). On the other hand, chronic activation of IRE1 during prolonged increases in insulin biosynthesis, however, may lead to β-cell death through ­IRE1-­mediated activation of JNK (Urano et  al. 2000). UPR restores tissue homeostasis following ER stress, whereas prolonged ER stress triggers β-cell apoptosis through the UPR-activated transcription factor CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) in T2DM (Mihailidou et al. 2014). Hyperglycemia-­ induced ER stress causes CHOP induction in β-cells. Furthermore, CHOP plays an important role in β-cell death and promotes the progression of T2D (Oyadomari et al. 2002). As another metabolic disturbance, elevated levels of free fatty acids (FFAs) in circulation are

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also an important risk factor for the development of T2D (Kahn et al. 2006). Thus, palmitate is one of the most abundant dietary saturated FFAs (SFFAs) inducing β-cells apoptosis. Decreased β-cell mass is increasingly recognized as one of the main factors in the pathogenesis of T2D.  Chronically palmitate overload of ER in β-cells causes ER stress and leads to apoptosis via eIF2α, ATF4, and CHOP induction. β-cell functions and survival are altered by the palmitate-­induced rapid phosphorylation of the ER Ca2+ sensor protein kinase, PERK and subsequent ER stress. However, high levels of stearoyl-­ CoA (stearoyl-coenzymeA) desaturase-1 decrease susceptibility of these cells to the cytotoxic effects of palmitate (Butler et  al. 2003; Gwiazda et  al. 2009; Karaskov et  al. 2006; Lai et al. 2008). In T2D, elevated levels of SFFAs and glucose contribute to a state of glucolipotoxicity. β-cells do not tolerate chronic elevations in SFFA levels. Prolonged exposure to FFAs has cytostatic and pro-apoptotic effects on human pancreatic β-cells. β-cell death induced by SFFAs is related to 2 to fivefold increase in caspase-2 activity, but not to caspase-3 activation. Indeed, SFFAs are lipotoxic whereas long chain mono-unsaturated fatty acids can provide cytoprotection (Fürstova et al. 2008; Lupi et al. 2002; Morgan and Dhayal 2010). In this context, stearic and palmitic acids induce apoptosis in pancreatic β-cells, whereas unsaturated fatty acids, oleic and palmitoleic acids maintain β-cell viability (Šrámek et  al. 2016). SFFAs are shown to induce ER stress in pancreatic β-cells. During the cellular adaptation to ER stress, UPR transmits the information about the protein folding status at the ER to the nucleus and cytosol to restore ER homeostasis. Activation of IRE1α leads to JNK activation by phosphorylation. Eventually, ER stress can mediate cell death induction (Biden et al. 2014; Hetz et  al. 2011; Sano and Reed 2013). JNK, PKC, p38 MAPK, ERK, and Akt kinases and their pathways involve in apoptosis induction by SFFAs (Šrámek et al. 2016). Hydrolysis of inositol phospholipids by phospholipase C is the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular events

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through activation of PKC (Nishizuka 1992). vation and translocation from cytosol to the Glucolipotoxicity inhibits ceramide utilization nucleus is necessary for apoptosis induction of for complex sphingolipid biosynthesis, thereby β-cells by SFFAs in contrast to unsaturated FFAs reducing the flow of ceramide from the ER to (Eitel et al. 2003). In ER-stressed β-cells, inducgolgi (Hanada et  al. 2009). Two sphingolipid tion of TRB3 results in β-cell apoptosis via the transfer proteins, ceramide transfer protein activation of NF-κB (Fang et al. 2014). Cytokines (CERT) and phosphate adaptor protein 2 (FAPP2) favor JNK activation via the mixed-lineage proregulate lipid metabolism on the cytosolic side tein kinase3 (MLK3)-mitogen-activated protein (Yamaji et al. 2008). Consequently, glucolipotox- kinase kinase 7 (MKK7)-JNK-JNK-interacting icity impairs ceramide traffic from the ER to the protein 1 (JIP1) (scaffold protein in β-cells) sigGolgi apparatus, thus promoting the accumula- naling pathway (Tournier et al. 2001; Whitmarsh tion of ceramide in the ER in β-cells. PI3K/Akt et al. 2001). Indeed, MLK3 is a pivotal kinase in pathway regulates ceramide metabolism by con- cytokine-­activated β-cell death. In the β-cell, actitrolling the vesicular transport in β-cells (Gjoni vation of MLK3 not only regulates JNK activaet al. 2014). tion but also lowers Akt-dependent resistance to Lipid metabolites generated by signal-induced apoptosis via pseudokinase TRB3. Thereby, hydrolysis of membrane phospholipids, such as TRB3 is a negative regulator of Akt, which is a ceramide and phosphatidylinositol-3,4,5-­ key mediator of MLK3 effects. In this process triphosphate, have the potential to mediate exter- MLK3 stabilizes TRB3 protein levels to suppress nal signals (Musashi et al. 2000). PKC-associated Akt survival kinase and facilitate proinflammaapoptotic signals induce rapid post-translational tory effects of IL-1β (Humphrey et  al. 2010). modification of PKC-delta (PKC-δ) in the regu- Change in kinetics of MLK3-JNK activation can latory domain, which facilitates translocation of impact β-cell inflammation and survival. the kinase from the cytoplasm to the nucleus. Cytokines are required at a threshold of proinActive caspase 3 also accumulates in the nucleus flammatory signal activation for inducing β-cell resulting in caspase cleavage of PKC-δ and gen- death (Humphrey et al. 2014). In this case, both eration of a constitutively activated form of PKC- the functional instability of GSIS and stressδ. Nuclear accumulation of PKC-δ is essential induced apoptotic/necrotic β-cell death due to for apoptosis (Reyland 2007). Tribbles homolog MLK3- and TRB3-­ driven mitochondrial dys3 (TRB3) is a pseudokinase inhibiting Akt, a key function emerge as hallmarks of T2D (Humphrey mediator of insulin signaling, and contributes to et al. 2010; Szabadkai and Duchen 2009). insulin resistance in insulin target tissues. The In fact, glucolipotoxicity contributes to a overexpression of TRB3 by pancreatic β-cells decline in insulin-producing β-cell mass through leads to exacerbated apoptosis triggered by activation of the NF-κB and signal transducer SFFAs. In this case, PKC-δ activation mediates and activator of transcription 1 (STAT1). This apoptosis induction by palmitate. Indeed, activa- data indicates that, tumour necrosis factor reception and nuclear accumulation of PKC-δ is tor (TNFR5) signaling has a major role in triggerenhanced by TRB3 expression. Increased TRB3 ing glucolipotoxic β-cell death (Bagnati et  al. expression results in SFFAs-­induced β-cell apop- 2016). The atypical activation of the non-­ tosis via PKC-δ pathway. Inhibition of the TRB3/ canonical NF-κB pathway by proinflammatory PKC-δ axis is relevant for preservation of β-cell cytokines constitutes a novel mechanism that mass and is important in the conditions of contributes to the pro-apoptotic effect of NF-κB increased serum SFFA levels (Qin et  al. 2014). in β-cells (Meyerovich et  al. 2016). β-cells Palmitate induced apoptosis in β-cells is accom- respond to palmitate via the SFFA-toll like receppanied by nuclear translocation of PKC-δ. This tor 4 (TLR4)/MyD88 pathway and produce chetranslocation is prevented by the phospholipase C mokines that recruit CD11b(+)Ly-6C(+) M1-type inhibitors, which also substantially reduces apop- proinflammatory monocytes/macrophages to the tosis. These evidences confirm that PKC-δ acti- islets. M1-like macrophage-induced islet inflam-

8  Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes

mation contributes to palmitate-induced β-cell dysfunction without affecting insulin sensitization (Eguchi et  al. 2012). In fact, macrophages are present within healthy islets and are important for the maintenance of β-cell mass and proliferation of β-cells in response to increased workload (Banaei-Bouchareb et al. 2006; Eguchi and Nagai 2017). TLR2 and TLR4 together with the nucleotide-binding oligomerization domain, Leucine-rich Repeat and Pyrin domain containing-­3 (NLRP3) inflammasome are the triggers of pancreatic islet inflammation in T2D.  Thereby, NLRP3 mediates the activation of macrophages and triggers β-cell dysfunction (Westwell-Roper et al. 2014). In this context, blocking pathologic T2D islet inflammation to ameliorate β-cell dysfunction while promoting physiologic immune cell function to enhance β-cell proliferation may be an effective strategy in T2D (Eguchi and Nagai 2017). Sustained SFFA-induced stress results in mitochondrial damage in β-cells. Damaged mitochondria are eliminated by mitophagy. Palmitic acid-induced mitophagy deficiency is driven by decreased Rheb (Ras-like small GTPase) and Kinesin superfamily protein (KIF5B) expression, and disassociation from mitochondria. Palmitic acid-ROS-NLRP3 pathway is strongly associated with mitophagy dysfunction, proinflammatory responses and insulin resistance (Yang et al. 2014). On the other hand, the expression of mitochondrial Rho GTPase (Miro1) is reduced in human T2D. Deficiency in Miro1-mediated mitophagy triggers mitochondrial ROS.  Inhibited mitophagy causes ­mitochondrial dysfunction, and unbalanced activation of MKK-JNK pathway, which results in insulin resistance via IRS-Akt-FoxO1 inhibition (Chen et al. 2017). Thereby mitophagy dysfunction due to SFFA-induced stress contributes to the etiology of T2D. Cytokines induce a NF-κB activation and nitric oxide-dependent disruption of the mitochondrial membrane potential and this may be a necessary event for both β-cell apoptosis and necrosis. Overexpression of the anti-apoptotic protein Bcl-2 may prevent β-cell death by counteracting mitochondrial permeability transition (Barbu et  al. 2002). Thus, high-mobility group

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box-1 (HMGB1) can signal through receptor for advanced glycation end products (RAGE) and TLRs to activate NF-κB signaling pathway, which results in increased production and release of cytokines via the p38 MAPK, PI3K/Akt, and ERK1/2 pathways (Klune et al. 2008; Park et al. 2003). Thus, HMGB1  in patients with T2D is significantly promoted by the glucose, in an association with an upregulation of pro-inflammatory cytokines, via activating NF-κB signaling pathway. In these cases, HMGB1 inhibition reduces the pro-inflammatory cytokines in response to high glucose (Chen et al. 2015; Wang et al. 2016). In palmitate-exposed β-cells, interaction between TLR and MyD88 enhances. Concomitant with TLR/MyD88 interaction, the level of phospho-­ JNK increases, whereas the level of IκBα decreases. This finding suggests that JNK inhibitors significantly protect against palmitate-­ induced β-cell death, contrarily IKK inhibitor does not (Lee et  al. 2011). Double-stranded RNA-dependent protein kinase R (PKR) involves both in insulin resistance and in downregulation of pancreatic β-cell function in a kinase-­ dependent manner in the progression of T2D. In contrast, protein-binding function of PKR is associated with the proliferation of pancreatic β-cells through TRAF2/RIP1/NF-κB/c-Myc pathways. Therapeutic approach for T2D may arise due to kinase catalytic function of PKR (Gao et  al. 2015). GSIS in pancreatic β-cells depends on coordinated glucose uptake, oxidative metabolism, and Ca2+-triggered insulin exocytosis. NF-κB is activated by an increase in intracellular Ca2+ in β-cells. Attenuation of NF-κB activation in β-cells impairs GSIS (Norlin et  al. 2005). Mitochondrial oxidative damage plays a key role in pancreatic β-cell failure in the pathogenesis of T2D.  In glucolipotoxicity, the expression levels of mitochondrial antioxidant enzymes, β-cell apoptosis, lipogenic enzymes, oxidative stress, ER stress, mitochondrial membrane depolarization, NF-κB and sterol regulatory element binding protein 1c (SREBP)-1c are all increased. Mitochondria-targeted antioxidants protect pancreatic β-cells against oxidative stress, promote their survival, and increase insulin secretion in glucolipotoxicity associated T2D (Lim

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et  al. 2011). Palmitate induces the SREBP-1c expression along with the downregulation of pancreatic and duodenal homeobox (Pdx)-1 and GLP-1 receptor (GLP-1R). These are two essential effectors for the β-cell function. Either SREBP-1c deprivation or Pdx-1 overexpression relieves palmitate-induced GSIS impairment. SREBP-1c-Pdx-1-GLP-1R signaling pathway involves in the SFFA-induced GSIS impairment and the development of T2D (Shao et  al. 2009, 2010). Since dietary SFFAs stress increases the expression level of SREBP-1c, suppression of IRS-2/Akt pathway is a part of the downstream mechanism for the SREBP-1c-mediated insulin secretion defect. Palmitate-induced impairment of insulin secretion is restored by supplement of polyunsaturated fatty acids through SREBP-1c and uncoupling protein 2 (UCP2). The onset of T2D because of the intake of excess SFFAs causes both insulin resistance and impaired insulin secretion in β-cells (Kato et al. 2008). On the other hand, excessive eIF2α phosphorylation is poorly tolerated by β-cells and exacerbates SFFA-induced apoptosis. The synergistic activation of the PERK-eIF2α sensors of the ER stress response and ATF6 pathways lead to a marked induction of ATF4 and the pro-apoptotic transcription factor CHOP.  Proapoptotic marker CHOP plays an important role in palmitate-­ induced β-cells apoptosis. CHOP transcription in response to cytokines and palmitate depends on the binding of ATF4 and activator protein (AP)-1 to the CHOP promoter (Cnop et  al. 2007; Karaskov et  al. 2006; Pirot et  al. 2007). The IRS-2 branch of the insulin/insulin-like growth factor (IGF)-signaling pathway is a common element in peripheral insulin response and pancreatic β-cell growth and function. The loss of compensatory hyperinsulinemia over long periods of peripheral insulin resistance is associated with the failure of IRS-2 signaling (White 2002). Fluctuations of blood glucose promote β-cell survival via regulation of IRS-2 expression and a subsequent parallel protein kinase B activation (Lingohr et  al. 2006). Control of basal IRS-2 gene transcription is critical to normal β-cell function. Disruption of IRS-1 retards growth, but diabetes does not develop because insulin secre-

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tion increases to compensate for the mild resistance to insulin. Disruption of IRS-2 impairs both peripheral insulin signaling and pancreatic β-cell function. Dysfunction of IRS-2 may contribute to the pathophysiology of human T2D (White 2002; Withers et al. 1998). GK promotes β-cell growth and prevents glucotoxic β-cell apoptosis. GK regulates β-cell mass as well as β-cell function. IRS-2 is found to be involved in GK-mediated β-cell hyperplasia in high fat diet–fed subjects. This approach may compensate for β-cell loss in patients with T2DM (Oh et  al. 2014; Terauchi et al. 2007). GK upregulates IRS-2 expression in β-cells. GKA suppresses the expressions of CHOP and Bax and protects against β-cell apoptosis under ER stress in an ERK1/2-dependent, IRS-2-independent manner (Shirakawa et  al. 2013). GKA promotes β-cell growth and prevent glucotoxic β-cell apoptosis via upregulation of IRS-2 and subsequent activation of AKT/PKB phosphorylation. Anti-apoptotic effects of GKA are against glucotoxicity-, oxidative stress- and ER stress-induced β-cell death. Additionally, GKA increases beta-catenin and cyclin D2 mRNA expression and inactivates GSK3β by increasing phosphorylation (Oh et al. 2014; Wei et al. 2009). GSK3β is also involved in palmitate-­ induced caspase-3 dependent apoptosis (Huang et  al. 2014). IRE1-JNK signaling, CHOP, and GSK3β contribute to the β-cell loss. Activation of the UPR is critical for the survival of insulin-­ producing pancreatic β-cells with high secretory protein production. Any disruption of ER homeostasis in β-cells can lead to cell death and contribute to the pathogenesis of T2D (Fonseca et  al. 2009). Indeed, a third signaling component, GSK3β, also plays a role in β-cell death caused by ER stress. GSK3β is a substrate of the survival kinase, Akt. GSK3β is inhibited by serine phosphorylation in response to insulin by either MAP kinase-activated protein (MAPKAP) kinase-1 or p70 ribosomal S6 kinase (Cross et al. 1995). The MKK3/6-p38 MAPK-MAPKAPK/2 pathway is activated in response to pro-apoptotic concentrations of SFFAs in human pancreatic β-cells. In this condition, SFFAs inhibit ERK1/2 kinase (Šrámek et  al. 2016). ERK1/2 are serine-­ threonine kinases and are well-known members

8  Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes

of the MAPK kinase family. It is a part of the c-Raf-MEK1/2-ERK1/2 signaling pathway (Bramanti et al. 2015). Chronic exposure to elevated levels of palmitate has an inhibitory effect on proinsulin, through the generation of high levels of intracellular ceramide. This leads to a significant decrease in the levels of activated ERK.  The ceramide-induced inactivation of the ERK is stimulated by PP2A activity (Guo et al. 2010). However, Watson et  al. claimed that palmitate-­induced defect in insulin release from β-cells is dependent on reduced calcium/ calmodulin-­ dependent protein kinase II (CaMKII) and ERK activation rather than the changes in ceramide content or mitochondrial fatty acid accumulation (Watson et al. 2011). Inhibition of ERK1/2 partially prevents the inhibition of insulin gene expression in pancreatic β-cells in the presence of palmitate or ceramide. Glucose-induced expression of the serine/threonine Per-Arnt-Sim domain-containing kinase (PASK) mRNA and protein levels is reduced in the presence of palmitate. PASK is a novel mediator of glucolipotoxicity on the insulin gene in pancreatic β-cells (Fontés et  al. 2009). The PASK regulates insulin gene transcription via Pdx-1. PASK phosphorylates and inactivates GSK3β, thereby preventing Pdx-1 serine phosphorylation and alleviating GSK3β-mediated Pdx-1 protein degradation in pancreatic β-cells (Semache et  al. 2013). Actually, ER stress– induced apoptosis is associated with activation of JNK and reduces insulin signaling, evidenced by attenuated Akt phosphorylation and resultant dephosphorylation of GSK3β (Srinivasan et  al. 2005). However, down-regulation of GSK3β protects cells from ER stress-induced apoptosis without altering the level of ER stress proapoptotic marker CHOP.  Collectively, ER stress-­ induced apoptosis is mediated by signaling through the PI3K/Akt/GSK3β pathway (Kim et al. 2005a; Srinivasan et al. 2005). Reduction in GSK3β expression creates resistance to ER stress-induced apoptosis. Therefore, GSK3β, rather than CHOP, may be a more promising novel therapeutic target for T2DM to promote β-cell survival (Huang et  al. 2014; Srinivasan et al. 2005). Mechanistically, increase in insulin

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biosynthesis overwhelms the folding capacity of the ER, leading to chronic activation of the UPR.  Chronic ER stress signaling results in IRE1-JNK signaling, CHOP, and GSK3β activation. Eventually, all these contribute to β-cell dysfunction and death (Fonseca et  al. 2009). Contrarily, JNK inhibition results in increased adaptive UPR and reduced cell death in islets from diabetic subjects. Restored adaptive UPR protects against apoptotic UPR gene expression and β-cell death and dysfunction (Chan et  al. 2015). If the effects of IRE1 on insulin are mentioned briefly, the IRE1 pathway is important in insulin biosynthesis, where transient increases in insulin production lead to IRE1 activation. Phosphorylation of the ER resident kinase, IRE1α, which occurs as a result of acute exposure of the β-cell to high glucose, is coupled to insulin production. Chronic exposure of β-cells to high glucose causes ER stress and hyperactivation of IRE1, leading to the suppression of insulin gene expression. Consequently, IRE1 signaling is therefore a potential target for therapeutic regulation of insulin biosynthesis (Lipson et al. 2006). IRE1/X-box binding protein 1 (XBP1s), and PERK/ATF4/CHOP mediates the UPR-induced sensitization of pancreatic β-cells to the proinflammatory effects of cytokines (Eizirik et  al. 2013). Induction of IRE1α-interacting protein ubiquitin D (UBD) is mostly an effect of interferon-­gamma (IFN-γ), with a minor contribution by IL-1β. Deficiency of both UBD and IRE1α reverse the up-regulation of JNK phosphorylation and the increase in apoptosis, confirming the regulatory function of UBD on IRE1α-dependent JNK activation in β-cells apoptosis (Brozzi et  al. 2016). Furthermore, during the glucolipotoxicity, mTORC1 inhibition in β-cells decreases IRE1α protein expression and JNK activity. In this respect, TORC1 regulates the β-cell stress response to glucose and fatty acids. These data elucidate the β-cell failure-­ associated T2D emerging due to glucolipotoxicity (Bachar et  al. 2009). FFA load, when exceeding β-cell esterification capacity, might impair ER functions and trigger an ER stress

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response, contributing to β-cell toxicity. Increasing fatty acid concentrations lead to early JNK activation that precedes induction of ER stress markers and apoptosis. Induction of JNK and FoxO1 activation plays an important role in fatty acid-induced apoptosis (Martinez et  al. 2008). On the contrary, FoxO1 deficiency in β-cells results in enhanced insulin secretion at high glucose concentrations, indicating the role of FoxO1 on GSIS and β-cell proliferation. In this respect, the impaired insulin secretion and decreased β-cell mass in T2D can be controlled through the FoxO1 (Miyazaki et al. 2012). The JNK-activated prostaglandin E2 (PGE2) induces β-cell dysfunction. Accordingly, PGE2-mediated JNK1 activation, through dephosphorylation of Akt and FoxO1, leads to nuclear accumulation of FoxO1 and nucleocytoplasmic shuttling of Pdx-1, finally resulting in defective GSIS in β-cells (Meng et al. 2009). Additionally, Pdx-1 is downregulated by deletion of another forkhead transcription factor, FoxA2 (Lee et al. 2002). FoxO1 and FoxA2 share common DNA-binding sites in the Pdx-1 promoter. Thus, FoxO1 may inhibit the transcription of Pdx-1 through competing the binding sites with FoxA2 (Shao et al. 2013). Activation of the PI3K/Akt pathway prevents palmitate-­ induced toxicity of pancreatic β-cells. Acylated ghrelin and unacylated ghrelin promote survival of β-cells. This effect is mediated by cAMP/PKA, ERK1/2, and PI3K/Akt pathways (Granata et al. 2007). Various studies further identify that FoxO1 is an important mediator in the PI3K/Akt pathway, which could be phosphorylated on Thr24, Ser256, and Ser316 by Akt, resulting in transport of FoxO1 from the nucleus to the cytoplasm (Kim et al. 2005b). GLP-1 receptor agonists potentiate GSIS and improves hyperglycemia by increasing proliferation rate of β-cells and preventing β-cells from apoptotic cells death in diabetes. The proliferative action of GLP-1 receptor agonist in β-cells is mediated by activation of PI3K/Akt, which results in inactivation of FoxO1 (Fang et al. 2012; Tamura et al. 2015). FoxO1-related negative regulation of Pdx-1 and the direct correlation between lipid stress and FoxO1 activation indi-

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cate the worsening effect of the PI3K/Akt/FoxO1 pathway on β-cell lipoapoptosis and GSIS impairment (Shao et al. 2013). On the other hand, Increasing IRS-2 expression in β-cells can distinctly increase the rate of glucose- and IGF-1-­ induced β-cell mitogenesis, implicating a significant role for IRS-2  in expanding β-cell mass (Lingohr et al. 2002). Inhibition of PI3K or Akt (PKB) significantly increases IRS-2 levels in β-cells. However, FoxO3a mediates the IRS-2 transcriptional control in β-cells via an IRE in the IRS-2 promoter. Nuclear translocation of FoxO3a in β-cells is regulated by IRS signaling. The inhibitory effect of insulin/IGF-1 on β-cell IRS-2 expression is achieved by introduction of constitutively activated FoxO3a. These evidences indicate that FoxO3a is the major driver of basal IRS-2 expression in β-cells (Tsunekawa et  al. 2011). Since β-cell death is a major contributing factor to T2D, to eliminate underlying mechanisms that decrease cell survival is extremely important. Glucose-dependent insulinotropic polypeptide (GIP), acting via the PI3K/PKB signaling pathway, decreases nuclear FoxO1 interaction with the FoxO1 response element (FHRE) in β-cells, resulting in reduction of glucolipotoxicity-­induced apoptosis. This means that PI3K/Akt/FoxO1-mediated transcriptional regulation of Bax expression is an important pathway for β-cell survival (Kim et al. 2005b). As mentioned above, FoxO1 inhibits β-cell proliferation through suppression of the Pdx1 transcription factor, by competing with FoxA2 and protects against β-cell failure, which is induced by oxidative stress through insulin gene transcription factors, such as NeuroD (β-cell E box transcription factor; BETA2) and MafA induction. Nuclear redistribution of FoxO1 is associated with increased expression of these transcription factors. In β-cells exposed to oxidative stress FoxO1 translocates from the cytoplasm to the nucleus, while Pdx1 translocates in opposite direction. Consequently, FoxO1 may be a logical approach in the treatment of T2D. Phosphorylation of nuclear FoxO1 contributes to β-cell protection because of its nuclear exclusion (Buteau and Accili 2007; Kitamura and Ido Kitamura 2007). Akt-dependent phosphory-

8  Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes

lation of FoxO1 at S256 (P-FoxO1) enables its binding to 14–3-3 dimers and nuclear export. Dephosphorylated FoxO1 enters nuclei and activates pro-apoptotic genes. In accordance with the nuclear accumulation of P-FoxO1, PKC-δ overexpression alone does not increase apoptotic β-cell death (Gerst et al. 2015). Overexpression of kinase-negative PKC-δ protects β-cells from palmitate-induced mitochondrial dysfunction and inhibits nuclear accumulation of FoxO1. The inhibition of nuclear accumulation of FoxO1 is accompanied by an increased phosphorylation of FoxO1 and a significant reduction of FoxO1 protein (Hennige et al. 2010). In pathophysiological oxidative stress conditions, FoxO1 can maintain insulin gene expression as well as key transcription factors MafA and NeuroD/Beta2 that are necessary for normal β-cell function (Gross et al. 2008; Kitamura and Ido Kitamura 2007). FoxO1 can play key role in the β-cell’s adaptive responses to differing in  vivo metabolic conditions. Thereby, it is thought that manipulation of IRS-2 expression in β-cells could be a means to promote β-cell survival and as such, a potential therapeutic target to treat T2D (Rhodes 2005; White 2002). GK and IRS-2 are critical requirements for β-cell hyperplasia that occur in response to glucolipotoxicity-­ induced insulin resistance (Terauchi et al. 2007). Nakamura et al. claimed that GKA-stimulated IRS2 production affects β-cell proliferation but not β-cell function. During the glucolipotoxicity, oxidative stress diminishes the effects of GKA on the changes in expression of genes involved in β-cell function and proliferation (Nakamura et al. 2012). However, during the insulin signaling via IRS-2 in β-cells, a reduction in nuclear exclusion of FoxO1 contributes to the insufficient proliferative response of β-cells to insulin resistance (Takamoto et al. 2008).

5

 -Cell Differentiation β and Dedifferentiation in T2D

The concept of β-cell dedifferentiation in diabetes is not well defined. β-cells in the diabetic state lose components of their differentiated state (Weir et al. 2013). Interestingly, it is claimed that

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loss of β-cell mass is due to β-cell dedifferentiation, not death (Talchai et al. 2012). Pancreatic β-cells consist of stem cells with a complex cellular differentiation. Mature β-cells can lose their differentiated phenotype and cellular identity and regress to a less differentiated state via various adaptive mechanisms to escape cell death under stress conditions (Bensellam et  al. 2018). The increased rate of apoptosis in β-cells is less, when compared to the impairment of β-cell function, however the average β-cell mass is about 39% lower in T2D subjects compared with matched controls and gradually decreases with the duration of clinical diabetes (Butler et  al. 2007; Rahier et  al. 2008). Recent studies have clarified the mechanisms of conversion to dedifferentiated phenotype of the pancreatic β-cells under stress conditions. Endocrine cells are organized in islets of Langerhans comprising 5 cell types, α, β, δ, ε, and pancreatic polypeptide (PP) cells, which produce the hormones glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide, respectively (Collombat et  al. 2007; Gao et  al. 2014). Recent findings have revealed number of genetic determinants underlying endocrine cell genesis. In this context, a complex network of transcription factors is activated to progressively and differentially specify the endocrine subtype lineages. These transcription factors include mainly the homeodomain-containing proteins Nkx6.1, Aristaless-related homeobox gene (Arx), paired box gene 4 (Pax4), and Pdx1 (Collombat et al. 2007). There are at least five different evidences that α-cells and β-cells share a close developmental relationship. Firstly, during the pancreatic development both glucagon and insulin expressing cells differentiate. Pdx1 is expressed by both adult β-cells, and α-cell’s progenitors (Herrera 2000). Secondly, when near-­ total of β-cells are killed, α-cells can be converted into new β-cells. It has been proposed that this spontaneous adult cell conversion could be used as a method of producing β-cells for T2D therapy (Thorel et al. 2010). Third, propagation of DNA methylation during cell division is essential for repression of α cell lineage determination genes to maintain β-cell identity. β-cell-specific dele-

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tion of DNA methyltransferase1 results in their conversion to α-cells through Nkx2.2-­dependent expression of the α-cell determination factor Arx. The repressor activity of Nkx2.2 on the methylated Arx promoter in β-cells is an indispensible event required for maintaining β-cell identity (Dhawan et al. 2011; Papizan et al. 2011). Fourth, the newly formed α-cells acquire a β-cell phenotype through the ectopic expression of Pax4 thereby a functional β-cell mass is restored (Collombat et al. 2009). Finally, Pdx1-­containing glucagon/Arx-producing cells demonstrate glucagon-insulin double positivity. Thus, these cells are indistinguishable from normal β-cells. Consequently, excessive expression of Pdx1  in embryonic endocrine progenitor cells results in conversion of α-cells into β-like-cells through an intermediate stage (Yang et  al. 2011). Enhancement of human β-cell proliferation is one potential approach to restore β-cell mass to prevent or cure T2D. In this respect, eight candidate molecules are proposed as β-cell mitogen (Shirakawa and Kulkarni 2016). A non-specific endocrine cell mitogen, WS6 (IκB kinase and Erb3 binding protein-1 inhibitor) stimulates not only β-cell but also α-cell proliferation, while maintaining the differentiated β-cell phenotype without disturbing the intrinsic β-cell-to-α-cell ratio. Due to its importance as a potential diabetes therapy, WS6 does not induce the significant dedifferentiation of human β-cells, considering the proportion of insulin-positive cells per islet, and expression of insulin and key β-cell ­transcription factors (Boerner et  al. 2015). The inhibition of dual-specificity tyrosine-regulated kinase-1a (DYRK1A) and Cdc-like kinases (CLKs) promotes human β-cell proliferation. In addition, 5-iodotubercidin (5-IT) is a potent and selective inhibitor of the DYRK1A and CLKs families. These evidences suggest that inhibition of DYRK1A may be a valuable therapeutic strategy to restore human functional β-cell mass in T2D (Dirice et al. 2016; Shirakawa and Kulkarni 2016). T2D islets display decrease in GSIS compared to normal islets. Reduced levels of GSIS are accompanied with the decreased MafA expression levels (Guo et al. 2013). MafA (RIPE3b1) is

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member of the Maf family of basic leucine zipper. It is identified as a transcription factor that specifically binds to a conserved insulin enhancer element RIPE3b/C1-A2 and activates insulin gene expression (Zhu et al. 2017). In addition to MafA, there are also selective decreases in Nkx6.1 and Pdx1 expressions in islet cells of human with T2D.  Especially, MafA very sensitive target of oxidative stress, potentially manifests with postprandial hyperglycemia in prediabetic individuals. Thus, MafB, a MafA-­ related transcription factor expressed in human β-cells, is also extremely compromised (Guo et  al. 2013). Indeed, sustained expression of MafA results in significantly lower plasma glucose levels, higher plasma insulin, and augmented islet β-cell mass (Matsuoka et al. 2015). Arx deficiency in neonatal α-cells results in an α-to-β-like conversion through an intermediate bihormonal state. Furthermore, these Arx-­ deficient converted cells express β-cell markers including Pdx1, MafA, and glucose transporter 2 (Glut2) (Wilcox et al. 2013). Indeed, Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. The loss of Arx in α-cells regenerates a functional β-cell mass and thereby reverses diabetes, which occur following β-cell depletion (Courtney et al. 2013). While Pdx1 is normally restricted to β-cells, early overexpression in α-cells results in a loss of glucagon-expressing α-cells with a concomitant gain of insulin-producing β-cells demonstrating an α-to-β-cell fate conversion (Yang et al. 2011). The transcription factor encoded by the Pdx1 gene is the earliest tissue-selective transcriptional regulator, as it has fundamental actions in the formation of all pancreatic cell types, islet β-cell development, and adult islet β-cell function (Offield et al. 1996). MafA potentiates the ability of Pdx1 to induce β-cell formation from neurogenin 3 (Ngn3)-positive endocrine precursors and enable Pdx1 to produce β-cells from α-cells (Matsuoka et al. 2017). Thus, Pdx1 acts as a master regulator of β-cell fate and β-cell-specific removal of Pdx1 results in severe hyperglycemia (Gao et al. 2014). In fact, there are two independently controlled pathways for β-cell differentiation. Disruption of

8  Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes

homeobox gene Nkx6.1 leads to loss of β-cell precursors and blocks β-cell neogenesis (Sander et  al. 2000). The early expression of Pax4  in a subset of endocrine progenitors is essential for the differentiation of the β- and δ-cell lineages (Sosa-Pineda et al. 1997). The antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts (Collombat et al. 2003). Once cell fate has been established, additional transcription factors such as Insulin gene enhancer protein 1 (Isl1), Pax6, MafA, MafB, and Pdx1 act to maintain the phenotype of specified islet cells. MafB is not only important to islet α-cell function but also be involved in regulating genes required in both endocrine α- and β-cell differentiation. MafA is a potent insulin activator that is likely to function downstream of Nkx6.1 during islet insulin-producing cell development. Whereas, Pax6 transactivates the glucagon and insulin promoters (Artner et al. 2006; Matsuoka et  al. 2004; Offield et  al. 1996; Sander et  al. 1997). Both Arx and Pax4 act as transcriptional repressors that control the expression level of one another, thereby mediate proper endocrine fate allocation. During early islet cell specification, endocrine progenitor cells exhibit a potential to adopt a β−/δ- or an α-cell fate, the alternative destinies requiring the activities of Pax4 and Arx, respectively. Cells exhibiting all known δ-cell characteristics develop at the expense of β- and α-cells (Collombat et al. 2005). This suggests a secondary requirement of Pax4  in β−/δ-cell ­progenitors for the specification of the β-cell fate (Collombat et al. 2007). On the basis of loss-of-function phenotypes, Pax4 appears necessary firstly for the β−/δ-cell fate and later for the β-cell lineage allocation (Collombat et al. 2007). PP expression is a hallmark of the β-cell lineage. Since neither glucagon nor insulin gene-expressing cells are essential for the differentiation of the other islet endocrine-­ cell types, PP cell ablation results in accompanying loss of β- and δ-cells (Herrera et  al. 1994). Arx is ectopically expressed in mature insulin-­ producing cells as soon as these initiate hormone production during embryogenesis, or in adult β-cells. In both instances, a lethal hyperglycemia

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is observed concurrently with a dramatic reduction in the β-cell content and a proportional increase in the glucagon- or PP-producing cell numbers. These data support the notion that Arx converts the endocrine progenitors toward α or PP cell fate or induce the conversion of β-cells into α- or PP-cells. The misexpression of Arx converts differentiated β-cells into cells displaying α and PP-cell characteristics, but it also points to the existence of a plasticity of differentiated β-cells (Collombat et al. 2007). β-cell loss in T2D due to dedifferentiation involves two critical point. Firstly, in T2D patients, approximately threefold increase is found in the number of pancreatic islet cells that no longer produce any of the four major pancreatic hormones, which retain endocrine features. Moreover, transcription factors FoxO1 and Nkx6.1, markers of the β-cell, are either decreased or mislocalized in β-cells from persons with diabetes. Secondly, these transcription factors ectopically localize in glucagon-producing “α-like”-cells or somatostatin-immunoreactive cells of individuals with T2D, respectively. These data indicate that the insulin-producing β-cells become dedifferentiated and undergo conversion to “α-like”-cells or “δ-like” cells during the course of T2D (Cinti et al. 2016). FoxO1 deletion in β-cells results in hyperglycemia that is associated with a loss of β-cell identity and conversion into other endocrine cell types, particularly α-cells (Talchai et al. 2012). β-cell dedifferentiation occurs commonly in T2D and is associated with an acquired loss of FoxO1 function. In fact, an inverse relationship between FoxO1 and β-cell differentiation exists when T2D develops. In addition, striking increases are detected in Neurog3, octamer-binding transcription factor 4 (Oct4), Nanog, and L-Myc expressions. Eventually, FoxO1 is required to maintain β-cell identity and prevent β-cell conversion into non-β pancreatic endocrine cells in response to chronic pathophysiological stress (Talchai et al. 2012). The misexpression of Pax4 and/or the inhibition of Arx might promote the exclusive specification of endocrine progenitors toward the β-cell lineage and/or convert endocrine cells into β-cells. This concept may propose an interesting

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perspective for therapeutic purpose (Collombat et  al. 2007). Interestingly, senescence does not alter α-cell plasticity, thereby α-cells can be reprogrammed to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after β-cell loss (Chera et al. 2014). On the other hand, the ectopic expression of Pax4 in δ-cells is sufficient to induce their conversion into functional β-cell phenotype (Druelle et al. 2017).

these new therapeutic options based on protein kinases signaling pathways.

References

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type 2 diabetes in mice. Nature. 1998;391:900–4. https://doi.org/10.1038/36116 Xia G, Zhu T, Li X, Jin Y, Zhou J, Xiao J. ROS-mediated autophagy through the AMPK signaling pathway protects INS-1 cells from human islet amyloid polypeptide-induced cytotoxicity. Mol Med Rep. 2018;18:2744–52. https://doi.org/10.3892/ mmr.2018.9248. Xu J, Lin S, Myers RW, Addona G, Berger JP, Campbell B, Chen H-S, Chen Z, Eiermann GJ, Elowe NH, Farrer BT, Feng W, Fu Q, Kats-Kagan R, Kavana M, Malkani S, McMasters DR, Mitra K, Pachanski MJ, Tong X, Trujillo ME, Xu L, Zhang B, Zhang F, Zhang R, Parmee ER. Novel, highly potent systemic glucokinase activators for the treatment of type 2 diabetes mellitus. Bioorg Med Chem Lett. 2017;27:2069–73. https://doi.org/10.1016/j.bmcl.2016.10.085 Yamaji T, Kumagai K, Tomishige N, Hanada K.  Two sphingolipid transfer proteins, CERT and FAPP2: their roles in sphingolipid metabolism. IUBMB Life. 2008;60:511–8. https://doi.org/10.1002/iub.83 Yang Y-P, Thorel F, Boyer DF, Herrera PL, Wright CVE. Context-specific α- to-β-cell reprogramming by forced Pdx1 expression. Genes Dev. 2011;25:1680–5. https://doi.org/10.1101/gad.16875711 Yang S, Xia C, Li S, Du L, Zhang L, Zhou R. Defective mitophagy driven by dysregulation of rheb and KIF5B contributes to mitochondrial reactive oxygen species (ROS)-induced nod-like receptor 3 (NLRP3) dependent proinflammatory response and aggravates lipotoxicity. Redox Biol. 2014;3:63–71. https://doi. org/10.1016/j.redox.2014.04.001 Zhang S, Liu J, Dragunow M, Cooper GJS. Fibrillogenic amylin evokes islet beta-cell apoptosis through linked activation of a caspase cascade and JNK1. J Biol Chem. 2003;278:52810–9. https://doi.org/10.1074/ jbc.M308244200 Zhang Y, Warnock GL, Ao Z, Park YJ, Safikhan N, Ghahary A, Marzban L.  Amyloid formation reduces protein kinase B phosphorylation in primary islet β-cells which is improved by blocking IL-1β signaling. PLoS One. 2018;13:e0193184. https://doi. org/10.1371/journal.pone.0193184 Zhu Y, Liu Q, Zhou Z, Ikeda Y.  PDX1, Neurogenin-3, and MAFA: critical transcription regulators for beta cell development and regeneration. Stem Cell Res Ther. 2017;8:240. https://doi.org/10.1186/ s13287-017-0694-z Zimmet P, Alberti KG, Shaw J.  Global and societal implications of the diabetes epidemic. Nature. 2001;414:782–7. https://doi.org/10.1038/414782a Zraika S, Hull RL, Verchere CB, Clark A, Potter KJ, Fraser PE, Raleigh DP, Kahn SE. Toxic oligomers and islet beta cell death: guilty by association or convicted by circumstantial evidence? Diabetologia. 2010;53:1046– 56. https://doi.org/10.1007/s00125-010-1671-6

9

Bile Acid Toxicity and Protein Kinases Atilla Engin

Abstract

If the bile acids reach to pathological concentrations due to cholestasis, accumulation of hydrophobic bile acids within the hepatocyte may result in cell death. Thus, hydrophobic bile acids induce apoptosis in hepatocytes, while hydrophilic bile acids increase intracellular adenosine 3′,5′-monophosphate (cAMP) levels and activate mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways to protect hepatocytes from apoptosis. Two apoptotic pathways have been described in bile acids-induced death. Both are controlled by multiple protein kinase signaling pathways. In mitochondria-­controlled pathway, caspase-8 is activated with death domain-independent manner, whereas, Fasdependent classical pathway involves ligandindependent oligomerization of Fas. Hydrophobic bile acids dose-dependently upregulate the inflammatory response by further stimulating production of inflammatory cytokines. Death receptor-mediated apoptosis is regulated at the cell surface by the receptor A. Engin (*) Department of General Surgery, Faculty of Medicine, Gazi University, Ankara, Turkey

expression, at the death-inducing signaling complex (DISC) by expression of procaspase-8, the death receptors Fas-associated death domain (FADD), and cellular FADDlike interleukin 1-beta (IL-1β)–converting enzyme (FLICE) inhibitory protein (cFLIP). Bile acids prevent cFLIP recruitment to the DISC and thereby enhance initiator caspase activation and lead to cholestatic apoptosis. At mitochondria, the expression of B-cell leukemia/lymphoma-2 (Bcl-2) family proteins contribute to apoptosis by regulating mitochondrial cytochrome c release via Bcl-2, Bcl-2 homology 3 (BH3) interacting domain death agonist (Bid), or Bcl-2 associated protein x (Bax). Fas receptor CD95 activation by hydrophobic bile acids is initiated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) signaling. However, activation of necroptosis by ligands of death receptors requires the kinase activity of receptor interacting protein1 (RIP1), which mediates the activation of RIP3 and mixed lineage kinase domain-like protein (MLKL). In this chapter, mainly the effect of protein kinases signal transduction on the mechanisms of hydrophobic bile acids-induced inflammation, apoptosis, necroptosis and necrosis are discussed.

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_9

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Keywords

Bile acid · Hydrophobic bile acids · Glycochenodeoxycholic acid (GCDCA) · Na+/taurocholate (TC) cotransporter (NTCP) · Apical sodium bile acid cotransporter (ASBT) · Cholangiocytes · Canalicular bile salt export pump (BSEP) · Ursodeoxycholic acid (UDCA) · Tauroursodeoxycholic acid (TUDCA) · Transmembrane G-protein-­ coupled receptor (TGR5)

1

Introduction

Bile acids induce cytotoxic effects, depending on their hydrophobic properties. The retention and accumulation of hydrophobic bile acids in hepatocytes during cholestasis is a major cause of liver damage. Hydrophilic bile acids may induce apoptosis and interleukin-8 (IL-8) synthesis, but not the cytolysis (Araki et  al. 2001; Attili et  al. 1986). Therefore, the bile salt-induced liver damage is thought to be depend on the total amount, and the hydrophobic-hydrophilic balance of bile salt species (Morita et al. 2011). The cytotoxicity of hydrophobic bile acids may be ameliorated by hydrophilic bile acids under certain conditions. Nevertheless, the cytotoxicity caused by bile salts in hepatocytes is largely determined by bile salt species, bile salt concentrations, cholesterol and cell membranes phospholipids, increased membrane fluidity and permeability (Araki et al. 2003; Ikeda et al. 2017; Morita et al. 2019). Biliary cholesterol solubilization is dependent not only on the concentration of cholesterol itself but also on the phospholipid/bile salt ratio. Increase in the cholesterol/phospholipid ratio makes them more resistant to the solubilizing action of bile salts (Ikeda et al. 2017). The relative cytotoxic effect of bile salts is likely due to both amounts of bile salts accumulated in the cell and direct toxic effects of bile salts including the abilities to disrupt cell membranes and to alter intracellular signaling (Ikeda et  al. 2017). In addition, the bile salt molecules modulate canalicular cell membrane phospholipids and membrane fluidity by

penetrating into the phospholipid bilayers, related with their hydrophobic properties (Asamoto et al. 2001). Eventually, hydrophobic bile acids stimulate reactive oxygen species (ROS) generation, release of cytochrome c and apoptosis-inducing factor. Thereby, in human hepatic mitochondria exposed to excess hydrophobic bile acids in cholestasis, ROS generated the mitochondrial permeability transition (MPT) induction leads to mitochondrial pathway-related cell death (Rodrigues et al. 1998; Sokol et al. 2005). In fact, MPT induction is a critical intracellular event that triggers both the apoptotic and necrotic forms of cell death in hepatocytes (Botla et  al. 1995; Lemasters et  al. 1998; Sokol et  al. 2005; Yerushalmi et al. 2001). Hydrophobic bile acids induce hepatocyte apoptosis by activating the death receptor via extrinsic pathway. This pathway involves ligand-­ independent oligomerization of Fas, recruitment of Fas-associated death domain (FADD), activation of caspase 8, and subsequent activation of all effector proteases, including downstream caspases (Faubion et  al. 1999). Furthermore, induction of apoptosis through tumor necrosis factor (TNF)-related apoptosis-inducing ligand- receptor 2 (TRAILR2)/death receptor 5 (DR5) expression by bile acids is Fas-independent but it is dependent on death receptors (Higuchi et al. 2001). After death receptor activation and death-inducing signaling complex (DISC) formation, caspase 8 is activated and the pro-apoptotic protein, BH3 interacting domain death agonist (Bid) is cleaved and translocated to the mitochondria. Subsequently, MPT pores open and the cytochrome c is released. A caspase cascade then initiates and, finally, activation of the effector caspases, leads to irreversible hepatocyte death (Higuchi et  al. 2001; Reinehr et al. 2004; Scaffidi et al. 1998; Yang et al. 2007). In intrinsic pathway of apoptosis, bile acids are also able to induce apoptosis through the mitochondrial pathway, in which intracellular oxidative stress causes mitochondrial dysfunction and the subsequent release of proapoptotic factors. ROS generation, MPT induction, and cytochrome c release from mitochondria are critical steps in the induction of apoptosis by bile acids. Antioxidants may reduce liver injury

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caused by low levels of bile acids by preventing the generation of oxidant stress (Rodrigues et al. 1998; Yerushalmi et  al. 2001). This chapter debates the control mechanisms of protein kinases that are effective on opposite signaling pathways leading to cell death and survival via hydrophobic and hydrophilic bile acids, respectively.

2

 ile Acid Biosynthesis B and Transporters

Human liver synthesizes about 200 to 600  mg bile acids per day. A total bile acid pool is approximately 3 g, and consists of approximately 40% cholic acid (CA), 40% chenodeoxycholic acid (CDCA), 20% deoxycholic acid (DCA), and trace amount of lithocholic acid (LCA). All these are recycled 4–12 times a day (Chiang 2013). Primary bile acids, CA and CDCA are synthesized from cholesterol in the liver via two multistep biosynthetic pathways (Gonzalez 2012). In the neutral bile acid pathway (or classic pathway), steroid ring modification precedes side-chain cleavage, whereas in the acidic pathway (or alternative pathway) side-chain cleavage precedes steroid ring modifications (Chiang 2004). Both the classical and the acidic pathways are responsible for the production of at least 95% of the bile acids, and these two pathways contribute about equally to bile acid synthesis in humans (Pellicoro and Faber 2007). The sequential conversion of cholesterol molecules to bile acids via classical neutral synthesis pathway is accomplished by seventeen enzymes, which are located in the cytosol, endoplasmic reticulum (ER), mitochondria, and peroxisomes (Russell 2003). This pathway is responsible for synthesis of approximately 80% of bile acids in human livers and their formation starts with a 7α-hydroxylation of cholesterol by the microsomal cholesterol 7α-hydroxylase (cytochrome P450 7A1; CYP7A1) enzyme. Thus, 7α-hydroxylase (CYP7A1) is the only rate-­limiting enzyme in bile acid synthesis, and synthesizes two primary bile acids, CA and CDCA in human liver. The alternative pathway starts with a hepatic or extra-

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hepatic sterol 27-hydroxylation by CYP27A, which is a mitochondrial cytochrome P450 enzyme, and widely found in most tissues and macrophages (Duane and Javitt 1999; Norlin and Wikvall 2007). Transcription of CYP7A1 is downregulated by the bile acid-activated farnesoid X receptor (FXR). When bound to bile acids, FXR represses transcription of the gene encoding CYP7A1 and activates the gene encoding intestinal bile acid-­ binding protein (IBABP) (Makishima et al. 1999; Parks et al. 1999). The acidic pathway may contribute to relatively little, the amount of bile acid synthesis that takes place via the 27-hydroxylation pathway in healthy humans is about 9% of total bile acid synthesis in human hepatocytes (Duane and Javitt 1999). Conjugated bile acids are secreted into bile via the canalicular bile salt export pump (BSEP), forming mixed micelles with cholesterol and phosphatidylcholine in the gallbladder to prevent precipitation of cholesterol and to protect gallbladder epithelium cells from bile acid toxicity. In humans, most bile acids are amino conjugated at the carboxyl group (amidation), with the ratio of glycine-to taurine-­bile acid conjugates in bile is about 3.5:1, dependent on dietary intake of amino acids. At first step, the rate-limiting microsomal synthetase catalyzes the formation of the bile acid-coenzyme A (CoA) thioester, followed by conjugation with taurine or glycine catalyzed by the bile acid-­ CoA:amino acid N-acyltransferase in cytosol (Hardison and Grundy 1983; Solaas et al. 2000). Once formed, bile acids undergo extensive enzyme-catalyzed taurine and glycine conjugation to form four different “amidated” bile acids. Eventually, taurocholic acid (TCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), and taurochenodeoxycholic acid (T-CDCA) are synthesized. This mixture is actively transported out of the liver into the bile. Glucuronidated and sulphated bile acids contribute to the serum bile acids by 14–32%, 16–44%, to urine bile acids by 4–11% and 61–82% and biliary bile acids by 0.2–1% and 0.3–2%, respectively (Takikawa et  al. 1985). GCDCA is the most abundant bile acid of the total pool in human liver with the concentration of 31% and represents a notable differ-

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ence from rodents. Conjugated forms with glycine and taurine of CDCA, a primary bile acid in humans, represent about 50% of the total bile acid pool in the human liver, whereas the CA and muricholic acid (MCA) forms are more than 80% of the total volume in rodents. These interspecies differences in the hepatic bile acid pool composition should be taked into consideration in pathological conditions occurred due to specific bile acid accumulation (García-Cañaveras et al. 2012; Humbert et al. 2012). About 90% of the bile acids secreted into the intestine are reabsorbed into portal blood and are then efficiently taken up back by hepatocytes. The ileum is the major site of reabsorption. Bile acids continue to cycle between the intestine and the liver, creating an enterohepatic cycle (Ballatori et al. 2009). In the distal intestine, bacterial 7α-dehydroxylases convert the conjugated-CA and CDCA from CA and CDCA to DCA and LCA, respectively. DCA and LCA are the secondary bile acids. LCA induces its own detoxification by activating nuclear receptors to promote sulfotransferase, which conjugate the LCA by sulfation in the enterocyte. Subsequently, conjugated LCA is effluxed back into the intestinal lumen. In fact, sulfation is the major pathway for detoxification of hydrophobic bile acids in humans (Hofmann 2004). The excretory pole of the hepatocyte forms the border of the bile canaliculus. The basolateral and canalicular membranes differ in their biochemical composition and functional characteristics (Boyer 1980). Basolateral bile salt transport systems are essential for bile formation since major portion of bile salts excreted into bile by the liver are reabsorbed on each pass through the intestine and undergo an “enterohepatic circulation”. Bile salts are taken up by human hepatocytes via the basolateral membrane Na+/ taurocholate (TC) cotransporter (NTCP) and organic anion transporting proteins (OATPs) (Hagenbuch and Meier 1994; Hofmann 1999). The bile salt enterohepatic circulation is mainly regulated by three different bile salt transport proteins. These are the canalicular BSEP (ABCB11), the ileal Na+-dependent bile salt transporter ISBT (SLC10A2) and the hepatic sinusoidal Na+-TC cotransporting polypeptide

A. Engin

NTCP (SLC10A1). Other additional transporters include the OATPs (SLC21A) and the multidrug resistance-associated proteins 2 and 3 (MRP2,3: ABCC2,3) (Kullak-Ublick et  al. 2000; Trauner and Boyer 2003). During bile secretory failure (cholestasis), bile salt transport proteins undergo adaptive responses that serve to protect the liver from bile salt toxicity. They facilitate extrahepatic routes of bile salt excretion (Trauner and Boyer 2003). Firstly; small amounts of bile acids may escape into the systemic circulation, reabsorbed when passing through the renal tubules in the kidney, and are then circulated back to the liver through systemic circulation. In the renal tubule the apical sodium bile acid cotransporter (ASBT) acts as a salvage mechanism to prevent urinary excretion of bile acids that undergo glomerular filtration (Barnes et al. 1977; Christie et al. 1996). On the other hand, dianionic conjugated bile salts are secreted into bile with the MRP2. In bile ductules, a minor portion of protonated bile acids and monomeric bile salts are reabsorbed by nonionic diffusion and the ASBT transports back into the periductular capillary plexus by MRP3 or a truncated form of Asbt (tAsbt), and subjected to cholehepatic shunting (Trauner and Boyer 2003). In cholehepatic shunt, bile acids are absorbed in the biliary tract, secreted into the periductular capillary plexus, and carried directly back to the hepatocyte for secretion. Alternatively, ASBT functions to sense bile acid concentration in bile. Thereby, intracellular signaling systems in cholangiocytes is activated via bile acid transport (Xia et  al. 2006). Canalicular efflux of divalent sulfated or glucuronidated bile salts is mediated by the MRP2, which is strongly decreased in cholestasis in human. Decreased MRP2 expression leads to compensatory increases in the basolateral expression of MRP1 and MRP3 in liver (Borst et  al. 1999; Kullak-Ublick et  al. 2000). Intestinal epithelial cells reabsorb majority of the secreted bile acids through both ASBT and sodium independent OATPs. Cytosolic IBABP mediates the transcellular movement of bile acids to the basolateral membrane across which they exit the cells via organic solute transporters (OST). They enter portal venous circulation by a Na+-independent mechanism. Subsequently,

9  Bile Acid Toxicity and Protein Kinases

majority of conjugated bile acids are efficiently reabsorbed from portal venous blood into hepatocytes, mainly via the NTCP (Alrefai and Gill 2007). In humans, three liver-specific OATPs (OATP-A, OATP-C, and OATP-8) transport bile acids (Kullak-Ublick et al. 2001). For the hepatic uptake of bile acids, OATP-C is the most relevant isoform and is exclusively localizes in liver. Within the hepatocyte, bile acids are bound to cytosolic proteins and then traverse by diffusion. Transport across the canalicular membrane is the rate-limiting step in overall hepatocellular bile acid excretion. This is mediated by the BSEP (Kullak-Ublick et  al. 2000; Tamai et  al. 2000). The major portion of biliary bile acids are reabsorbed by five different ways, which are included apical OATP3, the ASBT, cytosolic intestinal bile acid-binding protein (cIBABP), and basolateral Mrp3/MRP3 and tAsbt (Meier and Stieger 2002). Intracellular trafficking and membrane insertion of bile salt transporters is regulated by lipid, protein, and extracellular signal-related kinases (ERK) in response to physiologic stimuli such as cyclic adenosine 3′,5′ monophosphate (cAMP) or TC (Kullak-Ublick et  al. 2004). cAMP activates protein kinase C zeta (PKC-ζ) in the phosphatidylinositol 3-kinase (PI3K)-dependent manner without inducing translocation of PKC-ζ to the plasma membrane. Inhibition of cAMP-induced PKC-ζ activity results in inhibition of cAMP-induced increases in TC uptake and NTCP translocation. Neither dominant-negative protein kinase B (PKB, Akt) nor constitutively active PKB affects cAMPinduced activation of PKC-ζ. Consequently, cAMP-induced NTCP translocation involves the activation of the PI3K/PKC-ζ signaling pathway. This is followed by the activation of the PI3K/ PKB signaling pathway (McConkey et al. 2004). Size and function of cholangiocytes are variable according to their location along the biliary tract. Conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT. The apical Na+-dependent bile acid transporter (ABAT) and the cIBABP messenger RNAs are detected in large cholangiocytes. These proteins mediate bile acid uptake from the duct lumen in large ducts. Bile acid uptake by ABAT and the

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PI3K pathway are important for bile acids to signal cholangiocyte proliferation. Thus, in bile duct obstruction, increased biliary bile acid concentration and ABAT expression initiate increased cholangiocyte proliferation and secretion (Alpini et  al. 1997, 2002b; Kip et  al. 2004; Lazaridis et al. 1997). A specific BSEP is present at the level of the hepatocyte canalicular membrane. This member of the adenosine triphosphate (ATP)-binding cassette (ABC transporter) family of proteins is the primary transporter of bile acids from the hepatocyte to the biliary system. Transport of bile salts is dependent on ATP hydrolysis (Kubitz et al. 2012; Soroka and Boyer 2014). The activity of this transporter regulates the formation of the “bile acid-dependent bile flow”. One of the important functions of bile acids is their feedback regulation on their own synthesis in the hepatocytes according to their specific concentration in the cell by inhibiting CYP27A1. This mechanism is obtained by direct regulation of FXR by bile acids in hepatocytes. In fact, increased level of bile acids in the hepatocytes activates FXR, which is a strong inhibitor of CYP27A1, the rate limiting enzyme in bile acids synthesis (Makishima et  al. 1999). Canalicular ABC (ATP-binding cassette) transporters consist of multidrug resistance gene 1 (MDR1), MDR2, and sister of P-glycoprotein (SPGP). BSEP is exclusively expressed in the liver on the hepatocyte apical, canalicular membrane, evenly distributed throughout the lobular domains. BSEP traffics out of the Golgi directly into a subapical, vesicular compartment where it can reside before moving to the apical plasma membrane. Whereas, there is a direct route from Golgi to the canalicular membrane for trafficking of MDR1, which is an ATP-dependent transporter of organic cations. In hepatocytes, PI3K regulates bile acid secretion and intracellular trafficking of MDR1 (Kipp and Arias 2000; Sai et  al. 1999). The half-life of BSEP in the apical membrane is approximately 4–6  days and constitutively recycles between the plasma membrane and subapical vesicles (endosomes). Polarization of hepatocytes requires recruitment of rab11a and myosin Vb to intracellular membranes that

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contain apical ABC transporters (Lam et al. 2007; Wakabayashi et  al. 2005). Hepatocytes take up bile acids and secrete them again into bile for ongoing enterohepatic circulation. Uptake of bile acids into hepatocytes occurs largely in a sodium-­ dependent manner by the NTCP. BSEP transports primarily monovalent bile salt species, including taurine and glycine conjugates of primary bile salts, CA and CDCA, and the secondary bile salt, DCA, as well as ursodeoxycholic acid (UDCA). BSEP constitutes the rate limiting step of hepatocellular bile salt transport and drives enterohepatic circulation of bile salts (Stieger 2011). In this context, the intestine also plays an active role in bile acid-mediated suppression of bile acid synthesis in liver. FXR induces the fibroblast growth factor intestine 19 (FGF19). This growth factor activates the cell-surface receptor tyrosine kinase, fibroblast growth factor receptor 4 (FGFR4) signaling, thus inhibiting CYP7A1 mRNA expression levels in human hepatocytes through a c-Jun N-terminal kinase (JNK)-dependent pathway (Holt et  al. 2003). Regulation of canalicular bile salt efflux through BSEP, basolateral elimination through organic solute transporters alpha and beta (OSTα/OSTβ) and inhibition of hepatocellular bile salt uptake through basolateral NTCP provides critical steps in protection from bile salt toxicity of hepatocyte (Baghdasaryan et  al. 2014). When intracellular bile acid concentration is less than 10 μmol/L in both hepatocytes and stellate cells, it functions as intracellular signaling molecule triggering wide variety of protein kinases. The expression of bile acid transporter is required for bile acid signaling, and the degree of bile acid transporter expression determines the sensitivity of cells to bile acid signaling. UDCA and tauroursodeoxycholic acid (TUDCA)-inhibited ABAT expression is dependent on Ca2+-induced PKCα activation in cholangiocytes. Bile acids alter Ca2+, cAMP, PKC and PI3K intercellular signaling systems in cholangiocytes. Reduced cholangiocyte ABAT expression decrease endogenous bile acid stimulation of cholangiocyte growth and secretion. Furthermore, TUDCA increases membrane translocation of the Ca2+-dependent PKCα and inhibits the activity of mitogen-­

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activated protein kinase (MAPK), but this response is independent from Raf proteins and MAPK p38 and JNK/stress-activated protein kinases (Alpini et al. 2001, 2002a, 2004; LeSage et al. 2001). Both solute carrier (SLC) and ABC drug transporters can be regulated by PKCs-­ related signaling pathways (Mayati et al. 2017). Moreover, p38 MAPK and PKC signal transduction pathways are important in the stimulated exocytosis of BSEP by TUDCA from a vesicular compartment to the plasma membrane. In this context, p38 MAPK regulates BSEP trafficking from the Golgi to the canalicular membrane. The Golgi serves as a BSEP pool in cholestasis, when p38 MAPK activity is inhibited. Thereby, activation of p38 MAPK by tauroursodesoxycholate (TUDC) recruits Golgi-­ associated BSEP. TUDC-induced stimulation of TC excretion is accompanied by a p38 MAPK-­ dependent insertion of subcanalicular BSEP into the canalicular membrane. Thereby, TUDC induces a transient and concentration-dependent activation of p38 MAPK and of ERK-2 (Kubitz et al. 2004; Kurz et al. 2001). Both ERK and p38 MAPK activation is required for the choleretic effects of both TUDC and hypo-osmotic cell swelling (Kurz et  al. 2001). G protein-and tyrosine kinase-dependent, PKC-independent activation of MAPKs are involved in the regulation of TC excretion by liver cell hydration changes. Hyperosmolarity induces retrieval of BSEP from the canalicular membrane, correlating with cholestasis, whereas, hypo-osmolarity is accompanied by a rapid recruitment of intracellular BSEP to the canalicular membrane. Hypotonic swelling of cells acts through activation of ERK-1/2 and p38 MAPK, and results in stimulated bile acid excretion. In this procedure, BSEP- and MRP2-specific vesicles participate in the short-term osmoregulation of canalicular secretion. This is due to a cause-effect relationship between bile salt excretion and transporter localization (Häussinger et  al. 2000; Noé et al. 1996; Schmitt et al. 2001). ABC transporters located in the hepatocyte canalicular membrane of mammalian liver are critical players in bile formation and detoxification. Direct Golgi-to-apical membrane

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trafficking of ABC transporters provides specific physiological regulation of transporter. However, the PI3K/PKB (AKT) signaling pathway contributes to the biliary secretory failure through the internalization of canalicular transporters endocytosis via classical PKC (cPKC) in cholestasis (Boaglio et al. 2010; Kipp and Arias 2002; Roma et al. 2000). Whereas, PI3K pathway is shown to be involved in exocytosis of ATP-dependent canalicular transporters. PI3K inhibition results in decreasing protein levels in canalicular membrane vesicle and sinusoidal membrane vesicle fractions. PI3K is required for intracellular trafficking of itself, as well as of ATP-dependent canalicular transporters (Misra et al. 1998). Bile acid secretion induced by cAMP and TC is associated with recruitment of several ABC transporters to the canalicular membrane. However, trafficking of BSEP and MRP2 to the canalicular membrane in response to cAMP is independent of PI3K activity (Misra et al. 2003). Hepatocellular canalicular network formation, an important component of hepatocyte polarization, requires activation of the liver kinase B1 (LKB1) and adenosine monophosphate (AMP)-activated protein kinase (AMPK). The serine/threonine kinase LKB1, which is activated by the bile acid TC and, in turn, activates AMPK1/2 has emerged as a key determinant of hepatic polarity. AMPK and LKB1 also participate in network maintenance through preventing low-Ca2+-mediated disruption of the canalicular network and tight junctions (Fu et al. 2010; Treyer and Müsch 2013). In fact, LKB1 exerts its effects by phosphorylating and activating 14 different protein kinases, all related to the AMPK. LKB1 is required for establishment of a fully polarized canalicular network. Maintenance of polarity may require subsequent activation of other kinases (Alessi et al. 2006). In brief, LKB1 is an upstream kinase that is activated by bile acids and facilities the establishment of apical polarity in the hepatocyte. Additionally, its activity is required for microtubule-dependent trafficking of canalicular ABC transporter, ABC subfamily B member 11 (ABCB11) to the canalicular membrane (Fu et al.

