Tissue-Specific Cell Signaling [1st ed.] 9783030444358, 9783030444365

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Joana Vieira Silva Maria João Freitas Margarida Fardilha   Editors

Tissue-Specific Cell Signaling

Tissue-Specific Cell Signaling

Joana Vieira Silva Maria João Freitas Margarida Fardilha •

Editors

Tissue-Specific Cell Signaling

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•

Editors Joana Vieira Silva Laboratory of Signal Transduction, Department of Medical Sciences Institute of Biomedicine—iBiMED, University of Aveiro Aveiro, Portugal

Maria João Freitas Laboratory of Protein Phosphorylation and Proteomics, Department of Cellular and Molecular Medicine, Faculty of Medicine KU Leuven Leuven, Belgium

i3S—Instituto de Investigação e Inovação em Saúde University of Porto Porto, Portugal Laboratory of Cell Biology, Unit for Multidisciplinary Research in Biomedicine (UMIB) Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto Porto, Portugal

Margarida Fardilha Laboratory of Signal Transduction, Department of Medical Sciences, Institute of Biomedicine—iBiMED University of Aveiro Aveiro, Portugal

ISBN 978-3-030-44435-8 ISBN 978-3-030-44436-5 https://doi.org/10.1007/978-3-030-44436-5

(eBook)

© Springer Nature Switzerland AG 2020 Chapters 3 and 15 are 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 chapters. 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

Preface

Cells have evolved to sense external chemical and physical cues, transduce the signals intracellularly through molecular cascades, and generate an appropriate response. The outcome can be an altered gene-expression pattern or a change in cell shape or metabolism to optimize the use of nutrients or to avoid toxins. In multicellular organisms, signal transduction is also a key determinant of cell fate (proliferation, differentiation, or apoptosis) and cell–cell interaction. Over the last few decades, numerous signaling cascades have been unraveled. They have revealed how signals are sensed at the molecular level and transduced to generate an integrated cellular and organismal response. Most recent textbooks on cell signaling highlight the components, design principles, and crosstalk of signal transduction cascades that are common to (most) cell types. This book takes a more top-down approach and focusses on signaling processes that are cell-type or organ specific. These specialized signaling processes are key to understanding organ specialization and integration, but also the development of diseases. Chapter 1 describes neurotransmission in neurons and specific signaling pathways in glial cells that contribute to brain homeostasis. Glial cells support neurons (astrocytes), mediate myelination (oligodendrocytes), but can also function as macrophages (microglial cells). This is followed by an overview of inter- and intracellular communication in major endocrine glands, including the pituitary (Chap. 2) as well as the thyroid, parathyroid, and adrenal glands (Chap. 3). These glands secrete hormones into the bloodstream to coordinate complex peripheral processes. Subsequently, transduction cascades are described that are triggered through danger sensing in innate immune cells (Chap. 4) and culminate in the activation of T- and B-lymphocytes (Chap. 5). This activation also entails adhesion molecules and co-receptors that modify the transcriptional landscape in lymphocytes. Chapter 6 is devoted to the regulation of urine formation by the kidneys and urine transport in the urogenital tract. Distinct signaling cascades have evolved in the specialized epithelium (urothelium) of the upper and lower urinary tract. An adjacent organ is the prostate, an exocrine gland that nourishes and protects sperm (Chap. 7). Prostate development and homeostasis are regulated by androgens, mesenchymal–epithelial interactions, and growth factors. Chapter 8 details the main v

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functions of testis in testosterone (steroidogenesis) and sperm production (spermatogenesis), but also covers the regulation of the blood–testis barrier. This is followed by an update of the signaling events that control sperm motility and oocyte recognition (Chap. 9). Another hormone-regulated organ is the female mammary gland, which can differentiate into a milk-producing gland (Chap. 10). This differentiation entails a massive remodeling of mammary tissue after each pregnancy and is controlled by both steroid and peptide hormones. Chapter 11 discusses signaling in female gametogenesis, including oocyte differentiation from a germ-cell precursor and oocyte maturation. Specialized signaling also occurs in the heart, where cardiomyocytes, endothelial cells, vascular smooth muscle cells, and fibroblasts work together to regulate cardiac function (Chap. 12). Likewise, homeostasis of the adult lung is maintained by complex growth-factor signaling in epithelial and mesenchymal cells (Chap. 13). Chapter 14 discusses transduction pathways that determine skeletal muscle plasticity, in particular involving regulation of skeletal muscle mass, contractile activity, and metabolism (Chap. 14). The final chapter deals with adipocyte-specific signaling related to adipogenesis and adipocyte browning (Chap. 15). Leuven, Belgium

Mathieu Bollen

Acknowledgments

The authors acknowledge the support of Institute of Biomedicine—iBiMED, University of Aveiro (UIDB/04501/2020); Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto (Pest-OE/SAL/UI02015/2019); and Foundation for Science and Technology for the individual grant to J.V.S. (SFRH/BPD/123155/2016).

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Introduction

Unicellular and multicellular organisms depend on efficient communication with the surrounding environment. The evolutionary development of a plethora of signaling components has ensured efficient ways of extra-, intra- and inter-cellular communications. This process is called Cell Signaling or Cell Signal Transduction. The basic cell signaling unit shared by most signaling pathways contains core functional components: the signals, their receptors, and effectors (Fig. 1a). Cells communicate mainly through chemical signals usually secreted into the extracellular milieu from a sending cell and received by the target cell which must have the appropriate receptor for the initial signal. Binding to the receptor, the signaling molecule induces alterations on the receptor activity or conformation which is recognized in the interior of the cell triggering a chain of events that both carries and amplifies the signal leading to a certain response (Fig. 1b). The biochemical mechanisms of signal transduction are quite similar among cells: similar proteins and protein modules are used to detect and respond to signals. For example, cAMP acts as a second messenger in bacteria, fungi and animals. Nonetheless, while the basic signal components are the same or similar they may be used in very different arrangements and biological processes. In fact, cAMP is synthesized through different and distantly related enzymes in bacteria, fungi and animals and acts on different proteins. In the simplified picture of a cell signaling pathway described above the signaling components act sequentially and a certain signal induces a cell response. Today, it is well established that signaling pathways are much more complex. Signaling molecules/components act in different signaling pathways (pathway crosstalk) and may have multiple downstream partners (pathway branching). Pathway branching and crosstalk originate highly synchronized complex signaling networks conveying plasticity, robustness and variability. Tissue/organ specialization adds another level of complexity in multicellular organisms. This depends on the synchronized regulation of many physiological

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events such as cell growth and division, cell morphology, metabolism, differentiation and development. Thus, the tight coordination of gene expression, protein translation programs (including splicing, post-translational modifications and stability) and subcellular localizations is of utmost relevance. The availability of signaling components and their interconnections that confer cell-specific signal transduction patterns is the basis for the present textbook on Tissue-Specific Cell Signaling.

(a)

(b)

Fig. 1 Cell Signal Transduction refers to all processes involved in receiving and translating an extracellular signal, allowing the cell to respond appropriately. a Most signaling molecules (with some exception such as thyroid and steroid hormones and nitric oxide) act on plasma membrane receptors without penetrating the cell. Cells can synchronize and process multiple signals to generate a response. b The response to an extracellular signal depends on the presence of a receptor and on the cellular context of the target cell. For instance, one cell-type can reply to the same signal with secretion, other with contraction and other with decreased rate of contraction. Cells that lack the appropriate receptor do not respond. If a target cell secretes a signal, then the target cell can affect the cell it received the signal from

Contents

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Beyond Brain Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cátia D. Pereira, Filipa Martins, Fernanda Marques, João Carlos Sousa, and Sandra Rebelo

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Cell Signalling Within Pituitary, the Master Gland of the Endocrine System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sofia S. Pereira, Carolina B. Lobato, and Mariana P. Monteiro

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Cell Signaling Within Endocrine Glands: Thyroid, Parathyroids and Adrenal Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sofia S. Pereira, Carolina B. Lobato, and Mariana P. Monteiro

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Signaling Pathways Governing Activation of Innate Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bruno M. Neves and Catarina R. Almeida

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Cell Activation and Signaling in Lymphocytes . . . . . . . . . . . . . . . . 133 Alexandre M. Carmo and Sónia N. Henriques

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Signaling Pathways Involved in Kidney and Urinary Tract Physiology and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 João Lobo and Rui Henrique

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More Than Androgens: Hormonal and Paracrine Signaling in Prostate Development and Homeostasis . . . . . . . . . . . . . . . . . . . 195 Juliana Felgueiras, Vânia Camilo, Margarida Fardilha, and Carmen Jerónimo

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Testicular Signaling: Team Work in Sperm Production . . . . . . . . . 225 Joana Santiago, Daniela Patrício, and Joana Vieira Silva

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Sperm Signaling Specificity: From Sperm Maturation to Oocyte Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Maria João Freitas, Daniela Patrício, and Margarida Fardilha

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10 Hormone Signaling Pathways in the Postnatal Mammary Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Fátima L. Monteiro, Inês Direito, and Luisa A. Helguero 11 Oogenesis Signaling from Development to Environmental Plasticity and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 Bruno Marques, Ricardo Matos, and Rui Gonçalo Martinho 12 Key Signaling Pathways in the Cardiovascular System . . . . . . . . . . 337 Fábio Trindade, Inês Falcão-Pires, Andreas Kavazis, Adelino Leite-Moreira, Daniel Moreira-Gonçalves, and Rita Nogueira-Ferreira 13 Growth Factor Signaling in the Maintenance of Adult Lung Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 Henrique Araújo-Silva, Jorge Correia-Pinto, and Rute S. Moura 14 The Signaling Pathways Involved in the Regulation of Skeletal Muscle Plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 Alexandra Moreira-Pais, Francisco Amado, Rui Vitorino, Hans-Joachim Appell Coriolano, José Alberto Duarte, and Rita Ferreira 15 Adipocyte Specific Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409 David F. Carrageta, Pedro F. Oliveira, Mariana P. Monteiro, and Marco G. Alves

Editors and Contributors

About the Editors Joana Vieira Silva Laboratory of Signal Transduction, Department of Medical Sciences, Institute of Biomedicine—iBiMED, University of Aveiro, Aveiro, Portugal; i3S—Instituto de Investigação e Inovação e Saúde, University of Porto, Porto, Portugal; Laboratory of Cell Biology, Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal. e-mail: [email protected] Maria João Freitas Laboratory of Protein Phosphorylation and Proteomics, Department of Cellular and Molecular Medicine, Faculty of Medicine, KU Leuven, Leuven, Belgium. e-mail: [email protected] Margarida Fardilha Laboratory of Signal Transduction, Institute for Research in Biomedicine—iBiMED, Medical Sciences Department, University of Aveiro, Aveiro, Portugal. e-mail: [email protected]

Contributors Catarina R. Almeida Department of Medical Sciences, iBiMED—Institute for Biomedicine, University of Aveiro, Aveiro, Portugal Marco G. Alves Laboratory of Cell Biology, Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), Department of Microscopy, University of Porto, Porto, Portugal Francisco Amado QOPNA & LAQV, Department of Chemistry, University of Aveiro, Aveiro, Portugal

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Henrique Araújo-Silva Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal; ICVS/3B’s - PT Government Associate Laboratory, University of Minho, Braga/Guimarães, Portugal Vânia Camilo Cancer Biology and Epigenetics Group—Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal Alexandre M. Carmo IBMC—Instituto de Biologia Molecular e Celular, Porto, Portugal; i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal David F. Carrageta Laboratory of Cell Biology, Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), Department of Microscopy, University of Porto, Porto, Portugal Hans-Joachim Appell Coriolano Physiology and Anatomy, German Sport University, Cologne, Germany Jorge Correia-Pinto Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal; ICVS/3B’s - PT Government Associate Laboratory, University of Minho, Braga/Guimarães, Portugal; Department of Pediatric Surgery, Hospital de Braga, Braga, Portugal Inês Direito Hormones and Cancer Research Group, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal José Alberto Duarte Faculty of Sport, CIAFEL, University of Porto, Porto, Portugal Inês Falcão-Pires Department of Surgery and Physiology, Cardiovascular R&D Center, Faculty of Medicine of the University of Porto, Porto, Portugal Margarida Fardilha Laboratory of Signal Transduction, Department of Medical Sciences, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal; Laboratory of Protein Phosphorylation and Proteomics, Faculty of Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Juliana Felgueiras Laboratory of Signal Transduction, Department of Medical Sciences, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal; Cancer Biology and Epigenetics Group—Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal Rita Ferreira QOPNA & LAQV, Department of Chemistry, University of Aveiro, Aveiro, Portugal

Editors and Contributors

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Maria João Freitas Laboratory of Protein Phosphorylation and Proteomics, Faculty of Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Luisa A. Helguero Hormones and Cancer Research Group, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal Rui Henrique Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal; Cancer Biology and Epigenetics Group, Research Center of Portuguese Oncology Institute of Porto (GEBC CI-IPOP) and Porto Comprehensive Cancer Center (P.CCC), Porto, Portugal; Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS-UP), Porto, Portugal Sónia N. Henriques IBMC—Instituto de Biologia Molecular e Celular, Porto, Portugal; i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; Programa Doutoral em Biologia Molecular e Celular (MCbiology), Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal Carmen Jerónimo Cancer Biology and Epigenetics Group—Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal; Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal Andreas Kavazis School of Kinesiology, Auburn University, Auburn, AL, USA Adelino Leite-Moreira Department of Surgery and Physiology, Cardiovascular R&D Center, Faculty of Medicine of the University of Porto, Porto, Portugal; Department of Cardiothoracic Surgery, Centro Hospitalar Universitário São João, Porto, Portugal Carolina B. Lobato Endocrine, Cardiovascular and Metabolic Research, Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, Porto, Portugal; Department of Anatomy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal João Lobo Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal; Cancer Biology and Epigenetics Group, Research Center of Portuguese Oncology Institute of Porto (GEBC CI-IPOP) and Porto Comprehensive Cancer Center (P.CCC), Porto, Portugal; Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS-UP), Porto, Portugal

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Editors and Contributors

Bruno Marques Center for Biomedical Research (CBMR), Universidade do Algarve, Faro, Portugal Fernanda Marques Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, Braga, Portugal; ICVS/3B’s, PT Government Associate Laboratory, Braga/Guimarães, Portugal Rui Gonçalo Martinho Center for Biomedical Research (CBMR), Universidade do Algarve, Faro, Portugal; Department of Medical Sciences, Institute for Biomedicine (iBiMED), Universidade de Aveiro, Aveiro, Portugal; Instituto de Medicina Molecular, Universidade de Lisboa, Lisbon, Portugal Filipa Martins Neuroscience and Signaling Laboratory, Department of Medical Sciences, Institute of Biomedicine (iBiMED), University of Aveiro, Aveiro, Portugal Ricardo Matos Center for Biomedical Research (CBMR), Universidade do Algarve, Faro, Portugal Fátima L. Monteiro Hormones and Cancer Research Group, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal Mariana P. Monteiro Endocrine, Cardiovascular and Metabolic Research, Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, Porto, Portugal; Department of Anatomy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal Daniel Moreira-Gonçalves Department of Surgery and Physiology, Cardiovascular R&D Center, Faculty of Medicine of the University of Porto, Porto, Portugal; Faculty of Sport, CIAFEL, University of Porto, Porto, Portugal Alexandra Moreira-Pais Faculty of Sport, CIAFEL, University of Porto, Porto, Portugal Rute S. Moura Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal; ICVS/3B’s - PT Government Associate Laboratory, University of Minho, Braga/Guimarães, Portugal Bruno M. Neves Department of Medical Sciences, iBiMED—Institute for Biomedicine, University of Aveiro, Aveiro, Portugal

Editors and Contributors

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Rita Nogueira-Ferreira Department of Surgery and Physiology, Cardiovascular R&D Center, Faculty of Medicine of the University of Porto, Porto, Portugal Pedro F. Oliveira Laboratory of Cell Biology, Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), Department of Microscopy, University of Porto, Porto, Portugal; Faculty of Medicine, Department of Genetics, University of Porto, Porto, Portugal; i3S—Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal Daniela Patrício Laboratory of Signal Transduction, Department of Medical Sciences, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal; Department of Chemistry, CICECO, Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal Cátia D. Pereira Neuroscience and Signaling Laboratory, Department of Medical Sciences, Institute of Biomedicine (iBiMED), University of Aveiro, Aveiro, Portugal Sofia S. Pereira i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), Porto, Portugal; Endocrine, Cardiovascular and Metabolic Research, Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, Porto, Portugal; Department of Anatomy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal Sandra Rebelo Neuroscience and Signaling Laboratory, Department of Medical Sciences, Institute of Biomedicine (iBiMED), University of Aveiro, Aveiro, Portugal Joana Santiago Laboratory of Signal Transduction, Department of Medical Sciences, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal Joana Vieira Silva Laboratory of Signal Transduction, Department of Medical Sciences, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal; i3S—Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal; Laboratory of Cell Biology, Unit for Multidisciplinary Research in Biomedicine (UMIB), Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal João Carlos Sousa Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, Braga, Portugal; ICVS/3B’s, PT Government Associate Laboratory, Braga/Guimarães, Portugal

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Fábio Trindade Department of Surgery and Physiology, Cardiovascular R&D Center, Faculty of Medicine of the University of Porto, Porto, Portugal; Department of Medical Sciences, iBiMED–Institute of Biomedicine, University of Aveiro, Aveiro, Portugal Rui Vitorino Department of Medical Sciences, iBiMED, University of Aveiro, Aveiro, Portugal

Chapter 1

Beyond Brain Signaling Cátia D. Pereira, Filipa Martins, Fernanda Marques, João Carlos Sousa, and Sandra Rebelo

Abstract The brain is considered the most complex organ of the human body, being composed by a complex network of neurons and glial cells. Neurons communicate with each other through synapses, while glial cells essentially support both the structure and function of neurons, but also participate in the transmission of signals within the central nervous system. Neuronal signaling comprises electrical signaling that culminates with synaptic transmission, with consequent release of a chemical neurotransmitter at the presynaptic terminal. Neurotransmitter binding to a specific receptor originates postsynaptic signal transduction events that are specific of the type of receptor activated; these are summarized along this chapter. Glial cells are very active and, depending on the cell type, its specific functional demands and partners, their activity is regulated by distinct signaling pathways, also detailed throughout this chapter. In essence, this chapter summarizes the signaling pathways crucial for both neurons and glial cells, as well as the cross talk between these two building blocks of the nervous system.

C. D. Pereira · F. Martins · S. Rebelo (B) Neuroscience and Signaling Laboratory, Department of Medical Sciences, Institute of Biomedicine (iBiMED), University of Aveiro, 3810-193 Aveiro, Portugal e-mail: [email protected] C. D. Pereira e-mail: [email protected] F. Martins e-mail: [email protected] F. Marques · J. C. Sousa Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus Gualtar, 4710-057, Braga, Portugal e-mail: [email protected] J. C. Sousa e-mail: [email protected] ICVS/3B’s, PT Government Associate Laboratory, Braga/Guimarães, Portugal © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_1

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Keywords Brain · Central nervous system · Neurons · Glial cells · Electrical signaling · Chemical signaling · Neurotransmitters · Metabolic signaling · Immune signaling · Myelination signaling

Abbreviations 5-HT AC AMPA ARC ARG1 ATP BDNF Ca2+ Cl– cAMP CD CX3CL1 CX3CR1 CNS CREB DAG DAMP ER ECF ECM EGR1 EPSP Erk1/2 FIZZ1 GABA GDP GIRK GFAP GLUT1 GPCR GTP HCAR1 IFN IL IP3 IPSP K+

5-hydroxytryptamine Adenylate cyclase Amino-methylisoxazole propionic acid Activity-regulated cytoskeleton-associated protein Arginase 1 Adenosine triphosphate Brain-derived neurotrophic factor Calcium ion Chloride ion Cyclic adenosine monophosphate Cluster of differentiation Fractalkine CX3C chemokine receptor 1 Central nervous system cAMP response element binding protein Diacylglycerol Damage-associated molecular pattern molecule Endoplasmic reticulum Extracellular fluid Extracellular matrix Early growth response protein 1 Excitatory postsynaptic potential Extracellular-related kinase 1/2 Flammatory zone 1 γ-aminobutyric acid Guanosine diphosphate G protein-gated inwardly rectifying K+ channel Glial-fibrillary acidic protein Glucose transporter type 1 G protein-coupled receptor Guanosine triphosphate Hydrocarboxylic acid receptor 1 Interferon Interleukin Inositol triphosphate Inhibitory postsynaptic potential Potassium ion

1 Beyond Brain Signaling

LDH1 Mg2+ MCT MHC MS Na+ NAD+ NADH NMDA NO NOS NS OPC PAMP PIP2 PKA PKC PLC PNS ROS TCA TLR TNF TRP TrkB TREM2 YM1/2

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Lactate dehydrogenase 1 Magnesium ion Monocarboxylate transporter Major histocompatibility complex Multiple sclerosis Sodium ion Oxidized nicotinamide adenine dinucleotide Reduced nicotinamide adenine dinucleotide N-methyl-D-aspartate Nitric oxide Nitric oxide synthase Nervous system Oligodendrocyte precursor cell Pathogen-associated molecular pattern Phosphatidylinositol diphosphate cAMP-dependent protein kinase Protein kinase C Phospholipase C Peripheral nervous system Reactive oxygen species Tricarboxylic acid Toll-like receptor Tumor necrosis factor Transient receptor potential Tropomyosin-related receptor kinase B Triggering receptor expressed on myeloid cells 2 Chitinase 3-like 3

1.1 Introduction The brain is considered the most complex organ of the human body, being composed by a complex network of nerve cells called neurons and glial cells. While neurons transmit communication signals, glial cells mostly support both the structure and function of neurons. The brain is the central organ of the nervous system (NS) and, together with the spinal cord, constitutes the central nervous system (CNS). The brain consists of three major parts: the cerebrum, the brainstem and the cerebellum. The cerebrum comprises two cerebral hemispheres separated by a deep longitudinal fissure, and the diencephalon hidden by the former structures. Each cerebral hemisphere contains cerebral cortex, the subcortical nuclei and white matter. In turn, the diencephalon includes the thalamus and the hypothalamus. The brainstem is composed by the midbrain, the pons and the medulla oblongata, which extends from the diencephalon to the spinal cord. The cerebellum is located back of pons and medulla

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and is sustained by cerebellar peduncles [1–3]. In general, the CNS is responsible for the integrative functions of the NS. Information regarding internal structures (e.g. visceral structures) and other structures around us is transported to the spinal cord and subsequently to the brain, where the information is distributed, interpreted and a decision made. At this moment, the neurons involved in the body response send relevant information back, normally via the spinal cord for the motor response (if transmitted by the limbs). However, the formation of a motor response presumes an intricate prior processing, which includes sensation, perception, attention, memory, emotion and learning. The spinal cord, in addition to communication functions, is also involved in some basic integrative functions, for example related to reflexes. Additionally, the NS is also composed by the peripheral nervous system (PNS), which is constituted by neurons that are located outside the CNS, some with cell bodies in the PNS and processes in both the PNS and CNS, and by the axons of other neurons with cell bodies in the CNS [1–3]. The functional role of neuronal cells is to send and receive electrical impulses that communicate messages about sensory, motor and cognitive events throughout the brain. Glial cells are the connective tissue of the NS, filling the empty gaps between neurons and neuronal processes in both the CNS and PNS. They have important supportive, nutritive and protective functions. Further, they participate in signal transmission within CNS. This chapter will summarize the structure and function of neurons and glia cells that are the building blocks of the NS. Furthermore, the signaling pathways of neurons (i.e. neurotransmission signaling) will be firstly described, followed by the description of the signaling pathways of glial cells, namely metabolic signaling, immune signaling and myelination signaling.

1.2 Neurons and Glial Cells Neurons are the structural and functional unit of the NS. Structurally, all neurons have a cell body (or soma) and most have numerous dendrites emerging from the cell body, being the major information-gathering sites, and a single axon, which transports signals to other neurons/organs, ending as axon terminals, where the synapses occur. Interestingly, in some neurons, the dendrite shafts are smooth and, in others, they present short spines. Dendrites receive synaptic contacts from other neurons, some on the spines and others on the shafts. Functionally, neurons are responsible for information-handling through highly specialized neuronal parts. Dendrites are responsible for collecting information from someplace, either from other neurons, internal organs or the outside. Dendrites, together with the cell body and the initial part of the axon, are responsible for information processing, while the axon conducts the processed information along its extension for impulse propagation. At the axon terminals, the transmission of information onward occurs via synaptic transmission. Neurons are polarized cells, where electrical signals travel in only one direction at physiological conditions. Neurons assume a huge variety of sizes and shapes when

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compared to other tissue cells (discussed below). Despite this enormous diversity, they use the same organelles and physiologic processes used by other cells. The soma supports the metabolic and synthetic needs of the rest of the neuron and contains the nucleus and the cytoplasm or perikaryon. The cytoplasm comprises clusters of rough endoplasmic reticulum (ER) known as Nissl bodies, as well as the Golgi complex, free ribosomes, mitochondria and smooth ER. Neuronal cells present an elongated and delicate structure and the preservation of their integrity is achieved by an internal cytoskeleton consisting of a network of filamentous proteins, namely microtubules, microfilaments and neurofilaments, identical to those observed in other cells. The anterograde and retrograde transport along the microtubules allow the transportation of the needed material from the cell body to the axon and dendrites, and the unnecessary material to be returned to the soma, respectively [2, 3]. As mentioned above, neurons present a huge variability in terms of shapes and sizes. Based on the pattern of dendritic and axonal projections, the structural classification of neurons comprises unipolar, bipolar and multipolar neurons. Multipolar neurons are the most abundant neuronal type, being largely distributed in the NS. Bipolar neurons appear in some sensory epithelia, like the retina and olfactory epithelium. Unipolar neurons are common in invertebrates and very few are found in vertebrates. However, pseudounipolar neurons appear in vertebrate sensory ganglia, having a unipolar appearance but acting as bipolar neurons. The length and destination of the axon is the base of the functional classification of neurons as sensory (afferent) neurons, motor (efferent) neurons and interneurons. Sensory neurons transport the information to the CNS. Motor neurons have axons that directly end on muscle, glands or ganglionic neurons of the PNS. Interneurons are the most abundant neuronal type and are exclusively present in the CNS, being responsible for the interconnection of other neurons [2, 3]. While neurons are the key players in NS function through enabling chemical and electrical transmission of information, glial cells are essential for the neuronal function, as they perform relevant tasks such as support, nutrition, myelin formation and brain tissue immune surveillance, while still participating in the transmission of signals in the CNS. Astrocytes, oligodendrocytes and microglia are all glial cells [4]. We will next provide brief information on the role of each glial cell type. Astrocytes are the most abundant cells in the mammalian brain, exceeding neuron numbers by far. They have common cytological and immunological properties that allow for their identification in the nervous tissue: overall, they display a star-shaped morphology, large bundles of intermediate filaments composed of glial-fibrillary acidic protein (GFAP) and glial end-feet surrounding brain capillaries. Despite the description of its star-shaped form, astrocytes are a more morphologically and functionally diverse group of glial cells. They are essential to neuronal homeostasis by, for instance, providing nutrients, removing waste products and producing antioxidants. As components of the blood-brain barrier, together with endothelial cells of the brain blood vessels, astrocytes help to keep the brain parenchyma’s extracellular fluid (ECF) microenvironment separated from the fluctuations in the concentration of molecules in circulation. Astrocytes also respond during certain brain diseases and injuries, mostly those with an associated inflammatory process; in these cases,

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they hypertrophy, proliferate and secrete cytokines and other soluble factors. Most notably, astrocytes modulate neuronal activity and synaptic transmission [5]. During neurotransmission, neurons release neurotransmitters and ions at high concentrations towards the synaptic cleft. The rapid removal of these molecules is essential to maintain the extracellular milieu that surrounds synapses and, thus, not interfere with future synaptic transmission. The role of astrocytes and astrocytic signaling for synaptic transmission is so relevant that it led to the coining of the term tripartite synapse (and, presently, multipartite synapse) [6]. We will explore this aspect in further detail later in this chapter. Microglial cells are, although still disputed, of myeloid origin and, thus, considered the macrophages of the brain. Microglia display rod-shaped somas with numerous processes extending symmetrically. Microglia can circulate and migrate within different brain regions and phagocyte, process and present antigens. In response to changes in the CNS microenvironment, such as during pathological conditions, microglia respond by quickly becoming reactive, which results in changes in both morphology and upregulation of macrophage-type molecules. Hence, their primary function and signaling pathways are related to immune homeostasis and response. The immune mediators produced by microglia are the leading messengers able to interact and stimulate the adjacent cells. Simultaneously, microglial cells possess a variety of receptors that enable them to respond to brain insults [7, 8]. Oligodendrocytes are the glial cells that produce and ensheath neuron axons with myelin in the CNS. In the PNS, this role is performed by Schwann cells, which are the most abundant glial cells of the PNS. Myelin corresponds to the membrane (highly enriched in particular lipids and proteins) of oligodendrocytes and Schwann cells enwrapped around axons and is essential for insulation, permitting rapid electrical conduction down axons in vertebrates [9].

1.3 Neuronal Signaling The most important functional property of neurons is their ability to receive, conduct and transmit signals [1]. Neuronal signaling consists in the communication of information within neurons and between neurons and target cells (either other neurons or non-neuronal cells), and depends on a combination of electrical and chemical signals. Neurons can generate electrical signals that allow for the rapid conduction of information within the cell, whereas the transmission of information between neurons and target cells occurs at localized sites where specialized functional contacts—the synapses—are established [1, 10, 11]. Two types of synapses can be distinguished based on the mechanism of signal transmission. On the one hand, electrical synapses permit the nearly instantaneous, passive flow of electrical current from one neuron directly to another through gap junctions, consisting of tightly packed membrane channels—the connexons—that form pores between dendrites or somas of two adjacent nerve cells. Additionally,

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chemical synapses allow for signal transmission when there is no intercellular continuity between the presynaptic neuron and the postsynaptic target cell, which is accomplished via secretion of chemical messengers—the neurotransmitters—into the synaptic cleft separating both cells [1–3].

1.3.1 Electrical Signaling Neurons have the ability to generate electrical signals that transmit information from one part of the cell to another by altering their electrical potentials. In resting conditions, neurons maintain a negative electrical membrane potential—the resting membrane potential—caused by differences in the concentrations of ions inside and outside the cell, ranging from –60 to –80 mV in different neurons [3]. Neurons have two particular features that contribute to the maintenance of electrical potentials: (1) the neuronal cell membrane is selectively permeable to ions due to the presence of ion channels; and (2) different ion concentration gradients exist across the neuronal cell membrane, which are maintained by active transporters that move ions into or out of cells against their concentration gradients [1, 10]. In particular, neurons contain higher concentrations of potassium (K+ ) ions inside the cell and lower concentrations of sodium (Na+ ), calcium (Ca2+ ) and chloride (Cl– ) ions in comparison to the ECF [3, 4, 10]. The resting membrane potential is primarily generated by the differences in Na+ and K+ ion concentration inside and outside the cell. In neurons, there is a constant influx of Na+ ions and efflux of K+ ions along their concentration gradients and the stability of the resting potential is maintained by the activity of Na+ /K+ pumps that export Na+ ions and import K+ ions [3, 12]. Alterations in the membrane potential of neurons can be classified according to their effect on the electrical gradient: (1) depolarization occurs when the membrane potential becomes less negative (the cell’s interior is less negative); (2) hyperpolarization occurs when the membrane potential becomes more negative (the cell’s interior is more negative); and (3) repolarization consists in a change in the electrical gradient in which the cell returns to the resting membrane potential [4, 12]. The action potential consists in large and brief alterations of the membrane potential across the cell membrane, which is propagated along the axon. When the neuron receives a stimulus, such as a chemical signal from another neuron or environmental stimulus, receptors in its postsynaptic membrane are activated, which, in turn, activates ion channels that originate reversible alterations of the membrane potential across the plasma membrane. A region of the neuronal membrane could receive multiple and simultaneous stimuli, both inhibitory and excitatory inputs. When the sum of all the individual inputs (gradual potentials) makes the cell membrane potential reach a certain potential threshold (or depolarization threshold), an action potential could be triggered [3, 10, 12]. Briefly, the action potential could be divided in three phases.

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Depolarization

The stimulus causes depolarization of the membrane, which opens voltage-gated Na+ channels, allowing for the entry of Na+ ions into the cytosol down their concentration and electrical gradients. If the depolarization is large enough, more voltage-gated Na+ channels open and additional Na+ ions enter the cell, resulting in the generation of a complete all-or-nothing action potential. At the peak of depolarization, voltage-gated K+ channels open, which permits K+ ions flood out of the cell.

1.3.1.2

Repolarization

K+ ions move down their concentration gradient from the inside of the cell to the ECF and the membrane potential returns to its resting potential. The voltage-gated Na+ channels start to close and become temporarily inactivated no matter how strong a stimulus is applied to the neuron. Therefore, at this stage, it is impossible to generate another action potential—absolute refractory period.

1.3.1.3

Hyperpolarization

As the voltage-gated Na+ channels close and the voltage-gated K+ channels remain open, K+ ions continue to leave the cell for a short period and the repolarization continues past the resting membrane potential (i.e. the membrane becomes more negative). At this stage, it is only possible to generate new action potentials if the triggering event is bigger than the common to achieve the depolarization of a resting neuron—relative refractory period. Then, the voltage-gated K+ channels close and the resting membrane potential is re-established by the activity of Na+ /K+ pumps [3, 4, 12]. Action potentials initiate at the beginning of the axon (i.e. the initial segment) as the cell membrane depolarizes and are propagated by depolarizing neighboring regions of the membrane along the axon to reach the terminals, where they can initiate neurotransmitter release. Briefly, as Na+ ions enter the cell and depolarize the membrane, more voltage-gated Na+ channels are opened, spreading the depolarization [3, 4, 12]. Conduction of action potentials along unmyelinated axons is continuous (conduction speed ≤1 m/s), whereas along a myelinated axon is saltatory (conduction speed up to 120 m/s). In the latter, the voltage-gated Na+ channels are very abundant at the nodes of Ranvier and the myelin acts as an electrical insulator, speeding up the action potential conduction. Therefore, in this type of conduction, the action potential jumps from node to node [1, 3, 12, 13].

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1.3.2 Chemical Signaling or Synaptic Transmission Signal transmission at chemical synapses represents the main pathway used for neuronal communication. In contrast to electrical synapses, which are uncommon in the mammalian NS, there are over 100 different neurotransmitters that enable a tremendous diversity of physiological responses through chemical signaling [1, 3]. Neurotransmitters can be categorized into two primary classes based on their size: the small-molecule neurotransmitters and the relatively larger neurotransmitters termed neuropeptides. Small-molecule neurotransmitters usually mediate the transmission of a rapid intersynaptic signal, whereas neuropeptides tend to exert a more prolonged effect on postsynaptic functions, namely through modulation of the actions of smallmolecule neurotransmitters [1, 3, 14]. A brief description of the signal transmission mechanism operating at chemical synapses is provided below, followed by the discussion of different signal transduction cascades activated by neurotransmitters that permit the communication between presynaptic neurons and postsynaptic target cells.

1.3.2.1

Chemical Synaptic Transmission Process

The sequence of events characterizing signal transmission at chemical synapses includes four classical steps: (1) neurotransmitter synthesis and packaging in the presynaptic neuron; (2) neurotransmitter release into the synaptic cleft; (3) neurotransmitter binding to specific receptors in the postsynaptic target cell; and (4) rapid neurotransmitter removal from the synaptic cleft and/or degradation at the synaptic cleft. Briefly, the process is initiated with the synthesis of the neurotransmitter, which occurs either locally in the presynaptic terminal—the case of small-molecule neurotransmitters—or in the neuronal cell body, followed by axonal transport to the presynaptic terminal—the case of neuropeptides. The neurotransmitter is then loaded into synaptic vesicles adjacent to the presynaptic membrane, where they are protected from enzymatic degradation, forming highly concentrated packets ready for quick release. When an action potential arrives to this region, the consequent membrane depolarization elicits the opening of voltage-gated Ca2+ channels, resulting in Ca2+ influx to the presynaptic terminal. The transient increase in Ca2+ intracellular concentration causes the fusion of synaptic vesicles with the plasma membrane of the presynaptic neuron, leading to release of the neurotransmitter into the synaptic cleft and subsequent retrieval of synaptic vesicle membranes to the presynaptic terminal for reuse or degradation. After being exocytosed, the neurotransmitter diffuses through the synaptic cleft and binds to a specific receptor in the plasma membrane of the postsynaptic target cell (e.g. another neuron, muscle fiber or gland cell). The neurotransmitter receptor can simultaneously function as an ion channel—the ionotropic receptor—or, alternatively, can modulate ion channels indirectly via activation of intracellular signaling molecules—the metabotropic receptor. Binding of the neurotransmitter to either type of receptor culminates with the opening or closing of ion channels, generating a current flow that can increase (excitatory effect) or decrease

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(inhibitory effect) the membrane potential of the postsynaptic terminal. The summation of all excitatory and inhibitory electrical signals received at a given moment by the target cell determines whether or not it will fire an action potential. At the same time the neurotransmitter is binding to the appropriate receptor to produce a postsynaptic response, different mechanisms are acting to remove it from the synaptic cleft and terminate its action, thus preparing the chemical synapse for another cycle of signal transmission. Removal of the neurotransmitter can occur through simple diffusion out of the synaptic cleft into the cerebrospinal fluid, via recycling by direct reuptake into the presynaptic terminal or indirect uptake into nearby glial cells using specific transporter proteins and/or through enzymatic degradation to an inactive substance within the synaptic cleft [1–4].

1.3.2.2

Neurotransmitter-Mediated Signal Transmission Within Neurons

A minor group of neurotransmitters consists of small soluble molecules, including some amino acids (e.g. glutamate, aspartate, γ-aminobutyric acid (GABA) and glycine), a few purine derivatives (e.g. adenosine triphosphate (ATP) and adenosine) and several biogenic amines that can be subdivided into acetylcholine, catecholamines (e.g. dopamine, epinephrine and norepinephrine) and monoamines (e.g. serotonin and histamine). Neuropeptides compose the vast majority of neurotransmitters and can be loosely categorized into brain–gut peptides (e.g. substance P and vasoactive intestinal peptide), opioid peptides (e.g. encephalin and α-endorphin), pituitary peptides (e.g. oxytocin and vasopressin), hypothalamic-releasing peptides (e.g. thyrotropin-releasing hormone and somatostatin-14) and miscellaneous peptides (e.g. angiotensin-II and neurotensin) [1, 3]. Upon their release by the presynaptic neuron into the synaptic cleft, neuropeptides bind to metabotropic receptors, whereas small-molecule neurotransmitters interact with ionotropic and/or metabotropic receptors expressed by the postsynaptic target cell. Ionotropic receptors (also termed ligand-gated ion channels) are multimers composed of four to five protein subunits, each consisting of an extracellular domain that binds to the neurotransmitter and a membrane-spanning domain forming the pore of the ion channel. On the other hand, metabotropic receptors (also termed G proteincoupled receptors, GPCRs) are monomeric proteins that comprise a neurotransmitterbinding extracellular domain, seven transmembrane domains and an intracellular domain linked to a heterotrimeric guanosine triphosphate (GTP)-binding protein (i.e. G protein), which acts as an intermediate transducing molecule [1–3]. Interaction of neurotransmitters with these two classes of postsynaptic receptors activates different intracellular molecular mechanisms in the target cell that modify its electrical behavior and are ultimately transduced into a cellular response. Of note, some neurotransmitters also bind to receptors located presynaptically and/or in extrasynaptic sites, triggering a negative feedback response that inhibits additional neurotransmitter release by the presynaptic/extrasynaptic neuron, but these mechanisms will not be considered here. The main signal transduction cascades activated during

Major influx of Na+ and Ca2+ ions

AMPA and kainate receptors

Major influx of Na+ ions

Fast EPSP

CNS (neocortex, striatum, hippocampus, amygdala, nucleus accumbens, olfactory bulb, cerebellum, thalamus)

Learning and memory storage

[1–3, 14]

Postsynaptic ionotropic receptor

Signal transduction mechanism

Postsynaptic effect

Synapse location

Physiological response

References

[1–3, 14]

Learning and memory storage

CNS (cerebral cortex, basal ganglia, olfactory bulb, hippocampus, hypothalamus)

Slow EPSP

NMDA receptor

Glutamate

Small-molecule neurotransmitter

[1–3, 14, 93]

Learning and memory storage, control of sleep, anxiety and pain processing Control of motor activities

CNS (cerebral cortex, thalamus, hippocampus, hypothalamus, forebrain, brainstem), autonomic ganglia and peripheral tissues (skeletal muscle)

Fast EPSP

Major influx of Na+ and Ca2+ ions

Nicotinic receptor

Acetylcholine

[1–3, 14, 93] (continued)

Modulation of pain perception and anxiety response

CNS (hippocampus, entorhinal cortex, amygdala, nucleus accumbens, spinal cord) and autonomic ganglia

Fast EPSP

Major influx of Na+ and Ca2+ ions

5-HT3 receptor

Serotonin

Table 1.1 Signal transduction pathways of neuronal communication associated with interaction of small-molecule neurotransmitters with ionotropic receptors. HT, 5-hydroxytryptamine; AMPA, amino-methylisoxazole propionic acid; ATP, adenosine triphosphate; Ca2+ , calcium ion; Cl– , chloride ion; CNS, central nervous system; EPSP, excitatory postsynaptic potential; GABA, γ-aminobutyric acid; IPSP, inhibitory postsynaptic potential; Na+ , sodium ion; NMDA, N-methyl-D-aspartate

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Fast EPSP

CNS (spinal cord, thalamus, hypothalamus, hippocampus, cerebellum) and autonomic ganglia

Mechanosensation and modulation of pain perception

[1–3, 14]

Postsynaptic effect

Synapse location

Physiological response

References

ions

Major influx of

Ca2+

Signal transduction mechanism

and

P2X1 –P2X7 receptors

Postsynaptic ionotropic receptor Na+

ATP

Small-molecule neurotransmitter

Table 1.1 (continued)

ions

[1–3, 14, 94]

Memory storage and anxiety response

CNS (widespread), autonomic ganglia and peripheral tissues (liver, lung)

Fast IPSP

Major influx of

Cl–

GABAA receptor

GABA

[1–3, 14]

Modulation of motor neuron excitability

CNS (spinal cord, brainstem)

Fast IPSP

Major influx of Cl– ions

Gly receptor

Glycine

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neuronal communication will be described in the subsequent sections “Postsynaptic Signal Transduction Events Driven by Ionotropic Receptors” and “Postsynaptic Signal Transduction Events Driven by Metabotropic Receptors”, with particular focus on signaling events mediated by small-molecule neurotransmitters (Tables 1.1 and 1.2; Figs. 1.1, 1.2, 1.3, and 1.4).