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2010; Homolya et al. 2014). Thus, LKB1 plays a critical role in bile acid homoeostasis. Lack of LKB1  in the liver results in cholestasis (Woods et  al. 2011). As mentioned above, TC affects hepatocyte polarity by activating the cAMPEapc-Rap1-mitogen-activated protein extracellular signal-regulated kinase (MEK)-LKB1-AMPK pathway. TC accelerates canalicular network formation and concomitantly increases cAMP. Similarly, activation of exchange protein directly activated by cAMP (Epac), (cAMP downstream kinase), accelerates canalicular network formation, similar to the effects of TC. Inhibition of Epac downstream targets, Rasrelated protein 1 (Rap1) and mitogen-activated protein kinase kinase (MEK), block the TC effect. TC rapidly activates MEK, LKB1, and AMPK, which are prevented by inhibition of adenyl cyclase or MEK (Fu et  al. 2011). DCA stimulate ASBT gene expression acting on the down-stream activated protein-1 (AP-1) response element via the epidermal growth factor (EGF) receptor and MEK cascade (Duane et al. 2007). On the other hand, bile salts as signaling molecules, activate nuclear receptors in the hepatocyte and ileal enterocyte, as well as an increasing number of G-protein coupled receptors (Hofmann and Hagey 2008). Transmembrane G-protein-coupled receptor (TGR5) is activated by bile acids. TGR5 is present in several subcellular locations within the cholangiocyte, including cilia, plasma membranes, intracellular space, vesicles, and ER. TGR5 is expressed on diverse cholangiocyte compartments, including a primary cilium, and its ciliary localization determines the cholangiocyte functional response to bile acid signaling (Masyuk et  al. 2013). Hepatocytes do not express TGR5. However, TGR5 is an important mediator of bile acid-induced cholangiocyte proliferation as well as protects cholangiocytes from death receptor-mediated apoptosis via TGR5dependent CD95 receptor serine phosphorylation. These mechanisms protect cholangiocytes from bile acid toxicity under cholestatic conditions (Reich et al. 2016).

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3

 rotein Kinases-Bile Acids P Crosstalk

As mentioned above, subsets of both conjugated and unconjugated bile acids have been shown to activate multiple kinase signaling pathways such as PKC, ERK1/2, MAPK, p38 MAPK, JNK, and/or PI3K/AKT.  Bile acid receptors are not only important in the regulation of bile acid synthesis and their metabolism, but also regulate glucose homeostasis, lipid metabolism, and energy expenditure. In addition to their wellknown function as dietary lipid detergent, bile acids have emerged as important signaling molecules that regulate energy homeostasis. Bile acids modulate the activity of transmembrane and nuclear receptors by binding to the TGR5 and the FXR, respectively (Trabelsi et al. 2017). Bile acids may mediate a liver/β-cell axis by maintaining the glucose competence of these cells through an FXR-dependent mechanism. Consequently, bile acids or FXR increases glucose-stimulated insulin secretion in primary islets (Seyer et  al. 2013). Insulin induces the CYP7A1 gene expression in primary human hepatocytes, while it is repressed by glucagon. However, insulin has dual effects on human CYP7A1 gene transcription. Thus, physiological concentrations of insulin rapidly inhibit FoxO1 activity leading to stimulation of the human CYP7A1 gene, whereas prolonged insulin treatment induces sterol regulatory element-binding protein-1c (SREBP-1c), which inhibits human CYP7A1 gene transcription. Glucagon and cAMP strongly repress CYP7A1 mRNA expression in human primary hepatocytes. In this case, cAMP and PKA inhibit human CYP7A1 gene transcription at PKA-responsive region located within the hepatic nuclear factor 4α (HNF4α) binding site in the human CYP7A1 promoter (Li et  al. 2006b; Song and Chiang 2006). Glucose stimulates bile acid synthesis by increasing ATP levels to inhibit AMPK, while inducing HNF4α to stimulate CYP7A1 gene transcription (Li et al. 2010). Glucose and insulin rapidly induce CYP7A1 gene expression and bile acid synthesis leading to an enlarged bile acid pool and elevated circulating bile acids (Li et  al. 2012b). Insulin

signaling activates AKT, and possibly also the MAPK/ERK1/2 pathway to inhibit CYP7A1 gene transcription. On the other hand, glucagon and cAMP strongly inhibit CYP7A1 expression via activation of PKA, which phosphorylates HNF4α to abolish its DNA-binding activity. Consequently, both insulin and glucagon effects result in inhibition of CYP7A1 expression in human hepatocytes (Song and Chiang 2006). Indeed, bile acids activate specific nuclear receptors (FXR, preganane X receptor, and vitamin D receptor), TGR5, and JNK1/2/AKT/ERK1/2/ MAPK cell signaling pathways in the liver and gastrointestinal tract (Dent et al. 2005; Hylemon et al. 2009). Unconjugated bile acid, DCA activates the Raf-1/MEK/ERK and AKT signaling cascades in primary hepatocytes via an epidermal growth factor receptor (EGFR)/Ras-­ dependent mechanism. DCA and TDCA cause ROS generation in hepatocytes that is dependent on metabolically active mitochondria. The generation of ROS is essential for protein tyrosine phosphatase (PTPase) inactivation, receptor tyrosine kinase activation, and enhanced signaling down the ERK1/2 and AKT pathways (Fang et  al. 2004; Rao et  al. 2002). LCA, which is a hydrophobic secondary bile acid produced by colonic microflora, is the most toxic bile acid. The basal as well as inducible glutathione (GSH) levels are important determinants of cellular resistance to LCA toxicity. The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) induces the major hydroxylation enzymes CYP3As and antioxidative genes. Thereby kelch-­ like ECH-associated protein 1 (Keap1)-Nrf2-­ antioxidant responsive element (ARE) signaling represents an important adaptive mechanism of cellular defense against toxic LCA. In this case, Nrf2 provokes GSH biosynthesis (Kensler et al. 2007; Tan et al. 2007). Whereas, UDCA increases the GSH synthesis through an activation of the PI3K/Akt/Nrf2 pathway in hepatocytes (Arisawa et al. 2009). On the other hand, TUDCA increases the expression levels of the bile acid transporters and Nrf2, decreases the expression levels of glucose-regulated protein 78 (GRP78), protein kinase R (PKR)-like ER kinase (PERK), activating transcription factor 4 (ATF4), and CCAAT/

9  Bile Acid Toxicity and Protein Kinases

enhancer-binding protein (C/EBP)-homologous protein (CHOP) in hepatocytes (Zhang et  al. 2017). FXR-TC-JNK axis is a novel mechanism to protect hepatocyte against toxicant-induced acute hepatitis. In this cycle, disruption of FXR in hepatocytes impairs BSEP function and increases TC levels. It is thought that TC-induced JNK activation is mediated by sphingosine1-phosphate receptor 2 (S1PR2) like hydrophobic bile acids. Thereby JNK may promote hepatocyte cell death via S1PR2  in FXRdisrupted hepatocytes (Takahashi et  al. 2017; Webster and Anwer 2016). Furthermore, TC activates the signaling pathways of AKT by 9-fold and ERK1/2 by 3 to 5-fold, whereas it downregulates the mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G-6-Pase) (Cao et  al. 2010). In fact, AKT phosphorylates FoxO1 and inhibits PEPCK and G-6-Pase in gluconeogenesis. In addition, glycogen synthase kinase 3β (GSK3β) activity is inhibited by phosphorylating via AKT pathway and glycogen synthesis enhances in hepatocytes. Bile acids may mimic the insulin action in regulating glucose metabolism by stimulating glycogen synthesis and inhibiting gluconeogenesis (Fang et al. 2007). Activation of the PI3K/PDK-1/ PKC-ζ pathway is required for the optimal activation of FXR by TC, which induces the small heterodimeric partner (SHP). SHP, as a nuclear receptor protein interacts with HNF4α, FOXO1, CEBPα transcription factors and inhibits their functions (Cao et  al. 2010). Activation of the nuclear FXR induces the membrane G ­protein-­coupled receptor TGR5, which alters bile acid composition, and both FXR and TGR5 may coordinately stimulate GLP-1 secretion from intestinal L cells to improve hepatic glucose and lipid metabolism (Pathak et  al. 2017). In brief, bile acid-activated FXR and signal transduction pathways are also involved in the regulation of hepatic gluconeogenesis, glycogen synthesis and insulin sensitivity (Trauner et al. 2010). The activated FXR-SHP pathway regulates the enterohepatic recycling and biosynthesis of bile acids and underlies the downregulation of hepatic fatty

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acid and triglyceride biosynthesis. Indeed, SHP inhibits the activity of several nuclear receptors. Thereby, SHP induction negatively regulates biosynthesis and uptake of bile acids. The Bile acidTGR5-cAMP the cAMP-dependent thyroid hormone activating enzyme type 2 iodothyronine deiodinase (D2) signaling pathway increases energy expenditure (Wei et al. 2009). Conjugated bile acids activate the ERK1/2 and AKT signaling pathways via unidentified G protein alpha i (Gαi) protein coupled receptor(s) in primary hepatocytes, while unconjugated bile acids activate the ERK1/2 and AKT pathways by at least two different mechanisms. Unconjugated bile acids activate hepatocyte receptor tyrosine kinases and intracellular signaling pathways in a ROS-dependent manner. In contrast, conjugated bile acids primarily activate receptor tyrosine kinases and intracellular signaling pathways in a G protein-coupled receptors (GPCR) (Gαi)dependent and as well as ROS-dependent manner. Expression of dominant-negative Gαi reduces TCA-induced activation of AKT and of glycogen synthase (GS) in intact hepatocytes (Dent et al. 2005; Fang et al. 2007). The generation of ROS is essential for protein tyrosine phosphatases inactivation, receptor tyrosine kinase activation, and enhanced signaling down the ERK1/2 and AKT pathways. Thus, DCA activates the ERK1/2 and AKT pathways by stimulating the synthesis of superoxide ions, which is shown to inactivate phosphotyrosine phosphatase(s) resulting in the activation of the EGFR (Fang et  al. 2004). In addition, DCA, CDCA and T-CDCA can activate matrix metalloproteinase(s) (MMPs) that generate transforming growth factor β (TGF-β), an EGFR ligand in cholangiocytes (Werneburg et al. 2003). The GPCRs-activated by S1P have been linked to the activation of various cell signaling pathways, including ERK1/2 and AKT. Sphingosine kinase 2 (SphK2) is primarily located in the nucleus and is activated by phosphorylation by phospo ERK1/2 (pERK1/2) to produce S1P, a powerful inhibitor of histone deacetylase 1 and 2 (Hait et al. 2009).

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4

Bile Acids and Cell Death

At pathophysiologic concentrations, bile acids induce proinflammatory cytokine expression in human hepatocytes, but not in nonparenchymal cells or cholangiocytes. This liver-specific inflammatory response requires bile acid-entry into hepatocytes via basolateral transporter, NTCP. Pathophysiologic levels of bile acids induce the markers of ER stress and mitochondrial damage (Cai et  al. 2017). Not all bile acids are toxic. In contrast to hydrophilic bile acids, accumulation of hydrophobic bile acids within the hepatocyte induces cell death. Inhibition of BSEP causes hepatic accumulation of toxic bile acids, CDCA, glyco-deoxycholic acid (GDCA), and DCA, leading to hepatocyte death (Oizumi et  al. 2017). Toxic bile acids promote death receptor-mediated cell death signaling. Therefore, they induce oligomerization of cell surface death receptors either by ligand-independent or -dependent mechanisms. The cytotoxic signals downstream of the DISC differ between cell types and have been characterized as “type I” and “type II” cellular responses. In type II cells, overexpression of B-cell leukemia/lymphoma-2 (Bcl-2) or B-cell lymphoma-extra large (Bcl-xL) blocks caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis is accompanied by activation of large amounts of caspase-8 within the DISC, whereas in type II cells DISC formation is strongly reduced and activation of caspase-8 and caspase-3 are independent to DISC (Scaffidi et  al. 1998). Current concepts suggest that bile acid-associated hepatocyte apoptosis is death-receptor dependent. Glycochenodeoxycholate (GCDC)-induced hepatocyte apoptosis involves ligand-independent oligomerization of Fas, recruitment of FADD, activation of caspase 8, and subsequent activation of effector proteases, including downstream caspases and cathepsin B (Faubion et al. 1999; Higuchi and Gores 2003; Miyoshi et  al. 1999) (Fig.  9.1). On the other hand, mitochondrial generation of ROS, and increase in mitochondrial free Ca2+ promotes the MPT.  In fact, MPT is a causative factor in necrotic cell death. Therefore, onset of the MPT to increasing num-

bers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death (Lemasters et al. 1998).

5

 ile Acid-Induced Hepatic B Inflammation

Bile acid accumulation in the liver causes cholestatic liver injury that often leads to progressive liver damage and subsequent liver failure. Hepatocytes release inflammatory cytokines that stimulate neutrophil chemotaxis. This inflammatory response involves mitochondrial damage and toll like receptor 9 (TLR9) activation in hepatocytes. Bile acid-­ stimulated chemokine expression in hepatocytes is mediated by nonparenchymal cells. Deleterious effects of TCA on mitochondria and the ER are both dose- and time-dependent. As evidenced by cytochrome c, apoptosis-inducing factor (AIF) and 78-kDa glucose-regulated protein (Grp78) leakage into the cytosol, is positively correlated with bile acid induction of [C-C motif] chemokine ligand 2 (Ccl2) and chemokine (C-X-C motif) ligand 2 (Cxcl2) mRNA expression. NTCP plays a critical role in the pathogenesis of cholestatic liver injury since it is required for conjugated bile acid to be taken up into hepatocytes. By blocking of NTCP functions, hepatocytes injury is reduced (Cai et al. 2017). In response to cholestasis, activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and MAPK signaling, production of inflammatory cytokines, and recruitment of neutrophils occur. In acute cholestatic liver injury, loss of cellular FADD-­like interleukin 1-beta (IL-1β)–converting enzyme (FLICE) inhibitory protein (cFLIP) in hepatocytes promotes a rapid release of proinflammatory and chemotactic cytokines (IL-6, IL-1β, CCL2, CXCL1, and CXCL2), an increased accumulation of CD68+ macrophages and an influx of neutrophils. All these result in apoptotic and necrotic hepatocyte cell death in the liver (Gehrke et  al. 2018). ER stress or the unfolded protein response (UPR) contributes to hepatocyte cell death together with the alterations of lipid and fatty acid metabolism. Receptor-interacting pro-

9  Bile Acid Toxicity and Protein Kinases

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Fig. 9.1 Induction of death transduction pathways by receptor, APO-1, Fas receptor, CD95L: CD95 with its hydrophobic bile acid in hepatocyte. Hydrophobic bile natural ligand (CD178), cFLIP: The antiapoptotic caspase acids promote both Fas- and TRAIL-R2/DR5-mediated 8-homolog cellular FLICE inhibitory protein, CHOP: C/ cell death signaling by activating ligand-dependent EBP-homologous protein, Cyt c: Cytochrome c, DD: or -independent death receptor oligomerization. Moving Death domain, DED: Death effector domain, DISC: of Fas receptor to the plasma membrane by JNK- and Death-inducing signaling complex, DR: Death receptor, PKC- activation results in death receptor oligomerization EGFR: Epidermal growth factor receptor, elF2α: The and DISC formation. Hydrophobic bile acid-induced alpha subunit of the translation initiation factor 2, ER: NADPH oxidase-JNK pathway triggers the EGFR activa- Endoplasmic reticulum, FADD: Fas (The receptor CD95)tion, through Src kinase Yes. Following CD95 tyrosine associated death domain, Fas: The receptor CD95, IL-1β: phosphorylation and DISC formation, tBid/Bax-­ Interleukin-1β, IL23 Interleukin-23, IRE1α: Inositoldependent mitochondrial dysfunction and accompanied requiring enzyme 1 α, JNK: c-Jun N-terminal kinase, excessive ROS generation lead to either apoptosis or MAPK: Mitogen-activated protein kinase, M1: necrosis. Furthermore, hydrophobic bile acids may Macrophage type 1, MPT: Mitochondrial permeability directly target mitochondria, either through induction of transition, Mt.: Mitochondria, NADPH: Reduced nicotinthe MPT and ROS production or activation of pro-­ amide adenine dinucleotide phosphate, PERK: PKR-like apoptotic Bcl-2 family members. (Abbreviations: endoplasmic reticulum kinase, PKC: Protein kinase C, AA-Bcl-2: Antiapoptotik Bcl-2, AIF: Apoptosis-inducing PLAD: Pre-ligand binding assembly domain, ROS: factor, APAF1: Apoptosis peptide activating factor 1, Reactive oxygen species, SMAC: Second activator of ATF4: activating transcription factor 4, BA: Bile acid, mitochondrial apoptosis, tBID: Truncated BID, Th17: T BAX: BCL-2 associated X apoptosis regulator, BCL-2: helper 17, TNFα: Tumor necrosis factor-α, TRAIL: Tumor antiapoptotic B-cell leukemia/lymphoma 2 protein, BID: necrosis factor-associated apoptosis-inducing ligand, BH3-domain-only pro-apoptotic factor, BIM: Bcl-2 inter- UPR: Unfolded protein response) acting mediator of cell death, Casp: Caspase, CD95: death

tein (RIP) kinases have an important function in necrotic cell death in hepatocyte injury (Malhi et al. 2010). It is suggested that the death receptors Fas, TNF receptor 1 (TNF-R1), and tumor necrosis factor-­related apoptosis inducing ligand (TRAIL) receptor 1/2 (TRAIL-R1/2) are major mediators of the apoptotic pathway. Despite this idea, bile acids do not appear to enhance TNF-α/ TNF-R1 cytotoxicity, because, hepatocytes can-

not express TNF-α following GCDC treatment (Higuchi et al. 2001). Indeed, enhanced expression and oligomerization of this death receptor has been described in GCDCA-­induced apoptosis in Fas-deficient cell lines. Aggregation of TRAIL-R2/death receptor 5 (DR5) is observed following GCDC treatment of these cells. Bile acid increases expression of TRAIL-R2/DR5 mRNA and protein 10-fold while expression of

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TRAIL-R1 is unchanged (Higuchi et  al. 2001). The death receptors oligomerize and recruit different adaptor proteins which activate the initiator caspase 8 and likely caspase 10. Active caspase 8 cleaves the BH3-­only protein, BH3 interacting domain death agonist (Bid) and generates truncated Bid (tBid), following N-myristoylation (Zha et  al. 2000). Although Bcl-2 associated protein x (BAX)-induced cytochrome c release is not dependent on tBid, the two proteins could function synergistically. However, Bid induces cytochrome c release through a mechanism independent of mitochondrial permeability transition pore and Bax (Kim et al. 2000). Thus, an activation cascade of proapoptotic proteins from BID to Bcl-2 antagonist killer (BAK) or BAX integrates the pathway from surface death receptors to the irreversible efflux of cytochrome c (Korsmeyer et al. 2000). In brief, activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates the downstream caspases. This Bid-mediated pathway is critical in hepatocyte apoptosis induced by Fas/TNF-R1 engagement (Yin 2000). Since death receptors are ubiquitously expressed in the liver, hepatocytes are subject to death receptor-induced Bid cleavage with subsequent BAX/BAK activation and cell death. Adequate cellular levels of the antiapoptotic proteins myeloid cell leukemia 1 (Mcl1) and/or Bcl-xL are, therefore, necessary to counteract the deleterious consequences of constitutive Bid activation (Malhi et al. 2010). Elevated concentrations of bile acids during cholestasis increase expression of the transcription factor early growth response factor-1 (Egr-1) in hepatocytes. Egr-1 then upregulates proinflammatory mediators that causes neutrophils to accumulate in the liver and become activated to damage hepatocytes (Kim et al. 2006). Exposure of hepatocytes to bile acids increases levels of numerous mediators, including cytokines (IL-1β, IL-10), chemokines (keratinocyte chemoattractant; KC), interferon-­ γ-­inducible protein 10 (IP-10), interferon–inducible T cell alpha chemoattractant (I-TAC), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T cell expressed and pre-

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sumably secreted (RANTES), MCP-3, macrophage inflammatory protein-1 alpha (MIP-1α), MIP-2, MIP-3α, lipopolysaccharide-­induced CXC chemokine (LIX), scavenger receptor for phosphatidylserine and oxidized low density lipoprotein (SR-PSOX), MCP-3, B lymphocyte chemokine (BLC), adhesion molecules (intercellular adhesion molecule 1(ICAM-1), vascular cell adhesion molecule 1 (VCAM-1)), enzymes in arachidonic acid metabolism (cyclooxygenase-2; COX-2), and other proteins that influence immune cell levels and function (Allen et  al. 2011). Activation of MAPK signaling by bile acids is required for upregulation of Egr-1  in hepatocytes. Indeed, bile acids modulate gene expression in hepatocytes through activating the FXR-MAPK signaling. Thus, expression of Egr-1 in hepatocytes is upregulated by bile acids through FXR and MAPK-dependent mechanisms during cholestasis (Allen et  al. 2010, 2011). CHOP is a key component in ER stress-­ mediated apoptosis. Thus, cholestasis induces CHOP-mediated ER stress and triggers hepatocyte cell death. Over-expression of CHOP and BAX are downstream target in the CHOP-­ mediated ER stress pathway in cholestasis. However, in acute period of cholestasis, necroptosis mediates hepatic necroinflammation. In this case, the PERK/eukaryotic initiation factor 2 alpha (eIF2α) signaling pathway plays a role in the regulation of CHOP by insulin-like growth factor type 1 receptor (IGF-1R). IGF-1R contributes to cholestatic liver injury, promoting the involvement of both CHOP and BAX in this process (Afonso et al. 2016; Cadoret et al. 2009; Tamaki et  al. 2008). Conjugated bile acids can activate the S1PR2  in cholangiocyte plasma membranes which initiates an AKT and ERK1/2 signaling pathway that elicits an inflammatory and fibrotic response (Wang et al. 2017). In fact, neutrophil accumulation is the principal cause of hepatocyte toxicity in the early stages of cholestatic liver injury and inflammation. Neutrophils are activated and recruited to the liver by the pro-inflammatory mediators induced by high levels of bile acids (Li et  al. 2017). Monocytes and macrophages express TGR5, the

9  Bile Acid Toxicity and Protein Kinases

G-protein coupled bile acid receptor, that can be activated by both conjugated and unconjugated bile acids. Among these, LCA, DCA, CDCA, and CA are the most potent activators in a descending order (Perino and Schoonjans 2015). TGR5 activation leads to an increase in cAMP and PKA activation and then modulates bile acid homeostasis and inflammation (Guo et al. 2016a; Perino and Schoonjans 2015; Thomas et  al. 2008). TGR5 has been detected in cholangiocytes, Kupffer cells, and sinusoidal endothelial cells but not hepatocytes (Keitel and Häussinger 2012). Activation of TGR5 in macrophages reduces pro-­ inflammatory cytokines while maintaining anti-­ inflammatory cytokine expression thus promoting the development of an anti-inflammatory macrophage phenotype (Reich et al. 2017). The anti-inflammatory effect of TGR5  in macrophages is mediated by inhibiting the NF-κB and JNK signaling pathways. TGR5 activation alone induces the expression of IL-1β and TNF-α in macrophages. The TGR5-mediated increase in pro-inflammatory cytokine expression is suppressed by JNK inhibition. The induction of proinflammatory cytokine expression in Kupffer cells by TGR5 activation is correlated with the suppression of cholesterol 7α-hydroxylase (Cyp7A1) expression in hepatocytes. These results suggest that TGR5 mediates the bile acidinduced pro-inflammatory cytokine production in Kupffer cells through JNK-dependent pathway (Lou et al. 2014; Perino and Schoonjans 2015). Recent studies suggest that TGR5 is required for bile acid induced cholangiocyte proliferation and the activation of TGR5 protects cholangiocytes from death-­ receptor-­ mediated apoptosis by TGR5-dependent CD95 receptor serine phosphorylation in cholestasis (Keitel and Häussinger 2013; Reich et al. 2016). IL-1β and CDCA reduce CYP7A1 but induce c-Jun messenger RNA expression in human primary hepatocytes. However, IL-1β inhibits human CYP7A1 reporter activity via the HNF4α binding site. HNF4α is one of the two major transcription factors, which are driving CYP7A1 promoter activity in hepatocytes. IL-1β and CDCA inhibit HNF4α but induces c-Jun, which

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in turn blocks HNF4α recruitment of peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator-1α (PGC-1α) to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. Thereby, the JNK/c-Jun signaling pathway protects the hepatocytes against the toxic effect by inhibiting bile acid synthesis (Li et  al. 2006a). On the other hand, IL-17A signaling is a prerequisite for hepatic neutrophil accumulation during obstructive cholestasis (Meng et al. 2012). Bile acids activate ERK1/2  in hepatocytes, which stimulate up-regulation of Egr-1. Egr-1 then regulates production of MIP-2. The synergistic enhancement of MIP-2 expression by IL-17A and TCA is important for obstructive cholestasis (O’Brien et al. 2013). In fact, bile acids stimulate the production of IL-23 and IL-17A by hepatocytes and by T-helper 17 (Th17) cells, respectively. IL-17A enhances the bile acid-induced production of IL-23 and other inflammatory mediators by hepatocytes. In cholestatic liver, bile acid accumulation triggers the production of inflammatory mediators, which are induced by the IL-23/IL-17A axis in Kupffer cells. Thereby, IL-17A promotes hepatic inflammation during cholestasis by synergistically enhancing bile acid-induced production of proinflammatory cytokines by hepatocytes (Meng et  al. 2012; O’Brien et al. 2013). CDCA, as the major hydrophobic primary bile acid, dose-dependently induce nucleotide oligomerization domain (NOD)-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation and secretion of pro-­inflammatory cytokine-IL-1β in macrophages by promoting ROS production. CDCA is an endogenous danger signal to activate NLRP3 inflammasome and initiates liver inflammation during cholestasis (Gong et  al. 2016). Bile acids are endogenous danger-associated molecular patterns (DAMPs) that can activate both signal 1 and 2 of the NLRP3 inflammasome in inflammatory macrophages (Hao et  al. 2017). Receptor-interacting serine/ threonine-protein kinase 1 (RIPK1)-RIPK3MLKL-mediated necroptosis has been attributed to cause inflammatory response through the

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release of DAMPs (Moriwaki and Chan 2016). mRNA and protein expression of RIPK3 and MLKL, as well as MLKL phosphorylation, strongly increase in the cholestatic hepatocytes due to bile acid toxicity. RIP3 deficiency prevents necroinflammation induced by bile acids accumulation (Afonso et  al. 2016). DAMPs that are released from dead hepatocytes may activate inflammatory responses in immune cells and hepatic stellate cells. DAMPS result in the assembly of a cytosolic protein complex termed the inflammasome, which activates the serine protease caspase-1, resulting in activation and secretion of IL-1β and other cytokines (Kubes and Mehal 2012). Furthermore, inflammasomes sense DAMPs and pathogen-associated molecular patterns (PAMPs) via nucleotide-binding oligomerization domain NOD-like receptors (NLRs). DAMPs and PAMPs activate caspase-1 which then proteolytically activates IL-1β and IL-18. IL-1β amplifies the inflammatory response by further stimulating production of inflammatory cytokines. In cholestasis, bile acids inhibit NLRP3 inflammasome activation in macrophages via the TGR5-cAMP-PKA axis and phosphorylation of NLRP3 on Ser 291 (Guo et  al. 2016b; Li et  al. 2017). Interestingly, while the NLRP3 inflammasome aggravates inflammatory liver injury by inducing IL-1β, on the other hand, affects the epithelial integrity of cholangiocytes by inducing the production of pro-inflammatory cytokines. In addition, bile acids, including DCA and CDCA, can activate the NLRP3 inflammasome in macrophages (Wang et  al. 2019). In hepatocytes, inflammasome activation induces hepatocyte death via pyroptosis (Schroder and Tschopp 2010).

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dependent manner in cells exposed to bile acid (Perez and Briz 2009; Sokol et  al. 1995). Cholestasis is characterized by disruption of bile flow, resulting in intrahepatic and systemic retention of bile acids, with a concomitant toxic response in liver parenchymal cells. Cholestatic liver injury may arise from genetic disorders, drug toxicity, hepatobiliary malignancies or obstruction of the biliary tract. Inflammation and necrosis have an equal impact or one of them has more prevalent role (Castro and Rodrigues 2017). The accumulation of bile acids causes defective autophagic clearance, shown by the accumulation of insoluble p62 and ubiquitinated proteins and cell death accompanied by caspase-3 processing. Hepatocytes exposed to bile acids lead to the accumulation of autophagosomes due to suppressed autophagy flux (Kim et  al. 2018). Apoptosis is activated late after extrahepatic cholestasis in parallel with development of liver fibrosis, whereas it is widely accepted that apoptosis may trigger hepatic fibrogenesis (Luedde et al. 2014). Two apoptotic pathways are described as the mitochondrial pathway and the classical death receptor pathway. These two apoptotic pathways interact between caspase activation, mitochondrial dysfunction, and cellular distribution of Bcl-2-related proteins in cholestatic hepatocyte injury. GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a Fas-associated protein with death domain-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This is explained by cytokine-induced activation of NF-kB-regulated antiapoptosis genes like A1 and cellular inhibitor of apoptosis 2 (cIAP2) (Maher 2004). Bile acid-­ associated hepatocyte apoptosis occurs through the death receptors Fas. Glycochenodeoxycholate 6 Bile Acid-Induced Apoptosis (GCDC)-induced hepatocyte apoptosis involves ligand-independent oligomerization of Fas, Accumulation of bile acid within hepatocytes recruitment of FADD, activation of caspase 8 results in cell injury and death. The exposure to (Faubion et al. 1999). Toxic bile acids cause cell low concentration of bile acid leads to apoptosis, death, partly due to the activation of a protease while high concentration exposure causes cascade. The proximal signaling protease casnecrosis. The percent of cells undergoing pase 8 appears to be activated by toxic bile acids apoptosis increase in a time- and concentration-­ in a Fas-receptor-­dependent manner. However,

9  Bile Acid Toxicity and Protein Kinases

both play a critical role in bile acid-induced apoptosis (Faubion et  al. 1999). CD95 ligand (CD95L)-induced apoptosis involves a sphingomyelinase- and PKC-ζ-­dependent activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoforms. It has been demonstrated that bile acid-induced oxidative stress may trigger the activation of JNKs and PKC (Reinehr et al. 2005a). CD95L triggers activation of the Src kinase family member Yes, which requires NADPH oxidase-mediated oxidative stress as an upstream event. Yes-activation is also responsive to hydrophobic bile acids. CD95L-induced CD95 activation and DISC formation are dependent upon Yes signal. Hydrophobic bile acids activates Yes and the CD95 system and induces apoptosis (Reinehr et al. 2004). Hydrophobic bile acids, such as GCDCA and T-CDCA, could induce cytotoxicity by acting as detergents on cell membranes. However, serum bile acid levels in cholestatic patients are insufficient to produce a significant detergent effect. Therefore, bile acid-induced cell death occurs by either necrosis or apoptosis. In contrast to T-CDCA and TUDCA, GCDCA-­induced apoptosis occurs by a mitochondria-­controlled pathway in hepatocytes (Maher 2004). Indeed, pathophysiological concentrations of bile acids induce apoptosis by directly activating death receptors. However, GCDC-induced hepatocyte apoptosis involves ligand-independent oligomerization of Fas, recruitment of FADD, activation of caspase 8, and subsequent activation of effector proteases, including downstream caspases and cathepsin B (Faubion et al. 1999) and inducing oxidative damage and mitochondrial dysfunction. The percent of cells undergoing apoptosis increase in a time- and concentration-­ dependent manner in cells exposed to GCDC, and when compared to GCA is much lesser extent. ROS generation precedes the onset of apoptosis. MPT blockers, caspase-8 inhibition, and antioxidants prevent apoptosis. ROS generation is reduced in hepatocytes (Rodrigues et  al. 1998; Yerushalmi et  al. 2001). Toxic bile acids have been shown to induce apoptosis in a Fasdependent manner. Suppression of PKC prevents

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bile acid-stimulated cFLIP-L and -s phosphorylation, restors cFLIP binding to glutathione S-transferase (GST)-FADD, and attenuates bile acid-induced apoptosis (Faubion et  al. 1999; Higuchi et al. 2003). cFLIP is a phosphoprotein and that toxic bile acids potentiate phosphorylation of both cFLIP-L and -S by a PKC-dependent mechanism (Higuchi et al. 2003). The phosphorylation of cFLIP decreases binding of cFLIP to FADD, resulting in reduced recruitment to the DISC. Decreased cFLIP results in enhanced activation of pro-caspase-8/10 (Higuchi and Gores 2003). Recruitment of the FADD involves the activation of caspase-8 and Bid, as well as downstream effector caspases. Activation of death receptors by bile acids results in induction of their transport from the Golgi (Sodeman et  al. 2000). Oxidative stress due to bile acid accumulation ultimately leads to Fas activation, involving NADPH oxidase activation. Thus, interaction of bile acids with mitochondria directly generates ROS (Reinehr et al. 2005a). More recently, G-protein-coupled receptor TGR5 activation has also been implicated in Fas translocation and oligomerization. Bile acid-­induced JNK activation is dependent on bile acid activation of TGR5. JNK formed complexes with caspase 8, are reduced following bile acid treatment, but this reduction is prevented when TGR5 or JNK is inhibited. In conclusion, bile acids activate TGR5, which leads to JNK activation and reduced complex formation of JNK with caspase 8, thus facilitating caspase 8 recruitment to DISC (Yang et al. 2007). Fas expression at the cell surface via transport from the Golgi complex induce Fas-FADD binding (Bennett et al. 1998). In addition, p53 contributes to cyclin kinase inhibitorenhanced, bile-acid-­induced apoptosis via Fas. Blockade of DCA-­ induced ERK1/2 and AKT activation, with inhibitors of RAS, PI3K, or MEK1/2, increases apoptosis approximately 10-fold. In this case, apoptosis is dependent on bile acid-induced, ligand-independent activation of the CD95 death receptor. Increased expression of CD95 in hepatocytes overexpress p21 or p27. Overexpression of p21 or p27  in a p53-dependent fashion enhances basal plasma membrane levels of CD95 but do not further enhance bile

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acid stimulated CD95 activation. Increased expression and nuclear localization of p53 correlates with increased expression of multiple proapoptotic gene products (Zhang et  al. 2008). Hydrophobic bile acids are known to induce apoptosis in hepatocytes, in contrast, hydrophilic bile acids increase intracellular cAMP levels and activate MAPK and PI3K pathways to protect hepatocytes from apoptosis. Overexpression of p21 enhances p53 protein levels. p53 overexpression is evident in the enhanced apoptotic response. Thus, overexpression of p21 leads to elevated sensitivity to proapoptotic stimuli. Taurolithocholate-3-­ sulfate (TLCS) induces a JNK-dependent EGFR/CD95 association in the cytosol and moving of this protein complex towards the plasma membrane. Although inhibition of EGFR tyrosine kinase activity allows for cytosolic EGFR/CD95 association, it prevents targeting of the EGFR/CD95 complex to the plasma membrane. Furthermore, hydrophobic bile acids activate NADPH oxidase isoforms. Resultant oxidative stress triggers activation of the CD95 system, and apoptosis (Amaral et  al. 2009; Qiao et  al. 2002a; Reinehr et  al. 2005b). Accumulation of hydrophobic bile acids in the liver contributes to cholestatic liver injury. They dose-dependently induce NLRP3 inflammasome activation and secretion of pro-inflammatory cytokine-IL-1β in macrophages by promoting ROS production and K+ efflux. Hydroperoxide generation precedes the hepatocyte injury, and thiobarbituric acid-­reacting substances (TBARS) are accumulated, thereby mitochondrial pathways of cell death are stimulated. In this respect, CDCA triggers ROS formation in part through TGR5/EGFR downstream signaling, including PKB/AKT, ERK and JNK pathways (Gong et al. 2016; Sokol et al. 1995). On the other hand, bile acids inhibit NLRP3 inflammasome activation via the TGR5-cAMP-PKA axis. TGR5 bile acid receptor-induced PKA kinase activation leads to the ubiquitination of NLRP3, which is associated with the PKA-induced phosphorylation of NLRP3. Furthermore, PKA-induced phosphorylation of NLRP3 serves as a critical brake on NLRP3 inflammasome activation and subsequent apoptosis of hepatocytes (Guo et  al.

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2016b). Intracellular ROS generation by mitochondria appears to be an early event in hydrophobic bile acid-induced hepatocyte toxicity (Sokol et al. 1995). Bile acids also induce apoptosis through the mitochondrial or intrinsic pathway, in which intracellular stress causes mitochondrial dysfunction and the subsequent release of proapoptotic factors. Whereas, hydrophobic bile acids can induce the MPT. Thus, the GCDC-mediated hepatocyte necrosis via MPT induction is inhibited by a hydrophilic bile salt, UDCA. The critical common steps of apoptosis by bile acids involve ROS generation, MPT induction, cytochrome c release and decrease of antioxidants (Botla et al. 1995; Rodrigues et al. 1998; Yerushalmi et  al. 2001). The E3 ligases inhibitor of apoptosis (cIAP1/2) and the linear ubiquitin chain assembly complex (LUBAC) ubiquitinate RIP1 in the TNFR1 signaling complex (TNF-RSC). Polyubiquitinated RIP1 then engages downstream adaptors such as nuclear factor-kappa B essential modulator (NEMO) to activate IKK to promote NF-κB transcriptional activity, leading to cell survival, proliferation, and differentiation (Walczak 2011). When RIP1 ubiquitination is blocked by removal of the E3 ligases cIAP1 and cIAP2 RIP1 forms a secondary complex in the cytosol with FADD and caspase-8 termed the “Ripoptosome”, which contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. cFLIPL prevents ripoptosome formation to initiate apoptotic cell death. Ripoptosome is necessary but not sufficient for cell death (Feoktistova et al. 2011; Tenev et  al. 2011). Bile acids not only initiate ligand-­independent death-receptor oligomerization but also modulate the signaling pathway, causing a strong sensitization of the cells to death receptor-­ mediated apoptosis. Death receptormediated apoptosis is regulated at the cell surface by the receptor expression, at the DISC by expression of procaspase-8, FADD, or apoptosis inhibitor cFLIP. At mitochondria, the expression of proapoptotic or antiapoptotic Bcl-2 family proteins contribute to apoptosis. In this regard, Bcl-2, Bcl-xL, Bid, or Bax, regulate mitochondrial cytochrome c release. Bile acids promote death-­receptor oligomerization by either ligand-­

9  Bile Acid Toxicity and Protein Kinases

dependent or -independent mechanisms. In the absence of Fas, toxic bile acids increase the TRAIL-R2/DR5 on the cell surface. Whereas in the presence of Fas, bile acids induce the cell surface trafficking of Fas mRNAs, and they accumulate on the plasma membrane (Higuchi and Gores 2003). At the DISC level, bile acids do not alter expression of procaspases, FADD, or cFLIP.  However, bile acids appear to prevent cFLIP recruitment to the DISC and thereby enhance initiator caspase activation. Both caspase 8 and caspase 10 recruitment and processing within the TRAIL-DISC are greater in GCDCA-­ treated cells whereas recruitment of cFLIPL and cFLIPS is reduced (Higuchi et  al. 2003). Loss of cFLIP in hepatocytes promotes acute cholestatic liver injury early after extrahepatic cholestasis, which is characterized by a rapid release of proinflammatory and chemotactic cytokines, TNF, IL-6, IL-1β, CCL2, CXCL1, and CXCL2, an increased CD68+ macrophages and an influx of neutrophils into liver. These result in apoptotic and necrotic hepatocyte cell death (Gehrke et al. 2018). Due to bile acid toxicity, expression of Th1-related cytokines increases, and TNF-α stimulates the TRAIL-R2/DR5  in hepatocytes (Gomez-Santos et al. 2012). In Fasindependent apoptosis in Fas deficient cells, aggregation of death receptor, TRAIL-R2/DR5 mediates apoptosis in bile acids-exposed cells (Higuchi et al. 2001). TGR5 expression and its responsiveness to bile acids are confirmed in human hepatocytes. TGR5 inhibition attenuates bile acid-induced caspase 8 activation, which results from reduced bile acid-induced caspase 8 recruited to a DISC. Bile acid-induced JNK activation is dependent on bile acid activation of TGR5. JNK forms complexes with caspase 8, which are reduced following bile acid treatment, but this reduction is prevented when TGR5 or JNK is inhibited (Yang et  al. 2007). JNK activation may be important for bile acid-induced apoptosis by triggering ligand-independent CD95 surface trafficking and activation of apoptosis. Inhibition of JNK1 or PKC prevented taurolithocholic acid-3 sulfate (TLCS)-induced CD95 membrane trafficking and blunted the apoptotic response

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(Graf et  al. 2002). Induction of apoptosis by hydrophobic bile salts involves EGFR activation and EGFR-dependent CD95 tyrosine phosphorylation, which triggers CD95 membrane targeting and Fas-associated death domain/ caspase-8 recruitment. The latter step is apparently controlled by PI3K (Reinehr et  al. 2003). CD95 activation by hydrophobic bile acids is initiated by NADPH oxidase-dependent ROS signaling. Consequences of TLCS-induced oxidative stress is JNK activation and Src kinase family member Yes-dependent activation of the EGFR.  EGFR-catalyzed CD95 tyrosine phosphorylation and formation of the death-­ inducing signaling complex result in apoptosis. JNK-dependent EGFR-CD95 association in the cytosol allows transport of this protein complex to the plasma membrane (Reinehr et al. 2005b). EGFR-dependent CD95 tyrosine phosphorylation, triggers CD95 membrane targeting and Fas-associated death domain/ caspase-8 recruitment. The latter step is apparently also controlled by PI3K (Reinehr et al. 2003). The Src kinase Yes as an upstream target of proapoptotic bile acids, triggers EGFR activation (Reinehr et al. 2004). EGFR associates in a JNK-dependent pathway with CD95 and catalyzes CD95 tyrosine phosphorylation. Eventually, membrane trafficking of CD95 and subsequent DISC formation leads to apoptosis (Reinehr et al. 2003). Bile acid-induced NADPH oxidase activation not only triggers CD95 activation, but also hepatocyte shrinkage. Hydrophobic, proapoptotic bile salts induce hepatocyte shrinkage largely through NADPH oxidase-derived oxidative stress. A vicious cycle between oxidative stress and cell shrinkage propagates CD95 activation and apoptosis (Becker et al. 2007). Detached from their natural extracellular matrix, hepatocytes enter apoptosis (Pinkse et al. 2004). Hepatocyte adherence to the extracellular matrix is mediated by integrins. Integrins have cytoprotective effect through the activation of non-receptor dual tyrosine kinase complex, which consists focal adhesion kinase (FAK) and c-Src (Mitra and Schlaepfer 2006). Hydrophobic bile acid induced apoptosis is associated with

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augmented JNK and decreased FAK phosphorylation. cAMP-guanine exchange factor (cAMP-GEF) activation in hepatocytes increases the PI3K dependent phosphorylation of FAK.  Phosphorylation of this protein kinase is necessary for cytoprotective effect in hydrophobic bile acid-induced apoptosis via cAMP-GEF/ PI3K/FAK survival pathway in hepatocytes (Usechak et  al. 2008). Indeed, hepatocytes express three catalytic PI3K Class I isoforms (p110α/β/γ). PI3K p110γ (p110γ is associated with detrimental effects) is activated by hydrophobic, but not by hydrophilic bile salts. Bile salt-induced hepatocyte apoptosis is partly mediated via a PI3K p110γ dependent signaling pathway, potentially involving JNK (Hohenester et  al. 2010). PKC-δ silencing increases GCDC-­ induced JNK phosphorylation, decreases GCDC-­ induced AKT phosphorylation, and increases expression of Bcl-2 interacting mediator of cell death (BIM). GCDC translocate BIM to the mitochondria in hepatocytes. PKC-δ does not mediate GCDC-induced apoptosis in hepatocytes. Instead PKC-δ activation by GCDC stimulates a cytoprotective pathway that involves JNK inhibition, AKT activation, and downregulation of BIM (Webster et  al. 2014). GCDC, the predominant circulating bile acid in humans, paradoxically elicits prodeath as well as prosurvival signals in hepatocytes (Dent et  al. 2005; Hohenester et  al. 2010; Rust et  al. 2000, 2009; Schoemaker et al. 2004). Prodeath pathways converge on sustained phosphorylation of JNK. The role of JNK in hepatocyte death is supported by results that show that chemical or genetic inhibition of this kinase. JNK attenuates bile acidinduced apoptosis (Hohenester et al. 2010; Kluwe et  al. 2010; Reinehr et  al. 2005b). Bile acidinduced prosurvival signaling involves activation of PI3K and AKT.  Inhibition of either kinase results in increased cell death in response to GCDC (Cullen et  al. 2004; Schoemaker et  al. 2004). Sustained JNK activation is a prodeath signal in bile acid-induced apoptosis in hepatocytes (Hohenester et  al. 2010; Reinehr et  al. 2005b), whereas activation of PI3K/AKT is prosurvival (Hohenester et  al. 2010; Webster et  al. 2002). Our studies show that genetic silencing of

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PKC-δ results in an increase in GCDC-induced phosphorylation of JNK and apoptosis while decreasing GCDC phosphorylation of AKT.  These opposing effects would both promote hepatocyte cell death (Webster et al. 2014). DCA-induced JNK2 signaling is cytoprotective whereas DCA-induced JNK1 signaling is cytotoxic. DCA-induced ERK1/2 activation was responsible for increased DNA binding of C/ EBPβ, cAMP response element-binding protein (CREB), and c-Jun/AP-1. Inhibition of C/EBPβ, CREB, and c-Jun function promotes apoptosis following DCA treatment, and the level of apoptosis is further increased in the case of CREB and c-Jun, but not C/EBPβ, by inhibition of MEK1/2. The combined loss of CREB and c-Jun function or of C/EBPβ and c-Jun function enhance DCAinduced apoptosis above the levels resulting from the loss of either factor individually; however, these effects are less than additive. Loss of c-Jun or CREB function correlates with increased expression of FAS death receptor and p53 upregulated modulator of apoptosis (PUMA) and decreased expression of c-FLIPL and c-FLIPS, proteins previously implicated in the modulation of the cellular apoptotic response. Collectively, these data demonstrate that multiple DCAinduced signaling pathways and transcription factors control hepatocyte survival (Qiao et  al. 2003). Cholestatic liver disorders are accompanied by the hepatic accumulation of cytotoxic bile acids that induce cell death. Increases in cAMP protect hepatocytes from bile acid-induced apoptosis by a cAMP-guanine exchange factor (GEF)/PI3K/AKT pathway. The hydrophobic bile acid-induced phosphorylation of the proapoptotic kinase JNK is inhibited by GSK inhibition or cAMP-GEF activation. The hydrophobic bile acids-induced apoptosis is accompanied by phosphorylation of the ER stress markers pIEF2α and IRE-1, and cAMP-GEF analog or GSK inhibitors prevent this phosphorylation. cAMP/ cAMP-GEF/Rap1/PI3K/AKT/GSKβ survival pathway in hepatocytes inhibits bile acid-induced JNK phosphorylation (Johnston et al. 2011). Bile salt-­ induced intracellular signaling pathways play important roles in modulating hepatocyte apoptosis. GCDCA induces ER-related calcium

9  Bile Acid Toxicity and Protein Kinases

release. Significant increases in activities of calpain and caspase-12 are observed after GCDCA treatment. Bip and CHOP mRNA expressions are increased with the dose- and incubation timedependently. Cytochrome c release from mitochondria suggest that ER stress is induced by GCDCA (Tsuchiya et al. 2006). GCDC-induced activation of JNK, ERK, and AKT, relies on the presence of the bile salt transporter NTCP. In order to achieve this, either GCDC enters the cell to modulate intracellular kinase phosphorylation or binds to NTCP, which is necessary to induce kinase phosphorylation (Webster and Anwer 2016). GCDC leads to activation of sphingosine kinase (SphK) and then generation of sphingosine-1-phosphate (S1P).  The S1P is transported out of the cell by an ABC transporter and binds to S1PR2, leading to activation of Gαi and inhibition of cAMP production. S1PR2 is the predominant S1P receptor expressed in cholangiocytes. Intracellular mediators of inflammatory damage, induced by S1PR2 stimulation, are the ERK1/2/ AKT/NF-kB axis, with increased COX-2 activity at the biliary level (Baiocchi et al. 2019; Salomone and Waeber 2011). The hepatotoxic bile acid GCDC modulates hepatocyte cell death through activation of JNK, AKT, and ERK. The nonhepatotoxic bile acid TC activates Akt and ERK through the S1PR2. S1PR2 inhibition attenuates GCDC-induced apoptosis and inhibits and augments GCDC-induced JNK and AKT phosphorylation, respectively. S1PR2 activation is proapoptotic in GCDC-induced cell death (Webster and Anwer 2016). Transfer of calcium to mitochondria is required for SphK2-induced apoptosis, as cell death and cytochrome c release is abrogated by inhibition of the mitochondrial Ca2+ transporter. Targeting SphK1 to the ER converts it from anti-apoptotic to pro-apoptotic state (Maceyka et al. 2005). Thus, SphK1 plays a pivotal role in mediating bile salt-induced apoptosis in hepatocytes in part by interfering with intracellular [Ca2+] signaling and activation of S1PR1 (Karimian et al. 2013; Maceyka et al. 2002). The S1PR2 inhibition attenuates GCDC-­ induced apoptosis and inhibits and augments GCDCinduced JNK and Akt phosphorylation, respectively. In addition, GCDC must enter hepatocytes

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to mediate cell death or activate kinases. These results suggest that S1PR2 activation is proapoptotic in GCDC-induced cell death but that this effect is not due to direct ligation of the S1PR2 by the bile acid (Webster and Anwer 2016).

7

 ntiapoptotic Effect of Bile A Acids

Bile acids are known to be involved in hepatocyte cell survival pathways. In this regard, hydrophobic bile acids have been reported to be cytotoxic and to induce apoptosis in hepatocytes (Amaral et al. 2009). In contrast, hydrophilic bile acids such as UDCA and conjugates have been reported to prevent apoptosis possibly via the activation of the AKT and ERK1/2 signaling pathways (Qiao et al. 2002b; Schoemaker et al. 2004). It is noted that TLR4 is not required for the initiation of acute inflammation during cholestasis. Bile acids do not directly cause cell toxicity but increases the expression of numerous proinflammatory mediators, including cytokines, chemokines, adhesion molecules, and other proteins that influence immune cell levels and function in cholestasis. Bile acids activate Erk1/2  in hepatocytes which stimulates up-regulation of Egr-1. Egr-1 then regulates production of inflammatory mediators (Allen et al. 2011). Since its ability to reduce the apoptotic threshold in several cell types, UDCA, is an endogenous hydrophilic bile acid in clinical use for the treatment of certain liver diseases (Beuers et  al. 1998; Lazaridis et  al. 2001). UDCA and its amidated conjugates, TUDCA and glycoursodeoxycholic acid, are shown to modulate mitochondrial membrane perturbation, pore formation, Bax translocation, cytochrome c release, caspase activation, and subsequent substrate cleavage. UDCA inhibition of apoptosis involves an interplay of events in which both depolarization and channel-forming activity of the mitochondrial membrane are inhibited (Rodrigues et  al. 1999). In addition, TUDCA can directly stabilize mitochondrial membranes, having a profound effect on Bax channel formation in isolated mitochondria. TUDCA is a potent

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inhibitor of Bax association with mitochondria. Thus, TUDCA modulates apoptosis by suppressing mitochondrial membrane perturbation (Rodrigues et  al. 2003). TUDCA significantly decreases taurolitholic acid (TLCA)-induced upregulation of cFos and JunB.  Furthermore, TUDCA inhibits TLCA-­ induced AP-1 transcriptional activity and reduces TLCA-­induced apoptosis. Thus, TUDCA modulates apoptosis, which is induced by toxic bile acids, through inhibition of the human transcription factor AP-1 (Pusl et al. 2008). AP-1 is a dimer consisting of proteins belonging to Jun, Fos, ATF, or musculoaponeurotic fibrosarcoma (MAF) families of transcription factors, which are involved in the regulation of cell proliferation, transformation, and death (Shaulian and Karin 2002). The p38/ ERK/MAPK and PI3K pathways are involved in protection of TUDCA against GCDCAinduced apoptosis in hepatocytes (Schoemaker et al. 2004). The NTCP is crucial for conjugated bile salt-induced hepatocyte apoptosis. TUDCA protection against GCDCA-­ induced apoptosis is dependent on activation of p38/ERK/MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF-kB activation, and transcription (Schoemaker et  al. 2004). In addition, UDCA inhibits TGF-β1-­induced degradation of NF-kB and its inhibitor inhibitor kappa B (IkappaB). In conclusion, these results demonstrate that UDCA inhibits E2F-1 transcriptional activation of hepatocyte apoptosis, thus modulating p53 stabilization, NF-kB degradation, and expression of Bcl-2 family members. Moreover, UDCA modulates transcription factors, E2F-1 and p53, independent of its effect on mitochondria and/or caspases (Sola et  al. 2003). UDCA reduces p53 transcriptional activity, thereby preventing its ability to induce Bax expression, mitochondrial translocation, cytochrome c release, and apoptosis in hepatocytes. Interestingly, p53 is a key molecular target in UDCA prevention of cell death, where the finely tuned, complex control of p53 by mouse double minute 2 homolog (Mdm-2) represents a prime target for UDCA modulation of p53-mediated cell death (Amaral et al. 2007). Indeed, UDCA improves liver function by three major mecha-

nisms of action, including protection of cholangiocytes against the cytotoxicity of hydrophobic bile acids, stimulation of hepatobiliary secretion, and inhibition of liver cell apoptosis (Solá et al. 2006). Similar to glucocorticoid receptors (GR), mineralocorticoid receptors (MR) contribute to the protective effect of UDCA against the E2F-1/Mdm-2/p53 apoptotic pathway induced by TGF-β1 in hepatocytes (Solá et al. 2004).

8

Bile Acid-Induced Necroptosis and Necrosis

In addition to apoptosis, different regulated necrotic cell death forms may emerge in bile acid-exposed cells. These are defined as genetically controlled cell death processes with morphological hallmarks of oncotic necrosis. Although RIPK1- and RIPK3-MLKL-mediated necroptosis is the most understood form of regulated necrosis, cell death mechanisms known as parthanatos, oxytosis, ferroptosis, NETosis, pyronecrosis and pyroptosis can be observed (Vanden Berghe et  al. 2014). Necroptosis, the most well-studied pathway of regulated necrosis, depends on RIPK3 activity. RIP1 and RIP3 engage in physical interactions upon activation of death receptors. The expression of RIP3 in different cell lines correlates with their responsiveness to necrosis induction. The kinase activity of RIP3 is essential for necrosis execution. Upon induction of necrosis, RIP3 is recruited to RIPK1 to form a necrosis-inducing complex (He et  al. 2009), creating a filamentous amyloid protein complex called necrosome (Li et al. 2012a). The MLKL is a functional RIP3 substrate that binds to RIP3 through its kinase-like domain but lacks kinase activity of its own. Upon phosphorylation by active RIP3, MLKL oligomerizes and translocates to cellular membranes, hence compromising their ability to preserve ionic homeostasis. The membrane localization of MLKL is essential for Ca2+ influx, which is an early event of TNFinduced necroptosis (Cai et al. 2014; Wang et al. 2014). In agreement with a role of necroptosis in cholestatic liver injury, combined ablation of hepatocyte-­specific caspase-8 and NF-κB essen-

9  Bile Acid Toxicity and Protein Kinases

tial modulator results in spontaneous massive liver necrosis and cholestasis, with a concomitant formation of necrosome complexes in the foci of necrotic areas (Liedtke et  al. 2011). Activation of necroptosis by ligands of death receptors requires the kinase activity of RIP1, which mediates the activation of RIP3 and MLKL, two critical downstream mediators of necroptosis. Blocking the kinase activity of RIP1 inhibits the activation of necroptosis and allows cell survival and proliferation in the presence of death receptor ligands. Necroptosis is suppressed by caspase-8/FADD-mediated apoptosis (Zhou and Yuan 2014). Cholestasis results in necroptosis, concomitant with progressive bile duct hyperplasia, multifocal necrosis, fibrosis and inflammation. MLKL phosphorylation increases and insoluble aggregates of RIP3, MLKL and RIP1 form in hepatocytes (Afonso et al. 2016). In fact, although RIP3 and MLKL progressively increase in both liver soluble and insoluble protein fractions, the phosphorylated MLKL/MLKL ratio is particularly increased in insoluble fraction (Afonso et al. 2016). RIP3 deficiency attenuates development of jaundice and cholestasis in severe chronic hepatitis induced by conditional deletion of TGF-β-activated kinase-1  in liver parenchymal cells (Vucur et  al. 2013). Hepatocytes are protected from cell death by only two anti-apoptotic proteins, Bcl-xL and Mcl-1. ER stress or the UPR contributes to hepatocyte cell death during alterations of lipid and fatty acid metabolism. Finally, the current information implicating RIP kinases in necrosis provides an approach to more fully address this mode of cell death in hepatocyte injury (Malhi et al. 2010).

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induces the dual specificity MAP kinase phosphatase 1 (MKP-1), which prevents GCDC-­ induced phosphorylation of MKK4 and JNK activation. Thereby, TUDC exerts its anti-­ apoptotic effect via preventing hydrophobic bile acids-induced CD95 activation at the levels of JNK activation and CD95 serine/threonine phosphorylation (Sommerfeld et al. 2015). Some protein kinases mediate both the prevention and stimulation of bile acids related cell death. Fyn activation mediates cholestasis, Src kinase Yes triggers CD95 activation and apoptosis (Reinehr et al. 2013). Hydrophobic bile acid-activated Yes triggers EGFR activation, subsequent CD95 tyrosine phosphorylation, and apoptosis. In contrast, cAMP exerts antiapoptotic effect via protein kinase A-dependent inhibition of Yes/ EGFR pathway (Reinehr et al. 2004).

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N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia

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Atilla Engin and Ayse Basak Engin

Abstract

Although stroke is very often the cause of death worldwide, the burden of ischemic and hemorrhagic stroke varies between regions and over time regarding differences in prognosis, prevalence of risk factors, and treatment strategies. Excitotoxicity, oxidative stress, dysfunction of the blood-brain barrier, neuroinflammation, and lysosomal membrane permeabilization, sequentially lead to the progressive death of neurons. In this process, protein kinases-related checkpoints tightly regulate N-methyl-D-aspartate (NMDA) receptor signaling pathways. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca2+ overload and ultimately neuronal death. Thus, reduced expression of postsynaptic density-95 protein and increased protein S-nitrosylation in neurons is responsible for neuronal vulnerability in cerebral A. Engin Department of General Surgery, Faculty of Medicine, Gazi University, Ankara, Turkey A. B. Engin (*) Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey

ischemia. In this chapter death-associated protein kinases, cyclin-­dependent kinase 5, endoplasmic reticulum stress-induced protein kinases, hyperhomocysteinemia-­related NMDA receptor overactivation, ephrin-Bdependent amplification of NMDA-evoked neuronal excitotoxicity and lysosomocentric hypothesis have been discussed. Consequently, ample evidences have demonstrated that enhancing extrasynaptic NMDA receptor activity triggers cell death after stroke. In this context, considering the dual roles of NMDA receptors in both promoting neuronal survival and mediating neuronal damage, selective augmentation of NR2A-­ containing NMDA receptor activation in the presence of NR2B antagonist may constitute a promising therapy for stroke. Keywords

Stroke · N-methyl-D-aspartate (NMDA) receptor · Cerebral ischemia · Excitotoxicity · Tyrosine receptor kinase B (TrkB) · Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase · Postsynaptic density-95 (PSD-95) · Ephrin-B (EphB) · Death-­ associated protein kinase 1 (DAPK1) · Cyclin-dependent kinase (CDK)

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_10

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1

Introduction

the blood-brain barrier (BBB), and inflammatory responses, which altogether lead to the progresAlthough stroke is the second leading cause of sive death of neurons, glial and endothelial cells death worldwide and the fifth-leading cause of (Dirnagl et al. 1999). In stroke, energy depletion death in the USA, there is also no effective ther- causes ionic pump failure and disrupts ionic apy to improve the structural and functional out- homeostasis. Imbalance between the influx of comes of this disorder. Unfortunately, a vast Na+ and Cl− ions and the efflux of K+ ions creates majority of stroke patients are not able to receive a transmembrane osmotic gradient, with ensuing the acute treatments because of the narrow time movement of water into the cells. Finally, cerewindows (Feigin et  al. 2014; George and bral edema and BBB disruption develops (Loh Steinberg 2015; Writing Group Members et  al. et al. 2019). Under conditions of cerebral hypoxia 2016). Worldwide, the burden of ischemic and and/or ischemia, three distinct mechanisms, that hemorrhagic stroke increased significantly are characteristic of ischemic stroke, contribute between 1990 and 2010. The absolute number of to neuronal damage. Thereby, mitochondrial people with ischemic and hemorrhagic stroke response to hypoxia is followed by reactive oxyincreased by 37% and 47%, respectively, while gen species (ROS) generation, and excessive oxithe number of deaths increased by 21% and 20%. dative stress due to reoxygenation. Each occurs Nevertheless, in the past two decades in high-­ at a different stage of ischemia and reperfusion income countries, incidence of ischemic stroke (Abramov et al. 2007). reduced significantly by 13%, mortality by 37%, In the past several decades, excitotoxicity, a and mortality-to-incidence ratios by 21%. By type of neurotoxicity mediated by glutamate has contrast, in low-income and middle-income drawn much attention in stroke research. The countries, a significant increase of 22% in inci- N-methyl-D-aspartate (NMDA) type of glutadence of hemorrhagic stroke and a non-­significant mate receptor processes extracellular glutamate increase in the incidence of ischemic stroke have signals into diverse intracellular signaling outbeen noted. Even so, mortality rates for ischemic puts. Indeed, cerebral ischemia leads to a masstroke fell by 14%, and mortality-to-incidence sive release of glutamate, which stimulates the ratio by 16% (Krishnamurthi et  al. 2013). NMDA receptor and induces calcium influx Ischemic stroke develops due to either cerebral through these receptors. Consequently, the infarction, or cerebral hemorrhage. In majority calcium-­dependent activation of death-signaling of cases, cerebral ischemia results in secondary proteins of these receptors result in neuronal brain injury and neuronal death (Bayraktutan death (Lai et al. 2014). There are increasing evi2019; Pluta et  al. 2018; Sternberg and Schaller dences that protein kinases are important regula2020). Approximately 30–50% of survivors after tors of developing ischemic cerebral stroke. stroke remain functionally disabled (Sladojevic Thus, it is a fact that, protein kinases are critical et  al. 2017). The lack of blood flow during the modulators of a variety of intracellular and stroke leads to neural cell loss with multiple extracellular signal transduction pathways mechanisms, including excitotoxicity, mitochon- (Jiang et al. 2018; Wang et al. 2014). It is thought drial dysfunction, free radical release, protein that, identifying individuals who are at risk for misfolding, and inflammatory changes. ischemic stroke, and understanding the interacTherefore, ischemic brain injury is a complicated tions between protein kinases-related checkdisease affecting a variety of brain regions, points and NMDA receptor signaling pathways resulting in disruption of numerous neural cir- may provide a wider therapeutic window regardcuits and involving complex injury response ing the fall-­down of morbidity and mortality. In (George and Steinberg 2015). Ischemic stroke this context, different aspects of NMDA recepdue to the occlusion of a main cerebral artery tor-mediated excitotoxic neuronal death pathleads to a critical shortage of oxygen, glucose ways and their protein kinases-related and further nutrients in the affected brain areas. checkpoints have been discussed in ischemic Excitotoxicity, oxidative stress, dysfunction of cerebral stroke.

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NMDA-Mediated Excitotoxicity and Protein Kinases

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several mechanisms. Receptor tyrosine kinases, protein kinase C (PKC), and mitogen activated protein kinases (MAPK) pathways are modulated through activation of key transcription facNMDA receptors related neuronal death or sur- tors, activator protein-­ 1 (AP-1) and nuclear vival is dependent on the stimulation of distinct factor kappa-light-chain-enhancer of activated B NMDA receptor subunits. The GluN2AR and cells (NF-κB) (Chen et  al. 2011). In ischemic GluN2BR NMDA receptor subtypes cause neu- stroke, activity of γ-secretase and the resulting ronal survival and neuronal death, respectively. Notch activation endanger neurons by inducing Synaptic GluN2AR protects neurons against NF-κB and hypoxia-inducible factor 1-alpha excitotoxic neuronal death mediated by synaptic (HIF-1α) pathways in addition to MAPK-related GluN2BR (Liu et al. 2007). Similarly, opposing pathways (Cheng et  al. 2014). Both the NF-κB action of NR2B and NR2A in mediating cell and MAPK signaling pathways regulate the death and cell survival is evident in focal isch- expression and activation of nucleotide-binding emic stroke (Liu et al. 2007). Synaptic GluN2A-­ oligomerization domain (NOD)-like receptor containing and extrasynaptic GluN2B-containing (NLR) Pyrin domain containing 1 and 3 (NLRP1 NMDA receptors have different co-agonists: and NLRP3) inflammasomes in primary cortical D-serine for synaptic NMDA receptors and gly- neurons and brain tissue following ischemic cine for extrasynaptic NMDA receptors. Under stroke (Fann et  al. 2018). Signaling through hypoxic conditions, it is likely that the failure of NLRP1 and NLRP3 inflammasomes produces synaptic glutamatergic transmission, the impair- caspase-1 and pro-inflammatory cytokines (Fann ment of the GluN2A-activated neuroprotective et al. 2013). Acute brain injury causes the release cascade, and the persistent over-activation of of damage associated molecular patterns extrasynaptic GluN2B-containing NMDA recep- (DAMPs) and the contents from dying and tors lead to excitotoxicity (Vizi et  al. 2013). necrotic neurons into the extracellular environExtrasynaptic NMDA receptors activate cell ment, and subsequently trigger intense innate death pathways and play a key role in glutamate-­ immune response involving microglia and infilinduced excitotoxic neurodegeneration and trating leukocytes (Anrather and Iadecola 2016). apoptosis. Accordingly, the function of protec- The proinflammatory signals from immune tive pathways is impaired by the concomitant mediators rapidly activate post-­stroke neuroinblockade of GluN2A-containing receptors flammation. Thus, oxidative stress, excitotoxic(Brassai et  al. 2015). Thus, diminished oxygen ity, matrix metalloproteinases (MMPs), and glucose availability in cerebral stroke elicit high-mobility group box 1 (HMGB1), arachiincreased neuronal glutamate release which in donic acid metabolites, MAPK, potentially perturn causes overexcitation of neurons postsynap- petuate ischemic brain damage subsequent to the tically (Prentice et al. 2015). Glutamate receptor acute damage (Jayaraj et al. 2019). activation contributes to calcium overload, which Whatever the reason, oxidative stress in ischin turn activates calcium dependent enzymes, emic tissues compromises the integrity of the increasing ROS and reactive nitrogen species genome, resulting in DNA lesions, cell death in (RNS) and triggering cascades of cell death neurons, glial cells, and vascular cells, and (Abramov and Duchen 2008). Following cere- impairments in neurological recovery after bral ischemia and restoration of the blood sup- stroke (Li et  al. 2018). In patients pre-stroke ply, the activation of reduced nicotinamide period, vascular medial hypertrophy, luminal adenine dinucleotide phosphate (NADPH) oxi- narrowing, and increased vascular resistance are dase and xanthine oxidase generated ROS and detected. These changes then result in vascular RNS, and loss of mitochondrial membrane remodeling and permanent hypoperfusion. As potential result in catastrophic cell death the vessels become functionally less responsive (Abramov et  al. 2007). Under these circum- and more extensively filled with atherosclerotic stances, ROS contribute to cell signaling through plaque, there is risk for vascular compression

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and thrombi. In these cases, following stroke induced excitotoxicity results in a decrease of and ischemic lesion development, severe sub- full-length TrkB (TrkB-FL), the production of acute cerebral edema and BBB disruption occur truncated TrkB-FL and the upregulation of inac(Henning et  al. 2010) (Fig.  10.1). Pro-­ tive TrkB (TrkB-T1) in stroke patients. Although inflammatory activation of glial and endothelial the TrkB-FL/TrkB-T1 balances and protects cells is accompanied by the release of cytokines, neurons from excitotoxic death, combination of chemokines and ROS in ischemic brain tissue. TrkB-FL downregulation and TrkB-T1 upreguDestruction of the BBB induced by stroke pro- lation are significant causes of neuronal death in motes the migration of immune cells to the excitotoxicity (Vidaurre et  al. 2012). TrkB-FL brain. Recruitment and transmigration of periph- receptors are degraded under excitotoxic condieral immune cells such as neutrophils and mono- tions with focal cerebral ischemia, and excitocytes across the disrupted BBB cause secondary toxicity also upregulate a truncated form of the brain damage (Jin et al. 2010; Petrovic-Djergovic TrkB receptor. Glutamate stimulation upreguet al. 2016). Interleukin 1 beta (IL-1β) increases lates TrkB-T1 and TrkB-T2 mRNA in hippoNMDA receptor function through activation of campal neurons, with a concomitant decrease in tyrosine kinases and subsequent NR2A/B sub- the number of transcripts for TrkB-FL. Activation unit phosphorylation. These effects may contrib- of TrkB-FL and TrkB-T receptors by BDNF ute to glutamate-mediated neuronal death induces independent signaling activity and, (Viviani et al. 2003). Combined tumor necrosis therefore, activation of the latter receptors may factor alpha (TNF-α) and NMDA stimulation have distinct modulatory effects on excitotoxic results in a transient increase in activity of extra- neuronal damage. The upregulation of TrkB-T cellular signal-regulated kinases (ERKs) and under excitotoxic conditions correlates with the c-Jun N-terminal kinases (JNKs). TNF-α recep- induction of a BDNF-­induced inhibition of Ras tor, TNFRII stimulates NMDA receptor-depen- homolog family member A (RhoA) response in dent calcium influx and death (Jara et al. 2007). cultured hippocampal and striatal neurons One of the major hallmarks of cerebral ischemia (Gomes et  al. 2012). RhoA mediates the Ca2+is excitotoxicity, characterized by overactivation dependent activation of p38 MAPK, which is of glutamate receptors leading to intracellular coupled to neuronal death under excitotoxic Ca2+ overload and ultimately neuronal death. conditions. The expression of active RhoA alone Therefore, C-terminal domain of GluN2B-­ not only activates p38α but also induces neuroNMDA receptors subunits are crucial in promot- nal death (Semenova et al. 2007). Therefore, the ing neuronal death in cerebral ischemia (Vieira inhibition of RhoA by TrkB-T accounts for neuet al. 2016). In these cases, excessive stimulation roprotection by BDNF during the period of exciof extrasynaptic NMDA receptors, cause cal- totoxic stimulation. Activation of TrkB-T cium overload and mitochondrial dysfunction, receptors constitute an endogenous neuroprotecwhich trigger a range of downstream neurotoxic tion under conditions characterized by excitocascades that impair neuronal function or lead to toxic cell death in brain ischemia (Gomes et al. cell death due to glutamate excitotoxicity 2012). The Rho small guanosine triphosphate (Hardingham 2009; Stanika et al. 2009). In con- proteins (GTPase), RhoA activates the Rhotrast, subtoxic NMDA exerts the neuroprotective associated coiled-coil containing kinase effect via activation of prosurvival phos- (ROCK), which belongs to the family of serine/ phoinositide 3-kinase (PI3K)/protein kinase B threonine kinases (Matsui et  al. 1996). ROCK (Akt) ­ pathway against ischemic brain injury. family members (ROCK1 and ROCK2) is genThus, brain-derived neurotrophic factor erally implicated in cell death and survival (Shi (BDNF)- tyrosine receptor kinase B (TrkB) sig- and Wei 2007). Overactivation of ROCK during naling and Ca2+-dependent calmodulin cascade acute cerebral ischemia contributes to early contributes to NMDA induced activation of worsening of cerebral injury, by stimulating the PI3K/Akt pathway (Xu et  al. 2007). NMDA- inflammatory response. The activation of ROCK

10  N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia

Fig. 10.1  NMDAR-related excitotoxic neuronal death in cerebral ischemic stroke. Many protein kinasesrelated distinctive deadly checkpoints are activated during the cerebral ischemic stroke. Following cerebral ischemia, ROS cause breakdown of the BBB by upregulating inflammatory mediators. In addition, cerebral ischemia results in the higher extracellular glutamate concentrations that stimulate the extrasynaptic GluN2Bcontaining NMDAR, and trigger excitotoxic neuronal death via PTEN, CDK5, and DAPK1. Following activation DAPK1 combines with the extrasynaptic NMDARs, phosphorylates the NR2B subunit and mediates cell death. In this context, the pS23 and pS23/CypD interaction induces neuronal death through mitochondriadependent pathways. Furthermore, excitotoxic death due to NMDAR overactivity also requires activation of MEK stimulation by glutamate. On the other hand, calcium influx via overactivation of NR2B-containing NMDARs activates PKC, which phosphorylates the p47phox-p22 assembly of the active NOX complex. The activation of NOX complex at the cell surface leads to neuronal death via production of superoxide. PSD95 induces Ca2+/CaM-dependent enzyme nNOS and indirectly binds to PP1 and PP2B via AKAP79, resulting in the overproduction of RNS.  Simultaneously, Rho-­ induced overactivation of ROCK contributes to early worsening of cerebral injury, by stimulating the inflammatory response and ROS production via NOX. A p38 MAPK pathway substrate, cytosolic phospholipase A2 causes cell membrane phospholipid hydrolysis and supports brain damage in stroke. (Abbreviations: AKAP79: A-kinase anchor protein; AMPAR: Alpha-amino-3-­ hydroxy-5-methylisoxazole-4-propionate receptor; AP-1; adaptor protein-1; BBB: Blood-brain barrier;

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CaM: Ca2+/Calmodulin; CaMK: CaM-dependent protein kinase; CBP: CaM: Ca2+/Calmodulin; CREB-binding protein; CREB: Cyclic adenosine monophosphate response element; Cypd: Cyclophilin D; DAPK1; Death-associated protein kinase 1; ENMDAR: Extrasynaptic NMDAR; EphB2: Ephrin-B2; GluA1: Extrasynaptic site of AMPAR at the plasma membrane, permeable to Ca2+; GluA2: The constitutive site of AMPA receptors, impermeable to Ca2+; IFNγ: Interferongamma; MAPK: Mitogen-activated protein kinase; MD: Mitochondrial dysfunction; MEK: MAPK/extracellular signal-regulated kinase (ERK) kinase; NLRP: Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing inflammasomes; NMDA: N-methyl-D-aspartate; NMDAR: NMDA-type of glutamate receptor; nNOS: Neuronal nitric oxide synthase; NO: Nitric oxide; NOX: reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; O2·−: Superoxide anion; ONOO−: Peroxynitrite; PI3K: Phosphoinositide 3-kinase; PKA: Protein kinase A; PKC: Protein kinase C; p22: Membrane bound subunit of NOX; p38: MAPK family kinase; p47: Cytosolic subunit of NOX; PP1: protein phosphatase 1; PP2B: Calcineurin; pS23: Phosphorylated p53 at serine 23; PSD95: Post-synaptic density protein-95; PDZ: postsynaptic density-95/Discs large/Zonula occludens 1 domain; PTEN: phosphatase and tensin homolog; RhoA: Small guanosine triphosphatases; RN2B: GluN2B subunit-­ containing NMDA receptor; RN2A; GluN2A subunit-containing NMDA receptor; ROCK: Rho-­ associated coiled-coil containing protein kinase; ROS: Reactive oxygen species; SNMDAR: Synaptic NMDAR; Tat-­ NR2B9c: Tat-conjugated peptide comprising the nine C-terminal residues of NR2B)

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stimulates NADPH oxidase (NOX) activation and ROS production. Increased expression of adhesion molecules secondary to the reduction of endothelium-derived nitric oxide (NO) during ischemic injury is mediated by ROCK, as ROCK is an upstream negative regulator of endothelial nitric oxide synthase (eNOS) (Sladojevic et  al. 2017). The neuroprotection provided by ROCK inhibition has similar magnitude as protection by NMDA receptor blockade due to crosstalk between ROCK induced pathways and NMDA receptor related excitotoxicity (Gisselsson et al. 2010). The GTPase are also coupled to the activation of the stress kinase JNK (Marinissen et al. 2004) and there is evidence that the JNK/cJun signaling pathway is important for neuronal death induced by excitotoxicity. Indeed, glutamate toxicity may involve the JNK group of MAPKs (Yang et  al. 1997). In postischemic brain tissue, activation of the three MAPK signaling pathways, including ERK1/2 or the stress-activated protein kinase (p38/SAPK2 or JNK/SAPK) pathways are important. These protein kinases phosphorylate intracellular enzymes, transcription factors, and cytosolic proteins transducing stress-related signals which are involved in apoptosis and inflammatory cytokine production (Irving and Bamford 2002). A p38 MAPK pathway substrate, cytosolic phospholipase A2, causes cell membrane phospholipid hydrolysis and mediates arachidonic acid metabolism, leading to brain damage (Adibhatla et  al. 2003). High level of extracellular glutamate causes excitotoxicity, which further exacerbated ischemic brain injury. Massive oxidant generation after excitotoxicity is an important mechanism of excitotoxic cell damage (Lipton 1999). Indeed, NADPH oxidase is the primary source of NMDA-induced superoxide production. NOX plays a central role in oxidant generation under excitotoxic conditions. Ca2+ influx through NMDA receptors activates PKCζ, which in turn phosphorylates p47phox and triggers the translocation of p47phox, finally initiating the activation of the NOX (Brennan et al. 2009). Although increased NMDA receptor activity is dependent upon elevated intracellular calcium levels, excitotoxic death also

requires activation of MAPK/ERK kinase (MEK) induced by glutamate. The activities of MAPK/ERK substrate mitogen- and stressresponse kinase 1 (MSK1), and its substrate, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) are essential components of neuronal cell death (Hughes et al. 2003; Satoh et al. 2000). NMDA induces JNK activation and selective phosphorylation of MAPK kinase 7 (MKK7) only. Following NMDA stimulation, MKK7 strongly translocates into the nuclei of dying neurons. NMDA stress causes a strong degradation of MAPK-activating death domain-containing protein/differentially expressed in cells (MADD/ DENN), and a nuclear translocation. Death effector domain is found in the cytoplasm and transferred into the nucleus upon stimulation of CD95. Nuclear accumulation of MADD has been shown to trigger apoptosis (Stegh et  al. 1998). D-JNKI1 prevents the JNK-mediated phosphorylation of MADD.  As a marker of active neuronal cell death, JNK-mediated phosphorylation of MADD prevents its entry into the proteasome pathway (Centeno et al. 2007).

3

 MDA and PSD-95 Crosstalk N in Cerebral Stroke

Among the family of membrane-associated guanylate kinase proteins, postsynaptic density-95 (PSD-95) is involved in NMDA receptor signaling via its PSD-95/disc large/zonula occludens-1 (PDZ) domains. The PDZ domains of PSD-95 and PSD-93 are structurally similar, but N-terminal PSD-93 PDZ domains show an affinity for a N2B-derived C-terminal octapeptide (Fiorentini et  al. 2013). Following brain ischemia, movement of NMDA receptor from lipid rafts to PSDs is proportional with increased tyrosine phosphorylation of NR2A and NR2B subunits in rafts. In this case, NR1, NR2A and NR2B levels are elevated in PSDs, while reducing in lipid rafts (Besshoh et al. 2005). Reduced expression of PSD-95 in neurons is responsible for neuronal vulnerability mediated by direct activation of alpha Ca2+/calmodulin-dependent

10  N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia

protein kinase II (CaMKII) transduction pathway in the postsynaptic compartment. Therefore, the NMDA channel blockers fail to have any effect on neuronal survival in PSD-95 deficient neurons (Gardoni et  al. 2002). PSD-95 Ser73 phosphorylation causes NR2A dissociation from PSD-95, while it does not interfere with NR2B binding to PSD-95 (Gardoni et al. 2006). Upon NMDA receptor activation, NO is generated from neuronal nitric oxide synthase (nNOS) which is linked to the NMDA receptor through shared interaction with PSD-95 (Brenman et al. 1996; Nakamura et al. 2013). Coupling of PSD95 and neuronal nNOS plays an important part in neuronal damage caused by stroke. A smallmolecular inhibitor of nNOS-PSD-95 interaction, SCR-4026, exhibits neuroprotective activities in NMDA-­induced neuronal damage in primary cortical neurons (Mo et  al. 2016). Stimulation of NMDA receptors and L-type calcium channels facilitate formation of a Fyn kinase-PSD95-NR2A complex during transient brain ischemia followed by reperfusion. Enhancing NMDA receptor function increases ischemic neuronal cell death (Hou et al. 2002). The non-receptor tyrosine kinases, Src, Fyn, focal adhesion kinase, and proline-rich tyrosine kinase 2 are involved in the excitotoxicity, which is induced by the overactivation of NMDA receptor following cerebral ischemia (Sun et al. 2016). PSD-95 is critical for the Src family kinases-mediated tyrosine phosphorylation of NMDA subunit NR2A in the postischemic brain. During transient brain ischemia and reperfusion, Src family protein tyrosine kinases bind to NR2A.  Coupling of PSD-95 facilitates the NR2A tyrosine phosphorylation. In fact, increasing in interactions between cerebral ischemia/ reperfusion-­ induced Src, NR2A and PSD-95 delay neuronal cell death (Hou et  al. 2005, 2007). Furthermore, gamma-aminobutyric acid A (GABA(A)) receptor agonists attenuate Src activation and interactions among NR2A, PSD95 and Src, resulting in the suppression of NMDA receptor tyrosine phosphorylation. So that the NR2A-PSD-95-Src signal module disintegrates (Xu et  al. 2008). In cerebral stroke, dynamic balance between excitation and inhibi-

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tion by coactivation of the GABA receptors attenuate the excitatory NMDA receptor via inhibiting postsynaptic NMDA receptor/Srcmediated signal amplification, the “NMDA receptor-Ca2+/PKC/Src/NMDA receptor-Ca2+” cycle (Zhang et  al. 2007). PSD-93 serves as a membrane-anchored substrate of Fyn kinase and plays a role in the regulation of Fyn-mediated modification of NMDA receptor function (Nada et al. 2003). PSD-93 tightly binds the C-terminal tails of the NMDA receptor, which is critical for NMDA receptor activity in ischemic stroke. The inhibitory effect on Fyn-mediated phosphorylation of NR2B caused by PSD-93 deficiency confers neuroprotection against ischemic brain injury (Zhang et al. 2014).

4

Protein S-Nitrosylation

Protein S-nitrosylation stimulates activation of death-signaling pathways and inhibits the survival-­signaling pathways in cortical neurons exposed to excitotoxicity by NMDA receptor stimulation. NO plays an important role in excitotoxicity and under conditions of excessive glutamate receptor activation NO interacts with O2·− (superoxide) to form the toxic molecule peroxynitrite (ONOO−) (Chen et  al. 2011). NMDA receptor activity is regulated by a variety of post-­ translational modifications, including nitrosylation and phosphorylation. Proteintyrosine phosphorylation regulates NMDA receptors related neuronal toxicity (Choi et  al. 2000; Wang and Salter 1994). NMDA exposure transient focal cerebral ischemia results in increased levels of S-nitrosylated SHP-2 (SNO)Src homology region 2-containing protein tyrosine phosphatase-­ 2 (SHP-2). SNO-SHP-2 inhibits its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. This suggests that formation of SNOSHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke (Shi et al. 2013). Apoptosis signal-regulating kinase 1 (ASK1), and proline-­ rich tyrosine kinase 2

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(PYK2) are activated by NO via increased S-nitrosylation during cerebral ischemia-reperfusion. Modulation of NMDA receptor activity and Ca2+ dynamics by these protein kinases result in ischemic neuronal cell death (Liu et al. 2013; Yan et al. 2015). NO S-nitrosylated cellular protein aggravates neuronal injury. In this context, receptor-interacting protein 3 (RIP3) is a sensor molecule regulating cell apoptosis and necrosis. Cerebral stroke induces both RIP3 S-nitrosylation and RIP3 activation. Interaction of RIP3 with RIP1 facilitates cerebral ischemic injury. NMDA receptor antagonists or nNOS inhibitors diminish RIP3 S-nitrosylation and reduce neuronal damage (Miao et  al. 2015). Increase in the tyrosine phosphorylation of the 2B subunit of the NMDA receptor, NR2B contributes to the maintenance of long-term potentiation. Thus, the endogenous tyrosine kinase, Src is important for neuronal damage (Rostas et  al. 1996; Yu et  al. 1997). In cerebral stroke, crucial nNOS activation produces excessive amounts of NO, which, in turn, covalently modifies c-Src by S-nitrosylation. In fact, key regulatory enzymes governing cell survival and cell death are aberrantly modified and regulated by calpains, NO and ROS in affected neurons. The aberrantly activated Src family kinases are functionally linked to the glutamate receptors and increase the excitotoxic neuronal death caused by stroke (Hossain et al. 2012).

A. Engin and A. B. Engin

bral stroke DAPK1 combines with NMDA receptors leading to NMDA receptors phosphorylation and over-activation (Tu et  al. 2010) (Fig.  10.1). Subsequently the intracellular calcium overload via the glutamate-operated channels during the ischemic period triggers the cell death (Benveniste et al. 1988). This group of protein kinase induces distinct death pathways of apoptosis, autophagy and programmed necrosis. Protein kinase D (PKD), Beclin 1, and the NMDA receptors are among the substrates. Furthermore, DAPK is required for cell death under oxidative stress in a process that displays the characteristics of caspase-­independent necrotic cell death (Bialik and Kimchi 2014; Eisenberg-Lerner and Kimchi 2007). Cerebral ischemia recruit DAPK1 into the NMDA receptor NR2B protein complex in the cortex. A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance (Tu et al. 2010). The disruption of the DAPK1-NMDA receptors complex resulted in a decrease in NMDA receptors phosphorylation. Then the glutamate-stimulated Ca2+ influx is inhibited and intracellular Ca2+ overload was alleviated, which blocked the release of cytochrome c and cell death (Tian et al. 2014). In lethal conditions, B-cell leukemia/lymphoma-2 (Bcl-2) associated protein × (Bax)/Bcl-2 antagonist killer (Bak) insert the outer mitochondria membrane. Dramatic change occurs in the intracellular localization of Bax promote cell death (Wolter et al. 1997). The BH3/Bax/lipid interaction promoted formation of mitochondrial permeability transi5 Death-Associated Protein tion pores mediate cytochrome c release (Kuwana Kinase and Stroke et  al. 2002). The cyclophilin D mitochondrial Death-associated protein kinase (DAPK) 1 has membrane permeability transition pore is critical important role in cerebral ischemic damage, in ischemic brain injury (Wang et al. 2010). The whereby DAPK1 potentiates NMDA receptor-­ mitochondrial swelling induced by Ca2+ increases mediated excitotoxicity through interaction with gradually with the increasing calcium concentrathe NR2BR subunit. DAPK1 also mediate a tion. This swelling is mediated by the upregularange of activities from autophagy, membrane tion of the cyclophilin D protein. Ultimately, blebbing and DNA fragmentation ultimately increase in cyclophilin D leads to the destruction leading to cell death (Nair et  al. 2013). In fact, of the mitochondrial membrane potential and the DAPK is the founding member of a family of generation of excessive ROS (Li et al. 2012). In highly related, death associated Ser/Thr kinases fact, mitochondrial permeabilization is primarily that belongs to the calmodulin (CaM)-regulated Bax-­ dependent. Bax-inhibiting peptide (BIP) kinase superfamily (Shiloh et al. 2014). In cere- prevents the glutamate-induced Bax transloca-

10  N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia

tion to the mitochondria and the release of cytochrome c from the mitochondria (Iriyama et  al. 2009). However, the decreased Bcl-2 or Bcl-xL to Bax ratio causes Ca2+ independent cytochrome c release from mitochondria which is a trigger of apoptosis (Kuwana et  al. 2002; Wolter et  al. 1997). DAPK1 not only combines with NMDA receptors, but also interacts with Beclin-1 (Zalckvar et  al. 2009b). The activated form DAPK phosphorylates beclin 1 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl-XL and induces autophagy (Zalckvar et  al. 2009a, b). The induction of Fas ligand (FasL) and Bcl-2 mRNA transcripts, RE1 protein silencing transcription factor (REST) contributes to glutamate-­induced excitotoxic neuronal death by modulating GluR2 expression. FasL and Bcl-2 over expression and their subsequent down regulation also suggests that FasL is the direct effector of apoptosis in the cerebral cortex. On the other hand, the presence of Bcl-2 attenuates the survival signals in neurons under neurotoxic conditions (Segura Torres et al. 2006).

6

Cyclin-Dependent Kinase 5

Excitotoxicity has been associated with neuronal death in brain ischemia through the deregulation of specific cell signaling pathways (Aarts et al. 2003; Arundine and Tymianski 2003). Multiple cyclin-dependent kinase (CDK) members may also participate in neuronal death (Copani et al. 2001). Among them, aberrant activation of the CDK5, is directly associated with neuronal death following stroke. CDK5, a serine/threonine kinase is highly expressed in the central nervous system, particularly following ischemic stroke and its aberrant activation is directly associated with neuronal apoptosis and death. The excitotoxic component of ischemia/hypoxia is predominately regulated by CDK5 and its activator p35 (Rashidian et  al. 2005; Slevin and Krupinski 2009). Calpain proteases cleave the p35 to a smaller more stable and mislocalized p25 form. This, in turn, converts CDK5 into a death inducer. Such inappropriate activation of

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CDK5 induces stroke (Rashidian et al. 2005). As an alternative hypothesis, ischemia induced phosphorylation of the NMDA receptor 2A subunit at Ser1232 is catalyzed by CDK5 (Rashidian et  al. 2005; Wang et  al. 2003). The aberrant activity of the protein kinase CDK5 is a principal cause of neuronal death during stroke. Ischemia induced cerebral ischemia causes calpain-dependent conversion of the CDK5activating cofactor p35 to p25 (Meyer et  al. 2014). Overactivation of NMDA receptors leads to calpain activation, and calpain blockade protects neurons from excitotoxicity (Higuchi et al. 2005). Furthermore, calpain-mediated cleavage of the NR2B subunit in neurons gives rise to active NMDA receptor forms present on the cell surface after excitotoxic glutamatergic stimulation (Simpkins et  al. 2003). A membrane-­ permeable peptide, Tat-CDK5-CTM that specifically disrupts the binding of CDK5 and NR2B and then leads to the degradation of CDK5 by a lysosome-mediated pathway in stroke (Zhou et  al. 2019). GluN2B-containing NMDA receptors induce neuronal injury by calcium-­dependent activation of the death-promoting protease calpain. Moreover, during the cerebral ischemia, the extracellular glutamate concentration rises abruptly, and stimulates the GluN2B-containing NMDA receptor in the extrasynaptic sites, and triggers excitotoxic neuronal death via phosphatase and tensin homolog (PTEN), CDK5, and DAPK1 (Lai et  al. 2014). NMDA receptors are a key player in cerebral stroke. Phosphorylation of NMDA receptor subunits at their cytoplasmic carboxyl termini is an important mechanism to regulate the receptor function. CDK5 is responsible for regulating phosphorylation and function of NMDA receptors. GluN2B subunit at its cytoplasmic carboxyl termini is regulated by CDK5 in neuronal ischemia (Lu et al. 2015). Ischemic stroke leads to cleavage of p35 into the more stable p25 by the calcium-dependent protease calpain and to increased CDK5 activity (Su and Tsai 2011). A modified 24-aa peptide (Lys254-­Ala277) derived from p35, TFP5 decreases the p25/p35 ratio in the ischemic hemisphere (Bosutti et al. 2013; Ji et al. 2017). The NR2A- and NR2B-containing

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NMDA receptor subtypes (NR2ACNRs and NR2BCNRs) are the predominant NMDA receptors expressed in the forebrain. NR2BCNRs are predominantly distributed on the extrasynaptic membrane (Rumbaugh and Vicini 1999; Tovar and Westbrook 1999). NR2ACNRs and NR2BCNRs may exert opposing effects on neuronal death. DJ-1 (PARK7, Parkinsonism associated deglycase) is involved in stroke-­ induced brain injury (Aleyasin et  al. 2007; Yanagisawa et  al. 2008), indicating that DJ-1 dysfunction may play a broad role in the central nervous system (CNS) damage. Loss of DJ-1 increases the sensitivity to excitotoxicity and ischemia. Importantly, DJ-1 expression decreases markers of oxidative stress after stroke. The suppression of DJ-1 increases the susceptibility to NMDAinduced neuronal death through the overactivation of PTEN/NR2BCNR-dependent cell death pathway (Chang et  al. 2010). Interestingly, PTEN forms a death-signaling complex with the NR1/NR2B subunits that are primarily located at extrasynaptic sites of NMDA receptors. Suppressing the protein phosphatase activity of PTEN, through downregulating extrasynaptic NMDA receptors, protects against ischemic neuronal death (Li et  al. 1998; Ning et  al. 2004; Stocca and Vicini 1998). Moreover, PTEN-­ induced kinase 1 (PINK1) acts with DJ-1  in a common pathway to regulate NMDA receptor-­ mediated neuronal death. PTEN upregulation mediates DJ-1 suppression-induced increase in NR2BCNR function and neuronal death. This finding suggests that DJ-1 may act upstream of PTEN to regulate NR2BCNR-dependent neuronal death. Our data indicate that the phosphatase PTEN may play bi-directional roles to regulate neuronal survival/death in DJ-1-dependent pathways DJ-1/PTEN/NR2BCNR and DJ-1/PTEN/ PINK1/NR2ACNR (Chang et  al. 2010). Enhancing the protein expression of PINK1 antagonizes oxygen-glucose deprivationinduced reduction of Akt phosphorylation. This indicated that Akt is a downstream target of PINK1 in ischemic neuron injury. The overactivation of NR2BRs contributes to ischemic neuron death through suppressing PINK1-dependent survival signaling (Shan et al. 2009).

7

Endoplasmic Reticulum Stress-Induced Cell Death

Indeed, NMDA receptors are critical for neuronal communication. Homeodomain interacting protein kinase 2 (HIPK2)-JNK-c-Jun signaling as a key mechanism that regulates the transcription of NMDA receptor subunits GluN2A and GluN2C.  These changes result in a significant increase of GluN2A/GluN2B ratio in synapse and mitochondria. Persistent activation of the ERK-CREB pathway and the upregulation of synaptic activity-regulated genes, collectively contribute to the resistance of HIPK2 deficient neurons to cell death (Shang et al. 2018). HIPK2 can be activated by stress conditions to promote cell death via the ataxia telangiectasia mutated (ATM) or JNK pathway in neurons. HIPK2 regulates transforming growth factor beta (TGF-β)induced JNK activation and apoptosis. Overexpression of HIPK2 leads to Daxx [interactor for the CD95 (Fas/apoptosis antigen 1 (APO-­1)) death receptor], phosphorylation, and ectopic expression of HIPK2 activates the JNK signaling pathway, which is enhanced by coexpression of Daxx. HIPK2 signals to JNK via a pathway using Daxx and the MKK4/SEK1 and MKK7 (Choi et al. 2013; Hofmann et al. 2003). HIPK2 as the essential link that promotes endoplasmic reticulum (ER)-stress-induced cell death in spinal motor neurons via the inositolrequiring enzyme 1α (IRE1α)-ASK1-JNK pathway. ER stress, induced by tunicamycin or by the accumulation of misfolded superoxide dismutase 1 (SOD1(G93A)) proteins, activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain (Lee et al. 2016). The ER is a subcellular compartment playing a central role in calcium storage and signaling. Disturbances of ER calcium homeostasis constitute a severe form of stress interfering with central functions of this structure including the folding and processing of newly synthesized membrane and secretory proteins (Paschen and Mengesdorf 2005). Indeed, the expressions of ER stress-related markers, immunoglobulin binding protein

10  N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia

(BiP), activating transcription factor-4 (ATF-4), and CCAAT/enhancer-binding protein (C/ EBP)-homologous protein (CHOP) are increased in the infarct region of brain (Morimoto et al. 2007). Impairment of ER function arises from depletion of ER calcium stores, oxidative stress, and from blocking of the proteasome for degrading unfolded proteins. ER stress is known to play a crucial role in hypoxia/ ischemia-induced cell damage. The accumulation of misfolded proteins in neurons is associated with excitotoxicity and is found in stroke. Thus, multiple damage mechanisms associated with brain following cerebral ischemia and reperfusion alter unfolded protein response and contribute to a pro-apoptotic phenotype in neurons. The RNA-dependent protein kinase (PKR)-like endoplasmic reticulum eukaryotic initiation factor 2 (eIF2) alpha (eIF2α) kinase (PERK) is responsible for eIF2α phosphorylation in the early post-ischemic brain (DeGracia and Montie 2004; Kumar et al. 2001). Following PERK activation and subsequent eIF2α phosphorylation, elevated ATF4 contributes to cell death processes by upregulating transcription of proapoptotic Bcl-2 family members in addition to the key transcription factor C/EBP homologous protein. Moreover, CHOP controls transcription of genes encoding both pro- and antiapoptotic Bcl-2 family members, thereby, CHOP is effective in cell fate decisions (Oyadomari and Mori 2004). While enhancing calcium influx through L-type calcium channels promotes the survival of neurons, NMDA receptor-­ mediated calcium influx can lead to excitotoxic death. L and NMDA receptor channel activities differentially regulate the transcription factor C/EBP beta to control neuronal survival. L channel-dependent calcium influx results in increased CaMKIV activity, which acts to decrease nuclear C/EBPbeta levels. Conversely, NMDA receptor-mediated influx rapidly elevates nuclear C/EBPbeta and induces excitotoxic death via activation of the calcium-dependent phosphatase, calcineurin. Moderate levels of ­α-amino-3-­hydroxy-5-methyl-4-isoxazolepropi onic acid (AMPA) receptor activity stimulate L

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channels to improve survival, whereas higher levels stimulate NMDA receptors and reduce neuronal survival (Marshall et  al. 2003). Cerebral ischemia initiates an ER-based stress response that results in the transcriptional upregulation and corresponding increased expression of caspase-12 protein. The enhanced expression of glucose-regulated protein 78 (GRP78), Caspase-12, CHOP/growth arrest and DNA damage 153 (GADD153), ATF4 and processing of the transcription factor, X-box protein 1 (xbp1) mRNA in the affected brain regions clearly indicate the critical involvement of ER-mediated cell death/survival mechanisms (Mouw et  al. 2003; Nakka et  al. 2010). Caspase-12 is essential for this ER stressinduced apoptosis. Furthermore, tumor necrosis factor receptor-­associated factor 2 (TRAF2) interacts with procaspase-­12 and promotes the clustering of procaspase-12 and its activation by cleavage in response to ER stress (Yoneda et al. 2001).

8

Hyperhomocysteinemia and Stroke

One of the most important causes of vascular remodeling is hyperhomocysteinemia (HHcy). Hypertrophy, collagen accumulation, matrix remodeling, elevated ROS and endothelial dysfunction are distinctive properties of HHcy related vascular remodeling (Bao et  al. 2010; Bhalodia et al. 2011; Rodionov et al. 2010; Sen et al. 2010; Steed et al. 2010). The homocysteine (Hcy) level is an independent risk factor for severe neurological impairment. Elevated Hcy levels independently predict severe neurological impairment, and stroke recurrence in the large artery atherosclerosis stroke subtype (Ji et  al. 2015). In addition, high total Hcy (tHcy) levels in the acute phase of an ischemic stroke can predict mortality (Shi et al. 2015). In hyperhomocysteinemic subjects with ischemic stroke more than five-fold mortality rate occurs as compared to controls. Inhibition of GluN2A-NMDA receptor signaling significantly reduces the ischemic damage in hyperhomocysteinemic brain.