Postsynaptic Signal Transduction Events Driven by Ionotropic Receptors Neurotransmitter binding to the appropriate ionotropic receptor induces a conformational change in the latter that causes its pore to open or close, thus altering the flux of specific ions across the plasma membrane, with the consequence of a change in the excitability properties of the postsynaptic target cell. For instance, some smallmolecule neurotransmitters (e.g. glutamate and acetylcholine) elicit excitatory postsynaptic potentials (EPSPs) by activating cation channels that increase the inward conductance of Na+ ions (Table 1.1 and Fig. 1.1a), while others (e.g. GABA and glycine) trigger inhibitory postsynaptic potentials (IPSPs) via activation of Cl– channels that allow for the influx of this negatively charged ion (Table 1.1 and Fig. 1.1b). Moreover, in some cases, the entry of Ca2+ ions also occur, which can act as a second messenger within the postsynaptic target cell (Table 1.1 and Fig. 1.1a). Once the neurotransmitter is removed from the synaptic cleft, its receptor returns to the original conformation. Given that ionotropic receptors combine the neurotransmitter receptor and the ion channel in the same macromolecule, neuronal chemical signaling mediated by these receptors results in immediate, though brief, postsynaptic electrical responses within the target cells [1, 3, 14].

Postsynaptic Signal Transduction Events Driven by Metabotropic Receptors Under basal conditions, the three-subunit G protein is maintained in an inactive state via binding of its α subunit (and, in turn, the β and γ subunits) to guanosine diphosphate (GDP). When the neurotransmitter interacts with and alters the conformation of a specific metabotropic receptor, the G protein couples to the latter and becomes transiently activated by exchanging GDP for GTP, resulting in dissociation of the α subunit from the βγ complex as well as from the receptor. Subsequently, the GTPbound α subunit and/or the free βγ complex can either bind directly to and activate ion channels present at the postsynaptic terminal or affect indirectly their open/closed state by altering the function of downstream effectors, leading to variations of the membrane potential in the target cell. These effector molecules typically comprise enzymes involved in the production of second messengers, which trigger complex cascades of intracellular biochemical mechanisms regulated by protein phosphorylation events. Among these signaling pathways, two of the most relevant include the cyclic adenosine monophosphate (cAMP) pathway and the phosphoinositol pathway. Termination of neuronal chemical transmission elicited by neurotransmitter– metabotropic receptor interaction involves the hydrolysis of GTP to GDP, causing

Glutamate

Group I (mGluR1, mGluR5) mGlu receptors

Activation of phosphoinositol pathway

Slow EPSP

CNS (hippocampus, lateral septum, olfactory bulb, thalamus, cerebellum, cerebral cortex, striatum, nucleus accumbens)

Learning and memory storage

[1–3, 14, 95]

Small-molecule neurotransmitter

Postsynaptic metabotropic receptor

Signal transduction mechanism

Postsynaptic effect

Synapse location

Physiological response

References

[1–3, 14, 95]

Learning and memory storage

CNS (cerebral cortex, hippocampus, accessory olfactory bulbs, cerebellum, thalamus, striatum)

Slow IPSP

Inhibition of cAMP pathway GIRK channel opening and voltage-gated Ca2+ channel closing

Group II (mGluR2, mGluR3) mGlu receptors

[1–3, 14, 96]

Learning and memory storage, control of motor activities Regulation of smooth muscle contraction, glandular secretion and blood vessel dilation

CNS (cerebral cortex, striatum, olfactory bulb, amygdala, hippocampus, nucleus accumbens, thalamus, brainstem), autonomic ganglia and peripheral tissues (smooth muscles, secretory glands)

Slow EPSP

Activation of phosphoinositol pathway

M1-class (M1 , M3 , M5 ) muscarinic receptors

Acetylcholine

[1–3, 14, 96] (continued)

Learning and memory storage, control of motor activities Regulation of smooth muscle contraction, glandular secretion and blood vessel dilation

CNS (thalamus, olfactory bulb, brainstem, hippocampus, cerebral cortex, striatum, cerebellum), autonomic ganglia and peripheral tissues (heart, lung)

Slow IPSP

Inhibition of cAMP pathway GIRK channel opening

M2-class (M2 , M4 ) muscarinic receptors

Table 1.2 Signal transduction pathways of neuronal communication associated with interaction of small-molecule neurotransmitters with ionotropic receptors. 5-HT, 5-hydroxytryptamine; ATP, adenosine triphosphate; Ca2+ , calcium ion; cAMP, cyclic adenosine monophosphate; CNS, central nervous system; EPSP, excitatory postsynaptic potential; GABA, γ-aminobutyric acid; GIRK, G protein-gated inwardly rectifying K+ channels; IPSP, inhibitory postsynaptic potential; K+ , potassium ion

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Dopamine

D1-like (D1 , D5 ) receptors

Activation of cAMP pathway

Slow EPSP

CNS (thalamus, hypothalamus, striatum, nucleus accumbens, substantia nigra, olfactory bulb, amygdala, cerebral cortex, hippocampus)

Control of locomotor activity, motivation, reward and reinforcement, cognition, learning and spatial memory, sleep and wakefulness, anxiety response and feeding behavior

[1–3, 14, 97]

Small-molecule neurotransmitter

Postsynaptic metabotropic receptor

Signal transduction mechanism

Postsynaptic effect

Synapse location

Physiological response

References

Table 1.2 (continued)

[1–3, 14, 97]

Control of locomotor activity, motivation, reward and reinforcement, cognition, learning and spatial memory, sleep and wakefulness, anxiety response and feeding behavior

CNS (striatum, hypothalamus, hippocampus, substantia nigra, cerebral cortex, thalamus, olfactory tubercle, nucleus accumbens, cerebellum, spinal cord)

Slow IPSP

Inhibition of cAMP pathway GIRK channel opening and voltage-gated Ca2+ channel closing (D2 )

D2-like (D2 –D4 ) receptors

[1–3, 14, 96]

Control of sleep and wakefulness, attention and alertness, mood and feeding behavior Regulation of heart rate, smooth muscle contraction, glandular secretion and blood vessel dilation

CNS (widespread) and peripheral tissues (heart, liver, spleen, kidney, smooth muscles, secretory glands)

Slow EPSP

Activation of phosphoinositol pathway

α1 receptor

Norepinephrine

[1–3, 14, 96] (continued)

Control of sleep and wakefulness, attention and alertness, mood and feeding behavior Regulation of heart rate, smooth muscle contraction, glandular secretion and blood vessel dilation

CNS (widespread) and peripheral tissues (heart, kidney, lung, liver)

Slow EPSP

Activation of cAMP pathway

β1 and β2 receptors

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Serotonin

5-HT1 family (5-HT1A , 5-HT1B , 5-HT1D , 5-HT1E , 5-HT1F ) receptors

Inhibition of cAMP pathway GIRK channel opening (5-HT1A )

Slow IPSP

CNS (striatum, cerebral cortex, amygdala, globus pallidus, substantia nigra, thalamus, hippocampus, hypothalamus, spinal cord)

Modulation of emotions, learning and cognition

[1–3, 14, 96, 98]

Small-molecule neurotransmitter

Postsynaptic metabotropic receptor

Signal transduction mechanism

Postsynaptic effect

Synapse location

Physiological response

References

Table 1.2 (continued)

[1–3, 14, 96, 98]

Motor control and control of feeding behavior

CNS (cerebral cortex, olfactory tubercle, striatum, globus pallidus, substantia nigra, hypothalamus, hippocampus, spinal cord, amygdala, nucleus accumbens, cerebellum)

Slow EPSP

Activation of phosphoinositol pathway

5-HT2 family (5-HT2A , 5-HT2B , 5-HT2C ) receptors

[1–3, 14, 96]

Learning and memory, control of sleep and thermoregulation

CNS (hippocampus, striatum, nucleus accumbens, olfactory tubercle, cerebral cortex, amygdala, hypothalamus, thalamus, globus pallidus, substantia nigra)

Slow EPSP

Activation of cAMP pathway

5-HT4 , 5-HT6 and 5-HT7 receptors

[1–3, 14, 99] (continued)

Regulation of synaptic plasticity

CNS (cerebral cortex, thalamus, cerebellum, spinal cord) and autonomic ganglia

Slow IPSP

Inhibition of cAMP pathway GIRK channel opening and voltage-gated Ca2+ channel closing

GABAB receptor

GABA

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Histamine

H1 and H2 receptors

Activation of phosphoinositol pathway Activation of cAMP pathway (H2 )

Slow EPSP

CNS (cerebral cortex, striatum, hippocampus, hypothalamus, thalamus, cerebellum)

Control of sleep and wakefulness, arousal and attention

[1–3, 14, 96]

Small-molecule neurotransmitter

Postsynaptic metabotropic receptor

Signal transduction mechanism

Postsynaptic effect

Synapse location

Physiological response

References

Table 1.2 (continued)

[1–3, 14, 96, 100]

Modulation of pain perception and neuroinflammation

CNS (cerebral cortex, striatum, nucleus accumbens, hippocampus, cerebellum)

Slow EPSP

Activation of phosphoinositol pathway Activation of cAMP pathway (P2Y11 )

P2Y2 , P2Y4 and P2Y11 receptors

ATP

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Fig. 1.1 Postsynaptic signaling events driven by interaction of small-molecule neurotransmitters with ionotropic receptors: excitatory (a) and inhibitory (b) signal transduction mechanisms. Abbreviations 5-HT, 5-hydroxytryptamine; AMPA, amino-methylisoxazole propionic acid; ATP, adenosine triphosphate; Ca2+ , calcium ion; Cl– , chloride ion; EPSP, excitatory postsynaptic potential; GABA, γ-aminobutyric acid; IPSP, inhibitory postsynaptic potential; K+ , potassium ion; Mg2+ , magnesium ion; Na+ , sodium ion; NMDA, N-methyl-D-aspartate; ↑, small increase; ↑↑, large increase

the α subunit to recombine with the βγ complex, thus reversing the G protein to its inactive, heterotrimeric form. Since metabotropic receptors modulate indirectly the gating of ion channels and stimulate cascades of signal transduction events that provide a means for amplification of the original signal, activation of these receptors elicits slow and long-lasting postsynaptic electrical responses within neurons and non-neuronal cells [1–3, 15]. Indirect Signal Transduction via the cAMP Pathway In the cAMP signaling pathway, the binding of neurotransmitters (e.g. dopamine and norepinephrine) to their cognate metabotropic receptors triggers the transient activation of a G protein subtype with stimulatory effect (i.e. Gs protein), leading to detachment of the GTP-bound αs subunit. The latter then moves along the inner surface of the postsynaptic membrane to activate adenylate cyclase (AC), an enzyme that catalyzes the conversion of cytosolic ATP into cAMP. The increased intracellular concentration of this second messenger stimulates membrane-bound cAMP-dependent protein kinase (PKA) to phosphorylate several target proteins, namely ion channels and transcription factors (Table 1.2 and Fig. 1.2a) [1, 3, 14]. For example, PKA action

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Fig. 1.2 Postsynaptic signaling events driven by interaction of small-molecule neurotransmitters with metabotropic receptors: indirect excitatory (a) and inhibitory (b) signal transduction mechanisms associated with the cAMP pathway. Abbreviations 5-HT, 5-hydroxytryptamine; AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; PKA, cAMP-dependent protein kinase; ↑, increase; ↓, decrease

Fig. 1.3 Postsynaptic signaling events driven by interaction of small-molecule neurotransmitters with metabotropic receptors: indirect excitatory signal transduction mechanisms associated with the phosphoinositol pathway. Abbreviations ATP, adenosine triphosphate; Ca2+ , calcium ion; DAG, diacylglycerol; EPSP, excitatory postsynaptic potential; ER, endoplasmic reticulum; IP3 , inositol triphosphate; PKC, protein kinase C; PLC, phospholipase C; ↑, increase

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Fig. 1.4 Postsynaptic signaling events driven by interaction of small-molecule neurotransmitters with metabotropic receptors: direct inhibitory signal transduction mechanisms. Abbreviations Ca2+ , calcium ion; GABA, γ-aminobutyric acid; GIRK, G protein-gated inwardly rectifying K+ channels; IPSP, inhibitory postsynaptic potential; K+ , potassium ion; ↑, increase; ↓, decrease

on Na+ -permeable channels induces their pore to open and the subsequent influx of Na+ ions results in depolarization of the postsynaptic terminal, producing an EPSP [3]. Moreover, PKA can phosphorylate and activate the cAMP response element binding protein (CREB), allowing for this transcriptional activator to modulate gene expression in the target cell [1]. In other situations, the interaction between neurotransmitters (e.g. glutamate and GABA) and metabotropic receptors induces the production of IPSPs by activation of a different G protein subtype with inhibitory effect (i.e. Gi protein), whose GTPbound αi subunit inactivates AC and, consequently, prevents postsynaptic signal transduction through the cAMP pathway (Table 1.2 and Fig. 1.2b) [1, 14]. Indirect Signal Transduction via the Phosphoinositol Pathway In the phosphoinositol pathway, the binding of neurotransmitters (e.g. serotonin and ATP) to specific metabotropic receptors initially releases the α subunit of another subtype of stimulatory G protein (i.e. Gq protein) and this activated, GTP-bound αq subunit induces the enzymatic activity of phospholipase C (PLC). As a result, two second messengers, namely diacylglycerol (DAG) and inositol triphosphate (IP3 ), are produced from the cleavage of phosphatidylinositol diphosphate (PIP2 ), a membrane phospholipid. On the one side, DAG remains within the postsynaptic membrane and recruits cytosolic protein kinase C (PKC), which is triggered to initiate phosphorylation of various protein substrates in the plasma membrane (e.g. ion channels) and intracellularly (e.g. metabolic enzymes). On the other side, IP3 diffuses to the smooth ER membrane to activate specific receptors constituting Ca2+ -releasing channels that permit the efflux of stored Ca2+ ions into the cytosol. The transient elevation in the cytoplasmic concentration of this second messenger stimulates the activity of a broad range of Ca2+ -dependent enzymes (e.g. Ca2+ /calmodulin-dependent protein kinases), which can ultimately modulate the state of ion channels and a diversity of other cellular processes, for example gene expression, in the target cell (Table 1.2 and Fig. 1.3) [1, 3, 14].

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Direct Signal Transduction Pathways In addition to the stimulation of second messengers and related intracellular signaling cascades that is driven by the GTP-bound α subunit of different G protein subtypes, the free βγ complex of activated G proteins can also interact with a variety of effector proteins, such as ion channels and enzymes, to mediate the production of electrical signals in the postsynaptic target cell. For instance, in response to the binding of some neurotransmitters (e.g. acetylcholine and dopamine) to metabotropic receptors, the βγ dimer is able to directly activate selective K+ channels and/or inhibit voltage-gated Ca2+ channels, leading to hyperpolarization of the postsynaptic membrane (Table 1.2 and Fig. 1.4) [3, 14, 15].

1.4 Glial Signaling Glia is composed of highly active cells that interact with each other and with neurons. Ions, small molecules and secreted factors mediate this communication through receptor activation, thus inducing the activity of distinct signaling pathways. Moreover, each glial cell type, given its specific functional nature and intercellular communication partner, preferentially uses diverse signaling pathways. For this reason, we will outline the most relevant glial cell signaling mechanisms independently for each glial cell type. Furthermore, and given the extensive variety of cellular signaling processes in glial cells, we will mostly focus on mature cells. Whenever considered most relevant to reflect the function of glial cells, we will discuss aspects of cell signaling during pathological conditions.

1.4.1 Astrocytic Signaling Astrocytic signaling is highly intertwined to those of neurons and is based on the close morphological and functional relationship between astrocytes and neurons. The role of astrocytes in maintaining neuronal function stems from metabolism to being considered an essential component of the synapse. In fact, the majority of synapses in the brain and spinal cord are multi-partite, being composed of (1) the presynaptic terminal; (2) the post-synaptic dendritic compartment; (3) the perisynaptic process of the astrocyte; (4) the extracellular matrix (ECM) present in the synaptic cleft; and, frequently, (5) the process of neighboring microglial cells that surveys the synaptic structure [16]. We will next describe the most relevant signaling pathways in astrocytes taking in consideration its close relationship to neuronal activity.

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Metabolic Signaling

Astrocytes are ideally located to have access to the glucose and other energetic substrates that are in the blood stream. Astrocyte end feet are surrounding the blood vessels that irrigate the brain, being part of the neurovascular unit. For that reason, most of the circulating glucose, when entering the brain, will need to pass though astrocytes. Once inside astrocytes, glucose enters the glycolytic pathway and gives rise to pyruvate, which, instead of entering in the tricarboxylic acid (TCA), originates lactate. This metabolic signaling pathway is crucial for neuronal functioning. In addition, lactate ensures adequate energy supply, modulates neuronal excitability levels and regulates adaptive functions in order to set the ‘homeostatic tone’ of the NS [17]. Lactate production in astrocytes derives from two pathways: glycogenolysis and glycolysis, as a consequence of a cell specific gene expression profile that favors the conversion of pyruvate to lactate rather than the use of pyruvate in the TCA cycle [18]. Both processes are triggered by activity-dependent neuronal signals: norepinephrine, vasoactive intestinal peptide, adenosine and K+ promote glycogenolysis, whereas glucose uptake and lactate production (aerobic glycolysis) are triggered by glutamate, ammonium, nitric oxide (NO) and K+ [18]. Lactate can be then transported from astrocytes (through monocarboxylate transporter (MCT) 1 and 4) to neurons (through MCT2) and, once inside neurons, lactate gives rise to pyruvate [19]. The conversion of lactate to pyruvate by lactate dehydrogenase 1 (LDH1) requires oxidized nicotinamide adenine dinucleotide (NAD+ ) and produces reduced nicotinamide adenine dinucleotide (NADH), which affects the redox state of the neuron [18]. The increase in NADH positively modulates the activity of N-methyl-D-aspartate (NMDA) receptors, which leads to enhanced Ca2+ currents, the activation of intracellular signaling cascades and the induction of the expression of plasticity-associated genes—for example, those encoding activity-regulated cytoskeleton-associated protein (ARC), early growth response protein 1 (EGR1) and brain-derived neurotrophic factor (BDNF). Pyruvate formed from lactate enters the mitochondria in the TCA cycle, where it will give rise to 32–36 ATP molecules. This ATP will support the energy demands of neurons and also modulate the activity of ATP-dependent K+ channels, resulting in neuronal depolarization [20]. Not all the lactate enters the neuron, and extracellular lactate can additionally signal neurons by acting on a GPCR known as hydrocarboxylic acid receptor 1 (HCAR1), which inhibits AC activity [21].

1.4.1.2

Calcium Signaling

During neuronal activity, astrocytes remove neurotransmitters released at synapses. After this uptake, astrocytes get activated via numerous mechanisms, including the GPCRs that regulate the cytosolic concentration of second messengers, such as Ca2+ and cAMP [22, 23]. In response to this activation, astrocytes are able to modulate the activity of neighboring cells, including neurons, by the release of diverse substances, such as classical neurotransmitters (e.g. glutamate, ATP and neuropeptides).

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Of interest, both excitatory and inhibitory signals cause global or focal Ca2+ elevations in astrocytes that precede gliotransmitter release, which might exert excitation or inhibition of neighboring synapses [23]. This activation and, hence, the responses by astrocytes are due to the fact that they express a variety of channels, transporters and receptors that provide a diversity of functions, both homeostatic and synaptic. Notably, astrocytes can express many of the same neurotransmitter receptors expressed by neurons and, consequently, are able to respond to a wide variety of neurotransmitters. Most of these responses occur via GPCRs that, upon stimulation by synaptically released neurotransmitters, trigger different intracellular signaling cascades, including Ca2+ mobilization from internal stores. Additional mechanisms of Ca2+ elevations include transmembrane Ca2+ flux through activation of transient receptor potential (TRP) channels [24–26]. By measuring changes in Ca2+ levels, astrocytes have been found to respond to glutamate [27, 28], acetylcholine [27, 29], ATP [30], GABA [31–33] and endocannabinoids [34–36]. While astrocytes can be activated by the most abundant neurotransmitters in the brain, evidence is still scarce for astrocyte responsiveness to neurotransmitters that have important roles in brain function, such as dopamine, histamine, serotonin and opioids [37–42].

1.4.1.3

cAMP Signaling

As stated above, astrocytes can respond to extracellular stimuli through activation of plasma membrane GPCRs, with an increase in intracellular levels of secondary messengers. One such messenger particularly relevant for astrocytic signaling is cAMP [43]. One relevant source of cAMP in astrocytes is the activation of β-adrenergic receptors. Astrocytes express the three types of β-adrenergic receptors, thus elevating intracellular cAMP levels upon activation. Furthermore, astrocytes also express the A2A and A2B adenosine receptors that are coupled to AC. Other neuromodulators, including serotonin, dopamine and histamine, can also activate AC-coupled GPCRs on astrocytes [44, 45]. Thus, astrocytes are equipped with the machinery to increase intracellular cAMP and also possess its downstream targets. cAMP signaling in astrocytes is transduced predominately via PKA and exchange factor directly activated by cAMP 1 (Epac1) [46]. cAMP in astrocytes controls diverse cellular functions. Specifically, cAMP has been shown to: (1) trigger glycogenolysis-related energy supply, namely the degradation of glycogen and enhancement of aerobic glycolysis and lactate production [47]; (2) regulate homeostasis-maintenance processes, such as glutamate uptake, extracellular K+ buffering and water permeability; (3) modulate astrocytic immune response by regulating the release of cytokines and inflammatory factors [44]; and (4) alter astrocyte morphology by triggering the formation of processes and inhibiting swelling of astrocytes upon trauma or lesion [48, 49].

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1.4.2 Microglial Signaling Microglia, together with perivascular, meningeal and choroid plexus macrophages, are often referred to as the resident macrophage cell population in the CNS, where they perform essential tasks during development and homeostasis. Microglial cells can be classified in resting and activated microglia based on cell morphology and functional characteristics. Ramified microglia were classically defined as resting cells, but recent observations showed that they are highly active and continually surveying their microenvironment with extremely motile processes and protrusions [50]. Regarding activated microglia, it is classically recognized that, upon stimulation with external or internal toxic compounds, microglial cells acquire an amoeboid-like shape characterized by a largely rounded soma with fewer, thicker and shorter processes [51, 52]. However, it is currently known that microglial cells are an extremely heterogeneous population within the CNS based on their transcriptome and functions [53], and probably a large continuum of microglial morphologies exists between these two extremes [54–60].

1.4.2.1

Resting Microglia Signaling

As the resident immune cells of the brain parenchyma, microglia act as central communicators between the NS and the immune system, as they are the first sentinels protecting against invading pathogens and tissue damage. Under physiological conditions, resting microglia play a crucial role in the immune surveillance of the brain, interacting with other brain cells and actively monitoring and remodeling impaired synapses [50, 61]. The fractalkine (CX3CL1)–CX3C chemokine receptor 1 (CX3CR1) signaling represents the most important communication channel between neurons and microglia. The expression of CX3CL1 in neurons and of its receptor CX3CR1 in microglia determines a specific interaction, playing fundamental roles in the regulation of the maturation and function of these cells. Indeed, microglia mediate prune supernumerary synapses and trophic-factor production through CX3CL1–CX3CR1 signaling in neurodevelopment [62, 63].

1.4.2.2

Activated Microglia Signaling

When considering only activated microglia, it is well known that, similarly with what happens with macrophages, microglial cells are able to secrete anti- or pro-inflammatory mediators that not only act as paracrine mediators of neuronal cell plasticity and survival, but may also promote the autocrine polarization into different states of activation [64, 65]. This polarization can also be due to factors produced by the neighboring cells, namely astrocytes, or by immune cells that can invade the brain parenchyma. Cytokines such as interferon (IFN)-γ, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF) activate the classical stimulation of

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microglia and induce their polarization into a pro-inflammatory phenotype, also known as M1 microglia. In turn, cytokines like IL-4 and IL-13 induce the alternative activation pathway that favors microglial differentiation into an anti-inflammatory phenotype able to induce tissue repair, known as M2 microglia. Each state of activation is associated with the production of specific factors and the gain or loss of particular functions. Although both M1 and M2 microglial cells are able to phagocytose and present antigens, the functional effects of classical activation are geared towards antigen presentation and the killing of intracellular pathogens. Therefore, upregulation of many associated receptors and enzymes reflects that purpose. Additionally, M1 microglial cells mainly produce pro-inflammatory cytokines (e.g. IL-1β, IL-6 and TNF), present high intracellular nitric oxide synthase (NOS) activity and increased production of reactive oxygen species (ROS) and NO. On the contrary, M2 microglial cells mainly produce IL-4, present decreased production of ROS and NO as well as increased levels of arginase 1 (ARG1), chitinase 3-like 3 (YM1/2), inflammatory zone 1 (FIZZ1), cluster of differentiation 206 (CD206), triggering receptor expressed on myeloid cells 2 (TREM2), CD163, Dectin-1 and CD301 [66]. Importantly, polarized microglia are not locked in a particular state; both microglia and macrophages are plastic cell types that can be altered if the cytokine environment changes [67]. However, in many acute injuries and chronic brain inflammation, the continued production of cytokines like IFNγ and TNFα maintains an M1 activation state, which can lead to increased tissue damage. When activated, microglia acquire an amoeboid phenotype and are able to phagocytose brain pathogens and debris through presenting those antigens to immune cells. To accomplish this, microglia express different receptors, most prominently the Toll-like receptors (TLRs), through which they are able to detect pathogens and tissue damage signaling [68, 69]. Tissue damage results in the release of intracellular proteins that comprise ‘damage-associated molecular pattern’ molecules (DAMPs), which are detected by TLRs and other cell surface receptors, eliciting an inflammatory response. Similarly, microbial pathogens express conserved pathogen-associated molecular patterns (PAMPs) that are common to microbes and allow for their rapid and efficient recognition by the innate immune system, primarily through TLRs specialized in recognizing these molecules. After stimulation of these receptors, there is an activation of intracellular signaling pathways that culminate with the migration of different transcription factors to the nucleus, with the consequent alteration in the expression levels of microglial target genes in an attempt to destroy the damaged cell or the invading pathogen.

1.4.3 Oligodendrocyte Signaling Oligodendrocytes’ principal function is the production of myelin, a lipid-rich membrane that insulates neuronal axons, allowing for the rapid transmission of action potentials in the CNS. Although myelin production is the well-described function attributed to oligodendrocytes, which is suggestive of a single lineage of cells, it is

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now known that oligodendrocytes and their precursor cells throughout the CNS have different morphologies and regional functional differences have been observed [70]. Even in demyelinating diseases, such as multiple sclerosis (MS), oligodendrocytes are not only the cells able to promote remyelination as we will describe below.

1.4.3.1

Axo-Myelinic Synapse Signaling and Coupling with Axons

Oligodendrocytes are fundamental for the functioning of the NS as they participate in several cellular processes, including axonal myelination and metabolic maintenance for neurons. Regarding myelination signaling, oligodendrocyte progenitors’ proliferation and myelination by mature oligodendrocytes was suggested to be regulated by glutamate from neuronal activity [71–74]. The presence of glutamate receptors in the oligodendroglial cell membrane throughout the lineage suggests that glutamate signaling is important for these cells both during their progenitor and mature stages. Oligodendrocyte precursor cells (OPCs) receive glutamatergic synaptic inputs from unmyelinated axons [75–81] and, particularly, they express the highest density of glutamate receptors compared to later lineage stages [82], which enable them to monitor and respond to changes in neuronal activity. These synaptic contacts seem to occur predominantly on entirely unmyelinated axons or unmyelinated segments of the axon [80, 81, 83], which raises the question of whether synaptic input is involved in prompting OPC differentiation and myelination. Moreover, myelinating oligodendrocytes respond to neuronal activity [84, 85] via an NMDA receptor-mediated Ca2+ rise in the myelin sheath [86]. In this model, depolarization of the internodal axolemma by traversing action potentials is detected by voltage-gated Ca2+ channels located in the juxtaparanode region, resulting in intra-axonal Ca2+ release. This, in turn, promotes the fusion of glutamatergic vesicles and release of glutamate into the periaxonal space, which then activates amino-methylisoxazole propionic acid (AMPA) and NMDA receptors in the innermost myelin leaflets, promoting Ca2+ influx into the myelin cytoplasm. As a consequence of myelinic receptor activation, there is an increase in glucose uptake, due to recruitment of glucose transporter type 1 (GLUT1), and the stimulation of glycolysis by oligodendrocytes, resulting in increased production of pyruvate and lactate. Pyruvate can be used as an energy substrate for myelinic mitochondria, while lactate is transported across the periaxonal space to the axon to fuel aerobic metabolism by axonal mitochondria for the efficient production of ATP at internodes [86]. Additionally to glutamate signaling, recent studies using mutant mice to selectively manipulate BDNF signaling in desired cell types, in combination with animal models of demyelinating disease, have demonstrated that BDNF not only potentiates normal CNS myelination in development, but also enhances recovery after myelin injury [87]. In this model, action potential firing by active neurons results in the release of BDNF along the axon. BDNF–tropomyosin-related receptor kinase B (TrkB) signaling could influence OPC survival and differentiation in development and after myelin injury, and promotes activity-dependent myelination by modulating glutamatergic (NMDA and AMPA receptors) neurotransmission. It is well

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established that BDNF–TrkB signaling via extracellular-related kinase 1/2 (Erk1/2) promotes the synthesis of myelin proteins and this influences myelin sheath thickness. Not known are the molecular mechanisms that underpin this effect and it is hypothesized that the BDNF–TrkB–Erk cascade results in transcriptional activation, controlling myelin protein expression [87].

1.4.3.2

Oligodendrocytes as Antigen Presenting Cells

Although extremely constant since childhood, oligodendrocytes start to proliferate when a demyelinating signaling occurs in the brain. MS is a good example of that. In MS, a chronic inflammatory disease of the CNS, the patient’s immune system starts to attack one or more components of myelin sheath, leading to neuronal demyelination, neurodegeneration and, ultimately, loss of vital neurological functions such as walking. The most common form of the disease is the relapse-remitting, in which patients show demyelinating acute attacks followed by period of recovery and of remyelination. Such remyelination, in animal models, is known to occur by newly generated oligodendrocytes, and remaining mature oligodendrocytes do not seem to contribute to this process [88–90]. However, in humans, the absence of new oligodendrocytes in shadow plaques suggests that remyelination of lesions occurs transiently or not at all, or that myelin is regenerated by pre-existing, and not new, oligodendrocytes in MS [91]. Independently of their relevance in remyelination, recent studies also showed that, in MS, oligodendrocytes may have additional new functions. By using single-cell transcriptomic analysis of isolated oligodendrocytes lineage cells from the MS mice model, it was found a unique oligodendrocyte lineage population that expressed genes involved in antigen processing and presentation to immune cells (via major histocompatibility complex (MHC) class I and II) and in immunoprotection [92]. Additionally, it was shown that OPCs are able to phagocytose myelin peptides and that MHC-II-expressing OPCs can activate CD4-positive T cells [92]. These new functions prompt the role of new signaling pathways activated in oligodendrocytes that can be modulated for a better control of the disease.

1.5 Conclusion Despite CNS huge complexity, it is mostly composed by two cell populations: neurons and glial cells. While neurons have the particularity of communicating with each other through synapses, the glial cells perform essential functions to neurons such as support, nutrition, neurotransmitter removal, myelin formation, immune surveillance, and are also involved in signal transmission in the CNS. The range of functions assigned to glial cells is increasingly diverse. The neuronal signaling comprises different well documented signal transduction cascades activated by neurotransmitters that allow for the communication of presynaptic neurons and postsynaptic target cells. The glial cell signaling mechanisms have

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been outlined for each cell type and good examples of interaction between neurons and astrocytes are described at the brain energy metabolism (metabolic signaling). The major future challenge will be the integration of both neuronal and glial signaling pathways to better understand the crosstalk between neurons and glial cells.

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Chapter 2

Cell Signalling Within Pituitary, the Master Gland of the Endocrine System Sofia S. Pereira, Carolina B. Lobato, and Mariana P. Monteiro

Abstract The endocrine system is responsible for ensuring whole body homeostasis through the regulation of multiple systemic functions. This goal is achieved by enabling a highly efficient intra and inter-cellular communication through the use of chemical messengers, commonly known as hormones. Given the unquestionable importance that the endocrine system assumes for survival, each endocrine function is tightly regulated through multiple molecular mediators organized in redundant signalling pathways that intermingle towards a single common aim, the continuous strive for body balance. In this section, promised of two parts/chapters, the signaling pathways responsible for the regulation of physiological functions within the endocrine organs are reviewed. This is the first section comprising the detailed description of the signalling pathways that were identified in the pituitary gland. Keywords Endocrine system · Signaling pathways · Pituitary gland · Hypothalamic-pituitary axis

S. S. Pereira Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Porto, Portugal e-mail: [email protected] Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), Porto, Portugal S. S. Pereira · C. B. Lobato · M. P. Monteiro (B) Endocrine, Cardiovascular and Metabolic Research, Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, Porto, Portugal e-mail: [email protected] Department of Anatomy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal e-mail: [email protected] © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_2

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Abbreviations [Ca2+ ]i AC ACTH ActRI ActRII AVP bFGF CaMKII cAMP CBP CREB CRH CRHR D2R D2RL D2RS DA DAG DHT E2 Egr-1 ER EREs ERK FGFR FoxL2 FS FSH GABA GH GHRH GHRHR GHSR GnIH GnRH GnRHR GR HPA HPG IGF-1 IP3 KISS1R

Intracellular calcium concentration Adenylate cyclase Adrenocorticotropic hormone Activin receptor type I Activin receptor type II Arginine vasopressin Basic fibroblast growth factor Calmodulin dependent protein kinase II Cyclic 3 ,5 adenosine monophosphate CREB binding protein cAMP response element binding Corticotropin releasing hormone Corticotropin releasing hormone receptor Dopamine D2 receptor Long dopamine D2 receptor Short dopamine D2 receptor Dopamine Diacylglycerol 5αdihydrotestosterone Estradiol Early growth response protein 1 Estrogen receptor Estrogen response elements Extracellular signal–regulated kinases Fibroblast growth factor receptor Forkhead box L2 Folliculostellate Follicles timulating hormone Gamma aminobutyric acid Growth hormone Growth hormone releasing hormone Growth hormone releasing hormone receptor Growth hormone secretagogue receptors Gonadotropin inhibitory hormone Gonadotropin releasing hormone Gonadotropin releasing hormone receptor Glucocorticoid receptor Hypothalamic pituitary adrenal axis Hypothalamic pituitary gonadal axis Insulin like growth factor 1 Inositol 1,4,5-triphosphate KISS1 receptor

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LH MAPK NKB NPY PAC1-R PACAP PIP2 Pit-1 PKA PKC PLC POMC PRL SST SSTR T3 T4 TGF-β TRH TSH VIP VGCC

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Luteinizing hormone Mitogen activated protein kinase Neurokinin B Neuropeptide Y Pituitary adenylate cyclase activating polypeptide type I receptor 1 Pituitary adenylate cyclase activating peptide Phosphatidylinositol 4,5-bisphosphate Pituitary specific transcription factor Protein kinase A Protein kinase C Phospholipase C Proopiomelanocortin Prolactin Somatostatin Somatostatin receptors Triiodothyronine Thyroxine Transforming growth factor beta Thyrotropin releasing hormone Thyroid stimulating hormone Vasoactive intestinal peptide Voltage gated Ca2+ channels

2.1 Introduction The endocrine system assumes the responsibility of ensuring body homeostasis by regulating the integration of multisystem functions. This mission is achieved by enabling inter-cellular and intra-cellular communication via chemical messengers that are commonly known by the designation of hormones. The pituitary gland is an ovoid structure located at the base of the skull that anatomically bonds to the central nervous system by the means of an infundibulum, which establishes the connection with the basal hypothalamus necessary for the thigh regulation of the vast majority of the endocrine system [1]. The pituitary gland is a multihormonal secreting gland responsible for the overall coordination of the endocrine system function. In this chapter, focus will be given to the anterior pituitary endocrine function, acting as the master of the endocrine system.

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2.2 Pituitary Gland 2.2.1 Anatomy and Histology of Pituitary Gland The pituitary gland is comprised of two lobes with distinct embryological origins, histological features and functionality. The neurohypophysis, pars nervosa or posterior lobe of the pituitary gland is an inferior extension of the hypothalamus through the infundibulum, which is responsible for the release of arginine vasopressin (AVP) and oxytocin produced by neurons located in the supra-optic and paraventricular hypothalamic nuclei into the systemic circulation. The adenohypophysis or anterior lobe of the pituitary arises later during embryogenesis from the pharyngeal epithelium and then migrates upwards through the Rathke pouch to join the neurohypophysis at the base of the skull, to form the co-joined pituitary gland [2, 3]. The anterior pituitary lobe is among one of the most densely irrigated mammalian tissues. Despite not receiving direct arterial blood supply, receives a dense capillary network of portal vessels arising directly from the hypothalamus, infundibulum and posterior pituitary that supply the anterior pituitary gland. This pituitary portal system provides not only blood supply to ensure tissue vitality but is also responsible for carrying hypothalamic hormone releasing factors from the hypothalamus into the anterior pituitary, essential for the functional integrity of the hypothalamic pituitary axis. Lastly, bi-directional cross-talk within the hypothalamic-pituitary anatomical axis is ensured by the venous drainage that allow pituitary hormones not only to reach the systemic circulation but also reaching the hypothalamus through capillaries with retrograde blood flow towards the median eminence of the hypothalamus [2]. Histologically, anterior pituitary cells are organized in nests and cords separated by fenestrated sinusoidal capillaries. Anterior pituitary cells derive from undifferentiated proliferative progenitor cells, which are able to differentiate into five different hormone producing cell types, namely lactotrophs or prolactinproducing cells, somatotrophs or growth hormone-producing cells, corticotrophs or adrenocorticotropin-producing cells, gonadotrophs or follicle stimulate hormone/luteinizing hormone-producing cells and thyrotrophs or thyrotropin-producing cells (Fig. 2.1). Endocrine cells with different hormone secretion pattern within the pituitary gland can be solely identified through immunocytochemical reactions, since, based on the staining properties of secretory vesicles, only three cell types can be identified with no correlation with the hormonal secretory profile, which are acidophil, basophil and chromophobe cells. In addition, the anterior pituitary lobe also presents non-endocrine cells folliculostellate (FS) cells, characterized by a starlike appearance, agranular cytoplasm and ability to form follicles. Although FS are considered non-endocrine cells, these contribute to regulate other pituitary endocrine cells functions through intercellular communication [4].

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Fig. 2.1 Hypothalamic-pituitary axis with focus on anterior hypophysis regulation. Abbreviations ACTH—adrenocorticotropic hormone; CRH—corticotropin-releasing hormone; DA— dopamine; FSH—follicle-stimulating hormone; GH—growth hormone; GHRH—growth hormonereleasing hormone; GnRH—gonadotropin-releasing hormone; IGF-1—insulin-like growth factor 1; LH—luteinizing hormone; PRL—prolactin; TRH—thyrotropin-releasing hormone; TSH— thyroid-stimulating hormone receptor. Notes Dashed red arrows represent negative feedback pathways

2.2.2 Pituitary General Secretion Control Anterior pituitary hormone secretion is regulated by a multitude of circulating factors. In this section, focus shall be given to the hypothalamic-pituitary axis receptormediated hormone secretion, as specific hypothalamic hormone releasing factors induce increased gene transcription, mRNA stability and hormone secretion in each pituitary cell line through common central signaling pathways [1]. Indeed, hypothalamic hormone releasing factors via protein kinase A (PKA) activation increase cyclic 3 ,5 -adenosine monophosphate (cAMP) production, which promotes the increase of intracellular calcium concentrations ([Ca2+ ]i ) either by inducing the release of calcium (Ca2+ ) stored in the endoplasmic reticulum or by inducing conformational changes in voltage-gated Ca2+ channels (VGCCs), while cell membrane becomes depolarized. These pathways activation ultimately leads to [Ca2+ ]i -mediated growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH) and thyroid-stimulating hormone (TSH) exocytosis [1]. In addition,

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gonadotropin-releasing hormone (GnRH) and thyrotropin-releasing hormone (TRH) also activate phospholipase C (PLC)/protein kinase C (PKC) pathway, leading to the release of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and TSH [5]. Furthermore, hypothalamic inhibitory hormones, such as dopamine (DA) and somatostatin (SST), are able to suppress pituitary hormone secretion by reducing intracellular Ca2+ availability and also through adenylate cyclase (AC) inhibition, with subsequent cAMP levels reduction [1].

2.2.3 Corticotroph Cells Corticotrophs are the pituitary cells responsible for ACTH synthesis and secretion, which is regulated via multiple stimuli including hypothalamic regulatory factors [corticotropin-releasing hormone (CRH), AVP and DA]; pituitary cytokines and glucocorticoid hormones secreted by the adrenal cortex [6].