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GluN2A-­NMDA receptor dependent increase in ERK-MAPK phosphorylation under hyperhomocysteinemic conditions is due to Hcy-induced neurotoxicity (Jindal et al. 2019). Hcy is a high affinity agonist of NMDA receptors for the GluN1/2A subunit. Hcy induced NMDA receptor signal in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDA receptors. This implies that in HHcy, Hcy contributes to post-synaptic responses mediated by GluN2A-­ containing NMDA receptors (Sibarov et  al. 2016). Level of HHcy is a risk factor for recurrent cerebral infarction. Further, particular demographic and clinical outcomes including age, relative “National Institutes of Health Stroke (NIHSS) scores”, and circulating triglyceride levels were markedly associated with the occurrence of cerebral infarction (Anniwaer et  al. 2019). Hcy level is an aggravating factor in atherosclerosis, which is positively associated with high risk of intracerebral hemorrhage (Zhou et  al. 2018). Hcy metabolism leads also to the redox imbalance and increased oxidative stress resulting in elevated lipoperoxidation and protein oxidation, the products known to be included in the neuronal degeneration (Lehotsky et al. 2015). Glutamatergic excitotoxicity appears to be associated with brain damage caused by Hcy. This amino acid induces neurodegeneration via NMDA receptor overstimulation and/or by reducing glutamate uptake (da Cunha et al. 2012; Matté et al. 2010). HHcy, a common metabolic disorder of the methionine cycle, is an independent risk factor for multiple neurodegenerative disorders (Obeid and Herrmann 2006; Sacco et  al. 1998; Seshadri et  al. 2002). Oxidative injury and neuronal cell death associated with elevated levels of extracellular Hyc involves stimulation of the NMDA subtype of ionotropic glutamate receptors (Kruman et al. 2000, 2002; Lipton et  al. 1997; Mattson and Shea 2003; Poddar and Paul 2009). Overactivation of NMDA receptors is known to be involved in glutamatemediated excitotoxic cell death. Although it is proposed that Hcy activates NMDA receptors analogous to glutamate, contrarily, the effects of Hcy and glutamate on NMDA receptor activation are quite different. Glutamate-mediated excitotoxic cell death has been primarily attributed to

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activation of NR1/NR2B subunit containing NMDA receptors (NR2B-NMDA receptor) (Poddar and Paul 2009). Excitotoxic neuronal death in stroke results in part from superoxide produced by neuronal NOX2. Indeed, calcium influx through NR2B-containing NMDA receptors triggers mitochondrial depolarization, NOX2 activation, superoxide formation, and cell death. PI3K couple NMDA receptors to PKCζ activation, NOX2 activation, and excitotoxic cell death (Brennan-Minnella et  al. 2013). NMDA receptor-CaMKII cascade is functionally coupled to acid-sensing ion channels (ASICs) and contributes to acidotoxicity during ischemia. Ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by CaMKII activity, as a result of activation of NR2B-containing NMDA receptors during ischemia (Gao et al. 2005). Synaptic NMDA receptors have anti-apoptotic activity, whereas stimulation of extrasynaptic NMDA receptors cause loss of mitochondrial membrane potential and cell death. Calcium entry through synaptic NMDA receptors induces both CREB activity and BDNF gene expression. In contrast, calcium entry through extrasynaptic NMDA receptors activates CREB shut-off pathway that blocked induction of BDNF expression and increases neuronal apoptosis. The synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDA receptors in brain and is required for inhibition of NMDA receptor-dependent ERK activation. Coupling of NR2B to SynGAP weakens synaptic transmission through Ras-ERK inhibition (Hardingham et  al. 2002; Kim et  al. 2005; Li et  al. 2002; Liu et  al. 2007). In contrast, Hcy-­ mediated neuronal cell death involves stimulation of NR1/NR2A containing NMDA receptor (Poddar and Paul 2009). Although the NMDA receptors that are generally thought to be involved in cell survival, in the molecular basis, have dual roles in promoting neuronal survival and mediating neuronal damage (Hetman and Kharebava 2006; Liu et  al. 2007). The dual nature of the NMDA receptor as a mediator of excitotoxic cell death and activity-dependent cell survival likely

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results from divergent patterns of protein kinase CaMKII, protein-tyrosine kinases (PTK) and activation, transcription factor activation, and MEK1/MEK2 (ERK1/ERK2 kinase), respecgene expression (Lee et  al. 2005). Hyc-NMDA tively. Combined inhibition of CaMKII and receptor induced neuronal cell death involves MEK1/MEK2 has an additive effect. Glutamate-­ sustained activation of ERK MAPK, which also induced apoptotic-like death is promoted by inhidiffers from glutamate-NMDA receptor medi- bition of protein phosphatase (PP) 1 and protein ated transient activation of ERK MAPK. NMDA-­ tyrosine phosphatase (PTP), respectively. mediated influx of Ca2+ leads to activation of the Consequently, in glutamate-induced cortical neuCa2+-dependent phosphatase calcineurin and the rotoxicity ERK1/ERK2 activation be mainly dephosphorylation and activation of striatal-­ mediated by NMDA receptor. Moreover, a pathenriched protein tyrosine phosphatase, which way dependent on both PKC and PTK is mainly limits the duration of ERK activity as well as its involved, which is also responsible for ERK1/ translocation to the nucleus (Paul et  al. 2003). ERK2-mediated apoptotic-like death. In this ERK and p38 MAPKs are activated following case, CaMKII-dependent pathway is relatively NMDA receptor stimulation by multiple agonists mildly involved (Jiang et al. 2000). (Poddar et al. 2010; Poddar and Paul 2009). The Overstimulation of ionotropic glutamate opposing effects of ERK MAPK on neuronal sur- receptors (iGluRs), such as the NMDA and vival and death following exposure to glutamate AMPA receptors, produces excitotoxicity in sevand Hcy could be attributed to the differential eral brain regions. The molecular composition of activation of the NMDA receptor subunits. Hcy-­ those receptors and their regulation by intracelNMDA receptor induced activation of p38 lular signaling systems are determinants in the MAPK plays a crucial role in neuronal cell death development of progressive neuronal damage in (Poddar and Paul 2013). On the other hand, the CNS.  Initial neuronal death is due to an NMDA receptor activation have differential excessive Ca2+ influx through NMDA receptors, effects on AMPA receptor trafficking, depending whereas the further damage process is mediated on the subunit composition of NMDA receptors. by AMPA receptors through p38 signaling NR2A-NMDA receptors promote, whereas (Rivera-Cervantes et al. 2004). NR2B-NMDA receptors inhibit, the surface Cytotoxic brain edema triggered by neuronal expression of GluR1. Activation of ERK MAPK swelling is the main cause of mortality following following stimulation of NR2A-NMDA recep- cerebral ischemia (Rungta et al. 2015). Neuronal tors induces trafficking of AMPA receptor sub- swelling owing to ischemic stress is rapidly initiunits to the surface, while inhibition of ERK ated by aberrant depolarization and entry of MAPK following stimulation of NR2B-NMDA sodium and chloride ions, primarily caused by receptors inhibits AMPA receptor surface inser- reduced activity of the Na+/K+ adenosine triphostion. Inhibition of NMDA receptor activation or phatase (ATPase) and accumulation of extracelphosphorylation of ERK p38 MAPK attenuates lular glutamate due to synaptic release/spillover Hcy-induced decrease in surface expression of (Rungta et al. 2015). GluA2-AMPA receptor subunit (Kim et al. 2005; Poddar et  al. 2017). Small GTPases Ras and Rap1 are important for signaling synaptic AMPA 9 Ephrin-B-Dependent receptor (−R) trafficking. Rap2, which shares Amplification of NMDA-­ 60% identity to Rap1, is present at excitatory Evoked Neuronal synapses. Rap2-JNK pathway, which opposes the Excitotoxicity action of the NR2A-containing NMDA-R-­ stimulated Ras-ERK1/2 signaling and comple- There is a potential relationship between neuroments the NR2B-containing NMDA-R-stimulated nal Ephrin-B (EphB) 2 function and the extent of Rap1-p38 MAPK signaling (Zhu et  al. 2005). glutamate excitotoxicity during ischemic stroke. Glutamate-induced apoptotic-like death is largely NMDA receptors, glutamate and voltage-gated prevented by inhibition of NMDA receptor, PKC, ion channels permeable for calcium, are central

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for the pathological processes that underlie gluta- et  al. 2002; Yamaguchi and Pasquale 2004). mate excitotoxicity (Lai et al. 2014). Any shift in Inappropriate levels of Ca2+ influx through the balance to reduce synaptic or enhance extrasyn- NMDA receptor can contribute to neuronal loss aptic NMDA receptor signaling may be detri- in acute ischaemia and traumatic brain injury. mental to neuronal viability (Parsons and Worse, NMDA receptor blockade can promote Raymond 2014). Extrasynaptic NMDA receptors neuronal death outright or render neurons vulnermay be in close contact with mitochondria, able to secondary trauma. Thus, responses to whereas the postsynaptic scaffold, and also the NMDA receptor activity follow a classical horspine structure as such, keeps mitochondria at a metic dose-response curve: both too much and distance to synaptic NMDA receptors. Thus, too little can be harmful (Hardingham 2009). upon stimulation of extrasynaptic, but not synap- EphB2 promotes the immediate response to tic NMDA receptors, mitochondria are exposed ischemia-­reperfusion event in the CNS by pro-­ to high and possibly damaging calcium rises inflammatory activation of astrocytes via EphB-­ (Bading 2017). The EphB-induced phosphoryla- dependent signaling. Simultaneously tion of a single tyrosine (Y504) in the extracel- NMDA-evoked neuronal excitotoxicity occurs lular domain of EphB2 leads to direct recruitment (Ernst et al. 2019). of NR1 and its associated subunits NR2A and NR2B to EphB2. EphB activation of EphB receptor promotes an association of EphB with NMDA 10 Lysosomal Membrane receptors that may be critical for synapse funcPermeabilization and Stroke tion (Dalva et  al. 2000; Hanamura et  al. 2017). EphB modulates the functional consequences of Ca2+ overload following glutamate treatment NMDA receptor activation. In this mechanism induces the activation of calpain and the producactivity-independent and activity-dependent tion of ROS, which are two major contributors to ­signals converge to regulate the synaptic connec- neuronal death in glutamate-mediated excitotoxtions. Thus, along with binding and clustering of icity (Yan et  al. 2016). A metabolic sequence NMDA receptors, EphB2-mediated activation of including NMDA receptor activation, activation EphB2 receptor also enhances glutamate-­ of phospholipase A2 (PLA2) and production of stimulated Ca2+ influx through the NMDA recep- free radicals, and also the activation of calpain tor (Takasu et  al. 2002). Neuronal EphB2 is a are shown to be critical in ischemic cell death. positive regulator of extrasynaptic NMDA recep- This has been defined as “lysosomocentric,” tors. Loss of EphB2 in neurons selectively dimin- hypothesis or lysosomal-dependent cell death in ishes the mitochondrial Ca2+ load upon activation stroke (Lipton 2013; Wang et  al. 2018). ROS of NMDA receptor. EphB2 is rapidly activated in arising from arachidonic acid metabolism and the ischemic brain. Moreover, brain-specific loss NOX leads to activation of ERK1/2, phosphoryof EphB2 reduces the extent of cerebral tissue lation of cytosolic PLA2. Activation of PLA2 in damage in the acute phase of ischemic stroke cerebral ischemia increases lipid peroxidation (Ernst et al. 2019). As mentioned above, a large (Muralikrishna Adibhatla and Hatcher 2006). family of receptor tyrosine kinases, the Both lysosomal membrane permeabilization erythropoietin-­ producing human hepatocellular (LMP) and ROS production are blocked by EphB receptor/ephrin-B ligand system has NMDA channel-blockers and by inhibitors of important roles in stroke pathology. EphB2 stim- MAPK kinase, calcium-dependent/independent ulation of EphB modulates the functional conse- PLA2 (cPLA2 and iPLA2, respectively) quences of NMDA receptor activation and (Windelborn and Lipton 2008). The aspartic suggests a mechanism whereby activity-­protease, cathepsin D is redistributed from lysoindependent and activity-dependent signals con- somes to the cytosol upon oxidative stressverge to regulate the development and remodeling induced apoptotic cell death (Roberg and of synaptic connections (Pasquale 2008; Takasu Ollinger 1998). Cathepsin release may result in

10  N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia

caspase-­ dependent or -independent cell death with or without involvement of mitochondria. LMP causes the proteolytic activation of BH3 interacting domain death agonist (Bid), which is cleaved by the two lysosomal cathepsins B and D.  Subsequently, Bid induces mitochondrial outer membrane permeabilization, resulting in cytochrome c release and apoptosome-dependent caspase activation (Boya and Kroemer 2008). Caspases-2 and -8, PLA2, sphingosine kinase-1, and the Bcl-2 family proteins Bid, Bcl2, Bcl-X-­Bax, and Mcl-1 may all be downstream targets of cathepsins (Johansson et  al. 2010). Bid-­phosphatidic acid interactions in the lysosomal membranes form “lipidic pores,” which lead to the permeabilization of lysosomal or lysosomal-­like membranes (Zhao et  al. 2012). During neuronal excitotoxicity the expression of the GluN2A-containing NMDA receptor is specifically up-regulated, and as a result, the ratio of GluN2A- versus GluN2B-containing NMDA receptors is altered. If GluN2A-containing NMDA receptor is inhibited without affecting the basic expression of both GluN2A- and ­GluN2B-­containing NMDA receptors, increasing endogenous ROS and loss of mitochondrial membrane potential induce the expressions of Bcl-2-Bax and apoptosis-inducing factor (Zhu et al. 2018). LMP is regulated by kinase-dependent signaling events at large. Bcl-2 interacting mediator of cell death (Bim) and active Bax colocalized to lysosome. Lysosomal permeabilization contributes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Activation of JNK promotes lysosomal breakdown in response to TRAIL via activation of the BH3-only-domain protein, Bim (Werneburg et al. 2007). Activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) is accompanied by the decrease of phosphorylated-mammalian target of rapamycin (p-mTOR), phosphorylated-AKT (p-AKT) and phosphorylated-forkhead box O3a (p-FOXO3a), which induces FOXO3a translocation into the nucleus and up-regulates the expression of Bim and cleave caspase 3 (Li et  al. 2017). Because the lysosomal membrane composition plays a key role in the maintenance of lysosomal integ-

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rity, sphingosine-cathepsin B interaction is important in LMP dependent cell death (Kågedal et  al. 2001; Werneburg et  al. 2002). Therefore, arachidonic acid and its metabolites function as second messengers, and modulate neuronal activity. Moreover, they are transsynaptic modulators in the NMDA receptor-induced long-term potentiation. Increases in arachidonic acid following cerebral ischemia are mediated by the NMDA receptor, while NMDA-mediated calcium entry enhances cPLA2 activity (Lazarewicz et  al. 1992; Lucas and Dennis 2005; MrsićPelcić et  al. 2002; Shen et  al. 2007). NMDAmediated calcium entry, followed by metabolism of phospholipids to arachidonic acid, followed by metabolism of arachidonic acid by cyclooxygenase-­2 result in the superoxide production via LMP pathway. Consequently, LMP occurs as one of the important causes of ischemic cell damage in stroke (Windelborn and Lipton 2008).

11

Conclusion, and Future Perspectives in Therapeutic Interventions

Current use of NMDA receptor antagonists as neuroprotective agents has been disappointing in clinical trials (Lipton 2004). One possible explanation is that these antagonists, while suppressing NMDA receptor-mediated neurotoxicity, block the physiological effects of NMDA receptors. However, it has been demonstrated that enhancing extrasynaptic NMDA receptors triggers cell death while activating synaptic NMDA receptors promotes neuronal survival. In fact, blocking NR2B-mediated cell death is effective in reducing infarct volume only when the receptor antagonist is given before the onset of stroke. Whereas, activation of NR2A-mediated cell survival signaling with administration of either glycine alone or in the presence of NR2B antagonist significantly attenuates ischemic brain damage. Because of the dual roles of NMDA receptors in both promoting neuronal survival and mediating neuronal damage, selective augmentation of NR2A-containing NMDA receptor activation may constitute a promising therapy for stroke

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(Hardingham et al. 2002; Liu et al. 2007). A sys- standing of the role of CDK5 inhibition in neurotematic understanding of the damaging mecha- protective mechanisms will help to develop nisms modulating the chain of events leading to selective, safe and efficacious pharmacological cellular survival/damage may help to generate inhibitors of CDK5 for therapeutic use (Mushtaq the potential strategies for neuroprotection. This et al. 2016). Suppressing lipid and protein phosarea covers the stroke pathophysiology to iden- phatase activity of PTEN, respectively, activates tify potential molecular targets. Among them Akt and inhibits extrasynaptic NMDA receptor PSD-95/NMDA receptor interaction, certain key activity and thereby protects against ischemic protein kinases involved in oxidative stress, neuronal death. Downregulation of two distinct CaMKs and MAPKs signaling, inflammation PTEN phosphatase activities present the possiand cell death pathways are remarkable alterna- bility of the use of PTEN as a potential therapeutives. However, selecting promising targets from tic target for stroke treatment via regulation of various signaling cascades, for drug discovery extrasynaptic NMDA receptors activity (Ning and development is very challenging (Mehta et al. 2004). et al. 2007). Since overactivation of NMDA glutamate receptors contributes to neuronal death after stroke, nonetheless very promising agents References are NMDA receptor antagonists and are described as future therapeutics for stroke patients (Martin Aarts MM, Arundine M, Tymianski M.  Novel concepts in excitotoxic neurodegeneration after stroke. Expert and Wang 2010). Disrupting nNOS-PSD-95 Rev Mol Med. 2003;5:1–22. https://doi.org/10.1017/ interaction via overexpressing the N-terminal S1462399403007087. amino acid residues prevents glutamate-induced Abramov AY, Duchen MR.  Mechanisms underlying the loss of mitochondrial membrane potenexcitotoxicity and cerebral ischemic damage. tial in glutamate excitotoxicity. Biochim Biophys These types of drugs block the ischemia-induced Acta. 2008;1777:953–64. https://doi.org/10.1016/j. nNOS-PSD-95 association selectively. They may bbabio.2008.04.017. have potent neuroprotective activity and amelio- Abramov AY, Scorziello A, Duchen MR.  Three distinct mechanisms generate oxygen free radicals in neurons rate focal cerebral ischemic damage (Zhou et al. and contribute to cell death during anoxia and reoxy2010). The activation of GluN2A/AKT/ERK genation. J Neurosci. 2007;27:1129–38. https://doi. pathways protects neurons against post-­ org/10.1523/JNEUROSCI.4468-06.2007. ischaemic neurovascular injury (Huang et  al. Adibhatla RM, Hatcher JF, Dempsey RJ.  Phospholipase A2, hydroxyl radicals, and lipid peroxidation in 2017). Analyzes of NR2B downstream signaling transient cerebral ischemia. Antioxid Redox Signal. in neuronal death after stroke may provide evi2003;5:647–54. https://doi.org/10.1089/1523086037 dences for developing better NMDA receptor70310329. based therapeutics targeting downstream proteins Aleyasin H, Rousseaux MWC, Phillips M, Kim RH, Bland RJ, Callaghan S, Slack RS, During MJ, Mak (Shu et al. 2014). In this context, the inhibitory TW, Park DS.  The Parkinson’s disease gene DJ-1 is effect on Fyn kinase-mediated phosphorylation also a key regulator of stroke-induced damage. Proc of NR2B caused by PSD-93 deletion confers Natl Acad Sci U S A. 2007;104:18748–53. https://doi. org/10.1073/pnas.0709379104. profound neuroprotection against ischemic brain injury (Zhang et al. 2014). Furthermore, inhibit- Anniwaer J, Liu M-Z, Xue K-D, Maimaiti A, Xiamixiding A. Homocysteine might increase the risk of recurrence ing S-nitrosylation of Fyn kinase can exert neuin patients presenting with primary cerebral infarction. roprotective effects against cerebral ischemia/ Int J Neurosci. 2019;129:654–9. https://doi.org/10.10 80/00207454.2018.1517762. reperfusion injury, potentially via NMDA receptor-­mediated mechanisms (Hao et al. 2016). Anrather J, Iadecola C. Inflammation and stroke: an overview. Neurotherapeutics. 2016;13:661–70. https://doi. Inhibition of PSD-95 decouples NMDA receptor org/10.1007/s13311-016-0483-x. downstream signaling and results in neuropro- Arundine M, Tymianski M.  Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxtection after focal cerebral ischemia (Bach et al. icity. Cell Calcium. 2003;34:325–37. https://doi. 2012, 2019). On the other hand, a better underorg/10.1016/s0143-4160(03)00141-6.

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cerebral ischemia. Neurobiol Dis. 2014;68:104–11. https://doi.org/10.1016/j.nbd.2014.04.010. Zhao K, Zhou H, Zhao X, Wolff DW, Tu Y, Liu H, Wei T, Yang F. Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis. J Lipid Res. 2012;53:2102–14. https:// doi.org/10.1194/jlr.M027557. Zhou L, Li F, Xu H-B, Luo C-X, Wu H-Y, Zhu M-M, Lu W, Ji X, Zhou Q-G, Zhu D-Y. Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95. Nat Med. 2010;16:1439–43. https://doi.org/10.1038/nm.2245. Zhou Z, Liang Y, Qu H, Zhao M, Guo F, Zhao C, Teng W.  Plasma homocysteine concentrations and risk of intracerebral hemorrhage: a systematic review and meta-analysis. Sci Rep. 2018;8:2568. https://doi. org/10.1038/s41598-018-21019-3. Zhou Y-F, Wang J, Deng M-F, Chi B, Wei N, Chen J-G, Liu D, Yin X, Lu Y, Zhu L-Q.  The peptide-directed lysosomal degradation of CDK5 exerts therapeutic effects against stroke. Aging Dis. 2019;10:1140–5. https://doi.org/10.14336/AD.2018.1225. Zhu Y, Pak D, Qin Y, McCormack SG, Kim MJ, Baumgart JP, Velamoor V, Auberson YP, Osten P, van Aelst L, Sheng M, Zhu JJ.  Rap2-JNK removes synaptic AMPA receptors during depotentiation. Neuron. 2005;46:905–16. https://doi.org/10.1016/j. neuron.2005.04.037. Zhu J, Xu S, Li S, Yang X, Yu X, Zhang X. Up-regulation of GluN2A-containing NMDA receptor protects cultured cortical neuron cells from oxidative stress. Heliyon. 2018;4:e00976. https://doi.org/10.1016/j. heliyon.2018.e00976.

Alzheimer’s Disease and Protein Kinases

11

Ayse Basak Engin and Atilla Engin

esis, neuroinflammation, oxidative stress, granulovacuolar degeneration, loss of Wnt signaling, Abeta-related synaptic alterations, prolonged calcium ions overload and NMDAR-related synaptotoxicity, damage signals hypothesis and type-3 diabetes are discussed briefly. In addition to clinical perspective of AD pathology, recommendations that might be effective in the treatment of AD patients have been reviewed.

Abstract

Alzheimer’s disease (AD) is the most common neurodegenerative disorder and accounts for more than 60–80% of all cases of dementia. Loss of pyramidal neurons, extracellular amyloid beta (Abeta) accumulated senile plaques, and neurofibrillary tangles that contain hyperphosphorylated tau constitute the main pathological alterations in AD. Synaptic dysfunction and extrasynaptic N-methyl-D-aspartate receptor (NMDAR) hyperactivation contributes to excitotoxicity in patients with AD.  Amyloid precursor protein (APP) and Abeta promoted neurodegeneration develop through the activation of protein kinase signaling cascade in AD. Furthermore, ultimate neuronal death in AD is under control of protein kinases-­related signaling pathways. In this chapter, critical check-points within the cross-talk between neuron and protein kinases have been defined regarding the initiation and progression of AD. In this context, amyloid cascade hypothA. B. Engin (*) Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey A. Engin Department of General Surgery, Faculty of Medicine, Gazi University, Ankara, Turkey

Keywords

Alzheimer’s disease (AD) · N-methyl-D-­ aspartate receptor (NMDAR) · Amyloid precursor protein (APP) · Amyloid beta (Abeta) · Tau · Abeta-derived diffusible ligand (ADDL) · Ephrin B receptor · Cytosolic p21-activated kinase (PAK) · Neurofibrillary tangle (NFT) · Type 3 diabetes

1

Introduction

The worldwide prevalence of dementia is increasing rapidly. It was estimated that 35.6 million people lived with dementia in 2010. Expected numbers of patients will be doubled every 20 years, to 65.7 million in 2030 and 115.4

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_11

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million in 2050. In 2010, 58% of all people with dementia lived in countries with low or middle incomes. This proportion will be anticipated to rise to 63% in 2030 and 71% in 2050 (Prince et al. 2013). In this context, Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the World and accounts for more than 60–80% of all cases of dementia (Alzheimer’s Association 2014). Furthermore, it affects 35–45% of those aged 85 years or older (Rojas-­Fernandez et al. 2002). The predominant hypothesis of AD is neurotoxic effects due to abnormal accumulation of the beta-amyloid (Abeta). Normally soluble proteins, Abeta and tau accumulates into amyloid-like filaments and make up the plaques of AD (Goedert and Spillantini 2006; Rojas-Fernandez et al. 2002). Initial deposits form non-fibrillar diffuse plaques, but are progressively transformed into fibrils, giving rise to the characteristic amyloid plaques. The severity of dementia is positively related to the number of neurofibrillary tangles (NFTs) in neocortex and hippocampus rather than the degree of senile plaque (SP) deposition. NFTs form inside the neuronal cells that die during the course of the disease and consist primarily of abnormal paired helical filaments (PHFs) (Arriagada et al. 1992). In this case, tau becomes hyperphosphorylated at multiple sites and integrate into PHFs, losing its physiological functions (Lee et  al. 1991). Tau-tubulin kinase (TTBK) I and II are enzymes in phosphorylation of tau protein at specific Serine/Threonine residues responsible for the hyperphosphorylation of PHF-tau. Thereby, TTBKI converts native tau to PHF-tau. It is noted that four phosphorylation sites of tau are identified for each kinase. These are the major phosphorylation sites of PHF-tau. TTBK1  in AD brain is one of the underlying mechanisms inducing cyclin-dependent kinase (CDK) 5 and calpain activation, N-methyl D-aspartate receptor (NMDAR) subunit 2B (NR2B) downregulation, and subsequent memory dysfunction (Imahori et al. 1998; Sato et al. 2008). Collectively, loss of pyramidal neurons, extracellular accumulation of senile plaques that contain Abeta deposits, and NFTs that contain hyperphosphorylated tau constitute the main

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pathological alterations in AD (Grundke-Iqbal et  al. 1986; Selkoe 2001). AD is characterized by a progressive loss of cognition, in addition to the formation of SPs and NFTs. Besides the dysregulation of glutamate receptor trafficking and the p21-activated kinase/LIM kinase pathway, drebrin loss is another hallmark of AD.  Mechanistically, reduced synaptic transmission and loss of dendritic spines occurs prior to the formation of amyloid plaques and neuronal cell loss. In this case, Abeta-derived diffusible ligands (ADDLs) bind to the postsynaptic site and induce the drebrin loss in dendritic spines. This process occurs at the prodromal stage of AD (Ishizuka and Hanamura 2017; Pozueta et al. 2013). Drebrin is an actin-­binding protein that is located at mature dendritic spines and it is closely associated with loss of cognitive functions in ADDL-induced synaptic defects (Ishizuka et al. 2014). Considering the evidences, it can be argued that the popular “amyloid cascade hypothesis” is mediated by multiple kinases (Utton et al. 1997). In this context, tau hyperphosphorylation is also regulated by major protein kinases. In all tauopathies, expression of stress-activated protein kinase (SAPK), c-Jun N-terminal kinase (JNK), p38 kinase and CDKs increase in the brain. Thus, in these patients, SAPK, JNK and p38 immunoreactivities are found in hyperphosphorylated tau containing-neurons and glial cells (Ferrer et  al. 2005; Kobayashi et  al. 1993). In addition, glycogen synthase kinase 3 (GSK3) (Utton et  al. 1997), mitogen-activated protein kinase (MAPK)/extracellular signal-­ regulated kinase (ERK), and calcium/calmodulin-­ dependent kinase II (CaMKII) may participate in tauopathies during phosphorylation (Ferrer et al. 2001). Amyloid precursor protein (APP) expression and neuronal exposure to oligomeric Abeta42 enhance Ras/ERK signaling cascade and GSK3 activation. So, APP plays a central role in promotion of neurodegeneration, through activation of Ras-ERK signaling axis as well as GSK3 (Chaput et al. 2012; Kirouac et al. 2017). The maintenance of the normal balance between the formation and degradation of cellular proteins is provided with autophagy. Autophagy

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is strongly associated with AD pathogenesis and autophagic lysosome reformation is regulated via the peroxisome proliferator-activated receptor-­ gamma (PPAR-γ)/adenosine monophosphate (AMP) activated protein kinase (AMPK)/ mammalian target of rapamycin (mTOR)/p70 ribosomal S6 Kinase (S6K) signaling pathway (Zhang et  al. 2017). As mentioned above, the “amyloid cascade hypothesis” is the most popular current opinion in AD pathogenesis. On the recovery of memory and recognition disorders, and neuronal apoptosis, as well as in reducing amyloid beta 1–42 (Aβ1–42)-suppressed cell viability several treatment strategies are tested. Unfortunately, currently available drugs are ineffective to prevent the AD progression and rather they act on the symptoms, therefore mechanismoriented drug explorations are intensively investigated (Singh et  al. 2018). It is thought that, reappraisal of AD related-signaling cascades will ensure to take into consideration the novel therapeutic options in AD. In this respect, the contradictory functions of major protein kinases, which lead to AD will be evaluated.

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reaches a critical level that triggers the amyloid cascade. Pre-dementia phase of AD is an earlystage pathology that presents, ranging from mild neuronal dystrophy to early-stage Braak pathology. This period may continue for several years according to individual resilience and brain reserve. Clinically defined dementia phase of AD is characterized with cognitive and functional impairment. At this stage, there is significant accumulation of neuritic plaques and NFTs in affected brain areas (De-Paula et  al. 2012). Impairment of APP metabolism leads to increased production of Abeta. It is generally thought that Abeta is secreted into the extracellular space and aggregates to form amyloid plaques. In fact, APP and its cleavage product, Abeta acts as homeostatic regulators of synaptic activity. However, the precise processes of plaque formation are still not well known. Extracellular aggregates, Abeta and amyloid plaques, all are toxic to the surrounding neurons. Subsequently, the intraneuronal accumulation of Abeta causes synaptic dysfunction, cognitive impairment, and leads to the formation of amyloid plaques (Takahashi et al. 2017; Wang et al. 2012). Neuritic/synaptic Abeta accumulation is the nidus of plaque forma2 Abeta Hypothesis tion. The APP is normally transported down to the neurites and turns into Abeta at synapses. in Alzheimer’s Disease Synapses are sites of early Abeta accumulation Intraneuronal Abeta42 immunoreactivity pre- and aberrant tau phosphorylation in AD, which cedes both NFTs and Abeta plaque deposition. alter the synaptic composition at early stages of Thus, intracellular Abeta42 accumulation is an the disease (Gouras et  al. 2014). In this stage, early event for neuronal dysfunction. It is well-­ phospho-tau increases in Abeta-­positive synaptoknown that preventing intraneuronal Abeta42 somes. Soluble oligomers in surviving neocortiaggregation is an important therapeutic option cal synaptic terminals are signs of the dementia for the treatment of AD (Gouras et  al. 2000; onset. This event is consistent with “amyloid casGustafson et al. 2007). Abeta dimers are closely cade hypothesis” in which oligomeric Abeta associated with fibrillar Abeta. Moreover, Abeta drives phosphorylated tau accumulation and syndimers can be extracted from insoluble Abeta aptic spread (Bilousova et  al. 2016). Jackson deposits. Soluble Abeta-protein is the main com- et  al. demonstrated that accumulation of tau in ponent of dementia in Alzheimer-type pathology AD worsens synapse loss. Endogenous tau plays (Mc Donald et al. 2010; Shankar et al. 2008). The an important role in the neurotoxicity of tau and intracellular and membrane bound Abeta42 accu- Abeta at earlier stages of the disease (Jackson mulation in the temporal neocortex is the most et al. 2016). Abeta and hyperphosphorylated tau closely associated problem with AD symptoms have both direct and indirect cytotoxic effects (Steinerman et al. 2008) (Fig. 11.1). Pre-clinical that affect neurotransmission, axonal transport, AD may last for several years until the overpro- signaling cascades, organelle function, and duction and accumulation of Abeta in the brain immune response. All these lead to synaptic loss,

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Fig. 11.1  Synapse dysfunction in Alzheimer’s disease. Amyloid-β activates stress-related protein kinases via NMDARs, and causes oxidative stress. The phosphorylation of ERK1/2 and MEK1/2 via MAPK results in apoptosis. Amyloid-β provokes mitochondrial dysfunction either through NMDAR activation or directly multiple protein kinases activation. Bcl-2-CypD-Caspase pathway leads to synapse loss and AD.  Amyloid-β acts on mitochondria either directly or indirectly by causing elevated cytoplasmic Ca2+ levels and oxidative stress. Following depolarization of the membrane, NMDAR and VDCC open and flux toxic amounts of Ca2+ into the cytoplasm. The mitochondrial oxidative stress and the release of cytochrome c, activation of caspase-9, and subsequently of caspase-3 cause neuronal damage. Amyloid-β oligomers activate microglia for production of cytokines, which promote both the inhibition of brain insulin signaling and peIF2α, pJNK, pPKR, pIKKβ increase. Both events act to promote insulin resistance and contribute to synapse loss and impaired long-term potentiation. (Abbreviations: AD: Alzheimer’s disease, AβPP: Amyloid beta precursor peptide, Aβ42: Amyloid beta 42 peptide, AβO: Amyloid beta oligomer, ADDL: Aβ-derived diffusible ligand, Akt: Protein kinase B, AMPK: AMP activated protein kinase, Bcl2: antiapoptotic B-cell leukemia/lymphoma 2 protein, CaMKII: Ca2+-calmodulin dependent protein kinase, CDK: cyclin-dependent kinase, Chk2: Checkpoint kinase 2, Cp: Caspase, CREB: cAMP regulatory-element-binding protein, CypD: Cyclophilin D, EbhR: ephrin receptor, elF2α: eukaryotic initiation factor-2α, ERK: extracellular

signal-regulated kinase, FAK: Focal adhesion kinase, FynK: Src family tyrosine kinase, GLUN2B: subunit of NMDAR, mainly reside at extrasynaptic sites, GSK3β: glycogen synthase kinase 3β, IGFR: insulin-like growth factor receptor, IGF: insulin-like growth factor, IKKβ: inhibitor of kappa B kinaseβ, IR: Insulin receptor, IRS-1: insulin receptor substrate-1, JNK: c-Jun N-terminal kinase, LTP: long-term potentiation, mTOR: mammalian target of rapamycin, mTORC1: mTOR complex 1, MARK2: microtubule-affinity regulating kinase 2, MAPK: mitogen-activated protein kinases, MEK: mitogen-activated protein kinase or extracellular signal-­ regulated kinase kinase, Mt: mitochondria, NFT: neurofibrillary tangle, NMDAR: N-methyl-D-aspartate receptor, PI3K: phosphatidylinositol 3-kinase, PHF: abnormal paired helical filament, PKA: cAMP-dependent protein kinase A, PKB: protein kinase B, PKC: protein kinase C, p38K: p38 kinase, PKR: RNA-dependent protein kinase, PrP: The host-encoded cellular prion protein, ps473: Akt (phospho), pTyr: phospho tyrosine, RNP: ribonucleoprotein, ROS: reactive oxygen species, SAPK: stress-activated protein kinase, Ser: Serine, SFK: Src family kinases, Tau: Human Tau is encoded on chromosome 17q21 and occurs mainly in the axons of the CNS and consists largely of six isoforms generated by alternative splicing, Thr: Threonine, TNFR Tumor necrosis factor (TNF)-receptor, TTBK I/II: Tau-tubulin kinase I/II, VDCC: voltage-dependent Ca2+ channels, Wnt: Wingless-Int)

and dysfunctions in neurotransmitter release (Rajmohan and Reddy 2017). Synaptic activity reduces intraneuronal Abeta accumulation and

provides a protection against Abeta-related synaptic alterations (Tampellini et al. 2009). Varying amounts of endogenous Abeta42 accumulates in

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both glutamatergic and gamma-­ aminobutyric acid (GABA)ergic APP/presenilin-1 (PS1) processing primary neurons. As well as being endocytosed from the extracellular compartment, Abeta is also produced within neurons, after gamma-secretase cleavage of APP C-terminal fragments (Willén et  al. 2017). After Abeta42 exposure autophagy is suppressed. Activation of autophagy increases phosphorylated ERK1/2 (pERK1/2) level and rescue the dysfunction of hippocampal neurons. Since diminished GABAergic tone starts from the preclinical stage of AD, some GABAergic stress test may be effective for identifying cognitively normal elder adults (Yin et al. 2017). In these cases, Sirtuin 1 (silent information regulator 1; SIRT1) regulates many processes that are responsible for development of AD, including APP processing, neuroinflammation, neurodegeneration, and mitochondrial dysfunction (Chong et  al. 2012; Rizzi and Roriz-Cruz 2018). Indeed, SIRT1 is essential in maintaining normal learning, memory and synaptic plasticity without alterations in basal synaptic transmission or N-methyl-Daspartate receptor (NMDAR) function (Michán et al. 2010). In AD patients, a decrease in SIRT1 is present in the parietal cortex, which is closely associated with the accumulation of Abeta and tau protein with cognitive impairment (Julien et  al. 2009). Whereas, activation of SIRT1 reduces Abeta toxicity and decreases nuclear factor kappa-light-­ chain-enhancer of activated B cells (NF-κB) transcriptional activity (Teng and Tang 2010).

3

Abeta Toxicity on N-Methyl-­ D-Aspartate Receptor in Alzheimer’s Disease

In AD, dysregulation of intracellular Ca2+ signaling is an early event, which occurs prior to the presence of clinical symptoms. It is a crucial factor contributing to AD pathogenesis. Abeta oligomers enhance the NMDAR-mediated 2+ postsynaptic Ca signaling in response to presynaptic stimulation by increasing the availability of extracellular glutamate. The abnormal Ca2+

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response leads to impairments in long-term potentiation (LTP), which is an important process in memory formation. Furthermore, internalization of synaptic NR2A-NMDAR creates damage with the postsynaptic Ca2+ response (Liang et  al. 2017). Abeta selectively depresses excitatory synaptic transmission on to neurons that overexpress APP.  This depression depends on NMDAR activity and can be reversed by blockade of neuronal activity (Kamenetz et  al. 2003). Synaptic dysfunction is associated with NMDAR hyperactivation and oxidative stress which ultimately results in AD pathology. Mutations in APP and PS1 lead to elevated secretion of Abeta, especially the more amyloidogenic Abeta42. These toxic forms of Abeta enhance Ca2+ influx into neurons by inducing membraneassociated reactive oxygen species (ROS) or by forming an oligomeric pore in the membrane. Thereby renders neurons vulnerable to excitotoxicity and apoptosis (Bezprozvanny and Mattson 2008; Gouras et al. 2000). The NMDARs are cationic channels induced by the neurotransmitter glutamate, having critical roles in excitatory synaptic transmission. Oligomeric Abetainduced Ca2+ influx occurs through postsynaptic NMDAR.  Simultaneously, excessive formation of ROS and oxidative stress occur. Prevention of excessive NMDAR activity has been considered as a potential route for treatment of AD-associated memory loss (De Felice et  al. 2007; Wilcock 2003). Glutamate kills neurons by a two-stage mechanism. Initial phase is mediated by NMDARs, and the second phase is “oxidative pathway”, which includes Ca2+ as an inducer of oxidative stress (Rodrigues et  al. 2014). The moderate level of NMDAR activity is considered to be beneficial for neurons, whereas excessive activation of NMDARs, with subsequent “Ca2+ overload” is deleterious (Hardingham and Bading 2010). Ca2+ influx evoked by intense synaptic NMDAR activation renders neurons more resistant to apoptotic and oxidative stress, whereas stimulation of extrasynaptic NMDARs causes loss of mitochondrial membrane potential, which is an early marker for glutamate-induced neuronal damage, and subsequent cell death occurs (Hardingham et al. 2002). Concerning the

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receptor location, in death versus survival decisions, findings show that extrasynaptic NMDARs alone account for the large majority of Ca2+ accumulation in cell death. Excitotoxicity, free radical generation, and altered synaptic function encouraged by oxidative stress play an important role in AD pathogenesis. Oxidative stress mediators produced through NMDAR activation and their interaction with other molecules are driving forces for tau hyperphosphorylation and synapse dysfunction (Kamat et al. 2016). Tau mRNA localizes in the somato-dendritic component of primary hippocampal cells. A sub-toxic concentration of glutamate enhances the local translation and hyperphosphorylation of tau. Tau mRNA is in a dendritic ribonucleoprotein (RNP) complex that includes CaMKIIα mRNA. This protein kinase is translated locally in response to glutamate stimulation. Thereby, stimulation of NMDAR redistributes tau to the somato-dendritic region of neurons where it may trigger neurodegeneration (Kobayashi et  al. 2017). The increase in phosphorylated tau levels results in increase of total tau. However, extrasynaptic-NMDAR activation mainly induces overexpression but not hyperphosphorylation of tau. Whereas an uncompetitive, low-affinity, open NMDAR channel blockers significantly prevent extrasynaptic-NMDAR activation-induced elevation of total, phosphorylated and dephosphorylated tau (Sun et al. 2016). NMDAR-NR2B receptor-dependent cytotoxicity is partially derived from ERK1/2 activation. ERK1/2 activation has a dual role both in neuronal survival and death. Selective synaptic NMDAR activation induces ERK activation, whereas extracellularNMDAR activation shuts off ERK signaling pathway. Tau protein inhibits the activity of ERK-activating protein kinase (MAPK kinase; MEK), and promotes ERK dephosphorylation (Amadoro et al. 2006; Léveillé et al. 2008; Sun et al. 2016). Investigations on the specific role of the NR2B subunit of NMDARs in brain functions revealed that the presence of 179 differentially expressed genes involve in ion channel activity and neurotransmission, signal transduction, cytoskeleton transcription, and growth fac-

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tor activity. It was found that increase in NMDARs activity through NR2B overexpression in the forebrain promotes the serine/threonine phosphorylation of MAPK/ERK-cyclic adenosine monophosphate (cAMP) regulatory-­ element-­ binding (CREB) (Li et  al. 2014). Receptor localization is important for protein kinase activity. In this respect, synaptic localization of NMDARs is regulated by ephrin (Eph)B2 receptor (Simón et al. 2009). One of the largest family of receptor tyrosine kinases, the Eph receptor family are subdivided into EphA and EphB receptors (EbhBRs). EbhBRs physically associate with the NR1 subunit of NMDARs (Calò et al. 2006). Thus, Ephrin-A5 inhibits ERK but activates JNK in primary hippocampal neurons. Myosin light chain kinase (MLCK), protein kinase G (PKG), protein tyrosine phosphatase (PTP) as well as the Src family kinases, are required for the stimulation of hippocampal neurons with ephrin-A5 (Yue et  al. 2008). Ephrins and the Eph receptors (EphRs) are involved in regulation of excitatory neurotransmission. EphB2 receptors are reduced in the hippocampus before the development of impaired object recognition and spatial memory. Thus, a reduction in EphR levels have been found in postmortem hippocampal tissue from patients with incipient AD. Although no change in surface expression of NMDAR subunits is apparent, decreasing in EphR signaling disrupts synaptic activity (Long et al. 2006; Simón et al. 2009). The protein levels and the phosphorylation status of the NMDAR subunits GluN1, GluN2A and GluN2B are shown to correlate with cognitive performance. Therefore, NMDAR subunits selectively and differentially are reduced in areas of AD brain (Sze et  al. 2001). The binding of EphB to EphBR recruits other proteins to the EphBR/NMDAR complex, including CaMKII, which is known to phosphorylate the NMDAR. Thereby, the major phosphorylation site on subunit NR2B contains an abundant protein kinase at postsynaptic sites (Omkumar et  al. 1996). EphB2 activation of EphBR in primary cortical neurons amplifies NMDAR-­dependent influx of calcium through the NMDAR tyrosine phosphorylation, via activation of the Src family of tyrosine kinases.

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Eventually, EphB2Rs potentiate NMDAR func- of extracellular fibrillar Abeta in amyloid plaques, tions (Dalva et al. 2000; Takasu et al. 2002). Src ADDLs as soluble oligomers of the Abeta pepfamily kinases (SFKs) are main mediators in tide stimulate tau phosphorylation of hippocampathways leading to the generation of the Ca2+ pal neurons. Tau phosphorylation is blocked by signal (Anguita and Villalobo 2017). EphBR the Src family tyrosine kinase inhibitor 4-aminoactivation decreases Ca2+-dependent desensitiza- 5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d] tion of NR2B-­containing NMDARs. These find- pyrimidine (PP1), and by the ings suggest that EphBRs are one of the main phosphatidylinositol-­3-­kinase inhibitor (PI3K) regulators of synaptic localization of NMDARs (De Felice et al. 2008). PI3K inhibition protects (Nolt et al. 2011). In humans, defective EphBR- neurons from oxidative stress via suppression of dependent regulation of NMDAR localization ERK activation (Levinthal and DeFranco 2004). and function is associated with AD (Sheffler- ADDLs induce excessive formation of Collins and Dalva 2012). In contrast, increasing ROS.  These soluble oligomers directly bind to EphB2R expression reverses deficits in NMDAR-­ NMDARs or attach in close proximity to recepdependent LTP and memory impairments. tor localization. In both manners, they trigger Depletion of EphB2R is critical in Abeta-induced neuronal damage through NMDAR-dependent neuronal dysfunction in AD (Cissé et al. 2011). calcium flux (De Felice et al. 2007). Binding of On the other hand, Abeta impairs NMDAR-­ ADDL to neuronal target receptors leads to aberdependent LTP by inducing internalization of rant activation of trophic signaling, tau hyperNMDARs. Dephosphorylation of the NMDAR phosphorylation and neuronal dysfunction. In subunit, NR2B at phospho-tyr1472 correlates this context, significant increases are found in the with the Abeta-dependent receptor endocytosis. levels of phosphorylation of total protein kinase This process can cause synaptic dysfunction and B (Akt) substrates such as, GSK3 beta (Ser9), tau the cognitive deficits in AD (Kurup et al. 2010; (Ser214), mTOR (Ser2448), despite the decreased Snyder et  al. 2005). As it is well-known, Abeta level of the Akt target p27(kip1). Loss of phosoligomers disturb the function of GluN2B-­ phorylated-Akt and phosphatase and tensin containing NMDARs that means impairing both homolog (PTEN)-containing neurons occur as the glutamatergic transmission and synaptic plas- prominent findings of end stage-AD brain (De ticity. In contrast, overexpression of EphB2 not Felice et  al. 2008; Griffin et  al. 2005). These only rescues the impaired GluN2B-containing effects of ADDLs seem likely to be related to NMDA receptors trafficking induced by ADDLs early AD memory failure. Focal adhesion kinase in hippocampal neurons, but also ameliorates the (FAK) serves as an adaptor protein, a high-affinimpaired cognitive functions and GluN2B-­ ity binding site for SH2 domains of Src proteins containing NMDARs trafficking. Indeed, ADDLs including Fyn and PI3K.  Exposure of neuronal play key role in cognitive deficits of AD. Thereby, cells to Abeta promotes the stable association of it is thought that regulating the EphB2-ADDLs-­ FAK with Fyn kinase (Chen and Guan 1994). NMDAR interaction by small interfering pep- Evidences suggest that alterations in p59Fyn tides may be a promising strategy for AD kinase, a Src family tyrosine kinase, contribute to treatment (Hu et al. 2017; Shi et al. 2016). While AD pathogenesis. Fyn levels are decreased in the depletion of EphB2 contributes to the Abeta- synapses, whereas they increase in the neuronal induced decrease in NMDAR expression, cell where it is colocalized with neurofibrillary increasing EphB2 expression markedly restores tangles. These alterations cause synapse loss (Ho LTP and memory even in the presence of high et al. 2005b). Consequently, Fyn kinase plays an Abeta levels. Increased EphB2 levels augment important role in the regulation of Abeta producNMDAR expression. Thus, increasing in the neu- tion and mediates Abeta-induced synaptic loss ronal EphB2 expression reverses Abeta-induced and neurotoxicity. In this case, Fyn also induces deficits in NMDAR-mediated synaptic function tyrosine phosphorylation of tau (Yang et  al. (Cissé et al. 2011). Independent of the presence 2011). The host-encoded cellular prion protein

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(PrPC)-dependent activation of Fyn induced by Abeta dimers leads to tau hyperphosphorylation at tyrosine 18. PY18-tau is found in neurofibrillary tangles in AD brain. The proline-rich region of tau and the SH3 domain of Fyn kinase mediates this process (Lee 2005). Abeta oligomers bind with high affinity to PrPC locus on neuronal cell surfaces. These oligomers trigger the activation of SFKs, through a transmembrane PrPC partners, such as caveolin, metabotropic glutamate receptor 5 (mGluR5), neural cell adhesion molecule 1 (NCAM) or epidermal growth factor receptor 9 (EGFR9). In this case, neuroreceptors, such as NMDAR, α-amino-3-­hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) receptor (AMPAR), γ-aminobutyric acid type A receptors (GABAAR) and nicotinic acetylcholine receptor (nAChR) and adhesion proteins such as E- and N-cadherin, are directly involved in synaptic function. Thereby, the PrP/SFK/cadherin pathway contributes to synaptic dysfunction in AD (Málaga-Trillo and Ochs 2016). The modulation of E-cadherin and NMDAR endocytosis by PrP and SFKs is only part of a larger molecular network, which can be blocked by Abeta oligomers to impair synapses and trigger neurodegeneration. Abeta oligomers might lead to synaptic damage by six different mechanisms. These are; formation of pore-like structures with channel activity; alterations in glutamate receptors; circuitry hyper-excitability; mitochondrial dysfunction; lysosomal failure and alterations in signaling pathways-related synaptic plasticity; and neurogenesis. In this context, several signaling proteins, including Fyn kinase, GSK3β and CDK5 are involved in the neurodegenerative progression of AD (Crews and Masliah 2010). As another option, soluble Abeta binds to PrPC at neuronal dendritic spines and it forms a complex with Fyn. This complex results in the activation of Fyn kinase. In contrast, abolishing of Fyn activation and Fyn-dependent tau hyperphosphorylation induced by endogenous oligomeric Abeta regulates Abeta-induced Fyn kinase/tau functions (Larson et al. 2012). There are ample evidences related to the implication of the Src tyrosine kinase, Fyn in Abeta-induced neuronal dysfunction. In this regard, it is thought that genetic abla-

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tion of Fyn can abolish oligomeric Abeta-mediated toxicity (Lambert et al. 1998). Fyn kinase-dependent pathways are critical in AD-related synaptotoxicity. Increased Fyn kinase expression is sufficient to trigger prominent neuronal deficits in the context of even relatively moderate Abeta levels. Whereas, reducing the tau levels increase inhibitory currents and normalize excitation/inhibition balance and NMDAR-mediated currents. In AD, Abeta, tau, and Fyn kinase jointly impair synaptic and network function. Briefly, alterations of Fyn expression and activation induced by Abeta have an important role in synaptic and cognitive impairment in AD (Chin et  al. 2004, 2005; Roberson et  al. 2011). Fyn-dependent Abeta redistribution and accumulation in lipid rafts results in ADDL-induced cell death. In this case, Fyn mediates Abeta/tau-induced toxicity. Tau deprivation prevent memory deficits and improve survival in AD.  Furthermore, memory deficits are fully prevented with uncoupling the Fyn-­mediated co-action of NMDAR and excitatory postsynaptic density-95 (PSD-95) (Haass and Mandelkow 2010; Ittner et  al. 2010; Williamson et al. 2008). Whether the endogenous soluble extracellular Abeta activates the intracellular Fyn kinase is still not known. However, PrPC-­mediated Fyn activation may play a determining role in late stages of AD, when Abeta dimers are at the maximum concentrations in the brain (Larson et al. 2012). The plasma membrane transporters are critically important for regulation of the magnitude and time course of synaptic signaling. The control of endocytic trafficking via tyrosine phosphorylation signals influences the function of neuronal receptors, ion channels and neurotransmitter transporters (Buckley et  al. 2000). Cytosolic p21-activated kinase (PAK) deficits and aberrant PAK translocation in AD is secondary to neuron loss and tau accumulation. Abeta oligomer-induced abnormal PAK activation and translocation could disrupt the normal PAK signaling pathways in oligomer-­binding excitatory neurons. The active phospho-­PAK of membrane is significantly elevated in comparison to cytosol in AD patients. PAK signaling defects and cotranslocation of PAK- the Rho family small gua-

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nosine triphosphate proteins (GTPases) (Rac1- cell division control protein 42 (Cdc42)) complex in AD brain are caused by Abeta oligomers. Rac activation following Abeta oligomer accumulation is one of the initial events in AD. Thereby, activation and translocation of Rac1 and PAK contributes to synaptic dysfunction and excitatory synapse loss in AD. Whereas, the Src/Fyn kinase inhibitor PP2 effectively prevents oligomer-induced Rac/PAK translocation (Chromy et al. 2003; Hayashi et al. 2002; Ma et al. 2008). Indeed, PAK and its activity are markedly reduced in AD. Abeta directly affects PAK signaling deficits and drebrin loss in hippocampal neurons. Therefore, drebrin loss and related memory impairment is associated with the PAK inhibition in cognitive deficits in AD (Zhao et al. 2006).

4

Neuroinflammation in Alzheimer’s Disease

Neuroinflammation contributes to cognitive impairment and plays a significant role in AD progression. Matrix metalloproteinase (MMP)-9 regulates blood-brain barrier (BBB) permeability via release of cytokines and free radicals in AD.  MMP9-mediated breakdown of the basal lamina and destruction of gap junctions in the neurovascular unit result in increased central nervous system (CNS) permeability and inflammation (Candelario-Jalil et  al. 2009; Reijerkerk et  al. 2006; Verslegers et  al. 2013). Intercellular adhesion molecule-5 (ICAM-5) mediates the regulation of dendritic spine elongation and maturation. This molecule is cleaved by MMP9, upon activation of NMDARs. Reductions in ICAM-5 have been linked to changes in dendritic spine morphology that are related to LTP.  Consequently, MMP9 has an important role in synaptic function (Conant et al. 2010; Tian et  al. 2007). Indeed, MMP9 is significantly lower in AD patients who have decreased levels of Abeta42 and Abeta40, and increased total tau levels compared to healthy controls (Mroczko et  al. 2014). Abeta42 and Abeta40 levels of cerebrospinal fluid (CSF)

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change in parallel with a reduction of MMP9. Activated microglia secretes macrophage-derived chemokine (MDC) that induces chemotaxis of T helper 2 (Th2) cells. This means that MDC produced by microglia promotes neuro-­ inflammation via accumulation of Th2 cells into the injury site (Columba-Cabezas et al. 2002). In neurons, Abeta40 and Abeta42 formation occurs in the trans-Golgi Network (TGN) and endoplasmic reticulum (ER), respectively. Abeta42 is made and retained within the ER in an insoluble state. The earliest event taking place in AD most probably is the generation of Aβ1–42 in the ER (Greenfield et al. 1999; Hartmann et al. 1997; Xu et  al. 1997). Thus, the increased Abeta42 accumulation in vulnerable distal neurons and synapses lead to their destruction. The subsequent formation of more amorphous plaques reflects remnants of this destruction, which is shaped by activated inflammatory cells (Gouras et al. 2010). CD36, as a major pattern recognition receptor, mediates microglial response to Abeta. CD36 plays a key role in the proinflammatory events associated with AD.  Uptake of Abeta leads to secretion of the interleukin 1-beta (IL-1β) and activation of the NACHT, LRR and PYD domains containing protein3 (NALP3) inflammasome. In addition, Abeta induces CD14, toll like receptor 4 (TLR4), TLR2, Src-Vav-RAC signaling cascade, as well as p38 MAPK in microglia. This process drives production of ROS and activation of the canonical pro-inflammatory transcription factor NF-κB (El Khoury et al. 2003; Halle et al. 2008; Reed-­Geaghan et al. 2009). Vav1 leads to a reduction in T-cell receptor (TCR) signaling. Reduced TCR signaling leads to transient Erk activation and favors Th2/regulatory T cell (Treg) differentiation. Decrease of calcium flux and of Erk, Akt and p38 activities cause the drop down of the production of interferon-gamma (IFN-γ), IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by CD4+ T cells. Vav1 has an adaptor function in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation (Kassem et  al. 2016).The Vav, as a guanine nucleotide exchange factor (GEF) for Rac is phosphorylated at tyrosine site in response to fibrillar Abeta pep-

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tides. Vav activity is required for fibrillar Abetastimulated intracellular signaling. Rac1, which is a component of signaling cascade, is a critical regulator for both of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and of microglial phagocytosis. Abeta induces the microglial NADPH oxidase-­derived ROS production through an ensemble of cell surface receptors that initiate a tyrosine kinase-VavRac1-based signaling cascade (Wilkinson et  al. 2006). Abeta deposition in the brain through the interaction of fibrillar forms of amyloid with cell surface receptors causes the activation of intracellular signal transduction cascades in the microglia (Bamberger and Landreth 2001). Thus, exposure of microglia and THP1 monocytes to fibrillar Abeta leads to time- and dose-dependent increases in protein tyrosine phosphorylation. The tyrosine kinases Lyn, Syk, and FAK are activated on exposure of microglia and THP1 monocytes to Abeta, resulting in the tyrosine kinase-induced generation of superoxide radicals (McDonald et  al. 1997). It is still unclear how Abeta peptides induce damage to cells causing their death. Sepulveda et al. proposed that Abeta oligomers may disrupt cellular membranes and increase their permeability to ions. Abeta perforations produce a wide range of toxic effects ranging from synaptotoxicity to cell death (Sepulveda et al. 2010). The early phase of cell death is energy dependent and involves the externalization of phosphatidylserine (PS) residues in the inner leaflet of the plasma membrane. This is a signal for engulfing and disposing of injured cells by inflammatory cells (Schutters and Reutelingsperger 2010). This process occurs with the expression of the phosphatidylserine receptor (PSR) on microglia during oxidative stress (Kang et al. 2003). Neurons can expose PS, which acts as ‘eat me’ signals provoking phagocytosis via microglial receptors (Vilalta and Brown 2018). PS decreases cholinesterase, improves memory, and ameliorates hippocampal inflammation injury in AD brains by increasing antioxidant capacity (Zhang et al. 2015). Damaged neurons, highly insoluble Abeta peptide deposits and NFTs generate stimuli for inflammation from early preclinical stage to terminal stages of

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AD. Th1 cells increase microglial activation and Abeta deposition. These changes are related to impaired cognitive function. In addition, Abeta peptide directly and independently activate the classical complement pathway as well as the alternative complement pathway. These events trigger the formation of C3 complement; generate cytokine-like C5a complement-activation fragment; and mediate formation of the proinflammatory C5b-9 membrane attack complex (Akiyama et al. 2000; Bradt et al. 1998; Browne et  al. 2013; Cooper et  al. 2000). On the other hand, the JNK/signal transducer and activator of transcription 3 (STAT3) signaling pathway in patients with AD also contributes to the activation of Abeta-induced neuro-­ inflammatory responses in microglia (An et  al. 2018). JNK activity is increased in Abeta42-­ expressing brains, and the Abeta42-induced defects are rescued by reducing JNK or caspase activity through genetic modification or pharmacological treatment in AD. In addition, these impairments may also be restored by repressing forkhead box subgroup O (FoxO) activity (Hong et al. 2012). Both apoptosis as well as the prior activation and proliferation of microglial cells relies upon the presence of FoxO3a. Aβ1–42 exposure maintains FoxO3a in an unphosphorylated “active” state and facilitates the cellular trafficking of FoxO3a from the cytoplasm to the cell nucleus. This transcription factor activates “pro-apoptotic” programs (Shang et  al. 2009). The JNK/FoxO signaling pathway seems to be essential in death evoked by Abeta. Therefore, it may have therapeutic potential for AD. The prostaglandin-E2 (PGE2) signals show anti-inflammatory effect through a class of four E-prostanoid (EP) receptors. In the presence of EP receptors, inflammation is repressed via decreasing Akt and NF-κB inhibitor (IκB) kinase (IKK) phosphorylation, and preventing nuclear translocation of p65 and p50 NF-κB subunits. The PGE2-EP4 receptor levels decrease significantly in human cortex with progression of AD. Consequently, the EP4 receptor is expected to suppress the microglial inflammatory responses of Abeta42 peptides, while the anti-­ inflammatory effect of the EP4 receptor is abol-

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ished (Andreasson 2010; Shi et  al. 2010; Woodling et  al. 2014). Evidences indicate that chronic activation of microglia, presumably via the secretion of cytokines and reactive molecules, may exacerbate plaque pathology as well as enhance the hyperphosphorylation of tau and the subsequent development of neurofibrillary tangles (Kitazawa et al. 2004). Expression of ubiquilin-1 is upregulated in nucleoplasm, or its translocation from the cytoplasm to the nucleus during neurofibrillary tangle formation changes in the hippocampus of patients with AD. This is a potentially protective response to increased tau phosphorylation in hippocampal neurons, however the failure of such a response contributes to neuronal degeneration in end-stage AD (Mizukami et al. 2014). Activated microglia not only secretes cytokines, chemokines or ROS, but also involves increased turnover of neuroprotective endogenous substances (Regen et  al. 2017). The interaction of CD40, which is expressed on the surface of microglia, and T-cell receptor CD40 ligand (CD40L) promotes pro-­ inflammatory microglial cell activation in response to Abeta (Tan et al. 2002). Peripheral T cells of AD patients overexpress CXCR2 (CXC chemokine receptor 2). This receptor is intracerebral microglial tumor necrosis ­factor-­alpha (TNF-α)-dependent and is associated with T-cell adhesion on brain endothelium, and transendothelial migration (Liu et al. 2010). Abeta binding and activation of cell surface immune and adhesion molecules such as CD45, CD40, CD36 and integrins, subsequent recruitment of Src family tyrosine kinases such as Fyn, Lyn and Syk kinases are achieved by microglial activation. In later stage, activation of ERK and MAPK pathways induces proinflammatory gene expression and leads to the production of cytokines and chemokines. These molecules contribute to synaptic dysfunction, damage and loss. The production of inflammatory mediators leads to microglial activation, and further secretion of proinflammatory molecules and amyloid. So, this cycle is maintained. Simultaneously with the inflammation, direct neuronal injury from amyloid-induced signaling contributes to neurodegeneration of AD patients (Ho et  al. 2005a;

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Weisman et  al. 2006). The microglia-­mediated neuroinflammation plays an important role in the pathogenesis of AD.  Advanced glycation end products (AGEs)/receptor for advanced glycation end products (RAGE) or Rho/Rho kinase (ROCK) are both involved in the development of non-specific inflammation. The AGEs/RAGE/ Rho/ROCK pathway could intensify the non-specific inflammation of AD (Chen et  al. 2017). AGEs are formed as a result of the Maillard reaction and these molecules promote amyloid oligomer aggregation in AD (Bucala and Cerami 1992; Woltjer et al. 2003). In addition, lymphocyte-specific protein tyrosine kinase (LCK) is a lymphoid-specific, Src family protein tyrosine kinase that is known to play a pivotal role in T-cell activation and interact with the T-cell coreceptors, CD4 and CD8. LCK is significantly down-regulated in AD (Zhong et  al. 2005). The lack of correlation between Abeta plaques and cognitive decline in AD is related to the difference in the ability of inflammatory cells from person to person to effectively remove Abeta plaques from the brain. On the other hand, while tau is normally localized to axons, Abeta42 accumulates into distal dendrites (Gouras et al. 2010). Pro-inflammatory cytokines produced by Abeta-activated microglia exacerbate the hyperphosporylation of tau proteins that forms NFTs in AD pathology. Thus, activated microglia are abundant in the AD brain, and concentrate around senile plaques. The Abeta burden in affected brains may be removed largely by microglial clearance. However, microglia mediate synapse loss through a complement-­ dependent mechanism or via activation of neurotoxic astrocytes. Therefore, microglial dysfunction causes AD progression (Hansen et al. 2018; Santos et al. 2016; Tan et al. 2012). Although a correlation could not be demonstrated between distribution and burden of Abeta plaques and cognitive scores with autopsy and tau positron emission tomography (PET) imaging studies, the spread of NFTs through neuronal networks has a strong correlation to neuronal loss and cognitive decline. NFTs is best represented by Braak staging system (Braak and Braak 1991; Giannakopoulos et  al. 2009;

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Suemoto et  al. 2017). In addition, receptor- the Abeta-triggered production of IL-1β, IL-4 interactive protein kinase-1 (RIPK1) activity is and IL-6 in astrocytes and ameliorates AD proresponsible for the substantial portion of gression. Deletion of TRPA1 decreases PP2B transcriptomic changes in AD.  Indeed, activity but increases the phosphorylation of Akt necroptosis is executed by the mixed lineage in brain tissues (Lee et  al. 2016). The kinase domain-like (MLKL) protein, which is intermediate-­conductance Ca2+-activated K+ triggered by RIPK1 and RIPK3. Furthermore, channel3 (KCa3) expression is markedly associnecroptosis is positively correlated with Braak ated with ER stress and unfolded protein response stage, and inversely correlated with cognitive (UPR) in both Abeta-stimulated primary astroscores in human AD brains (Caccamo et  al. cytes and brain lysates of AD patients. Gene 2017). Overexpression of the RIPK1 induces deletion or blockade of KCa3.1 restores Akt/ both NF-ĸB activation and apoptosis (Hsu et al. mechanistic mTOR signaling, moreover, glial 1996). RIPK3 may indirectly promote nucleotide-­ activation and neuroinflammation are attenuated. binding and oligomerization domain (NOD)-, Memory deficits and neuronal loss are reversed leucine-rich repeat (LRR)- and pyrin domain-­ through elimination of KCa3 (Yu et al. 2018). containing protein 3 (NLRP3) inflammasome activation through stimulating mitochondrial ROS production or triggers inflammation through 5 Oxidative Stress NF-κB-dependent cytokine gene transcription and Alzheimer’s Disease and ripoptosome/inflammasome-mediated pro-­ IL-­1β (Moriwaki and Chan 2016). Peripheral T The excessive Ca2+ entry through NMDA and cells could exert their effects on the pathogenesis AMPA receptors induced by Abeta oligomers of AD without entering the CNS.  On the one cause mitochondrial dysfunction, oxidative stress hand, pro-inflammatory cytokines, which are and mitochondrial membrane depolarization. secreted by T-cells, enter the CNS and activate Mitochondrial damage underlies the neurotoxicity microglia and astrocytes, on the other hand, induced by Abeta oligomers and thus, oxidative peripheral activated T cells promote activation of stress is a critical component of AD (Alberdi myeloid cells such as monocytes, macrophages et  al. 2010; Maiese 2014). Inhibition of the and dendritic cells. Consequently, pro-­expressions of APP and Aβ1–40 in the cortex and inflammatory cytokines such as TNF-α, IL-1β hippocampus, restores the activities of superoxide and IL-6 increase in CNS (Morel et  al. 2009). dismutase and glutathione peroxidase and Activated T cells also exist as infiltrates in the decreases the production of malondialdehyde in brains of AD patients (Itagaki et al. 1988; Togo the cortex. Abolishing of oxidative stress et al. 2002). Protein C kinase 2 (CK2) in astro- improves the histopathological changes in the cytes is involved in the neuroinflammatory cortex and hippocampus and downregulates the response in AD. CK2 immunopositive astrocytes expressions of JNK2, p53 and cleaves caspase are associated with amyloid deposits 3  in the hippocampus. Reduction of oxidative (Rosenberger et al. 2016). Abeta oligomers dis- stress in conjunction with reduction in Abeta rupt ER Ca2+ homeostasis, which induces ER expression results in significantly improved stress that leads to astrogliosis in AD (Alberdi cognitive function (Wang et  al. 2013). Nuclear et al. 2013). Transient receptor potential ankyrin abnormalities, tau deposition and caspase 1 (TRPA1) is a sensor for detecting ROS and key activation in the postsynaptic density fractions of gatekeeper in regulating the inflammatory AD cases are related to the synapse degeneration response. TRPA1-Ca2+-protein phosphatase 2B during disease progression. Therefore, absolute (PP2B) signaling pathway regulates Abeta-­ neuronal density is also decreased in AD (Broe triggered activation of NF-κB and nuclear factor et  al. 2001; Louneva et  al. 2008). Presynaptic of activated T cells (NFAT) and inflammation in terminals are vulnerable to ER-stress depending astrocytes. Functional loss of TRPA1 abrogates on Abeta. The ER-resident caspase-12 is a

11  Alzheimer’s Disease and Protein Kinases

mediator of Abeta neurotoxicity. Thereby, mitochondrial failure accompanied by a reduction in actin levels via caspase-12 activation participates in Abeta-induced synaptic toxicity. Indeed, neuronal death in the brains of patients with AD can be initiated by the local activation of caspases within the synaptic compartment (Quiroz-Baez et al. 2011). The proapoptotic protein B-cell leukemia/ lymphoma-2 (Bcl-2) interacting mediator (Bim) of cell death is an essential mediator of Abeta-­ induced neurotoxicity. The cell cycle molecule cdk4 and its downstream effector B-myb, are required for Abeta-dependent Bim induction and death in neurons. Bim have been identified as vulnerable region of brain in AD patients (Biswas et  al. 2007). Autophagic Abeta clearance in brain attenuates cellular apoptosis in hippocampus and is accompanied by an improvement in spatial learning and memory abilities in AD (Jiang et al. 2014). The “Aging Alzheimer Cascade” progresses during late life. It is influenced by ROS and reactive nitrogen species (RNS), oxidative stress, redox transition metals and increased deposition of Abeta. Accumulation of Abeta is due to increased levels of APP, and tau. In patients with genomic vulnerability to AD, oxidative stress-­ induced brain damage triggers specific antioxidant defenses. In these cases, increased levels of Abeta and aggregation of hyper-­phosphorylated tau result in paired helical filaments (PHFs) and impaired functions related to the apolipoprotein Eε4 isoform (Rodrigues et  al. 2014). Human umbilical cord mesenchymal stem cells (hUCMSCs) transplantation significantly ameliorate cognitive function without altering Abeta levels in hippocampus, by reducing oxidative stress. The improved cognitive function is linked to the up-regulation of neuronal synaptic plasticity related proteins levels including Sirt1, brainderived neurotrophic factor (BDNF) and synaptophysin (Cui et  al. 2017). Especially BDNF is essential to protect against neurodegeneration, through control of the amyloidogenic pathway, which increases Abeta production in hippocampal neurons, and through stimulating neural stem cells (Blurton-Jones et  al. 2009; Matrone et  al.

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2008). BDNF binding triggers its transmembrane receptor, tropomyosin-­related kinase B (TrkB), that activates three main downstream phosphorylation cascades. Phospholipase Cγ (PLCγ), PI3-K/Akt, and MAPKs pathways together act as the predominant regulators by BDNF/TrkB (Benarroch 2015). BDNF exerts neuroprotective effects by antagonizing NMDAR triggered excitotoxicity, promoting dendritic regeneration, and possesses anti-apoptotic effects via Bcl-2 protein and/or by post-translational modifications of apoptosis-­related proteins such as Bad and Bim (Wu et al. 2010). Contrarily to BDNF, the precursor form of BDNF (proBDNF) induces apoptosis through the specific interaction with p75 and its co-receptor, sortilin. Neurotoxic signaling of proBDNF in AD pathology is resulted by an increase of proBDNF stability due to ROS, by the increase of expression of the p75 co-receptor and the increase of the basal levels of p75 processing (Fleitas et al. 2018). BDNF/TrkB signaling exerts its protective effect by regulating superoxide anion homeostasis via inhibition of p47(phox) phosphorylation (Tsai et  al. 2012). PPAR-γ coactivator 1alpha (PGC-1α), a key effector of ROS detoxifying enzyme expression, as well as mitochondrial biogenesis, is shown to be induced in a CREB-­ dependent manner (St-Pierre et al. 2006). Moreover, the neuroprotective effect of BDNF against ROS-mediated cell death is abolished by disruption of CREBmediated transcription (Lee et al. 2009). BDNF/ TrkB signaling exerts negative-feedback regulation on oxidative stress through inhibition of p47(phox) phosphorylation, preservation of mitochondrial electron transport capacity, and upregulation of mitochondrial uncoupling protein 2 (UCP2) (Chan et al. 2010). In fact, BDNF induces the synthesis of activity-­regulated cytoskeleton-associated protein (Arc), which modulates synaptic plasticity. Contrarily, Abeta impairs BDNF-induced Arc expression. Collectively, BDNF induces activation of four different pathways at least: PI3K-Akt- mTOR signaling pathway, the phosphorylation of eukaryotic initiation factor 4E binding protein (4EBP1) and p70 ribosomal S6 kinase (p70S6K), the dephosphorylation of eukaryotic

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elongation factor 2 (eEF2), and the expression of Arc. However, interrupting the PI3K-AktmTOR signaling pathway only, prevents all the effects of BDNF (Chen et  al. 2009). Oxidative stress induces apoptosis of neuronal cells by inhibiting the mTOR-mediated phosphorylation of ribosomal p70 S6K1 and 4EBP1. ROS simultaneously inhibits the upstream kinases, Akt and phosphoinositide-dependent kinase 1 (PDK1), and activates the negative regulator, AMPK-α. In this case, inhibition of mTOR signaling is at least in part through activation of AMPK (Chen et al. 2010). mTOR functions with PI3K, Akt, FoxO transcription factors, PRAS40, and p70S6K to prevent apoptotic cellular death in AD. Repression of Abeta production requires inhibition of Akt and mTOR with the concomitant activation of AMPK (Maiese 2014).

6

Alzheimer’s Disease and mTOR Pathway

Alzheimer’s disease may be discussed from two different points of view; neuronal damage or cognitive impairment. Based on recent data, the mTOR pathway arises as a versatile subject whose modulation may impact on mechanisms of both neuronal viability and cognition (Franco et al. 2017). In the CNS, mTOR and its signaling components are present in brain endothelial cells (Galan-Moya et  al. 2011), neurons (Cota et  al. 2006), inflammatory microglia (Dello Russo et  al. 2009; Shang et  al. 2011). mTOR binding protein, Raptor is an essential component of the mTOR complex1 (mTORC1), and functions to recruit the mTOR substrates the eukaryotic initiation factor 4EBP1 and the serine/threonine kinase ribosomal protein p70S6K to the mTORC1 complex (Hara et al. 2002; Kim et al. 2002). The binding of Raptor to mTOR is necessary for the mTOR-catalyzed phosphorylation of 4EBP1 and raptor strongly enhances mTOR kinase activity toward p70S6K (Hara et al. 2002). Thus, the protein expression of p-mTOR, mTOR-mediated phosphorylation of 4EBP1 and S6K1 pathways are amplified in AD. Blocking mTOR enhances activities of IL-6 and TNF-α signaling pathways. Simultaneously, increasing in Caspase-3 activity

causes cellular apoptosis and worsens learning performance (Wang et al. 2016). In patients with AD, a decrease in mTOR activity in peripheral lymphocytes appears to correlate with the progression of AD.  The levels of mTOR, the double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor 2alpha (eIF2α) in lymphocytes follow the cognitive decline in AD.  Phosphorylated PKR and eIF2α levels are significantly increased in lymphocytes of AD patients. These are also significantly correlated with cognitive and memory scores in AD patients (Paccalin et al. 2006). PKR accumulates in degenerating neurons and is activated by Aβ1– 42 neurotoxicity. It modulates Abeta synthesis through beta-site APP cleaving enzyme-1 (BACE 1) induction. Moreover, PKR increases in cerebrospinal fluid from patients with AD and induces the activation of pro-inflammatory pathways leading to TNF-α and IL1-β production (Hugon et  al. 2017). The control of translation through PKR/eIF2α signaling pathway is altered in AD lymphocytes as it was observed in brains of AD patients. PKR in the cell nuclei of brain tissue from AD patients, and the level of phosphorylated PKR are significantly higher than in the disease-free controls. There is a sound relationship between the activation of PKR and neuronal cell death in AD (Chang et al. 2002; Onuki et al. 2004). An intense immunoreactivity for phosphorylated eIF2α is observed in AD brain tissue. Most of the phosphorylated eIF2α immunoreactive neurons have also high immunoreactivity for phosphorylated tau. Phosphorylation of eIF2α is associated with the degeneration of neurons in AD. This phenomenon is also observed in phosphorylated PKR, the upstream kinase for eIF2α. Therefore, PKR-eIF2α pathway has been thought to be pro-apoptotic (Chang et  al. 2002). These two pathways: PKR-eIF2α pathway (pro-­ apoptotic) and mTOR kinase-4EBP1-S6K (anti-­ apoptotic) pathway control the initiation of neuronal death in AD patients (Morel et al. 2009). Moreover, p53, regulated in development and DNA damage responses 1 (Redd1) genes and tuberous sclerosis complex 2 (TSC2) are molecular links between the up-regulation of PKR and the down-regulation of mTOR. Although PKR is not specific to AD, it could be an early biomarker

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of the neuronal death or a critical target for a therapeutic approach in AD (Morel et al. 2009). Loss of mTOR signaling also has been shown to impair LTP and synaptic plasticity in AD and can be restored through the upregulation of mTOR signaling. The mTOR pathway modulates Abetarelated synaptic dysfunction in AD (Ma et  al. 2010). Since a reduction in mTOR signaling occurs in patients with AD due to detrimental effects of Abeta exposure on mTOR, a minimum level of activity of the PI3K, Akt, and mTOR pathways is required to prevent the onset and progression of AD. Increased Ras homolog enriched in brain (Rheb) GTPase activity that is known to increase mTORC1 activity, is necessary to degrade the BACE1. BACE1 promotes Abeta accumulation in AD. In contrast, over-expression of Rheb depletes BACE1 and reduces Abeta generation. Unfortunately, the BACE level of the brain maintains its high level during the progression of AD (Shahani et al. 2014). Abnormal tau hyperphosphorylation is major step of insoluble tau aggregates deposition in neurons of AD brains. In the temporal cortex of patients with AD, increased levels of phosphorylation for Akt, GSK3β, mTOR signaling, and tau contribute to the pathology of AD. While the levels of active phosphorylated-Akt in particulate fractions increase, in cytosolic fractions Akt levels significantly decreases in AD. Loss of phosphorylatedAkt and PTEN-containing neurons represent the end stage-AD (Griffin et  al. 2005). Constitutive overexpression of mTOR increases total tau, whereas constitutive deletion of mTOR or constitutive overexpression of the inactive form of mTOR and S6K decrease the total tau (Tang et al. 2013). In fact, memory impairment is a result of accumulation of Abeta peptide and mitochondrial damage in the brain of patients with AD.  Decreased PTEN gene-induced putative kinase 1 (PINK1) expression is associated with AD. Restoring neuronal PINK1 function significantly reduces Abeta levels, oxidative stress, as well as mitochondrial and synaptic dysfunction (Du et al. 2017). As part of the mTOR signaling cascade, phosphorylated p70 S6 kinase (Thr389 or Thr421/Ser424), total tau, and hyperphosphorylated-tau significantly increases in AD brain as

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compared to control cases (An et  al. 2003). Decreasing in BACE1 as a rate limiting enzyme for Abeta generation, promotes Abeta clearance by enhancing autophagy through Akt/mTOR signaling inhibition and AMPK/Raptor pathway activation (Zhu et al. 2013). In fact, autophagy is a major degradation pathway for organelles and aggregated proteins. In case of Abeta deposition, it may function as a cytoprotective mechanism (Rubinsztein et  al. 2007). Beclin 1 (BECN1-adaptor protein) regulates autophagy and APP processing. The function of BECN1 is inhibited through four different mechanisms during the progress of AD.  These include, reduction of BECN1 expression, sequestration of BECN1 to non-­ functional locations, formation of inhibitory complexes between BECN1 and antiapoptotic Bcl-2 proteins, inhibition of the BECN1/vacuolar protein sorting 34 (Vps34) complex through the activation of CDK1 and CDK5 (Salminen et  al. 2013). If autophagy is increased in AD, it facilitates the clearance of Abeta, aggregation-prone proteins and promote neuronal survival. Reduction of BECN1 mRNA in AD brain tissue increases intraneuronal Abeta accumulation, extracellular Abeta deposition, and neurodegeneration and causes microglial dysfunctions (Pickford et  al. 2008). Epidermal growth factor receptor related protein (ErbB2) effectively suppresses autophagic flux by physically dissociating BECN1 from the Vps34- phosphoinositide 3-kinase regulatory subunit 4 (Vps15) complex independent of its kinase activity (Wang et  al. 2017). ErbB2 is a member of the epidermal growth factor receptor (EGFR)/ErbB family membrane tyrosine kinases and is tightly associated with neuritic plaques in AD (Chaudhury et al. 2003). Inhibition of ErbB2 expression can effectively decrease Abeta production through activating autophagy. Therefore, an ErbB2 kinase-targeted therapeutic strategy may render cognitive improvement without eliciting complications (Wang et al. 2017). Moreover, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites. Both the BECN1-­APP association and BECN1-dependent APP endocytosis are negatively regulated by Akt activation

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(Swaminathan et al. 2016). Increase in autophagosome numbers in neurons from AD brains also show increased levels of soluble, proteolytically processed microtubule-associated protein 1 light chain 3-I (MAP1LC3-I) and MAP1LC3-II (Yu et al. 2005). When autophagy is induced, microtubule-associated protein 1 light chain 3 β (MAP1LC3β/LC3B) localizes to the site of autophagosome nucleation and then conjugates to the membrane lipid phosphatidylethanolamine. Thereby, this protein becomes membraneassociated to drive vacuolar elongation with other autophagy related proteins (Geng and Klionsky 2008; Xie and Klionsky 2007). In this manner, LC3B turns into from soluble (LC3B-I) form to lipid-modified form (LC3B-II), which aggregates to autophagosomes in cytoplasm (He et al. 2015). Although increased autophagy facilitates the clearance of a­ ggregation-­prone proteins and promote neuronal survival, excessive autophagic activity is detrimental as well and leads to cell death. A possible explanation for this apparent contradiction is that reduced BECN1 levels lead to changes in autophagosomal flux. This could impair endosomal-lysosomal degradation cycle (Jaeger and Wyss-Coray 2009). In fact, lysosomes have three basic functions, namely digestion and autophagic functions, signal transduction and membrane trafficking (Cheng et  al. 2010). Autophagosomes contain the enzymes necessary for processing of APP into Abeta and are potential producers of this toxic peptide. More APP-rich substrates are diverted into the macroautophagy pathway and macroautophagy contributes substantially to intracellular Abeta accumulation. The inefficient extracellular elimination of autophagy-generated Abeta implies greater pathogenicity. Because intracellular Abeta is more cytotoxic than extracellular Abeta. Furthermore, induction or inhibition of macroautophagy in neurons by modulating mTOR kinase elicits parallel changes in autophagosome proliferation and Abeta production (Yu et  al. 2005). Disturbances in initiation of autophagy due to insufficient BECN1 levels could cause expansion of the endosomal-­ lysosomal system. This process produces a high load of potentially Abeta generating vacuoles

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(Jaeger and Wyss-Coray 2009). Production of Abeta protein in autophagic vacuoles promotes its extracellular deposition in neuritic plaques. Autophagy is widely regarded mechanism for the removal of the detrimental Abeta peptides and Tau aggregates. Nonetheless, some data indicate that autophagy has an unfavorable function in facilitating the production of intracellular Abeta (Tung et al. 2012). Endocytosed Ca2+ plays a role in the fusion of late endosomes and lysosomes (Pryor et  al. 2000). Intracellular membrane Ca2+ channels actually play active roles in membrane trafficking. In this context, intracellularly-localized transient receptor potential (TRP) channels actively participate in regulating membrane traffic, signal transduction, and vesicular ion homeostasis (Dong et al. 2010). Similarly, transient receptor potential mucolipin-1 (TRPML1) is widely expressed in mammalian cell lysosomes or in the endosome membrane (Cheng et  al. 2010). It is the main channel for lysosome Ca2+ release and the key regulator for lysosomal storage and transportation. However, trafficking and maturation of lysosomes can be precisely regulated by dynamic changes in small GTPases and membrane lipids, as well as Ca2+ signaling. In this case, TRPML1 plays a role in lysosomal membrane trafficking similar to voltage-gated Ca2+ channels in neurotransmission (Li et  al. 2013). Overexpression of TRPML also results in an inhibition of autophagy (Kim et  al. 2009). TRPML1 mutations do not only induce the occurrence of neurodegenerative lysosomal storage disorders but also affect the accumulation of autophagy. Therefore, TRPML1 is considered to be the regulator of autophagy (Cheng et  al. 2010; Curcio-Morelli et  al. 2010). In fact, the AMPK/mTOR/S6K pathway is the predominant signaling pathway that regulates autophagy (Li et  al. 2015). On the other hand, attenuation of autophagy correlates with the initiation of autophagic lysosome reformation (ALR). The degradation of macromolecules and release of intracellular substituents following autophagy appears to trigger mTOR reactivation, which inhibits autophagy and stimulates the recycling of proto-lysosomal membrane components (Yu

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et  al. 2010). While autophagy is promoted by AMPK, the mTOR/S6K signaling pathway negatively regulates autophagy. AMPK functions as an energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mTORC1, a central cell-growth regulator. Mechanistically, mTORC1 inhibits Unc-51 like autophagy activating kinase (Ulk1) activation by phosphorylating Ulk1 Ser 757 and disrupts its interaction with AMPK (Kim et al. 2011). Indeed, upon autophagy induction, the ULK1 complex, which is a serine/threonine protein kinase, translocates to autophagy initiation sites and regulates the recruitment of a second kinase complex, the VPS34 complex (Zachari and Ganley 2017). TRPML1 overexpression reduces the expression levels of ALR-related proteins, recovers both memory and recognition impairments and attenuates neuronal apoptosis. In addition, TRPML1 overexpression attenuates Aβ1–42-suppressed cell viability, Aβ1–42-­ decreased lysosomal Ca2+ ion concentration and Aβ1–42-induced ALR-related protein expression levels. Furthermore, while TRPML1 overexpression simultaneously decreases the protein expression level of phospho-AMPK (p-AMPK), it induces the protein expression levels of p-S6K and p-mTOR.  TRPML1 plays an important role in the pathogenesis of AD through the regulation of autophagy via the PPARγ/ AMPK/mTOR signaling pathway (Zhang et  al. 2017). As mentioned above, TRPML1 interacts directly with the autophagic mechanisms. In addition to channel activity required for chaperone-mediated autophagy, TRPML1 may also act as a docking site for intralysosomal heat shock cognate protein 70 (Hsc70) (ly-Hsc70) (Venugopal et al. 2009). Several kinases such as microtubule-affinity regulating kinase 2 (MARK2), protein kinase A, CaMKII, and checkpoint kinase 2 (Chk2) are known to phosphorylate tau on calmodulin Ser262. Among these, MARK2 significantly interacts with tau in AD brain. In this respect, MARK2 has an important role in early phosphorylation of tau (Gu et  al. 2013). In contrast, Lund et al. have claimed that MARK1

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and MARK2 were abundantly expressed in neuronal cytoplasm, but that expression levels did not increase in AD (Lund et  al. 2014). Granulovacuolar degeneration (GVD) is characterized by the presence of vacuolar cytoplasmic lesions in nerve cells. GVD bodies as late-stage autophagic markers, are double membrane vacuoles present in neurons, that suggests the failure to complete the autolysosome formation (Funk et al. 2011). GVD accumulation is extremely specific to AD. The GVD stages are significantly correlated with NFT stages, neuritic plaque pathology, Abeta-protein deposition phases, cerebral amyloid angiopathy stages, and high clinical dementia rating (CDR) scores (Thal et al. 2011). Although the expression of MARK4 is below the detection level in normal brain tissue, phosphorylated form of MARK4 co-localizes with phosphorylated tau (p-tau) Ser262  in GVD bodies that progressively accumulate in AD. This process is thought that a part of the cellular defense mechanism remove activated MARK and p-tau Ser262 from the cytosol (Lund et al. 2014). MARKs regulate tau-­ microtubule binding and play a crucial role in neurons. Because of their role in hyperphosphorylation of tau makes them potential druggable target for AD therapy (Annadurai et  al. 2017). Phospholipid-binding modules play crucial roles in location-dependent regulation of many protein kinases. Thus, protein kinases translocate to membranes via lipid-­ binding domains. Phospholipid-engagement of the kinase associated-1 (KA1) domain, which is included in the C-terminal tail of MARK/partitioning defective1 (PAR1) kinases, plays a role in the activation and binding of MARK/PAR1 kinases at particular membrane locations of neurons in AD (Leonard and Hurley 2011; Moravcevic et al. 2010). Wingless-Int (Wnt) signaling is important in regulation of hippocampal synapses, in addition to impact in learning and memory. Modulation by Wnt signaling is essential for alpha7-nAChR (α7nAChR) expression and function in synapses. Among different Wnt ligands, Wnt-7a stimulates clustering of presynaptic proteins, induces recycling and exocytosis of synaptic vesicles, and

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increases neurotransmitter release (Cerpa et  al. 2008; Farías et  al. 2007; Inestrosa and VarelaNallar 2014). In the brain of AD patients, loss of Wnt activity, decreased β-catenin levels and increased tyrosine-216 phosphorylation of GSK3β are observed. GSK3β is a dynamic switch in protein levels and activity, especially in the orbitofrontal cortex and the medial frontal gyrus (Folke et al. 2019). Therefore, loss of Wnt signaling activity could be a factor in triggering the onset and progression of AD (Tapia-Rojas and Inestrosa 2018). Inhibition of the Wnt/β-catenin pathway promotes amyloidogenic APP processing, increases the Aβ1–42 concentration and the Abeta42/Abeta40 ratio, favoring the formation of Abeta oligomers. In contrast, activation of the Wnt pathway favors non-amyloidogenic APP processing, reduces Aβ1–42 peptide formation and aggregation, with the concomitant increment in Aβ1–40 peptide. Therefore, loss of the Wnt signaling pathway may contribute to the pathogenesis of AD (Tapia-Rojas et al. 2016). In fact, Wnt proteins are a family of cysteine-­ rich glycosylated proteins that function through a canonical pathway targeting β-catenin or through non-canonical β-catenin independent pathways. Protection by Wnt1 during Aβ1–42 exposure contributes to the specific prevention of apoptotic genomic DNA fragmentation, which represents a significant component for neuronal cell loss during AD (Chong et  al. 2005). Furthermore, downregulation of Wnt/β-catenin, through activation of GSK3β by Abeta, and inactivation of PI3K/Akt pathway signaling causes oxidative stress in AD (Vallée et  al. 2017). In AD brain tissue APP and β-catenin shows cellular co-localization, thereby reduction in the levels of nuclear β-catenin detected in AD brain is simultaneous with the decrease in the expression of the inactive phosphorylated GSK3β (Zhang et  al. 2018a). Bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes showed that CaMKIV is regulated by the Wnt signaling pathway. Since Wnt-3a increases the binding of β-catenin to the CaMKIV promoter in hippocampal neurons, its expression plays a role in the neuroprotective

function of the Wnt signaling against Alzheimer’s amyloid peptide (Arrázola et al. 2009). Activation of the Wnt signaling improves spatial memory impairment and restores the expression of CaMKIV in AD.  Wnt1 and its integration with Akt1, GSK3β, and β-catenin increases neuronal cell survival by repressing inflammatory microglial activation during Aβ1–42 exposure (Chong et  al. 2007). The protective capacity of Wnt1 against Aβ1–42 toxicity involves the maintenance of genomic DNA integrity, blockade of cellular membrane phosphatidylserine externalization and the inhibition of inflammatory cell activation during AD (Chong et  al. 2007). As mentioned above, tau phosphorylation is mediated by diverse protein kinases. Among these, GSK3β phosphorylates practically all tau residues described in AD (Hernandez et al. 2013; Kremer et al. 2011). Indeed, GSK3β is a central component of Wnt signaling and its activity is mediated by the function of the Wnt/β-catenin pathway (Metcalfe and Bienz 2011). Inhibition of Wnt signaling favors GSK3β-mediated tau hyper phosphorylation (Hooper et  al. 2008; Scali et al. 2006).

7

Synaptic Activity in Alzheimer’s Disease

As a rule, synapses are formed by connections between two neurons that allow a neuronal cell to pass a signal to another cell. This channel usually gets damaged or lost in most neurodegenerative diseases (Shahani et  al. 2014). Synapse degeneration in AD is characterized by the worsening of cognitive function, synapse loss, and neuronal cell death. Neuroinflammatory process and oxidative stress occurs earlier than the apoptosis, however does not affect memory function (Rai et al. 2014). APP is transported down from both axons and dendrites. Inhibition of clathrinmediated endocytosis immediately lowers the brain interstitial fluid Abeta levels. Approximately 70% of interstitial fluid Abeta arises from endocytosis-associated mechanisms, the vast majority of this pool also dependent on synaptic activity

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(Cirrito et  al. 2008). Pathological accumulation of Abeta and hyperphosphorylation of tau develop concomitantly within synaptic terminals (Takahashi et al. 2010). In contrast, Abeta selectively depresses excitatory synaptic transmission onto neurons that overexpress APP (Kamenetz et al. 2003). APP is transported on vesicles distinct from the β-secretase components and that Abeta cannot be generated when transport is blocked by tau (Goldsbury et al. 2006). Synaptic activity increases extracellular Abeta and decreases intracellular Abeta. Reduction of intraneuronal Abeta protects against Abeta-related synaptic alterations. Synaptic activation promotes anterograde transport of APP from dendrites to synapses (Tampellini et al. 2009). The relationship between the intracellular and extracellular pools of Abeta is complex, however intraneuronal Abeta serves as a source for some of the extracellular amyloid deposits (Oddo et al. 2006). Targeting and functional disruption of particular synapses by Abeta oligomers may provide a molecular basis for the specific loss of memory function in early AD.  The impact on memory ultimately would depend on the number of synapses targeted. Because of these complexities, it is difficult to predict a simple relationship between the oligomer levels, synaptic reserve and day-to-day fluctuations in cognitive functions (Lacor et al. 2004). The coexistence of neuronal mitochondrial pathology and synaptic dysfunction is an early pathological feature of AD.  Cyclophilin D (CypD), an integral part of mitochondrial permeability transition pore (mPTP), is involved in Abeta-provoked mitochondrial dysfunction. ROS induces Abeta-­ mediated inactivation of neuronal protein kinase A (PKA)/CREB signal transduction pathway and causes loss of synapse. CypD-blockade of neurons abolishes synaptic dysfunction by preventing Abeta-disrupted PKA/CREB signaling via increasing PKA activity (Du et  al. 2008, 2014). The activated autophagy provides significant neuroprotection against Aβ1–42-­induced synaptic dysfunction. In this case, autophagy is an integrated part of PI3K/Akt1/mTOR/CREB signaling pathway. Autophagic activation improves the oxidative defense mechanism and

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neurodegenerative damages. Thereby, the integrity of synapse and neurotransmission is maintained in AD (Singh et  al. 2017). Synaptic and extrasynaptic NMDARs have distinct compositions and couple with different signaling pathways: while synaptic NMDARs tend to promote cell survival, extrasynaptic NMDARs promote cell death (Li and Ju 2012). The synaptic or extra-synaptic cellular location of NMDARs controls neuronal viability. Selective extra-synaptic NMDAR activation induces mitochondrial membrane potential breakdown and triggers cell body and dendrite damages, whereas synaptic NMDAR activation generates physiological stimuli through ERK activation. Since extra-synaptic NMDAR activation contributes to excitotoxicity, selective targeting of the extra-synaptic NMDARs seems to be a therapeutic strategy (Léveillé et al. 2008). Intracellular Ca2+ signaling contribute to the major symptoms of AD and may be the predominant cause of the neurodegeneration in AD. The APP intracellular domain alters the expression of key signaling components such as the ryanodine receptors (RyRs). It is proposed that this remodeling of Ca2+ signaling will result in the learning and memory deficits that occur early during the onset of AD (Berridge 2010). Abeta enhances Ca2+ release from the endoplasmic reticulum (ER) through both the inositol 1,4,5-triphosphate receptor (IP3R) and the RyR (Ferreiro et  al. 2004). Alterations of RyR-­dependent Ca2+ signals contribute to the progression of AD pathogenesis through the amplification of Abeta peptide production and memory decline. In these contexts, RyR is implicated in both initiation and progression of AD (Oulès et  al. 2012). On the other hand, NMDARs play roles in Ca2+ signaling as plasma membrane Ca2+ channels and in the formation of the CaMKIIα -NMDAR complex. This complex is a critical modulator in LTP induction in the postsynaptic density, which is an important regulator of synaptic function (Otmakhov et al. 2004). NMDAR is a heterotetramer receptor, which is mostly composed of two NR1 subunits and two NR2 subunits. Synaptic and extrasynaptic NMDARs are gated by different endogenous co-agonists, D-serine and glycine,

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respectively (Papouin et al. 2012). In mature synapses, NR2A-­containing NMDARs are predominant at the synaptic sites, and comprise of approximately 60% of the total synaptic NMDARs. In fact, glutamate receptors in the PSD proteins are assembled into large protein complexes. Moreover, NMDAR subunits are substantially more abundant than AMPA receptors in the PSD protein fraction (Cheng et  al. 2006). Synaptic and extrasynaptic NMDARs have opposite physiological roles in mediating intracellular signaling and death pathways. Abeta oligomers affect glutamatergic synaptic transmission by increasing the availability of extracellular glutamate. Abeta oligomer-dependent disturbances on synaptic glutamatergic transmission mainly affect Ca2+ signaling in the dendritic spine only. In contrast, Abeta oligomer-­induced non-synaptic glutamate release and elevation in extrasynaptic glutamate concentration mainly affect the Ca2+ dynamics of the whole cell. Loss of synaptic membrane NR2B-NMDAR will inhibit LTP induction by disrupting CaMKII-­ NMDAR formation. Therefore, selective inhibition of synaptic NR2B-NMDARs internalization in AD is thought to be a useful therapeutic approach that may prevent the loss of synapses and memory decline (Liang et  al. 2017). However, Birnbaum et  al. proposed that Abeta induces the activation of p38 MAPK and subsequent synaptic loss through Ca2+ flux- and G protein-independent mechanisms (Birnbaum et al. 2015). Chronically elevated concentrations of glutamate and neuronal inflammation have been associated with increased sensitivity and/or activity of the glutamatergic system, resulting in neuronal dysfunction and cell death in AD. Thus, the prolonged Ca2+ overload initially leads to loss of synaptic function, followed by synaptotoxicity and ultimately cell death, which correlates with the loss of memory function and learning ability deficit in AD patients (Danysz and Parsons 2012). NMDA and possibly nicotinic receptors are critically involved in mediating the disruptive effect of Abeta and that targeting muscarinic receptors can indirectly modulate Abeta’s actions (Ondrejcak et al. 2010).

8

 lucose Metabolism, Insulin G Resistance and Alzheimer’s Disease

Currently, AD is defined as “type 3 diabetes” and it is clarified that this neurodegenerative disease has multiple shared pathology with diabetes mellitus (DM) (Bartl et  al. 2013; Kandimalla et  al. 2017; Narasimhan et al. 2014). However, insulin resistant brain state-associated molecular changes are not related to peripheral insulin resistance. Thus, administration of insulin directly to the brain ameliorates selected cognitive parameters targeted in AD.  These findings are the base of “Damage Signals Hypothesis” in AD pathogenesis (Kuljiš and Salković-Petrišić 2011). Extensive disturbances in brain insulin and insulin-like growth factor (IGF) signaling mechanisms represent early and progressive molecular changes and histopathological lesions in AD. Therefore, “type 3 diabetes” reflects the fact that AD represents a form of diabetes that selectively involves the brain. Furthermore, molecular and biochemical features of glucose metabolism overlap with both type 1 and type 2 DM (T2DM) in AD (de la Monte and Wands 2008). High glucose and insulin lead to neuronal insulin resistance. Glucose transport into the neurons is achieved by regulatory induction of surface glucose transporter-3 (GLUT3). While GLUT3-stimulated glucose transport and oxidative stress is increased, mitochondrial metabolic activity is significantly reduced (Engin et al. 2017). In this context, AD is fundamentally a metabolic disease that results in progressive impairment in the brain’s capacity to utilize glucose and respond to insulin and IGF stimulation. Chronic hyperinsulinemia links to cognitive impairment and neurodegeneration with increased APP-Abeta accumulation versus reduced clearance in the CNS (de la Monte 2012a). In insulin-resistant states, the brain is initially exposed to excessive insulin. High insulin levels provoke excessive increases in activities of Akt and atypical protein kinase C (aPKC). This leads to decreases in FoxO proteins and PGC-1α levels and increases in levels of Aβ1–40/42 peptides, p-tau, interneuronal plaques and intraneu-

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ronal tangles. These changes create a reasonable link between insulin-resistance and AD development (Caccamo et al. 2018; Sajan et al. 2016). It was demonstrated that both insulin receptor (IR) and insulin are present in the brain, and insulin is actively transported across the BBB, in addition insulin might also be produced locally in the brain. However, neuronal glucose uptake is not insulin-dependent (Schulingkamp et al. 2000). In the cerebellar cortex of AD cases even without diabetes, markedly reduced response to insulin signaling in the IR/insulin receptor substrate 1 (IRS-1)/PI3K signaling pathway also decreases the response to IGF-1 in the insulin-like growth factor type 1 receptor (IGF-1R)/IRS-2/PI3K signaling pathway. IGF-1 resistance and associated IRS-1 dysfunction triggered by Abeta oligomers is an early and common feature of AD (Talbot et al. 2012). In the temporal cortex of AD patients, IGF-1R levels increase, and the levels of IGF-1binding protein-2 (IGFBP-2) decrease. Despite the overall increase in IGF-1R levels, a significantly lower number of neurons express IGF-1R in AD.  Thereby, aberrantly distributed IGF-1Rs in AD neurons are especially evident in those with NFTs. Moreover, significant decreases in IRS-1 and IRS-2 levels are identified in AD neurons. For these reasons, it is thought that neurons may be resistant to IGF-1R/IR signaling in AD (Moloney et al. 2010). Indeed, IGF-1 is protective against the development of Abeta pathology. Inverse relationship between serum IGF-1 and brain Abeta levels reflects the ability of IGF-1 to induce clearance of brain Abeta, (Carro et  al. 2002). Although insulin triggers ADDL internalization, IGF-1 keeps ADDLs on the cell surface. Nevertheless, both insulin and IGF-1 reduce ADDL binding, protect synapses from ADDL synaptotoxic effects, and prevent the ADDLinduced surface insulin receptor loss. Contrarily, dysfunctions of brain insulin and IGF-1 receptors contribute to Abeta aggregation and subsequent synaptic loss (Zhao et al. 2009). IRs regulate neurotransmitter release and receptor recruitment at synapse. In the NMDAR subunit, NR2 tyrosine phosphorylation site is responsible for potentiation of NMDAR activity by insulin signaling (Abbott et  al. 1999; Christie et  al. 1999).

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NMDARs are simultaneously required for synaptic targeting of Abeta oligomers and insulin signaling. Although oligomer-attacked and non-­attacked neurons exhibit similar surface levels of NMDARs, oligomers cannot bind directly to NMDARs. Insulin down-regulates the oligomer-­binding sites of the neuron even in the absence of a parallel reduction in surface levels of NMDARs (Decker et  al. 2010). Dysfunction of the insulin/PI3K/Akt signaling pathway, which regulates glucose metabolism in the brain, can lead to tau hyperphosphorylation in the brain of AD patients. Furthermore, GSK3β is considered as a common kinase in insulin signaling transduction and tau protein phosphorylation (Zhang et al. 2018b). Following activation of PI3K/Akt signaling pathway, phospho-Akt phosphorylates and inhibits the GSK3β. In fact, the “GSK3 hypothesis of AD” reveals that the overactivity of GSK3β accounts for several features of AD such as memory impairment, Tau phosphorylation, increased amyloid production, microglia-­ mediated inflammation, and neuronal death. In fact, GSK3β mediates the hyperphosphorylation of tau and produces impairments in learning and memory by preventing the induction of LTP through NMDARs. According to this hypothesis, overexpression of GSK3β results in neurodegeneration and AD (Balaraman et al. 2006; Peineau et  al. 2007). As mentioned above, insulin resistance may lead to dephosphorylation and activation of GSK3β (Henriksen and Dokken 2006). Collectively, these abnormalities are associated with reduced levels of IRS mRNA, tau mRNA, IRS-associated PI3K, and phospho-Akt (activated, ps473), and increased GSK3β activity and APPmRNA expression. Therefore, “type 3 diabetes” reflects the pathogenic mechanism of neurodegeneration (Steen et  al. 2005). On the other hand, insulin depletion leads to increased tau phosphorylation in the brain. Insulin deficiency results in PKA activation and tau phosphorylation. Strikingly, both active PKA and induced tau phosphorylation are reversed upon insulin treatment (van der Harg et  al. 2017). Both insulin resistance and oxidative stress may promote the transcriptional activity of FoxO proteins, resulting in hyperglycaemia and a further increased

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production of ROS. The consecutive activation of JNKs and inhibition of Wnt signaling result in the formation of Abeta plaques and tau protein phosphorylation. Insulin resistance/Wnt inhibition/FoxO activation/ROS formation/neuronal loss creates a vicious cycle in AD (Engin et  al. 2017; Manolopoulos et  al. 2010). High glucose can regulate tau hyperphosphorylation and neuronal apoptosis via TLR9-p38MAPK signaling pathway (Sun et  al. 2017). APP expression and neuronal exposure to oligomeric Abeta42 enhance Ras/ERK signaling cascade and GSK3β activation (Kirouac et  al. 2017). In this context GSK3β can be targeted as a therapeutic approach in AD. The concomitant and mutual alteration of energy metabolism-­ mTOR signaling-protein homeostasis represents a self-sustaining harmful event that triggers the degeneration and death of neurons and the development and progression of AD (Di Domenico et  al. 2017). Expression of selected active kinases, including SAPK/JNK and p38 kinase, in brain homogenates increase in all the tauopathies as well as in dystrophic neurites of senile plaques in AD. These findings support the “amyloid cascade hypothesis” (Ferrer 2004). Abeta oligomers inhibit neuronal AMPK activity through a NMDAR-dependent mechanism, and decrease adenosine triphosphate (ATP) and glucose transporter levels. Abeta oligomers exposed neurons present an acute reduction in surface levels of GLUT3 and GLUT4. Accumulation of Abeta oligomers in brain initially inhibits AMPK activity, triggering early brain and cognitive damage. As AD progresses, however, homeostatic mechanisms may lead to persistent and excessive brain AMPK activity that could, in turn, further compromise synapse integrity and function. Thus, the effects of Abeta oligomers on AMPK may contribute to AD-linked brain metabolic and synaptic defects (Seixas da Silva et al. 2017). The adverse effect of insulin resistance on AD pathology occurs through a two-stage process. First is the chronic overactivation of Akt/AMPK(Ser-485) hyperphosphorylation and second is inhibition of AMPK-mediated tau dephosphorylation cycle (Kim et al. 2015). AMPK is a physiological tau

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kinase and can increase the phosphorylation of tau at Ser-262. Although AMPK activation decreases mTOR signaling activity, it has no neuroprotective property and may lead to Abeta generation (Cai et  al. 2012). AMPK inhibitors activate GSK3β. GSK3β inhibitors also attenuate adenylate kinase 1 (AK1)-mediated tau phosphorylation. Considering that GSK3β is over-activated in AD, AK1 expression is markedly increased in the brains of AD patients (Park et  al. 2012). Green coffee bean extract improves cognition, decreases serine phosphorylation of IRS, increases PI3K activity and Akt gene expression, decreases GS3Kβ gene expression and tau hyperphosphorylation (Mohamed et  al. 2020). As mentioned above, insulin resistance is one of the important risk factors for the onset of Alzheimer’s disease. Chronic hyperglycemia accelerates the formation of AGEs favoring a neurodegenerative milieu. Thus, AGEs generated by chronic hyperglycemia are found in postmortem AD brain (Culberson 2017). IR/IRS-1 signaling pathway appears to be the proximal cause of insulin resistance in AD dementia and amnestic cognitive impairment. This is due to serine inhibition of IRS-1, triggered by oligomeric Abeta (Talbot and Wang 2014). Indeed, it is estimated that 46% of AD patients have impaired fasting glucose and data suggests that majority of AD patients have central insulin resistance. Furthermore, brain insulin resistance is an early and common feature of AD, a phenomenon accompanied by IGF-1 resistance and closely associated with IRS-1 dysfunction. Thereby, it is showed that impaired insulin signaling is an important part of AD pathological process (Janson et  al. 2004; Steen et  al. 2005; Talbot et  al. 2012). It is thought that insulin-­ degrading enzyme (IDE) is the main mechanism in clearing Abeta from the brain. Insulin regulates IDE expression and can directly compete with Abeta for binding to IDE. If there is a high level of insulin in the brain, IDE can be diverted to degrade insulin, consequently allowing APP-­ Abeta accumulation due to decrease in Abeta degradation (Schuh et al. 2011). Really, the IDE has the property of catabolizing insulin and Abeta, and play a critical role in Abeta clearance

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in the brain as Abeta scavenger protease (Ling et al. 2003; Qiu et al. 1998). In brief, the accumulation of Abeta exacerbates the problem because Abeta disrupts insulin signaling by competing with insulin or reducing the affinity of insulin binding to its own receptor. Since they are both substrates of the IDE, insulin and Abeta share a common system for degradation and disposal. Thereby, Abeta is a direct competitive inhibitor of insulin binding and action. Consequently, Abeta can directly interfere with IR signaling, by inhibiting the autophosphorylation of IRs. The increased levels of Abeta in AD are directly linked to the brain insulin resistance (Ling et al. 2002; Xie et al. 2002). Moreover, prolonged high cerebral insulin concentrations cause microvascular endothelium proliferation, chronic hypoperfusion, and energy deficit, triggering Abeta oligomerization and tau hyperphosphorylation (Schuh et al. 2011). In T2DM, chronic hyperglycaemia, hyperinsulinaemia, oxidative stress, accumulation of AGEs, increased expression and activation of IDE, increased production of proinflammatory cytokines, and cerebral microvascular disease associated with peripheral insulin resistance could result in cognitive impairment and neurodegeneration (Whitmer 2007). On the other hand, AbetaPP oligomers inhibit neuronal transmission of insulin-stimulated signals by desensitizing and reducing the surface expression of insulin receptors. Furthermore, intracellular AbetaPP-Abeta directly interferes with PI3K activation of Akt, which leads to impaired survival signaling of neuron, increased activation of GSK3β, and hyperphosphorylation of tau (de la Monte 2012b). Simply reducing peripheral insulin resistance is ineffective, as indicated by the failure of many T2D treatments to reduce AD risk or improve cognition in AD dementia (Talbot and Wang 2014).

9

Clinical Perspective

Neuropathological processes related to AD in persons without dementia may represent a preclinical stage of the illness. Indeed, it has been suggested that up to 40% of cognitively normal individuals

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have some neuropathological criteria for AD (Price et al. 2009). Although the original neuropathological guidelines for AD are built on the correlation of amyloid plaques and NFT counts to cognition, many previous studies have established that amyloid plaques are less well correlated to the clinical and anatomical progression of AD than other pathologies, including synapse loss and NFTs (Morris et  al. 2018; Terry et  al. 1991). Evidences suggest the appearance of NFTs precedes Abeta pathology in vast majority of affected brain regions. In this regard, it is argued that the abnormal hyperphosphorylation and intraneuronal aggregation of tau protein are early events in the evolution of the AD-related neurofibrillary pathology (Rüb et  al. 2017). More recently, the use of novel in vivo tau selective PET tracers shed further light on the spatial and temporal relationship of these pathologies. Thus, it is demonstrated that Abeta accumulation is a protracted process that can extend for more than two decades before the onset of clinical AD (Villemagne et al. 2018). Thereby, approximately 30% of apparently healthy older people, and 50–60% of people with mild cognitive impairment, present with cortical 11–labeled Pittsburgh Compound B (11C-PiB) retention in PET scan. In these groups, Abeta burden does correlate with episodic memory and rate of memory decline. These observations suggest that Abeta deposition is not part of normal ageing, but Αbeta deposition occurs well before the onset of symptoms, and represents the asymptomatic individuals and prodromal AD.  Consequently, all these confirm the “amyloid cascade hypothesis” (FoderoTavoletti et al. 2009; Villemagne and Rowe 2013).

10

Conclusion

AD is characterized by a progressive loss of cognition, in addition to the formation of SPs and NFTs. So, the “amyloid cascade hypothesis” still maintains its popularity in the explanation of the pathogenesis of AD.  The intraneuronal accumulation of Abeta oligomers and p-tau cause synaptic dysfunction and cognitive impairment. Abeta oligomers enhance NMDAR hyper-

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activation and oxidative stress in response to extrasynaptic stimulation by increasing the availability of extracellular glutamate ultimately results in AD pathology. In this respect, many protein kinases contribute to synaptic and cognitive impairment as well as memory deficits. Particularly, neuroinflammation plays important role in cognitive impairment and in AD progression. While loss of mTOR signaling impairs LTP and synaptic plasticity in AD, loss of Wnt signaling activity triggers the onset and progression of AD. “Damage signals hypothesis” in AD pathogenesis involves type 3 diabetes with insulin resistance. Brain insulin resistance in AD is accompanied by IGF-1 resistance and closely associated with IRS-1 dysfunction. Since current drugs are not effective enough to prevent AD progression, new treatment methods targeting check-points protein kinases can be developed.

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321 gliosis involves upregulation of KCa3.1 and inhibition of AKT/mTOR signaling. J Neuroinflammation. 2018;15:316. https://doi.org/10.1186/ s12974-018-1351-x. Yue X, Dreyfus C, Kong TA-N, Zhou R. A subset of signal transduction pathways is required for hippocampal growth cone collapse induced by ephrin-A5. Dev Neurobiol. 2008;68:1269–86. https://doi.org/10.1002/ dneu.20657. Zachari M, Ganley IG.  The mammalian ULK1 complex and autophagy initiation. Essays Biochem. 2017;61:585–96. https://doi.org/10.1042/ EBC20170021. Zhang YY, Yang LQ, Guo LM. Effect of phosphatidylserine on memory in patients and rats with Alzheimer’s disease. Genet Mol Res. 2015;14:9325–33. https://doi. org/10.4238/2015.August.10.13. Zhang L, Fang Y, Cheng X, Lian Y, Xu H, Zeng Z, Zhu H.  TRPML1 participates in the progression of Alzheimer’s disease by regulating the PPARγ/AMPK/Mtor signalling pathway. Cell Physiol Biochem. 2017;43:2446–56. https://doi. org/10.1159/000484449. Zhang N, Parr CJC, Birch AM, Goldfinger MH, Sastre M. The amyloid precursor protein binds to β-catenin and modulates its cellular distribution. Neurosci Lett. 2018a;685:190–5. https://doi.org/10.1016/j. neulet.2018.08.044. Zhang Y, Huang N-Q, Yan F, Jin H, Zhou S-Y, Shi J-S, Jin F. Diabetes mellitus and Alzheimer’s disease: GSK-3β as a potential link. Behav Brain Res. 2018b;339:57– 65. https://doi.org/10.1016/j.bbr.2017.11.015. Zhao L, Ma Q-L, Calon F, Harris-White ME, Yang F, Lim GP, Morihara T, Ubeda OJ, Ambegaokar S, Hansen JE, Weisbart RH, Teter B, Frautschy SA, Cole GM. Role of p21-activated kinase pathway defects in the cognitive deficits of Alzheimer disease. Nat Neurosci. 2006;9:234–42. https://doi.org/10.1038/nn1630. Zhao W-Q, Lacor PN, Chen H, Lambert MP, Quon MJ, Krafft GA, Klein WL.  Insulin receptor dysfunction impairs cellular clearance of neurotoxic oligomeric A{beta}. J Biol Chem. 2009;284:18742–53. https:// doi.org/10.1074/jbc.M109.011015. Zhong W, Yamagata HD, Taguchi K, Akatsu H, Kamino K, Yamamoto T, Kosaka K, Takeda M, Kondo I, Miki T.  Lymphocyte-specific protein tyrosine kinase is a novel risk gene for Alzheimer disease. J Neurol Sci. 2005;238:53–7. https://doi.org/10.1016/j. jns.2005.06.017. Zhu Z, Yan J, Jiang W, Yao X, Chen J, Chen L, Li C, Hu L, Jiang H, Shen X.  Arctigenin effectively ameliorates memory impairment in Alzheimer’s disease model mice targeting both β-amyloid production and clearance. J Neurosci. 2013;33:13138–49. https://doi. org/10.1523/JNEUROSCI.4790-12.2013.

Bacterial Protein Kinases

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Evren Doruk Engin

Abstract

Keywords

Bacteria are able to inhabit and survive vastly diverse environments. This enormous adaptive capacity depend on their ability to perceive cues from the micro-environment and process this information accordingly to mount appropriate metabolic responses and ultimately sustain homeostasis. From systems perspective, microbial cells conceal significant degree of organismal complexity, which may only be managed by continuous bulk cellular information flow and processing, inside the cell, between other cells and the environment. In this respect, reversible covalent modification of proteins is one of the universal mode of information flow mechanism used to regulate metabolism in all organisms. More than 30 types of post translational modifications have been identified, where phosphorylation constitutes nearly half of them. Bacterial cells possess several modes of phosphoprotein mediated information flow mechanisms. Histidine kinases and two component systems, bacterial tyrosine kinases, Hanks type serine/threonine kinases, atypical serine kinases and arginine kinases have been identified in many species.