2.2.3.1

Adrenocorticotropic Hormone

ACTH plays a key role in the major physiological stress response system, the hypothalamic-pituitary-adrenal axis (HPA). In response to stress, CRH is released by neurons of the hypothalamic paraventricular nucleus into the hypophysis portal blood, to stimulate ACTH synthetizes and secretion by the anterior pituitary corticotrophs, which in turn will stimulate the adrenal cortex to synthetize and release glucocorticoids [7].

2.2.3.2

ACTH Regulation via CRH

CRH biological actions are exerted by coupling to CRH receptors (CRHR), which belong to the class II/secretin-like family of the G-protein coupled receptors superfamily. In mammals, there are two CRHRs expressed: CRHR1 and CRHR2. The predominant receptor in pituitary corticotroph cells is CRHR1 [8]. CRH binds to CRHR1 activating Gαs protein. Gαs activation results in AC activation and thus generation of cAMP. In turn, cAMP binds to the PKA regulatory subunit to release and activate its catalytic subunit. Then, PKA phosphorylates downstream targets, such as cAMP response element binding (CREB), which lead to the transcription of pro-opiomelanocortin (POMC), the prohormone precursor of ACTH. Besides, PKA also activates two other transduction pathways: the Ca2+ -dependent pathway which involves Ca2+ influx; and the mitogen-activated protein kinase (MAPK) pathway, an independent transduction pathway. PKA activates Rap1 and the signal is passed via phosphorylation through the kinase cascade (b-Raf/MEK/ERK). Extracellular signal–regulated kinases 1 and 2 (ERK1/2) regulate the induction and activity of target transcription factors involved in the transcriptional regulation of POMC, such as

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Nur77 and Nurr1 [9, 10]. Activated PKA also acts on membrane VGCCs triggering Ca2+ entry into the cell with a subsequent activation of Ca2+ /calmodulin-dependent protein kinase II (CaMKII) [11]. These signaling events lead to ACTH secretion and concomitant activation of POMC gene expression in corticotroph cells [12].

2.2.3.3

ACTH Regulation via Glucocorticoids

ACTH acts in the adrenal cortex to stimulate glucocorticoid synthesis. In turn, glucocorticoids maintain a regulatory feedback control of ACTH secretion, indirectly by inhibiting hypothalamic CRH and directly by inhibiting pituitary ACTH synthesis and secretion [6]. Indeed, glucocorticoids are able to cross the corticotroph cell membrane to bind the cytoplasmatic glucocorticoid receptor (GR). GR is then translocated into the nucleus to bind the negative glucocorticoid response elements of the POMC gene, downregulating gene transcription [13, 14]. Besides that, glucocorticoids inhibit ACTH secretion via CRH-mediated signaling pathways inhibition, a nongenomic GR-dependent mechanism [15].

2.2.4 Thyrotroph Cells Thyrotroph cells are the less abundant cell type in the anterior pituitary, representing only approximately 5% of functional cells. Thyrotroph cells are responsible for TSH synthesis and secretion, which is mainly stimulated by the hypothalamic TRH. Furthermore, TSH secretion is negatively regulated by the hypothalamic factor SST and thyroid hormones [16].

2.2.4.1

Thyroid-Stimulating Hormone

TSH is the intermediate hormone in the hypothalamic-pituitary thyroid (HPT) axis. Stimulated by TRH, TSH is released into the bloodstream to reach thyroid follicular cells and induce synthesis and secretion of thyroid hormones: thyroxine (T4) and triiodothyronine (T3) [17]. TSH is a glycol-protein structurally composed by two different subunits (α- and β-subunit) transcripted by two different genes: α-GSU gene and TSH-β gene. The α-subunit is expressed in excess in the pituitary to facilitate formation of the intact hormones, as this is shared by TSH and gonadotropins (FSH and LH), whereas β-subunit differs among the different hormones conferring to each one an individual physiological activity [18, 19].

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TSH Regulation via TRH

TRH has a dual effect on TSH, leading to rapid release of pre-synthesized stored TSH and also to an increase of the genes expression that are responsible for TSH synthesis. TRH binds to the membrane G protein-coupled type1 TRH receptor, which is coupled to Gq/11 proteins [20, 21]. Gq proteins activate PLC that subsequently hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2), thus inducing the release of second messengers: inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) [22]. In turn, DAG activates PKC, which induces gene expression, partially via the transcription factor activating protein-1 and by phosphorylating members of the MAPK family. Simultaneously, IP3 raises intracellular calcium concentration ([Ca2+ ]i ) levels by releasing Ca2+ from the intracellular pools, which activates the movement of secretory granules into the cell surface leading to TSH exocytosis [23, 24]. Besides that, Ca2+ can couple to calmodulin kinase, which activates CREB-binding protein (CBP) inducing the transcription of both α-GSU and TSH-β genes [23, 25]. Although, pituitary-specific transcription factor (Pit-1) is the most reported candidate protein to mediate the phenomena [25, 26], the precise molecular mechanisms by which these mechanisms occur, are still unclear. CBP and Pit-1 were found to act synergistically to stimulate the TSH-β promoter, while TRH mediated α-GSU stimulation appears to involve CBP and Pou1F1-like (PLIM) protein in a Pit-1-independent way [25].

2.2.4.3

TSH Regulation via Somatostatin

SST is predominantly produced in the hypothalamic periventricular nucleus, in addition to paraventricular, arcuate and ventromedial hypothalamic nuclei. SST binds to high-affinity SST receptors (SSTR) in the anterior pituitary gland to inhibit basal and TRH-stimulated TSH secretion [27]. SSTR are members of the Rhodopsin family of GPCRs and comprise five receptor subtypes (SSTR1-SSTR5) [28, 29]. In the thyrotrophs, the predominant SSTR subtypes are SSTR1 and 5 [29]. SSTR are coupled to Gi/o proteins that when activated lead to AC inhibition, thus decreasing intracellular cAMP and blocking TSH synthesis [28, 29]. Besides that, SST may also exert some cAMP-independent effects through decreasing [Ca2+ ]i [30]. SST acts directly on Ca2+ channels blockade and potassium (K+ ) channels opening, which result in cell membrane hyperpolarization and subsequent inhibition of VGCCs [31]. Intracellular cAMP and Ca2+ reduction prevent Ca2+ -regulated TSH exocytosis from the thyrotrophs [29, 30].

2.2.4.4

TSH Regulation via Thyroid Hormones

Thyroid hormones (T3) exert a powerful feedback inhibition over TRH response system by inhibiting synthesis and processing of TRH and decreasing TRH receptors in the hypothalamus and also by inhibiting TSH synthesis in the anterior pituitary

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gland [17, 32]. Although T4 concentrations in circulation are higher than T3, T4 is converted into metabolically active T3 by the anterior pituitary deiodinases [33, 34]. T3 mechanism of action involves interaction with its nuclear receptor acting predominantly at transcriptional level [35]. There are several different thyroid hormone receptors (THR) isoforms. THRα1, THRβ1 and THRβ2 are considered to be the relevant isoforms for T3 mediated TSH regulation in thyrotrophs [17]. Some reports found that the T3 binding to its receptor inhibits the expression of both α-GSU and TSH-β genes at the transcriptional level [35, 36]. However, the T3-induced repression of the TSH-β gene seems to be greater than that of α-GSU genes [37]. Although abundant information exists on the mechanisms involved in positive gene regulation by T3 [37], the molecular mechanisms involved in negative T3 regulation, such as for TSH subunit genes, are still not entirely understood. T3 binding directly to THR negative T3-responsive element (nTRE) in α-GSU and TSH-β genes has been postulated to inhibit TSH gene transcription [35, 38], while other authors proposed as an alternative explanation the unligated THR interaction with corepressors, including nuclear receptor co-repressor or silencing mediator for retinoid and thyroid hormone receptors on the target promoter. Upon T3 binding, THR corepressors would be released, resulting in the association with the DNA-binding transcription factor, repressing transcription [39]. Both theories have raised several questions that need to be answered before T3 negative regulatory mechanisms on thyrotrophs can fully be understood [37].

2.2.5 Gonadotroph Cells Gonadotroph cells comprise 10–15% of pituitary functional cells [40]. Gonadotroph cells can be mono- (15%) or bihormonal (70%) secreting either FSH or LH, or co-secreting both hormones, as suggested by immunohistochemical studies. FSH and LH together in the ovaries and the testis regulate gametogenesis (oogenesis or spermatogenesis), gonadal sex steroids (estrogen, progesterone and testosterone) and peptide hormones (inhibins) synthesis and secretion. Along with hypothalamic releasing factors, afore mentioned signaling molecules constitute the hypothalamicpituitary-gonadal axis (HPG) [41]. LH and FSH are heterodimers constituted by a common αGSU subunit and LHβ or FSHβ, which are encoded by chromosomes 6, 11 and 19, respectively [42]. These hormones display different actions and depict partially independent secretion patterns under regulation of specific signaling pathways, mediated by hypothalamic factors, such as GnRH and gonadotropin-inhibitory hormone (GnIH); intra-pituitary peptides acting in a paracrine manner, namely activin, follistatin and pituitary adenylate cyclase-activating peptide (PACAP); in addition to peripheral gonadal hormones feedback loops [43].

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Gonadotropin-Releasing Hormone

GnRH stands out as the main regulator of gonadotropins (FSH and LH) release. GnRH is released by the hypothalamus in a pulsatile manner into the pituitary portal system [44]. Depending on GnRH pulse frequency and amplitude, the gonadotropins secretion profile elicited will vary [45]. GnRH binds to the GnRH receptor (GnRHR) on gonadotrophs. GnRHR is a Gcoupled membrane receptor that activates Gq/11 subunit, which in turn activates PLC. PLC hydrolyzes PIP2 into DAG and IP3 leading to calcium release from intracellular pools and ultimately triggering FSH and LH exocytosis. Additionally, DAG activates PKC that increases the cell sensitivity to Ca2+ and also mediates FSH and LH exocytosis. Moreover, long-term stimulation by GnRH also activates phospholipase A2 and phospholipase D [46, 47]. Lastly, Gq subunit activation triggers FSHβ gene transcription whereas Gs activation suppresses FSH synthesis and induces LHβ expression [48]. Sustained GnRH stimulation tends to decrease [Ca2+ ]i in gonadotroph cells. Ca2+ repletion is assured by activated Ca2+ -dependent K+ channels leading to membrane hyperpolarization after GnRH stimulation. In turn, hyperpolarization triggers an action potential responsible for the activation of membrane high-voltage activated L-type Ca2+ channels, thus allowing the repletion of intracellular Ca2+ pools [49].

Regulation of Gonadotropin-Releasing Hormone Action GnRH secretion being pulsatile [50] enables a discontinuous gonadotroph stimulation that turns out to be is essential for physiological gonadotropin secretion [51]. GnRH pulsatile secretion is ensured by autocrine regulation via Ca2+ -dependent and cAMP signaling pathways in result of Gq/11 and Gs subunits activation, respectively. Gq activates PLC increasing the formation of IP3 that mediates Ca2+ release from intracellular storages [52]. Additionally, GnRH-binding activates Gs subunit and consequently the cAMP pathway [53]. However, in the presence of higher GnRH concentrations, Gi subunit is activated to suppress GnRH neuronal activity and GnRH secretion. This ultimately implies that GnRH secretion is the end result of pulsatile Ca2+ - and cAMP-mediated GnRH release, which is highly dependent on G-subunits selective activation [50]. GnRH secretion is further regulated by circulating estrogens, capable of dosedependent activation of estrogen receptors α and β (ERα and ERβ) present on hypothalamic neurons. Indeed, via ERα Gi-subunit, estradiol (E2 ) inhibits action potential, cAMP-pathway and GnRH secretion [54]. However, at higher E2 concentrations, GnRH secretion is positively regulated via ERβ [50]. Worth mentioning, GnRH secretion is also upregulated by other molecules, including kisspeptins [55], glutamate and norepinephrine and downregulated by GnIH, CRH, neuropeptide Y (NPY), urocortin 2 (Ucn2), gamma-aminobutyric acid (GABA) and opioids [56].

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Along with neurokinin B (NKB), kisspeptins are a family of peptides co-expressed in the hypothalamus. Kisspeptins are encoded by the KISS1 gene and comprise kisspeptin 54 (Kp-54), formerly named metastin, and other smaller peptides, namely Kp-14, Kp-13 and Kp-10 [57]. Kisspeptins share a C-terminal argininephenylalanineNH2 sequence that binds to the specific KISS1 receptor (KISS1R) to activate the Gcoupled protein Gpr54 ubiquitously [58]. In turn, Gpr54 activation promotes GnRH secretion in addition to simulating gonadotropin release via PLC, PKC, MAPK and consequently trough the increase of [Ca2+ ]i [59]. GnIH is a hypothalamic peptide that suppresses GnRH release via Gpr147 receptor [60]. A similar rationale applies to NPY that, via Y1 and Y5 receptors, which expression is under control of estrogens, inhibits GnRH-induced downstream pathways and LH secretion [61, 62]. In addition, CRH, a central regulator of the HPA axis, is able to suppress GnRH pulses via CRH1 and CRH2 by decreasing mRNA levels and GnRH secretion, respectively. Moreover, Ucn2, a member of CRH family, suppresses gonadotropins production via gonadotrophs CRH2 receptor [63].

2.2.5.2

Pituitary Adenylate Cyclase-Activating Peptide

PACAP is a hypothalamic peptide produced by gonadotroph and FS cells. PACAP secretion and receptor (PAC1-R) expression are upregulated by low frequency GnRH pulses. In turn, PACAP stimulates directly and indirectly LH secretion, either through PAC1-R downstream pathways via cAMP and IP3 or through GnRH-mediated, respectively [64, 65].

2.2.5.3

Activins, Follistatin and Inhibins

In the absence of GnRH stimulation, LH secretion is rapidly suppressed whereas FSH secretion proceeds in a pulsatile manner under regulation of inhibin, activin and follistatin local secretion [66]. Activins are β-subunit dimers with four identified forms (βA, βB, βC and βE) among which βA- and βB-subunits are best characterized, which can give rise to activin A (βAβA), activin B (βBβB) or activin AB (βAβB) [67]. Indeed, activins are known to regulate tissue growth and differentiation, while stimulating FSH secretion via paracrine and autocrine actions [68]. In turn, inhibins are dimeric proteins, members of the transforming growth factorβ (TGF-β) family with a shared α-subunit and a βA or a βB subunit. Inhibins are mainly secreted peripherally by the gonadal tissues. Inhibin A is secreted by dominant follicles and corpus luteus in the ovarium, whereas inhibin B is secreted in testis, as well as in the ovary during early follicular and late luteal phases of the ovarian cycle [68]. In addition, inhibins are secreted by gonadotrophs, thyrotrophs, somatotrophs and FS cells [69]. In mammals, activin and inhibin, are responsible for increasing or decreasing FSH release, respectively. Activin and inhibin release depends on the relative intracellular

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availability of α and β subunits, as a predominance of β-subunits leads to activin release while high levels of α-subunits trigger activin secretion [16]. Activins binding-mediated activin receptor type II (ActRII) activation induces activin receptor type I (ActRI) phosphorylation, which then activates intracellular SAMD proteins/forkhead box L2 (SMAD/FoxL2) signaling pathways that regulate gene transcription and function [70]. Ultimately, activins increase FSHβ transcription [71] and gonadotrophs sensitivity to GnRH by increasing GnRHR expression [72, 73]. In contrast, inhibins competitively bind to ActRII to form inactive receptor heterodimers thus decreasing activin actions [66]. Inhibins are able to suppress FSH [68] by acting directly on gonadotroph cells via TGF-β3 receptor to reduce FSHβ synthesis and secretion [74], through still poorly characterized downstream pathways [66]. Lastly, follistatin is a structurally independent monomeric peptide mainly produced by gonadotrophs and FS cells in addition to other pituitary endocrine cell lines, which binds activin reducing the affinity to target receptors and ultimately selectively reducing FSH secretion [68, 72].

2.2.5.4

Sex Steroids-Mediated Feedback Loops

Gonadal steroid hormones are responsible for direct regulation of gonadotropins release by acting via specific receptors on gonadotroph cells [45]. These mechanisms are distinctly regulated in the female and male gonadal axis. In females, during the early follicular phase of the ovarian cycle, rising estrogen levels act directly on the ERα of gonadotroph cells to decrease αGSU, LHβ and FSHβ subunits mRNA transcription and ultimately LH and FSH secretion, in a negative feedback loop. The E2 suppressive effects are extended to the hypothalamus and mediated by kisspeptin neurons via ERα [75]. In late follicular phase, triggered by high estrogen and low progesterone levels, estrogens feedback effect on the hypothalamic-pituitary axis shifts from negative to positive [76], increasing hypothalamic GnRH secretion pulses frequency and reducing circulating inhibin levels, thus allowing the ovulatory FSH and LH surge to occur [45]. Herein, E2 acts on somatotrophs via ERα [77] to increase gonadotroph cells sensitivity to GnRH and also the proportion of active secretory gonadotroph cells during the gonadotropin surge [78]. During luteal phase, the increase of progesterone levels downregulates GnRH pulses frequency that consequently also reduces LH pulse frequency [45, 79]. In males, not only testosterone, but also 5α-dihydrotestosterone (DHT), a reduced form of testosterone and E2 , an aromatized testosterone compound, are able to suppress FSH and LH secretion, either by suppressing GnRH neurons or directly action on gonadotroph cells [80]. This phenomena is mostly achieve by GnRHinduced alterations in [Ca2+ ]i [81], which ultimately suppresses LH synthesis [82] but controversially increases FSHβ mRNA levels [83] and FSH secretion [84].

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2.2.5.5

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Leptin

Leptin acting as a surrogate marker of energy availability is also able to stimulate the HPG by increasing GnRHR expression in gonadotrophs in addition to possibly raising intracellular levels mRNA of activins subunits [85].

2.2.5.6

Gonadotropins Differential Secretion

FSH and LH although being mostly synthetized and stored by the same gonadotroph cells, secretion is partially independent and mediated by different molecular pathways. Noteworthy, both FSH and LH are both secreted in a pulsatile manner. Despite the detailed underlying molecular mechanisms are still lacking clarification, slow GnRH pulses every two to four hours were shown to lead to FSH secretion via PKA and CREB [86], whereas more frequent GnRH pulses every half an hour to one hour, seem to mediate LH synthesis and secretion via transcription of early growth response protein 1 (Egr-1) that increases LHβ protein expression [87, 88]. However, since FSH half-time life is longer than the LH, FSH secretion pattern is more subtle and somehow difficult to ascertain [89].

2.2.6 Somatotroph Cells GH is a 191-amino-acid hormone synthetized and released by pituitary’ somatotroph cells. GH primarily induces linear growth and plays a core role in regulating metabolism throughout lifetime [90]. GH stimulates cartilage formation by increasing liver insulin-like growth factor 1 (IGF-1) release. In addition, GH antagonizes insulin effects by stimulating amino-acid uptake and protein synthesis, by increasing free fatty acids mobilization from adipose tissue and by decreasing glucose uptake [90]. GH secretion is mostly regulated by two hypothalamic hormones (Fig. 2.2)—GHreleasing hormone (GHRH) and SST, also known as GH-inhibiting hormone—in addition to ghrelin, which have direct action on somatotrophs via specific receptors [91, 92].

2.2.6.1

Up- and Down-Regulation of the Signaling Pathways Involved in GH Secretion by Somatotrophs

GHRH is secreted by the arcuate nuclei neurons of the hypothalamus [93]. GHRH suppresses its own receptor mRNA levels via cAMP pathway [94] and stimulates somatotrophs proliferation [95], GH gene transcription and GH release [96]. GHRH peripheral plasma concentrations are extremely low, hence regulation of pituitary GH secretion occurs locally via the portal system [97].

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Fig. 2.2 Overall regulation of growth hormone (GH) secretion. GH acts systemically via hepatic production of insulin-like growth factor 1 (IGF-1) and it is regulated not only by hypothalamic factors—GH-releasing hormone (GHRH) and somatostatin—but also by IGF-1 circulating levels and by multiple endogenous and exogenous stimulus. Abbreviations VIP—vasoactive intestinal peptide. Notes Dashed red arrows represent negative feedback pathways

In somatotrophs, GHRH receptor (GHRHR) is G-protein coupled and activated not only by GHRH but also by vasoactive intestinal peptide (VIP) and secretin [98]. In addition, glucocorticoids, thyroid hormones, estrogens and androgens also influence GHRHR expression [97]. The GHRHR is a seven-domain transmembrane receptor coupled with G proteins [97, 99]. GHRHR-coupled α subunit (Gs) upregulates AC which increases intracellular cAMP levels [98]. In turn, cAMP activates PKA that prompts membrane depolarization by triggering an increase in [Ca2+ ]i [100], hence leading to GH secretion via Ca2+ /CaMKII [101]. Moreover, cAMP via PKA phosphorylates CREB protein [97] that in turn increases the expression of Pit-1 gene. Ultimately, Pit-1 activates GH [102] and GHRHR [103] genes transcription. Gq subunit of GHRHR triggers PLC to convert PIP2 to DAG and IP3. In turn, DAG and increased [Ca2+ ]i activate PKC that phosphorylates GH-secreting peptides [104] and increases GHRHR expression [97]. PKC also triggers phospholipases A and D activity that amplify PKC activity by hydrolyzing phosphatidylinositol [105]. In an opposite direction, SST suppresses GH and TSH secretion, by direct action on somatotrophs via SSTR [106] that blocks the described intracellular pathways [107]. The SSTR-mediated SST actions are the same as previously described when focusing on TSH regulation.

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Additionally, GH secretagogue receptors (GHS-R) are activated by an endogenous agonist which is ghrelin, in addition to multiple exogenous agonists besides being also allosterically activated by GHRH [108], all contributing to enhance GHRH activity [109]. In somatotrophs, ghrelin activates multiple signaling pathways, namely PLC, PKC and cAMP/PKA, which ultimately leads to a rise in [Ca2+ ]i [110] and strongly stimulates GH secretion in humans [111]. However, ghrelin-mediated GH secretion despite also depending on extracellular Ca2+ influx is independent from the GHRHinduced secretion [112].

2.2.6.2

Regulation of GH and GHRH Synthesis and Release

Along with SST, GHRH determines a pulsatile release of GH from somatotrophs [113]. Moreover, GH induces hepatic synthesis of IGF-1, which together with GH contribute to downregulate GHRH synthesis, GH gene transcription and GH secretion [114, 115]. Additionally, IGF-1 increases mRNA levels and stimulates SST release, ultimately suppressing GHRH expression [116]. Altogether, high GH and IGF-1 levels ultimately decrease GH secretion [117], whereas high GH and GHRH stimulate SST secretion [97]. Although GH receptors are present at the arcuate nucleus [118], whether GH is able to directly regulate GHRH neurons or indirectly via NPY-containing on neighbor neurons [119]. Additionally, other hormones were shown to interfere in pathways responsible for GH hormone synthesis and release, including thyroid hormones, as high circulating thyroid hormone levels decrease GHRH mRNA and increase SST mRNA expression [120]; glucocorticoids that are able to directly regulate GHRH-producing neurons [121] and gonadal steroids [122]. Furthermore, molecules such as α1 and β2 adrenergic agonists inhibit GH secretion, whereas α2 adrenergic agonists [123], prostaglandins [124] and leptin [125] are able to increase circulating GH levels.

2.2.7 Lactotroph Cells Lactotroph cells, also known as mammotrophs, are PRL-producing cells that constitute 15–25% of the total functional cell mass in the anterior pituitary [126]. PRL is also secreted by mammosomatotrophs that were described to be transitional pituitary cells capable to alternate between somatotroph and lactotroph secretory functions [127]. The number of PRL producing cells varies during the menstrual cycle and increases significantly during pregnancy and lactation [128–130].

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Prolactin

PRL is a polypeptide hormone structurally related to GH. PRL was shown to be involved in numerous processes, including reproduction, metabolism, immunology and behavior. The most well described PRL action is promoting growth and development of the mammary gland, as well as initiation and maintenance of lactation [131]. Pituitary lactotrophs have a high basal secretory activity, which is rather unique among endocrine cells. To maintain PRL homeostasis, PRL secretion is under a hypothalamic tonic inhibitory control, predominantly mediated by DA [132].

2.2.7.2

Inhibitory Control of PRL Synthesis and Secretion

DA is released from the ventral tegmental area of the substantia nigra, midbrain and the arcuate nucleus of the hypothalamus into hypophysis portal veins [132]. Besides that, the anterior pituitary through short portal veins also receives DA from the posterior pituitary [133]. DA inhibits PRL synthesis and secretion by binding to dopamine D2 receptors (D2R) [6, 134, 135]. D2R belong to the seven-transmembrane domain of G-protein coupled receptor class, which exists in two main variants: the long isoform named long D2R (D2RL) and the short isoform (short D2R, D2RS). The two D2R isoforms have different functional characteristics, as these differ in a cytoplasmic loop that plays a crucial role in determining the G protein coupling specificity and consequently in activating different signaling pathways [135–138]. DA signaling via Gαi protein inhibits AC activity resulting in diminished intracellular cAMP levels. In the absence of cAMP, the PKA catalytic subunit remains inactive and does not phosphorylates target proteins, thus preventing activation of PRL gene transcription and PRL release [139–142]. Besides that, DA also signals via Gα0 that leads to K+ channels opening, VGCCs inhibition and subsequent decrease of [Ca2+ ]i [143]. Furthermore, D2R activation also inhibits PLC, which reduces Ca2+ mobilization from endoplasmic reticulum, also decreasing [Ca2+ ]i [138, 144]. Low Ca2+ levels inhibit the formation of PRL storage vesicles and release [141]. PLC inhibition prevents PKC activation, which in a similar mode to what occurs with PKA, when inactivated prevents PRL gene transcription (Fig. 2.3) [132].

2.2.7.3

Stimulatory Control of PRL Synthesis and Secretion

Since PRL is involved in the regulation of numerous processes, such as reproduction, metabolism, immunology and behavior, PRL positive regulation is complex and includes several hormones (Table 2.1). Only the most important and well stablished PRL releasing stimulus are described in further detail. The most powerful physiological stimulus for PRL release is nipple suckling, responsible for triggering the release of lactotroph cells from tonic suppression. This mechanism occurs through an afferent neural pathway leading to hypothalamus dopaminergic neurons inhibition. Besides that, suckling also increases VIP

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Fig. 2.3 Inhibitory control of prolactin (PRL) via dopamine (DA). (1a) DA signaling via Gαi protein inhibits the adenylate cyclase (AC) activity; (2a) it results in a decrease of intracellular cyclic 3 ,5 -adenosine monophosphate (cAMP) levels; (3a) without cAMP, the catalytic subunit of protein kinase A (PKA) remains inactive; (1b) DA also signals via Gα0 which leads to potassium (K+ ) channels opening and inhibition of calcium (Ca2+ ) channels; (2b) it also inhibits phospholipase C (PLC), blocking diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) formation, ultimately reducing calcium (Ca2+ ) mobilization from endoplasmic reticulum and (3b) protein kinase C (PKC) activation; (4) Together, these pathways suppress PRL gene transcription and ultimately PRL release. Notes Stop arrow represent inhibitory pathways

and oxytocin hypothalamic levels, which may also act as PRL releasing hormones (PRH) [141, 145, 146], although the signaling pathways involved in VIP- and oxytocin-mediated PRL secretion are not completely understood.

Estrogens During pregnancy, estrogen levels are high and the number of PRL-secreting cells and serum PRL levels increase. Estrogens control PRL expression in lactotroph cells, primarily by a transcription-dependent mechanism [16]. Estrogen responses are mediated by the ER, a nuclear receptor that interacts with specific DNA sequences, the estrogen response elements (EREs). PRL promoter contains EREs responsible for mediating estrogen effects on gene transcription. However, for that to occur the PRL enhancer/promoter needs to be bounded to Pit-1, a key transcription factor that regulates basal PRL expression [147–149]. Besides this pathway estrogens also regulate PRL expression via transforming growth factor beta (TGF-β) and basic fibroblast growth factor (bFGF). Lactotrophs secrete both TGF-β1 and TGF-β3. After estrogens stimulation TGF-β1 levels decrease, while TGF-β3 levels increase. This effect occurs not via EREs but via an alternative estrogen-sensitive sequence. Estrogen-induced TGF-β3 is released from lactotrophs to act on neighboring FS

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Table 2.1 Summary of the signaling pathways modulated by different positive regulators on the PRL secretion Hormone

Signaling pathway

References

Estrogens

PLC/Ca2+

Perez-Castro et al. [16], Day et al. [147], Simmons et al. [148], Holloway et al. [149], Sarkar [153], Ishida et al. [154], Chaturvedi and Sarkar [151], Jaye et al. [152]

AC/cAMP MAPK/ERK

Thyrotropin releasing hormone

PLC/Ca2+ MAPK/ERK

Kanasaki et al. [158], Tashjian et al. [159], Noel et al. [160], Perez-Castro et al. [16], Gershengorn [161]

Oxytocin

PLC/Ca2+

Peter et al. [163], Johnston and Negro-Vilar [164], Samson et al. [145], Chiodera et al. [146]

Vasoactive intestinal polypeptide

AC/cAMP

Nicosia et al. [165], Oliva et al. [166]. Onali et al. [167], Chiodera et al. [146]

Arginine vasopressin

PLC/Ca2+ AC/cAMP

Peter et al. [163], Nagy et al. [168], Pan and Mai [169], Shin [170]

Angiotensin II

PLC/Ca2+ AC/cAMP

Enjalbert et al. [171], Audinot et al. [172], Login et al. [173], Moreau et al. [174]

Neurotensin

PLC/Ca2+ AC/cAMP Extra-cellular Ca2+ mobilization

Memo et al. [175], Enjalbert et al. [176], Memo et al. [177]

Leptin

AC/cAMP Extra-/intra-cellular Ca2+ mobilization PI3K

Tennekoon et al. [178], Sarmento-Cabral et al. [179]

Adiponectin

AC/cAMP Extra-/intra-cellular Ca2+ mobilization PI3K

Sarmento-Cabral et al. [179]

Melatonin

AC/cAMP PLC/Ca2+ Extra-cellular Ca2+ mobilization

Ibanez-Costa et al. [180]

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cells. On FS cells, TGF-β3 induces bFGF synthesis that binds to specific tyrosine kinase receptors found on lactotrophs membrane, fibroblast growth factor receptors (FGFR) [150–152]. Activated FGFR then activate PLC that hydrolyzes PIP into DAG and I3P, leading to PKC stimulation. PKC activates the MAPK/ERK pathway and consequently ERK1/ERK2 phosphorylation, which in turn activates transcription factor Ets-1. PRL promoter contains Ets-1 binding sites that acts synergistically with Pit-1 to regulate PRL transcription. Besides that, FGFR is also able to directly activate Src kinase, which signal is passed via phosphorylation through the kinase cascade (bRaf-MEK1/2-ERK1/2), by which ERK1/2 induces activation of transcription factors involved in lactotroph cell proliferation [151–154]. Estrogens also decrease of lactotroph response to DA acting on GTP-binding proteins of the AC-transducing system. Estrogens lead to Gαi and Gα0 G-proteins phosphorylation, which are then uncoupled from the D2R and so downregulating receptor activity [153, 155]. Moreover, by modifying alternative splicing, estrogens favor D2RL isoform instead of D2RS expression, which has also consequences for D2R interaction with G-proteins [16, 156, 157].

TRH Although TRH was originally solely reported as a TSH-releasing hormone, there is now considerable evidence that this factor is also involved in other pituitary functions. In lactotrophs, TRH stimulates PRL synthesis and release of through different signaling cascades [158–160]. As previously described for TSH regulation, TRH binds to Gq protein-coupled TRH receptor and activates MAPK/ERK pathway directly or indirectly via PLC. PLC activation hydrolyzes PIP2 into IP3 and DAG. DAG activates PKC which in turn passes the signal via phosphorylation through the MAPK/ERK cascade [161]. Activated ERK increases PRL gene transcription through activation of Est-1 and Pit-1 transcription factors [162]. Besides that, IP3 increases intracellular Ca2+ levels leading to secretory granules movement the cell surface and PRL exocytosis [158, 161].

2.3 Conclusion This chapter describes the main signaling pathways involved in anterior pituitary secretory regulation. Through the multiple harmoniously articulated metabolic pathways hereby described, either by autocrine, paracrine or endocrine pathways, thigh physiological control is achieved in an ever-changing synchronized orchestra towards body homeostasis, coordinated by pituitary gland. Acknowledgments Unit for Multidisciplinary Research in Biomedicine (UMIB) is funded by grants from FCT (UID/Multi/00215/2016 and UID/MULTI/0215/2019).

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164. Johnston CA, Negro-Vilar A (1988) Role of oxytocin on prolactin secretion during proestrus and in different physiological or pharmacological paradigms. Endocrinology 122(1):341–350. https://doi.org/10.1210/endo-122-1-341 165. Nicosia S, Spada A, Borghi C, Cortelazzi L, Giannattasio G (1980) Effects of vasoactive intestinal polypeptide (VIP) in human prolactin (PRL) secreting pituitary adenomas. Stimulation of PRL release and activation of adenylate cyclase. FEBS Lett 112(2):159–162 166. Oliva D, Vallar L, Giannattasio G, Spada A, Nicosia S (1984) Combined effects of vasoactive intestinal peptide and dopamine on adenylate cyclase in prolactin-secreting cells. Peptides 5(6):1067–1070 167. Onali P, Schwartz JP, Costa E (1981) Dopaminergic modulation of adenylate cyclase stimulation by vasoactive intestinal peptide in anterior pituitary. Proc Natl Acad Sci USA 78(10):6531–6534 168. Nagy G, Mulchahey JJ, Smyth DG, Neill JD (1988) The glycopeptide moiety of vasopressinneurophysin precursor is neurohypophysial prolactin releasing factor. Biochem Biophys Res Commun 151(1):524–529 169. Pan JT, Mai LM (1990) Dopamine antagonism does not potentiate the effects of oxytocin and vasopressin on prolactin secretion. Life Sci 47(26):2443–2450 170. Shin SH (1982) Vasopressin has a direct effect on prolactin release in male rats. Neuroendocrinology 34(1):55–58. https://doi.org/10.1159/000123277 171. Enjalbert A, Sladeczek F, Guillon G, Bertrand P, Shu C, Epelbaum J, Garcia-Sainz A, Jard S, Lombard C, Kordon C et al (1986) Angiotensin II and dopamine modulate both cAMP and inositol phosphate productions in anterior pituitary cells. Involvement in prolactin secretion. J Biol Chem 261(9):4071–4075 172. Audinot V, Rasolonjanahary R, Bertrand P, Priam M, Kordon C, Enjalbert A (1991) Involvement of protein kinase-C in the effect of angiotensin-II on adenosine 3 ,5 -monophosphate production in lactotroph cells. Endocrinology 129(4):2231–2239. https://doi.org/10.1210/endo129-4-2231 173. Login IS, Judd AM, Kuan SI, MacLeod RM (1991) Role of calcium in dopaminergic regulation of TRH- and angiotensin II-stimulated prolactin release. Am J Physiol 260(4 Pt 1):E553–E560. https://doi.org/10.1152/ajpendo.1991.260.4.E553 174. Moreau C, Rasolonjanahary R, Audinot V, Kordon C, Enjalbert A (1994) Angiotensin II effects on second messengers involved in prolactin secretion are mediated by AT1 receptor in anterior pituitary cells. Mol Cell Neurosci 5(6):597–603. https://doi.org/10.1006/mcne.1994. 1073 175. Memo M, Carboni E, Trabucchi M, Carruba MO, Spano PF (1985) Dopamine inhibition of neurotensin-induced increase in Ca2+ influx into rat pituitary cells. Brain Res 347(2):253–257 176. Enjalbert A, Arancibia S, Priam M, Bluet-Pajot MT, Kordon C (1982) Neurotensin stimulation of prolactin secretion in vitro. Neuroendocrinology 34(2):95–98. https://doi.org/10.1159/ 000123284 177. Memo M, Castelletti L, Valerio A, Missale C, Spano PF (1986) Identification of neurotensin receptors associated with calcium channels and prolactin release in rat pituitary. J Neurochem 47(6):1682–1688 178. Tennekoon KH, Eswaramohan T, Karunanayake EH (2007) Effect of leptin on prolactin and insulin-like growth factor-I secretion by cultured rat endometrial stromal cells. Fertil Steril 88(1):193–199. https://doi.org/10.1016/j.fertnstert.2006.11.115 179. Sarmento-Cabral A, Peinado JR, Halliday LC, Malagon MM, Castaño JP, Kineman RD, Luque RM (2017) Adipokines (leptin, adiponectin, resistin) differentially regulate all hormonal cell types in primary anterior pituitary cell cultures from two primate species. Sci Rep 7:43537. https://doi.org/10.1038/srep43537 180. Ibanez-Costa A, Cordoba-Chacon J, Gahete MD, Kineman RD, Castano JP, Luque RM (2015) Melatonin regulates somatotrope and lactotrope function through common and distinct signaling pathways in cultured primary pituitary cells from female primates. Endocrinology 156(3):1100–1110. https://doi.org/10.1210/en.2014-1819

Chapter 3

Cell Signaling Within Endocrine Glands: Thyroid, Parathyroids and Adrenal Glands Sofia S. Pereira, Carolina B. Lobato, and Mariana P. Monteiro

Abstract Despite the fact that there can be argued that no single cell in the human body can be devoid of molecular tools that fit into the broad definition of an endocrine function, some organs are primarily dedicated to hormone secretion and are therefore designated endocrine glands. Under regulation by pituitary gland (reviewed on the previous chapter), three peripheral organs are exclusively devoted to endocrine functions: the thyroid, the parathyroid and the adrenal glands. This Chapter on endocrine system will cover the signaling pathways implied in these three organs, with identification of their particular and shared features. Keywords Endocrine system · Signaling pathways · Thyroid gland · Parathyroid gland · Adrenal gland

S. S. Pereira Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Porto, Portugal e-mail: [email protected] Institute of Molecular Pathology and Immunology, University of Porto (IPATIMUP), Porto, Portugal S. S. Pereira · C. B. Lobato · M. P. Monteiro (B) Endocrine, Cardiovascular and Metabolic Research, Unit for Multidisciplinary Research in Biomedicine (UMIB), University of Porto, Porto, Portugal e-mail: [email protected] C. B. Lobato e-mail: [email protected] Department of Anatomy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal © The Author(s) 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_3

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Abbreviations 17βHSD5 1α,25(OH)2D 2-AG [Ca2+ ]i [Ca2+ ]e [(PO4 )3− ]e AA AADC AC Ach ACTH ANG I ANG II AT1R ATF ATP CaMK CaMKII cAMP CaSR CREB CRH CT CYB5A CYP11A1 CYP11B1 CYP11B2 CYP17A1 CYP21A2 DA DAG DBH DHEA DHEA-S Epi ERK FGF23 GH GR Hh HPA HSL

17β-hydroxysteroid dehydrogenase 1α,25-dihydroxyvitamin D 2-arachidonoylglycerol Intracellular calcium concentration Extracellular ionized calcium concentration Phosphate serum concentration Arachidonic acid L-aromatic amino acid decarboxylase Adenylate cyclase Acetylcholine Adrenocorticotropic hormone Angiotensin I Angiotensin II Angiotensin II receptor type 1 Activating transcription factor Adenosine triphosphate Calmodulin-dependent protein kinases Calmodulin-dependent protein kinase II Cyclic 3 ,5 -adenosine monophosphate Calcium-sensing receptor cAMP response element binding Corticotropin-releasing hormone Calcitonin Cytochrome B5A Cholesterol side chain cleavage enzyme 11β-hydroxylase Aldosterone synthase 17α-Hydroxylase 21α-hydroxylase Dopamine Diacylglycerol Dopamine β-hydroxylase Dehydroepiandrosterone Dehydroepiandrosterone sulfate Epinephrine Extracellular signal-regulated kinase Fibroblast growth factor 23 Growth hormone Glucocorticoid receptor Hedgehog signaling pathway Hypothalamic-pituitary-adrenal axis Hormone-sensitive lipase

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IP3 LDL LO MAG MC2R MAPK MCT8 NCoR NE NIS NO PI3K PIP2 PNMT PKA PKC PLA2 PLC PLD (PO4 )3− PRL PTH PTHrP SF1 Shh StAR SULT2A1 T3 T4 TG THSR TPO TRH TRHR1 TSH TSHR VMAT1 VGCC

Inositol 1,4,5-triphosphate Low-density lipoproteins Lipoxygenase Monoacylglycerol lipase Melanocortin receptor 2 Mitogen-activated protein kinase Monocarboxylate transporter 8 Nuclear receptor co-repressor Norepinephrine Sodium-iodide symporter Nitric oxide Phosphoinositide-3-kinase Phosphatidylinositol 4,5-bisphosphate Phenylethanolamine N-methyltransferase Protein kinase A Protein kinase C Phospholipase A2 Phospholipase C Phospholipase D Phosphate Prolactin Parathormone Parathyroid hormone-related protein Steroidogenic factor 1 Sonic hedgehog Steroidogenic acute regulatory protein Sulfotransferase 2A1 Triiodothyronine Thyroxine Thyroglobulin Thyroid-stimulating hormone receptor Thyroid peroxidase Thyrotropin-releasing hormone Thyrotropin-releasing hormone receptor 1 Thyroid-stimulating hormone Thyroid-stimulating hormone receptor Vesicular monoamine transporter 1 Voltage-gated Ca2+ channels

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3.1 Introduction There can be no argue that there is no single cell in the human body devoid of functionalities that could fit in this broad definition of an endocrine function, some cell types present a specific histological endocrine differentiation and are primarily committed to synthesize and secrete hormones. Endocrine differentiated cells can be found either scattered, isolated or in cell aggregates in organs pertaining to different physiological systems, such as endocrine cells along the gastro-intestinal system which comprise part of the “diffuse endocrine system”, or in alternative can be organized in endocrine tissues within organs that are dedicated to the secretion of specific hormones, also known as endocrine glands. Hormones can be classified according to three different molecular classes based on their chemical structure: peptide hormones, which include proteins and polypeptides; steroids, which are lipidderived hormones and amino acid-derived, namely tyrosine derived hormones [1]. The focus of this chapter will be to describe the signaling pathways so far identified in the three peripheral glands of the endocrine system, namely within thyroid, parathyroid and adrenal gland.