Histidine kinases · Two component systems · Serine threonine kinases · Bacterial tyrosine kinases · Phosphotransferase system · Information flow · Signal processing · Signal integration · Operational amplifier · Response regulator

E. D. Engin (*) Ankara University, Biotechnology Institute, Gümüşdere Campus, Keçiören, Ankara, Turkey

1

Introduction

In his influential work “What’s Life?”, Erwin Schrödinger have coined out his “order – from – order” principle (Egel 2012). In line with this, living cells entail complex system behavior, in a way that, they generate massive proteomic diversity, far exceeding the coding capacity of the corresponding genome  – in order to break the genomic imprisonment. This proteomic expansion may easily reach three orders of magnitude of complexity that can be presumed from underlying genomic repertoire. Vast majority of this proteome diversification may be attributed to three major mechanisms (Prabakaran et al. 2012): • diversification at transcriptional level, alternative splicing • post-translational cleavage of peptide chains • post-translational covalent modifications of proteins

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_12

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Fig. 12.1  Post translational modifications of proteins (Walsh et al. 2005)

Post-translational modification of proteins is an array of covalent modification of amino acids, which allow the adaptive changes in the functional properties of the proteins. Phosphorylation is probably far most well studied one (Cain et al. 2014). More than 30 types of post translational modifications (PTMs) have been identified. As of march 2020, more than 1,250,000 experimental and putative PTMs have been published in nearly 90,000 scientific papers (http://dbptm.mbc.nctu. edu.tw/statistics.php#resource) (Huang et  al. 2019). In most cases, this covalent modification occurs at an amino acid residue with nucleophilic side chain. These nucleophilic groups are attacked with electrophilic molecule fragments. Phosphorylation is probably the most well studied form of PTM and constitute approximately half of these modifications (Jørgensen and Linding 2008). Reversible phosphorylation of protein substrates is a dynamically regulated process and may occur on certain amino acid residues. Directional interaction networks that mainly

depend on protein phosphorylation, provide flow of information within the cell (Prabakaran et al. 2012). Figure 12.1 summarizes the most prevalent type of covalent modification seen in proteomes which belong to all three kingdoms of life. The hallmark of these modifications is their reversibility. The components of this signal processing and propagation pipeline can be considered as units which take signals from its inverting and non-inverting inputs and display a differential output signal. In the case of phosphoryl modification of proteins, protein kinase may be considered as the non-inverting input, whereas, phosphatase in the reciprocating counterpart (Mijakovic et  al. 2016; Mijakovic and Macek 2012). The resultant signal is displayed as another phosphorylation event, binding and forming complexes with other biomolecules, such as proteins and DNA (Fig. 12.2a). Merely for modeling purposes, this module formed by kinase, phosphatase, the phosphryl acceptor protein and other interactants can be considered as homologous to operational amplifiers. In these terms, vast experience exists with operational

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Fig. 12.2 The counterbalancing actions of protein kinases and phosphatases in signalling networks. (a) Protein kinase and phosphatase act on certain residues of protein substrate S. The phosphorylated substrate further interacts with another or series of other proteins (response

regulators) to yield an output. (b) This post translational signal relay module modelled as an operational amplifier. (c) Input  – output signal characteristics depend on the internal of the particular module (“Operational amplifier” 2020; Prabakaran et al. 2012)

amplifiers as the building blocks of analogous computers, which are capable of performing linear, non-linear and frequency dependent mathematical operations (Fig.  12.2b). Just like their electronic counterparts, the kinase/phosphorylase modules have their distinct response characteristics which may inherently depend on the specific features of its sub-components. Additionally, the response characteristics may also be modulated by certain additions to the circuit. The output response may be gradual, which conform Michaelis – Menten curve. Alternatively, in the presence of multiple cooperative phosphorylation sites, the curve may present “steeper” logistic curve properties, or a supersensitive on/ off type circuitry (Fig.  12.2c) (Altszyler et  al. 2017; “Operational amplifier” 2020). In contrast, uncontrolled oxidation and nitrosylation of proteins are in irreversible in nature. These class of modifications are frequently associated with protein damage, misfolding and even-

tually aggregation. Notably, after cells recover from the proteotoxic assault and start proliferating, protein aggregates become inherited to over generations. These aggregates act as an inheritable epigenetic memory element and render the cells impervious to impending proteotoxic effects (Govers et al. 2018). Historically, the importance of protein phosphorylation has been realized as a major player in the regulation of the cellular metabolism. In other words, most of the information flow that handles the controls of the regulation of cellular machinery involves protein phosphorylation. Therefore, eukaryotic protein kinases constitute a significant fraction of the proteome. Accordingly, most prevalent phosphryl-modification occurs in threonine, serine and tyrosine residues (Depardieu et al. 2016). Tyrosine phosphorylation essentially induce electrostatic repulsion and allosteric transitions on the acceptor site. Furthermore, it provides a

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docking site for well known phospho-tyrosine binding domains (SH2, PTB) (Pereira et  al. 2011). Receptor tyrosine kinases (RTKs) are type 1 membrane proteins with the following structural features (Hanks et al. 1988; Hunter 2014): • extracellular ligand-binding domains • transmembrane domain • intracellular tyrosine kinase catalytic domain and unstructured carboxy-terminal tail with autophosphorylation sites. Upon ligand binding, receptor tyrosine kinase activity increases, which in turn triggers a sequence of events (Hunter 2014): • • • •

conformation of existing RTK dimer is altered or, dimer formation is induced the catalytic domains get juxtapositioned activation in trans is ensued

Once kinase activity commences, autophosphorylation progressively takes place in additional sites. This is followed by the recruitment of phospho-tyrosine binding proteins with SH2 and PTB domains. Alternatively, direct phosphorylation of substrates and downstream propagation of signal takes place (Hunter 2014). Phospho-tyrosine turns over so rapidly that, half life is measured at a magnitude of a few seconds. The bonding energy of phospho-tyrosine is about 8–10 kcal, which is comparable to phospho-­ serine and phospho-threonine. Phosphorylation of serine and threonine occurs at β-OH groups. Distinct from the former two, tyrosine is phosphorylated at O4 position of the phenolic ring, which protrudes further away from the peptide backbone. Accordingly, this allows phospho-­ tyrosine binding domains to have deeper binding pockets, a feature which significantly augments the binding energy via π-bonding and hydrophobic interactions (Kaneko et al. 2012). Cellular processes are under tight control of regulatory networks, in order to fine tune the metabolism to maintain the homeostatic balance, in response to continually changing environment.

Table 12.1  Chemical properties of reversible phosphoryl-­ modifications that occur in signalling networks (Sajid et al. 2015) Group Alcohol Phenolic Basic

Acidic

Other

Amino acid Serine Threonine Tyrosine Histidine Arginine Lysine Aspartic acid Glutamic acid Cysteine

Phospho-modification type Phosphate ester

Phosphoamidate

Phosphate carboxylate acid anhydride

Phosphate thioester

Unarguably, transcription layer has a deep impact on the metabolic axis of the cell, albeit, with much larger latency, compared to allosteric control and post-translational modification of proteins. The latter two provide much confined but faster responses (Prabakaran et al. 2012). The phosphoryl type of post-translational modifications occur at the side chains of serine, threonine, tyrosine, histidine, arginine, aspartic acid, glutamic acid and cyteine residues of proteins (Table 12.1) (Sajid et al. 2015). Accordingly, the distinct signaling systems which depend on phosphryl-post translational modifications in kingdom bacteria are as follows (Cain et al. 2014): • Histidine kinases and two component systems (TCS) • Bacterial tyrosine kinases (BY-kinases) • Hanks type serine/threonine kinases • Atypical serine kinases • Arginine kinases

2

 istidine Kinases and Two H Component Systems

Histidine kinases have been identified in a wide range of organisms in all three kingdoms, namely, bacteria, archaea, protozoa, fungi, plants. Additionally, bacteriophages and viruses also encode histidine kinase systems. Albeit, mammalian genomes have been found to be

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devoid of typical histidine kinase TCSs (Ferris et al. 2012). BRENDA enzyme information system entry EC 2.7.13.- includes three sub entries EC 2.7.13.1, 2 and 3, which are assigned for protein-­ histidine pros-kinase, protein-histidine tele-­ kinase and protein-histidine kinase respectively (https://www.brenda-enzymes.org/ecexplorer. php?browser=1&f[nodes]=132,153&f[action]=o pen&f[change]=304). Most of the protein histidine kinases are assigned to EC 2.7.13.3, where the exact phosphorylation mechanism is can not be attributed to tele- or pros- phosphorylation: The equation for the reaction is essentially denoted as reversible in nature. The histidine kinase component also acts as the cognate phosphatase. However, clearly this is not the case for the response regulator components, where a distinct set of response regulator phosphatases exist. Its worth noting that, far more kinases have been identified than phosphatases. Therefore, in many cases the components of the suggested op-amp

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Histidine kinases constitute the most well identified signalling mechanisms. They mostly appear as two component systems. In most cases, histidine kinase component is one pass or multi pass transmembrane proteins, which have cytoplasmic tail with ATPase activity. Upon receipt of stimulus via sensor domain, cytoplasmic domain hydrolyses ATP to phosphorylate the histidine residue which is embedded at the His-Box motif. Phosphate group is ultimately transferred to aspartate of the cognate response regulator protein. The response regulator, in turn, binds to and activates pertinent DNA-binding protein. Therefore, stimulus induced histidine autophosphorylation starts the phosphorelay signalling cascade, which finally ends up in transcription of relevant response genes (Behr et al. 2014). Histidine kinase two component systems can be classified according to topological domains of the sensor kinases. Two pass variants have extracellular/periplasmic input domain between two transmembrane domains and intracellular histidine kinase domain (Fig. 12.3). Upon receipt of

ATP + protein L-histidine  ADP + protein N-phospho-L-histidine model may be obscure, incomplete or ambiguous (Kannan et al. 2007). Structurally, the sensor kinases are membrane anchored proteins. Both prokaryotic and eukaryotic versions of these two component ­phosphorelay systems are made up of canonical modules. Just like multifunctional receptor tyrosine kinase molecules, most of the histidine kinases are membrane anchored proteins with the following domains/subdomains (Ferris et  al. 2012; Grebe and Stock 1999): • A variable extracellular N-terminal sensor domain, which collects environmental cues, such as pH, ion concentration and pheromone molecules. • A transmembrane portion. • Central (transmitter) region consists of both histidine kinase A and phoshoacceptor histidine H-box.

stimulus, histidine kinase domain autophosphorylates at histidine residues. Later on, the phosphoryl group is transferred on aspartate of the response regulator. This modification induces a conformation change to allow response regulator to bind with DNA-binding proteins. Subsequently, this complex binds to appropriate promoters to start transcription (Fig. 12.3). A few examples of these two component systems are as follows: • Virulence sensor histidine kinase PhoQ (UniProt:P0DM80) of Salmonella typhimurium is a component of PhoP/PhoQ system, which regulate expression of virulence genes, adaptation to low pH and low magnesium ion concentrations. Immediate intracytoplasmic part of this histidine kinase includes HAMP domain (Histidine kinase, Adenyl cyclase, Methyl-accepting proteins and Phosphatases  – InterPro:IPR003660) which transmits conformational changes

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Fig. 12.3  Histidine kinase two component systems

from the periplasmic ligand binding domain to cytoplasmic kinase domain and regulates the phosphorylation or methylation of homodimeric receptors (Bearson et  al. 1998). • Sensor histidine kinase DpiB (UniProt:P77510) of Escherichia coli is involved in citrate fermentation. In the presence citrate, sensor kinase DpiB phosphorylates response regulator DpiA.  PAS (Per- period circadian; Arnt- Ah receptor nuclear translocator protein; Sim- single minded protein  – InterPro:IPR000014)

domain is involved in signal transduction into the cytoplasmic part (Yamamoto et al. 2009). • Sensor histidine kinase EnvZ (UniProt:P0AEJ4) of Escherichia coli is involved in osmoregulation (Mizuno et  al. 1982). • Sensor histidine kinase HprS of Escherichia coli belongs to HprR/HprS system and is involved in hydrogen peroxide and redox state sensing (Mijakovic et al. 2002). • Sensor histidine kinase MtrB (UniProt:P9WGK9) of Mycobacterium tuber-

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•

•

•

•

culosis regulates cell division, survival inside the macrophage (Plocinska et al. 2012). Sensor-type histidine kinase PrrB (UniProt:P9WGK7) of Mycobacterium tuberculosis supports early intracellular multiplication of the bacterium. Includes HAMP domain (Haydel et al. 2012). Bacillus subtilis sensor histidine kinase WalK (UniProt:Q45614) is involved in osmoregulation and cell wall metabolism. Includes HAMP, PAS and PAC domains (Bisicchia et al. 2007). Brucella abortus sensor kinase CckA (UniProt:P0DOA0) allows intracellular survival and growth of the bacterium (Willett et al. 2015). Bacillus subtilis sporulation kinase D, KinD (UniProt:O31671) controls sporulation and biofilm formation (Castilla-Llorente et  al. 2008).

Multi-pass histidine kinases have five, six or seven membrane spanning domains with short extracellular/periplasmic sensor loops. The organization of intracellular kinase domain resembles the two-pass variant (Fig. 12.3). • Staphylococcus aureus sensor histidine kinase/phosphatase LytS (UniProt:Q53705), regulates genes involved in autolysis, programmed cell death, biofilm formation and cell wall metabolism. LytS includes GAF (InterPro:IPR003018) domain for signal transduction to the cytoplasmic domain (Lehman et al. 2015). • Escherichia coli sensor histidine kinase YpdA (UniProt:P0AA93) is the sensor kinase component of YpdA/YpdB which is involved in a nutrient sensing network. Consecutive phosphorylation of YpdA and YpdB takes place in response to high extracellular pyruvate concentrations. YpdA contains GAF transmitter domain (Behr et al. 2014). • Sensor histidine kinase DesK (UniProt:O34757) of Bacillus subtilis contains five transmembrane segments instead of six. This two component sensor system is involved in the regulation of membrane

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­ uidity, via control of the expression of Delta5 fl acyl-lipid desaturase (Hunger et al. 2004). • Vibrio campbellii autoinducer 1 sensor kinase/ phosphatase LuxN (UniProt:A7MRY4) is anchored to the cytoplasmic membrane by seven membrane spanning domains. LuxN acts as Autoinducer 1 receptor, where it autophosphorylates in the absence of this pheromone. As the cell density of the culture increases, AI-1 concentrations also build up, which in turn, enhances dephosphatase activity of LuxN (Freeman et al. 2000). • Sensor histidine kinase LnrJ (UniProt:P94438) of Bacillus subtilis, is another histidine kinase with five transmembrane domains. It senses membrane perturbations and provides resistance against linearmycins secreted by streptomycetes (Stubbendieck and Straight 2017). Single-pass histidine kinases may occur in two flavors, either with separate phosphoryl acceptor histidine and kinase domains, or acceptor and kinase groups on same peptide chain. • Mycobacterium tuberculosis sensor histidine kinase component HK2 (UniProt:O07777) is the member of the HK1/HK2/TcrA system. Acceptor domain HK1 requires the HK2 domain. The complementary activity of both are necessary to relay the signal to the response regulator TcrA (Shrivastava et al. 2007). Intracellular histidine kinase two component systems does not have extracellular/periplasmic sensory domain. Instead, they monitor intracellular signals. • Escherichia coli sensory histidine kinase/ phosphatase NtrB (UniProt:P0AFB5) provides metabolic control under nitrogen deprivation (Jiang and Ninfa 1999). • Oxygen sensor histidine kinase NreB (UniProt:Q7WZY5) of Staphylococcus carnosus functions as an oxygen sensor via its iron sulfur ([4Fe-4S]) cluster. NreB/NreC is involved in the expression of enzymes responsible for nitrate/nitrite reduction in the absence of oxygen (Kamps et al. 2004).

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3

Bacterial Tyrosine Kinases

BY-kinases comprise a distinct family of signal relay proteins, which display either very limited or no homology with eukaryotic receptor tyrosine kinases. Instead of canonical Hanks-type motifs, most bacterial tyrosine kinases include Walker A and B ATP/GTP-binding motifs. There is whole bunch of information that, Hanks-type serine/threonine kinases are abundant in microbial genomes. Most probably, last universal common ancestral Hanks-kinases were able to process serine and threonine, just before the bacterial and eukaryotic branches had separated. Afterwards, appearently bacteria have tracked a distinct path to evolve phosphotyrosine mediated information flow to circumvent metabolic regulation (Grangeasse et  al. 2012). BY-kinase of firmicutes and proteobacteria have very similar structures, yet with a few distinct features. Both proteins are anchored to the plasma membrane via two transmembrane segments. Firmicute sensory hairpin domain protrudes directly outside of the cell as a compact loop structure. Whereas, proteobacterial sensory hairpin loop is more bulky and located in the periplasmic space (Fig. 12.4). Intracellular domain is made up of a phosphate binding P-loop (Walker A and A′ motifs), Walker B motifs with nucleoside triphosphate hydrolase activity (InterPro:IPR027417). The domain gets autophosphorylated at the short tyrosine rich segment which comprises three to seven tyrosine residues. Firmicute intracellular domain is most commonly expressed as a s­ eparate protein from the multipass transmembrane sensory domain. Whereas, proteobacterial BY-kinase is produced as a single peptide chain (Grangeasse et al. 2012). BY-kinases were discovered as part of extracellular polysaccharide biosynthetic system. A non-exhaustive list includes Wzc (Escherichia coli K12, UniProt:P76387; Salmonella typhi, UniProt:Q8Z5G6), Etk (Escherichia coli K12, UniProt:P38134), Ptk (Acinetobacter johnsonii,

UniProt:O52788), EpsB (Pseudomonas solanacearum, UniProt:Q45409), AmsA (Erwinia amylovora, UniProt:Q46631) (“BY-Kinases: ec:2.7.10.- taxonomy:‘Bacteria [2]’” is queried at https://www.uniprot.org/). Dephosphorylated form of tyrosine-protein kinase Wzc (UniProt:P76387) of Escherichia coli is involved in colanic acid (CHEBI:60963) export. This polyanionic heteropolysaccharide, also called as M-antigen, is freely secreted to the medium to form the loose mesh like structure of capsular material, in biofilm formation (Grangeasse et al. 2002; Morona et al. 2000). Mass spectrometric analysis of immunoaffinity enriched Escherichia coli lysates yielded 512 phospho tyrosine sites on 342 proteins. Hence, tyrosine phosphorylation plays key role in the regulation of the metabolism (Lai et  al. 2016). BY-kinase family may include a number of atypical members, based on the presence of Walker motifs. • Escherichia coli septum site  – determining proteins MinD/Mrp displays ATPase activity. However, modulators and signal molecules are yet to be identified for kinase mode of action (Li and Young 2012). • Large ribosomal subunit assembly protein BipA of Escherichia coli demonstrate tyrosine autophosphorylation (Choi and Hwang 2018). • In contrast to conventional BY-kinases, Myxococcus xanthus social motility protein MasK and Pseudomonas aeruginosa lipopolysaccharide synthesis protein WaaP share conserved tyrosine kinase functional motifs with eukaryotic protein kinases (Thomasson et al. 2002; Zhao and Lam 2002). • Caulobacter crescentus cell division and growth protein DivL (UniProt:Q9RQQ9) of is classified as histidine kinase (EC:2.7.13.3) of a classical two component system. Yet, upon activation, Y550 is autophosphorylated instead of histidine (Wu et al. 1999).

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Fig. 12.4  Bacterial tyrosine kinases. Firmicutes and proteobacteria has slightly different versions

4

PEP Group Translocation – Phosphotransferase System – PTS

UniProt:P08838, PtsI of Bacillus subtilis) activity which transfers the phosphate from PEP to paracentrally located histidine active site to form tele-phosphohistidine intermediate. Later on, the Bacterial phosphoenolpyruvate-dependent phos- phosphate is relayed to the phosphocarrier prophotransferase systems (PTSs) are wide spread, tein HPr (Fig. 12.5) (Mijakovic et al. 2002). except a few diverse species. Genes that encode Phosphocarrier protein HPr (UniProt:P08877, PTS constitute approximately 3% of host PtsH of Bacillus subtilis) is phosphorylated at genome. PTS primarily involves the allosteric His15 residue (InterPro:IPR001020; regulation of enzymes for carbohydrate utiliza- Phosphotransferase system, HPr histidine tion. Enzyme I (EI) has phosphoenolpyruvate-­ phosphorylation site) This N-terminal histidine protein phosphotransferase (EC:2.7.3.9, acts as phosphryl acceptor from EI. Afterwards,

332

E. D. Engin

Fig. 12.5  Phosphotransferase system

the phosphate group of phospho-HPr is transmitted to EIIA/B/C sugar specific permease complex (Herzberg et al. 1992). A conserved serine residue (Ser46) in the central part of the HPr peptide chain is present both in gram negative and positive versions. Yet, this serine amino acid is marked as modifiable (InterPro:IPR002114 Phosphotransferase system, HPr serine phosphorylation site) residue only in the gram positive HPr UniProt entries. According to String-DB (https://string-db.org) homologues for HPr kinase/phosphorylase (HPrK/P) is highly prevalent in firmicutes, where

they have been suggested to play role in regulation of carbohydrate transport inside the cell (Deutscher et  al. 1994). In proteobacteria phylum, HPrK/P homologues are confined to betaand to a lesser extend alphaproteobacteria. Appearently, phospho-PTS proteins have much subtle interactions with transcription factors and other target proteins that regulate nitrogen and phosphorus metabolism, intracellular potassium homeostasis, glycogen and β-hydroxybutyrate accumulation, biofilm formation, virulence and directed transposon insertion mutagenesis (Barabote and Saier 2005).

12  Bacterial Protein Kinases

Enzyme IIBCA PTS transport complex is histidine phosphorylated from C-terminal EIIA domain. Upon transfer of the phosphate to EIIB N-terminal cysteine residue, carbohydrate molecule passes through EIIC transmembrane channel and acquires the phosphoryl group. Bacillus subtilis ManP (EC:2.7.1.191, UniProt:O31645) is the permease complex for mannose. Many other transporter complexes for various carbohydrate molecules have been identified and annotated in BRENDA Enzyme Information System. A comprehensive list of the prototypical versions of these proteins can be located with accessions through EC:2.7.1.191 (mannose) to EC:2.7.1.211 (sucrose) in BRENDA database (https://www.brenda-enzymes.org/ ecexplorer.php?browser=1&browser=1&f[node s]=132,153&f[action]=open&f[change]=154 &ec_id=7209#7209). In starvation conditions where no transportable carbohydrates exist, (i.e. low glucose levels), phospho-EIIA accumlate, which in turn triggers membrane bound adenylate cyclase activation to increase cAMP levels. Consequently, catabolite activator protein (CRP) is allosterically activated to bind the consensus sequence (5′-AAATGTGATCTAGATCACATTT-3′) that is present in the promoters of numerous operons in the bacterial genome. The net effect of CRP on the transcription of individual operons mainly depend on the relative binding position of this global transcriptional regulator to the RNA polymerase. Activation, repression, coactivation and corepression are the possible outcomes (Youn et al. 2006). In high glucose conditions, the phosphate relayed via phosphotransferase system is consumed by transported glucose molecules, leaving EIIA in unphosphorylated state. Overall, this mechanism provides the ability to rapid adaptation to feeding on various nutrients. The uptake of glucose and other readily metabolizable sugars actuate carbon catabolite repression signalling to prevent the overhead of “investments” required for the catabolism of more complex alternative carbon sources.

333

5

Serine/Threonine Kinase

Hanks type bacterial serine/threonine kinases (STKs) and their cognate phosphorylases (STPs) represent a broad group of signal transduction molecules that exert control on numerous aspects of the cellular metabolism and functions. Biosynthesis of the cell wall, cell division, regulation of the central metabolism, stress response, biofilm formation, virulence, antibiotic resistance, translation, transcription, DNA replication are shown to be under control of STKs in numerous organisms (Stancik et al. 2018). The use of mass spectrometric methods to explore bacterial phosphoproteome has provided evidences of the significant contribution of STKs/ STPs in regulation tasks. Its not uncommon to detect more than 100 serine or threonine phosphorylated proteins in proteomes of many bacteria from both gram negative and positive lineage (Mijakovic and Macek 2012). The literature is significantly biased over STKs. The number of identified STKs are approximately three times more than STPs. A UniProt query “EC:2.7.11.1 taxonomy:‘Bacteria [2]’” generates 218 Swiss-Prot and over 32,000 TrEMBL entries, indicating the popularity of these proteins. Among numerous examples, Mycobacterium tuberculosis serine/threonine kinase PknA is involved in the phosphorylation of an array of proteins including FtsZ, Wag31, GlmU, FhaB, PstP, EmbR and Rv1422, which are responsible for cell growth, shape and division (Fig.  12.6). PknA also activates proteosomal complex via threonine phosphorylation, in order to survive oxidative stress caused by host immune cells. Another serine/threonine kinase is PknB, which phosphorylates PbpA. PstP is the only phopsphatase that has been shown to counterbalance the signalling network (Nagarajan et al. 2015). Serine/threonine-protein kinase toxin HipA and antitoxin HipB are the components of a type II toxin – antitoxin module. Under normal conditions, HipA is neutralized by HipB. Upon serine phosphorylation of Glu-tRNA-ligase by

E. D. Engin

334

Fig. 12.6  Mycobacterium tuberculosis serine threonine kinase signalling network

HipA, charging of tRNA(Glu) is disturbed. Amino acid starvation and RelA/SpoT mediated stringent response ensues. Increased levels of (p) ppGpp leads to impaired transcription, translation and biosynthesis of cell wall. Consequently, as metabolism and growth ceases, cells become

refractory against the toxic effects of antimicrobials. This persistence phenotype is transient, no antibiotic resistance mutations can be detected in those cells. Certain mutations in HipA, which presumably weaken the interaction with HipB, cause Hip (high persistence) phenotype. Hip

12  Bacterial Protein Kinases

335

Fig. 12.7  The regulation of glyoxylate shunt

cells present with 1000–10,000 times higher persistence rates upon antimicrobial exposure (Schumacher et al. 2015). Citric acid (TCA) cycle acts as a central metabolic hub, which links carbohydrate, amino acid and fatty acid catabolism, as well as, production of intermediary precursors for biosynthetic activities. Cataplerotic and anaplerotic fluxes of this pathway are primarily determined by the growth state of the cell. This dual role requires tight con-

trol over the concentrations of TCA intermediates, in order to retain the homeostasis of the cell. Increased demand on biosynthetic precursors may disturb this balance, where, anaplerosis is required to replenish TCA cycle intermediates. Isocitrate dehydrogenase (Icd, EC:1.1.1.42), along with products of aceABK operon play a central role in this anaplerotic reactions. Together, these enzymes enable glyoxylate pathway to bypass TCA cycle. Elevated cAMP levels con-

E. D. Engin

336

verts Icd phosphatase AceK into Icd kinase, which in turn serine phosphorylate Icd. Phospho-­ Icd can no longer catalyse the conversion of cis-­ aconitate to 2-oxoglutarate. Instead, cis-aconitate flux is diverted to glyoxylate and malate by the virtue of isocitrate lyase (AceA) and malate synthase (AceB) enzymes (Fig.  12.7) (Crousilles et al. 2018; Huergo and Dixon 2015). The expression of ace operon is under negative control of DNA-binding transcriptional dual regulator Cra and integration host factor (IHF) α/β. The expression levels are augmented by phosphorylated DNA-binding dual regulator ArcA; CRP-cAMP DNA-binding dual regulator and IclR-pyruvate DNA-binding transcriptional repressor (Fig. 12.7) (Crousilles et al. 2018).

6

Conclusion

Post-translational modifications of peptides in bacteria have long been neglected. However, especially protein phosphorylation occurs as a principle mechanism for the metabolic regulation in virtually all species. Protein kinases provide a control layer which is responsible for mounting immediate responses against perturbations which disturb homeostasis. Here I suggest that protein kinase/phosphatase, phospho-modifiable peptide substrate and possible response regulators all together form control modules which can be modelled much like operational amplifiers. Numerous operational amplifier modules, each with specific signal transmission and integration characteristics may form complex circuit models, which may add some perspective to augment our understanding of the decision making mechanisms and the stochastic behaviour of the individual cells.

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Indoleamine 2,3-Dioxygenase Activity-Induced Acceleration of Tumor Growth, and Protein Kinases-Related Novel Therapeutics Regimens

13

Ayse Basak Engin and Atilla Engin

Abstract

Indoleamine 2,3-dioxygenase (IDO) is overexpressed in response to interferon-gamma (IFN-γ). IDO-mediated degradation of tryptophan (Trp) along the kynurenine (Kyn) pathway by immune cells is associated with the anti-microbial, and anti-tumor defense mechanisms. In contrast, IDO is constitutively expressed by various tumors and creates an immunosuppressive microenvironment around the tumor tissue both by depletion of the essential amino acid Trp and by formation of Kyn, which is immunosuppressive metabolite of Trp. IDO may activate its own expression in human cancer cells via an autocrine aryl hydrocarbon receptor (AhR)– interleukin 6 (IL-6)-signal transducer and activator of transcription 3 (STAT3) signaling loop. Although IDO is not a unique marker, in many clinical trials serum IDO activity is suggested to be an A. B. Engin (*) Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey A. Engin Department of General Surgery, Faculty of Medicine, Gazi University, Ankara, Turkey

important parameter in the pathogenesis of cancer development and growth. Measuring IDO activity in serum seems to be an indicator of cancer growth rate, however, it is controversial whether this approach can be used as a reliable guide in cancer patients treated with IDO inhibitors. Thus, IDO immunostaining is strongly recommended for the identification of higher IDO producing tumors, and IDO inhibitors should be included in post-operative complementary therapy in IDO positive cancer cases only. Novel therapies that target the IDO pathway cover checkpoint protein kinases related combination regimens. Currently, multimodal therapies combining IDO inhibitors and checkpoint kinase blockers in addition to T regulatory (Treg) cell-modifying treatments seem promising. Keywords

Indoleamine 2,3-dioxygenase 1 (IDO1) · Tryptophan (Trp) · Kynurenine (Kyn) · Nitric oxide (NO) · Aryl hydrocarbon receptor (AhR) · Tryptophan 2,3-dioxygenase (TDO) · IDO inhibitors · 1-methyl-D-tryptophan (1-MT) · Indoleamine 2,3-dioxygenase 2 (IDO2) · Interferon-gamma (IFN-γ)

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_13

339

A. B. Engin and A. Engin

340

1

Introduction

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first and rate-limiting step of kynurenine (Kyn) pathway along the major route of tryptophan (Trp) catabolism. Degradation of Trp along the Kyn pathway via IDO induction is related to a variety of physiological and pathophysiological processes, including anti-microbial and antitumor defense (Thomas and Stocker 1999). In this context, IDO1 activity is markedly increased in sepsis, constituting an independent predictor of severity and fatality of diseases (Huttunen et  al. 2010). Thus, increased IDO1 activity and Kyn shows a strong positive correlation with markers of infection and inflammation, as well as in sepsis and community-acquired pneumonia severity score. In a multivariate regression analysis adjusted for age and comorbidities, higher IDO1 activity and lower Trp levels are strongly associated with short-term adverse outcome of infections (Meier et  al. 2017; Schefold et  al. 2016). In addition, IDO1 activity and serum levels of Trp catabolites of the Kyn pathway increase with chronic kidney disease (CKD) severity. In CKD, induction of IDO1 may primarily be a consequence of chronic inflammation (Schefold et al. 2009). The same situation is also observed in hepatitis B virus infections. Suppressor capacity of IDO1 on hepatitis B virus is abrogated in IDO1-knockout cells and recovered by the reinduction of IDO1 in these cells. Actually, IDO1 is an anti-hepatitis B virus effector and an indicator of subsequent immune response, which is operative during the early phase of infection (Yoshio et  al. 2016). On the other hand, IDO1 activity also shows significant alterations in various metabolic disorders. In accordance with this, secretion of interferon-­gamma (IFN-γ) is significantly higher in the obese than in the control subjects. This might be partly dependent on the action of adipocyte-­ derived leptin, that shifts T-helper (Th) cells toward a Th1 phenotype. Thus, in obese children, a shift to Th1-cytokine profile is dominated because of the excess production of IFN-γ (Pacifico et al. 2006). Activation of IDO1 shifts Trp metabolism from serotonin synthesis to formation of Kyns, which might con-

tribute to development of metabolic syndrome (Brandacher et al. 2006b). Therefore, decrease in Trp levels and subsequent reduction in serotonin production provoke satiety dysregulation that ultimately leads to increased caloric uptake and obesity (Brandacher et  al. 2007). Eventually, IDO1 mediated Trp catabolism due to chronic immune activation in morbidly obese patients is the major cause of reduced plasma levels of Trp. However, decreased plasma Trp concentration is independent of weight reduction or dietary intake in obesity (Brandacher et al. 2007). On the other hand, increased IDO1 activity also is a sensitive and an early marker of atherosclerosis in obesity (Niinisalo et al. 2008; Pertovaara et al. 2007). In this respect, IFN-γ up-regulates the expression of inducible nitric oxide (NO) synthase (iNOS), which produces large amounts of NO. IDO1 and iNOS represent important components of the innate immune response (Bogdan 2001). Moreover, there are precise evidences that the iNOS and IDO1 pathways are interrelated (Thomas and Stocker 1999). The simultaneous activation of superoxide synthesis along with NO transforms the biological actions of NO to oxidative stress by forming peroxynitrite (Pacher et al. 2007). Thus, NO is effectively removed from microenvironment via peroxynitrite formation, reactive oxygen species (ROS) generation and decreased endothelial NO production. Key molecular events in atherogenesis are oxidative modification of lipoproteins and phospholipids, endothelial cell activation, and macrophage infiltration. The fact that enhanced oxidative stress and reduced endothelial NO production is indicator for the roles of ROS and NO in atherosclerosis (Förstermann et al. 2017). It was demonstrated that excessive ROS/reactive nitrogen species (RNS) production in the Helicobacter pylori (H. pylori)-infected stomach by activated neutrophils and H. pylori itself can damage DNA in gastric epithelial cells, implying its involvement in gastric carcinogenesis (Handa et  al. 2010). In this case, NO causes a bimodal effect in IDO1 function in IFN-γ-stimulated cells; while high concentrations of NO decrease IDO1 activity, low concentrations of NO increase IDO1 activity (López et  al. 2006). Thus, serum

13  Indoleamine 2,3-Dioxygenase Activity-Induced Acceleration of Tumor Growth…

nitrite levels are significantly lower in H. pylori seropositive cancer patients. Reduction of NO most probably might be due to the formation of peroxynitrite and other RNS. H. pylori seropositive colorectal cancer patients with significantly higher serum IDO1 activity and reduced NO suggest that H. pylori might support the immune tolerance leading to cancer development (Engin et al. 2015). Thereby, the scientific interest in the enzyme has been growing since the observations of the involvement of IDO1 in the mechanisms of immune tolerance and in the concept of tumor immuno-editing process (Macchiarulo et  al. 2009). More than a decade, it is well-known that chronic stimulation of Th1-mediated immunity may cause enhanced IDO1 activity in malignant diseases (Widner et  al. 2000). In this context, activation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway and the induction of IDO1 are two regulatory mechanisms that are present in most cancer cells (Arumuggam et  al. 2015; Constantinescu et  al. 2008). Indeed, STAT3nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-IDO1 pathway displays potent immunosuppressive function on T cells immunity and efficiently promotes lymph node metastasis in patients with breast cancer (Yu et al. 2014). In addition, IFN-γ-induced JAKprotein kinase C delta (PKC δ)-STAT1 signaling pathway also stimulates IDO1 expression (Cheng et  al. 2010). Immune checkpoint inhibitors are now widely accepted as a key component of the therapeutic strategies in cancer (Collin 2016). Although IDO1 activity plays a critical function in cancer progression, it is not a tumor-specific marker. Therefore, many synthesized IDO1 inhibitors have not entered routine clinical practice. As mentioned above, current paradigm in tumor immunology is that tumor cells may escape from immune control due to “adaptive resistance” mediated by IFN-γ, which induces programmed death-ligand (PD-L)1 and IDO expression in tumor cells (Carbotti et al. 2015). Therefore, in this chapter, considering the protein kinase-IDO1 crosstalk in the molecular mechanisms underlying immune escape, whether the use of IDO1 inhibitors in combination with pro-

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tein kinase-related immune checkpoint regulators may have therapeutic efficiency in IDO1 overexpressing tumors is discussed.

2

I DO1 Activity in Cancer Progression

IDO1 and tryptophan 2,3-dioxygenase (TDO) are the primary enzymes that catalyze the rate-­ limiting cleavage of the Trp indole ring 2,3-­double bond and incorporation of molecular oxygen. The product of this reaction is N-formylkynurenine, which is rapidly and spontaneously converted into L-Kyn. L-Kyn is converted into 3-hydroxy-­ L-kynurenine, 3- hydroxyanthranilate and quinolinic acid, which also impact immune responses. Thus, the combined actions of IDO1, direct suppression of effector T-cell activity and concurrent expansion of T-regulatory (Treg) cells, highlights its pleiotropic functions in immune suppression (Harden and Egilmez 2012). Pro-inflammatory signals including IFN-γ, cytosine-phosphate-­ guanosine (CpG) oligodeoxynucleotides and lipopolysaccharide are potent inducers of IDO1 expression (Fujigaki et  al. 2006; Mellor et  al. 2005; Taylor and Feng 1991). Cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and IL-1β, synergize with each other to dramatically increase IDO1 expression. Other IDO1 modulators include soluble glucocorticoid-­ induced TNF receptor, prostaglandin E2 and oncogenic KIT exon (Balachandran et al. 2011). Interesting new data suggests that Wnt5α also mediates IDO1 activity through β-catenin signaling in lymph node-­ derived dendritic cells (DCs). Wnt5a-conditioned DCs promote the differentiation of Tregs in an IDO-dependent manner (Holtzhausen et  al. 2015), while maintaining continuous expression through an aryl hydrocarbon receptor (AhR)-IL-­ 6-­ STAT3 signaling loop in some cancer cell lines. Inhibition of the AhR-IL-6-STAT3 signaling loop restores T-cell proliferation in the presence of IDO-expressing human cancer cells (Litzenburger et al. 2014) (Fig. 13.1). Similar to IDO1, TDO mRNA expression has also been found in human tumors. Enzymatically active

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Fig. 13.1  The IDO1-AhR-IL-6-STAT3 signaling loop in tumor cell. In the cytoplasm, IDO1 metabolizes Trp to Kyn in Kyn pathway. Kyn directly induces the apoptosis of the effector cytotoxic lymphocytes (CD3+CD8+ cells). Thus, IDO1 controls the activation of Tregs in the tumor microenvironment. Kyn interacts with the AhR in naïve CD4+ T cells, and results in the induction of CD4+CD25+FOXP3+ Treg via phosphorylation of the IDO1 by TGF-β signaling. Kyn also activates the AhR in tumor cells. Thereafter, the Kyn and AhR translocates into the nucleus. This complex dimerizes with ARNT and binds to DRE in the promoter of target gene. While peripheral IDO activity supports anti-tumor immunity by regulating cytotoxic activity of immune cells, overexpression of IDO1 activity in cancer cells facilitates tumor growth and invasion. IDO1-positive tumor cells with constitutively phosphorylated STAT3 expresses IL-6 mRNA and secretes IL-6. IL-6 is released from the tumor cells and binds to its receptor. Upon activation of the IL-6R, JAK phosphorylates STAT3. Simultaneously, HAT acetylates STAT3. Phosphorylated and acetylated STAT3 dimers translocate into the nucleus, where they bind to the promoter of target genes. Constitutive IL-6 release drives IDO1 expression in human cancers via STAT3. STAT3 activity mediates constitutive IDO1 expression and activity in human cancer cells. IDO1 may activate its own expression in human cancer cells via an autocrine AhR–IL-6-STAT3 signaling loop. The IDO1-AhR-IL-6-STAT3 signaling regulatory loop

maintains IDO1 expression in human cancers (Litzenburger et  al. 2014), and provides a tolerogenic tumor microenvironment. (Abbreviations: AAADC: Aromatic L-amino acid decarboxylase or 5-hydroxytryptophan decarboxylase; α7-nAChR: alpha-7 nicotinic acetylcholine receptor; AhR: Aryl hydrocarbon receptor; ARNT: Aryl hydrocarbon receptor nuclear translocator; APC: Antigen presenting cell; BH4: Tetrahydrobiopterin; DRE: Dioxin responsive elements; FoxP3: Forkhead lineage-transcription factor; Fyn: Immune receptor signaling status control kinase; HAT: Histone acetyltransferases; IFNγ: Interferon-gamma; IFNγR: Interferon- gamma receptor; IL-6: Interleukin-6; IL-6R: Interleukin-6 receptor; IDO1: Indoleamine 2,3-dioxygenase; IKK: inhibitor of NF-κB (IκB) kinase; IRF-1: Interferon response factor-1; JAK: janus kinase; KAT: Kynurenine amino transferase; Kyn: Kynurenine; NAD+: nicotinamide adenine dinucleotide; NFκB: Nuclear factor-κB; NMDAR: N-methyl-D-aspartate receptor; p38 MAPK: Mitogen-activated protein kinase; PKCδ: Protein kinase C delta; qBH2: quinonoid dihydrobiopterin; QPRT: Quinolinic acid phosphoribosyl transferase; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; SHP: SH2 domain containing protein tyrosine phosphatase; STAT: Signal transducer and activator of transcription; TDO: tryptophan-2,3-dioxygenase; TGF-β: Transforming growth factor-beta; TME: Tumor microenvironment; Treg: regulatory T cell; Trp: Tryptophan)

TDO is expressed in a significant proportion of human tumors (Pilotte et  al. 2012). Dominant factors that affect TDO expression and/or activity include estrogen, progesterone,

8-­bromoadenosine- 3′,5′-cyclic adenosine monophosphate (8-bromo-cAMP) (Li et al. 2014) and the glucocorticoid response element (Comings et al. 1995). New preclinical data suggest that all,

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three mammalian Trp catabolic enzymes, IDO1, IDO2 and TDO, are expressed in brain tumors. Both IDO2 and TDO contribute to the regulation of immunity (Metz et al. 2014; Wainwright et al. 2014). In contrast, the newest member of the Trp catabolic family, IDO2, has yet to be confirmed as a critical contributor to Kyn accumulation and tumor immunity (Zhai et al. 2015). IDO1 expression in various human carcinoma cases is modulated with the infiltration of activated Th1-polarized T cells (Godin-Ethier et al. 2009). In contrast, the Kyn pathway, where IDO is the key enzyme, serves as a negative feedback loop for Th1 cells (Xu et al. 2008). Not only infiltrating cells such as DCs and monocytes, but also tumor cells can be sources of IDO1 (Zamanakou et al. 2007). Thereby, IDO1-induced Trp depletion from the tumor microenvironment may be the result of elevated levels of the enzyme and augmented Trp consumption by both tumor cells and antigen presenting cells (APCs) of the host (Zamanakou et  al. 2007). Whereas, increase in IDO1 activity and the accumulation of Trp metabolites results in a strong inhibitory effect on the development of immune responses by blocking T cell activation, inducing T cell apoptosis and promoting the Treg cell differentiation (Liu et al. 2009a). It is fact that, IDO1-mediated Trp depletion is an integrated molecular switch. This process establishes an immunosuppressive environment by amplifying tolerogenic APCs, expanding CD4+ Treg population, downregulating cytotoxic T-cell (CTL) activity, and promoting other cells that provide critical support to inflammatory carcinogenesis. Consequently, IDO1 is a key immunosuppressive factor that facilitates tumor progression in the setting of chronic inflammation. IDO as an integral component of “cancer-associated” inflammation, changes the immune system in a manner of promoting tumor progression (Muller et  al. 2010; Smith et al. 2012). When Trp levels are reduced following induction of IDO1 creates a tolerogenic milieu in the tumor and the tumor-draining lymph nodes, both by direct suppression of T cells and enhancement of local Treg-mediated immunosuppression (Munn and Mellor 2007). IDO expression in regulatory DCs is prompted

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by an autocrine interferon process controlled by cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) pathway receptors on Treg. This reduces capacity of DCs to present antigens to T cells. IDO+ plasmacytoid dendritic cells (pDCs) create a profoundly suppressive microenvironment within tumor draining lymph nodes via constitutive activation of Tregs (Sharma et  al. 2007). However, IDO+ pDCs are also able to prompt CD4+ T cells to become Tregs. If this occurs in a tumor-draining lymph node, IDO drive the production of Tregs and regulatory DCs which will further suppress immunity against tumor cells (Murphy and Zheng 2015). The induced IDO1-mediated inhibitory activity in the pDCs suppress the T cell stimulatory activity of other DCs that comprise the major fraction of local draining lymph nodes (Muller et al. 2008). Even if it is not a unique marker, IDO1 causes immunosuppression through breakdown of Trp. Elevation of IDO1 activity in aggressive tumors and its involved regional lymph nodes suppresses T cell immunity in the tumor microenvironment. Hence, increase in IDO1 activity in tumor cells may be used as an indicator of tumor invasion capacity (Muller and Prendergast 2007; Soliman et al. 2010). Because the tumor cell IDO1 activity is important in the immune escape mechanisms, serum IDO1 activity is still used as a parameter in the cancer development and growth. The relationship between serum Kyn/Trp ratio and neopterin concentrations indicates not only the cellular immune activation of cancer cases but also tumor-mediated IDO-activity (Schroecksnadel et  al. 2005). In addition to the increased Trp degradation rate in patients with gynecological cancers compared to controls, higher serum Kyn concentrations and a higher Kyn/Trp ratio due to IDO activity indicate the immune escape mechanism that plays an important role in cancer progression. Thereby, tumor derived higher IDO expression is correlated with poor clinical outcome (de Jong et  al. 2011; Ino 2011). Accelerated Trp degradation represents an immune escape mechanism indirectly, while IDO is a fundamental immune escape mechanism for tumor cells. Thus, increase in serum IDO activity in cancer patients is associated with more

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advanced stages of disease. These results suggest that increased IDO activity is involved in cancer progression, most likely through its immunosuppressive effect (Suzuki et  al. 2010). Also, Trp degradation rate by tumor cells and high serum IDO activity may be critical for the balance between host immune response and the capacity of the tumor to evade normal host immune defense in non-small cell lung cancer patients (Engin et  al. 2010). Accordingly, high Kyn/Trp ratio in patient’s sera are associated with high rate of lymph node metastasis, larger tumor size, adjacent tissue invasion and poor disease-specific survival (Zhai et al. 2015). Indeed, the Kyn/Trp ratio in serum samples is recently validated as a prognostic tool in cervical cancer patients. Low concentration of Trp and high concentration of Kyn is associated with greater size of tumor and lymph node metastases (Ferns et  al. 2015). Although the expression of IDO1 by normal cells infiltrating the peritumoral stroma may exert an immunosuppressive action on cancer cells, IFN-γ-mediated induction of tumor IDO1 activity is sufficient for evasion from host immune defense, and cancer progression (Astigiano et al. 2005; Yasui et al. 1986). In fact, serum Trp levels and the Kyn/Trp ratio are influenced by both IDO1 and the liver enzyme TDO. However, IDO1 expression in the tumor cells, rather than tumor microenvironment, leads to intracellular Trp depletion and production of immune inhibitory metabolites in tumor cells (Ferns et  al. 2015). In this context, recent evidences indicate that the increase in IDO1 activity, and IDO1induced Trp degradation products in tumor tissue suppress antitumor immunity of host. It has been shown that IDO1 is constitutively expressed by many tumors and creates an immunosuppressive microenvironment within the tumor tissue both by depletion of the essential amino acid Trp and by formation of Kyn, which is immunosuppressive metabolite of Trp (Platten et  al. 2012). Consistent with these findings, higher level of IDO1 expression in colorectal cancer cells is an indicator of a poor prognosis of these patients. Furthermore, IDO1-related high immunoreactivity is significantly correlated with the frequency of liver metastases (Brandacher et  al. 2006a).

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Similarly, IDO mRNA levels of bladder urothelial carcinoma are significantly higher than that of normal bladder. Furthermore, IDO1 activity, histological grade and tumor–node–metastasis (TNM) stage are closely associated with diseasefree survival. These results indicate that IDO1 is related to the progression of bladder urothelial carcinoma and might be one of the crucial prognostic factors for this type of cancer (Yang et al. 2015). In fact, IDO1 activity is characterized best by the Kyn/Trp ratio which correlates with concentrations of immune activation marker, neopterin (Schröcksnadel et  al. 2006). As stated in many studies, this positive correlation indicates that IDO1 is responsible for the increase in Kyn/Trp ratio in cancer cells (Hascitha et al. 2016). It is concluded that in more than 50% of the cases, cancer cells show high IDO1 expression, which is significantly correlated with a reduced number of CD8+ tumor-infiltrating lymphocyte. Overall and progression-free survival is significantly reduced in patients with tumor expressing excess IDO1 compared to patients without IDO1 expression (Inaba et al. 2009). The Trp metabolites Kyn and kynurenic acid are efficient agonists and acts as a native ligand for the AhR of human tumor cells. Following ligand binding, AhR translocates into the nucleus and dimerizes with AHR nuclear translocator (ARNT) (DiNatale et al. 2010; Opitz et  al. 2011b). In breast cancer cells, long-term effect of estrogen causes not only down regulation of estrogen receptor but also overexpression of AhR.  The AhR induces tyrosine kinases. Thereby, AhR provides escaping from apoptosis elicited by a variety of apoptosis inducing agents in breast cancer. Correspondingly, Kyn acts as an AhR ligand, and enhances its anti-apoptotic response in breast cancer cells (Bekki et  al. 2015). Kyn, produced by IDO in tumor cells, activates the AHR, thereby inducing IL-6. STAT3 phosphorylation correlates with IL-6 protein expression in human non-small cell lung carcinoma. IL-6  in turn drives IDO expression via STAT3 activation. IDO may activate its own expression in human cancer cells via an autocrine AhR–IL-6-STAT3 signaling loop (Litzenburger et  al. 2014) (Fig.  13.1). However, these models

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possess limited usefulness when considering the potential effects that the standard treatment have on IDO activity and/or expression, as well as the potential change of expression between primary and recurrent tumors (Zhai et al. 2015).

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The staging of colorectal cancer has prognostic value in terms of making decisions about adjuvant therapy and follow up after surgical intervention due to current practice. The TNM staging system, which is recommended by American Joint Committee on Cancer (AJCC), (Compton et al. 2000) places patients into one of 3 IDO Immunostaining Is four stages according to tumor size, invasion depth of bowel wall, lymph node metastasis and a Reliable Guide distant metastasis. However, the accuracy of curin Complementary Cancer rent prognostic criteria may change, when the Therapy ultra-staging concept has been used for the stratiAs mentioned above, results of previous studies fication of patients. Because of the difficulties to reveal three basic points: Firstly, high IDO1 apply the ultra-staging in routine practice, expression is observed in majority of high-grade patients are evaluated by conventional histopathundifferentiated carcinoma tissues. Secondly, ologic analysis and IDO immunostaining has tumor-derived IDO expression is inversely cor- been ignored. In a rare sample of IDO1 immunorelated with the T-cell infiltration. Finally, in staining studies, cancer tissue showed a high total patients with a high IDO expression and low IDO immunostaining score. This result is a strong T-cell infiltration, survival rates are significantly predictor for immune tolerance, lymphatic invareduced (Ben-Haj-Ayed et al. 2016). These evi- sion and subsequent lymph node metastasis dences indicate that IDO is not only an important (Engin et  al. 2016). IDO immunostaining is mediator of peripheral immune tolerance but also strongly recommended for the identification of closely related to the other pathways involving higher IDO producing tumors. In this context carcinogenesis. Considering clinical practice, the staging of IDO positive cancer patients should be serum IDO activity is influenced by various met- re-evaluated by taking IDO immunostaining abolic conditions of either cancer cells or tumor intensity into account (Engin et al. 2016). Suitable infiltrating cells. Although measuring IDO activ- complementary therapy modalities are needed to ity in serum seems to be an indicator of cancer be selected regarding the revised staging. IDO growth rate, it is controversial whether this inhibitors should be included in post-operative approach can be used as a reliable guide in cancer complementary therapy in IDO positive cancer patients treated with IDO inhibitors and in ultra-­ cases only. staging of cancer cases (Soliman et  al. 2010). Therefore, it is clear, that the tumor cell IDO activity is more informative than serum IDO 4 IDO1-Protein Kinases level. Considering these data, previous evidences Crosstalk in Inhibition raise the following question; Is the evaluation of of Cancer Progression IDO activity of tumor tissue an effective method to predict the invasiveness of tumor cells? To identify novel IDO inhibitors suitable for Assessment of tumor cell IDO immunostaining drug development, 1597 compounds in the intensity has not taken place currently in routine National Cancer Institute Diversity Set III histopathological evaluation concept (Liu et  al. Library were tested for inhibitory activity against 2009b). Unfortunately, studies related to immu- recombinant human IDO (Tomek et  al. 2017). nostaining of IDO expression in cancer tissues Although, many novel IDO inhibitor scaffolds are very scanty. Due to insufficient data, it is not have been described, only few potent compounds fully known in clinical practice whether cancer have entered clinical trials. Moreover, instead of cell derived IDO activity is still responsible for selective binding of IDO inhibitor to IDO tumor immune evasion. expressed in cancer cells, preference of systemic

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IDO inhibition and its undesirable side effects are serious problem in using IDO1 inhibitors (Röhrig et al. 2015). Because of the insufficient clinical trials, the clinical benefit and autoimmune toxicity of IDO inhibitors are still not clear (Soliman et al. 2010). As observed in many clinical studies, IDO is a major inducer of immune tolerance during tumor development; therefore, inhibition of the IDO pathway seems to be an important modality for cancer treatment (Jiang et  al. 2017). Except trametinib, no other IDO inhibitor is currently approved by the US Food and Drug Administration. It is hypothesized that IDO inhibitors would increase the effectiveness of the T-cell response against tumors, leading to growth inhibition (Flaherty et  al. 2012; Iversen et  al. 2014). IDO1 inhibitors break the tumor’s immune escape mechanisms and inhibit the Trp depletion. The inhibition of IDO1 leads to a reduction of anti-inflammatory Trp metabolites, which counteracts the formation of an immunosuppressive microenvironment. Whereas, the pro- and anti-­carcinogenic properties of mechanistic target of rapamycin (mTOR) and/or AhR activation create a serious adverse effect depending on the type of cancer, the tumor environment and the concurrent anticancer treatment (Günther et  al. 2019). IDO-inhibitor drugs do not kill tumor cells directly. Thus, the role for these drugs in the clinic is to enhance the immune responses triggered by chemotherapy, vaccine or checkpoint inhibitors. Fortunately, in most studies the toxicity of IDO-inhibitor drugs appears to be low, even with sustained administration. In on-going clinical trials, IDO-inhibitor drugs are given by combination with chemotherapy or immune-­modulator (Munn and Mellor 2016). Nutrient-signaling responses to Trp availability have independent roles for mTOR and general control non-­ depressible 2 (GCN2) kinases in cancer cells (Metz et al. 2012; Prendergast et al. 2014). GCN2 is a stress-response kinase that is activated by elevations in uncharged tRNA. Trp depletion as caused by IDO overactivation leads to an accumulation of uncharged Trp-tRNA in cells. This activates the integrated stress response kinase GCN2. Then, GCN2 inhibits the eukaryotic translation initiation factor 2α (eIF2α),

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blocks the protein synthesis and arrests cell growth (Harding et al. 2003; Munn et al. 2005). Consequently, Trp deprivation caused by IDO generates signals sensed by the GCN2 kinase that inhibits eIF2α and blocks translation. However, IDO can regulate mTOR and autophagy independently from GCN2 kinase control (Metz et  al. 2012). When Trp levels are sufficient (Trp sufficiency concept), activated mTOR phosphorylates ribosomal S6 kinase, which phosphorylates downstream ribosomal protein S6 (pS6) (Inoki et al. 2012). S6 kinase activation turns on cellular growth, proliferation, and protein synthesis (Magnuson et al. 2012). In this respect, it is reasonable that the regulation of mTOR-S6K axis signaling networks may enable the development of novel therapeutics. On the other hand, alternate mechanisms involving nutrient sensor 5′ adenosine monophosphate (AMP)-activated protein kinase (AMPK) can control mTOR by activating mTOR inhibitory complexes (Li et  al. 2015; Luo et al. 2010). In fact, AMPK serves as a key metabolic regulator in maintaining energy homeostasis during cellular stress in cancer cells (Luo et  al. 2010). Thus, AMPK functions to restore depleted ATP levels (Blagih et al. 2015) and becomes activated when upstream liver kinase B1 (LKB1) tumor suppressor is phosphorylated (Luo et  al. 2010). Moreover, activated AMPK has different downstream functions, including phosphorylation of 6-phosphofructo-­2kinase/fructose-2,6-biphosphatase 2 (pPFKFB2), an enzyme involved in glycolytic flux (Ros and Schulze 2013). AMPK has been shown to have both pro-tumorigenic and anti-­tumorigenic roles in cancer (Faubert et al. 2015). Because the activation of AMPK reprograms cellular metabolism and enforces metabolic checkpoints by acting on mTORC1, p53, fatty acid synthase, it may be used together with the IDO inhibitors for cancer therapy as a metabolic tumor suppressor (Luo et  al. 2010). There is a reciprocal relationship between IDO and AMPK activation in the tumor microenvironment (TME). Since the myeloidderived suppressor cells (MDSCs) are the main IDO contributors in the TME, reduction of IDO in MDSCs may influence AMPK activation in CD8+ T cells (Schafer et al. 2016). MDSCs rep-

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resent a heterogeneous population of immunosuppressive cells in many cancer types, thus display potent immunosuppressive activity on T cells (Yu et  al. 2013). IDO deficiency not only diminishes tumor burden, but also significantly limits the infiltration of MDSCs in tumor tissue. Therefore, IDO expression in human MDSCs has been attributed to tumor progression and immune escape. Indeed, MDSCs suppress T-cell activation and induce suppressive Tregs cells. The MDSC-mediated modulation of T cells could be attributed to their increased IDO activity (Jitschin et  al. 2014). In this context, direct targeting of MDSCs and cancer cells by combination therapy can inhibit IDO signaling and tumor cell proliferation pathways, while reducing T cell exhaustion and promoting cytotoxic CD8+ T cells (Schafer et al. 2016). In fact, checkpoint blockade agents have been ineffective as single agents in IDO-overexpressing cancer models because the therapeutic effect of these immunotherapies is mainly dependent on direct reactivation of intratumoral CD8+ T cells (Spranger et al. 2014). On the other hand, the cytotoxic activity of natural killer (NK) cells is reduced by IDO inhibition. NK cell activity is reduced in a dose-­dependent manner by the IDO inhibitor, 1-methyl-DL-tryptophan. Eventually, these results indicate that circulating IDO plays an important role in anti-tumor immunity by regulating cytotoxic activity of NK cells (Kai et al. 2003). The IDO mRNA is expressed in T lymphocytes, B lymphocytes and NK cells and that expression is increased upon activation with IFN-γ. Interestingly, the cytotoxicity of NK cells against tumor cells is reduced by IDO inhibitors. Thus, to hinder NK cells to generate killing activity against cancer cells seems to be an important adverse effect of IDO inhibitors (Kai et al. 2004). High c-­mesenchymalepithelial transition factor (c-Met) expression is closely associated with poor prognosis in cancer patients. The (c-Met) receptor tyrosine kinase inhibitors have shown anti-tumor activity in non-­ small cell lung cancer both in preclinical and in clinical trials. Small molecule inhibitors of c-Met kinase have emerged as a promising target for cancer drug development (Lv et al. 2019; Pasquini and Giaccone 2018). IDO activity regulator NK4

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suppresses cancer cell growth via suppressing IDO expression. Moreover, it promotes NK cell accumulation in the tumor stroma. NK4 can suppress IDO expression via the receptor tyrosine kinase inhibitor, c-Met, signaling pathway. Since the phosphoinositide 3-kinase (PI3K)-Protein kinase B (AKT), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase1/2 (ERK1/2), and the JAK-STAT pathways are known as signal pathways downstream of c-Met, NK4 can exert own antitumor activity by inhibiting IDO expression via a c-Met -PI3K-­ AKT signaling pathway (Wang et al. 2016). Many immunotherapy options have been focused on vaccine-based, monoclonal antibody therapies and checkpoint inhibitors. However, recently small molecule inhibitors of IDO1 have been emerged as a set of distinct compounds, which function as immunomodulators (Murphy and Zheng 2015). Of these, Epacadostat is more than 100-fold selective for IDO1 against TDO and represents a highly specific agent with competitive inhibitory kinetics for Trp binding. Navoximod is approximately 20-fold selective for IDO1 against TDO and exhibits non-­ competitive inhibitory kinetics for Trp binding. BMS-986205 is an irreversible inhibitor of IDO1 that is highly specific for that enzyme. Although alternative action, and the pathophysiological role of IDO2 are still unclear, none of these agents inhibit IDO2 appreciably (Fatokun et  al. 2013; Prendergast et al. 2017). Preclinical studies of 1-methyl-tryptophan (1-MT) showed that it reduces tumor growth but did not prevent tumor progression. However, 1-MT exists in two stereoisomers with potentially different biological properties. While the L isomer is the more potent inhibitor of IDO activity, the D isomer is significantly more effective in reversing the suppression of T cells created by IDO-expressing DCs (Hou et  al. 2007). Although IDO2 is expressed in human tumors, Trp degradation is entirely provided by IDO1. Importantly, amongst two isomers, only 1-methyl-L-tryptophan (1-L-MT) is able to block IDO1 activity in cancer cells (Löb et al. 2009). Interestingly, 1-methyl-D-­tryptophan (1-D-MT) increases the Kyn production of cancer cells with IDO1 activity due to upregulation

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of IDO1 mRNA and protein expression. The upregulation of IDO1 expression is observed only in cancer cells with either constitutive or IFN-γ-induced IDO1 expression (Opitz et  al. 2011a). In fact, 1-MT is a competitive inhibitor of IDO1 that can break tolerance. It has been reported that 1-MT modifies the polarization of DCs by modulating MAPK. The action of 1-MT correlates with an increased phosphorylation of p38 and ERK MAPKs and sustained activation of the transcription factor c-Fos (Agaugué et  al. 2006). Inhibition of p38 MAPK phosphorylation prevents the increase in IDO1 mRNA and Kyn production by 1-D-MT treatment, suggesting that p38-MAPK participates in 1-D-­ MT mediated signaling. In addition, inhibition of c-Jun N-terminal kinase (JNK) signaling reduces the induction of IDO1 mRNA and Kyn release in the presence of 1-D-­MT (Opitz et al. 2011a). In line with this result, p38 MAPK has previously been shown to contribute to the induction of IDO1 in a leukaemia cell line and in DC (Fujigaki et  al. 2006). 1-D-­MT has transcriptional effects that may promote rather than suppress anti-tumor immune escape by increasing IDO1 in the cancer cells (Opitz et al. 2011a). Imatinib mesylate is a small molecule inhibitor of cKIT, platelet-derived growth factor receptor A (PDGFRA), Abelson (ABL), and break point cluster region (BCR)ABL tyrosine kinases. Imatinib selectively reduces intra-­tumoral Tregs by inhibiting IDO expression in gastrointestinal stromal tumor (GIST). Indeed, the intra-tumoral CD8+ T cell to Treg ratio correlates with intra-tumoral IDO expression in patients. Consistent with this finding, the CD8+ T cell to Treg ratio in human GISTs with acquired resistance to imatinib is significantly lower than in sensitive tumors and resistance is associated with increased IDO expression when compared to sensitive tumors. This may be an option for combined molecular and immune therapy in human GIST (Balachandran et  al. 2011). A small molecule, sodium butyrate (NaB) has received much attention as a potential chemopreventive agent for cancer treatment due to its protective action against intracellular events including IFN-­ γ-­ mediated signaling transduction. NaB represses

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the activity of the STAT1 to inhibit STAT1-driven transcriptional activity of IDO (Jiang et al. 2010). NaB inhibits IFN-γ-induced IDO expression via reduced nuclear translocation of STAT1. Therefore, NaB may be a potential anti-tumor drug to reduce the IDO-induced immune tolerance in cancer therapy (He et al. 2013). Moreover, sphingosine kinase (SphK) 1 enhances colon cancer cell proliferation and invasiveness, meanwhile suppressing cell apoptosis. SphK1 increases the constitutive expression of ERK1/2 but reduces the constitutive expression of p38 MAPK. Modulation of these molecules increases the sensitivity of colon cancer cells to the NaB (Liu et al. 2012; Xiao et al. 2014). Bortezomib, as a proteasomal inhibitor like NaB, blocks the STAT1-interferon regulatory factor-1 (IRF1) pathways activation, which are necessary for IDO expression. Thus, bortezomib suppresses STAT1-driven IDO transcription in nasopharyngeal carcinoma cells by downregulating IFN-γ-­ induced IDO expression (Jiang et al. 2017). It is fact that STAT1 works directly by binding to the IFN-γ-activated sites within the IDO promoter. Also, it acts indirectly by inducing IRF-1, which binds to the IDO promoter (Ramsauer et  al. 2007). However, as an adverse effect, bortezomib induces protective autophagy in pancreatic and colorectal cancer cells through activating AMPKUnc-51 like autophagy activating kinase (Ulk1) signaling. In this respect, it is reasonable, to use AMPK or autophagy inhibitors as an adjunct or chemo-sensitizer for bortezomib (Min et  al. 2014). On the other hand, bortezomib-mediated inhibition of cell proliferation of chronic myelogenous leukemia cells is associated with down-­ regulation of S-phase kinase protein 2 (SKP2) (Iskandarani et  al. 2016). Within the inflammatory microenvironment IL-6-mediated activation of STAT3 is one of the principal pathways implicated in promoting tumorigenesis. This transcription factor regulates the expression of numerous critical mediators of tumor formation and metastatic progression (Sansone and Bromberg 2012). On the other hand, suppressor of cytokine signaling (SOCS)3 is known to interact with phosphotyrosine-­ containing peptides and be selectively induced by IL-6. In this case, IL-6

13  Indoleamine 2,3-Dioxygenase Activity-Induced Acceleration of Tumor Growth…

binds to IDO and targets the IDO/SOCS3 complex for subsequent proteasomal degradation of IDO in CD8-DCs. Consequently, IDO undergoes regulatory proteolysis in response to immunogenic stimuli (Orabona et al. 2008; Pallotta et al. 2010). A second-generation MAP-ERK kinase, the selective mitogen-activated extracellular signal-­ regulated kinase (MEK) inhibitor, trametinib, which potently inhibit phosphor-ERK1/2 (p-ERK1/2), has been approved by the Food and Drug Administration (FDA) for its use in unresectable or metastatic melanoma. Trametinib improves progression-free and overall survival compared to chemotherapy (Flaherty et al. 2012; Lugowska et al. 2015). Similar to the other IDO inhibitors, MEK inhibitors have limited efficacy as monotherapy and require combination with conventional chemotherapeutics.

5

Combination Therapy with IDO Inhibitors

The number of clinical trials focusing on IDO has recently increased. These reports, in addition to preclinical data suggest that, combining IDO targeting with chemotherapy, radiotherapy and/ or immunotherapy, may be an effective tool against a wide range of malignancies (Zhai et al. 2015). Induction of IDO1 creates a tolerogenic milieu in the tumor and the tumor-draining lymph nodes. Enhancement of local Tregmediated immunosuppression is characterized by expression of the master regulatory transcription factor forkhead box P3 (FoxP3). Treg cells prevent the development of effective antitumor immunity in tumor-­ bearing patients, and promote tumor progression. Therefore, directly or indirectly targeting Treg cells in combination with immune checkpoints inhibitors should be crucial to improve the treatment outcomes of cancer immunotherapy (Munn and Mellor 2007; Shitara and Nishikawa 2018). Because the high infiltration by Treg cells is associated with poor survival in various types of cancer, it is thought that strategies to deplete Treg cells and control of Treg cell functions to increase antitumor immune

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responses are urgently required in the cancer immunotherapy field (Ohue and Nishikawa 2019). It is known that cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the carcinogenesis. Thereby, selective CDK4/6 inhibitors not only induce tumor cell cycle arrest, but also promote anti-­carcinogenic immunity. Thereby, these inhibitors markedly suppress the proliferation of Treg cells and take place in new combination regimens (Goel et al. 2017). Palbociclib, a specific CDK4/6 inhibitor, is developed to arrest cell cycle progression in proliferating tumor cells. However, clinical studies indicate that palbociclib as a single agent fails to provide durable responses, and suggest to combine with other agents. Thus, combination therapies with CDK4/6 and mTOR inhibitors are more successful in cancer therapy, and provide maximal efficacy through synergistic drug-drug interaction while using minimal doses to reduce adverse effects. While palbociclib inhibits both ERK1/2 and p70S6K activity, everolimus blocks mTOR (Olmez et  al. 2017). In fact, the RAS-RAFMEK-ERK signaling pathway also enhances CDK4/6 activity by driving cyclin D1 transcription (Goel et  al. 2018). The cytotoxic effect of CDK inhibitor is dependent upon activation of apoptosis stress-inducing kinase 1 (ASK1), MAPKs JNK; MAPK8 and p38 MAPK, and subsequent induction of NOXA, a pro-apoptotic B-cell leukemia/lymphoma-2 (BCL-2) family protein (Paiva et al. 2015). The p38 MAPK, and the JAK-STAT pathways can modulate IDO expression in response to a variety of stimuli. However, only JNK/c-JUN pathway strongly contributes to IDO gene expression (Jung et al. 2007). STAT1 phosphorylation appears to play an important role in the control of IDO expression by IFN-γ. Curcumin ((1,7-bis(4-hydrosy-­3methoxyphenyl)-1,6-heptadiene-3,5-dione), a phenolic natural product) suppresses STAT1 activation by directly inhibiting Janus-activated kinase 1/2 and PKC-δ phosphorylation in bone marrow-derived DCs, suppressing the subsequent translocation and binding of STAT1 to the IFN-γ-activated site (GAS) element of the IRF1 promoter. Coincident with these inhibitory

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effects on IFN-γ-induced IDO expression, curcumin reverses IDO-mediated suppression of T-cell responses. Therefore, curcumin can be used for new combination regimens therapeutically in the control of cancers (Jeong et al. 2009). Administration of 1-MT significantly suppresses tumor growth, and 1-MT and temozolomide (TMZ) present synergistic antitumor effects. Combining IDO inhibition with standard TMZ treatment could be an encouraging therapeutic strategy for patients with malignant glioma (Hanihara et al. 2016). It is shown that 1-MT displays marked synergy with number of clinically relevant chemotherapeutic agents when used in combined chemo-immunotherapy regimens. Thus, the combination of 1-MT with cyclophosphamide, cisplatin, doxorubicin, or paclitaxel cause regression of established tumors (Muller et al. 2005). When 1-MT combined with cyclophosphamide, an additional anti-tumor effect is provided compared to chemotherapy alone (Hou et  al. 2007). Furthermore, preclinical evidences show that combined administration of indoximod, the D isomer of 1-MT, and docetaxel, the chemotherapeutic agent, has synergistic effect. This combination therapy has been well tolerated with no increase in expected toxicities or pharmacokinetic interactions in patients with metastatic cancer (Soliman et al. 2014).

6

Conclusion

IDO1 induction is associated with the chronic immune activation states, including antimicrobial and anti-tumor defense. In this context, endogenous IDO1 activity facilitates tumor outgrowth by creating peripheral tolerance and immunosuppressive environment against tumor antigens (Muller and Prendergast 2007). Nevertheless, IDO1 activity is not a tumor-specific marker and serum IDO1 activity may not be completely informative in the case of cancer progression. Therefore, serum IDO1 seems not a reliable indicator in estimating prognosis of cancer patients especially in the presence of co-morbid ­conditions. IDO1 is constitutively expressed by many tumor cells and creates an immunosuppressive microenvironment around the cancer tissue.

These immunomodulatory effects of Trp catabolites are most notably due to Kyn and its effect is mediated by the nuclear AhR (Platten et al. 2012). Therefore, IDO1 immunostaining of cancer cells seems to be a more rational method for prediction of immune tolerance, lymphatic invasion and subsequent lymph node metastasis of cancer cells. Novel therapies that target the IDO1 pathway, while reversing IDO1-mediated suppression of T-cell, should cover checkpoint protein kinases related combination regimens. Currently, multi-­ modal therapies combining checkpoint kinase inhibitors with T cell-modifying treatments look promising (Schafer et al. 2016). However, there are some controversial results regarding the efficacy of IDO1 inhibitors for IDO1 inhibition and cancer treatment (Günther et al. 2019). Although the clinical outcomes of most immunotherapeutic strategies have been less effective than anticipated because of the immune tolerance, the majority of preclinical analyses aimed IDO1 inhibition are currently ongoing.

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A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases Can Be A Potential Therapeutic Target for Anti-cancer Therapy

14

Basak Celtikci

Abstract

While protein tyrosine kinases (PTKs) play an initiative role in growth factor-mediated cellular processes, protein tyrosine phosphatases (PTPs) negatively regulates these processes, acting as tumor suppressors. Besides selective tyrosine dephosphorylation of PTKs via PTPs may affect oncogenic pathways during carcinogenesis. The PTP family contains a group of dual-specificity phosphatases (DUSPs) that regulate the activity of Mitogen-activated protein kinases (MAPKs), which are key effectors in the control of cell growth, proliferation and survival. Abnormal MAPK signaling is critical for initiation and progression stages of carcinogenesis. Since depletion of DUSP-­ MAPK phosphatases (MKPs) can reduce tumorigenicity, altering MAPK signaling by DUSP-MKP inhibitors could be a novel strategy in anti-cancer therapy. Moreover, Cdc25A is, a DUSP and a key regulator of the cell cycle, promotes cell cycle progression by dephosphorylating and activating cyclin-­dependent kinases (CDK). Cdc25A-CDK pathway is a novel mechaB. Celtikci (*) Hacettepe University, Faculty of Medicine, Department of Medical Biochemistry, Ankara, Turkey

nism in carcinogenesis. Besides the mammalian target of rapamycin (mTOR) kinase inhibitors or mammalian target of rapamycin complex 1 (mTORC1) inhibition in combination with the dual phosphatidylinositol 3 kinase (PI3K)/mTOR or AKT kinase inhibitors are more effective in inhibiting the phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and cap-dependent translation. Dual targeting of the Akt and mTOR signaling pathways regulates cellular growth, proliferation and survival. Like the Cdc2-like kinases (CLK), dual-specific tyrosine phosphorylation-­regulated kinases (DYRKs) are essential for the regulation of cell fate. The crosstalk between dual-specific phosphatases and dual- specific protein kinases is a novel drug target for anti-cancer therapy. Therefore, the focus of this chapter involves protein kinase modules, critical biochemical checkpoints of cancer therapy and the synergistic effects of protein kinases and anti-cancer molecules. Keywords

Kinases · Phosphatases · Carcinogenesis · DUSP · Cdc25A · CDK · PI3K · mTOR · AKT · MAPK

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_14

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B. Celtikci

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1

Introduction

Intracellular protein phosphorylation is in a balance of protein kinases’ and phosphatases’ functions. Both of them are key components of intracellular signal transduction pathways, through which cells respond to extracellular stimuli, such as growth factors, hormones, and environmental stresses. Our focus will be protein kinase family, also known as phosphotransferases, and its downstream signaling of the effectors that are controlling the cell division, the molecular mechanisms of cell fate decisions, and mainly the effect of the protein kinases on carcinogenesis. In the literature, several studies reviewed the clinical use of protein kinases as anti-cancer agents (Rask-Andersen et  al. 2014; Wu et  al. 2015, 2016; Zhao and Bourne 2018; Roskoski Jr 2019a, 2019b, 2019c, 2019d). All agreed that the development of small molecule kinase inhibitors is one of the most broadly explored areas of drug discovery. The focus of this chapter is mainly protein kinases, because they are one of the most studied drug targets in pharmacological research, according to many clinical trials involving kinase inhibitors. These clinical trials are triggered by the discovery of imatinib (Gleevec) against BCR-­ ABL, a constitutively active mutant kinase, in 2001. Since then, there are more than 3000 approved and experimental drugs in current clinical use and ongoing clinical trials. Other kinase inhibitors in current clinical use and ongoing clinical trials are PI3K/mTOR or AKT kinase inhibitors, and CLK kinases and DYRKs inhibitors. On the other hand, in the protein phosphatase family, DUSPs are essential modulators in carcinogenesis, through their regulatory activity on MAPK pathway, a well-known cellular proliferation and survival pathway. MAPK inhibitors are clinically used in anti-cancer therapy. In addition, Cdc25A is an important DUSP, a cell cycle regulator and a promoter of cell cycle progression via dephosphorylating and activating CDKs in carcinogenesis (Wang et al. 2008). Finally, DUSP1 (MKP-1) and DUSP4 (MKP2) may be potential therapeutic targets against the resistance to chemotherapy.

Overall, a crosstalk between dual-specific phosphatases and dual-specific protein kinases can be a potential therapeutic target for anti-­ cancer therapy.

2

Protein Kinases

Protein kinases are one of the largest classes of proteins encoded by human genome, although they cover only 5% of protein-coding genes. They are crucial enzymes for cell cycle regulation, proliferation, survival, apoptosis, metabolism, differentiation, transcription, and signaling. Their mechanism of action is based on reversible phosphorylation. Briefly, one phosphate from ATP is transfered onto hydroxyl groups of lipids, sugars, or amino acids. The phosphorylation of serine, threonine, or tyrosine moieties on their target proteins results in a conformational shift, changing the activity of the protein (Wang et al. 2014; Wu et al. 2015). Protein kinases are classified by their target amino acid of their protein substrates. They, mainly phosphorylate tyrosine, such as ALK, BCR-ABL, EGFR family, PDGF alfa/beta, VEGF family, c-MET, RET, BTK, JAK family and SRC family. ALK, EGFR family, PDGF alfa/ beta, VEGF family, c-met and RET are examples of receptor tyrosine kinases. These have gain or loss of mutations in several disorders, which make them drug targets in these diseases. CDK family, BRAF and MTOR are examples of protein kinases that phosphorylate serine/threonine, and lastly MEK 1/2 is an example of a dual specificity kinase (Roskoski Jr 2016a). Since protein kinases act as downstream modulators and effectors of cellular signaling, growth, proliferation and survival, the clinical use of these inhibitors is in not only anti-cancer therapy, but also asthma, autoimmune, cardiovascular, inflammatory, nervous diseases, ophthalmology, analgesia, central nervous system disorders (Alzheimer’s disease and Parkinson’s disease), diabetic complications, osteoporosis, and otology. These therapeutic kinase-related drug ­targets are not only protein kinases but also receptor protein kinaseinteracting ligands, lipid kinases, ­ glucokinase

14  A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases…

and pyruvate kinase. Most of them are small molecules, as they can pass the cellular membrane to ease the interaction with its target and specifically bind to ATP-binding pocket of protein kinases (Rask-Andersen et al. 2014) (Table 14.1). Among 48 clinically approved kinase inhibitors, most of them (25 inhibitors) are protein receptor tyrosine kinase inhibitors, 10 of them are protein non-receptor tyrosine kinases, and 13 of them are protein serine-threonine kinases (Table 14.1). 43 of them are used in the treatment of malignancies (37 of them against solid tumors including lymphomas and 8 of them against leukemias; Ibrutinib and imatinib are used in the treatment of both solid and non-solid tumors). 7 of them are used in the treatment of non-­ malignancies, such as asthma, autoimmune, cardiovascular, inflammatory and nervous diseases. At least 18 of these drugs are multikinase inhibitors. Most of them are reversible. Only 6 of them are covalent and irreversible. EGFR have the highest number of clinically approved inhibitors, followed by BCR-ABL, ALK, VEGF and BRAF. Kinases are very similar in their catalytically active kinase domain where their ATP-binding pocket is located. The most pronounced group is multitarget inhibitors, having targets ranging from three to nine. The second is the selective inhibitors with only one main target or inhibitors that target two structurally similar kinases in the same group. Those inhibitors and their targets are vemurafenib (BRAF), ceritinib (ALK), Idelalisib (PI3Kdelta), lapatinib (EGFR, HER2) and trametinib (MEK1/2). The third group is promiscuous (less selective) inhibitors as those having no less than ten validated targets. Those are sorafenib, dasatinib, pazopanib, bosutinib, regorafenib, ponatinib and lenvatinib. Regarding selectivity of these inhibitors, even though some considered as relatively more selective as ­lapatinib, erlotinib, and imatinib, some considered as less selective as dasatinib and sunitinib, fortunately some off-target effects of imatinib indicates it in the treatment of gastrointestinal stromal tumors. Another example, sunitinib and cabozantinib have potent Axl off-target activity, increasing their clinical effectiveness (Karaman et al. 2008; Davis et al. 2011).

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BCR-ABL inhibitor imatinib (reversible non-­ receptor tyrosine kinase inhibitor) is the first approved kinase inhibitor, used in the treatment of chronic myeloid leukemia (Steelman et  al. 2004). This breakthrough discovery was followed by EGFR inhibitors gefitinib and erlotinib (reversible receptor tyrosine kinase inhibitors), ALK/MET inhibitor crizotinib (reversible receptor tyrosine kinase inhibitor), BRAF inhibitor vemurafenib and dabrafenib (reversible serine-­ threonine kinase inhibitors) and other BCR-ABL inhibitors. First JAK1/2 inhibitor (reversible non-­ receptor tyrosine kinase inhibitor) ruxolitinib was approved and used in the treatment of myeloproliferative disorders in 2011. JAK pathway is initiated by cytokines and growth factors to phosphorylate signal transducer and activator of transcription (STAT) proteins. STATs are involved in cellular proliferation, survival and carcinogenesis. Their specific inhibitors are also used in anti-­ cancer therapy. First approved CDK inhibitor palbociclib (reversible serine-threonine kinase inhibitor) is selective for CDK4 and CDK6, and used in the treatment of breast cancer. In contrast, afatinib (EGFR/HER2 inhibitor), ibrutinib (BTK inhibitor), osimertinib (EGFR inhibitor), neratinib (EGFR/HER2 inhibitor), dacomitinib (EGFR) and acalabrutinib (BTK) are irreversible inhibitors. Trametinib, cobimetinib and binimetinib (MEK1/2 inhibitors) are allosteric kinase inhibitors (Wu et al. 2015). Most of these protein kinase inhibitors are type I inhibitors, which bind only the adenine site of the ATP pocket, targeting the active state of the kinase. Lenvatinib (VEGFR inhibitor) has both type I and II inhibitor features (targeting both the active and the inactive state of the kinase), is called type V inhibitor. Of 48 FDA-approved inhibitors, only trametinib, cobimetinib and binimetinib do not bind into the ATP binding pocket, instead bind into an adjacent allosteric pocket (targeting an allosteric site of the kinase), are called type III inhibitors, an allosteric MEK1/2 kinase inhibitors (Table 14.2). Type IV allosteric inhibitors, like sirolimus, bind into a pocket remote from the ATP binding pocket (targeting a pocket distant from the ATP-binding site, a hydrophobic pocket, or a pocket on the surface of

Small Small Small Small

CML

BC

RCC, STS

MTC NSCLC

Myelofibrosis

Melanoma CML RCC

Nilotinib (Tasigna)

Lapatinib (Tykerb/ Tyverb) Pazopanib (Votrient)

Vandetanib (Caprelsa) Crizotinib (Xalkori)

Ruxolitinib (Jakafi/ Jakavi) Vemurafenib (Zelboraf) Bosutinib (Bosulif) Axitinib (Inlyta)

MTC CC, GIST CML, ALL Melanoma Melanoma NSCLC

Small

CML, ALL

Dasatinib (Sprycel)

Cabozantinib (Cometriq) Regorafenib (Stivarga) Ponatinib (Iclusig) Dabrafenib (Tafinlar) Trametinib (Mekinist) Afatinib (Gilotri) (Tovok) (irreversible)

Small

RCC, GIST

Sunitinib (Sutent)

Small Small Small Small Small Small

Small Small

Small

Small

Small

Small Small Small

NSCLC NSCLC, PC RCC, HC, TC

Gefitinib (Iressa) Erlotinib (Tarceva) Sorafenib (Nexavar)

Agent Small

Indication CML, GIST, ALL

Drug Imatinib (Gleevec)

Molecule Molecule Molecule Molecule Molecule Molecule

Molecule Molecule Molecule

Molecule

Molecule Molecule

Molecule

Molecule

Molecule

Molecule

Molecule

Molecule Molecule Molecule

class Molecule

Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor

Inhibitor Inhibitor Inhibitor

Inhibitor

Inhibitor Inhibitor

Inhibitor

Inhibitor

Inhibitor

Inhibitor

Inhibitor

Inhibitor Inhibitor Inhibitor

Pharmacological action Inhibitor

Table 14.1  The list of FDA-approved protein kinase interacting agents and their therapeutic molecular targets

2012 2012 2012 2013 2013 2013

2011 2012 2012

2011

2011 2011

2009

BRAF BCR-ABL1 FLT1, FLT4, KDR, KIT, PDGFRA, PDGFRB, VEGFR KDR, MEK, VEGFR2, MET KDR, TEK, VEGFR BCR-ABL1 BRAF MEK1/2 ERBB2, EGFR

FLT1, FLT4, KDR, KIT, PDGFRA, PDGFRB, VEGFR FLT1, FLT4, KDR, EGFR, RET, VEGFR ALK, MET, EML4–ALK fusion protein, ROS JAK1/2

FDA approval Target(s) 2001 BCR-ABL1, ABL1, CSF1R, DDR1, KIT, NTRK1, PDGFRA, PDGFRB, RET 2003 EGFR 2004 EGFR 2005 BRAF, FLT3, FLT4, KDR, KIT, PDGFRB, RAF1, VEGFR 2006 FLT1, FLT3, FLT4, KDR, KIT, PDGFRA, PDGFRB, RET, VEGFR 2006 ABL1, ABL2, EPHA2, FYN, KIT, LCK, PDGFRB, SRC, YES1 2007 ABL1, KIT, LCK, EPHA3, EPHA8, DDR1, DDR2, PDGFRB, MAPK11, ZAK 2007 EGFR, ERBB2

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Drug Ibrutinib (Imbruvica) (irreversible) Nintedanib (Ofev) (vargatef) Idelalisib (Zydelig) Ceritinib (Zykadia) Alectinib (Alecensa) Lenvatinib (Lenvima) Palbociclib (Ibrane) Osimertinib (Tagrisso, Tagrix) Cobimetinib (Cotellic) Neratinib (Nerlynx) Brigatinib (Alunbrig) Abemaciclib (Verzenio) Acalabrutinib (Calquence) Midostaurin (Rydapt) Ribociclib (Kisqali) Baricitinib (Olumiant) Binimetinib (Mektovi) Dacomitinib (Visimpro) Encorafenib (Braftovi) Fostamatinib (Tavalisse) Gilteritinib (Xospata) Larotrectinib (Vitrakvi) Lorlatinib (Lorbrena) Netarsudil (Rhopressa) Trastuzumab (Herceptin) Cetuximab (Erbitux) Bevacizumab (Avastin) Panitumumab (Vectibix) Small Small Small Small Small Small Small Small Small Small Small Small Molecule Small Molecule Small Molecule Small Molecule Small Molecule Small Molecule Small Molecule Small Molecule Small Molecule Small Molecule Small Molecule Monoclonal antibody Monoclonal antibody Monoclonal antibody Monoclonal antibody

CLL, Follicular B-NHL, SLL NSCLC NSCLC HCC, RCC, TC BC NSCLC

Melanoma BC NSCLC BC MCL

AML, Mast cell leukemia BC Rheumatoid arthritis Melanoma NSCLC Melanoma Ch imm. thrombocytopenia AML Solid tumors NSCLC Glaucoma BC CC, SCCHN CC, NSCLC, RCC, GM CC

Molecule Molecule Molecule Molecule Molecule

Molecule Molecule Molecule Molecule Molecule Molecule

Molecule

Small

Idiopathic pulmonary fibrosis

class Molecule

Agent Small

Indication MCL, CLL

Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor

Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor

Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor

Inhibitor

Pharmacological action Inhibitor

2017 2017 2018 2018 2018 2018 2018 2018 2018 2018 2018 1998 2004 2004 2006

2015 2017 2017 2017 2017

2014 2014 2015 2015 2015 2015

2014

FLT3 CDK4/6 JAK1/2/3, tyk MEK1/2 EGFR BRAF Syk FLT3 TRK ALK ROCK1/2 ERBB2 EGFR VEGF EGFR

MEK1/2 ERBB2/EGFR ALK/EGFR CDK4/6 BTK

PI3K ALK ALK VEGFR CDK EGFR

VEGFR/FGFR

FDA approval Target(s) 2013 BTK

(continued)

14  A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases… 361

Small molecule Recombinant platelet-­ derived growth factor (PDGF) Antibody fragment Aptamer Recombinant keratinocyte growth factor (KGF) Recombinant insulin-like growth factor 1 (IGF1) Recombinant insulin

Rheumatoid arthritis Diabetic foot ulcer

AMD AMD Oral mucositis

Growth failure

DM

Type 2 DM AMD RCC Immunosuppressant, BC, SEGA, RCC, PTEN

Ranibizumab (Lucentis) Pegaptanib (Macugen) Palifermin (Kepivance)

Mecasermin (Increlex)

Insulin

Metformin (Glucophage) Sirolimus (Rapamune) Temsirolimus (Torisel) Everolimus (Zortress/ Certican)

Activator Inhibitor Inhibitor Inhibitor

Activator

Activator

Inhibitor Inhibitor Activator

Inhibitor Activator

Inhibitor

Pharmacological action Inhibitor

1982 (Humulin) 1995 1999 2007 2009

2005

2006 2004 2004

2012 1997

2013

AMPK MTOR MTOR MTOR

INSR

IGF1R

VEGF VEGF FGFR

JAK3 PDGFR

ERBB2, tubulin

FDA approval Target(s) 2011 VEGF

Their commercial name, their indicationa, the agent class, their pharmacological action, the year of their FDA approval and their protein kinase targets are shown in the table. Irreversible inhibitors are emphasized in parentheses a Abbreviations: ALL, acute lymphoblastic leukemia; AMD, age-related macular degeneration; BC - breast cancer; CC, colorectal cancer; CLL, chronic lymphocytic leukemia; CML, chronic myelogenous leukemia; DM, diabetes mellitus; GIST, gastrointestinal stromal tumors; GM, glioblastoma multiforme; MCL, mantle cell lymphoma; MTC, medullary thyroid cancer; NSCLC, non-small-cell lung carcinoma; PC, pancreatic cancer; PTEN, progressive neuroendocrine tumors of pancreatic origin; RCC, renal cell carcinoma; hepatocellular carcinoma; SCCHN, squamous cell carcinoma of the head and neck; SEGA, subependymal giant cell astrocytoma; STS, soft-tissue sarcoma; TC, thyroid carcinoma; TS-SEGA, subependymal giant cell astrocytoma associated with tuberous sclerosis; WMD, wet macular degeneration

Small molecule Small molecule Small molecule Small molecule

Antibody–drug conjugate

BC

class

Agent Fusion protein

Indication WMD, CC

Drug Aflibercept (Eylea/ Zaltrap) Trastuzumab emtansine (Kadcyla) Tofacitinib (Xeljanz) Becaplermin (Regranex)

Table 14.1 (continued)

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14  A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases… Table 14.2  The list of protein kinase inhibitor types Type I inhibitors Bosutinib (SRC) Brigatinib (ALK) Crizotinib (ROS1) Dasatinib (Abl) Erlotinib (EGFR) Gefitinib (EGFR) Palbociclib (CDK6) Fostamatinib (Syk) Tofacitinib (JAK1) Vandetanib (RET) Type I½A inhibitors Dabrafenib (B-Raf) Lapatinib (EGFR) Lenvatinib (VEGFR) Vemurafenib (B-Raf) Type I½B inhibitors Abemeciclib (CDK6) Alectinib (ALK) Ceritinib (ALK) Crizotinib-ALK Erlotinib (EGFR) Palbociclib (CDK6) Ribociclib (CDK6) Type IIA and IIB inhibitors Axitinib (VEGFR) Imatinib (Abl) Nilotinib (Abl) Ponatinib (Abl) Sorafenib (VEGFR) Bosutinib (Abl) Sunitinib (VEGFR2) Sunitinib (Kit) Type III inhibitors Trametinib (MEK1/2) (allosteric) Cobimetinib (MEK1/2) (allosteric) Binimetinib (MEK1/2) (allosteric) Type VI inhibitors Afatinib (EGFR) (covalently bound to its target; irreversible) Ibrutinib (BTK) (covalently bound to its target; irreversible) Osimertinib (EGFR) (covalently bound to its target; irreversible) Neratinib (EGFR/HER2) (covalently bound to its target; irreversible) Dacomitinib (EGFR) (covalently bound to its target; irreversible) Acalabrutinib (BTK) (covalently bound to its target; irreversible) (continued)

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Table 14.2 (continued) These inhibitors are classified as type I, I½A, I½B, IIA, IIB, III and VI inhibitors. The specific protein kinases that they inhibit are shown in parentheses. Type III inhibitors are allosteric and type VI inhibitors are covalently bound to its target and irreversible inhibitors. These are also emphasized in parentheses

the kinase). Overall, allosteric inhibitors do not bind to the active site of their target enzyme, but in contrast to type III allosteric inhibitors that bind within the deep cleft of protein kinases, type IV allosteric inhibitors act by binding elsewhere to non-active site residues. In addition, afatinib (EGFR/HER2), ibrutinib (BTK), osimertinib (EGFR), neratinib (EGFR/ HER2), dacomitinib (EGFR) and acalabrutinib (BTK) form a covalent adduct; because of this covalent inhibition, they have better efficacy, less drug resistance, and are called covalent inhibitors (type VI) (Table 14.2). Four of the six clinically FDA-approved covalent kinase drugs target EGFR (Wu et al. 2016; Kooistra et al. 2016). From type I inhibitors, 5 of them (bosutinib, dasatinib, imatinib, nilotinib and ponatinib) inhibit the activated chimeric BCR-ABL protein tyrosine kinase in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemias (Ph + CML). Bosutinib and dasatinib are multitarget inhibitors, also inhibit SRC family, enhancing their efficacy. Another 5 of them (alectinib, brigatinib, ceritinib, lorlatinib and crizotinib) inhibit ALK in the treatment of ALK-driven Non-small cell Lung carcinoma (NSCLC). Crizotinib is a multitarget inhibitor, also inhibits ROS and MET, enhancing its efficacy. Brigatinib’s kinase spectrum is relatively wider than crizotinib, so it is used in crizotinib resistant NSCLC (Roskoski Jr 2016a, 2019c). Among type I inhibitors, gefitinib and erlotinib are EGFR reversible inhibitors in the treatment of EGFR positive NSCLC, and Palbociclib is CDK4/6 inhibitor in the treatment of ER/HER2 receptor positive breast cancer (Roskoski Jr 2016b) (Table 14.2). From type II inhibitors, axitinib, sorafenib and sunitinib inhibit VEGFR.  Axitinib (PDGF and ABL inhibitor), sorafenib and sunitinib are all multitarget inhibitors, also inhibit KIT, enhancing their efficacy. Imatinib, nilotinib, ponatinib

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and bosutinib inhibit ABL.  Imatinib (PDGF inhibitor) and ponatinib are multitarget inhibitors, also inhibit KIT, enhancing their efficacy (Roskoski Jr 2016a) (Table 14.2). On the other hand, to boost the immune response against cancer, tumor-specific protein kinases, such as EGFR, MET-HGF, and HER2 can be used as therapeutic anti-cancer vaccines. In several cancers, protein kinase silencing via small interfering RNA can also be another strategy in protein kinase inhibition, as anti-cancer therapy.

mTOR inhibition. TSC1 or TSC2 gene mutation results in non-effectiveness of an upstream negative regulator of mTOR and the constitutive activation of the mTOR pathway, eventually unregulated cellular growth (Roskoski Jr 2019b).

2.2

CDK Inhibitors

CDK family is 21 protein serine-threonine kinases and regulates cell growth, proliferation and transcriptional regulation via affecting cell cycle. In this family, CDKs 1–6, 11 and 14–18 regulate the cell cycle and CDKs 7–13, 19 and 20 2.1 mTOR Inhibitors regulate transcription. Loss of regulation of the cell cycle (CDK11/14) and transcription (CDK mTOR, is a protein serine-threonine kinase, regu- 8/9), one of the hallmarks of cancer, results in lates cell growth, proliferation, survival, autoph- carcinogenesis. In several malignancies, there are agy, metabolism, and cytoskeletal organization. increased levels of (1) CDKs, (2) cyclins or (3) Its activity is dysregulated in many diseases, decreased levels of endogenous CDK modulaincluding cancer. Three clinically approved anti-­ tors/inhibitors, such as INK4 or CIP/KIP. cancer/immunosuppressant mTOR inhibitors are There is an overexpression of CDK1 in diffuse sirolimus, temsirolimus and everolimus. They are large Bcell lymphomas, melanomas; CDK2/4/8 in all natural product based macrolides, and bind to colorectal carcinomas; CDK4 in cervical cancer, FK binding protein (FKBP)-12 in order to gener- liposarcomas, osteosarcomas, rhabdomyosarcoate a complex that inhibits mTOR. mTOR is in mas, melanomas, colorectal cancer, and NSCLC; two multiprotein complexes, mTORC1 and CDK6  in gliomas, leukemias, lymphomas, and mTORC2; only mTORC1 is sirolimus sensitive. medulloblastoma via p53 and Rb; CDK8  in Sirolimus binds to FKBP-12 that binds to mTOR colorectal and gastric carcinomas; CDK9 in panfor its inhibition. mTOR initiates the ribosomal creatic neuroendocrine tumors and neuroblastotranslation of mRNA into proteins for cellular mas; CDK11  in osteosarcomas, and CKD14  in growth, cell cycle progression, and metabolism, esophageal carcinomas (Roskoski Jr 2019a). via its downstream effectors 4E-BP1 and S6K Therefore, several CDK inhibitors can be used as (Chiarini et al. 2015; Tavares et al. 2015). 4E-BP1 anti-cancer therapy. As an example, Palbociclib, is a key effector of the oncogenic activation of the a selective CDK4/6 inhibitor, is used in the treatAKT and ERK signaling pathways that integrates ment of ER+/HER2- advanced breast cancer, their function in several tumors (She et al. 2010). similar to other FDA-approved abemaciclib and mTOR is regulated positively by growth factors ribociclib in the treatment of HR+/HER2and their receptors, like Insulin growth factor-1R advanced breast cancer (Roskoski Jr 2016b; (IGF-1R), EGFR, VEGFR1/2/3. mTOR is regu- Whittaker et al. 2017). lated negatively by phosphatase and tensin In MAPK pathway, ERK1/2 activation prohomolog (PTEN), TSC1 (tuberous sclerosis motes the transcription of the D-type cyclins via ­ complex-1 or hamartin), and TSC2 (tuberin). the activity of FOS- and JUN-related transcripTSC1 and TSC2 form a complex that inhibits tion factors in the G1-phase of the cell cycle. The mTOR. Inhibiting antigenic and cytokine (IL-2, D-type cyclins activate with CDK4 and CDK6 IL-4, and IL-15)-induced T lymphocyte activa- (regulators of the G1-S transition) to generate tion and their proliferation, and inhibiting anti- active kinase complexes that directly phosphorybody production are the effects of sirolimus via late the retinoblastoma protein (RB). Histone

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acetylation and activated transcription of many genes, such as FOS and MYC. CDK8 phosphorygenes, including cyclin E, occur following the lates STAT1, suppresses natural killer cells, so its dissociation of both Histone deacetylase (HDAC) inhibition is cytotoxic to cancer cells. CDK8 also 1 and the E2F transcription factors from phosphorylates SMAD and NOTCH1 proteins. RB. CDK4/6 also phosphorylate the transcription CDK8 may function as an oncogene, especially factor FOXM1, promoting the G1/S transition in the development of colorectal cancer. Both and tumorigenesis, and suppressing senescence. CDK8 and CDK19 are potential therapeutic tarFollowing RB phosphorylation, cyclins E1 and gets in advanced prostate cancer, where siRNA E2 bind and activate CDK2. E2F1 also stimulates knockdown or small molecule inhibition the transcription of genes coding for proteins decreased invasion and migration (Bragelmann involved in DNA replication, such as cyclin A, et al. 2017). CDK8 activates β-catenin in gastric which becomes the predominant cyclin bound to and colon cancers. In addition, CDK8 acts by CDK2 in S phase and binds CDK1 in the late S/ phosphorylating E2F1, preventing it from proG2 phase. Activation of the transcription factors moting the degradation of β-catenin. CDK10 FOXM1 and FOXK2 through CDK1/cyclin A binds the N-terminal domain of the ETS2 tranthat is followed by CDK1/cyclin B, promotes the scription factor and suppresses the ETS2 transacmitotic progression via gene expression. CDK2-­ tivation domain. As a tumor suppressor, it inhibits cyclin A, CDK1-cyclin A, and CDK1-cyclin B the oncogenic potential of MAPK signaling. are crucial for S-, G2-, and M-phase progression. CDK11 is highly expressed in triple negative Cyclin A2 peaks at the G2-phase and cyclin E1 breast cancers and is associated with both an peaks at G1-phase (Whittaker et al. 2017). advanced stage and poor clinical prognosis (Zhou The first checkpoint G1-S enzymes include et al. 2016). Silencing of CDK11 expression sigCDK4, CDK6, and the D-type cyclins. The nificantly inhibits migration and proliferation, CDK2-cyclin E complex is required for the tran- and induces apoptosis in breast cancer, osteosarsition to the S-phase. G2-M is the second check- coma, liposarcoma and multiple myeloma cells. point as the M-phase CDK1-cyclin A/B complex Elevated CDK11 expression is associated with is activated, so carrying the cell to metaphase poor prognosis in osteosarcoma, liposarcoma and during mitosis. The metaphase to anaphase tran- multiple myeloma cells. LRP6 activation is a key sition is the third checkpoint, which leads to regulator for WNT signaling. This is mediated by sister-­ chromatid segregation, completion of the CDK14/cyclin Y complex, which phosphorymitosis, and cytokinesis as a single cell com- lates LRP6 at the plasma membrane in pletes cell division and forms two daughter cells. G2/M.  CDK14 is associated with increased Progression results when the M-phase cyclin-­ motility and metastatic potential, highly CDK complexes stimulate the anaphase-­ expressed in several malignant tumors, such as promoting complex, which results in the hepatocellular carcinoma, esophageal cancer, proteolytic destruction of proteins that hold the breast cancer, and gastric cancer. It has an imporsister chromatids together. tant role in the regulation of the cell cycle, tumor DNA damage checkpoints occur at the G1/S, proliferation, migration, and invasion (Yang et al. G2/M and S-phase. CDK1 activity is a major 2016). determinant of cell cycle progression and the Most of these small molecule CDK inhibitors DNA damage response via BRCA1, ATM and are type I inhibitors, that bind at the ATP–binding ATR. ATM or ATR catalyzes the phosphorylation site, are ATP-competitive and target the kinase in checkpoint kinase-1 (Chk1). The physiological its active state. Alvocidib (inhibits CDKs 1, 2, 4, CDK antagonist p21/CIP/WAF1 is induced 6, 7 and 9; in clinical trials for the treatment of through downstream of ATM or ATR and can breast, endometrial, and hematological maligarrest the cell cycle at checkpoints by inhibiting nancies) and seliciclib (inhibits CDKs 1, 2, 5, 7 CDK-cyclin complexes. In addition, CDK12 reg- and 9) are the first panCDK inhibitors in clinical ulates the expression of several cancer-associated use. Among these drugs’ effects, CDK7 or CDK9

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inhibition is responsible for the transcriptional inhibition of cell cycle (G1/G2 arrest) and apoptosis. Dinaciclib is a multitarget inhibitor of CDK1, CDK2, CDK5 and CDK9, blocks DNA replication and used in clinical trials for the treatment of breast and pancreatic cancer and several leukemias. Roniciclib is an another CDK multikinase inhibitor (inhibits CDK1, 2, 3, 4, 7 and 9) in clinical trials for the treatment of ovarian, thyroid and lung cancer. Trilaciclib (CDK 4/6 inhibitor; for breast/lung cancer) and voruciclib (CDK9 inhibitor; for CLL/melanoma) are CDK inhibitors that are currently in clinical trials for the treatment of several cancers. Riviciclib is a CDK1/4/9 inhibitor in clinical trials for the treatment of several cancers. Palbociclib, the first selective CDK inhibitor, inhibits CDKs 4 and 6, RB phosphorylation and the proliferation of human breast, colon, lung and leukemia cells. It is clinically used in the treatment of ER+/HER2– advanced breast cancer. Another selective inhibitor of CDK4 and CDK6, Abemaciclib inhibits phosphorylation of RB and RNA polymerase II and induces G1 arrest in HR+/HER2– advanced or metastatic breast cancer, KRAS mutant NSCLC, melanoma, colorectal cancer and dedifferentiated liposarcomas, especially in BRAF inhibitor (vemurafenib)-resistant cells. Ribociclib is a selective CDK4/6 inhibitor, inactivates RB, causes G0/G1 arrest, and inhibits proliferation in liposarcoma and HR+/HER2- advanced or metastatic breast cancer (Roskoski Jr 2016a, 2019b). Transcription-regulating CDKs are CDK7 and CDK9. CDK7 initiates transcription by phosphorylating the RNA polymerase II. CDK7 also mediates the phosphorylation and activation of other CDKs, known as a CDK-activating kinase. ‘Achilles cluster’ of super-enhancer-regulated and CDK7-dependent genes are required for ­cancer cell survival, so the tumor cells are dependent on or addicted to CDK7 for their survival (Wang et al. 2015). Besides CDK9 regulates the transcription of genes encoding short-lived anti-­ apoptotic proteins, such as MCL1 and XIAP, which are critical for the survival of transformed cells. For example, CDK9 inhibitor wogonin blocks RNA polymerase II phosphorylation and induces apoptosis via reduced MCL1 expression

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in leukemic T-cells (Polier et  al. 2011, 2015). CDK7/9 inhibitor CDKI-73 is more potent than the pan-CDK inhibitor alvodicib in chronic lymphocytic leukemia with overexpressed the anti-­ apoptotic BCL2 family genes. It is selective to primary leukemia cells and synergistic with fludarabine (Walsby et al. 2011). CDK 8/19 inhibitors are sorafenib, cortistatin A, and senexin A/B. Sorafenib is the first Type II inhibitor among CDKs. Cortistatin A has anti-­ leukemic activity through inducing the expression of super-enhancer-associated genes (Pelish et al. 2015). Both Senexins inhibit p21-activated transcription and oncogenic beta-catenin activity, induce EGR1 mRNA and increase the efficacy of chemotherapy in human lung carcinoma (Porter et  al. 2012). Senexin B has improved solubility and potency, compared to A (Rzymski et  al. 2015). CDK12 and 13 regulate DNA damage response and super-enhancer-associated gene expression. CDK12/13 inhibitor THZ531 induces apoptosis in leukemic T cells and inhibits the aberrant transcription and genomic instability that are the hallmarks of cancer. The synthetic lethality between CDK12/13 inhibitors and tamoxifen or PARP inhibitors has a novel potential in the combination treatment of human breast and ovarian cancers. Among all CDK inhibitors, CDK1 inhibitors are more sensitive in the treatment of KRAS mutant tumors, and CDK2/9 inhibitors have synthetically lethal effect in the treatment of MYC-­ addicted tumors (Costa-Cabral et al. 2016; Poon 2016). Palbociclib, ribociclib, and abemaciclib are FDA-approved CDK4/6 inhibitors in the treatment of breast cancer (Roskoski Jr 2016b). Cyclin D1 amplification occurs in 15–40% of all cancers, including melanomas and breast, lung, and oral carcinomas, via enhanced activation of CDK4 and CDK6, leading to G1-S progression. Increased Cyclin E expression is in enhanced malignant behavior of breast, colorectal, ovarian, and pancreatic carcinomas, chronic lymphoblastic leukemia, lymphomas through increased transition to the S-phase. Increased Cyclin A expression is correlated with poorer outcomes in patients with soft tissue sarcomas

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and endometrial, esophageal, hepatocellular, and thyroid tumors. Cyclin A also interacts with CKD2 to facilitate transit through the S-phase. Moreover, cyclin B expression has correlation with poorer outcomes in breast, esophageal and gastric cancers, and NSCLCs, since the CDK1-­ cyclin B complex supports entry into the M-phase of the cell cycle (Roskoski Jr 2019a). The INK4 group of CDK inhibitors acts on CDK4 and 6 to pass through the G1-phase of the cell cycle. The CIP/KIP family can inhibit most CDKs, especially increasing CDK4/6-cyclin D activity. p16/INK4 deletion/decrease is correlated with poorer patient survival in acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, NSCLC, melanoma, osteosarcoma, and retinoblastoma. In addition, reduced p21/CIP/WAF1 expression is associated with metastases and diminished survival in breast, colon, endometrial, and gastric carcinomas and Hodgkin lymphoma, because p21/CIP/ WAF1 slows the entry into the S- and M-phase of the cell cycle. Reduced p27/ KIP1 is correlated with tumor recurrence and poorer survival in breast, colon, esophageal, gastric, prostate carcinomas, and pancreatic neuroendocrine tumors. Moreover, aberrations in p57/ KIP2 expression occurs in ALL, bladder, colorectal, hepatocellular, ovarian, and pancreatic carcinomas (Roskoski Jr 2019a).

2.3

CLK and DYRK1A/B Inhibitors

The CLKs and DYRKs are protein serine/threonine kinases, which are activated by intramolecular tyrosine phosphorylation and both function in gene splicing. Especially, CLKs phosphorylate the serine- and arginine-rich proteins, those are the major components of the spliceosome. DYRK1A accumulates in nuclear speckles, where it activates splicing factors. It is involved in cellular processes related to proliferation and differentiation of neuronal progenitor cells. Therefore, their inhibitors can control splicing, proliferation and differentiation. Splicing defects have been linked to several diseases, including cancer. For example, DYRK1A is overexpressed in several malignancies, such as haematological

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and brain cancers (Soundararajan et  al. 2013). The DYRK1A kinase is involved in cellular repair of cancer cells that are impaired by chemotherapy and the resistance of cancer cells to pro-­ apoptotic stimuli. It enhances proliferation, migration, and decreases apoptosis, leading to aggressive cancers. It is also involved in neurological diseases and neoangiogenic processes (Ionescu et  al. 2012; Abbassi et  al. 2015). DYRK1A is a potent inhibitor of apoptosis (Fujita et al. 2015; Guo et al. 2010; Laguna et al. 2008; Seifert et  al. 2008; Seifert and Clarke 2009), an important component of signaling pathways in tumor suppression (Fernandez-­ Martinez et  al. 2015; Li et  al. 2014; Liu et  al. 2014), and has a capacity to mediate the activity of proteins in the progression of carcinogenesis (di Vona et  al. 2015; Zhang et  al. 2016; Liang et al. 2008b). DYRK1B is also overexpressed in several cancers, such as pancreatic cancer, ovarian cancer, lung cancer, rhabdomyosarcoma and breast cancer, via maintaining cellular quiescence by counteracting G0/G1-S phase transition. Specifically, DYRK1B controls the S phase checkpoint by stabilizing the CDK inhibitor p27Kip1 and inducing the degradation of cyclin D.  DYRK1B regulates cell cycle exit and promotes cell cycle arrest in an irreversible post-­ mitotic state during differentiation of muscle cells, or reversible quiescent state in cancer cells. DYRK1B also stabilizes the DREAM complex that represses cell cycle gene expression in G0 arrested cells. In addition, DYRK1B enhances cell survival by upregulating anti-oxidant gene expression and reducing intracellular levels of reactive oxygen species. Inhibiting DYRK1B promotes cell cycle re-entry and enhances apoptosis of those quiescent cancer cells with overexpressed DYRK1B, so small molecule DYRK1B inhibitors sensitize cells to the cytotoxic effects of anticancer drugs, such as cisplatin or anthracyclines, which target proliferating cells via inducing oxidative stress. Overall, inhibiting DYRK1B disrupts the quiescent state, reverse the chemoresistance of noncycling cancer cells, thus may enhance the anti-tumor effect of chemotherapeutic drugs (Friedman 2007, 2013; Becker 2018).