3.2 Thyroid Gland The thyroid is an endocrine gland located in the anterior part of the lower neck [2]. The functional unit of the gland is the thyroid follicle that consists of a central core of colloid surrounded by an epithelium with a single layer of follicular cells (Fig. 3.1). In the thyroid gland parenchyma there are two predominant cell types: the epithelial follicular cells, which are the vast majority of thyroid tissue cells [3] and the parafollicular cells, also known as C cells that reside in the periphery of the follicle [3, 4].

3.2.1 Follicular Cells Thyroid follicular cells are the ones responsible for the synthesis and secretion of thyroid hormones thyroxine (T4) and triiodothyronine (T3). The most important regulator of thyroid hormones synthesis is the thyroid-stimulating hormone (TSH) secreted by the anterior pituitary gland under the influence of the hypothalamic thyrotropin-releasing hormone (TRH). TSH is very sensitive to small fluctuations in serum thyroid hormones levels [5]. Besides that, as iodine is a limiting substrate for thyroid hormone synthesis, dietary iodine availability is also an important regulatory factor [6].

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Fig. 3.1 Human thyroid gland stained by hematoxylin and eosin (H&E) (200x); C—Colloid, FC— Follicular cells, PC—Parafollicular cells

3.2.1.1

Regulation of Thyroid Hormones Synthesis by TSH

TSH is the main physiological hormone implicated in thyroid function regulation. TSH acts on the follicular thyroid cell by activating the TSH receptor (TSHR), a member of the glycoprotein G coupled-receptor family (Fig. 3.2) [7]. TSHR is located at the basolateral membrane of thyroid follicular cells and mediates the activation of two regulatory pathways: cyclic 3 ,5 -adenosine monophosphate (cAMP) and phospholipase C (PLC) cascades [8, 9]. This dual-activation is rendered by the ability of TSHR being capable to interact with all Gα subtypes, in particular with the Gs and Gq subtypes [10, 11]. After TSH-mediated receptor activation TSHR couples predominantly to Gs [12, 13]. Gs activation then leads to cAMP production that binds to the regulatory subunit of protein kinase A (PKA), releasing and activating its catalytic subunit. Activated PKA regulates the iodine uptake and the transcription of genes involved in thyroid hormone production: sodium-iodide symporter (NIS), thyroglobulin (TG) and thyroid peroxidase (TPO) [4, 9, 14]. In the PLC cascade, the TSHR activation causes the activation of Gq protein that stimulates PLC [8, 9]. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). IP3 binds to its endoplasmic reticulum receptors, which act as channels for the release of the Ca2+ stored in this organelle. Increased intracellular calcium concentration [Ca2+ ]i is followed by an increase of Ca2+ from the extracellular medium. In thyroid cells, Ca2+ activates calmodulin-dependent protein kinases (CaMK) that regulate the iodide apical efflux, H2 O2 generation through Dual oxidase 2 (DUOX-2) activation, TG iodination and constitutive activation of nitric oxide (NO) synthase [9, 12, 15]. In

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Fig. 3.2 Signaling pathways activated by thyroid stimulating hormone (TSH) in thyroid follicular cells. (1) TSH binds to the thyroid stimulating hormone receptor (THSR), activating two Gα protein subtypes: Gs and Gq. These proteins activate two different regulatory pathways: cAMP and phospholipase-C (PLC) pathways, respectively. cAMP pathway: (2a) Gs activates adenylate cyclase (AC); (3a) AC converts adenosine triphosphate (ATP) to cAMP; (4a) cAMP binds to the regulatory subunits (R) of protein kinase A (PKA), releasing and activating the catalytic subunits (C) of this protein (5a) Activated PKA activates transcription of genes involved in the thyroid hormone production: sodium-iodide symporter, thyroglobulin and thyroid peroxidase. PLC pathway: (2b) Gq activation stimulates PLC that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3); (3b) IP3 binds to its endoplasmic reticulum receptors releasing the Ca2+ stored in this organelle; (4b) Increased intracellular Ca2+ is followed by an increase of Ca2+ from the extracellular medium and calmodulin-dependent protein kinases (CaMK) activation; (5b) DAG activates the protein kinase C (PKC); (6b) PLC pathway, through CaMK and PKC activation, regulate the iodide apical efflux, H2 O2 generation, thyroglobulin iodination and constitutive activation of nitric oxide synthase. Notes Dotted arrows depict particles movement

addition, DAG, the other molecule that results from PIP2 hydrolysis, is responsible for the activation of protein kinase C (PKC) which in turn activates protein kinase D (PKD) that enhances iodination and also activates the transcription of genes involved in the thyroid hormone production, such as DUOX-2 [4, 16, 17]. A cross-signaling between PIP2 and cAMP cascades has been reported. The activation of CAMK through the PIP2 cascade inhibits cAMP accumulation and thus the cAMP cascade. Besides that, the PKC activation enhances cAMP response to TSH [4, 18].

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Regulation of Thyroid Hormones Synthesis by Iodine

Iodine is the main substrate used by thyroid follicular cells for the synthesis of thyroid hormones [6]. The thyroid gland has the capacity to maintain synthesis and secretion of thyroid hormones when iodine availability becomes scarce by shifting the synthesis of hormones from T4 to T3, which synthesis requires less iodine besides being more potent [19]. In contrast, iodide excess decreases the thyroid response to TSH, thus inhibiting the thyroid hormones secretion. This phenomenon, known as the Wolff–Chaikoff effect, was first described in 1948 by Wolff and Chaikoff after the observation that rats exposed to high amounts of iodide, presented decreased levels of organic form of iodide [20]. Although the molecular mechanisms underlying the acute Wolff– Chaikoff effect are not completely understood, some studies reported that iodide is able to inhibit the first steps of both TSH regulatory pathways, cAMP and PLC pathways, inhibiting the downstream effects and thus the thyroid hormones secretion [21–24]. In normal physiological conditions, iodide-mediated thyroid function inhibition is transient and this phenomenon is termed “Wolff-Chaikoff effect adaptation”, which can be explained by the downregulation of the NIS and thus inhibition of iodide transport into the thyroid follicular cells [21].

3.2.1.3

Synthesis and Secretion of Thyroid Hormones

Thyroid hormones synthesis requires two precursors: iodide and TG (Fig. 3.3). First step consists in the transport of iodide into the follicular cell, via NIS. NIS activity is dependent on the Na+ gradient created by the Na+ /K+ -ATPase [25, 26]. Through the intracellular electrochemical gradient, iodide goes to the apical surface of the cell and is then transported into the colloid mainly through pendrin channels [26, 27]. Iodine is then oxidized by the enzyme TPO in the presence of hydrogen peroxide, which is generated by a NADPH oxidase, the enzyme DUOX2 [28, 29]. In addition, TG, a hormone containing about 120 tyrosine residues is synthesized on ribosomes, glycosylated in the endoplasmic reticulum, translocated to the Golgi apparatus and packaged in secretory vesicles, which is excreted into the colloid by exocytose [30]. In a process that is also catalyzed by TPO, oxidized iodide is then bounded to the tyrosyl residues of TG to form monoiodotyrosine (MIT) or diiodotyrosine (DIT), containing one or two iodine molecules, respectively [31, 32]. MIT and DIT are then combined to form T3 and T4, which in turn contain respectively three or four iodine molecules. Colloid, consisting of a reservoir of iodinated TG containing the thyroid hormones is engulfed in vesicles, by pinocytosis and internalized into the follicular cells. Then the vesicles are digested by lysosomes, which generates T4 and T3 to be released into the bloodstream, via monocarboxylate transporter 8 (MCT8) [33]. In contrast, MIT and DIT are retained in the cell and deiodinated by the iodotyrosine dehalogenase 1. Iodine is then recycled for further thyroid hormone synthesis [34, 35].

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Fig. 3.3 Synthesis and secretion of thyroid hormones by the thyroid follicular cell. (1) Iodide enters the follicular cell, via sodium-iodide symporter (NIS); (2) iodide (I− ) goes to the apical surface of the cell, where it is transported to the colloid mainly through pendrin channels; (3) thyroglobulin (TG) is produced and excreted to the colloid, by exocytose; (4) DUOX2 produce hydrogen peroxide (H2 O2 ); (5) In the presence of H2 O2 , thyroid peroxidase (TPO) oxides iodide and attaches it to TG to form monoiodotyrosine (MIT) or diiodotyrosine (DIT), then MIT and DIT combine to form thyroxine (T4) and triiodothyronine (T3); (6) TG complex is then internalized into the follicular cell and form intracellular vesicles; (7) vesicles are digested by lysosomes (Ly), which generates T4 and T3; (8) T3 and T4 are released into the bloodstream. Notes: Dotted arrows depict particles movement

3.2.2 Parafollicular Cells Parafollicular cells are present in the interfollicular connective tissue stroma of the thyroid gland. Contrarily to follicular cells that arise from the endoderm, parafollicular cells derive from the neural crest cells and belong to the neuroendocrine system [2]. Parafollicular cells secrete calcitonin (CT) hormone, which participates in the regulation of Ca2+ homeostasis, although having a minor role [36].

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Synthesis and Secretion of Calcitonin

CT is secreted in response to increased extracellular [Ca2+ ]e , through the activation of the calcium-sensing receptor (CaSR), a class C G-protein-coupled receptor [37–39]. Ca2+ -CaSR interaction activates Gq/11 protein that in turn stimulates PLC. PLC hydrolyzes PIP2 into DAG and IP3. IP3 acutely increases [Ca2+ ]i levels, in a similar process as previously described for the follicular cell, which initiates the processes of CT release from the secretory vesicles into the bloodstream [19, 40]. Besides that, DAG activates PKC that regulates the transcription of key genes for Ca2+ homeostasis, such as CaSR and the gene that encodes CT and CT gene-related peptide [41, 42]. Contrarily to what occurs with other CaSR responsive cells, such as parathyroid cells, CaSR-mediated CT release from thyroid parafollicular cells seems to occur independently of extracellular signal-regulated kinase (ERK) 1/2 signaling activation and cAMP synthesis suppression [42, 43]. In addition to Ca2+ , some gastro-intestinal hormones, including glucagon, gastrin and cholecystokinin were also demonstrated to carry the ability of stimulating CT release from parafollicular cells [36, 44]. The physiological effects of CT consist in decreasing blood Ca2+ levels through inhibition of the osteoclast activity in the bones, inhibition of Ca2+ reabsorption by renal tubuli and inhibition of Ca2+ absorption in the intestine [36].

3.3 Parathyroid Glands The human parathyroid glands are most often four ovoid infra centimetric glands located behind each pole of the lateral lobes of the thyroid gland, although anatomical variations in the number and location of the parathyroid glands can frequently occur [2]. The parathyroid tissue is comprised of two functional cell lines, the chief cells and the oxyphil cells. Chief cells are the predominant parathyroid cell type, which are responsible for synthesis and secretion of parathormone (PTH). The function of oxyphil cells remains controversial, despite recent evidence suggesting that these cells result from chief cells deactivation to preserve the PTH secreting potential, in addition to secreting parathyroid hormone-related protein (PTHrP) [45].

3.3.1 PTH Actions PTH is a key player in Ca2+ homeostasis, along with 1α,25-dihydroxyvitamin D [1α,25(OH)2 D] and fibroblast growth factor 23 (FGF23). PTH interacts with membrane-specific receptors on target organs, predominantly on kidney and bone to increase circulating Ca2+ levels [46].

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In the kidney, PTH-mediated Ca2+ reabsorption in the distal convoluted tubule ensures a thigh control over Ca2+ urinary excretion, despite the majority of filtered Ca2+ being reabsorbed along with sodium in the proximal convoluted tubule [47]. Additionally, PTH downregulates sodium-phosphate cotransporters and inhibits sodium–hydrogen antiporter in the proximal convoluted tubule, ultimately leading to decreased reabsorption of phosphate ((PO4 )3− ) and bicarbonate, respectively [48]. Still in the kidney, PTH activates the 25-hydroxyvitamin D3-1α-hydroxylase gene promoter, the enzyme responsible for the conversion of calcifediol (25hydroxycholecalciferol) into 1α,25(OH)2D [49]. In turn, 1α,25(OH)2D mediates dietary Ca2+ absorption by intestinal mucosa, with PTH ultimately promoting the alimentary Ca2+ absorption [50]. In the bone, PTH enhances bone turnover, leading to bone mineral matrix reabsorption that results in the release of Ca2+ and (PO4 )3− into circulation [51], in addition to stimulate new bone formation [52].

3.3.2 PTH Biosynthesis The PTH gene is located on chromosome 11 that when transcribed yields pre-proPTH, the PTH precursor [53]. Pre-pro-PTH consists of 115-amino-acids containing the mature PTH (1-84) sequence along with a 6-amino-acid pro-hormone sequence and a 25-amino-acids signal (“pre”) sequence at its N-terminus [54]. Mature PTH is stored in vesicles and granules, which are secreted into the extra-cellular fluid in the presence of low circulating calcium levels [55]. Moreover, PTH is co-stored in granules along with cathepsin B and H [56], capable of degrading PTH into C-terminus PTH fragments that hold no action over PTH/PTHrP receptors, which are selectively secreted under conditions of hypercalcemia rather than mature PTH(1-84) [55].

3.3.3 PTH Secretion and Its Regulation The most important regulator of PTH secretion is the negative feedback loop elicited by extracellular ionized Ca2+ concentrations ([Ca2+ ]e ) [57]. In parathyroid cells, an increase in [Ca2+ ]i reduces the fusion of preformed PTH storage vesicles with the cytosolic membrane, thus suppressing PTH secretion, instead of stimulating hormonal secretion as commonly observed in the other endocrine glands [58], although the mechanisms that underlie this phenomena are still poorly characterized [59]. [Ca2+ ]e binds to the seven-loop transmembrane CaSR on the extracellular membrane of parathyroid chief cells, which is coupled to Gq and Gi proteins [57]. Gq activates PLC that increases the formation of IP3 and DAG from PIP2. In turn, IP3 induces Ca2+ mobilization from intracellular reticular storages, thus increasing

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[Ca2+ ]i [60]. High [Ca2+ ]i activates Ca2+ -dependent K+ channels, which lead to cytoplasmic membrane hyperpolarization [61] that ultimately suppresses the fusion of PTH vesicles with the membrane and exocytosis [62]. Moreover, increased [Ca2+ ]i indirectly activates PLA2 and PLD through PKC activation [63]. DAG is converted by DAG lipase into 2-arachidonoylglycerol (2-AG) that is then hydrolyzed into arachidonic acid (AA) by monoacylglycerol lipase (MAG). AA is also produced by PLA2 that becomes activated in the presence of elevated [Ca2+ ]e and is ultimately modified by cyclooxygenase, lipoxygenase (LO) and epoxygenase (cytochrome P450) [64]. LO products from AA metabolism are strong inhibitors of PTH secretion when CaSR is activated by high [Ca2+ ]e [65, 66]. Moreover, Gi activation suppresses magnesiummediated adenylate cyclase (AC) activity, thus reducing adenosine triphosphate (ATP) conversion into cAMP [67, 68]. As cAMP is a known mediator for PTH secretion [69], reduction of intracellular cAMP arises as an additional pathway leading to Ca2+ -induced PTH secretion suppression (Fig. 3.4) [46].

Fig. 3.4 Suppression of PTH secretion by hypercalcemia. (1) Circulating free calcium binds calcium-sensing receptor on parathyroid cell membrane, activating its subunits Gq and Gi; (2a) Gq activates phospholipase-C (PLC); (2b) PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3); (2c) IP3 induces calcium (Ca2+ ) release from the endoplasmic reticulum. (3) In turn, high intracellular Ca2+ concentrations activate calcium-dependent potassium (K+ ) channels, which determines K+ outflow and consequently membrane hyperpolarization, suppressing membrane fusion and PTH exocytosis; (4a) Via DAG lipase and monoacylglycerol lipase (MAG), DAG is converted into 2-arachidonoylglycerol (2-AG) and arachidonic acid (AA), respectively; (4b) additionally, via protein kinase C (PKC), increased intracellular Ca2+ levels lead to phospholipases D and A2 activation (PLD and PLA2), also resulting in increased AA formation; (4c) AA is converted by lipoxygenase (LO) and its products also suppress PTH release; (5) lastly, subunit Gi inhibits adenylate cyclase (AC), suppressing cAMP pathway. Together, these pathways suppress PTH secretion and parathyroid cell proliferation, ultimately reducing PTH circulating levels in conditions in hypercalcemia. Notes Red lines represent inhibitory pathways and dotted arrows depict particles movement

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Aside from [Ca2+ ]e , other molecules are involved in PTH synthesis and secretion. 1α,25(OH)2 D extracellular levels contribute to regulate PTH secretion by inhibiting PTH gene transcription and parathyroid proliferation, in a Ca2+ -independent fashion [70, 71]. Additionally, hypermagnesemia is also able to suppress PTH secretion [69, 72], while hypomagnesemia displays more complex effects [73]. Moreover, calmodulin, a Ca2+ -binding protein shared by most eukaryotic cells, and calmodulin-dependent protein kinase II (CaMKII) both found in human parathyroid cells, also seem to play a role in regulating calcium homeostasis and PTH secretion [74]. While the role of calmodulin in human parathyroid cells is apparently not directly implied in Ca2+ -mediated PTH secretion, levels of active CaMKII decrease in the presence of high [Ca2+ ]e ultimately leading to decreased PTH secretion [74]. Lastly, FGF23 decreases PTH secretion and PTH mRNA levels probably through mitogen-activated protein kinase (MAPK)/ERK pathway activation, in a feedback loop between bone and parathyroid glands [75]. PTH secretion is stimulated by low circulating calcium levels, which lead to increased levels of mRNA coding pre-pro-PTH probably by increasing mRNA stability [76]. High phosphate serum concentration [(PO4 )3− ]e , decrease AA levels also leading to PTH secretion [77]. However, even in the presence of high [(PO4 )3− ]e , PTH secretion is suppressed by high [Ca2+ ]i [78].

3.4 Adrenal Gland The adrenal glands are a pair of endocrine organs located above the superior pole of each kidney in the retroperitoneal space. Each gland has two distinct parts: an outer region, near the adrenal capsule, designated adrenal cortex that comprises 80% of the adrenal gland mass, and an inner region, so called adrenal medulla [79]. The adrenal cortex and medulla are separate tissues that have different embryological origin and distinct morphological and functional characteristics [2]. In order to accomplish the physiological roles attributed to the adrenal glands, these rely on a rich arterial blood supply derived from three different branches of the abdominal aorta: inferior phrenic artery, middle adrenal artery and renal artery. The arterial blood enters in the adrenal gland through the capsule and flows centripetally through the adrenal cortex into the medulla [80].

3.4.1 Adrenal Cortex The adrenal cortex is responsible for the adrenal steroid production and it is divided into three distinct morphological layers with different functionality. The layers are the glomerulosa, the fasciculata and the reticularis layers (Fig. 3.5). These three layers present specific enzymatic features that are needed for the production of different steroid hormones [79, 81].

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Fig. 3.5 Human adrenal gland stained by Masson tricromium (100x); Ca—Capsule; ZG—Zona glomerulosa; ZF—Zona fasciculata; ZR—Zona reticularis; M—Medulla

3.4.1.1

Steroidogenesis

Adrenocortical steroid hormones are essential in the processes of body homeostasis. Cholesterol is the common precursor of all steroid hormones and is mostly (80%) obtained from the plasma low-density lipoproteins (LDL) [81, 82]. Besides that, de novo cholesterol synthesis from acetate can also occur in steroidogenic tissues [83]. Cholesterol uptake by steroidogenic cells is performed through receptorsmediated endocytosis and the number of receptors expressed by the cells depend on the presence of stimulus for steroid production [84]. Briefly, after cellular uptake, cytoplasmic cholesterol is transferred from the outer to the inner membrane of the mitochondria, by the steroidogenic acute regulatory protein (StAR) [85]. Once in the mitochondria, cholesterol is hydroxylated twice and cleaved by the cholesterol side chain cleavage enzyme (CYP11A1) to generate pregnenolone [86] (Fig. 3.6). After leaving the mitochondria, pregnenolone is oxidized and isomerized to form progesterone. From this step of the steroidogenic cascade, due to zone-specific enzyme expression, steroidogenesis differs among the different adrenal cortex layers [86, 87]. At the glomerulosa, progesterone is converted into 11-deoxycorticosterone and transferred back into the mitochondria and is successively hydroxylated by aldosterone synthase (CYP11B2) enzyme to originate aldosterone [88]. At the fasciculata, 17α-hydroxylase (CYP17A1) converts pregnenolone into 17α-hydroxypregnenolone, which is then oxidized to 17αhydroxyprogesterone and afterwards hydroxylated by 21α-hydroxylase (CYP21A2) to originate 11-deoxycortisol. At this point, 11-deoxycortisol reenters into the mitochondria to be converted by 11β-hydroxylase (CYP11B1) into cortisol [87, 89].

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Fig. 3.6 Steroidogenesis in the different layers of the adrenal cortex. 17βHSD5—17βhydroxysteroid dehydrogenase type 5; CYB5A—Cytochrome B5A; CYP11A1—cholesterol side chain cleavage enzyme; CYP11B1–11β-hydroxylase; CYP11B2—aldosterone synthase; CYP17A1—17α-Hydroxylase; CYP21A2–21α-hydroxylase; dehydroepiandrosterone (DHEA); dehydroepiandrosterone sulfate (DHEA-S); StAR—steroidogenic acute regulatory protein; SULT2A1—Sulfotransferase 2A1

At the zona reticularis, pregnenolone is hydroxylated by CYP17A1 to yield 17hydroxypregnenolone and then into dehydroepiandrosterone (DHEA). Adrenal reticularis layer can also synthesize low levels of testosterone through the action of the enzyme 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5) [86, 87, 90]. Due to the lipophilic properties of the steroid hormones, they are not stored in the cells, being only synthesized upon stimulation and immediately secreted [91]. 3.4.1.2

Adrenocortical Stem Cells

In early studies, Ingle et al. described the regeneration of the adrenal cortex after adrenal enucleation (removal of the inner content of the adrenal gland) by only leaving the capsule and underlying subcapsular cells intact, suggesting the existence of stem/progenitor cells in the periphery of the adrenal cortex. Furthermore, this finding also corroborated the hypothesis of a centripetal migration and differentiation of the adrenal cortex, previously described [92].

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The hedgehog signaling pathway (Hh) is a conserved pathway involved in adult tissue maintenance and renewal. Sonic hedgehog (Shh), an Hh family member, is present in a subpopulation of cells organized in clusters under the capsule of the adrenal gland. Lineage-tracing studies revealed that adrenocortical cells are derived from Shh positive cells, suggesting that those cells are the progenitor/stem cells of the adrenal cortex cells. Shh positive cells transduce the signal to the overlying steroidogenic factor 1 (SF1) negative cells present in the adrenal gland capsule triggering the expression of Gli1 molecule. During the adrenal development, Gli1+ capsular cells migrate to the adrenal cortex and behave as stem/precursors cells, since these give rise to the SF1+ /Shh+ progenitor cell pool that will lead to differentiated steroidogenic cells [93, 94].

3.4.1.3

Glomerulosa Layer

The glomerulosa layer is the outer layer of the adrenal cortex. It is the only layer that expresses CYP11B2 and thereby the single capable of synthetizing mineralocorticoids [95]. The most important and potent mineralocorticoid hormone is the aldosterone. Aldosterone is a key element of the renin-angiotensin-aldosterone system being responsible for regulating sodium homeostasis and thereby helping to control fluid volume and arterial pressure [96, 97].

Aldosterone Synthesis The main extracellular stimuli for aldosterone synthesis are angiotensin II (ANG II) and high K+ levels. Besides that, adrenocorticotropic hormone (ACTH) is also able to regulate aldosterone synthesis, although it has a minor contribution [88, 98].

Renin-Angiotensin-Aldosterone System A reduction on the renal perfusion pressure leads to renin synthesis by the kidney juxtaglomerular cells. Renin is an enzyme that cleaves a protein synthesized and secreted by the liver, the angiotensinogen, to form angiotensin I (ANG I) [99]. Then, angiotensin converting enzyme (ACE) converts ANG I into ANG II [100]. In the glomerulosa cells, ANG II binds to the ANG II receptor type 1 (AT1R) which is linked to the G-protein Gq/11 that couples the receptor to its effector PLC (Fig. 3.7). PLC activation leads to the hydrolysis of PIP2 to produce DAG and IP3 [88, 101, 102]. IP3 diffuses into the cytoplasm and binds to its endoplasmic reticulum receptors, which act as channels for the release of the Ca2+ stored in this organelle. It results in a transient increase of the Ca2+ cytoplasmic concentration [88, 98]. [Ca2+ ]i leads to the activation of CaMK [103]. The activation of different CaMK were shown to lead to different results: CaMK I is able to increase of the CYP11B2 through the activation of the transcription factors [Activating transcription factor (ATF)/cAMP

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Fig. 3.7 Aldosterone synthesis regulation via angiotensin II (ANG II). (1) ANG II binds to type 1 ANG II receptor (AT1R) which is coupled to phospholipase C (PLC) through the G-protein (Gq/11); (2) PLC-β is activated and hydrolyses phosphatidylisitol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3); (3) IP3 diffuses into the cytoplasm and binds to its endoplasmic reticulum receptors, which act as channels for the release of the calcium (Ca2+ ); (4) Intracellular Ca2+ activates the calmodulin-dependent protein kinases (CaMK); (5) CaMK activation increases the transcription of the aldosterone synthetize enzyme through the activation of the transcription factors (ATF/CREB) and also shift the voltage of the Ca2+ membrane channels leading to Ca2+ entrance; (6) Simultaneously, DAG activates the protein kinase C (PKC); (7) PKC phosphorylates and activates the transcription factors (ATF/CREB); (8) ATF/CREB activate the transcription of genes involved in aldosterone synthesis. Notes: Dotted arrows depict particles movement

response element binding (CREB)]; and CaMKII, on the other hand, shifts the voltage of the voltage-gated Ca2+ channels (VGCCs) leading to the enhance of Ca2+ influx and then increasing the aldosterone synthesis [88, 98, 103]. In addition, DAG is responsible for the activation of PKC that activates the transcription factors (ATF/CREB) and thus leading to the transcription of StAR and CYP11B2 [88, 104]. ANG II is also able to activate the MAPK signaling pathway, however the mechanisms are not yet completely elucidated [88]. A mechanism already described is that the binding of ANG II to AT1R activates ERK that is able to phosphorylate and activate the enzyme responsible for cleaving the cholesteryl esters to yield the cholesterol [105]. Other evidences, described that ERK can phosphorylate StAR, leading to the transport of cholesterol to the mitochondria [106, 107]. Thus, the mechanisms

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through which MAPK/ERK leads to the aldosterone synthesis are ensuring the availability of cholesterol and its entrance in the mitochondria membrane in order to begin the process of steroidogenesis.

Potassium Glomerulosa cells, respond to minor changes in the K+ levels with the increase of aldosterone production [108]. High levels of K+ lead to depolarization of the glomerulosa cell membrane that lead to the activation of the VGCCs and thus the influx of the Ca2+ [88, 109]. Increased [Ca2+ ]i activate CaMKs and the subsequent pathways already described in the previous section. In addition, the ability of K+ to increase cellular cAMP levels through the 2+ Ca -sensitive AC was also reported. cAMP activates PKA which then activates the transcription factors (ATF/CREB) and thus leading to the transcription of StAR and CYP11B2 [88, 110].

ACTH ACTH binds to melanocortin receptor 2 (MC2R) on the cytoplasmic membrane of glomerulosa cells [111]. Thus, ACTH increases cAMP concentration and activates PKA which phosphorylates and activates hormone-sensitive lipase (HSL) and StAR protein, resulting in the release of cholesterol from lipid droplets and its transportation to the inner mitochondrial membrane [88]. PKA can also activate transcription factors ATF/CREB and then induce the transcription of StAR and CYP11B2 [112]. In addition, PKA stimulates the flow of Ca2+ ions into the glomerulosa cells and thereby increases the production of aldosterone by a mechanism involving CaMKs [113].

3.4.1.4

Fasciculate Layer

Fasciculate layer is the widest zone of the adrenal cortex. It lies under the glomerulosa layer and it is responsible for the synthesis of the glucocorticoids [114]. Cortisol is the most potent glucocorticoid in humans. Moreover, cortisol has a circadian rhythm characterized by a peak in the period before wakening and a gradual decline throughout the day [115]. In addition, cortisol secretion increases acutely in response to stressful stimuli [116, 117].

Cortisol Synthesis Cortisol secretion is indirectly controlled by the central nervous system. The hypothalamus releases the corticotropin-releasing hormone (CRH) to the long pituitary portal

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Fig. 3.8 Cortisol synthesis regulation. (1) Adrenocorticotropic hormone (ACTH) binds to the melanocortin receptor 2 (MC2R); (2) Gα subunit activate adenylate cyclase (AC); (3) AC converts adenosine triphosphate (ATP) into cyclic 3 ,5 -adenosine monophosphate (cAMP); (4) cAMP binds to the regulatory subunits (R) of protein kinase A (PKA), releasing and activating the catalytic subunits (C) of this protein; (5) PKA phosphorylates the cAMP response element binding (CREB) transcription factor; (6) CREB activates the transcription of genes involved in the production of cortisol; (7) Simultaneously, PKA also phosphorylates and activates the hormone-sensitive lipase (HSL) and the steroidogenic acute regulatory protein (StAR), initiating the steroidogenesis. Notes Dotted arrows depict particles movement

veins. CRH binds to its membrane receptors in the anterior pituitary stimulating the release of ACTH into the blood [117]. ACTH acts on the fasciculate zone cells through the binding to the MC2R and subsequently it induces adrenocortical expansion and cortisol production [111, 118, 119]. Upon ACTH binding, the receptor undergoes conformational changes that activate AC, leading to the conversion of ATP to cAMP (Fig. 3.8) [120]. In turn, cAMP binds to the regulatory subunits of PKA, releasing and activating the catalytic subunits of this protein, which then phosphorylates the CREB transcription factor that leads to increased expression of genes involved in the production of cortisol, such as CYP11B1 [112, 121]. Concomitantly, it also phosphorylates and activates HSL and StAR, initiating the production of cortisol, as previously described [85].

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Reticularis Layer

Reticularis layer is the innermost layer of the adrenal cortex. It is located between the fasciculata layer and the adrenal medulla and it is responsible for the production of the adrenal androgens [114].

Adrenal Androgens Synthesis Adrenal androgens synthesis is synchronized with cortisol synthesis in response to ACTH stimulation [122]. The mechanism by which ACTH stimulates androgen synthesis by adrenocortical cells is similar to the mechanism described for cortisol [123]. Like cortisol, plasma levels of DHEA, androstenedione and testosterone exhibit a circadian rhythm. On the contrary, levels of dehydroepiandrosterone sulfate (DHEA-S) do not exhibit a circadian rhythm, since this being a sulfated steroid results in a longer half-life [124, 125]. Other endocrine signals have been proposed as co-regulators of adrenal androgen secretion, such as prolactin (PRL), estrogens, prostaglandins, angiotensin, growth hormone (GH) and gonadotropins [122, 126–128]. However, its impact on androgen secretion is not considered as relevant as ACTH.

3.4.2 Adrenal Medulla The adrenal medulla is the innermost layer of the adrenal gland and is surrounded by the adrenal cortex [129]. The adrenal medulla has an embryonic origin in the neural crest and is composed by chromaffin cells that are structurally and functionally related to postganglionic neurons of the sympathetic nervous system [2]. These cells are responsible for the production of catecholamine: DA, epinephrine (Epi) and NE [130, 131].

3.4.2.1

Adrenal Catecholamine Synthesis and Secretion

Like adrenal cortex, adrenal medulla is a key tissue involved in the physiological adaptation to stress [132]. Whenever the central nervous system perceives a stress, two key effector pathways that are activated. These include the hypothalamicpituitary-adrenal axis (HPA), which indirectly stimulates the adrenal medulla to produce catecholamines, and the sympathetic-adrenal axis, which stimulates the adrenal medulla to secrete catecholamines, through a neural mechanism [131–133]. Due to a centripetal blood flow coming from the adrenal cortex, high levels of cortisol pass through the adrenal medulla [80, 134, 135]. As a lipophilic hormone, cortisol is able to easily cross the chromaffin cell membrane to bind the cytoplasmatic glucocorticoid receptor (GR) (Fig. 3.9). Prior to cortisol binding, GR is sequestered

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Fig. 3.9 Adrenal catecholamines synthesis and secretion. (A) cortisol crosses the chromaffin cell membrane and binds to the glucocorticoid receptor (GR). GR complex is dissociated and GR goes to the nucleus to bind in glucocorticoid response elements, activating the transcription of phenylethanolamine N-methyltransferase (PNMT); (1) Tyrosine is hydrolyzed by the enzyme tyrosine hydroxylase (TH), producing L-3,4-dihydroxyphenylalanine (L-DOPA); (2) L-DOPA is decarboxylated by the enzyme L-aromatic amino acid decarboxylase (AADC), converting it into DA; (3) DA is then incorporated in chromaffin cell vesicles through the vesicular monoamine transporter 1 (VMAT1); (4) DA is hydroxylated to produce NE by dopamine β-hydroxylase (DBH); (5) NE can be stored in the vesicles or it can goes to the cytoplasm; (6) in the cytoplasm it is methylated by PNMT to produce Epi; (7) Epi is incorporated in the chromaffin vesicle; (8) in the vesicle NE and Epi are in a complex with chromogranin (Cg), Ca2+ and adenosine triphosphate (ATP); (9) when stimulated by acetylcholine, the chromaffin cell membrane is depolarized and the catecholamines are released. Notes Dotted arrows depict particles movement

in the cytoplasm as a multiprotein complex [136]. After cortisol binding, the complex is dissociated and GR is translocated into the nucleus where it binds to glucocorticoid response elements (GRE) in the promoter regions of target genes directly or interacts with other transcription factor proteins [133, 136]. Cortisol was found to increase the transcription of genes involved in the biosynthesis of catecholamines, such as phenylethanolamine N-methyltransferase (PNMT) [131, 133, 137]. The biosynthesis of catecholamines begins with the hydroxylation of tyrosine, the catecholamines common precursor, by the enzyme tyrosine hydroxylase (TH), producing L-3,4-dihydroxyphenylalanine (L-DOPA) [133, 138]. After that, L-DOPA is decarboxylated by the enzyme L-aromatic amino acid decarboxylase (AADC), converting it into dopamine (DA) [139]. DA is then incorporated into chromaffin cell vesicles where is hydroxylated to produce NE by dopamine β-hydroxylase (DBH)

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[140]. NE can be stored in the vesicles until secretion or methylated in the cytoplasm by PNMT to produce Epi [131, 141]. This last step does not occur in the adrenergic neurons as these do not express the PNMT enzyme [131], being specific of the adrenal medulla that is exposed to high levels of cortisol due to the portal nature of the adrenal circulation described above [80, 134, 135]. Catecholamines form a complex with chromogranin, ATP e Ca2+ , inside of the chromaffin cell vesicles until being released [137, 142]. Being only produced in the adrenal medulla, Epi is the major secretory product of the adrenal medulla [133, 134]. Opposite to catecholamines synthesis, catecholamines release is mainly mediated by the neuropeptide acetylcholine (ACh) discharge from sympathetic nerve terminals [143]. ACh binds to plasma membrane receptors on chromaffin cells and stimulates Ca2+ -mediated depolarization of the cell membrane [133, 144]. Then the increase of [Ca2+ ]i levels leads to the release of catecholamines complexed with chromogranin stored in the chromaffin cell vesicles [142, 145].

3.5 Conclusion In this chapter the key signaling pathways involved in the peripheral endocrine organs’ maintenance and physiology are described. The knowledge of those pathways is essential for understanding the molecular mechanisms that might lead to endocrine disruption and disease providing important clues into multisystemic impact of endocrine physiology and pathology. Acknowledgments The work was supported by the Foundation for Science and Technology (PTDC/MEC-ONC/31384/2017). Unit for Multidisciplinary Research in Biomedicine (UMIB) is funded by grants from FCT (UID/Multi/00215/2016 and UID/Multi/00215/2019).

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Chapter 4

Signaling Pathways Governing Activation of Innate Immune Cells Bruno M. Neves and Catarina R. Almeida

Abstract In accordance to their functions, most cells from the innate immune system are equipped with multiple receptors that sense invading pathogens and endogenous danger signals resultant from damaged cells. Recognition of such “alarm signals” triggers complex signaling pathways that involve the recruitment of adapter molecules to the receptors and consequent activation of transducers such as protein kinases. Finally, these cascades culminate in the nuclear translocation of transcription factors that control the expression of inflammatory effector molecules like cytokines, chemokines and enzymes involved in oxidative burst and prostaglandin/leukotriene synthesis. Also, Natural Killer (NK) cells, a particular type of inate immune cells, express multiple activating and inhibitory receptors that act in concert to capacitate them to directly recognize and destroy transformed or viraly infected cells, to secrete cytokines or both. The knowledge on these intricate signaling networks is crucial for the comprehension of the physiopathology of inflammatory diseases as well as for identification of possible therapeutical targets. In this chapter we provide an overview of the transduction cascades triggered following danger sensing by innate immune cells, as well as examples evidencing the impact of their malfunction to human health. Keywords Macrophages · Dendritic cells · TLR · CLR · RLR · NLR · ALR · Cytosolic sensors · NK cells · Activating signaling · Inhibitory signaling

B. M. Neves (B) · C. R. Almeida (B) Department of Medical Sciences, iBiMED—Institute for Biomedicine, University of Aveiro, Aveiro, Portugal e-mail: [email protected] C. R. Almeida e-mail: [email protected] © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_4

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Abbreviations ADCC AICL AIM Akt ALR AMP AP-1 ASC ATP BCG Bcl BDCA BIR c-Abl CARD CBP CCL CD Cdc42 cGAMP cGAS CLEC CLR CpG ODN CRACC CRD CREB CTLD DAI DAMPs DAP10, 12 DC DCAL-2 DCIR DC-SIGN DDX DDX60 Dectin DHX DHX36 DNA

Antibody-dependent cellular cytotoxicity Activation-induced C-type lectin Absent-in-melanoma Protein kinase B AIM-like receptors Adenosine monophosphate Activator protein 1 Apoptosis-associated speck-like protein containing CARD Adenosine triphosphate Bacillus Calmette-Guérin B cell lymphoma Blood dendritic cell antigen Baculovirus inhibitor repeat Abelson tyrosine kinase Caspase recruitment domain protein CREB-binding protein C-C motif chemokine ligand Cluster of differentiation Cell division cycle 42 Cyclic-GMP-AMP cGAMP synthase C-type lectin-like receptors C-type lectin receptors CpG oligodeoxynucleotides CD2-like receptor activating cytotoxic cells Carbohydrate recognition domain cAMP response element binding C-type lectin-like domain DNA-dependent activator of interferon-regulatory factors Damage-associated molecular patterns DNAX activating proteins of 10 kDa, 12 kDa Dendritic cell Dendritic cell-associated C-type lectin 2 DC-immunoreceptor DC-specific intercellular adhesion molecule (ICAM)-3 grabbing nonintegrin DEAD-box helicase 41 DExD/H-box helicase 60 DC-associated C-type lectin-1 DExH-box helicase 9 DEAH-box helicase 36 Deoxyribonucleic acid

4 Signaling Pathways Governing Activation of Innate Immune Cells

DNAM-1 DNAPK dsDNA dsRNA EAT-2 EBV ER ERK FADD FcRγ GM-CSF GMP Grb2 HIN HIV-1 HLA HMGB1 hMGL HMGN1 HSPs ICAM iE-DAP IFI16 IFN IgG IKK IL ILCs IRAK IRF IRp60 ISG ITAM ITIM ITSM IκB JNK KACL KIR KLRG1 LDL LFA LIR LMW LOX-1

DNAX accessory molecule-1 DNA-dependent protein kinase Double stranded DNA Double stranded RNA Ewing’s sarcoma-associated transcript-2 Epstein-Barr vírus Endoplasmic reticulum Extracellular signal-regulated kinase Fas-associated protein with death domain Fc receptor γ chain Granulocyte and macrophage colony stimulating factor Guanosine monophosphate Growth factor receptor-bound protein 2 Hematopoietic interferon-inducible nuclear antigens Human immunodeficiency virus 1 Human leukocyte antigen High-mobility group box 1 Human Monoglyceride lipase High Mobility Group Nucleosome Binding Domain 1 Heat-shock proteins Intercellular adhesion molecule γ-D-glutamyl-meso-diaminopimelic acid IFN-inducible 16 Interferon Immunoglobulin G IκB kinase complex Interleukin Innate lymphoid cells IL-1 receptor-associated kinase protein Interferon regulatory factor Inhibitory receptor protein IFN-inducible gene Immunoreceptor tyrosine-based activation motif Immunoreceptor tyrosine-based inhibitory motifs Immunoreceptor tyrosine-based switch motifs Inhibitor of nuclear factor kappa B Jun N-terminal kinase Keratinocyte-associated C-type lectin Killer cell immunoglobulin-like receptor Killer cell lectin-like receptor G1 Low-density lipoproteins Lymphocyte function associated antigen Leukocyte inhibitory receptor Low molecular weight Lectin-like oxidized LDL receptor-1

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LPG2 LPS LRR MAL MALT MAPK MAVS MCL MDA5 MDL-1 MDP MEK MHC MICA MICB MICL Mincle MIP MNDA MR MRE11 MTOC mTOR MyD88 NAIP Nck ND NFAT NF-κB NIK NK NKG2D NKR-P1 NLR NLRC NLRP NOD NTB-A PAMPs PI3K PLC-γ PRRs PYD PYHIN1

B. M. Neves and C. R. Almeida

Laboratory of genetics and physiology 2 Lipopolysaccharide Leucine-rich repeat MyD88-adaptor-like Mucosa associated lymphoid tissue translocation protein Mitogen-activated protein kinase Mitochondrial antiviral signalling protein Macrophage C-type lectin Melanoma differentiation-associated gene 5 Myeloid DAP12-associating lectin Muramyl dipeptide MAPK/ERK kinase Major histocompatibility complex MHC class I chain-related A chain MHC class I chain-related B chain Myeloid inhibitory C-type lectin-like receptor Macrophage-inducible C-type lectin Macrophage inflammatory protein Myeloid cell nuclear differentiation antigen Mannose receptor Meiotic recombination 11 homolog A Microtubule organizing centre Mechanistic target of rapamycin Myeloid differentiation primary response gene 88 NLR family, apoptosis inhibitory protein Non-catalytic region of tyrosine kinase adaptor protein 1 Not determined Nuclear factor of activated T-cells Nuclear factor-kappa B NF-κB-inducing kinase Natural Killer Natural Killer group 2D Killer cell lectin-like receptor subfamily B, member 1 NOD-like receptors NOD-like receptor family CARD domain containing Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing Nucleotide-oligomerization domain NK, T and B cell antigen Pathogen-associated molecular patterns Phosphoinositide 3-kinase Phospholipase C gamma Pattern-recognition receptors Pyrin Pyrin and HIN domain-containing protein

4 Signaling Pathways Governing Activation of Innate Immune Cells

Rac-1 RANTES RICK RIG-1 RIP-1 RLR RNA ROS SAP SECTM1 SH2 SHIP-1 SHP SIGNR1 SLAM SLP-76 ssRNA STING Syk TAB TAK1 TBK1 TCR TGF TICAM TIR TIRAP TLR TNF TNFR TRADD TRAF TRAIL TRAM TRIF ULBP WAS WASP WIPF1 XLP ZAP70

Ras-related C3 botulinum toxin substrate 1 Regulated upon activation, normal T-cell expressed, and secreted RIP-like interacting CLARP kinase Retinoic acid-inducible gene-1 Receptor interacting kinase 1 RIG-1-like receptors Ribonucleic acid Reactive oxygen species SLAM-associated protein Secreted and transmembrane 1 Src homology 2 SH2 domain-containing inositol 5 phosphatase-1 Src homology 2 (SH2) domain-containing phosphatases Specific ICAM-3 grabbing nonintegrin-related 1 Signaling lymphocytic activating molecule SH2 domain containing leukocyte phosphoprotein of 76 kD Single-stranded RNA Stimulator of interferon genes Spleen tyrosine kinase TAK1 binding protein TGF-beta-activated kinase 1 TANK binding kinase 1 T cell receptor Transforming growth factor TIR-domain containing adaptor molecule Toll/IL-1R homology TIR-containing adaptor protein Toll-like receptor Tumor necrosis factor Tumor necrosis factor receptor TNF receptor-associated death domain TNFR-associated factor 6 TNF-related apoptosis-inducing ligand TRIF-related adaptor molecule TIR-containing adaptor inducing IFN-β UL-16 binding protein Wiskott-Aldrich syndrome Wiskott-Aldrich syndrome protein WAS/WASL interacting protein family member 1 X-linked lymphoproliferative disease Zeta-chain-associated protein kinase 70

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4.1 Introduction Innate immune cells are central players in immunity and tissue homeostasis. This is a heterogeneous group of hematopoietic cells that distribute between blood and tissues, being part of the early defense barriers. Innate immune cells include neutrophils, basophils, eosinophils, monocytes, innate lymphoid cells, dendritic cells, Langerhans cells, mast cells and macrophages. Among these, monocytes, macrophages and dendritic cells have particular relevance in sensing danger signals from invading pathogens or damaged cells, promoting then inflammation and orchestrating adaptive immune responses. This hability to detect potential harmful components in their micronvironment is provided by an extensive repertoire of intra and extracellular receptors (pattern-recognition receptors) that when engaged conduce to the expression of cytokines, chemokines and other effector molecules. Innate lymphoid cells (ILCs) are lymphocytes, but contrarily to T and B cells, do not express antigen receptors. These lymphocytes are considered the innate counterpart of T cells, and include ILC1, ILC2, ILC3 and NK cells. ILCs other than NK cells are mostly tissue resident cells which have several functions, being involved in maintance of tissue homeostasis, and contributing to several immune pathways. The latest knowledge on ILC biology has been recently reviewed elsewhere [1]. Here, we will focus on signaling pathways involved on NK cell recognition of target cells. NK cells are cytotoxic lymphocytes that lack adaptive antigen receptors, and thus mirror the functions of cytotoxic T cells. NK cells recognize alterations caused by infections, transformation or cellular stress. For that, these cells express an array of different activating and inhibitory receptors acting in concert, with which they can distinguish healthy from diseased cells and mount powerful and fast responses. In the next section we will start by describing the pathways triggered in innate immune cells such as monocytes, macrophages and dendritic cells by patternrecognition receptors engagement. We will then discuss the cascades regulating NK cell cytotoxicity. Finally, in the last section of this chapter we will provide examples of pathologies related with defects on these pathways.

4.2 Recognition of Microorganisms and Endogenous Danger Signals by Innate Immune Cells Innate immune cells are equipped with germline-encoded pattern-recognition receptors (PRRs) that allow them to sense microbial components, globally designated as pathogen associated molecular patterns (PAMPs), and endogenous molecules resultant from cellular stress or tissue injury, the so called damage-associated molecular patterns (DAMPs) [2, 3]. PAMPs are highly conserved components of microbial metabolism such as proteins, lipids, carbohydrates and nucleic acids. They tend to be markedly distinct from self-antigens, allowing the innate immune cells to discriminate self from non-self. Regarding DAMPs, they are endogenous danger signals

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linked to the activation of innate immune system during non-infectious inflammation. DAMPs include extracellular and plasma molecules such as serum amyloid A, heparin sulphate, fibrinogen and extracellular ATP, and intracellular components like high-mobility group box 1 (HMGB1), S100 proteins, heat-shock proteins (HSPs), calrreticulin and aberrant or mislocated DNA [4, 5]. Attending to their structure and specificity PRRs are categorized into six classes (Table 4.1): Toll-like receptors (TLRs), C-type lectin receptors (CLRs), retinoic acidinducible gene-1 (RIG-1)-like receptors (RLRs), nucleotide-oligomerization domain (NOD)-like receptors (NLRs), absent-in-melanoma (AIM)-like receptors (ALRs), and cytosolic nucleic acid sensors such as the cGAS-STING pathway [2, 6].

4.2.1 Toll-like Receptors Structurally, TLRs are type I membrane glycoproteins comprising a ligand-binding ectodomain with leucine-rich repeats, a transmembrane domain, and a cytoplasmic Toll/IL-1R homology (TIR) domain that initiates downstream signaling [15]. Mammalians have between ten and thirteen types of TLRs being so far identified ten functional receptors (TLR1-10) in humans. The receptors are either found at cell surface where they sense microbial proteins, lipids and peptidoglycans, or within intracellular compartments such as the endoplasmic reticulum, endosome, lysosome, or endolysosome where they recognize pathogen nucleic acids [16]. Binding of ligands to TLRs activates complex intracellular signaling pathways that culminate in the expression of effectors crucial for resolution of infections: proinflammatory cytokines, chemokines, growth factors, type I interferons (IFN), and co-stimulatory molecules. These signaling cascades are initiated by the recruitment of different TIR domain-containing adaptor molecules to the cytoplasmic TIR domains of the receptors. Among the possible recruited adaptors are the myeloid differentiation primary response gene 88 (MyD88), the TIR-containing adaptor protein/MyD88-adaptor-like (TIRAP/MAL), the TIR-containing adaptor inducing interferon-β (IFN-β)/TIR-domain-containing adaptor molecule 1 (TRIF/TICAM1) and the TIR-domain-containing adaptor molecule/TRIF-related adaptor molecule 2 (TRAM/TICAM2). With the exception of TLR3 that only recruits TRIF, all other TLRs recruit and initiate MyD88-dependent signaling cascades that converge to the transcription factor Nuclear Factor-kappa B (NF-κB), a master regulator of inflammation, to Interferon regulatory factors (IRFs), and to the mitogen-activated protein kinases (MAPKs). MyD88 is the unique recruited adapter in TLR5, TLR7 and TLR9 signaling, while TLR1, TLR2, and TLR6, also recruit TIRAP. TLR4 uses the four adaptors: MyD88, TIRAP, TRIF and TRAM [17]. Collectively, depending on the adaptor usage, TLR signaling cascades could be grouped into: (1) MyD88-dependent and (2) TRIF-dependent pathways.

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Table 4.1 PRRs, cellular location and respective ligands. Table was constructed based on information collected from references [6–14] PRR

Cellular location

Ligands (PAMPs and DAMPs)

Cell surface

PAMPs

Ligand origin

TLRs TLR1/2

TLR2

Cell surface

Tri-acyl lipopeptides

Bacteria, mycobacteria

Soluble factors

N. meningitides

PAMPs Diacyl lipopeptides

Mycoplasma

Triacyl lipopeptides

Mycobacteria

Peptidoglycan

Gram-positive bacteria

Lipoteichoic acid

Neisseria

Porins

Mycobacteria

Lipoarabinomannan

Staphylococcus epidermidis

Phenol-soluble modulin

T. Cruzi

tGPI-mutin

T. maltophilum

Glycolipids

Measles virus

Hemagglutinin protein

Fungi

Zymosan

C. albicans

Phospholipomannan

C. neoformans

Glucuronoxylomannan DAMPs

TLR3

Endolysosome

Biglycans

Extracelular matrix

Decorin

Extracelular matrix

Versican

Extracelular matrix

LMW hyaluronan

Extracelular matrix

S100 proteins

Cytosol

Heat shock proteins

Cytosol

A

Cytosol

Histones

Nucleus

HMGB1

Nucleus

PAMPs Viral double-stranded RNA

Vesicular stomatitis virus, lymphocytic choriomeningitis virus, reovirus

DAMPs Self RNA

Nucleus (continued)

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Table 4.1 (continued) PRR

Cellular location

TLR4

Cell surface

Ligands (PAMPs and DAMPs)

Ligand origin

PAMPs LPS

Gram-negative bacteria

Fusion protein

Respiratory syncytial vírus

Envelope proteins

Mouse mammary tumor virus

HSP60

C. pneumoniae

Manan

C. albicans

Glycoinositolphospholipids

Trypanosoma

DAMPs

TLR5

Cell surface

TLR6/2

Cell surface

Biglycans

Extracelular matrix

Decorin

Extracelular matrix

LMW hyaluronan

Extracelular matrix

Heparan sulphate

Extracelular matrix

Fibronectin

Extracelular matrix

Tenascin C

Extracelular matrix

S100 proteins

Cytosol

Heat shock proteins

Cytosol

Histones

Nucleus

HMGB1

Nucleus

HMGN1

Nucleus

Defensins

Granules

Granulysin

Granules

Syndecans

Plasma membrane

Glypicans

Plasma membrane

PAMPs Flagellin

Flagellated bacteria

PAMPs Diacyl lipopeptides

Mycoplasma

Lipoteichoic acid

Group B Streptococcus

Zymosan

S. cerevisiae

DAMPs Versican TLR7

Endolysosome

Extracelular matrix

PAMPs Viral single-stranded RNA

Several virus (continued)

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Table 4.1 (continued) PRR

Cellular location

Ligands (PAMPs and DAMPs) RNA

Ligand origin Bacteria from group B Streptococcus

DAMPs Self RNA TLR8

Endolysosome

Nucleus

PAMPs Viral single-stranded RNA

Several virus

DAMPs Self RNA TLR9

Endolysosome

Nucleus

PAMPs CpG-DNA

Bacteria and mycobacteria

dsDNA viruses

Herpes simplex virus

Hemozoin

Plasmodium

DAMPs

TLR10

Cell surface

Self DNA

Nucleus

Self mitochoncrial DNA

Mitochondria

PAMPs Unknown

Influenza virus L. monocytogenes

CLRs Mannose receptor (CD206)

Cell surface

PAPMs High-mannose oligosaccharides, Fucose, Sulphated sugars and N-Acetylgalactosamine

M. tuberculosis M. kansasii F. tularensis, K. pneumoniae, HIV-1 Dengue vírus C. albicans C. neoformans P. carinii Leishmania spp.

DEC205 (CD205)

Cell surface

PAMPs Plasminogen activator

Y. pestis

K12

E. coli

Class B CpG ODN

HIV

DAMPs (continued)

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Table 4.1 (continued) PRR

DC-SIGN (CD209)

Cellular location

Cell surface

Ligands (PAMPs and DAMPs)

Ligand origin

Keratins

Apoptotic and necrotic cells

oxLDL

oxLDL

PAMPs Mannose and fucose-bearing glycans (LeX , LeY , LeA , LeB )

M. tuberculosis, M.leprae BCG Lactobacilli spp. H. pylori E.coli HIV-1, measles and Dengue vírus Schistosoma egg antigen Leishmania spp. C.albicans Ixodes scapularis Salp15 protein

DAMPs LeX , LeY carbohydrates on carcinoembryonic antigen Langerin (CD207)

Cell surface

Plasma membrane of cancer cells

PAPMs High-mannose oligosaccharides, Fucose and N-Acetylgalactosamine

HIV-1 M.leprae Candida spp. Saccharomyces spp Malassezia furfur

CLEC5A

Cell surface

ND

MGL (CD301)

Cell surface

PAMPs Terminal N-Acetylgalactosamine

Dengue virus S. mansoni Filoviruses

DAMPs O-linked GalNAc antigens Dectin 1 (CLEC7A)

Cell surface

Plasma membrane of cancer cells

PAMPs β-1,3 glucans

Mycobacteria spp P. carinii C. albicans (continued)

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Table 4.1 (continued) PRR

Cellular location

Ligands (PAMPs and DAMPs)

Ligand origin M. tuberculosis A.fumigatus H.capsulatum

CLEC2 (CLEC1B)

Cell surface

PAMPs Podoplanin

HIV-1

Rhodocytin

Snake venon

DAMPs Podoplanin

Tumor cells

Rhodocytin MICL (CLEC12A)

Cell surface

DNGR1 (CLEC9A)

Cell surface

Dectin 2 (CLEC6A)

Cell surface

DAMPs Uric acid

Extracelular

DAMPs F-actin

Cytoskeletal exposure of necrotic cells

PAMPs High-mannose oligosaccharides α-mannans

A. fumigatus M. tuberculosis H.capsulatum C.albicans S. cerevisae C. neoformans T.rubrum P. brasiliensis Soluble components of S. mansoni eggs

Mincle (CLEC4E)

Cell surface

PAMPs α-mannose

Malassezia spp

Trehalose-6,6-dimycolate

M. tuberculosis

DAMPs β-glucosylceramide

C.albicans

SAP130

Cytosol of damaged necrotic cells Nucleus of damaged cells

BDCA2 (CD303)

Cell surface

PAMPs Gp120

HIV-1 (continued)

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Table 4.1 (continued) PRR

Cellular location

Ligands (PAMPs and DAMPs)

Ligand origin

DCIR (CLEC4A)

Cell surface

ND

HIV-1

RLRs RIG-I

Cytosol

PAMPs Short dsRNA

MDA5

Cytosol

Orthomyxovirus

ssRNA

Paramyxovirus

dsRNA generated by RNA polymerase III transcription of virus dsDNA

Hepatitis C Epstein-Barr vírus Adenovirus, Herpes simplex virus 1

PAMPs Long dsRNA

Poliovírus Encephalomyocarditis vírus Vaccinia virus

LPG2

Cytosol

ND

ND

NLRs NOD1

Cytosol

PAMPs γ-D-glutamyl-meso-diaminopimelic acid

NOD2

Cytosol

PAMPs Muramyl dipeptide

NLRP1

Cytosol

Gram-negative and few Gram-positive bacteria Gram-negative and Gram-positive bacteria

PAMPs Anthrax lethal toxin

B. anthracis

Muramyl dipeptide

Gram-negative and Gram-positive bactéria

ND

T. gondii

DAMPs ATP NLRP3

Cytosol

Reduction in cellular ATP levels

PAMPs LPS

Gram negative bacteria

Zymosan

Fungi

Pore forming toxins

S. hygroscopicus (continued)

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Table 4.1 (continued) PRR

Cellular location

Ligands (PAMPs and DAMPs)

Ligand origin

Several viral proteins

Influenza, poliovirus, encephalomyocarditis vírus, rhinovirus, human respiratory syncytial virus

DAMPs ATP

NLRC4

Cytosol

External ATP

Glycosaminoglycan hyaluronan

Extracellular matrix

Uric acid cristals

Extracellular

Calcium pyrophosphate dehydrate cristals

Extracellular

Cholesterol cristals

Extracellular

Amiloid fibrils

Extracellular

mtROS

Intracellular

PAMPs Flagelin type III secretion system analogs

S. typhimurium L. pneumophila S. flexneri L.monocytogenes P. aeruginosa Enterohemorrhagic E. coli

ALRs AIM2

Cytoso

PAMPs dsDNA

DNA from intracellular pathogens including viruses, bacteria, and parasites

DAMPs dsDNA IFI16

Cytosol

Aberrant and/or mislocated self DNA

PAMPs dsDNA

DNA from intracellular pathogens including viruses, bacteria, and parasites

DAMPs dsDNA

Aberrant and/or mislocated (cytosol) self DNA (continued)

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Table 4.1 (continued) PRR

Cellular location

Ligands (PAMPs and DAMPs)

Ligand origin

Cytosolic DNA sensors cGAS–STING

Cytosol

PAMPs dsDNA Stem-loop–forming ssDNA

Intracellular pathogens including viruses, bacteria, and parasites

DAMPs dsDNA STING

Cytosol

Mislocated (cytosol) self DNA

PAMPs c-di-AMP c-di-GMP

C.trachomatis, M. tuberculosis Gram positive bacteria

4.2.1.1

MyD88-Dependent Pathway

Following TLR engagement, MyD88 forms a complex with IL-1 receptor-associated kinase proteins (IRAKs) termed as myddosome [18]. This results in autophosphorylation of IRAK-1 that then leaves the complex and interacts with activation of tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). The IRAK-1/TRAF6 complex associates with TGF-β-activated kinase 1 (TAK1) and with the TAK1 binding proteins, TAB1 and TAB2 [19]. From this new formed complex, IRAK-1 is degraded whereas the remaining complex of TRAF6, TAK1, TAB1, and TAB2 is transported across the cytosol forming large complexes with E2 protein ligases such as the Ubc13 and Uev1A. As result, TRAF6 is polyubiquitinated and thereby induces TAK1 activation by transphosphorylation. TAK1 subsequently activates two divergent cascades that culminate in the activation of IκB kinases complex (IKK)-NF-κB and the MAPK pathways. Activation of IKK complex promotes the phosphorylation and subsequent ubiquitination of the NF-κB inhibitory protein IκBα, which undergoes proteasome degradation, allowing NF-κB to translocate into the nucleus where it induces the transcription of genes involved in inflammatory response [20]. TAK1 also activates the three MAPK family members: extracellular signal–regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38. This results in the activation of AP-1 family transcription factors, regulating both, the transcription of inflammatory genes and their mRNA stability (Fig. 4.1).

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Fig. 4.1 Schematic representation of intracelular signaling pathways triggered by TLRs engagement. TLR signaling cascades are triggered when PAMPs or DAMPs interact with cell membranelocalized TLRs, such as TLR4, TLR5, and TLR2 or with endosomal-localized TLRs like TLR3, TLR7, TLR8 and TLR9. Two major pathways can be established, according to the adaptor molecules involved: the MyD88-dependent pathway (plain lines) and the TRIF-dependent pathway (dashed lines)

4.2.1.2

TRIF-Dependent Pathway

After TLR3 engagement, TRIF interacts directly with the TIR domain of the receptor, whereas for TLR4 another TIR domain containing adaptor, TRAM/TICAM-2, acts as a bridging between TLR4 and TRIF [21, 22]. TRIF in turn interacts with TRAF6 and TRAF3, leading to two distinct signaling cascades. From the interaction with TRAF6 it results the recruitment of the kinase RIP-1, which activates the TAK1 complex leading to NF-κB and MAPKs downstream signaling. In turn, the interaction of TRIF with TRAF3 promotes the recruitment of TANK-binding kinase 1 (TBK1) and IKKi that phosphorylate IRF3 and IRF7, promoting their nuclear translocation and the consequent induction of type I IFN genes and co-stimulatory molecules [23] (Fig. 4.1). Type I IFNs (IFN-α and IFN-β), and IFN-inducible genes (ISGs) are

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among the most potent antiviral molecules and TRIF-dependent signaling cascades assume therefore a critical importance for the control of viral infections.

4.2.2 C-Type Lectin Receptors C-type lectin receptors (CLRs) are transmembrane proteins characterized by the presence of one or more C-type lectin-like domains (CTLDs), belonging to the large superfamily of C-type lectins. CLRs were initially described to bind carbohydrates in a calcium-dependent manner, but it is currently known that many also bind glycans, proteins or lipids in a calcium-independent fashion. There are more than 60 CLRs identified in human immune cells, playing central roles in the recognition of molecular signatures of fungi, bacteria, virus, nematodes, damaged (apoptotic and necrotic) or aberrantly altered host cells [24]. CLRs are sorted into type I or type II family according to their structural differences in recognition domains. Type I receptors are transmembrane proteins with multiple carbohydrate recognition domains (CRDs), being members of this group the mannose receptor (MR), DEC-205 (CD205), and Endo 180 (CD280), among others. Type II receptors are also transmembrane proteins, but they have a single CRD. DC-specific intercellular adhesion molecule (ICAM)-3 grabbing nonintegrin (DC-SIGN), Langerin, DC-associated C-type lectin-1 (Dectin 1), Dectin 2, DC-immunoreceptor (DCIR) and macrophage-inducible C-type lectin (Mincle) are examples of type II CLRs. CLRs engagement generally results in the activation of the cell endocytic and phagocytic machinery, thus promoting the uptake of pathogens or abnormal selfconstituents [25]. Additionally, some CLRs can trigger intracellular signaling cascades that directly activate transcription factors such as NF-κB. Finally, there are CLRs whose signals predominantly modulate the responses to other PRRs [26]. This is of utmost importance because while TLRs engagement results in proinflammatory and immunogenic signals, binding of ligands to CLRs often results in tolerogenic signals. Therefore, the cross-talk between TLRs and CLRs may fine-tune the balance between immune activation and tolerance during infection or tissue damage. According to their cytoplasmic signaling motifs and downstream cascades, CLRs are grouped into: (1) spleen tyrosine kinase (Syk)-coupled CLRs, (2) CLRs with immunoreceptor tyrosine-based inhibitory motifs (ITIM) domains, and (3) CLRs without immunoreceptor tyrosine-based activation motif (ITAM) or ITIM domains [8].

4.2.2.1

CLR Coupling to Syk

CLR coupling to Syk can be subdivided into (a) hemITAM-based CLRs such as Dectin-1, CLEC-2 and DNGR-1, and (b) ITAM-coupled CLRs that include amongst

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others Dectin-2, BDCA-2, Mincle and MDL-1. In hemITAM-based CLRs the coupling of receptor to Syk is direct via a single tyrosine-based motif found in the cytoplasmic domain while in ITAM-coupled CLRs it occurs through the adaptors Fc receptor γ chain (FcRγ) or DAP-12 [27]. Example of hemITAM-Based CLRs Signaling Dectin-1 is the paradigm of hemITAM-based CLRs being the first non-TLR PPR shown to possess intrinsic signaling properties both through dependent and Sykindependent pathways (Fig. 4.2) [28]. In the Syk-dependent pathway, upon binding to agonist ligands, Dectin-1 dimerizes and tyrosine residues in the hemITAM-like motif are phosphorylated by Src kinases. This promotes the recruitment and activation of Syk that in turn recruits the downstream transducer caspase recruitment domain protein (CARD)9, forming a complex with the B cell lymphoma 10 (Bcl10), and the mucosa associated lymphoid tissue translocation protein 1 (MALT1). The CARD9–BCL10–MALT1 module then activates the IκB kinase (IKK) complex

Fig. 4.2 Schematic representation of CLRs-dependent signaling cascades. CLRs are cell membrane receptors mainly involved in the recognition of carbohydrate antigens. According to their cytoplasmic signaling motifs and downstream signaling cascades, CLRs are grouped into: a Sykcoupled CLRs, which can further be subdivided in hemITAM-based CLRs such as Dectin-1, and ITAM-coupled CLRs such as Dectin-2; b CLRs with ITIM domains like DC-SIGN, and c CLRs without ITAM or ITIM domains such as DCIR

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for canonical NF-κB signaling and subsequent expression of cytokines/chemokines such as TNF-α, IL-1β, IL-2, IL-10, IL-6, IL-23, CCL2 and CCL3 [29, 30]. Dectin-1, in a Syk-dependent way, also activates the non-canonical NF-κB pathway (RelB) via the NF-κB-inducing kinase (NIK), and the phospholipase C gamma-2 (PLC-γ2) [31, 32]. PLC-γ2 then signals via several calcium-dependent and MAPKs-dependent pathways to activate the nuclear factor of activated T-cells (NFAT) and the NLRP3 inflammasome [33, 34]. Regarding the Dectin-1 Syk-independent pathway it involves the phosphorylation and activation of the serine-threonine kinase RAF1. Activated RAF1 specifically promotes the phosphorylation of NF-κB p65 subunit at Ser276 residue, facilitating its acetylation by the histone acetyltransferase CREB-binding protein. Acetylated p65 can then form inactive dimers or bind to p50 to become transcriptionally active [35]. Example of ITAM-Coupled CLRs Signaling Dectin 2 is expressed in macrophages, monocytes, neutrophils, several DC subsets and B cells. It is involved in the recognition of mannan-like or mannan-containing glycoproteins, glycolipids or oligomannosides present in fungal cell walls [36, 37], soluble schistosomal egg antigens [38] and Histoplasma capsulatum components [39]. In contrast to Dectin-1, Dectin-2 lacks a defined intracellular signaling motif, requiring the association of ITAM with the adaptor molecule Fc receptor γ chain (FcRγ) to transduce signals, through a Dectin 2-FcRγ-Syk-dependent pathway [40]. Following Dectin-2 engagement, FcRγ-ITAM motif is dually phosphorylated by Src kinases, promoting the recruitment and activation of Syk that in turn activates NF-κB and MAPKs pathways in a CARD9-dependent or independent fashion, respectively (Fig. 4.2) [37, 40, 41].

4.2.2.2

CLRs with ITIM Domains

Several CLRs such as DCIR, DCAL-2 and MICL express ITIM motifs that recruit phosphatases and thereby negatively regulate signaling through kinase-associated receptors. These receptors are normally devoid of intrinsic activity however they negatively modulate immune cell activation triggered by activatory receptors such as Syk-coupled CLRs and TLRs [42–44]. The knowledge about the signaling events triggered by CLRs with ITIM domains is mainly derived from studies with DCIR. DCIR is expressed at high levels in blood monocytes, myeloid and plasmacytoid DCs, macrophages and in a less extent in B cells. DCIR recognizes endogenous mannotriose and sulfo-Lewisa glycans expressed in cancer cells, and has also been reported to bind HIV-1 by recognition of gp140 glycoprotein [45–47]. At the molecular level, activation of DCIR by anti-DCIR antibodies leads to receptor internalization into endosomal compartments in a clathrin-dependent manner. Phosphorylation of internalized receptor at its ITIM motif promotes the recruitment of the phosphatases SH2-domain-containing protein tyrosine phosphatase 1 (SHP1) or SHP2, which, by an unidentified mechanism, causes the downregulation of TLR8

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and TLR9 signaling and consequent impairment of IL-1β, IL-6, IL-12, IFN and TNF expression (Fig. 4.2) [43, 44].

4.2.2.3

CLRs Lacking ITAM or ITIM Domains

Among CLRs without a defined ITAM or ITIM motif we can find mannose receptor, DEC-205, DC-SIGN, SIGNR1, Langerin, hMGL, CLEC-1, DCAL-1, MCL and LOX-1, among others. These CLRs are predominantly expressed in dendritic cells and macrophages, where they mediate the capture, processing and subsequent presentation of antigens to T cells [31]. The engagement of ITAM/ITIM independent CLRs per se normally does not induce immune cell activation although it can, in some cases, modulate the signaling cascades of other CLRs and TLRs. Among this family of CLRs, DC-SIGN is one of the most extensively studied. The receptor is primarily expressed in DCs, controlling the egress of DC-precursors from blood to tissues, DC-T-cell interactions and recognition of mannose and fucosebearing glycans present in viruses, bacteria, protozoa and self-molecules [48]. Sensing of M. tuberculosis, C. albicans and HIV-1, by DC-SIGN triggers several signals that converge to activate Raf-1. Upon TLR-induced nuclear translocation of NF-κB, activated Raf-1 mediates the phosphorylation of NF-κB subunit p65 at the Ser276, which in turn allows the binding of the histone acetyl-transferases CREB-binding protein (CBP) and p300 and the acetylation of p65. Acetylated p65 increases and prolongs the transcription of IL-10 gene with consequent increased production of the immunosuppressive cytokine IL-10 (Fig. 4.2). Therefore, DC-SIGN cannot activate NF-κB by itself but strongly modulates p65 activity induced by other receptors [49, 50]. DC-SIGN also negatively modulates TLR2 and TLR4-signaling cascades via activation of MAPK/ERK (MEK) kinase. Binding of Salp15 protein from the tick Ixodes scapularis to DC-SIGN activates RAF1 leading to (MEK) phosphorylation. MEK-dependent signaling enhances the decay of IL6 and TNF mRNAs and decreases IL-12p70 cytokine production by impairing nucleosome remodeling at the IL-12p35 promoter [51].

4.2.3 RIG-I-Like Receptors RIG-I-like receptors (RLRs) include retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). These receptors contain a DExD/H-box RNA helicase domain that specifically recognizes viral double-stranded RNA (dsRNA) [52]. RIG-I and LGP2 additionally contain a C-terminal regulatory domain that senses singlestranded RNA (ssRNA) containing 5 -triphosphate. The RIG-I distinction of self from viral ssRNAs is ensured by the predominantly nuclear localization of cellular 5 -triphosphate ssRNAs, that even if present in the cytoplasm are normally capped or processed. Finally, RIG-I and MDA5 also have N-terminal tandem caspase activation

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and recruitment domains (CARD), which interact with the CARD domain of mitochondrial antiviral signalling protein (MAVS) located in the outer mitochondrial membrane and in peroxisomes [53]. Peroxisome MAVS induce early expression of interferon-stimulating genes via transcription factor IRF1, while mitochondrial MAVS relay the signal to TBK1 that in turn activates IRF3/IRF7 leading to expression of ISGs and type I interferons (Fig. 4.3) [54, 55]. Interaction of CARD domain of RIGI and MDA5 with MAVS also leads to the activation of NF-κB, a process involving the recruitment of TRADD, FADD, caspase-8, and caspase-10 [56]. Finally, RIG-I can bind to the adaptor ASC to trigger caspase-1-dependent inflammasome activation and the consequent production of mature IL-1β [57]. LGP2, similarly to RIG-I and MDA5, possesses a DExD/H-box helicase domain but lacks the CARD domain, being therefore mainly considered a regulator of RIG-I and MDA5 signaling [58].

Fig. 4.3 Representation of intracelular signaling pathways triggered by NOD-like receptors and major cytosolic nucleic acid sensors. Pathogen-derived nucleic acids are recognized by intracellular RNA sensors such as RIG-1 and MDA5 or intracellular DNA sensors, including cGAS/STING. In turn, NOD-like receptors such as NOD1 and NOD2 recognize specific bacterial aminoacid and peptidoglycans, respectively. Binding of ligands to these sensors activates downstream signaling cascades, resulting in the production of type I IFNs and pro-inflammatory cytokines to induce appropriate immune responses

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4.2.4 NOD-Like Receptors Nucleotide-oligomerization domain (NOD)-like receptors (NLRs) comprise a large family of cytosolic sensors of microbial molecules. In humans, there are 23 genes coding for NLRs, being these receptors primarily expressed in lymphocytes, macrophages and dendritic cells [11]. They contain three characteristic domains: (a) C-terminal leucine-rich repeat (LRR) motifs, responsible for ligand sensing and activity modulation; (b) an intermediary nucleotide-binding oligomerization (NOD) domain, required for nucleotide binding and self-oligomerization and (c) an N-terminal effector binding region that consists of protein-protein interaction domains. Based on the N-terminal domains, NLRs can be classified into three subfamilies also referred to as: caspase recruitment domain (CARD)-containing NODs, pyrin (PYD)-containing NLRPs and baculovirus inhibitor repeat (BIR)-containing NAIPs. NOD1 senses γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), an amino acid that is predominantly found in gram-negative bacteria and in some gram-positive bacteria, such as Listeria monocytogenes and Bacillus spp. NOD2, in turn, senses muramyl dipeptide (MDP), a molecule found in the cell wall of nearly all grampositive and gram-negative bacteria [59–61]. Upon recognition of their respective ligands, both NOD1 and NOD2 self-oligomerize and recruit the serine-threonine kinase RICK that becomes polyubiquitinated. RICK then activates an intricate signaling network that converge to the activation of NF-κB and the MAP kinases p38, ERK and JNK (Fig. 4.3) [62–65]. Another important signaling process by which NLRs participate in host response to PAMPs and DAMPs is through their involvement in assembly of a large multiprotein complex termed inflammasome that serves as scaffold for caspase 1 activation [10]. Pyrin-containing NLRs such as NLRP3 and NLRP1 associate with caspase-1 through the adaptor molecule apoptosis-associated speck-like protein containing a Cterminal CARD (ASC) while NLRC4 is thought to directly associate with caspase-1 via CARD-CARD interactions. These molecular platforms are crucial for caspase-1 activation and subsequent processing of pro-IL-1β and pro-IL-18, resulting in the secretion of their mature biologically active forms [66, 67].

4.2.5 AIM-Like Receptors In humans, the family of absent in melanoma 2 (AIM2)-like receptors (ALRs) includes the proteins AIM2, IFI16, PYHIN1, and MNDA. These receptors are structuraly characterized by the presence of C-terminal DNA-binding HIN domains and an N-terminal pyrin (PYD) domain that belongs to the death domain superfamily of signaling modules. ALRs are sensors of aberrant or mislocalized self DNA molecules such as genomic or mitochondrial DNA released into the cytosol upon cell damage and dsDNA from intracellular pathogens including viruses, bacteria, and parasites [68].

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The signaling cascades evoked by these receptors lead to expression of type I IFN and inflammasome activation, being the AIM2 sensor the best characterized. The HIN200 domain of AIM2 binds to pathogen or self dsDNA causing receptor oligomerization. This facilitates the interaction of PYD domains with ASC and the assembly of AIM2 inflammasome. As result, pro-caspase 1 is recruited and activated leading to proteolitic processing of pro-IL-1β and pro-IL-18 [69, 70]. IFN-inducible 16 (IFI16) protein, in addition to form inflammasomes also signals for the induction of type I IFN, a process dependent of the ER-resident protein stimulator of interferon genes (STING) [71–73].

4.2.6 Cytosolic DNA/Cyclic Nucleotide Sensors Cytosolic DNA sensors are a large family of proteins that includes cyclic-GMPAMP (cGAMP) synthase (cGAS), STING, DAI, DDX41, DNAPK, DHX9, DHX36, DDX60, and MRE11. These intracellular sensors activate the interferon pathway in response to DNA from bacterial, viral, parasitic, or self-origins. Among these sensors the cGAS–STING sensing pathway assumes particular relevance as its ablation completely abrogates the cytosolic DNA-induced IFN production [73–75]. After interacting with dsDNA, cGAS produces cGAMP that functions as a second messenger, binding and activating STING protein. STING dimerizes and translocates from the ER to an ER-Golgi intermediate compartment where it recruits and activates TBK1 [76, 77]. TBK1 in turn phosphorilates STING and IRF3, thereby inducing the expression of type I IFN [78]. STING also activates the kinase IKK, which phosphorylates the IkB family of inhibitors leading to translocation of NF-κB to the nucleus where it cooperates with IRF3 to induce the expression of interferons, TNF-α, IL-1β and IL-6 (Fig. 4.3) [79]. Besides sensing cytosolic dsDNA in a cGAS-dependent way, STING can directly bind to cyclic-di-adenosine monophosphate (c-di-AMP) and cyclic diguanylate monophosphate (c-di-GMP), metabolites produced by viable intracellular pathogens such as Chlamydia trachomatis, Mycobacterium tuberculosis and Gram positive bacteria like Listeria monocytogenes [12, 80–83].

4.3 NK Cells Natural killer (NK) cells are large granular lymphocytes of the innate immune system that respond early to infection and interact with other immune cells. They participate in cytotoxicity, cytokine production, antibody-dependent cellular cytotoxicity (ADCC) and immune regulation. NK cells recognize tumours, viral-infected cells and certain parasites. There is also evidence that NK cells play a role in transplantation, human pregnancy and autoimmunity (reviewed in [84]). Human deficiencies of NK cells are rare, but diseases where patients lack functional NK cells have been

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described in a few young patients (see for example [85]). Analysis of these diseases indicate that human NK cell activity is particularly important in defence against herpesviruses [86]. NK cells response is determined by the integration of activating and inhibitory signals. NK cells can kill target cells prior to antigen stimulation. Lytic proteins can be released rapidly because they are stored in preformed secretory lysosomes. Upon encountering a susceptible target cell, secretory lysosomes polarize towards the target cell, where its contents are released (reviewed in [87]). NK cells can also kill upon engagement of FAS, tumour necrosis factor (TNF) or TNF-related apoptosisinducing ligand (TRAIL) [88]. Binding of these cell surface death receptors recruits adaptor molecules such as FADD, TRADD and apical pro-caspases, leading to caspase activation and apoptosis [87]. NK cells are also a major source of IFN-γ and TNF-α and also have the capacity to secrete GM-CSF, IL-5, IL-13, MIP-1 (α and β) and RANTES. IFN-γ activates macrophages and neutrophils, promotes T-helper 1 cell differentiation, enhances MHC expression and has anti-mycobacterial, antiviral and growth inhibitory effects, while TNF-α initiates proinflammatory cytokine cascades [84, 89]. Activation of NK cell cytotoxicity mediated by targeted release of lytic granules occurs in steps, all required for efficient lysis of the target cells [90–92]. When conjugating with target cells, NK cells need to form a stable contact that may lead to formation of the immune synapse. Adhesion to susceptible target cells is accompanied by polarization of the actin cytoskeleton, Golgi apparatus, microtubules and cytotoxicity granules towards the contact area, prior to target cell lysis [93–95]. This polarization requires an intact actin cytoskeleton [90]. Secretory granules containing perforin travel along microtubules towards the MTOC and thus can only be delivered to the target cell after MTOC polarization [96]. Upon polarization, directed degranulation of the lytic granules towards target cells must occur for efficient and specific target cell lysis [92, 97]. Indeed, degranulation by NK cells can occur in the absence of polarization of the granules, and vice versa, but both are required for efficient lysis of the target cells [98]. Thus, cooperation between different combinations of NK cell receptors, for adhesion, directed granule polarization and degranulation, is required for efficient target cells lysis [92]. Inhibition of an efficient NK cell response may occur at any stage of NK cell activation, but most likely during adhesion and polarization.

4.3.1 NK Cell Receptors NK cells possess on its surface an array of activating and inhibitory receptors (Table 4.2) whose combined action will dictate activation or inhibition of cytotoxicity or cytokine secretion. Interestingly, none of the activating receptors apart from CD16 (the Fc receptor involved in ADCC) activates NK cells on its own [99]. Indeed, different activating receptors require complementation with other receptors, and inhibition can occur at different levels. A complex network of signaling events tightly regulates whether NK cell activation can lead to either cytotoxicity, cytokine secretion or both, while avoiding inappropriate activation by healthy cells.