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2.4

MAPK/ERK/MEK Inhibitors

Thr kinases, including Raf, Mos and MEKK (for MEK kinase), all phosphorylate and activate Several growth factors activate the PI3K signal, MEK.  Overall, each kinase phosphorylates and causing activation of AKT, and the MAPK/ERK activates the subsequent kinase in the general pathway, leading to cell proliferation, survival, sequence MAPK kinase kinase kinase and dissemination. The PI3K/AKT pathway also (MAP4K)—MAPK kinase kinase (MAP3K)— promotes anabolism, while the MAPK/ERK MAPK kinase (MAP2K)—MAPK (Cerignoli pathway affects mainly cellular proliferation and et al. 2006; Keyse 2008a). RAS is activated via invasion. Activation of receptor tyrosine kinases either mutationally or triggered by protein recepand their downstream targets, PI3K, SRC, and tor tyrosine kinases. Following RAS activation, RAS upregulates MAPK/ERK pathway. RAF is recruited to the cell membrane where Activation of MAPK pathway (RAS-RAF-MEK-­ RAF is phosphorylated, leading stimulation of its ERK signal cascade) is essential in many signal serine-threonine kinase activity. Activated RAF transduction pathways, such as gene transcrip- triggers sequential phosphorylation and activation, protein synthesis, stability, localization, and tion of the MEK1/2 and ERK, which translocates enzyme activity, cellular proliferation, differenti- to the nucleus where they regulate the activity of ation, senescence, migration, survival, and apop- several transcription factors that induce the tosis (Roberts and Der 2007; Cseh et al. 2014). expression of multiple genes, which are required Normally, this pathway is responsible for tumor for survival and proliferation (Montagut and suppression by inducing senescence via cell Settleman 2009). cycle arrest, and ubiquitination and degradation Mitogens cause rapid phosphorylation and of proteins that are necessary for cell cycle activ- activation of ERK1/2, leading to cell proliferaity and survival. Abnormal activation of MAPK tion, survival and differentiation. In nucleus, they affects degradation of proteins, which are phosphorylate transcription factors and initiate required for both migration and progression DNA synthesis. Activated RAS causes malignant through the cell cycle. Activated MAPK func- transformation via activated ERK1/2 and their tions are proliferative responses to growth fac- substrates as transcription factors like Elk1, tors, cell cycle progression, differentiation, stress c-Jun, c-Myc and c-Fos, controlling genes in cell responses, gene regulation, cell death, cardiovas- cycle progression, such as cyclin D1 and p21 cular, neural and immune development, and cyto- (Adjei 2001a, 2001b). Transient ERK activation skeletal motility. Therefore, altered MAPK induces G1 and S phase progression via E2F signaling pathway is in cancer, diabetes, inflam- transcription factor and cyclin A, while continumatory and neurodegenerative disorders, since it ous ERK activation causes cell cycle arrest. phosphorylates and regulates growth factor ERKs also phosphorylate and activate keys receptors, transcription factors, cytoskeletal pro- enzymes in nucleotide and DNA synthesis, such teins and phospholipases. ERK (1/2), c-Jun as the carbamoyl-phosphate synthetase and the amino terminal kinases (JNK) (1/2/3), and p38 aspartate transcarbamylase. Moreover, ERK (α, β, δ and γ) subgroups are most common mem- involves in G2 phase regulation via MSK1- phosbers of the MAPK family. Constitutive activation phorylated histone H3. In addition, ERK activaof ERK and JNK induce differentiation, cell tion results in G2/M cell cycle arrest, following cycle arrest, and cell senescence (Keyse 2008a). DNA damage (Roskoski Jr 2012). Activation of MAPK requires phosphorylaIn the cytoplasm, ERK phosphorylates cytotion of both threonine and tyrosine residues of a skeletal proteins that affect cell movement and conserved signature T-X-Y motif within the acti- trafficking, metabolism and cell adhesion. On the vation loop of the kinase, which is catalyzed by a other hand, active ERK in the nucleus causes single protein kinase known as MEK. MEK itself activation of DNA synthesis and alteration of cell is activated by phosphorylation on two conserved cycle progression. In addition, by increasing the serine residues. Three distinct mammalian Ser/ expression of tumor necrosis factor alpha and

14  A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases…

inducible nitric oxide synthase, ERK affects innate immunity (Burotto et  al. 2014). Overall, all these functions of ERK are the components of the hallmarks of cancer. ERK is associated with the ability of cancer cells to grow independently of normal proliferation signals and is dysregulated in 30% of human tumors (Liu et al. 2018). P38 is also involved in inflammatory responses, apoptosis, embryonic development and cell cycle regulation. Phosphorylated transcription factor ATF2 causes transcriptional activation of cell cycle regulatory genes, such as cyclin A and cyclin D1 (Recio and Merlino 2002). P38 promotes G1/S transition via phosphorylated pRB and regulates the G2 phase, M phase and the spindle assembly checkpoint (Nath et al. 2003). Fibroblasts of p38α knockout mice are more susceptible to HRASV12-induced transformation, have decreased apoptosis, and tumor formation in their xenografts, so p38 acts as a tumor suppressor. Its deletion in mice leads to an immature and hyperproliferative lung epithelium that is highly sensitized to KRASG12V-induced tumorigenesis (Ventura et  al. 2007). Moreover, p38 promotes cellular senescence as a means of evading oncogene-induced transformation via p53 (Sun et al. 2007; Han and Sun 2007). Phosphorylated transcription factor ATF2 regulates JNK signal transduction pathway (Gupta et al. 1995). The cyclin-dependent kinase inhibitor p21 is a JNK’s substrate and JNKs are involved in all phases of the cell cycle. JNK1 is related with JNK kinase activity, while JNK2 is related with JNK kinase stability (Sabapathy et  al. 2004). JNK is also involved in stress-­ activated cell cycle checkpoints by ­phosphorylating p53 and inducing G1/S phase arrest, following DNA damage (Ronai 2004). JNK inhibitors cause cell-cycle arrest, an increase in DNA accumulation (4 N) and apoptosis; therefore, they can be used in the treatment of cancer. Several studies showed the effect of JNK activity in RAS-­ induced cell transformation in  vitro (Kennedy and Davis 2003), but RAS-transformed fibroblasts derived from mice lacking JNK 1/2 indicate that loss of JNK does not prevent tumor formation on their xenografts and their lung

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metastases were larger. JNK can act as a tumor suppressor in vivo (Kennedy et al. 2003). Finally, because of their critical roles in mediating stress-induced apoptosis, both p38 and JNK signaling are important determinants of cellular responses to conventional cancer therapies including chemotherapy and radiation (Dolado et al. 2007). MAPK/ERK signaling functions as either a tumor suppressor or an oncogene, depending on timing, duration, and intensity of its signal and the tissue having mutant activated MAPK (EGFR/RAS/BRAF). Genetic mutations, such as EGFR, KRAS and BRAF, constitutively activate the MAPK pathway. These mutations cause dysregulation of cell fate, genome integrity, and survival, leading to increased protein amplification, altered tumor microenvironment and induction and progression of tumorigenesis. Overall, targeting the MAPK pathway, the most frequently mutated signaling pathway in human cancer, is a promising strategy in the treatment of cancer, because of its effects on cancer cell survival, dissemination, and resistance to drug therapy. Both BRAF and MEK inhibitors are in clinical use, but acquired resistance due to cancer heterogeneity and genomic instability needs to be overcome to increase their efficacy. In the treatment of melanoma, BRAF and MEK kinase inhibitors are being used alone and in combination with inhibitors of the MAPK and other pathways to optimize treatment of many tumor types, such as colorectal cancer and ovarian cancer. For example, BRAF inhibitors, vemurafenib and dabrafenib, are used in patients with advanced stage, V600EBRAF-mutated metastatic melanoma. These patients had >80% inhibition of cytoplasmic ERK phosphorylation (efficacy of these drugs) and clinically partial remission (Bollag et al. 2010; Sosman et al. 2012; Chapman et al. 2011; Flaherty et al. 2010; Hauschild et al. 2012). The combination of dabrafenib (BRAFi) with trametinib (MEKi) in melanoma with BRAF V600 mutations caused less off-target effects (keratoacanthomas and squamous cell skin cancers), compared with dabrafenib alone (Flaherty et al. 2012a, 2012b; Robert et al. 2019). In addition, MEK inhibitor, selumetinib, is efficiently

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used in metastatic biliary cancer, metastatic papillary or poorly differentiated thyroid cancer and metastatic NSCLC with KRAS mutations (Bekaii-Saab et  al. 2011; Ho et  al. 2013; Janne et al. 2013).

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(GSK3β), the negatively regulated kinase by AKT phosphorylation. GSK3β activation results in phosphorylation and subsequent ubiquitin-­ mediated degradation of PHLPP (Li et al. 2009). AKT inhibition results in up-regulation of receptor tyrosine kinases (resistance to AKT inhibitors), through loss of mTORC1 signaling and 2.5 AKT Kinase Inhibitors FOXO-dependent transcriptional receptor tyrosine kinases increases (Chandarlapaty et al. 2011; AKT activity is regulated downstream of protein Garrett et al. 2011). receptor tyrosine kinases via activation of PI3Ks. PI3K/AKT pathway alterations are shown The PI3Ks phosphorylate phosphatidylinositol-­ almost 40% of all tumor types, with PTEN loss 4,5-bisphosphate (PIP2) to generate (30%), mutations in PIK3CA (13%), PTEN phosphatidylinositol-­3,4,5trisphosphate (PIP3). (6%) and AKT (1%) (Millis et al. 2016). AKT2 AKT binding to PIP3 at the plasma membrane is frequently amplified in cancer, mostly in paninduces a conformational change that results in creatic and endometrial cancer, since it is associserine-threonine phosphorylation and activation ated with the epithelial–mesenchymal transition of AKT. Active AKT phosphorylates FOXO pro- (EMT) and metastasis. AKT1 is amplified mostly teins, and by accumulating these proteins in cyto- in neuroendocrine prostate cancers, and AKT3 is plasm, AKT blocks their target genes in cell cycle amplified mostly in neuroendocrine prostate arrest and apoptosis. AKT inhibitors relocalize cancers and triple negative breast cancer. Even FOXO proteins to nucleus and induce cell cycle though AKT1 mutations (specifically arrest and apoptosis. Resistance to this induction AKT1E17K mutation) are 1% of all cancers, can be through Wnt/beta-catenin signaling AKT1 is the most frequently mutated isoform in (Tenbaum et  al. 2012) and Wnt inhibitors can tumors, characterized by constitutive activation overcome this resistance via beta-catenin reduc- at the cell membrane and can be suppressed by tion (Arques et al. 2016). Thus, nuclear FOXO3A both allosteric and catalytic inhibitors (Landgraf and beta-catenin can be potential predictive bio- et al. 2008). In addition, germline PTEN mutamarkers of drug response. tions result in Cowden’s syndrome; this synNegative regulation of the PI3K/AKT signal- drome is characterized by the development of ing pathway is by PTEN, PP2A (protein phos- multiple hamartomas and with increased risk of phatase 2A), PHLPP (PH domain and leucine carcinomas of the breast, thyroid, endometrium rich repeat protein phosphatase 1) and polyphos- and kidney. Somatic PTEN loss is most common phate 4-phosphatase type II (INPP4B), via in endometrial and prostate carcinoma and gliodephosphorylation of PIP3 (for PTEN), PIP2 (for blastoma through PTEN mutations, insertions INPP4B) and AKT (for PP2A and PHLPP). and deletions (Hollander et al. 2011). AKT1 is important for G1-S checkpoint Since PI3K/AKT pathway alterations are ­transition, proliferation, and tumorigenesis, while common in malignancies, AKT inhibitors are AKT2 regulates cell-cycle exit via p21, and pro- used in the treatment of several malignancies. motes tumor invasion and metastatic dissemina- There are two types of AKT inhibitors: first group tion. PTEN deficient tumor cells depend on is allosteric inhibitors of the AKT PH-domain AKT2 via p21 upregulation for survival in pros- that prevent localization of AKT to the plasma tate and breast cancer, and glioblastoma (Chin membrane, thereby blocking AKT phosphorylaet al. 2014). The mTORC1 complex inhibits AKT tion and activation. The second group of inhibithrough its dephosphorylation by up-regulating tors comprise ATP- competitive inhibitors of PHLPP1 translation (Liu et  al. 2011; Li et  al. AKT. 2011). AKT activity is also self-limiting through Afuresertib (GSK2110183) and GSK2141795 its substrate Glycogen synthase kinase 3β (GlaxoSmithKline) are ATP-competitive

14  A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases…

i­nhibitors of AKT1–3. ARQ 092 and ARQ 751 (ArQule) are both highly potent allosteric inhibitors of wild type and mutant AKT, block ATP binding to the kinase site and inhibit AKT recruitment to the plasma membrane (Wu et  al. 2010; Kostaras et  al. 2020). AZD5363 (Astrazeneca) is a potent competitive kinase domain inhibitor of AKT1–3, which inhibits phosphorylation of AKT substrates and tumor growth in xenograft models. The presence of an activating PIK3CA mutation, AKT1E17K mutation or PTEN loss, in the absence of a significant KRAS, is significantly correlated with sensitivity to AZD5363 (Davies et al. 2012, 2015). AKT1E17K mutation is a potential predictive biomarker for response to AZD5363 monotherapy. Ipatasertib (GDC-0068), a potent and highly selective inhibitor of AKT1–3 that causes dose-dependent AKT signaling inhibition in cancer cells and xenograft tumor models (Blake et  al. 2012; Lin et  al. 2013; Saura et  al. 2017). MK-2206 (Merck; MSD) is the most clinically advanced allosteric inhibitor of AKT, especially AKT1/2. An alkylphospholipid, perifosine (D-21266; KRX0401; Aeterna Zentaris) accumulates in the plasma membrane and non-selectively inhibits AKT activation by disrupting the interaction between AKT and the phospholipids (Kondapaka et al. 2003; Hideshima et al. 2006). Since DNA-PK is an essential kinase in non-­ homologous end-joining-DNA double strand break repair pathway (Jette and Lees-Miller 2015), AKT inhibition hypersensitizes tumor cells to chemotherapy (Davies et al. 2002; Hirai et al. 2010; Vanderweele et al. 2004). There is a synergy between PI3K inhibition and PARP inhibition in BRCA1/2 mutant mice (Juvekar et  al. 2012), because PI3K inhibition impairs homologous recombination DNA repair via the loss of BRCA1/2 (Ibrahim and Abdel-Rahman 2012). There is also a synergy among receptor tyrosine kinases (EGFR/HER2)/PI3K/AKT/mTOR Inhibitors (Garcia-Garcia et  al. 2012; Puglisi et al. 2014). Tumors with PI3K/AKT and MAPK mutations require inhibition of both to suppress tumor growth and survival, due to significant crosstalk and redundancy between these pathways (Davies 2012; She et al. 2010). In addition, in castrate resistant prostate cancer, there is a

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reactivation of both the androgen receptor and the PI3K pathways (Morgan et  al. 2009). The PI3K/AKT pathway is activated in 30–50% of prostate cancers, via PTEN loss or up-regulation of androgen receptor by PI3K/AKT inhibitors, leading to resistance (Carver et  al. 2011). Intermittent dosing of AKT inhibitors result in a greater inhibition of AKT and delay drug resistance (Lopez and Banerji 2017; Stewart et  al. 2015).

3

Lipid Kinases

Inhibition of lipid kinases, predominantly the PI3K enzyme family is used in anti-cancer therapy, similar to protein kinases, as small molecules. These inhibitors specifically target the four class I catalytic subunits of PI3K, which are p110a, p110b, p110g and p110d. These subunits are encoded by the genes PI3KCA, PI3KCB, PI3KCG, and PI3KCD, respectively. Some PI3K inhibitors can inhibit the activity of all four class I PI3Ks, these are called pan-PI3K inhibitors. Selective inhibitors inhibit only one or more catalytic subunits, and dual inhibitors inhibit PI3K/ mTOR or PI3K/HDAC (Garrett et al. 2011). The mechanism of action of PI3Ks is similar to protein kinases, except they are phosphotransferases with an alcohol group as an acceptor, and their substrates can be carbohydrates, nucleotides, and lipids. Phosphorylation of PIP2 by PI3K to form PIP3, induces cellular proliferation and survival via their downstream effectors, such as AKT and mTOR. Since tumor suppressor PTEN is a negative regulator of PI3K phosphorylation, PI3K/AKT/mTOR inhibitors are widely used in anti-cancer therapy of a broad range of cancer forms (Wu et al. 2015). The first approved small molecule PI3Kdelta inhibitor idelalisib is selective to p110d, similar to PIK39. It used in the combination treatment of chronic myeloid leukemia, in addition to monoclonal antibody rituximab. Inactivation of p110d destroys regulatory T cell mediated immune tolerance to cancer cells (Ali et al. 2014). PI3K inhibitors are more efficient in combined anti-cancer treatments, because the other combined drug can overcome

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the negative feedback inhibition, resulting in reactivation of downstream receptor signaling (Garrett et al. 2011). Among the lipid kinases, sphingosine kinases, which catalyze the formation of cellular growth and migratory regulator sphingosine 1-­phosphate, are used in anti-cancer therapy.

4

Protein Phosphatases

Similar to protein kinases, protein phosphatases are classified by their target amino acid of their protein substrates. They mainly dephosphorylate tyrosine, so they are called protein tyrosine phosphatases (PTPs). The second group dephosphorylates serine-threonine, and the third group has dual specificity (Karlsson et al. 2006). Imbalance between PTK and PTP activity results in aberrant tyrosine phosphorylation, malfunctions in cellular proliferation, differentiation, survival/apoptosis, and motility, leading to carcinogenesis. Cancer-related PTP alterations can be mainly loss of expression, point mutation and single amino acid substitution, and amplification, overexpression and ectopic expression of PTPs. Therefore, PTPs are novel drug targets for cancer therapy (Keyse 2008b). Inhibitors of PTP1B, downstream of HER2/Neu; SHP2, has two SRC homolog 2 domains and important for growth factor-mediated signaling; the Cdc25 phosphatases, positive regulators of cell cycle progression; and the phosphatase of regenerating liver (PRL), promoters of tumor metastases are examples of potent PTP inhibitors. Cdc25s are PTPs, which contain a catalytic cysteine residue and are essential regulators of cell cycle transitions. Cdc25a is in Cdc25 family and crucial for S phase entry and the initiation of mitosis. Cdc25a can dephosphorylate CDKs, such as Cdk2 and Cdk4, and the homeodomain transcription factor cut, the protooncogene Raf-1 (decrease in Raf-1 kinase activity), EGFR, ERK (Jiang and Zhang 2008). PTPs can be products of tumor suppressor genes, such as PTEN (mutant in brain, breast, and prostate cancers) and RPTPρ, RPTPγ, LAR, PTPH1, PTP-BAS, and PTPD2 (mutant in colon cancer). On the other hand, PTPs and PTKs have

synergistic effects on mitogenic signaling and overexpression of PTPα results in SRC activation, leading to cell transformation, so PTPα is a physiological positive regulator of SRC kinase. Moreover, SHP2 is critical for the RAS-­ dependent cellular proliferation and survival. In Noonan syndrome, leukemias and solid tumors occur, it is caused by activating (gain of function) mutations in SHP2 (Jiang and Zhang 2008). Besides PRL can be oncogenes that promote cell growth and tumor invasion. PTP1B dephosphorylates activated EGFR and PDGFR. It enhances signaling in downstream of growth factor receptors and integrins, and promotes SRC family kinase activation. It also stimulates p120RASGap activity, resulting in activation of the RAS/ERK pathway. PTP1B can promote both the SRC kinase and the RAS/ERK pathways, which constitute major components of HER2/Neu signaling. Therefore, PTP1B may represent a new therapeutic target in breast cancer. PTP1B is upregulated in HER2/Neu-­ transformed cell lines and almost all breast tumors. In addition, PTP1B is critical for HER2/ Neu-induced breast cancer. Its deficiency suppresses HER2/Neu-induced mammary tumorigenesis and protects from lung metastasis (Julien et  al. 2007; Bentires-Alj and Neel 2007). Moreover, PTP1B is also critical for colon cancer via SRC activation (Zhu et al. 2007). Therefore, selective inhibition of PTP1B may be novel treatment strategy for breast and colon cancer. Upon growth factor or cytokine stimulation, SHP2 is activated, this is essential for the RAS-­ dependent signaling and for the growth factor-­ stimulated cell proliferation in leukemias and some solid tumors, such as gastric cancer (Jiang and Zhang 2008). Therefore, selective inhibition of SHP2 may be novel treatment strategy for these malignancies. The Cdc25 phosphatases positively regulate cell cycle progression and checkpoint response to DNA damage via removal of the inhibitory phosphates from tyrosine and threonine residues of CDKs. Three isoforms Cdc25A, Cdc25B, and Cdc25C are important in different phases of the cell cycle. Overexpression of Cdc25A and Cdc25B results in the loss of cell cycle ­checkpoint

14  A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases…

control, uncontrolled cell proliferation, and genetic instability, leading to carcinogenesis in breast cancer, pancreatic ductal adenocarcinoma, prostate cancer, and nonHodgkin’s lymphoma (Wang et al. 2005). Cdc25 phosphatase inhibitors are novel drugs in cancer treatment. Overexpression of PRL is correlated with enhanced cellular proliferation and anchorage-­ independent growth, leading to tumorigenesis and especially metastasis via enhanced motility and invasiveness (Liang et  al. 2008a). Mainly, overexpression of PRL3 is critical for metastasis in tumorigenesis, so it can be a potential drug target to prevent and/or treat metastases.

4.1

Dual-Specificity Phosphatases (DUSPs)/MAPK Phosphatases (MKP)

DUSPs remove phosphate groups from tyrosine and serine/threonine residues on their substrates, in contrast to kinases. They are vital signal transduction enzymes that crosstalk with protein kinases. As DUSPs dephosphorylate many key signaling molecules, including the MAPKs, DUSPs are transcriptionally regulated as downstream targets of MAPK signaling. They can either act as classical negative feedback regulators or mediate cross talk between distinct MAPK pathways. They are up-regulated in response to both mitogenic and/or stress stimuli at the transcriptional level. Therefore, their dysregulation causes many disorders, including inflammation, obesity and cancer. The importance of negative regulatory mechanisms in modulating the activity of oncogenic MAPK signaling points out that MKPs are novel drug targets for cancer therapy (Caunt and Keyse 2013). Activation/inactivation of MAPK is under tight regulation of both protein kinases and phosphatases. Since ERK activation requires phosphorylation of both threonine and tyrosine residues in the activation loop, its inactivation is also mediated by serine/threonine phosphatases, tyrosine phosphatase, and DUSPs through dephosphorylation of threonine and/or tyrosine residues in the kinase activation loop. The largest

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group of protein phosphatases that specifically regulates MAPK is the DUSP family phosphatases (Seternes et al. 2019). DUSPs are critical for the regulation of MAPK activity through gene transcription, protein modification or protein stability. Post-translational protein modifications, such as ubiquitination, phosphorylation, methylation, and acetylation, play important roles in the regulation of its protein stability and activity. Ubiquitination is essential in controlling protein degradation, activation, and interaction. For DUSPs, ubiquitination induces degradation of eight DUSPs, namely, DUSP1 (MKP1), DUSP4 (MKP2), DUSP5, DUSP6 (MKP3), DUSP7 (MKPX), DUSP8, DUSP9 (MKP4), and DUSP16 (MKP7). In addition, protein stabilities of DUSP2 and DUSP10 (MKP5) are enhanced by phosphorylation and increased by ERK and mTORC2, respectively. Methylation-induced ubiquitination of DUSP14 (MKP6) stimulates its phosphatase activity (Chen et al. 2019). A subfamily of DUSPs contains the MAP kinase-binding (MKB) motif or the kinase-­ interacting motif (KIM) that interacts with the common docking domain of MAPKs to mediate the enzyme–substrate interaction. KIM mediates differential recognition and binding of MAPK substrates and has either nuclear localization (NLS) or export (NES) signals for subcellular localization, so it is for the specific binding and substrate selectivity of the MKP for the different MAP kinase isoforms. DUSPs containing the KIM domain are typical DUSPs or MKPs (nuclear and/or cytoplasmic), except DUSP2 (PAC1), DUSP5, and DUSP8. These ‘typical’ MKPs, such as PAC-1, MKP1, MKP2, MKP3, MKP4, MKP5, MKP7, hVH3, hVH5, PYST2 and MK-STYX, share a CDC25 homology (CH2) region for MAPK docking. On the other hand, DUSPs without the KIM domain are atypical DUSPs. DUSP14 (MKP6) and DUSP26 (MKP8) have no KIM domain, but have the ability of dephosphorylating and inactivating MAPKs. Alternatively, DUSPs can control functions of MAPKs by sequestering them in the cytoplasm or nucleus, or by competing with MAPK substrates for binding to MAPKs.

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Inducible nuclear MKPs are DUSP1 (MKP1), DUSP2 (PAC1), DUSP4 (MKP2) and DUSP5 (hVH-3). Except DUSP5, these MKPs display a rather broad specificity for inactivation of the ERK, p38 and JNK MAP kinases. The cytoplasmic (ERK-specific) MKPs are DUSP6 (MKP3), DUSP7 (MKP-X) and DUSP9 (MKP4). The final group of MKPs in both the cytoplasm and nucleus are DUSP8 (hVH-5), DUSP10 (MKP5) and DUSP16 (MKP7), which dephosphorylate the p38 and JNKs (Keyse 2008a; Kidger and Keyse 2016). DUSP1 (MKP1) is a nuclear phosphatase, mitogen and stress inducible MKP and binds to JNK and p38 with stronger affinity, compared to its binding to ERK, leading to their dephosphorylation and inactivation, but ERK induces its proteasomal degradation, and activation of ERK phosphorylates DUSP1. DUSP2 favours dephosphorylation of ERK1/2 and p38 MAPKs, compared to its binding to JNK.  DUSP4 (MKP2) specifically inhibits ERK and JNK. ERK activation phosphorylates and induces DUSP4. Oxidation of catalytic cysteine within the active site of DUSPs, such as DUSP1 and 4, inactivates them and triggers their proteasomal degradation under oxidative stress (Owens and Keyse 2007). DUSP5 is stress activated MAPKs phosphatase, acts as an ERK selective phosphatase and only dephosphorylates ERK, so its overexpression results in both inactivation and nuclear translocation of ERK.  Its transcriptional induction via ERK-dependent growth factor stimulation and it can also bind and sequester inactive ERK in the nucleus. Its deletion in cells ­expressing mutant RAS results in cell proliferation, but its deletion in cells expressing mutant BRafV600E causes ERK-dependent cell cycle arrest, senescence and prevents cell transformation by this oncogene. DUSP5 is the nuclear counterpart of the inducible cytoplasmic ERK specific phosphatase DUSP6 (MKP-3) in regulating the spatiotemporal activity of the RAS/ERK pathway (Caunt et al. 2016). DUSP6 also specifically inhibits ERK. DUSP6 is decreased by reactive oxygen species, so it causes high ERK activity and acts as a classical negative feedback regulator of ERK activity in

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development and cancer. Because of the increased DUSP6 expression in response to elevated ERK signaling in tumours with RAS or BRAF mutations, DUSP6 may act as a tumour suppressor, except Ph + ALL and BRAF mutant malign melanoma (Furukawa et al. 2003). The ERK-induced DUSP6 phosphorylation triggers ubiquitination and proteasomal degradation of DUSP6. mTOR signaling and cellular reactive oxygen species accumulation also induce DUSP6 phosphorylation, leading to its ubiquitination and proteasomal degradation. Additionally, DUSP7, an ERK phosphatase, is ubiquitinated under hypoxic stress. DUSP8 (MKP3/6) specifically inactivates JNK. DUSP9 (MKP4), an ERK phosphatase, is associated with maintenance of the stemness of embryonic stem cells. DUSP10 is phosphorylated by mTORC2, leading to stabilization of DUSP10 and inactivation of p38. DUSP14 (MKP6) inactivates JNK, ERK, and p38, and is a negative regulator of T-cell receptor signaling by directly inhibiting ERK in T cells. Methylation-­ induced ubiquitination of DUSP14 promotes DUSP14 activation against T-cell activation. DUSP16 specifically inactivates JNK and is regulated by ubiquitin-mediated proteasomal degradation, and their ubiquitinations are regulated by phosphorylation. ERK phosphorylates DUSP16, resulting in enhancement of DUSP16 protein stability by preventing ubiquitination. Induction of DUSP16 strongly suppresses JNK activation. Therefore, the activation of the ERK pathway can strongly inhibit JNK activation by stabilizing DUSP16. DUSP6 downregulation via epigenetic silencing and loss of heterozygosity is shown in human pancreatic adenocarcinoma and lung cancer (Okudela et  al. 2009; Furukawa et  al. 2003). However, its upregulation is shown in human glioblastoma and papillary thyroid carcinoma through increased cell proliferation, migration and invasion. Its upregulation is also correlated with enhanced growth and invasion in melanoma, and is dependent on both BCR-Abl and ERK activity as evidenced by sensitivity to a tyrosine kinase inhibitor (TKI) (Imatinib) or MEK inhibitor. DUSP6 mRNA levels are inversely correlated

14  A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases…

with survival in Ph + ALL and its inhibitors can be used in its treatment. DUSP6 downregulation also causes drug resistance to anti-EGFR therapy (gefitinib and erlotinib) in EGFR mutant lung cancer. This resistance is caused by increased ERK-dependent phosphorylation of proapoptotic BIM EL and decreased ERK induced transcription facto Ets1. Low DUSP6 levels are also correlated with ERK-dependent crizotinib resistant ELM4–ALK positive lung adenocarcinoma, MET TKI resistant gastric cancer and cisplatin resistant ovarian cancer (Kidger and Keyse 2016). DUSP2 downregulation induces stemness, increases ERK activation and causes chemoresistance in colon cancer (Hou et  al. 2017), some solid tumors, such as ovarian (poor outcome), and acute myeloid leukemia via constitutive ERK activation (Kim et al. 1999). DUSP3 downregulation is shown in NSCLC (Wang et  al. 2011). DUSP3 deficiency causes enhanced cancer cell migration (Chen et al. 2017). DUSP5 downregulation is shown in human gastric and colorectal cancers (Shin et al. 2013; Togel et al. 2018). Loss of DUSP5 expression is correlated with poor clinical outcome in advanced gastric and prostate cancers. Moreover, both DUSP5 and DUSP6 are elevated in cancers having mutant RAS and RAF oncogenes. Overexpression of DUSP6 is seen in response to activated RAS. This may represent a compensatory increase in the negative feedback control of the ERK1/2 MAPK pathway (Caunt and Keyse 2013). Moreover, ERKdependent serpinB2 expression is increased with DUSP5 loss in mutant HRASQ61L cancers. Loss of serpinB2 is completely reversed the sensitivity to carcinogenesis that is caused by DUSP5 loss. DUSP5 acts as a tumour suppressor and regulates both the activity and localisation of nuclear ERK signaling in mutant HRASQ61L cancer (Rushworth et  al. 2014). Similarly, loss of DUSP6 is associated with the progression from pancreatic intraepithelial neoplasia to invasive ductal carcinoma (Ishida et  al. 2008). DUSP9 downregulation is also shown in several tumors through its anti-apoptotic effect (Owens and Keyse 2007).

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Furthermore, overexpression of DUSP7 is shown in acute leukemias. Altered expression of DUSP1 (MKP1) is shown in breast, pancreas, gastric, ovarian (poor outcome), lung, skin and prostate cancer. Its overexpression is associated with resistance to cisplatin, gemcitabine, anthracyclins, doxyrubicin, taxanes and TKIs (imatinib) through its anti-apoptotic effect via blocking JNK.  The DUSP1 (MKP-1) gene is a transcriptional target of the p53 tumour suppressor and is up-regulated by oxidative stress, DNA-­ damaging agents and hypoxia. In addition, altered expression of DUSP4 (MKP2) is shown in pancreatic, lung, ovarian, breast (with DUSP1), liver, thyroid and colon cancer. Its overexpression is also associated with resistance to doxyrubicin in gastric cancers and to trastuzumab (anti-Her2 antibody) in Her2+ breast cancers (Kidger and Keyse 2016; Keyse 2008a).

5

Conclusion

Even though the whole human kinome has not been studied yet, recent advances in the inhibition of kinases in tyrosine kinase-like groups, including CDK, MAPK, GSK3, DYRK and CLK kinase, PKA, PKG, and PKC kinase, and the calcium/calmodulin-dependent protein kinases are promising. Overall, the most clinically approved kinase inhibitors inhibit tyrosine kinases, such as BCR-Abl, ErbBs, and VEGFRs, and only one lipid kinase inhibitor is in clinical use, when it is combined with other drugs. All kinase inhibitors are mainly used for anti-cancer treatment. Since trastuzumab (Herceptin) (monoclonal antibody against HER2) was first approved in 1998, protein kinases become the largest drug target group in current clinical use for anti-cancer therapy. Although some selective ones can be dual or multiple target inhibitors, most of them are similar in structure, reversible and less selective, due to the high sequence similarity around their ATP-­ binding pockets. In order to enhance the specificity, natural products, which have pharmacophores and scaffolds that differ from most synthetic kinase inhibitors, can be a useful source to construct libraries with expanded structural diversity.

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Most importantly, inhibitors with favorable selectivity or multiple target selectivity could be more suitable for cancer treatment, in order to balance efficacy and toxicity, reduce toxicity and overcome mutation-acquired drug resistance. Thankfully, the field of human genetics and efficient tumoral genotyping enhance the discovery of novel drug targets. Tumor cell biology differs during carcinogenesis, so drug sensitivity and efficacy depend on the stage of tumor progression. Tumors are also heterogeneous masses that contain several cancer cell populations. As genetic profiling of tumors help to develop novel treatments by identifying protein kinases as potential pathological drivers, combination therapies can overcome the risk of drug resistance through key points for tumor survival. For example, Dacomitinib and Osimertinib are used in specifically advanced/metastatic NSCLC with EGFR exon-21 L858R mutations or exon-19 deletions. Another example, BRAF mutations are associated with enhanced sensitivity to both BRAF and MEK inhibitors in melanoma, colorectal and thyroid cancers. Protein kinase inhibitors are perfect novel drug targets, with their crucial role in tumor development and their inherent pharmacological tractability. Inflammation is one of the hallmarks of cancer and essential for its progression, so the protein kinases that drive tumor development and those that drive inflammation (chronic inflammatory diseases) could be the same. Importantly, several therapies for cancer can improve clinical outcome in chronic inflammatory disease, and several anti-cancer protein kinase inhibitors can be used for inflammatory disease. Besides protein kinases have as well emerged as promising points of pharmacological intervention in a broad array of indications discussed previously: CNS disorders, ophthalmology, pain, cardiovascular disease, and complications of diabetes, osteoporosis, and hearing loss. For the future, the number of approved type III allosteric inhibitors (only binimetinib, cobimetinib and trametinib; MEK1/2 inhibitors) has to be increased, with kinases against different enzymes in different signal transduction modules. Additional irreversible inhibitors has to be

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discovered to target the dozens of enzymes carrying active thiol groups near their ATP-binding pocket. Besides altered cellular signaling via aberrant tyrosine phosphorylation is one of the hallmarks of cancer. Since both of them have crucial roles in cellular pathways that are regulated by tyrosine phosphorylation, both abnormal expression and/or activation of PTKs and PTPs are involved in carcinogenesis. Several PTK/PTP inhibitors have being used in clinical practice for cancer treatment. In addition to PK inhibitors, PTP1B, SHP2, Cdc25 and PRL phosphatases are potential targets for cancer therapy. An important group of phosphatases, DUSPs, are critical for dephosphorylating MAPKs and MAPKs control protein stability of DUSPs. The protein stability of DUSP1, DUSP4, DUSP5, DUSP6, DUSP7, DUSP8, DUSP9, and DUSP16 are regulated by ubiquitin-mediated proteasomal degradation via three ubiquitin E3 ligases and one deubiquitinase (ubiquitin-specific peptidase (USP)). The ubiquitination/proteasomal degradation of DUSPs are usually regulated by phosphorylation. The major kinase for DUSPs is ERK.  ERK phosphorylates DUSP1, DUSP4, DUSP6 and DUSP16. Both altered ERK activation and decreased DUSP1 protein levels are shown in cancer cells (Gioeli et al. 1999; Hoshino et al. 1999). Besides ERK, mTOR also regulates protein stability of DUSPs. mTORC2 phosphorylates DUSP10 and enhances its protein stability, leading to reduction of p38 activity. Moreover, mTOR signaling also induces DUSP6 phosphorylation, leading to its proteasomal degradation. Each MKPs can act as either tumor suppressor or proto-oncogenic regulators of RAS/ERK signaling and this may depend on the differing and tissue-specific thresholds of ERK activity that are either promote cell proliferation or cause cell cycle arrest/senescence or cell death. In malignancies that specific increased MKP promotes tumor growth, inhibitor of this specific MKP can be used in the treatment of its malignancy. As a part of future directions to improve tumoral response to chemotherapy, DUSP1 (MKP-1) and DUSP4 (MKP2) may be potential therapeutic targets against the resistance to chemotherapy. In

14  A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases…

summary, the crosstalk between protein phosphatases and protein kinases can be a novel drug target in the treatment of malignancies.

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Protein Kinases in Hematological Disorders

15

Mufide Okay and Ibrahim C. Haznedaroglu

JH2 domain of the JAK2 gene. The protein kinase inhibitor drugs targeting specific kinase molecules have already been developed and widely used in the field of Clinical Hematology. The existence of a local renin-angiotensin system (RAS) specific to the hematopoietic bone marrow (BM) microenvironment had been proposed two decades ago. Local BM RAS is important in hematopoietic stem cell biology and microenvironment. There are interactions among the local BM RAS and PK. For example, ACE2-ang(1-7)-Mas axis inhibits p38 MAPK/NF-КB signaling pathway. The Local BM RAS may have a role in the effect on PK in this biological spectrum. The aim of this review is to outline the functions of PKs in the pathobiology of hematologic neoplastic disorders.

Abstract

Cell signaling is an important part of the complex system of molecular communication that governs basic cellular activities and coordinates cell cycle machinery. Pathological alterations in the cellular information processing may be responsible for the diseases such as cancer. Numerous diseases may be treated effectively via the pharmacological management of cellular signaling. Protein kinases (PK) have significantly important roles in the cell signal transduction process. Protein kinases phosphorylate serine, threonine, tyrosine and histidine amino acids in a wide variety of molecular networks. Two main PK groups are distinguished; serine/threonine kinase and tyrosine kinases. MAPK (mitogen-activated protein kinases), ERK, EGFR (epidermal growth factor receptor), src, abl, FAK (focal adesion kinase), and JAK (janus family kinase) are considered as the main PK molecular networks. Protein kinases are closely related to the pathobiology of hematologic neoplastic disorders. For instance; JAKV617F point mutationcausing polycythemia vera and essential thrombocytosis occur at the position 617 in the M. Okay · I. C. Haznedaroglu (*) Hacettepe University, Medical School, Department of Hematology, Ankara, Turkey

Keywords

Protein kinases · Leukemia · Cancer · RAS · JAK-STAT · TKI · Signal transduction

1

Introduction and the Family of Protein Kinases

Cell signaling is an important part of complex system of communication that governs basic cellular activities and coordinates cell actions.

© Springer Nature Switzerland AG 2021 A. B. Engin, A. Engin (eds.), Protein Kinase-mediated Decisions Between Life and Death, Advances in Experimental Medicine and Biology 1275, https://doi.org/10.1007/978-3-030-49844-3_15

383

M. Okay and I. C. Haznedaroglu

384

Pathological alterations of the cellular information processing are responsible for numerous diseases including cancer. Protein kinases (PK) have essential molecules of the cell signal transduction in health and in disease. They perform protein phosphorylation and activation in the signal transduction process. PK are considered as two major groups; membrane specific PK and cytoplasmic tyrosine kinases. Those proteins are classified as the tyrosine and serine/treonine kinases based on their catalytic properties, i.e.; amino acid type which is phosphorylated (Pawson et al. 2002). Biologically active proteins within the membrane are called as the receptor tyrosine kinases (RTK). There are 56 trans-membrane proteins in the RTK family which can be subdivided into 20 different families (Kazi and Ronnstrand 2019). Those receptors include insulin receptor, growth factors (EGF, VEGF, the platelet-derived growth factor receptors (PDGFRA, PDGFRB), the stem cell factor receptor (SCFR-KIT) the colony stimulating factor 1 receptor (CSF1R), FMS-like tyrosine kinase 3 (FLT3), FGF, NGF) receptors and Ephrin receptors (Epha, EphB). RTKs contain a critical region responsible for the activation of cytoplasmic sections called as the tyrosine kinase region. In the resting cells, inactive and active conformations of RTK are in physiological equilibrium. Those receptors become active just after they connect with the stimulatory induction factor (Pawson 2002). Biological response modifiers such as interferon (IFN) acts on PK molecules via the mechanisms of apoptosis induction or cell cycle inhibition (Asmana Ningrum 2014) (Fig. 15.1). Serine/threonine protein kinases phosphorylate the serine or threonine amino acids. The activity of those protein kinases can be regulated by specific triggers such as DNA damage. Likewise, numerous chemical signals, including cAMP/cGMP, diacylglycerol, and Ca2+/calmodulin may have additional roles. One very important group of protein kinases are the MAP kinases (mitogen-­activated protein kinases) family. Other critical subgroups are the kinases of the ERK subfamily, typically activated by mitogenic signals, and the stress-activated protein kinases JNK

and p38. They interact with each other in (patho) biological events including in the genesis of hematological neoplasia. Cytoplasmic protein kinases are SRC, ABL, focal adhesion kinase (FAK) and Janus Family Kinases (JAK) proteins. In the resting cells, those proteins are inactive in the cytoplasm. They become active just after the stimulation of the cell via growth factors or cytokines, which are directed towards their targets in the cytoplasm or nucleotide (Blum-Jensen and Hunter 2001). Protein kinases can lead to the oncogenic transformation through four mechanisms; 1. Retroviral transduction of proto-oncogene 2. Genomic arrangements 3. “Gain of function (GOF)” mutations 4. Excessive synthesis of protein kinase in different neoplastic disorders. The mechanisms related to PK are also important in the genesis of hematological neoplastic disorders. Furthermore, the pharmacological development of the protein kinase inhibitor drugs targeting specific kinase molecules lead to the increased survival in a wide variety of hematological diseases (Table  15.1). The aim of this review is to outline the clinically relevant functions of PKs in the pathobiology of hematologic neoplastic disorders.

1.1

MAPK Signal Transduction Pathway

MAP kinase pathway acts as a cascade of kinases responsible for the receptor-mediated stimulation. The cascade system has a role for both the signal amplification and regulatory interactions. Signal transmission begins with the G-protein activation (Ras activation). Then, MAPKKK (MAP kinase kinase kinase), MAPKK (MAP kinase kinase) and MAPK (MAP kinase) activations lead to further cellular signaling events. MAPK activates cytoplasmic substrates including the cell skeleton elements, other protein kinases and/or transcription factors within the nucleus via the phosphorylation and related biological cellular responses (Kolch 2000).

15  Protein Kinases in Hematological Disorders

385

IFNa-2b Apoptosis

Antiproliferation

Stress/cytokine

UV

TNFa FAD

MAPKK 4/7

Akt P13K

IRS1/2

GTPase Rap15

Elk1 and AFT2

Bax

Sitokrom c P53

Raf

P53

Ras P2PA

P15

MEK1

Stress response

BID

Src kinase (ShC/Gbr2/SOS)

P38

JNK Apaf-1

Receptor

MAPKK 3/6

MEKK

Caspase-8

Growth factor

Bim

Caspase-9

P21

Cyclin D

ERK2

Cdc2

c-jun, Ets-1, Cmyc and P90 (CREB)

CDK2

Membrane blebbing

Caspase-3

pRb

S

G2

G1

M

Cyclin E DNA fragmentation

P27

CDK2

E2F Cyclin, cdc25 and PCNA

Cyclin E

Caspase-7

Cyclin E/A

CDK4

Cell cycle Caspase-6

P16

Cyclin A CDK2

Fig. 15.1  Molecular mechanism of apoptosis induction or cell cycle inhibition by biological response modifiers (such as IFN) in hematology. (Adapted from Asmana

Ningrum et  al. (2014) with the permission of copyright guidelines in Hindawi (https://www.hindawi.com/ journals/scientifica/guidelines/))

1.2

1.3

Ras/Raf/MEK/ERK Signal Transduction Pathway

This transmission pathway begins with the Ras activation and kinase cascade progresses with Raf (= MAPKK), MEK (= MAPKK) and Erk (= MAPKK) proteins, respectively. Ras and RAF are the proto-oncogenes involved within this process. Ras proteins must be placed in the membrane with proper modification, farnezylation after the translation to become biologically active. In the resting cells, Ras proteins are inactive, known as Ras-GDP. Upon the stimulation of the cell; GTP is connected, instead of GDP, and activated the triggered conformation, Ras-GTP.  Ras activation is a reversible process. There is an over-activation of Ras/Raf/MEK/ERK pathway in 30% of human cancers including hematological neoplasia (Kolch 2000; Lee Jr and Mccubrey 2002).

 I-3 Kinase/Protein Kinase B P Signal Transduction Pathway

The phosphoinositide-3 kinase (Pi-3K) family is responsible for the transmission of growth and life signals. Upon the stimulation of the receptor; PI-3K catalyzes the phosphorylation of inositol phospholipids in the cellular membrane. Phosphatidylinositol triphosphate (PIP3) is a lipid mediator which formed by this way. PIP3 is responsible for the activation of PIP 3-dependent kinases (PDK) and protein kinase B (PKB) (Blum-Jensen and Hunter 2001). Protein kinase B is a protein encoded by the AKT1 and AKT2 genes. Protein kinase B conformation affects the activity of various proteins within the cell. One of them is “rapamycin’s mammary target (mTOR)” protein (Vogt 2001). mTOR increases the translation of various proteins within the cellular compartments. The synthesized proteins are the

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Table 15.1  Protein kinase inhibitor drugs targeting specific kinase molecules in the field of clinical hematology Target kinase molecule BCR-ABL, c-kit BCR-ABL, src BCR-ABL c-kit, PDGF BCR-ABL, src BCR-ABL

Protein kinase inhibitor drug Imatinib Dasatinib Nilotinib

Primary clinical indication CML CML, ALL CML

Bosutinib Ponatinib

CML CML, ALL

BCR-ABL JAK1, JAK2

Radotinib Ruxolitinib

CML Myelofibrosis

Bruton kinase PI3K delta

Ibrutinib Copanlisib

CLL CLL

Bruton kinase PI3K alpha/delta

Acalabrutinib Idelalisib

CLL CLL

FLT3

Midostaurin

AML

FLT3

Gilteritinib

AML

Hedgehog

Glasdegib

AML

Mutant IDH-2

Enasidenib

AML

IDH1

Ivosidenib

AML

Proteasome Proteasome

Bortezomib Carfilzomib

MM MM

Proteasome

Ixazomib

MM

PDGF, c-kit

Sunitinib

GIST

Spleen tyrosine kinase

Fostamatinib

Immune thrombocytopenia

Clinical perspectives Increased survival in CML patients Increased survival in CML and ALL patients Increased survival in CML patients Increased survival in CML patients Increased survival in multi-TKI resistant CML and ALL patients Increased survival in CML patients Increased survival in patients with Myelofibrosis Increased survival in CLL patients Increased responses to chemoimmunotherapy in follicular lymphoma Increased responses in resistant CLL patients Increased responses to chemoimmunotherapy in CLL patients Increased responses to chemotherapy in AML patients Increased responses to chemotherapy in AML patients Increased responses to chemotherapy in AML patients Increased responses to chemotherapy in AML patients Increased responses to chemotherapy in AML patients Increased survival in MM patients Increased responses to chemotherapy in resistant MM patients Increased responses to chemotherapy in resistant MM patients Increased responses to chemotherapy in patients with GIST Candidate drug for chronic resistant ITP

Abbreviations: ALL acute lymphoblastic leukemia, AML acute myeloid leukemia, CLL chronic lymphocytic leukemia, CML chronic myelogenous leukemia, GIST gastrointestinal stromal tumor; ITP immune thrombocytopenic purpura

growth factors, oncoproteins, or regulatory proteins of the cell cycle.

1.4

Cytoplasmic (Non-receptor) Tyrosine Kinases Signal Transduction Pathway

The expression and/or activity of src tyrosine kinase increase during cancer development. Inactive endogenous src is activated by stimulus and kinase activity increase/restrictive factors on SH domains are eliminated. Signal transduction

mechanism triggered by src catalytic activity is important for cell growth, adhesion and migration (Jones et al. 2000).

1.5

 TAT Proteins and Signal S Transduction Pathway

“Signal transducer and activator of transcription (STAT)” pathway is a biologically important pathway in the cell. There are numerous STAT proteins in human cell (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6). The cytokine

15  Protein Kinases in Hematological Disorders

binds to the receptor on the cell surface (α-subunit) within the STAT pathway. The oligomerization occurs as either αα or αβ. The oligomerization process activates the JAK proteins associated with the receptor with cross phosphorylation. Activated JAK proteins also phosphorylate the relevant receptors. Those regions allow inactive STAT proteins in cytoplasm to interact with the receptor. STAT proteins are then separated from the receptor and come into the cell nucleus to interact with specific sequence of response elements on DNA to stimulate transcription of the target genes. STAT3 and STAT5 activities have impact on the malignant transformation of the cell. There are two main carcinogenic mechanisms; firstly, the uncontrolled sustained activation of STAT proteins, and secondly, the mutations within the c-terminal. In the disease of multiple myeloma, there are the uncontrolled neoplastic activations of IL-6 upon STAT 3 molecule (Puthier et  al. 2001). Cytokine synthesis, activation and stimulation of the JAK/STAT pathway (Fig. 15.2) by autocrine/paracrine pathway in leukemic blasts are among the mechanisms which can

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cause continuous STAT activation in malignancies (Benekli et al. 2003; Sasi et al. 2014; Quotti Tubi et al. 2016). BCR-ABL induces growth and transformation of the biochemical protein, independent of the growth factor in hematopoietic cells. This pathogenetic event can cause the JAK/STAT oncoprotein to be continuously active. The inhibition of apoptosis in relation to the STAT5 activity is triggered by imatinib mesylate with the decrements of the Bcl-xL expression. Moreover, STAT5 activity plays a role in the apoptosis resistance associated with the enhanced BCR-­ ABL function (Horita et al. 2000).

2

Protein Kinases in Hematological Neoplastic Disorders

Cellular signaling is an important essential part of the complex communication system governing the basic cellular activities and coordinates cell actions. Pathological alterations of the cellular information processing are responsible for

Cytokine Receptor

SOCS

1 JAK

JAK

P

P

2 3

STAT

Survival proliferation

STAT

Proteasome SOCS

Nucleus STAT dimer

Fig. 15.2  Activation and regulation of the JAK-STAT pathway. (Adapted from Sasi et al. (2014) with the permission of copyright guidelines in Hindawi (https://www.hindawi.com/journals/mbi/2014/630797/))

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diseases such as cancer. Protein kinases are closely related to the pathobiology of hematologic neoplastic disorders. For example, the JAK gene family, V617F point mutation can generate polycythemia vera, whereas essential thrombocytosis may take place with the position 617 in the JH2 domain of the JAK2 gene. The existence of a local renin-angiotensin system (RAS) specific to the hematopoietic bone marrow (BM) microenvironment had been proposed two decades ago. Local BM RAS is important in hematopoietic stem cell biology and microenvironment. The biological activities of angiotensin receptors, AT1 and AT2, and pathological alterations in their signaling could influence whether cancer cells undergo apoptosis or survive in response to RAS activation. RAS signaling has been implicated in enhanced survival and increased proliferation of cancer cells. The signaling through AT2 promotes apoptosis but the activation of AT1 could enhance anti-apoptotic signaling. AT1 signaling interacting with AT2 and/or the MAS receptor produces multiple regulatory, biological, inflammatory, angiogenic, proliferative and apoptotic actions. Hyperactivity of the RAS axis produces apoptosis in the heart. AT1-mediated apoptosis may be influenced by AT2. RAS inhibits ERK signaling for the activation of cellular death (Haznedaroglu and Malkan 2016; Haznedaroglu and Beyazit 2013). There are relations between local BM RAS and PK; such as ACE2-ag(1-7)-Mas axis inhibits p38 MAPK/ NF-КB signaling pathway. The Local BM RAS may have a role in the effect on PK in the biological spectrum. The hypothetical associations of RAS and PK shall be searched within this framework (Aksu et  al. 2006; Haznedaroglu and Malkan 2016; Haznedaroglu and Beyazit 2013). The Ph* chromosome was the product of a t(9;22)(q34;q11) reciprocal translocation between chromosomes. This translocation generates the BCR–ABL fusion oncogene. In the ABL gene, the break point is generally located in the second exon (a2), whereas in the BCR gene the breakage takes place usually in one of the three regions called major (M-bcr), minor (m-bcr), and micro-bcr (μ-bcr) break point

regions. Depending on the location of the chromosome breakage in the BCR gene, three different types of BCR–ABL proteins; p210 BCR–ABL gene (M-bcr) (CML), p190 BCR-­ ABL gene, p230 BCR-ABL gene (Zhou et  al. 2018).The p190 BCR–ABL form is mainly associated with Ph*-positive acute lymphoblastic leukemia (ALL) and rarely appears in patients with chronic myeloid leukemia (CML) and might correlate with an aggressive course of the disease. A third break point in the region of the BCR gene called μ-bcr results in the transcription of an e19/ a2 mRNA that codes a 230  kDa BCR–ABL protein. This form of fusion protein is associated with the rare Ph*-positive chronic neutrophilic leukemia (Zhou et al. 2018). Tyrosine kinase TKI group drugs can generate clinical, hematological, molecular, cytogenetic responses in CML (Saydam et al. 2018; Haznedaroglu 2013a, b). In acute myeloid leukemia (AML), the importance of tyrosine kinases has been well studied in recent years. The type III family of RTKs member FLT3 and cytoplasmic/ non-receptor tyrosine kinase family member SRC are critical for the genesis of AML (Kazi and Ronnstrand 2019).

3

Protein Kinases in CML

The acquisition of the BCR–ABL oncogene (especially the p210 BCR–ABL form) is the initiating step in the development of CML (Trela et  al. 2014) (Fig.  15.3). The BCR–ABL gene occurs in a single pluripotent leukemic HSC, which gains a proliferative advantage and/or aberrant differentiation capacity over its normal counterparts, giving rise to the expanded neoplastic myeloid compartment (Zhou et  al. 2018). This process is possible since the BCR– ABL onco-protein is constitutively active tyrosine kinase as a result of oligomerization via the coiled-coil region of BCR and a deletion of the inhibitory SH3 domain of ABL.  Auto-­ phosphorylation of p210 BCR–ABL on the Y177 tyrosine residue and leads to the phosphorylation of numerous downstream targets. The activation of various signaling pathways such as Ras/ mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), or signal

15  Protein Kinases in Hematological Disorders

Substrate Tyr PO4 ATP

ADP

BCR/ABL ATP

CML Fig. 15.3  BCR-ABL in the genesis of CML. (Adapted from Trela et al. (2014) with the permission of copyright guidelines in Hindawi (https://www.hindawi.com/ journals/isrn/2014/596483/))

transducer and activator of transcription 5 (STAT5) by BCR–ABL kinase leads to the tumor transformation in conjunction with the dysfunction of underlying cellular processes associated with the control of proliferation, differentiation, and survival (Zhou et  al. 2018). BCR–ABL-positive cells become independent of the presence of growth factors in the environment; those cells are characterized by the increased proliferation, apoptosis resistance, and genetic instability leading to the CML progression. Furthermore, impaired cell adhesion leads to their spread and the abnormal release of immature cells to the peripheral circulating blood in CML (Mauro and Druker 2001). Imatinib, the TKI, competitively inhibits the inactive configuration of the BCR-ABL1 protein tyrosine kinase by blocking the ATP binding site and thereby preventing a conformational switch to the active form (Savage and Antman 2002; Tsao et  al. 2002). Crystallographic analysis of BCR-ABL protein highlights a two-lobe catalytic domain: N- and C-lobes towards N- and C-terminus of the sequence, respectively. β-Sheets compose the former, whereas α-helices prevail in the latter. An important Wolker loop

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(also known as phosphate-binding or P-loop) links two β-sheets of N-lobe. The hinge region, which links the two lobes, also participates in ATP binding by two hydrogen bonds. Within the ATP-binding pocket, a “gatekeeper” residue, interacts with ATP, as well. Furthermore, the molecule plays a key role in conferring selectivity to some of the BCR-ABL inhibitors (Rossari et  al. 2018). Imatinib (STI571) inhibits cellular proliferation and tumor formation without inducing apoptosis, and produces a 92–98% decrease in CML colony growth in vitro without inhibiting normal colony growth (Holtz et  al. 2002; Druker et al. 1996). The second generation TKIs, including nilotinib, dasatinib, bosutinib inhibit both BCR-ABL1 and other signaling pathways. Dasatinib and bosutinib inhibit both BCR-ABL1 and SRC kinases. Nilotinib is an inhibitor of BCR-ABL1, KIT, and PDGFR. Both nilotinib and dasatinib are >100-fold more potent than imatinib in vitro. PKs are also important for the resistance to imatinib treatment. The resistance to treatment with tyrosine kinase inhibitors (TKIs) is divided into two major categories: primary and secondary (Shah 2007). The mechanisms of secondary resistance are varied but most commonly involve the reactivation of BCR-ABL1 signaling and/or the activation of other signaling pathways such as SRC kinase (Milojkovic and Apperley 2009). Nilotinib (AMN107) shows greater potency and effectiveness against almost the totality of resistance-conferring mutations, except for T315I and few others, in newly diagnosed Ph*+ CML patients (Kantarjian et al. 2011). Bosutinib (SKI-­ 606) has been developed from a leading Src inhibitor compound (Boschelli et  al. 2001). Third-generation TKI is ponatinib (AP24534), a dual Src/Abl inhibitor designed to especially overcome T315I mutation. Isoleucine in position 315 complicates BCR-ABL switching to inactive conformation and H-b formation with DFG-out inhibitors. Nonetheless, ethynyl linkage of ponatinib has indeed been inserted to accommodate isoleucine side chain without any steric interference also in inactive conformation (DFG-out) (Zhou et al. 2011).

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4

 rotein Kinases in Malignant P Myeloid Neoplasms

In acute myeloid leukemia (AML), the importance of tyrosine kinases has been understood in recent years. The type III family of RTKs member FLT3 and cytoplasmic/ non-receptor tyrosine kinase family member SRC are important for AML (Kazi and Ronnstrand 2019). FLT3 is expressed in hematopoietic progenitor cells of both myeloid and lymphoid lineages. Mutations of FLT3 are very common in AML.  They are found in about 25–35% of the patients with AML.  FLT3 monomeric receptors are inactive until extracellular domain interacts with the ligand. Juxtra-membrane domain and the kinase domain are important for the continuation of inactive state. Non-covalent ligand dimers bind a pair of receptors which is mediated through a single interaction site in the third Ig-like domain. Ligand-binding process could induce significant structural changes in the intracellular domain (Kazi and Ronnstrand 2019). The SRC family of non-receptor tyrosine kinase includes 11 different protein kinases. Those kinases are also important for the genesis of AML. BLK, FGR, FYN, HCK, LCK, LYN, SRC, YES are eight members of this family. The non-receptor tyrosine kinase regulates several signaling pathway downstream of FLT3. FYN, HCK, LCK, LYN, FGR and SRC interact with and to be activated by FLT3. In the 2016 revised WHO disease classification, Ph*+ AML is included as a new provisional entity (Norris and Stone 2008). Ph*+ AML is rare comprising