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Table 4.2 Human activating NK cell receptors, their adapter proteins and ligands

ITAM-bearing adapters

Receptor

Adapter

Ligand

Selected references

CD16

FcR γ, TCR ζ

IgG

[121]

NKp30

FcR γ, TCR ζ

B7-H6

[122]

NKp44

DAP12

Viral haemagglutinins

[100, 123, 124]

NKp46

FcR γ, TCR ζ

Viral haemagglutinins, vimentin

[100, 125–127]

KIR-short

DAP12

HLA-C, B and A (low affinity)

[128, 129]

KIR2DL4 (CD158d)

FcR γ

HLA-G (soluble)

[130, 131]

CD94/NKG2C

DAP12

HLA-E

[132, 133]

NKG2D

NKG2D

DAP10

ULBPs, MICA, MICB

[104, 134]

SLAM family

2B4 (CD244)

SAP and EAT-2

CD48

[135, 136]

CRACC (CD139)

EAT-2

CRACC (CD139)

[137, 138]

NTB-A

SAP and EAT-2

NTB-A

[139–141]

CD84

SAP and EAT-2

CD84

[110, 142]

DNAM-1 (CD226)

CD155, CD112

[143]

NKp65

KACL

[144]

NKp80

AICL

[145]

CD2

LFA-3 (CD58)

[146]

CD7

SECTM1, galectin

[147–149]

CD44

Hyaluronan

[150, 151]

CD59

C8, C9

[152]

CD160 (BY55)

HLA-C

[153]

Other receptors

4.3.2 Activating Pathways Different signaling pathways are involved in several aspects of NK cell biology, such as maturation, education or activation. Here, we will focus on the pathways that derive from surface receptors binding to their extracellular ligands, and leading to

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recognition of target cells. Overall, there is redundancy of the activating signaling pathways, which makes the system very robust (Fig. 4.4). Some activating receptors are associated with ITAM-bearing signaling molecules. CD16, NKp46 and NKp30 associate with FcR γ and/or TCR ζ, while NKp44 associates with DAP12, all ITAM bearing molecules [100]. The activating isoforms of KIR (killer cell immunoglobulin-like receptor) and CD94-NKG2C also signal upon association with DAP12 [101]. Signaling through ITAM also governs other receptors, such as TCR, and thus has been studied in detail [102]. ITAM contains two tyrosines that are phosphorylated by Src kinase family members such as p56lck. Phosphorylated ITAMs will then be bound by ZAP70 and Syk tyrosine kinases, through the Src homology 2 (SH2) domains. In the case of antibody-dependent CD16 signaling, this will lead to phosphorylation of SLP-76 at the Tyr113 and Tyr128, which can then bind to Vav1 and Nck [103]. The receptor NKG2D associates with DAP10, a transmembrane molecule carrying a tyrosine-based motif that is different from an ITAM [104]. Upon phosphorylation, DAP10 can bind either to the p85 subunit of phosphoinositide 3-kinase (PI3K), or to

Fig. 4.4 Schematic representation of key intracellular signaling events governing NK cell activation upon binding to a target cell. Binding of activating receptors triggers redundant activating signaling pathways, eventually leading to either Vav or PI3K activation and subsequently to actin polarization, synapse formation and NK cytotoxicity. However, binding of inhibitory receptors can block these cascades, and thus NK cell activation will be determined by the net outcome of intricate signaling pathways

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the small adaptor Grb2 associated with Vav1 [105–109]. PI3K can be activated upon binding of different receptors, resulting on recruitment of PLC-γ1 or 2, Akt and Vav. NKG2D signals through Vav1, while ITAM-based receptors signal mostly via Vav2 or Vav3. Receptors of the signaling lymphocytic activating molecule (SLAM) family share immunoreceptor tyrosine-based switch motifs (ITSMs), S/TxYxxL/I. NK cells express different members of the SLAM family (but not SLAM itself): 2B4 (CD244); NK, T, and B cell antigen (NTB-A); CD2-like receptor activating cytotoxic cells (CRACC, CD319); CD84 [110]. These receptors signal by associating with the SLAM-associated protein (SAP)-related adapters, which recruit the tyrosine kinase Fyn [110]. Upon activation via 2B4, Fyn can either induce phosphorylation of Vav1, or block recruitment of the inhibitory SH2 domain-containing inositol 5 phosphatase-1 (SHIP-1) [111]. Ewing’s sarcoma-associated transcript-2 (EAT-2) is another SAP adaptor, but it does not bind Fyn. SAP and EAT-2 combine and promote NK cell activation upon 2B4 binding [111]. Interestingly, binding of the receptor 2B4 leads to Fyn-dependent phosphorylation of SLP-76 at Tyr113, while binding of NKG2D or DNAM-1 can trigger phosphorylation of the tyrosine at position 128. Phosphorylation of the two tyrosines leads to recruitment of Vav1, which explains the synergy seen upon binding of 2B4 with either NKG2D or DNAM-1 but not upon binding of NKG2D and DNAM-1 [103]. In the end, PI3K-Akt and Vav1-Rac1 signaling lead to activation of ERK, which will culminate on granule polarization and release. There is limited information regarding other activating receptors. DNAM-1 associates with LFA-1 and the tyrosine kinase Fyn [112]. Both NKp80 and NKp65 have a hemi-ITAM, a tyrosine sequence motif corresponding to half an ITAM, in their cytoplasmic tails. NKp80 stimulates Syk phosphorylation and Syk-dependent cytotoxicity [113]. Integrins represent another class of signaling molecules. Contrarily to what occurs with T cells, in NK cells LFA-1 binding signals independently of inside-out signaling and is sufficient to induce granule polarization (but not degranulation). In human T cells, different signalling pathways are activated upon LFA-1 binding, and phosphorylation of the β2-chain on Thr-758 has a role in this signalling, upstream of Rac-1/Cdc42 [114]. Binding of NK cell LFA-1 to purified ICAM-1 leads to tyrosine phosphorylation of molecules such as PLC-γ and Syk (similarly to CD16 signaling) [115]. Engagement of LFA-1 delivers activation signals that lead to phosphorylation of Vav1, which occurs upstream of actin polymerization and clustering of lipid rafts [116]. On the other hand, engagement of the inhibitory KIR receptors leads to recruitment of SHP-1 molecules that can dephosphorylate Vav-1 [117]. Thus, upon binding of inhibitory ligands, LFA-1 accumulates only transiently at the contact and F-actin and the MTOC do not polarize [91, 94, 118–120]. Therefore, inhibitory signals seem to disrupt polarization at an early stage, not allowing assembly of a mature activating synapse.

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4.3.3 Inhibitory Pathways NK cell receptors for MHC class I molecules can be divided into two main types, according to their structure: immunoglobulin (Ig)-like receptors (including the killercell Ig-like receptors, KIRs, and leukocyte inhibitory receptors, LIRs) and C-type lectin-like receptors (the NKG2 family) (reviewed in [101]). KIRs with long intracytoplasmic tails generate inhibitory signals through intracellular ITIM domains. LIR-1 binds classical MHC class I molecules and the non-classical MHC class I molecule human leukocyte antigen (HLA)-G. NKG2A is the inhibitory member of the NKG2 family that forms heterodimers with CD94, recognizes the non-classical MHC class I molecule HLA-E, and generates signals in a similar way to KIRs. HLA-E surface expression reflects classical MHC class I expression as it requires leader peptides from HLA molecules for stabilization [121]. Inhibitory receptors for non-MHC class I molecules have also been identified, for example, KLRG1 (binds cadherins), NKRP1 (binds LLT1), IRp60 (unknown ligand), Siglec-7 and Siglec-9 (both bind sialic acid). There is a big heterogeneity in the NK cell repertoire of different individuals [122], especially due to haplotypic and allelic variability in the KIR receptors [123]. Also, different NK cells within the same individual express different combinations of receptors [123]. In spite of a big diversity in the extracellular ligand-binding domains, inhibitory receptors seem to use a common mechanism for inhibition [92]. The inhibitory receptors have at least one ITIM in their cytoplasmic tail. Upon receptor ligation, the tyrosine becomes phosphorylated, then facilitating recruitment of Src homology 2 (SH2) domain-containing phosphatases (SHP-1 or 2). SHP-1 or 2 can then dephosphorylate intracellular targets, inhibiting NK cell function [124]. These targets may include activating receptors, Vav-1 [117], SLP-76 [125], phospholipase C (PLC)-γ, ZAP-70, and Grb2 [124]. However, Vav-1 was the only protein detected as a substrate to a trapping mutant of SHP-1 fused to KIR2DL1 during conjugation of an NK cell line with HLA-C expressing target cells [117]. In an alternative pathway, ITIM-bearing receptors can also lead to phosphorylation of Crk, which associates with c-Abl [126]. It has been found that Crk is linked to cytoskeleton scaffold complexes and is required for the movement of microclusters in lipid membranes [127]. The missing self hypothesis states that NK cells are capable of sensing a decrease in expression of self MHC class I in target cells [128]. NK cell responses are then controlled by the integration of signals from activating and inhibitory receptors at the immunological synapse. And indeed, there is a specific threshold in the level of target cell HLA-C needed to inhibit cytotoxicity that correlates with segregation of HLA-C from ICAM-1 at the synapse [129]. Thus, it has been proposed that one way in which inhibitory signaling can control activation (e.g. mediated by NKG2D) is by blocking recruitment to membrane microdomains and regulating the proximity of activating and inhibitory receptors [130–132].

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4.4 Impact to Human Health of Malfunctions in Innate Immune Cell Signalling Networks The adequate sensing of danger by PRRs as well as the resultant signaling transduction cascades are crucial to adequate immune responses. Mutations that result in loss or gain-of-functions can have dramatic effects in human health, causing either increased susceptibility to infections or autoimmune diseases. For instance, patients with autosomal recessive deficiency in the TLR adapter molecule MyD88 present recurrent infections by pyogenic bacteria, particularly Streptococcus pneumoniae while being resistant to other pathogens [133]. The common TLR5 stop codon polymorphism 392STOP has been linked to increased susceptibility to flagellated bacteria such as Legionella pneumophila [134] and the Asp299Gly TLR4 polymorphism to an increased risk of infection by Gram negative bacteria [135]. Similarly, mutations in the CLR Dectin-1 or in its transducer signaling molecule CARD9 result in defective production of IL-17 and IL-6 with consequent susceptibility to mucocutaneous fungal infections [136, 137]. On other hand, gain-of-function mutations in NLRP3 gene result in serious auto inflammatory diseases: familial cold auto inflammatory syndrome, Muckle-Wells syndrome and chronic infantile neurologic cutaneous articular syndrome. These clinical conditions, collectively known as cryopyrinopathies, are the result of caspase 1 overactivation and subsequent excessive production of the IL-1 family cytokines IL-1β, IL-18 and IL-33 [138–140]. Similarly, mutations in genes coding for cytoplasmic DNA sensors such as TREX1, RNase H2, SAMHD1, MDA5 and STING lead to elevated expression of type I interferons and IFN regulated genes resulting in auto inflammatory conditions like Aicardi-Goutières syndrome and STINGassociated vasculopathy with onset in infancy [141, 142]. Finally, NOD2 gene mutations strongly increase the risk of Crohn’s disease and NOD1 polymorphisms are associated to inflammatory bowel disease susceptibility [143–145]. Human pathologies where patients have absent or dysfunctional NK cells while maintaining function of other cell types are rare. These NK cell deficiencies result in increased susceptibility to infection [146]. A higher number of pathologies include an effect on NK cell function, while also affecting other immune cell populations. Many diseases with impaired NK cell cytotoxicity are mostly related to defective protein trafficking, due to, for example, defective positioning of lytic granules or abnormal actin organization at the immunological synapse. The Wiskott-Aldrich syndrome (WASP) is an example of this, with patients that have a genetic defect on WASP or WIPF1 showing increased susceptibility to multiple infections, including to Herpesviruses [147, 148]. In humans, loss-of-function mutations in PI3K are very rare, but gain-of-function mutations are more common [149]. These result in hyperactivation of PI3K signaling, leading to hyperphosphorylation of S6, mTOR and AKT. These patients have combined immune deficiency, with impaired B, T and NK cell function, and, somewhat counterintuitively, have recurrent chest infections and herpesviral infections [150, 151]. The mechanism behind impaired NK cell function in these patients is

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still unclear but it has been suggested that hyperactivation of the mTOR-AKT pathway may lead to NK cell hyporesponsiveness [149, 152]. On a different example, in the X-linked lymphoproliferative disease (XLP), patients are highly susceptible to EBV, again due to defective NK cells. These patients have a mutation in SAP, which interferes with 2B4 signaling and subsequent cytotoxicity [153]. Information on mutations on human genes affecting innate immunity continues to emerge, contributing to a better understanding of human immunity.

4.5 Conclusion Innate sensing involves recognition of danger signals by specialized receptors. The structure and intracellular location of different classes of PRRs allow recognition of defined pathogens or alarm molecules. NK cells possess activating and inhibitory receptors on the cell surface that will allow detection of diseased target cells. In this chapter we described the signaling cascades involved in activation of innate immune cells. Broadly, activation of different receptors triggers recruitment of adapter proteins, which will lead to activation of kinases and nuclear translocation of transcription factors that regulate expression of inflammatory molecules. Several pathologies have been described that emerge upon loss or gain of function mutations on different players from these cascades. These mutations can result in either increased susceptibility to infection or autoimmune diseases. Thus, it is important to clearly understand the intricate and intertwining signaling events involved in innate immunity. Much of the current research is focused in filling in the gaps in our knowledge on these mechanisms and on finding novel immunomodulatory strategies by targeting key signaling nodes.

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Chapter 5

Cell Activation and Signaling in Lymphocytes Alexandre M. Carmo and Sónia N. Henriques

Abstract Lymphocyte activation and proliferation are a result of a functional encounter between a lymphocyte bearing a clonotypic receptor that recognizes unique nonself-antigens and a cell that presents those specific antigens at its surface. Positive recognition of antigen by the T cell receptor of T lymphocytes, or by the B cell receptor of B lymphocytes, is then signaled to inner components of the cell through biochemical activation pathways. A number of adhesion molecules and co-receptors help to strengthen the interaction between the lymphocyte and the antigen presenting cell, and some of these surface proteins transduce signals that complement or amplify those delivered by the antigen receptor in a concerted action that modifies cellular behavior. B and T cell receptor-proximal signaling involves the extensive phosphorylation of intracellular effectors and adaptors that help to assemble large multi-protein complexes that transmit and diversify the received stimuli into the activation of an array of transcription factors that modify the transcriptional landscape of the cell and change cell behavior. The majority of the signaling steps are common to those of many different mammalian cell types. However, the extreme complexity of the lymphocyte signaling networks with many additional checkpoints and controlling steps endows lymphocytes with a remarkable capacity of regulating the outcome of activation that goes beyond the simple response to increased amounts of agonist. On the outcome of controlled lymphocyte activation depends the building of an effective immune response towards foreign or external aggression while avoiding self-reactivity against own components and development of autoimmunity. Keywords T cell receptor · B cell receptor · Immunological synapse · Antigen presentation · Signalosome A. M. Carmo (B) · S. N. Henriques IBMC—Instituto de Biologia Molecular e Celular, Porto, Portugal e-mail: [email protected] i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal S. N. Henriques Programa Doutoral em Biologia Molecular e Celular (MCbiology), Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal e-mail: [email protected] © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_5

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Abbreviations AP1 APC BCL6 BCL10 BCR BLNK BTLA CARD11 CBL CD40LG CSK cSMAC CTLA4 DAG dSMAC ER ERK FYB1 FYN GRAP2 GRB2 ICAM1 IgSF IKKA IKZF1 IL2 INPP5D InsP3 IRF8 ITAM ITGAL ITIM ITK LAG3 LAT LCK LCP2 LYN MALT1 MAP2K1 mIg NCK1

Activator protein 1 Antigen presenting Cell B-cell lymphoma 6 protein B-cell lymphoma/leukemia 10 B Cell receptor B-cell linker protein B- and T-lymphocyte attenuator Caspase recruitment domain-containing protein 11 Casitas B-lineage lymphoma proto-oncogene CD40 ligand C-terminal Src kinase Central supramolecular activation cluster Cytotoxic T-lymphocyte protein 4 1,2-Diacylglycerol Distal supramolecular activation cluster Endoplasmic reticulum Extracellular signal-regulated protein kinase FYN-binding protein 1 FGR/YES novel protein GRB2-related adapter protein 2 Growth factor receptor-bound protein 2 Intercellular adhesion molecule 1 Immunoglobulin superfamily Inhibitor of nuclear factor κB kinase A Ikaros family zinc finger protein 1 Interleukin 2 Inositol polyphosphate-5-phosphatase D Inositol 1,4,5-trisphosphate Interferon regulatory factor 8 Immunoreceptor tyrosine-based activation motif Integrin alpha-L Immunoreceptor tyrosine-based inhibitory motif IL2-inducible T-cell tyrosine kinase Lymphocyte-activation gene 3 Linker for activation of T cells Lymphocyte-specific protein-tyrosine kinase Lymphocyte cytosolic protein 2 Lck/Yes-related novel protein tyrosine kinase Mucosa-associated lymphoid tissue lymphoma translocation protein 1 Mitogen-activated protein kinase kinase 1 Membrane immunoglobulin Non-catalytic region of tyrosine kinase adaptor protein 1

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NFAT NFKB NFKBI NTAL ORAI1 PAG1 PD1 PDPK1 PH PIK3 PIK3C PIK3R PKC PLCG PLCG1 PLCG2 pMHC POU2F1 PRDM1 PRKCB PRKCQ pSMAC PtdIns(4,5)P2 PtdIns(3,4,5)P3 PTK PTPN6 PTPN11 RASA1 RASGRP1 RASGRP3 RTK SH SIGLEC SOS1 SRCR SYK TCR TGF TIGIT TNFR TRAF6 XBP1 ZAP-70

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Nuclear factor of activated T-cells Nuclear factor κB NFKB inhibitor IκB Non-T cell activation linker Calcium release-activated calcium channel protein 1 Phosphoprotein associated with glycosphingolipid-enriched microdomains 1 Programmed cell death protein 1 3-Phosphoinositide-dependent protein kinase 1 Pleckstrin homology Phosphatidylinositol 3-kinase Catalytic subunit of phosphatidylinositol 3-kinase Regulatory subunit of phosphatidylinositol 3-kinase Protein kinase C Phospholipase C-γ Phospholipase C-γ1 Phospholipase C-γ2 Peptide-MHC complex POU domain, class 2, transcription factor 1 PR domain zinc finger protein 1 PKC isoform β PKC isoform θ Peripheral supramolecular activation cluster Phosphatidylinositol 4,5-bisphosphate Phosphatidylinositol 3,4,5-trisphosphate Protein tyrosine kinase Protein tyrosine phosphatase non-receptor type 6 Protein tyrosine phosphatase non-receptor type II RAS GTPase-activating protein 1 RAS guanyl-releasing protein 1 RAS guanyl-releasing protein 3 Receptor tyrosine kinases SRC homology Sialic acid binding Ig-like lectin Son of sevenless homolog 1 Scavenger receptor cysteine-rich Spleen tyrosine kinase T cell receptor Transforming growth factor T cell immunoreceptor with Ig and ITIM domains Tumor necrosis factor receptor TNF receptor-associated factor 6 X-box-binding protein 1 Z-associated protein-70

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5.1 Introduction There is nothing too particular or distinctive about the intracellular signaling pathways of lymphocytes when comparing with those of any other mammalian cell type. T and B lymphocytes (or T and B cells) can accommodate signal transduction via the Wnt-, Notch-, Hedgehog- and Hippo-mediated cascades, as well as through the transforming growth factor receptor, cytokine receptor and nuclear receptor pathways in many different aspects of lymphocyte biology such as cell differentiation, lineage commitment, receptor repertoire selection, subset polarization, migration, adhesion, survival, transcription regulation and tumor suppression, among others. Even the main axis translating the T or B cell receptor (TCR, BCR) recognition of antigens into effector functions and cell proliferation utilizes signaling mediators that are common to many types of cell, namely downstream effectors of receptor tyrosine kinases (RTK) such as phospholipase C-γ (PLCG), phosphatidylinositol 3-kinases (PIK3), SRC-type kinases and adaptors like the growth factor receptor-bound protein 2 (GRB2), among others. There are, however, a few characteristics that are unique to the physiological antigen-dependent lymphocyte activation pathways, amongst them the singularity of the receptors themselves and the fact that T or B cell antigen receptors do not recognize a single specified ligand. Differently from, for example, the platelet-derived growth factor receptor or epidermal growth factor receptor that bind and are activated specifically by platelet-derived growth factor and epidermal growth factor, respectively, the TCR and BCR are clonotypic receptors whose ligand-binding domains are hyper-variable as a result of gene rearrangements. Each T or B cell thus have unique TCRs and BCRs that, although potentially recognizing a few structurally different ligands with variable affinities, have optimal binding to a given 3D-structure consisting of the antigenic peptide and a membrane-bound antigen presenting protein complex. The BCR can directly recognize epitopes of naturally folded proteins, most often presented by antigen presenting cells (APCs) but also as free soluble or microbe surface-expressed structures, whereas in the case of the TCR, the ligand consists of a short antigenic peptide complexed to a polymorphic MHC molecule expressed at the surface of APCs. In non-antigenic conditions, the peptides coupled to MHC molecules are usually self-peptides that result from the natural proteolysis and turnover of endogenous proteins. However, when the APC is infected or has phagocytosed microbes, the self-peptides can be partly replaced by those that result from the degradation of the foreign products. These are the antigens that can be recognized by the TCR as specific. But a major consequence of this process and that usually gets unnoticed or is poorly discussed is that, differently from most other cellular receptors, the TCR can never become saturated with ligands and therefore does not respond necessarily in a linear fashion to variations in the concentration of the microbial contents inside the APC. Indeed, the digestion of a few microbial cells results in the distribution between MHC molecules of many different nonself peptides representative of the microbial

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proteome. However, only a few identical peptides are recognized by the specific TCRs. The increase in the amount of pathogenic material ingested by a professional APC (e.g., dendritic cell, macrophage) will not translate into a higher frequency of the TCR-specific peptides above a given threshold, though; after reaching a certain level, any additional amounts of the specific peptide will compete with all other microbial peptides and also the endogenous material for coupling with the cell’s MHC molecules. The overall result is that the maximum number of specific peptides available for a fixed number of identical TCRs is very limited and, therefore, the T lymphocyte must have ways to respond with great sensitivity to only a few peptides that are diluted within a multitude of non-specific self and nonself antigens, and still be able to mount a response. For bacterial, viral or cancer antigens, these exist at numbers typically bellow 100 copies of epitope per APC [1–3], presented to a T lymphocyte that can have in the order of 100,000 identical TCRs [4].

5.2 T Cell Activation 5.2.1 The TCR-Signaling and Regulatory Machinery 5.2.1.1

The Structure of the T Cell Receptor

The TCR is a heterodimer of clonotypic α and β chains [5, 6], or γ and δ in a small percentage of T lymphocytes [7]. All four polypeptides are derived from immunoglobulin superfamily (IgSF) genes rearranged through ontogeny and display one variable and one constant extracellular Ig-type domains. At the T cell surface, TCR heterodimers are found closely but non-covalently associated with the CD3γ, δ and ε chains, each containing a single Ig-like domain, and with a disulfide-linked ζ-family dimer (CD247). The stoichiometry and the spatial arrangement of the TCRαβ/CD3 complex is composed of eight type I integral membrane proteins organized in four sets of dimers, TCRαβ, CD3δε, CD3γε and ζ2 [8, 9] (Fig. 5.1). TCRγδ complexes are slightly different from those of TCRαβ as they usually contain a second CD3γε dimer instead of the CD3δε pair [10], and they recognize unprocessed antigens with no need of MHC presentation [11]. Assembly of the TCRαβ/CD3 complex takes place in the endoplasmic reticulum (ER) and is initiated by the association of the CD3 dimers with the CD3ω chain, a peptide that is displaced upon the association of the TCRβ and α chains [12, 13]. Complexes formed may display just one of the TCR chains, but only to complexes that contain the αβ dimer in addition to CD3δε-γε, do ζ chains bind. CD3 polypeptides contain sequences that target to lysosomal degradation or to ER retention which are masked at assembly of the complex, so while TCRαβ/CD3 devoid of ζ2 may reach the Golgi, only full intact TCRαβ/CD3δε-γε-ζ2 complexes (from here on referred to as the TCR complex) become expressed at the cell surface [14, 15]. With the exception of the TCRαβ chains that contain only a very short cytoplasmic domain, all the CD3 and CD247 subunits possess a conserved cytoplasmic motif

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Fig. 5.1 TCR-mediated activation. The TCR complex is a transmembrane multiprotein complex that includes eight different subunits: a heterodimer of TCR αβ chains (or γδ), four CD3 polypeptides (the dimers γε and δε), and two ζ chains (CD247). The N-terminal domains of the TCR are highly variable and contain the portion of the receptor responsible for antigen recognition. The remaining six subunits include in their cytoplasmic tail ITAM domains and are responsible for intracellular signal propagation. Antigen recognition by T cells can only take place when the antigenic peptide is presented by an antigen presenting cell (APC) on its surface through an MHC molecule (class I or II). When the TCR engages with an MHC-peptide complex, the T cell becomes activated. (1) Initially, CD4 in T helper cells (or CD8 in cytotoxic T cells, not shown) brings LCK closer to the TCR in order to phosphorylate the ITAM sequences of the complex. (2) ZAP70 is then recruited to doubly phosphorylated ITAMs and is, in turn, able to phosphorylate and recruit the adaptor molecule LAT. (3) At this point, LAT builds a platform for docking of many effector/adaptor proteins that activate different signaling pathways. One such protein is PLCG1 that cleaves PtdIns(4,5)P2 into InsP3 and DAG. InsP3 promotes the release of calcium from intracellular compartments and DAG contributes to the activation of PKC. GRB2 also associates directly with LAT, connecting the latter to SOS1, an important activator of RAS. The LAT signalosome further includes PIK3R and GRAP2 (which recruits LCP2), relevant in mobilizing other downstream effectors. These events ultimately lead to the nuclear translocation of transcription factors such as NFAT, NFKB and AP1 that promote transcription of activation-associated genes, such as in this case, IL2

consisting of the sequence YxxL/Ix(6-8) YxxL/I [16] (Fig. 5.1). This sequence, known as immunoreceptor tyrosine-based activation motif (ITAM), is common to other hematopoietic antigen receptor subunits such as the Igα and β chains (CD79) of the BCR and certain Fc receptors, such as the β and γ subunits of the FcεR1. Each TCR-associated ζ chain contains three such modules that are sufficient to mediate proximal and distal events of TCR-induced cell activation, but signaling can also proceed independently of the ζ chain motifs through the single ITAMs of the other CD3 subunits.

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The Co-receptors CD4 and CD8

CD4 is a single chain type I membrane glycoprotein, containing four extracellular IgSF domains, whereas CD8 consists of a disulfide-linked heterodimer (CD8αβ), although some cells express CD8 as an α chain homodimer. CD4 and CD8 are expressed on mutually exclusive populations of T lymphocytes, commonly referred to as helper and cytotoxic T cells, respectively, and help the TCR responding to antigen by interacting with non-polymorphic regions of MHC class II or class I molecules, respectively [17, 18] (Fig. 5.1). By virtue of binding to the same MHC/peptide complex on APCs, TCR complexes and CD4 or CD8 interact in the plane of the membrane, and the physical approximation of either co-receptor to the TCR complex results on the potentiation of the response. Coprecipitation of the TCR complex with CD4 or CD8 can only be seen when mild conditions of cell membrane disruption and sensitive detection methods are employed, suggesting that although CD4 or CD8 may not be tightly bound to the complex they must lie in the vicinity and can readily join when activation takes place [19].

5.2.1.3

SRC-Family Kinases LCK and FYN and SRC-Homology Domains

Lymphocyte-specific protein-tyrosine kinase (LCK) and FGR/YES novel protein (FYN) are members of the SRC family of protein tyrosine kinases (PTK) and, as such, share common features, namely a myristoylated glycine residue at position 2 and two palmitoylated cysteines residues (C3 and C5 of LCK; C3 and C6 of FYN) that enable the enzymes to associate with the plasma membrane, one SRC homology (SH) 2 and one SH3 domains, and a catalytic domain (SH1) located near the C terminus. The amino terminal region is unique to each kinase and contains sequences that may be responsible for specific interactions like that of LCK with CD4 or CD8 and FYN with the TCR complex. The association with CD4 and CD8 improves LCK targeting to the TCR complex vicinity to perform its main function, which is the phosphorylation of the conserved tyrosine residues of ITAMs. This results in the recruitment to the TCR complex of the 70 kDa ζ-associated protein (ZAP70), a PTK that contains two tandemly arranged SH2 domains specific for the double phosphorylated tyrosines of ITAMs [20] (Fig. 5.1). The function of FYN is apparently subtler as FYN already associates with the TCR complex. It has been suggested that FYN can be an activator of LCK, as inhibition of FYN expression markedly downregulates LCK activity [21]. Alternatively, it is possible that LCK and FYN drive the activation of different signaling cascades, with LCK being crucial in the main axis of activation that proceeds through the membrane-bound adaptor linker for activation of T cells (LAT) while FYN may signal mainly through a second adaptor, phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) [22]. SH2 domains are small compact spherical protein modules of about one hundred amino acids arranged to enclose a hydrophobic core, and that are characterized by

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the ability of binding to phosphotyrosine-containing proteins. Specificity of recognition of SH2 domains seems to be determined by the amino acids flanking the target tyrosine. For example, the SH2 domains of the regulatory subunit of phosphatidylinositol 3-kinases (PIK3R) bind with high affinity to a consensus sequence pYMxM, while the SH2 domain of SRC has a preference for the sequence pYEEI [23]. Some proteins have only one SH2 domain whereas others possess more, which, although homologous, may have some differences that are reflected in the different specificities for ligands. Proteins containing two such motifs may bind in tandem to the same receptor if this contains closely spaced phosphotyrosine residues or to two different receptors/adaptors; conversely, different phosphorylated tyrosine residues on the same molecule may bind to SH2 domains of different proteins. SH3 domains have a compact barrel-like shape, and the 50–75 amino acids that compose the domain form an exposed hydrophobic pocket where the ligand binds, while charged residues that also contact the ligand are contained in a loop that surrounds the hydrophobic region. SH3-binding proteins and peptides contain prolinerich sequences that form left-handed type II polyproline helices [24, 25]. Small differences within the proline rich sequence account for determination of specific interactions to different SH3 domains such that, for example, the SH3 domain of SRC binds to a consensus motif RxLPPLPR, whereas the SH3 domain of PIK3R binds to a RxLPPRPx motif. Although in general SH2 and SH3 domains define protein-protein binding specificities and therefore the re-localization of SH2/3-containing enzymes to mediate signal transduction, these domains can also autoregulate the activity of the enzymes. Like all SRC-family kinases, LCK and FYN contain two regulatory tyrosine residues that control their enzymatic activity depending on their phosphorylation status. An autophosphorylation site situated within the kinase domain is connoted with positive signaling, whereas a C-terminal tyrosine residue, Y505 in LCK and Y531 in FYN, is an inhibitory site. When phosphorylated, this C-terminal tyrosine residue interacts in cis with the kinase’s own SH2 domain, inducing a conformation that renders the enzyme inactive [26]. On the other hand, the SH3 domain of SRC-type kinases has a role in the regulation of the catalytic activity of the enzyme, helping to maintain the protein in the repressed configuration through intramolecular interactions. Phosphorylation of the negative regulatory tyrosine residues of LCK or FYN is catalyzed by the tyrosine kinase C-terminal SRC kinase (CSK) and results in a pY-SH2 bindingdependent inactive conformation and downmodulation of T cell responses [27]. On the contrary, dephosphorylation of this site by the tyrosine protein phosphatase CD45 sets the kinases in a primed state which can further develop into a full activation state once the regulatory site is phosphorylated [28–30].

5.2.1.4

Tyrosine Phosphatase CD45

CD45 is a transmembrane protein expressed on all leukocytes, that displays phosphotyrosine phosphatase activity in the cytoplasmic domain. It contains two tandem phosphatase domains, the first having catalytic activity while the second is relevant

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for substrate binding, for example to the ζ chains of the TCR/CD3 complex [31, 32]. CD45 dephosphorylates the negative regulatory C-terminal phosphotyrosine residues of LCK and FYN with relative ease and thus positively regulates the activity of the kinases. However, also the regulatory tyrosine of SRC kinases is targeted by CD45, albeit at lower efficiency. Accordingly, increases in CD45 expression, such as those observed in peripheral T cells comparing with developing thymocytes, have more impact in the dephosphorylation of the regulatory SRC tyrosines and suggest CD45 to be a rheostat controlling through its level of expression the intensity of signal transduction [33]. Other mechanisms of CD45 regulation of T cell activation have been suggested, such as a possible dimerization of the molecule and the appearance of alternative splicing-dependent isoforms. Various CD45 isoforms can be generated by alternative splicing of three exons at the 5 end of the coding sequence, and be differently expressed in different cell types and depending on the state of development and cellular activation. Although the different isoforms have been shown to associate with diverse T cell surface molecules [34], no correlation with differential T cell signaling has been observed. Also, the putative dimerization of CD45 seems not to impact on T cell activation [33]. An alternative suggestion is that the varying amounts of CD45 together with the different overall glycosylation content between the different isoforms may play a role in the distribution of CD45 during immunological synapse formation and thus selectively interact with the target enzymes [35].

5.2.2 Triggering of the TCR and the Immunological Synapse The exact mechanism of TCR triggering is still not fully understood. Analysis of several TCRs interacting with specific peptide-MHC complexes (pMHC) reveals little or no changes in the structure of the extracellular part of the TCR upon binding, suggesting that if there are any conformational changes these should be at the level of the whole complex or its interaction with the signaling machinery. Models have been proposed based on physical forces established between the TCR and MHC in the context of dynamic membrane movements, such as the piston-like model [36] and the receptor deformation model [37]; other models suggest changes at the level of the CD3 architecture, like the detachment of CD247 chains, embedded in the inner leaflet of the plasma membrane, from the membrane and consequent exposure of their ITAMs to phosphorylation [38], or the recruitment to proline-rich sequences of CD3ε of the non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1) that can then couple to additional signaling molecules [39]. Many other models explore the possible dimerization or oligomerization of MHC molecules at the APC surface in a manner to induce aggregation of the TCRs, like the permissive geometry model [40]; however, it is rather improbable that adjacent oligomerized MHC molecules would present identical peptides to induce the aggregation of responding TCRs. Furthermore, the monomeric/oligomeric state of the TCR itself has been the subject of many intense debates, but using the most sophisticated technologies the further

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that the field has advanced is to suggest that the TCR complex exists in monomeric units at the surface of T cells at the resting state [41–43]. In the particular case of lymphocyte activation, it can be considered that the agonist that promotes cell division is not a single molecule, like a hormone or growth factor binding to its receptor, but instead it is the whole interacting surface of another cell, that which presents the antigen. Therefore, the conditions of optimal T cell activation are constrained by the interface established between the membranes of the two cells, which is called the immunological synapse. Cellular adhesion is stabilized by the interaction of large-sized integrins that bind to their ligands on the opposing cell, forming an outer circle termed peripheral supramolecular activation cluster (pSMAC) that encircles an inner contact region, the central SMAC (cSMAC), composed of the TCR complexes, co-receptors, adhesion molecules like CD2 and costimulators such as CD28, binding to their respective ligands expressed at the APC surface [44]. In the cSMAC, the distance between the apposing membranes is ~15 nm, optimally suited for the small TCR complexes to scan the multitude of MHC molecules presenting different antigenic peptides. Such restricted environment is also suggested to be crucial for signaling initiation and propagations because it physically excludes the large phosphatases like CD45 and CD148 from the TCR complex. This segregation of the phosphatases to a distal SMAC region (dSMAC) allows kinase-mediated phosphorylation to prevail over the counteracting dephosphorylation, as suggested by the kinase segregation model [45] and the diffusion trapping model [46]. These models may also accommodate the suggestion of serial triggering of several TCR complexes by the same pMHC complexes that, due to the low affinity of binding, can dissociate from one TCR complex and bind and activate the next one, thus possibly explaining how a few specific peptides coupled to MHC complexes can give rise to robust T cell activation [47]. These conceptual designs combined to the more mechanistic heterodimerization model, where the receptor dimerization is the actual coupling of CD4/CD8-LCK to the TCR complex [48], or to the pseudodimerization model, in which CD4 or CD8 binding to the MHC induces the approximation of LCK to an adjacent TCR complex [49], illustrate yet additional ways to explain the extreme sensitivity of the TCR to promote signaling upon identifying the rare specific pMHC complexes. Such sensitivity is well substantiated by the observation that 10 pMHCs at the APC surface are sufficient to elicit strong calcium signaling whereas in the absence of a co-receptor the number of pMHCs required to give rise to identical responses is approximately fourfold greater [49].

5.2.3 Signal Transduction in T Lymphocytes 5.2.3.1

Signal Initiation

MHC/peptide recognition by the appropriate TCR is central to the process of T cell activation, but given the very low affinity of the interaction, cell to cell contact is initiated and temporarily stabilized by adhesion molecules [50]. Integrins like

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integrin alpha-L (ITGAL), binding to intercellular adhesion molecule 1 (ICAM1) and helping to form the pSMAC, and cSMAC-localized small adhesion molecules such as CD2 that binds to CD58, define the area of pMHC scanning and provide time for the TCRs to survey the presented complexes. Upon engagement of the TCR and the approximation of the CD4/8 co-receptors to pMHC, phosphorylation of CD3/CD247 chains on the two conserved tyrosine residues in each ITAM is catalyzed by LCK and recruits ZAP70 [51] (Fig. 5.1). ITAM sequences in the CD3/CD247 subunits seem to be tailor-made for the simultaneous binding of the tandemly arranged SH2 domains of ZAP70 [52], and even if only a fraction of the 10 ITAMs present in each individual TCR complex is occupied by ZAP70 molecules, this should nevertheless represent a concentration of tyrosine kinases superior in number to that found in the dimerization of conventional RTKs. Concomitantly with the binding to the ITAM sequences, ZAP70 is phosphorylated by LCK and also possibly through autophosphorylation, on two tyrosine residues of its activation loop and gains an open conformation and acquires an active state [53]. Activated ZAP70 phosphorylates tyrosine residues on downstream substrates, including the adaptor LAT [54]. Spleen tyrosine kinase (SYK), a kinase structurally homologous to ZAP70 and that plays an important role in the activation of B lymphocytes, may also be involved in the initial stages of T cell signaling but its expression is markedly down-regulated in peripheral T cells when compared with thymocytes, suggesting that possibly SYK plays a role in thymic development [55]. In contrast, the expression of ZAP70 seems crucial to normal T cell functioning as human patients that lack the kinase show severe combined immunodeficiency due to the absence of CD8+ T cells, while CD4+ T lymphocytes respond poorly to activation [56, 57]. TCR triggering and ZAP70 docking is initiated in an outermost ring of the forming immunological synapse, where the TCR complex, CD4 and LAT are expressed in distinct microclusters in the plasma membrane. Upon T cell triggering, the distinct microdomains coalesce, or LAT is recruited to the TCR domains, and the hotspots of activation containing the TCR complex, LCK, ZAP70 and LAT, are sustained in this peripheral area of the synapse [58–60]. The TCR complexes are then transported centripetally to the center of the synapse where the TCR is endocytosed and recycled.

5.2.3.2

Assembly of the LAT Signalosome, Signal Branching and Transcriptional Activation

TCR-mediated T cell activation translates into many different outcomes depending on the sub-type or development stage of the T cell and on the nature of the stimulus and the APC, but conventionally for a naïve T cell being presented with an antigenic peptide, a major result is the induction of cell division and expansion of that specific T cell clone. Accordingly, the signaling pathways activated downstream of the TCR complex will ultimately drive the activation of transcription factors that lead to the production of growth factors, amongst them the cytokine interleukin 2 (IL2) that promotes T cell proliferation. The promoter region of the IL2 gene spans 300 nucleotides upstream of the transcription initiation site and is packed with consensus motifs for

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the binding of transcription factors such as the activator protein 1 (AP1) composed of the subunits FOS and JUN, the nuclear factor of activated T-cells (NFAT) and the nuclear factor κB (NFKB), among others. Many of these transcription factors are activated precisely by signaling cascades that originate at the signalosome assembled by LAT. Directly phosphorylated by ZAP70, the nine tyrosine residues of LAT become docking sites for SH2 domain-containing enzymes and cytosolic adaptors, including phospholipase C-γ1 (PLCG1), PIK3R and GRB2, among others [54]. PIK3R couples with the catalytic subunit of the enzyme, PIK3C, a lipid kinase which phosphorylates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] at the inner leaflet of the plasma membrane to generate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3 ] [61]. The phosphorylated D3 position of the inositol ring becomes a docking site for enzymes containing pleckstrin homology (PH) domains, including PLCG1, the TEC-family IL2-inducible T-cell tyrosine kinase (ITK) and the serine/threonine-protein kinase AKT1. One other enzyme that targets PtdIns(4,5)P2 is PLCG1, but the resulting outcome is completely different. Once bound through its SH2 domain to LAT and via the PH domain to the membrane, PLCG1 is activated by phosphorylation and catalyzes the hydrolysis of PtdIns(4,5)P2 to give rise to the second messengers inositol 1,4,5trisphosphate (InsP3 ) and 1,2-diacylglycerol (DAG) [62]. While DAG diffuses in the membrane where it has an important role in the activation of the Ser/Thr protein kinase C (PKC) [63], InsP3 functions by promoting the increase of the cytoplasmic concentration of free calcium, released from the ER and other intracellular stores [64]. The sensing of decreased ER Ca2+ concentration is then signaled to plasma membrane channels such as the calcium release-activated calcium channel protein 1 (ORAI1), allowing for a greater influx of calcium into the cell [65]. One of the most important roles of calcium in T cell signaling is the activation of the Ca2+ /calmodulin-regulated serine/threonine phosphatase calcineurin, which is involved in the translocation and activation of NFAT. Dephosphorylation of cytosolic NFAT by calcineurin unmasks nuclear localization signals and results in the nuclear import of the transcription factor, which then cooperatively associates with AP1 [66]. Meanwhile, the T cell-specific PKC isoform θ (PRKCQ) is recruited to the plasma membrane at the immunological synapse where it associates with the increasingly available DAG. PRKCQ phosphorylates on serine residues the caspase recruitment domain-containing protein 11 (CARD11) that assembles with the B-cell lymphoma/leukemia 10 (BCL10), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and the TNF receptor-associated factor 6 (TRAF6), the CBM signalosome [67, 68]. This signaling complex activates the serine protein kinase inhibitor of nuclear factor κB kinase α (IKKA), which phosphorylates the NFKB inhibitor IκB (NFKBI), targeting this molecule to ubiquitination and proteasomal degradation. In non-activated cells, NFKB associates with and is suppressed by NFKBI, and the disassembly of this complex allows the translocation of active NFKB to the nucleus [69]. Also, docking to multiple tyrosine phosphorylated sites of LAT, the small adaptors GRB2 and GRB2-related adapter protein 2 (GRAP2) amplify and diversify the

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signals. Both adaptors consist basically of one SH2 and two SH3 domains, with GRAP2 connecting via the SH2 and one SH3 domain the cytosolic adaptor lymphocyte cytosolic protein 2 (LCP2) to LAT [70]. LCP2 helps to build with LAT the precise spatial orientation for the signalosome to accommodate correctly upand downstream effectors. Through its own phosphorylated tyrosines, LCP2 binds the adaptor NCK1, the guanine nucleotide exchange factor VAV and ITK, the latter having a crucial role in activating PLCG1 [71]. LCP2 also interconnects via specific sequences the SH3 domain of PLCG1 and the SH2 and SH3 domains of ITK, while its own SH2 domain binds to a phosphorylated tyrosine residue of yet another adaptor, FYN-binding protein 1 (FYB1), which links TCR signaling to the actin cytoskeleton [72]. In parallel, GRB2 is responsible for the integration within the LAT signalosome of the guanine nucleotide exchange factor son of sevenless homolog 1 (SOS1) [54, 73]. SOS1 catalyzes the release of GDP from the small GTPase RAS, of which the predominant isoform in T cells is NRAS, and makes way for GTP to bind and activate RAS [74]. In T cells, another guanine nucleotide exchange factor, RAS guanyl-releasing protein 1 (RASGRP1), precedes the activity of SOS1 [75]. RASGRP1 contains a DAG-binding domain and targets to the plasma membrane following the activation of PLCG1, and the cooperativity between RASGRP1 and SOS1 strengthens signal transduction via RAS. The activity of RAS ultimately depends not only on the stimulation of its activators but also on overcoming the effects of its inhibitors, such as the RAS GTPase-activating protein 1 (RASA1) that inactivates RAS through the hydrolysis of RAS-bound GTP [76]. The role of RAS in the induction of the serine/threonineprotein kinase RAF1 seems to be mainly the localization of RAF1 to the plasma membrane where it may interact with effector molecules as well as with putative activators. Membrane-localized RAF1 activates the dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) [77], which in turn activates the serine/threonine mitogen-activated protein kinases 1 and 3, also known respectively as extracellular signal-regulated protein kinases (ERK) 2 and 1 [78]. Substrates of these kinases include the transcription factors ELK1, and the components of the AP1 transcription complex, JUN and FOS. Apart from the AP1, NFAT and NFKB consensus binding sequences, the IL2 promoter contains binding sites for multiple additional transcriptional factors, including early growth response protein 1 (EGR1), POU domain, class 2, transcription factor 1 (POU2F1) and DNA-binding protein Ikaros family zinc finger protein 1 (IKZF1), among others, activated through additional or concurrent signaling pathways [79]. Evidence suggests that every known responsive element has to be occupied by the specific factor to give rise to full activation, as deletion of any of the motifs greatly impairs IL2 gene transcription. For example, a dominant negative of NFAT blocks activation of the promoter, whereas transcription is only initiated when the NFKB site is occupied [80–82]. The large number of transcription factors taking part in the activation of genes such as the IL2 gene and the lack of redundancy observed illustrates the complexity of the mechanisms involved to integrate signals from different sources received at the cell surface and to translate them into a concerted action to produce important mediators in the later stages of T cell activation.

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5.2.4 Costimulation Stimulation of T lymphocytes through the TCR complex alone in the absence of costimulatory signals induces unresponsiveness, or anergy, towards antigen. This situation can be avoided by the inclusion, during the initial stages of T cell activation, of accessory cells that express ligands for costimulatory receptors of T cells [83]. One major costimulator expressed by T cells is CD28, a type I transmembrane glycoprotein expressed as a disulfide-linked homodimer. CD28 is a member of the IgSF as are its physiological ligands CD80 and CD86, expressed on activated B and T cells, dendritic cells and other APCs. CD28 autonomous signaling can be maintained in cells lacking TCR expression and it is rather difficult to distinguish the molecular components or types of signal that are differentially activated at the onset of activation between TCR- and CD28-mediated pathways [84]. In fact, it has been found that signaling mediators such as LCP2, GRAP2, GRB2 and PIK3R, among others, bind to phosphorylated tyrosine residues of the cytoplasmic tail of CD28, and that ITK, LCK, GRAP2 and GRB2 interact via their SH3 domains with proline sequences of CD28. However, CD28-mediated signal transduction is resistant to inhibitors of the PKC and calcium pathways, whereas signaling proceeding from stimulation of the TCR complex is not [85, 86]. A major event occurring following binding to its ligand and CD28 becoming phosphorylated on tyrosine residues is the docking of PIK3R via SH2 domains [87, 88]. As mentioned above, the catalytic domain PIK3C phosphorylates PtdIns(4,5)P2 to PtdIns(3,4,5)P3 , to which bind PH domains of ITK, AKT, and also of 3phosphoinositide-dependent protein kinase 1 (PDPK1), a direct activator of AKT. AKT, regulates several pathways that enhance NFKB and NFAT translocation. On the other hand, CD28-bound ITK is instrumental in the activation of PLCG1 and calcium signaling, and consequently on NFAT translocation and activation. It has been thoroughly discussed whether a major focus of CD28 signaling would be the complementation of the pathways leading to AP1 assembly. However, like TCRmediated signaling, CD28 is also directly involved in the activation of both JUN and FOS subunits [89, 90]. Also, much has been speculated on whether costimulation provided by the CD28 antigen simply amplifies the strength of the signals elicited by the TCR complex or whether there is a qualitative difference in the signals. It is now clear that CD28 significantly augments TCR-mediated responses by using similar signaling components, but there are unique epigenetic, transcriptional, and post-translational changes induced in T cells upon CD28-mediated activation that are distinctive from those transmitted by the TCR complex [79, 91, 92]. These include active roles in decreasing cytosine methylation, inducing histone acetylation and chromatin remodeling [93–95], increase of alternative splicing of many transcripts [96], and the stabilization of IL2 mRNA levels and increased IL2 production [97, 98].

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5.2.5 Signaling Modulation and Inhibitory Checkpoints Productive T cell activation results from the convergence of multiple signaling pathways that are initiated at the TCR complex but the potency of signaling is also already controlled and modulated starting at the onset of activation. At multiple stages, there are mechanisms that counter balance the activation stimuli and restrain the amplitude of the signals, preventing exacerbated responses that could lead to autoimmunity. A first level of regulation is mediated by CD45 that controls the levels of phosphorylation of both the inhibitory and regulatory tyrosine residues of SRC-type kinases. An also immediate regulatory phase is dependent on constitutive inhibitory surface receptors, such as scavenger receptor cysteine-rich (SRCR) family proteins CD5 and CD6 [99, 100], whose tyrosine residues of the respective cytoplasmic tails are phosphorylated coincidentally with those of the TCR complex chains and by the same SRC kinases [101, 102]. But in contrast, many of these phosphorylated tyrosine motifs of inhibitory receptors are sites for the docking of repressor enzymes like protein tyrosine phosphatase non-receptor type 6 (PTPN6) and protein tyrosine phosphatase non-receptor type 11 (PTPN11), also known as SHP1 and SHP2, respectively, that dephosphorylate a plethora of targets at the TCR signalosome including the active site of LCK; RASA1, which hydrolyses GTP from active RAS; and Casitas B-lineage lymphoma proto-oncogene (CBL) and CBLB, ubiquitin ligases that promote ubiquitination and degradation of several signaling components [103, 104]. An additional layer of inhibitory mechanisms seems to utilize the integral membrane adaptor PAG1. Contrary to LAT that has a diversity of phosphotyrosine-dependent ligands, PAG1 has multiple tyrosine motifs to which the main binding enzyme is FYN. This kinase is responsible for the phosphorylation of a specific residue of PAG1, Y317, which constitutes the docking site for CSK, the PTK that phosphorylates the inhibitory site of LCK and FYN inactivating these enzymes [105]. Also, the CD28 costimulator is a crucial target for regulation. CD28 activation is induced primarily through binding to its ligands, predominantly CD86 but also CD80, but this binding specificity is also shared by the CD28-homologous cytotoxic T-lymphocyte protein 4 (CTLA4). CTLA4 is essentially an inhibitory receptor whose expression is induced upon activation. Given its higher affinity than CD28 for binding to the common ligands, CTLA4 displaces CD28, and its repressor activity overcomes the activation signals transmitted by CD28 [106]. CTLA4 is also able to reverse stop signals that an antigen-recognizing T cell receives upon binding to APCs, promoting T cell motility and thus overriding TCR-mediated activation [107]. CTLA4 is one of several checkpoint inhibitors, which also include programmed cell death protein 1 (PD1, PDCD1), T cell immunoreceptor with Ig and ITIM domains (TIGIT), lymphocyte-activation gene 3 (LAG3), and B- and T-lymphocyte attenuator (BTLA), that are strongly induced 1-2 days after TCR triggering and halt the progression or terminate cell activation [108–111]. The modus operandi of many of these inhibitors is similar to that of CTLA4 as they compete with co-stimulatory receptors for the same ligands [112, 113], and become phosphorylated in tyrosine residues of immunoreceptor tyrosine-based inhibitory motifs (ITIMs). These sequences consist

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of a single tyrosine residue within a poorly defined consensus of S/I/V/LxYxxI/V/L, but given the difficulty in accurately defining ITIMs in genome databases [114, 115], it was established that it is their inhibitory role that determines their inclusion in the group. For the checkpoint inhibitors, as well as for most inhibitory receptors, ITIMs are bound by SH2 domain-containing protein phosphatases such as PTPN6 and PTPN11, as well as lipid phosphatases like the SH2 domain-containing inositol polyphosphate-5-phosphatase D (INPP5D), that decrease cell activation [116–120].

5.3 B Cell Activation 5.3.1 The BCR-Signaling and Regulatory Machinery 5.3.1.1

The Structure of the B Cell Receptor

The BCR has many similarities with the TCR, being also a transmembrane multichain protein complex encompassing a variable region responsible for antigenspecific binding, and a set of invariant modules that propagate activation signals (Fig. 5.2). The variable part of the BCR is contained in the extracellular part of the membrane immunoglobulin (mIg), which consists of two heavy and two light chains interconnected by disulfide bridges [121]. Like the TCR, the BCR acquires its antigen specificity through recombination of gene segments that encode the amino terminal part of both chains. Genetic recombination takes place during development and results in the ability of a single cell, expressing many copies of the same BCR unit, to have optimal recognition of unique antigens [122]. The signaling part of the BCR complex consists of a heterodimer of Igα and Igβ chains (CD79A and B, respectively), bound together by a disulfide bridge. Each of these subunits contains a single ITAM that when phosphorylated is coupled by the ZAP70-related kinase SYK [123]. Assembly of the BCR takes place in the ER, with a first stage of assembly of the mIg tetramers, but the multiprotein complex only exits the organelle after the addition of the remaining subunits, CD79B being added first followed suit by coupling of CD79A, which is synthesized in smaller amounts. The disulfide bridge is then formed and, at this point, the complete assembled complex leaves the reticulum and is transported to the cell surface [124, 125].

5.3.1.2

The B Cell Co-receptor Complex and Accessory Effectors

The B cell co-receptor complex includes the IgSF transmembrane protein CD19, the tetraspanin CD81, the complement C3d receptor CD21, and CD225, a poorly characterized transmembrane protein belonging to the dispanin family. Within the complex, CD19 serves the purpose of signal propagation and diversification, as upon BCR engagement the cytoplasmic tail of CD19 becomes phosphorylated allowing

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Fig. 5.2 BCR-mediated activation. The BCR is the multiprotein transmembrane complex involved in the recognition of antigens by the B cell—whether soluble or membrane-associated. The BCR includes a membrane-bound immunoglobulin (mIg) and the Igαβ heterodimer (CD79). The mIg has a similar structure of circulating antibodies, two heavy and two light chains, but it contains a membrane insertion sequence for cell surface expression. The variable region of the mIg is responsible for antigen specificity, while the constant region is immutable. CD79 contains one ITAM in each Ig subunit, coupling antigen recognition to intracellular signal propagation. (1) Antigen recognition by the BCR triggers a signaling cascade that initiates with ITAM phosphorylation by LYN. This in turn recruits SYK to the doubly phosphorylated ITAMs (2), which will allow the activation of BLNK and its binding to CD79A directly. BLNK is responsible for anchoring a number of other effector/adaptor proteins like PLCG2 and GRB2 in order to activate different signaling pathways. BTK also binds BLNK and further activates PLCG2. The activation of these different signaling pathways promotes the translocation of different transcription factors to the nucleus, acting to enhance the transcription of genes related to B cell activation, leading either to affinity maturation of Ig in germinal centers, or to Ig secretion

its interaction with a series of signaling effectors, including LCK/YES-related novel protein tyrosine kinase (LYN), the most predominant SRC-family kinase of B cells, and PIK3, VAV, PLCG2 and GRB2 [126–129]. CD81 facilitates adhesion and determines localization within the membrane and CD21 is involved in the recognition of complement antigens through C3d [130–132]. The role of CD225 is not well established, possibly having the function of a tight junction protein in the complex [133]. The B cell co-receptor complex becomes associated with the BCR through common recognition of complement antigens. This simultaneous binding that occurs at the surface of the B cell lowers the threshold for B cell activation, facilitating an immune response [131, 134]. CD19 is, however, able to directly associate with the BCR and exert its stimulatory role without being a part the aforementioned complex [135–137]. Other proteins can also potentiate B cell activation by a specific antigen. One of such proteins, the transmembrane receptor CD40 which belongs to the tumor necrosis factor receptor (TNFR) family, is able to bind to T cell-expressed CD40 ligand (CD40LG) [138], and this results in the phosphorylation on the B cell side of LYN, PIK3 and PLCG2, activation of NFKB and, ultimately, contributes to increased survival and proliferation of B cells [139–141]. In parallel to these activating modules, other molecules work to restrain B cell activation. Amongst them is CD22 [142], a transmembrane protein that belongs

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to the sialic acid binding Ig-like lectin (SIGLEC) family and binds CD45 amidst other sialylated glycoproteins. CD22 contains three ITIMs in its cytoplasmic tail that once phosphorylated recruit PTPN6. However, CD22 also transduces inhibitory signals independent of PTPN6/ITIM [143–145]. Fittingly, B cells deficient for CD22 present increased reactivity [142]. LYN has a dual role in regulating B cell activation as apart from phosphorylating CD19 and the ITAMs of CD79 to induce activation, it is also the kinase responsible for CD22 phosphorylation and signal repression. One other well-characterized inhibitor of B cell activation is FcGRIIB (FCGR2B, CD32), a low affinity receptor for IgG that is also capable of restraining signal progression following ITIM phosphorylation and recruitment of secondary inhibitory phosphatases, which in turn dephosphorylate, among other targets, CD19 [146, 147].

5.3.2 Signal Transduction in B Lymphocytes 5.3.2.1

BCR-Proximal Signaling

The earliest event upon BCR engagement is the activation of LYN that phosphorylates the CD79 ITAMs, counting with the contribution of BCR complex-loosely associated and co-receptor-bound FYN, LCK, SYK and additionally BTK, a kinase that belongs to the TEC family of protein kinases [123, 148, 149] (Fig. 5.2). LYN is basally coupled to the BCR through the N-terminus but optimal activation of the kinase requires SH2-mediated binding to the BCR ITAMs, further enhancing their phosphorylation [150, 151]. The phosphorylated ITAMs are then tightly bound by SYK, crucial for the initiation of the signaling cascades. Considering the absence of an equivalent to the T cell CD4/CD8 co-receptors in B lymphocytes, the PTKs find different strategies to bind and activate the BCR. Several models have been put forward to address this question but no definite consensus has yet been achieved. One hypothesis proposes that upon engagement but prior to any phosphorylation events, the BCR complex translocates into lipid rafts, membrane microdomains enriched in cholesterol and sphingomyelin. Given that these regions are also enriched in LYN, this translocation will itself increase the propensity for the BCR to become phosphorylated and therefore to activate downstream signaling effectors [152]. One other model considers that the activity of basally bound kinases increases when several BCR units become aggregated after ligand binding, or that ligand binding induces conformational changes in the BCR that in turn activate proximal kinases [153]. A third theory is based on the exclusion of larger molecules on account of their size. Despite being originally described for T cell activation, the kinetic segregation model also accommodates the initiation of B cell activation, suggesting that when an APC engages in contact with the B cell the narrow space between the two membranes excludes large-sized phosphatases from the vicinity of the BCR, altering the balance of basal phosphorylation of the receptor [154, 155].

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The expression of CD45 is not as critical for signaling in B cells as it is for T cells, given that CD45 knockout mice display fewer defects of the developing B lymphocytes comparing with those of thymocytes. Nevertheless, the signaling attributes of CD45 are similar in both TCR- and BCR-dependent pathways [156, 157]. FYN was shown to only become phosphorylated in CD45-expressing cells and though BTK displayed some level of phosphorylation in CD45-deficient cells, it was considerably decreased [158]. Also, LYN presented high basal phosphorylation in the absence of CD45 but this was due to the targeting of the inhibitory tyrosine (Y508) that locked LYN in its inactive conformation, while also preventing its recruitment to the BCR. However, phosphorylation of SYK and of the ITAMs in the constant subunits of the BCR could still take place in these cells, indicating some level of signal propagation in CD45-deficient B cells [159]. Moreover, impairments in calcium mobilization following antigen-receptor stimulation seem to be less drastic in B than in T cells deficient for CD45 expression [160, 161]. The identification of the transmembrane protein tyrosine phosphatase CD148 came to shed some light on the different susceptibilities presented by T and B cells in the absence of CD45. Despite being expressed in both types of lymphocyte, CD148 appears to have a more relevant role in B cells. CD45 and CD148 were found to have slightly overlapping roles in controlling the phosphorylation status and activity of SRC-family kinases in B cells. However, considering that the proteins have important dissimilar structures, they may have non-overlapping roles that have not been fully uncovered yet [162].

5.3.2.2

Integrative View of the BCR-Mediated Signaling Pathways

Similarly to T cell models, B cell activation also relies on the recognition of one particular antigen. The affinity of the interaction between the BCR and its ligand is however much higher than the corresponding of the TCR. When the BCR is triggered by antigen, cell spreading occurs over the presenting cell in order to maximize antigen engagement, which is followed by a contraction phase in an effort to increase clustering and enhance signaling [163]. This binding leads to the aggregation of various BCR subunits, which in turn results in the activation of PTKs. The aggregation of BCR subunits has been confirmed to take place prior to further downstream signaling events [136] but, as mentioned previously, how BCR triggering results in PTK activation is still a matter of debate in the field. BCR ITAMs are rarely doubly phosphorylated in the initial stages of activation. In this situation, SYK binds only weakly and this precludes signal propagation [164]. Complete phosphorylation of the ITAMs is thus necessary for SYK coupling and full activation which is followed by the kinase-mediated phosphorylation of several substrates, including the B-cell linker protein (BLNK, SLP65), an adaptor with a similar function of its T cell-expressed paralogue LCP2 (Fig. 5.2). BLNK binds directly to CD79A and after being phosphorylated by SYK becomes a binding anchor for PLCG2, VAV, GBR2 and NCK1 [165]. PLCG2 is phosphorylated and activated by SYK and BTK and hydrolyzes PtdIns(4,5)P2 to generate InsP3 and DAG and

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consequent downstream signaling, including activation of the transcription factors NFAT and NFKB that participate in B cell proliferation [166]. Similarly to its family member ITK in T cells, BTK membrane targeting relies on its PH domain binding to PtdIns(3,4,5)P3 , generated by PIK3, which is itself recruited by associating with LYN-phosphorylated CD19 [148, 167]. PIK3 inhibition precludes normal BTK phosphorylation [127]. The activation pathways originating from the BCR-proximal multi-protein complex are very similar to those that are induced at the TCR/LAT/LCP2 signalosome and result in the activation of equivalent transcription factors. Yet, despite the many parallels observed between B and T cell signaling cascades, albeit utilizing cell specific isoenzymes—for example, PLCG2 in B cells [168] instead of T cell-expressed PLCG1 leading to NFAT activation; PKCβ (PRKCB) in B cells [169] and PKCθ (PRKCQ) in T cells controlling the activation of the CBM signalosome and NFKB nuclear translocation; and RASGRP3 in B cells [170], RASGRP1 in T cells, to activate RAS and the MAPK pathway steering downstream transcription activation—there are often important dissimilarities in the architecture of the assembled multi-protein complexes. For example, a functional difference exists between T cellexpressed LAT and the structurally closest adaptor in B cells, non-T cell activation linker (NTAL, LAT2). Although LAT2 contains 10 phosphorylatable tyrosine residues of which 5 encompass GRB2-binding sites, it does neither bind PLCG2 nor PIK3 [171, 172]. On the other hand, LAT2 binds CBL, which is connoted with protein degradation and signaling inhibition [171]. In the same direction, GRB2 in B cells has been described as inhibitory in particular contexts, once its overexpression results in decreased calcium mobilization upon BCR activation [173], and it has been shown to physically associate with the negative regulator of B cell activation CD22 forming a quaternary complex that also includes INPP5D and the adaptor SHC [174]. The outcome of B cell activation depends not only on the identification of the antigen and the affinity of BCR binding but ultimately on the plethora of B cell surface proteins that may amplify or differentiate intracellular signals generated upon binding to their respective ligands of the APC. The multiplicity of glycoproteins that decorate the surface of lymphocytes reflect the developmental or maturation stages of the cell, which will engage with a diversity of APCs depending on the microenvironment or the context of the immune response. The same holds true for the TCR. Consequently, the final result of activation is thus dictated by the combinatorial signaling induced by the BCR but also by accessory surface molecules that leads to a complex network of transcription factors which ultimately modify the transcription profile and decide the fate of the cell. Upon recognition of antigen, B cells proliferate rapidly and may then differentiate into long-lived memory B cells or into antibody-secreting plasma cells. This decision is made in germinal centers of secondary lymphoid organs, and the upregulation of transcription factors such as PR domain zinc finger protein 1 (PRDM1, BLIMP1) induced upon BCR triggering via PIK3 but possibly with the contribution of signals from CD40LG and IL21 [175, 176], and X-box-binding protein 1 (XBP1) which is activated by a mammalian mechanism known as unfolded protein response that

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senses the accumulation of unfolded proteins in the ER lumen [177], leads to a transcriptional program that arrests the cell cycle and promotes Ig secretion [178, 179]. By opposition, the upregulation of the transcriptional repressor B-cell lymphoma 6 protein (BCL6), itself induced by interferon regulatory factor 8 (IRF8), is necessary to induce somatic hypermutation of the Ig variable sequences optimizing the affinity of the BCR to antigens, and class-switch recombination [180, 181]. Furthermore, BCL6 represses PRDM1, among other targets, helping to drive the polarization of the B cell into a BCR high-affinity non-antibody secreting B cell [182].

5.4 Final Remarks The signaling cascades that immediately follow receptor triggering are quite similar in T and B lymphocytes. However, important aspects set these cell types slightly apart, namely pre- and post-signaling events that can be noticeably distinct. T cells only recognize their respective antigen if it is presented in complex with an MHC molecule on the surface of particular cell types. For CD8-expressing T cells, antigen presentation is carried out by MHC class I proteins, which are expressed by all nucleated cells in the organism, whereas to CD4-expressing T cells antigen is presented by MHC class II molecules, upregulated on the surface of professional pre-activated APCs such as dendritic cells, macrophages and B cells. By contrast, engagement of the BCR does not require antigen cleavage given that these cells are able to identify a specific antigen in its native form. Moreover, B cells do not actually need the third party molecule that is the MHC, to facilitate this interaction. Notwithstanding these and other differences, inherent to the fact that T and B cells are functionally different sets of lymphocytes, it is remarkable the similarity found between the signaling machineries and the strict regulatory mechanisms and checkpoints that lymphocytes undergo during their activation in response to antigens to assure the mounting and orchestration of effective immune responses while avoiding the development of autoimmunity. Acknowledgments This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-032296 (PTDC/MED-IMU/32296/2017). SNH is recipient of a studentship SFRH/BD/133312/2017 from FCT.

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Chapter 6

Signaling Pathways Involved in Kidney and Urinary Tract Physiology and Pathology João Lobo and Rui Henrique

Abstract The kidneys are responsible for maintaining homeostasis, keeping the internal milieu in conditions that sustain life. A number of signaling pathways are implicated in all these feedback loops of regulation, and their disruption or overactivation might contribute to several diseases culminating in kidney failure and may, also, lead to kidney cancer. Because specific signaling cascades are also activated in the various histological subtypes of renal cell carcinoma, type-specific tailoring of targeted therapies is desirable. The urinary tract functions as a conduit that drives urine produced in the kidneys as a means of detoxification, for storage in the bladder and finally excretion by the urethra. This micturition cycle is highly coordinated, with prominent influence from the nervous system and related signaling pathways. Changes in these receptors and mediators disrupt the cycle and produce lower urinary tract symptoms which aggravate patients’ quality of life. Also, the urothelium covering the upper and lower urinary tract is not the same, hence signaling mechanisms involved in bladder and upper tract urothelial carcinoma are necessarily different. In this chapter we present and discuss some of the most relevant signaling cascades involved in kidney and urinary tract physiology and pathology, including non-neoplastic diseases and cancer. Keywords Bladder · Cancer · Kidney · Physiology · Signaling pathways · Urinary system J. Lobo · R. Henrique (B) Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal e-mail: [email protected]; [email protected] J. Lobo e-mail: [email protected] Cancer Biology and Epigenetics Group, Research Center of Portuguese Oncology Institute of Porto (GEBC CI-IPOP) and Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto (ICBAS-UP), Rua Jorge Viterbo Ferreira 228, 4050-513 Porto, Portugal © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_6

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Abbreviations α-AR AC Ach ADPKD AKT/PKB ANP APC AR ATP β-AR b-FGF BlCa BPH BUC CAIX cAMP ccRCC CDH1 CDKN2A cGMP chRCC CK1 CKD COX CTLA-4 DAG DHT DKD EGF EGFR EMT EpCAM ER ET FGF FGFR3 FH FLCN FSP1 Fzd GLUT1 GSK3β

Alpha adrenoceptor Adenylyl cyclase Acetylcholine Autosomal dominant polycystic kidney disease Protein kinase B Atrial natriuretic peptide Adenomatosis polyposis colon Androgen receptor Adenosine triphosphate Beta-adrenoceptors Basic fibroblast growth factor Bladder cancer Benign prostatic hyperplasia Bladder urothelial carcinoma Carbonic anhydrase IX Cyclic adenosine monophosphate Clear cell renal cell carcinoma E-cadherin Cyclin-dependent kinase inhibitor 2A Cyclic guanosine monophosphate Chromophobe renal cell carcinoma Casein kinase 1 Chronic kidney disease Cyclooxygenase Cytotoxic T-lymphocyte-associated protein 4 Diacylglycerol 5α-dihydrotestosterone Diabetic kidney disease Endothelial growth factor Endothelial growth factor receptor Epithelial-to-mesenchymal transition Epithelial cell adhesion molecule Estrogen-receptor Endothelin Fibroblast growth factor Fibroblast growth factor receptor 3 Fumarate hydratase Folliculin Fibroblast-specific protein 1 Frizzled proteins Glucose transporter 1 Glycogen synthase kinase 3β

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HAT HB-EGF HGF Hh HIF IGF-I IL IL-6R IP3 KCa LUTS MAPK MET MIBC MMR MMP MSI mTOR mTORC MUC1 nAChRs NMIBC NO PAI-1 PDE PDGF PD-L1 PIP3 PI3K PKA PKC PKD PKG PLC PPARγ pRCC PTH RCC ROS SDH SIRT STAG2 STAT3 TAK1 TERT

Histone acetyltransferase Heparin-binding EGF-like growth factor Hepatocyte growth factor Hedgehog Hypoxia inducible factors Insulin-like growth factor I Interleukin Interleukin-6 receptor Inositol triphosphate Kidney cancer Lower urinary tract symptoms Mitogen activated protein kinase Mesenchymal-epithelial transition Muscle-invasive bladder cancer Mismatch repair Matrix metalloproteinase Microsatellite instability Mammalian target of rapamycin Mammalian target of rapamycin complex Mucin 1 Nicotinic-type cholinergic receptors Non muscle-invasive bladder cancer Nitric oxide Plasminogen activator inhibitor-1 Phosphodiesterase Platelet derived growth factor Programmed death ligand 1 Phosphatidylinositol-3,4,5-triphosphate Phosphoinositide 3-kinase Protein kinase A Protein kinase C Polycystic kidney disease Protein kinase G Phospholipase C Peroxisome proliferator-activated receptor-gamma Papillary renal cell carcinoma Parathyroid hormone Renal cell carcinoma Reactive oxygen species Succinate dehydrogenase Sirtuin Stromal antigen 2 Signal transducer and activator of transcription 3 TGF-β-activated kinase 1 Telomerase reverse transcriptase

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Transforming growth factor beta 1 Transmembrane receptor potential Transient receptor potential cation channel subfamily V member 1 Tuberous sclerosis complex Upper tract urothelial carcinoma Vascular endothelial growth factor Von Hippel Lindau Vasoactive intestinal peptide World Health Organization Wnt inhibitory factor 1 Yes-associated protein

6.1 Introduction The urinary system and male genital organs are very important for homeostasis and life cycle. The former is composed of the kidney, which displays many critical functions including regulation of blood pressure, maintaining overall fluid and ionic balance and filtering waste and toxic substances from blood; and the urinary tract, which stores and transports urine for elimination. The latter includes the prostate gland and seminal vesicles which secrete prostatic fluid, the major component of semen that transports sperm; and the testes, the male gonads, responsible for producing and storing sperm, assuring the reproductive capacity of the species, being also responsible for producing hormones involved in male development, called androgens. In this chapter we will focus on some of the major signaling pathways involved in the main physiological functions of the kidney and the urinary tract. Instead of exhaustively listing all players involved, we will focus on the ones considered to be the most relevant. We will also briefly address some changes in signaling pathways which occur when normal physiology is disrupted by neoplastic transformation (i.e., in cancer).

6.2 The Kidney 6.2.1 Normal Anatomy, Development and Physiology of the Kidney: Implications for Disease Status The kidneys are among the most important organs that sustain life; indeed, they orchestrate a serie of events and regulate several processes without which we could not function [1, 2]. In this section we will give a very general overview of the kidney most relevant roles in maintaining homeostasis.

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The kidneys are the “maestros” of systemic homeostasis—they keep the internal milieu (i.e. sodium levels, potassium levels, pH) in a physiological and functional range even when we change to different environments and face diverse dietary and hydro-electrolytic inputs [3]. They are the regulators of blood pressure (which they govern in conjunction with the cardiovascular and autonomic nervous systems). The connection among these systems is so intricate that when one part of it fails, the other does so too—this refers to the cardiorenal syndrome [4, 5]. The kidneys produce urine (by the orchestrated events of filtration, reabsorption and secretion), which is a fundamental route of eliminating toxins, drugs and waste products from the body. This can only be accomplished thanks to their particular anatomy: they are composed of an outer layer—the cortex—and an inner layer—the medulla— and their functioning unit is the nephron (with various segments and a wide range of ion transporters, namely sodium transporters), which is in tight proximity to a network of blood vessels, such as the glomerulus where filtration takes place [6]. Moreover, the kidney regulates the phosphate, calcium and vitamin D metabolism, again in consonance with other structures, the parathyroid glands (which release the parathyroid hormone—PTH), the bones (the deposits of these elements) and the intestines (where absorption from dietary intake takes place) [7, 8]. Both morphology and physiology of the kidney change with ageing [9]. Given the intricate interaction between the kidneys and many other organ systems, it is logical that acute kidney injury or chronic kidney disease might have life-threatening implications for the afflicted patients. Kidney development is very much determined by pathways such as Wnt, Notch and Hippo [10, 11]. It emerges thanks to an interaction of two structures: the epithelial Wolffian duct-derived ureteric bud and the metanephros mesenchyme. This eventually gives rise to the functional units of the kidney, the aforementioned nephrons, which are populated with endothelial cells that will give rise to a fully functional vasculature in the surroundings. Given the complexity and meticulous arrangements of the nephrons in a fully functioning kidney, a complex array of signaling cascades and factors are activated and contribute to nephrogenesis, including GDNF/RET, fibroblast growth factor (FGF), Six1 and Sall1, Pax2, various Wnt glycoproteins and BMPs [12, 13].

6.2.2 Non-neoplastic Kidney Diseases Generally, in progressive, chronic kidney diseases (CKD), fibrosis is a central, common, last event towards end-stage kidney failure, many times independently of the etiological insults that lie behind. Fibrosis can affect both the tubules and the glomerulus—tubulointerstitial fibrosis and glomerulosclerosis, respectively [14]. The transforming growth factor beta 1 (TGF-β1) signaling pathway is the key mechanism involved in fibrosis establishment in several situations, including in CKD. This makes this player an attractive target to therapies for a vast amount of patients [15]. TGF-β1, the most profibrogenic cytokine there is, firstly binds to

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type 2 receptors, which then joins in a complex with the type 1 receptors, leading to phosphorylation of the cytoplasmic domain of type 1 receptors and its subsequent activation. What is interesting is that this TGF-β1-induced activation leads to signaling through two pathways: the canonical Smad-dependent, and the non-Smad dependent, which includes signaling through the mitogen activated protein kinase (MAPK), PI3K/AKT and Rho-like GTPase cascades [16–18]. Importantly, the action of TGF-β-activated kinase 1 (TAK1) is of paramount importance in this pro-fibrotic cascade, transducing the signal to several downstream pathways [19]. A genomewide approach in kidneys of patients with and without fibrosis disclosed differential expression of several players belonging to the Notch, Wnt and Hedgehog (Hh) pathways [20]. The rationale for activation of these developmental-related pathways (which are critical for kidney development, see above) is that it constitutes an attempt of repairing tissue injury that occurs in the context of kidney disease. However, there is an overactivation of signaling through these cascades, which eventually leads to fibrosis and loss of function. Specifically, the Wnt and Hh pathways have a role in promoting fibroblast differentiation and myofibroblast transformation, while Notch and Wnt signaling impact on epithelial dedifferentiation [21]. Indeed, and besides being overactivated in tubule epithelium, the major targets of the Hh pathway (which also includes canonical and non-canonical mechanisms) are interstitial fibroblasts [22]. The Wnt pathway exerts its effects by interacting with the Frizzled (Fzd) proteins, and transducing signals both through the canonical (β-catenin-dependent) and non-canonical (β-catenin independent) pathways. Some of the targets of this cascade are fibronectin, fibroblast-specific protein 1 (FSP1), Snail, matrix metalloproteinase 7 (MMP-7) and plasminogen activator inhibitor-1 (PAI-1) [23]. Again, whatever the context, although a temporary activation of these healing-prone pathways is desirable, their sustained activation ends up in promoting kidney fibrosis. Another pathway implicated in kidney disease is the Hippo pathway. Its major effector is the Yes-associated protein (YAP), which when activated translocates to the nucleus, modulating target gene expression, being implicated in cell growth, differentiation and survival. Besides its implication in kidney embryogenesis, this Hippo-YAP signaling has recently been implicated in a variety of kidney diseases, including diabetic kidney disease (DKD), cystic kidney disease and kidney fibrosis [11]. Moreover, interleukin-6 (IL-6) signaling (both classic and trans-signaling ways) has been implicated in autoimmune and inflammatory diseases of the kidneys, with podocytes being the major target as they express its receptor (IL-6R) [24]. Overactivation of the JAK/STAT pathway, which contributes to kidney’s response to injury, has also been documented throughout the nephron and glomerulus in various kidney diseases, with roles in DKD, acute kidney injury and kidney fibrosis [25]. The major cause of CKD at present (and still rising) is DKD, in which glomerular damage plays a pivotal role, explaining microalbuminuria in these patients [26]. During the course of the disease, an intense crosstalk between glomerular cells (endothelial cells, mesangial cells and podocytes) takes place, by means of activation of several signaling pathways. The endothelial-mesangial communication is mediated by platelet derived growth factor (PDGF) signaling, while the endothelial-podocyte crosstalk is established though vascular endothelial growth

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factor (VEGF), angiopoietins and endothelin-1. By doing so, endothelial damage may lead to podocyte damage, and vice versa. Ultimately, this means that not only direct injury to the endothelium is responsible for the clinical manifestations and microalbuminuria onset, but also the secretion of mediators released upon this crosstalk indirectly induce more damage [27]. The hydro-electrolytic balance achieved by the kidneys depends on the interaction and efficient cooperation between several mechanisms, such as the ones dependent on angiotensin, atrial natriuretic peptide (ANP) and dopamine. These systems also regulate the redox state: the natriuretic and dopaminergic systems are antioxidant while the renin-angiotensin system is prooxidant; hence pathological scenarios that induce change these systems (such as hypertension) also disrupt this redox balance, leading to increased oxidative stress. Angiotensin II is the key player in renin-angiotensinaldosterone signaling and binds to both AT1R (the agonist receptor) and AT2R (the functional antagonist) receptors. AT1R is a G protein-coupled receptor, and binding of angiotensin II activates the phospholipase C (PLC) pathway, increasing levels of inositol triphosphate (IP3) and diacylglycerol (DAG) and ultimately leading to activation of the Rho kinase and MAPK pathways. Furthermore, the AT1R also mediates activation of NADPH oxidase, leading to increased levels of reactive oxygen species (ROS) and oxidative damage, mediated by other pathways such as the NF-kB [28, 29]. On the other hand, there is evidence that ANP has indeed an antioxidant role, by binding both to NPRA receptors (which increase levels of cGMP) and NPRC receptors (which increase levels of cyclic adenosine monophosphate [cAMP]), and by regulating iNOS activity. Finally, besides increasing natriuresis (as ANP does), dopamine also decreases ROS production via binding to D1-like receptors, which activates both protein kinase A (PKA) and protein kinase C (PKC) and ultimately inhibits NADPH oxidase activity; or to D2-like receptors, which activates an antioxidant complex of proteins [30]. Polycystic kidney disease (PKD) is a hereditary condition (the most common form being autosomal dominant—ADPKD) caused by germline mutations in polycistin-1 and polycistin-2 (PKD1 and PKD2, respectively). PKD1 is a transmembrane protein similar to a G-protein coupled receptor and PKD2 is a calcium-selective channel of the transmembrane receptor potential (TRP) family. The kidneys of these patients develop multiple large fluid-filled cysts, which grow over decades and eventually compromise organ function, leading to kidney failure [31]. Recent evidence has demonstrated that the emergence and progression of these cysts in ADPKD results from changes in activation of several signaling cascades, especially those involved in proliferation, apoptosis and metabolism. In fact, cAMP results in an enlargement of the cysts by activating several downstream cascades; PKD1 and 2 mutation leads to intracellular Ca2+ reduction, which decreases the activity of Ca2+ /calmodulindependent phosphodiesterases (PDE), ultimately rising cAMP levels [32]. This is the rationale for using PDE activators as treatment of these patients [33]. Increased cAMP also activates BRAF, which signals through the ERK/MEK pathway further increasing proliferation, meaning that agents targeting BRAF might have success in treating these patients [34].

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Besides being relevant in regulating many tumor-related processes (see below), epigenetic mechanisms are also implicated in non-neoplastic diseases. Indeed, changes in microRNAs (miRs) levels are witnessed during CKD of several etiologies, and they influence the activation of several pathways that ultimately contribute, for instance, to fibrosis, podocyte apoptosis, mesangial cell proliferation or autophagy, inflammation and oxidative damage [35–37].

6.2.3 Renal Cell Carcinoma Kidney cancer (KCa) is the 14th most incident neoplasm worldwide (with 403,262 new estimated cases in 2018) and the 16th most lethal cancer in the world (with 175,098 deaths attributed to this disease estimated in 2018) [38]. One of the major challenges, however, has to do with its diagnosis: because the kidney is a retroperitoneal organ, renal masses are asymptomatic until late in their natural history and tumors are diagnosed at advanced stages; whereas 65% of KCa are diagnosed at localized stage, exhibiting a 5-year survival of 92%, approximately 1/3 of the cases are detected in regional or disseminated stages, showing a much poorer survival (particularly for the metastatic disease, with a 5-year survival of only 11.6%) [39]. By far the most common KCa subtype is renal cell carcinoma (RCC), and, thus, epidemiology trends refer mostly to the natural history of this neoplasm (although many other tumors can have origin in the kidney, including sarcomas and urothelial carcinomas of the renal pelvis, the latter being discussed in the following section). RCC is a heterogeneous disease that covers many tumor entities; according to the World Health Organization (WHO) 2016 classification, the three most common subtypes include clear cell (ccRCC, 65–70%), papillary (pRCC, 15–20%) and chromophobe (chRCC, 5–10%) RCC [40]. Because RCC is not a single disease, there have been efforts to molecularly define these tumor entities from an integrated point of view, considering genomics, epigenomics and proteomics data. Recently, Ricketts et al. have established distinctive signatures for each tumor subtype, which can be used for uncovering specific treatments for this heterogeneous disease: ccRCC showing an increase in ribose metabolism pathway (associating with poor prognosis) and an overactivation of the immune signature; type 1 pRCC displaying PBRM1 mutations (also associating with poor prognosis) and increased messenger RNA (mRNA) signature for RNA splicing and cilium genes; type 2 pRCC, with activation of glycolysis, ribose metabolism and genes related to Krebs cycle; and chRCC, with the finding of a subgroup of metabolically divergent tumors displaying poorer survival [41]. Also, they have uncovered universal markers shared by the most common histological subtypes, such as loss of CDKN2A (in 16% of RCC) and increased DNA promoter hypermethylation of specific genes, which associated with poor survival (Fig. 6.1). Given the heterogeneity of this disease, and for the sake of this chapter, we will focus on the most biologically or clinically relevant signaling pathways involved in RCC [42–45].

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Fig. 6.1 Summary of recently uncovered subtype-specific molecular pathways involved in renal cell carcinoma. Adapted from [41]

Kidney tumors are particularly prone to show a hypoxic environment, which triggers a compensatory overactivation of angiogenic signaling (in fact, this is why these tumors so often disclose highly vascularized regions and eventually bleed). This is in part due to inactivating mutations in von Hippel Lindau (VHL); this gene normally associates with elongins B and C and cullins to form a complex with ubiquitin ligase activity, that targets hypoxia inducible factors (HIFs) for elimination [46–48]. HIFs (namely HIF-1α, the most biologically relevant subunit of the HIF complex) are transcription factors that regulate genes involved in angiogenesis (VEGF and PDGF), glucose metabolism (glucose transporter 1—GLUT1—and Endoglin), proliferation (endothelial growth factor receptor—EGFR) and invasion/metastasis (mucin 1— MUC1), hence the large number of players and signaling cascades they govern [49]. HIFs additionally target carbonic anhydrase IX (CAIX) and display crosstalk mechanisms with epigenetic phenomena, such as microRNAs (miR-210) [50, 51]. HIFs are tightly regulated and reflect the media concentration of oxygen: when hypoxic conditions are present, HIF-1α translocates to the cell nucleus, joins HIF-1β subunit and transcriptional coactivators p300/CBP, ultimately leading to expression of genes containing hypoxia-response elements such as VEGF; in the event of normoxia, HIF1α becomes hydroxylated by hydroxylases (proline and asparagine), which renders it amenable to be bound to VHL and to be subsequently targeted for destruction in the proteasome; finally, in RCC with loss of VHL, there is no feedback in this pathway and HIF-1α is constantly active, activating its downstream tumor promoting cascades [52–55]. The PI3K/AKT/mTOR pathway, which is a key signaling mechanism in several cancers, inducing proliferation, survival and angiogenesis, is also fundamental

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in RCC. This pathway has been shown to be overactivated in RCC compared to normal kidney tissue, thus constituting the rationale for exploring mammalian target of rapamycin (mTOR) inhibitors as treatment of this cancer [56]. Binding of the aforementioned players (VEGF, endothelial growth factor [EGF], PDGF etc.), which are triggered by HIFs, to their respective tyrosine kinase receptors ultimately leads to activation of this pathway, generating phosphatidylinositol-3,4,5-triphosphate (PIP3); this, in turn, recruits protein kinase B (AKT/PKB) to the membrane for activation through phosphorylation by mTOR (through its rapamycin-insensitive complex, TORC2) and PDK1. The signaling through AKT also ends up activating mTOR, meaning that it locates both up- and downstream of the AKT cascade [57, 58]. Another protein (folliculin—FLCN), known to suffer loss-of-function mutations in patients with Birt-Hogg-Dubé syndrome which are prone to develop RCCs with hybrid morphologies, also interacts with this pathway, as it inhibits mTORC1 [59, 60]. β-catenin is another key player in KCa pathogenesis. This protein represents the effector of the canonical Wnt pathway, which regulates cell differentiation and various other determinant cellular processes such as proliferation, so it is not surprising that it is frequently deregulated in cancer [61]. Similarly to what happens with HIFs, β-catenin is tightly regulated: in physiological conditions it is targeted for destruction in the proteasome because it is phosphorylated by its encasing complex, composed of casein kinase 1 (CK1), glycogen synthase kinase 3β (GSK3β), adenomatosis polyposis colon protein (APC) and axin; however, when the Wnt pathway is activated, β-catenin is no longer phosphorylated and does not face destruction—it accumulates and translocates to the nucleus, where it activates oncogenes such as MYC [62]. Besides this direct effect of β-catenin, there is also crosstalk with other signaling cascades: activation of the Wnt pathway ultimately leads to activation of the aforementioned mTOR pathway, by means of impeding the GSK3β-mediated phosphorylation of tuberous sclerosis complex 2 (TSC2), which works as an inhibitor of mTOR [63, 64]. Specifically in RCC, the Wnt pathway seems to be overactivated: upregulation of β-catenin induces tumor formation in mice and hypermethylation of APC occurs in a subset of these cancers [65, 66]. Moreover, and very interestingly, there is a connection between the well-known and previously discussed VHL signaling mechanism and the Wnt pathway: on the one hand, β-catenin is degraded by the E3-ubiquitin ligase activity of VHL, which is frequently lost in these tumors; on the other hand, Jade-1, another player with E3-ubiquitin ligase activity, is activated by VHL and induces β-catenin destruction, a process which is lost in the absence of VHL [67, 68]. Finally, several inhibitors of the Wnt pathway (such as Wnt inhibitory factor 1 [WIF-1]) are downregulated in RCC, by promoter hypermethylation-induced silencing [69]. Alterations of the HGF/MET pathway are also frequent among RCC, especially pRCC (c-Met germline mutations are even the cause of a hereditary form of pRCC) [70]. Once again the overactivation of this cascade establishes complex crosstalk with other signaling pathways: binding of hepatocyte growth factor (HGF) to its tyrosinekinase membrane receptor, the mesenchymal-epithelial transition (MET) protein, induces its phosphorylation, activating the recruitment of several proteins such as

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signal transducer and activator of transcription 3 (STAT3) and phosphoinositide 3kinase (PI3K), ultimately leading to activation of the PI3K/AKT and Ras/MAPK pathways, which promote RCC growth [71]; it also releases β-catenin from the encaging action of E-cadherin (CDH1), thus promoting its nuclear translocation to exert its action as a transcription factor [72]. Again, VHL was eventually shown to also command this mechanism, as it is a negative regulator of this HGF-mediated β-catenin signaling [68]. The association between hypoxia, angiogenesis and metastatic dissemination is well established, and RCC represents a paradigmatic tumor model. Indeed, an association between hypoxia, epithelial-to-mesenchymal transition (EMT) players and metastasis is documented for RCC. EMT is of paramount importance before any metastatic dissemination occurs, and is activated by a hypoxic medium, which upregulates EMT-inducing proteins such as Twist [73]. Also, all signaling pathways related to cell metabolism, such as the ones involved in Krebs cycle, are fundamental for RCC development (with germline mutations in fumarate hydratase—FH—and succinate dehydrogenase—SDH—for instance being responsible for the emergence of specific hereditary syndromes developing RCC) [53]. FH and SDH loss lead to accumulation of fumarate and succinate, respectively, which in turn inhibit prolyl hydroxylase and impedes HIFs degradation [74, 75]. Interestingly, this connection between metabolism/hypoxia and EMT is tightly regulated by epigenetic mechanisms [76–78]. Indeed, epigenetic mechanisms (both regarding methylation, protein-coding players and non-coding RNAs) are additional regulators of all the complex signaling cascades involved in RCC, being important for its biology, diagnosis and prognosis [79–82]. Very recently, RNA modifications have also emerged as relevant players in regulating signaling pathways involved in all kinds of cellular processes, including in urological tumors such as RCC [83].

6.3 The Urinary Tract 6.3.1 Normal Anatomy and Physiology of the Urinary Tract: Implications for Disease Status The urinary tract is a contiguous canal, starting proximally in the renal papillae and including the renal pelvis, ureters, urinary bladder, ending distally with the urethra. Its main function is to collect urine produced by the kidneys, transport it, store it and finally eliminate it when appropriate, in a highly coordinated fashion, hence assuring the removal of metabolic and toxic compounds filtered in the kidneys [84]. Despite being considered a sterile site (as opposing to the digestive and respiratory tracts), the urinary tract exits to an environment that is filled with microbial agents. Nevertheless, infections are relatively infrequent over the lifetime of a healthy individual, in part thanks to the anatomy and physiology of this system [85]. Urothelium integrity and repair is maintained by a complex array of signaling pathways, such as peroxisome proliferator-activated receptor-gamma (PPARγ), EGFR and Hh/Wnt [86, 87].

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The urinary tract is frequently divided into upper and lower portions: the former comprising the renal papillae and pelvis and the pair of ureters; the latter being composed of the bladder and urethra, which are under the control of both voluntary/somatic and involuntary/autonomic control. This separation is not merely academic: it reflects somewhat different functions and different clinical implications when there is a disease/pathology of this system. Urine is constantly flowing in the upper urinary tract while it is intermittently expelled by the lower urinary tract (eliminating any infectious agents that have possibly gained access) [88, 89]. The urinary tract is a continuous system, and its luminal surface is covered by a specialized type of epithelium: the urothelium. It is composed of a basal and intermediate layer of cells, which are less well differentiated and constitute the reservoir for epithelial regeneration; and a superficial layer of cells, called “umbrella cells”, which are larger, hexagonal, linked by tight junctions and hosting the majority of uroplakins [90, 91]. “Umbrella cells” are fundamental for bladder physiology, allowing it to maintain its barrier integrity despite facing abrupt changes in volume and pressure. Despite sharing the same epithelium lining along the whole tract, it has been shown that the urothelium covering the bladder is different from a molecular, biochemical and embryological standpoint from the one covering the ureters, for instance [92]. In fact, urothelial cells in these sites derive from different germinal layers (the upper tract from the mesoderm, the lower tract from the endoderm); they have different amounts of protein players such as uroplakins and distinct immunoexpression of keratins; they show different tendencies to keratinize; and neoplastic disease from these organs shows distinct genetic and epigenetic landscapes (such as microsatellite instability and methylation patterns) and natural history [93–98]. Indeed, there is evidence for the existence of at least three urothelial lineages: the one covering the renal pelvis and ureters; the one of the detrusor and bladder trigone; and finally the one covering the bladder neck and proximal urethra [99]. The logical implication of these findings is that studies on urothelium should discriminate among these two major subtypes (upper urinary-type urothelium and lower urinarytype urothelium) and avoid mixing them and drawing conclusions for both groups, as they are morphologically the same but biologically (from a genetic and epigenetic point of view) distinct. This could (and should) have potential impact in management of patients with urinary tract pathology [100]. The urothelium is also not a passive epithelium concerning transduction of stimuli: it has the ability to process visceral sensation and communicate stimuli to bladder nerve and muscle cells [101], due to its proximity to nerve fibers running in the sub-urothelial plexus.

6.3.2 Neurological and Hormonal Control of Urine Transport and Micturition The micturition cycle is an orchestrated chain of events that encompasses two major phases: urine storage/bladder filling; and urine voiding/bladder emptying. It involves a complex interaction between both central and peripheral nervous systems and

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anatomic parts of the urinary tract, namely muscular structures (which in the urinary tract have specific composition, energetics, metabolism and mechanic properties); hence, a disturbance in any of these components might result in dysfunction and symptoms (such as lower urinary tract symptoms—LUTS) [102, 103]. After exiting the kidney, the urine is propelled towards the bladder by action of the smooth-muscle excitation in a syncytial-like fashion. This urinary tract peristalsis assures the unidirectional flow of urine towards the bladder, for storage [104]. The bladder has unique elastic properties which allow it to accumulate urine volume at the expense of little change in detrusor (the bladder wall muscle) and intravesical pressures. During filling, there is an activation of the spinal sympathetic pathway (T12-L2) and modulation of parasympathetic ganglionic transmission, which ultimately leads both to increase in bladder outlet resistance and inhibition of detrusor contraction. A supplementary way of increasing resistance to voiding is by activating the somatic guarding reflex, which increases resistance in the voluntary external urethral sphincter. When the bladder is full and reaches its maximum capacity, afferent signals originating from tension and pain receptors are transmitted by Aδ and C fibers into the pelvic and pudendal nerves and spinal cord, and ultimately interpreted in the brain stem and cerebral cortex. If voiding is deemed appropriate, the efferent signals operating during the filling phase are reversed: there is relaxation of the sphincter (following inhibition of the sympathetic and somatic pathways) and bladder wall contraction (following activation of the parasympathetic pathway) [102, 105–108]. The urethra is important in controlling the sense of imminent micturition [99]. Contraction of the bladder wall is mainly mediated by cholinergic signaling— initiated by acetylcholine (Ach) binding to muscarinic-type receptors. ACh release is mediated by depolarization, and ultimately induces muscle contraction, an action that is enhanced by cholinesterase inhibitors and inhibited by anticholinergic agents such as atropine [109]. Although all subclasses of muscarinic receptors were demonstrated in the human detrusor muscle, the most abundant and biologically relevant are M2 and M3 receptors [110]. These receptors are coupled to downstream pathways (G proteins), but there are differences in the contribution of the different signaling cascades. For instance, while M2 receptors couple to Gi/o , which leads to inhibition of adenylyl cyclase (AC), M3 receptors couple preferentially to Gq/11 , which impacts on a different pathway, ultimately resulting in increased calcium (Ca2+ ) influx, which is of paramount importance in bladder contraction, by activating myosin light chain kinase [111, 112]. Hence, different muscarinic receptors convey activation of distinct pathways, all contributing to efficient bladder contraction. It has been suggested that M2 and M3 muscarinic receptors are operating differently: M3 receptors operate at high concentrations of ACh, while M2 receptors are activated at lower concentrations of the agonist; also, it has been proposed that receptors show different topography in the bladder wall, M2 receptors being more junctional and M3 receptors being extrajunctional [113]. The most studied signaling pathways are indeed the ones dependent on M3 receptors, which are responsible for normal micturition contraction. Binding of ACh to M3 receptors leads to Ca2+ influx both by activation of the aforementioned Gq/11 and also by activation of PLC, which leads to IP3 production and IP3 -dependent Ca2+ release

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from the sarcoplasmic reticulum [114]. Activation of M3 receptors also promotes contraction by inhibiting myosin light chain phosphatase; this effect is conveyed by activation of Rho-kinase and also by signaling through PLC-produced DAG, which activates PKC [102, 115]. M2 receptors signaling mechanisms are less clear. As stated above, by inhibiting AC (responsible for producing cAMP), it has been suggested that they ultimately oppose relaxation-prone sympathetic signaling mediated by β-adrenoreceptors and even by purines (see below). Additionally, they can activate PKC and ultimately activate cation channels and inhibit KATP channels; interaction with these transporters was suggested to enhance contraction initiated by M3 receptors activation [116–119]. Nicotinic-type cholinergic receptors (nAChRs) are also present throughout the urothelium, and contribute to modulate bladder reflexes [120]. Adrenergic receptors also play an important role in bladder function. Stimulation of alpha-adrenoceptors (α-AR), especially subtype α1-AR in the bladder wall also lead to detrusor muscle contraction, although the effect is minor compared to ACh effect on muscarinic receptors [121]. In fact, these receptors may gain relevance in pathological conditions, such as detrusor overactivity secondary to neurogenic bladder or outlet obstruction. Also, in these conditions, there may be a change in the balance between the contraction-prone α-AR and the relaxation-mediating betaadrenoceptors (β-AR), with upregulation of the former and downregulation of the latter [122, 123]. The human bladder expresses three subtypes of β-AR (β1, β2 and β3), the most relevant for relaxation being the β3 form [124, 125]. The ligand for these receptors is norepinephrine, released by depolarization of adrenergic nerves in the bladder; because β-AR are far more frequent in the bladder wall than α-AR, the major physiological response to norepinephrine is relaxation. Epinephrine might also have a role in activating β-AR, but this theory has not been demonstrated yet [126]. Binding of norepinephrine to β-AR leads to overactivation of AC, increased levels of cAMP and activation of PKA, which ultimately mediates relaxation [127]. Besides the contribution of muscarinic and adrenergic receptors, purinergic signaling also intervenes in bladder contraction. Adenosine triphosphate (ATP) acts on two types of purinergic receptors: the family of P2X ion channels and the family of P2Y G-coupled receptors. In the human bladder the most common and relevant receptor is P2X1, and, again, changes in the amount of these receptors have been demonstrated in pathological conditions of the detrusor muscle [128, 129]. For instance, increased release of ATP is noticed in interstitial cystitis [130]. Multiple signaling cascades are initiated by purinergic receptors activation: ATP binding to P2X receptors leads to Ca2+ influx, while uridine triphosphate and adenosine 5 -O-(2-thiodiphosphate) binding to P2Y2 and P2Y4 receptors also culminates on the same effect by activating PLC/IP3 pathway [131]. ATP may also be diffused in response to stretch (with amiloride-sensitive apical sodium channels, ENaC, controlling basolateral ATP release) and act on P2X2 and P2X3 receptors, in order to stimulate stretch-induced exocytosis [132, 133]. Also, nitric oxide (NO), derived from L-arginine, has been suggested to play a role in bladder relaxation, potentially by interfering with cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG) [134]. NO synthases (both constitutive

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and inducible forms) are present in lower urinary tract smooth muscle [135, 136]. However, there is still very little evidence pointing towards a relevant role of NO in bladder relaxation. Similarly, various neuropeptides have been demonstrated to be released in the lower urinary tract, but their functional relevance to contraction and/or relaxation remains to be proven. It is the case of vasoactive intestinal peptide (VIP), which binds to VPAC1 and VPC2 Gs -coupled receptors inducing relaxation [137]; endothelins (ETs), namely ET-1 and ET-3, which activate Ca2+ channels and PKC leading to contraction [138]; tachykinins, such as substance P, neurokinin A and neurokinin B, which act on NK1-3 receptors (the predominant in humans being NK2) also interfering with Ca2+ channels [139]; and angiotensins, namely angiotensin II, which induce contraction by binding to angiotensin receptors [140]. Transient receptor potential cation channel subfamily V member 1 (TRPV1) channels, which are activated by vanilloids such as capsaicin, were also demonstrated to be spread along the urothelium surface and nerves and interstitial cells. When activated, these lead to influx of Ca2+ and release of NO and ATP [141], possibly playing a role in bladder disease and response to injury. The role of other transient receptor potential channels, which are nonspecific cation channels generally permeable to Ca2+ , is far less understood, but has gathered attention of researchers focusing on novel ways to treat voiding disorders [142, 143]. Hormones like estrogens also exert their effects on the urothelium, by binding to estrogen-receptors alpha and beta (ER-α and ER-β). Upon ligand-binding, they travel into the nucleus and target gene expression, functioning as transcription factors. In the bladder, the ER-β form seems to be the most relevant in influencing filling sensation [144]. Finally, prostanoids (prostaglandins and thromboxanes), synthetized by cyclooxygenase (COX), have also been implicated in bladder contraction [145]. Hypertrophy and/or hyperplasia of the bladder smooth muscle may occur as an adaptive response to increased urinary tract outlet resistance. This condition is very frequent among older men suffering from benign prostatic hyperplasia (BPH). It may also occur as a result of spinal cord injury, due to decentralization. Outlet obstruction changes cholinergic functions of the urinary bladder. Despite not fully characterized, many signaling pathways seem to be involved in this adaptation phenomenon, such as insulin-like growth factor I (IGF-I) [146, 147], EGF [148], heparin-binding EGF-like growth factor (HB-EGF) [149], angiotensin receptors [150], basic fibroblast growth factor (b-FGF) [151] and Ca2+ mediated signaling [152].

6.3.3 Urothelial Carcinoma Urothelial carcinoma may derive from any part of the urinary tract covered with urothelium; given its lower prevalence (about 5–10% of urothelial malignancies) [97], upper tract urothelial carcinoma (UTUC) it less well characterized and studied, compared to its counterpart bladder urothelial carcinoma (BUC), including involved signaling pathways. In this section we will focus mostly on signaling cascades described for BUC; still, given the heterogeneity of the disease (see below), only

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the most relevant pathways will be covered. A thorough understanding of these pathways might help uncovering novel treatments urgently needed for these patients, especially for those with metastatic disease, for whom the standard of care has been cisplatin-based chemotherapy protocols, with variable efficacy and frequently with emergence of resistance [153]. Bladder cancer (BlCa) is the second most frequent urological malignancy after prostate cancer. Incidence is on the rise (with estimated 549,393 new cases diagnosed in 2018 and 990,724 new cases expected in 2040). It also represents an important cause of cancer-related mortality, with 199,922 deaths estimated in 2018 and 387,232 predicted for 2040 [38, 154]. Very importantly, BlCa is considered a very important economic burden, especially given its natural history trend for multiple recurrences, requiring continuous surveillance and treatment [155]. The most common form of BlCa by far corresponds to urothelial carcinoma (>90% of the tumor subtypes), derived from the urothelium. However, there are two major forms of the disease, which are very distinct in terms of clinics, pathobiology and (epi)genetics: non muscle-invasive BlCa (NMIBC), the most frequent form (75–80% of all cases), which shows a papillary architecture and a tendency for recurrence over time; and muscle-invasive BlCa (MIBC), often associated with the precursor lesion urothelial carcinoma in situ, which is biologically aggressive and responsible for most deaths, due to metastatic dissemination [156, 157]. A complex array of signaling pathways are involved in BlCa emergence and progression [158]. BlCa is among the group of tumors with the so-called high tumor mutational burden (TMB) [159], and hence it is naturally very heterogeneous and difficult to treat. Recently, there have been efforts to stratify patients into different groups based on molecular markers, and so urothelial carcinoma has been proposed to comprehend different disease phenotypes, from basal-like to luminal-like cancers, and others [160, 161]. More recently, Robertson et al. [162] pursued an integrated multiplatform analysis of BlCa and uncovered five different subgroups of the disease: the luminal-papillary subtype (35% of the cases), which shows a papillary histological architecture, low risk of progression, frequent fibroblast growth factor receptor 3 (FGFR3) mutations and activation of the sonic hedgehog pathway, being amenable to treatment with tyrosine kinase inhibitors directed at FGFR; the luminalinfiltrated subtype (19% of the cases), with high expression of EMT markers and miR-200, resistant to cisplatin but amenable to treatment with immune checkpoint inhibitors, given the expression of programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4); the luminal subtype (6% of the cases), which expresses luminal markers FOXA1 and GATA3 along with KRT20 and SNX31 and for which specific therapies are lacking; the basal-squamous subtype (35% of the cases), which shows squamous differentiation, expressing high levels of basal keratins and PD-L1/CTLA-4; and finally the neuronal subtype (5% of the cases), with high expression of neuronal and neuroendocrine genes, and overactivation of cell cycle and proliferation pathways. Tyrosine kinase receptor-related pathways are of paramount importance in BlCa. In fact, 35–55% of tumors display mutations, translocations, or amplifications of either PIK3CA, EGFR, ERBB2, ERBB3, or FGFR3 [153]. For instance, EGFR

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signaling (modulated by MDA-9/synthein) associates with poor prognosis and also with changes in EMT-related genes [163]. Moreover, the loss of endophilin A1 promotes this signaling by interfering with EGFR internalization [164, 165]. Also, overexpression of ERBB2/3 and related signaling cascades have a negative impact on patient survival and could be tackled with drugs such as trastuzumab, especially in the metastatic context [166, 167], as opposed to FGFR3 mutations, which were shown to associate with good prognosis, in a metanalysis [168]. FGFR3 mutations activate the Ras-MAPK and PLC increasing tumor cell survival [169]. Interestingly, microRNAs miR-99a and miR-100, which negatively regulate FGFR3, are downregulated in BlCa [170]. On the other hand, Met downregulation associates with unfavorable prognosis [171]. The PI3K-Akt-mTOR pathway is also implicated in various aspects of BlCa pathogenesis, and its activation associates with disease relapse [172]. Mammalian target of rapamycin complex 2 (mTORC2) is overexpressed in BlCa, as well as TSC1 [173]; these results prompt a logical response to drugs targeting this pathway, such as everolimus [174]. Finally, the aberrant accumulation of GSK-3β in the cell nucleus associates with poor prognosis [175]. VEGF plays a role in BlCa, with upregulation of related players associating with poor survival, paving the way for new treatments such as bevacizumab [176]. Also, cell cycle signaling is disrupted in BlCa, with co-expression of P16INK4a and Ki67 in high-grade tumors, and homozygous deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A) especially in FGFR3-mutated tumors, along with mutations in TP53, RB1 and MDM2 amplification [177–179]. Apoptotic mechanisms are also implicated, with the overexpression of survivin and lower levels of Smac/DIABLO resulting in poor prognosis [180, 181]. Recently, telomerase reverse transcriptase (TERT) promoter mutations were also found to frequently occur in BlCa, rendering tumor cells immortal [182], as well as mutations in stromal antigen 2 (STAG2), involved in DNA repair [183]. Since BlCa discloses high TMB, it is only natural that it also expresses immune checkpoint molecules such as PD-L1 and PD-1, which already have approved targeting drugs [184]. Also, several interleukins and chemokines are implicated in BlCa, namely IL-5, IL-8 and IL-20, which associate with migration and increased levels of MMP-9 and interfere with diverse signaling cascades, including ERK and JAKSTAT [185–187]. Overexpression of epithelial cell adhesion molecule (EpCAM) also relates to advanced disease, and is also a targetable player [188]. Changes in EMT players have a fundamental role in disease progression [189], and an interplay with epigenetic mechanisms (both protein-coding and non-coding RNA players) has been established [190]. Indeed, methylation of several genes (including CDH1, RASSF1A, RUNX3, SOX9, etc.), post-translational modifications of several proteins (induced by several players, such as sirtuins—SIRTs) and miRs were found to associate with disease prognosis and could be explored as diagnostic and prognostic biomarkers [191–193]. Similar to the kidney, the recent field of epitranscriptomics is also uncovering relevant players involved in BlCa biology [83]. Androgen receptor (AR) is a key player in the pathogenesis and progression of prostate cancer; still, its signaling pathways have implications in other tumor subtypes as well, and BlCa is no exception. Upon interacting with its ligand (5α-dihydrotestosterone—DHT—which is converted from testosterone by action

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of cytochrome P450 enzyme 5α-reductase) it translocates to the nucleus, binding to DNA and recruiting chromatin remodeling complexes (SWI/SNF), coactivators (SRC) and factors with histone acetyltransferase (HAT) activity, which ultimately leave chromatin much more accessible for binding of RNA polymerase II, initiating transcription of target genes (ligand-dependent pathway). Additionally, other players such as growth factors and chemokines also activate the receptor via oncogenic signaling pathways like PI3K/AKT and ERK/MAPK (non-ligand dependent pathways) [194]. It has been shown that AR signaling promotes the drug resistant phenotype of the disease, and so treatments targeting the AR might be effective in treating these patients [195]. Still, the association of AR with clinicopathological features varied greatly among studies, the same happening with ERα/β, which were associated both with pro- and anti-tumor effects [196–198]. In UTUC there is a prominent impact of mismatch repair (MMR) proteins and microsatellite instability (MSI), with Lynch syndrome patients showing a proclivity for UTUC [199]. Also, epigenetic phenomena may also help detecting this disease subtype, which is generally asymptomatic and hence mostly diagnosed at advanced stages [200]. A brief summary of the most relevant signaling pathways in the urinary tract is depicted in Table 6.1.

6.4 Conclusion We have summarized the most relevant signaling pathways involved in the major biological processes of two of the main urogenital organs, both in normal tissue and in cancer. The kidneys are major organs of the human body and the great keepers of homeostasis. They control several biological processes indispensable to life. All this regulation can only be met at the expense of complex crosstalk signaling among several pathways. Indeed, while some of these cascades are relevant in embryogenesis or in response to kidney injury, their sustained and uncontrolled overactivation can result in loss of function and fibrosis. KCa is not a single disease; it comprises a complex array of distinct tumor entities, with their different molecular backgrounds, both at the genetic, metabolic and epigenetic levels. There is an urgent need to uncover novel therapies that specifically target the most relevant signaling pathways overactivated in these subgroups of patients, and that can only be achieved by exploring subtype-specific disease mechanisms. Physiological control of urinary bladder contraction and relaxation, allowing for a proper micturition cycle, involves a complex array of signaling cascades, of which the cholinergic pathway through muscarinic receptors seems to be determinant. However, there is evidence of changes in this balanced signaling crosstalk in disease states. A whole understating of these phenomena is needed in order to uncover novel ways to pharmacologically influence micturition and treat these patients. BUC is a highly mutated tumor model and mutations in tyrosine kinase receptor-related pathways are

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Table 6.1 Summary of most relevant signaling pathways involved in the urinary tract Major player/pathway

Major effect

Normal physiology PPARγ, Hedgehog/Wnt, EGFR

Urothelium integrity and repair

ACh (muscarinic receptors—M2, M3)

M3—bladder contraction M2—contraction enhancement, relaxation inhibitor

ACh (nicotinic receptors)

Modulating bladder reflexes

Norepinephrine (α-adrenoceptors—α1-AR)

Bladder contraction

Norepinephrine (β-adrenoceptors—β3-AR)

Bladder relaxation

Purinergic (ATP) signaling (P2X and P2Y receptors)

Bladder contraction

NO signaling

Bladder relaxation

Others (neuropeptides, TRP channels, hormones…)

Less understood role in contraction/relaxation

Cancer (urothelial carcinoma) Tyrosine kinase receptor pathways—EGFR

Poor prognostic features

Tyrosine kinase receptor pathways—ERBB2/3

Poor prognostic features

Tyrosine kinase receptor pathways—FGFR3

Good prognostic features

PI3K-Akt-mTOR pathway

Poor prognostic features

VEGF-related pathways

Poor prognostic features

Cell cycle (p16, Ki67, CDKN2A, TP53, RB1, MDM2), apoptosis (Survivin, Smac/DIABLO), telomere maintenance (TERT), DNA repair (STAG2) deregulation

Poor prognostic features

Interleukins (IL-5/8/20)

Poor prognostic features

EpCAM

Poor prognostic features

EMT pathway

Poor prognostic features

Hormones (AR and ER)

Variable association with prognostic variables

Epigenetic mechanisms (methylation, non-coding RNAs, post-translational modifications)

Modulation of disease progression interfering with other pathways

amongst the most common. However, activation and/or deregulation of several other signaling cascades, including players involved in cell cycle and apoptosis, immune evasion, hormone receptors, DNA repair, telomere maintenance among others is frequent in this heterogeneous cancer. Also, besides genetic events, epigenetics also plays a role in disease biology. A better understanding of these pathways and their interplay is needed for uncovering novel targeted therapies suitable for these patients, which commonly end up showing resistance to standard chemotherapy protocols.

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Acknowledgments JL is supported by an FCT—Fundação para a Ciência e Tecnologia—fellowship (SFRH/BD/132751/2017).

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Chapter 7

More Than Androgens: Hormonal and Paracrine Signaling in Prostate Development and Homeostasis Juliana Felgueiras, Vânia Camilo, Margarida Fardilha, and Carmen Jerónimo Abstract The prostate is the major exocrine gland of the male reproductive system. The prostatic epithelium secretes an alkaline fluid, the prostatic fluid, that constitutes about 20–30% volume of the seminal fluid. It provides proteins and ions essential to control the ejaculation process and to regulate proteins involved in sperm maturation (e.g. human kallikrein-related peptidases, phosphatases, polyamines, pepsinogen II, citrate, glucose, and Zn2+ , among others). The prostate exhibits some particularities when compared to other organs: it accumulates the highest levels of Zn2+ of any soft tissue; epithelial cells can produce energy by glycolysis (similarly to highly proliferative cells); and, it is the only gland that tends to grow with aging, being associated with disorders of elderly, such as benign prostatic hyperplasia and carcinoma. Prostate development starts early in embryogenesis, but prostate maturation is only concluded in puberty. Specification of the prostate during human embryogenesis occurs before clear morphological evidence of a developing structure and involves the expression of signaling molecules that drive cells from the urogenital sinus to a prostatic cell fate. Prostate development and homeostasis are regulated by several hormones and growth factors and are highly dependent on autocrine and paracrine signaling. Efforts have been made to identify the mediators of prostate signaling as

J. Felgueiras · M. Fardilha Laboratory of Signal Transduction, Department of Medical Sciences, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal e-mail: [email protected] M. Fardilha e-mail: [email protected] J. Felgueiras · V. Camilo · C. Jerónimo (B) Cancer Biology and Epigenetics Group—Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal e-mail: [email protected] V. Camilo e-mail: [email protected] C. Jerónimo Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_7

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revised in this chapter, however this has been compromised by experimental constrains. Furthermore, most of the studies has been performed in rodent models, which makes extrapolations to other species difficult, given the inter-species variability on prostate anatomy and morphology. Keywords Steroid hormones · Androgens · Growth factors · Epithelial-stroma interactions

Abbreviations ACPP ADT AKT AR ARE BAD BAG1 BAX BCL2 BMP BPH CASP1 CDKN1B CGRP CRCP CTNNB CYP17A1 DHT DLL DNA E E2 ER EGF EGFR ERBB2 FGF FGFBP FGFR FOS FOXO1 GPCR GPER

Prostatic acid phosphatase Androgen deprivation therapy RAC-alpha serine/threonine-protein kinase Androgen receptor Androgen response element Bcl2-associated agonist of cell death BAG family molecular chaperone regulator 1 Apoptosis regulator BAX Apoptosis regulator Bcl-2 Bone morphogenetic proteins Benign prostate hyperplasia Activating caspase-1 Cyclin-dependent kinase inhibitor 1B Calcitonin gene-related peptide Castration-resistant prostate cancer Catenin beta-1 Steroid 17-alpha-hydroxylase/17, 20 lyase 5α-dihydrotestosterone Delta-like protein Deoxyribonucleic acid Estrogens 17β-estradiol Estrogen receptor Epidermal growth factor EGF receptor Tyrosine-protein kinase erbB-2 Fibroblast growth factor FGF-binding proteins FGF receptor Proto-oncogene c-Fos Forkhead box protein O1 G protein-coupled receptor G protein-coupled estrogen receptor

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GOT2 HGF HIF1A HSP IGF IGFBP IL JAG JAK JNK JUN KGF KLK KLK3 MAPK NKX3-1 NOTCH OXT OXTR PCa PDGF PG PGR PI3K PRKCA PRKCE PRL PRLR PTC PTK RALA ROS SFRP1 SHBG SHH SMAD SOX9 SRC SRD5A STAT T T3 T4 tfm TGF

Mitochondrial aspartate aminotransferase Hepatocyte growth factor Hypoxia-inducible factor 1-alpha Heat shock protein Insulin-like growth factor IGF-binding proteins Interleukin Jagged protein Tyrosine-protein kinase JAK c-Jun NH2-terminal kinase Transcription factor AP-1 Keratinocyte growth factor Kallikrein-related peptidase Prostate-specific antigen (commonly known as PSA) Mitogen-activated protein kinase Homeobox protein Nkx-3.1 Neurogenic locus notch homolog protein Oxytocin OXT receptor Prostate cancer Platelet-derived growth factor Progesterone PG receptor Phosphatidylinositol 3-kinase Protein kinase C alpha type Protein kinase C epsilon type Prolactin PRL receptor Protein patched Focal adhesion kinase 1 Ras-related protein Ral-A Reactive oxygen species Secreted frizzled-related protein 1 Sex hormone-binding globulin Sonic hedgehog protein Mothers against decapentaplegic homolog Transcription factor SOX-9 Proto-oncogene tyrosine-protein kinase Src 3-oxo-5-alpha-steroid 4-dehydrogenase 1 Signal transducer and activator of transcription Testosterone Triiodothyronine Thyroxine Testicular feminization Transforming growth factor

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Thyroid hormone Thyrotropin-releasing hormone Tyrosine Urogenital sinus epithelium Urogenital sinus mesenchyme Urogenital sinus Vascular endothelial growth factor Wild-type

7.1 Introduction Most male internal genitalia derive from the embryogenic Wolffian duct (mesodermal origin), but not the prostate. The prostate gland has endodermal origin from a complex and heterogeneous part of the urogenital sinus (UGS) [1]. Prostate development follows gonads differentiation and involves several stages (Fig. 7.1) [2, 3]. Gonadal-derived fetal androgens and interactions between UGS epithelium (UGE) and mesenchyme (UGM) are determinant throughout all prostate organogenesis. The underlying molecular mechanisms are far from being completely understood,

Fig. 7.1 Stages involved in prostate organogenesis, principal signaling pathways implicated in each stage, and cellular composition of the adult gland. The mediators of the signaling pathways involved in each stage were mostly found in knockout studies using rodent models [2, 3]. Figures were produced using Servier Medical Art (https://smart.servier.com)

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but these requirements appear to be universal despite the anatomical dissimilarities observed in prostates from different species [4]. Several molecular pathways have been implicated in the embryonic development of the prostate (Fig. 7.1) [5]. Most of the findings, nonetheless, resulted from studies using rodent knockout models and still lack confirmation in humans. The multiple reports on the restricted expression of the signaling mediators to one of the compartments (epithelial or mesenchymal) strengthened the hypothesis of vital paracrine signaling in the regulation of prostate growth and differentiation. Paracrine signaling occurs in both ways and secreted signaling molecules include members of diverse pathways: hedgehog, fibroblast growth factor (FGF), Wnt, Notch, among others (Fig. 7.1) [6]. Despite the intensive prostate growth observed prenatally, the gland does not become static after birth. Prostate maturation is concluded during puberty, whereas androgens-mediated paracrine signaling between mesenchymal/stromal and epithelial compartments are maintained throughout life. This explains, at least in part, the tendency for prostate growth with ageing and its association with elderly disorders, such as benign prostate hyperplasia (BPH) and carcinoma [7, 8]. Some of the developmental genes are also expressed in mature prostatic ducts. These include the homeobox protein Nkx-3.1 (NKX3-1) and the transcription factor SOX-9 (SOX9), which can be observed in luminal and basal cells, respectively, though their expression is lower when compared with phases of active growth [9]. The human adult prostate is a histologically heterogeneous gland. Several alternative models had emerged over decades to describe the adult prostate anatomy [10]. The currently accepted model considers three distinct glandular zones—peripheral, central, and transition—which are surrounded by a fibromuscular stroma [11]. The peripheral zone is the largest (~70% of the prostatic glandular tissue), constituting the site of origin of most prostate carcinomas (70–80%) and other prostatic disorders (e.g. chronic prostatitis and post-inflammatory atrophy) [8]. The central zone is the area that surrounds the ejaculatory ducts, whereas the transition zone surrounds the urethra and is the main responsible for prostate enlargement in BPH [8, 11]. The mature human prostate is composed by 30–50 tubuloalveolar glands specialized in the production and excretion of prostatic fluid. The central lumen is lined by a stratified epithelium of secretory tall columnar or luminal cells (major functional component of the prostate) and basal cells (Fig. 7.1). Other much less frequent cells include stem cells, which are believed to lay primarily in the basal cell population, and neuroendocrine cells, that can result from the differentiation of precursor stem cells. The luminal secretory cells are androgen-dependent and highly differentiated to produce prostatic fluid.1 On the other hand, basal cells are not entirely androgen-dependent (despite being androgen-sensitive) and are believed to sustain ductal integrity and survival of luminal cells [1, 12]. The epithelial compartment is surrounded by a mesenchymal-derived stroma (Fig. 7.1), that differs between the different zones of the prostate and is affected by ageing. Although, the knowledge on

1 AR-mutant

mice, insensitive to androgens, that do not have prostate.

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stromal differentiation is still limited, smooth muscle cells and fibroblasts have been consistently referred as the main components. Immune cells, as well as vascular and neural components can also be found within the stroma [13].

7.2 Prostate Dependence on Androgens and Mesenchymal/Epithelial Interactions: Observations from Tissue Recombinant Experiments Androgens act by binding to the androgen receptor (AR), a ligand-dependent nuclear transcription factor that belongs to the nuclear steroid receptor superfamily. In the absence of functional AR or androgen deficiency, prostate development is largely impaired or nonexistent [2, 4]. As shown by tissue recombinant experiments, the development of a fully functional gland occurs when wild-type (wt)-UGM and wtUGE are combined (Table 7.1). In contrast, androgen-insensitive UGM from testicular feminization (tfm) mouse models (see Footnote 1) originates vagina-like structures when combined with UGE, and the prostate originated from tfm-UGE/wt-UGM combination lacks secretory function [2, 4]. Hence, for prostate adequate growth, the mesenchyme must express AR and be androgen target organ (also corroborated by experiments that used mesenchyme from seminal vesicle and skin origin) (Table 7.1). Interestingly, wt-UGM was reported to induce the differentiation of bladder epithelium (a highly specialized non-glandular, AR-negative epithelium) into prostatic epithelium [4]. Altogether, these findings suggest the androgenic effects on prostatic Table 7.1 Experimental evidences of the requirement of androgens and mesenchymal-epithelial interactions for prostate development in male rodent models. tfm, testicular feminization; UGE, urogenital sinus epithelium; UGM, urogenital sinus mesenchyme; wt, wild-type Tissue recombination

Epithelium None

Mesenchyme

Seminal vesicle

Bladder

None

Undifferentiated epitheliu

Seminal vesicle

Prostate

Skin

Keratinized epithelium

UGM

wt tfm

Fibromuscular tissue

Seminal vesicle

Prostate

UGE wt

tfm

Prostate

Prostate

Vagina

Vagina

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epithelium morphogenesis are mediated through mesenchymal-epithelial interactions, rather than epithelial AR signaling itself. In line with this thought, AR expression was shown to be restricted to the UGM prior to and during prostatic bud formation [4]. Recently, the ability of prostatic epithelium in inducing UGM differentiation into smooth muscle and to regulate smooth muscle architecture was also reported [4]. Two models have been proposed to explain how epithelial budding into the surrounding UGM initiates: the andromedin model and the smooth muscle model [14]. According to the andromedin hypothesis, AR-mediated signaling in the UGM leads to the production of one or more paracrine signaling factors, known as andromedins, that act on the UGE to promote growth and differentiation (Fig. 7.2a). Several molecules have been suggested as candidate andromedins, such as growth factors and members of the Wnt signaling pathway (see Sect. 7.5). The smooth muscle hypothesis defends the existence of localized and reciprocal mesenchymal-epithelial signaling and the presence of a smooth muscle layer that acts like a barrier between UGM and UGE to block excessive budding and outgrowth. In this model, androgens control epithelial budding by regulating the differentiation of smooth muscle [14]. The models are not mutually exclusive and might occur in simultaneous.

Fig. 7.2 Androgen-mediated genomic signaling in the prostate gland. a Circulating testosterone (T) can act directly in epithelial cells by binding to the androgen receptor (AR) or they can activate the AR signaling in stromal cells, resulting in the secretion of growth factors and survival molecules that act in luminal and basal cells from the epithelial compartment. b Androgen-mediated genomic pathway. Figures were produced using Servier Medical Art (https://smart.servier.com)

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7.3 Androgens and Androgen-Mediated Pathways in the Prostate Two natural androgens exist in mammals: testosterone (T), the major androgen secreted from testes, and 5α-dihydrotestosterone (DHT), the main androgen in prostate. T can be found in circulation bound to sex hormone-binding globulin (SHBG) (or other transporter proteins, such as albumin) or in its free and active form, which is able to translocate into prostatic cells (Fig. 7.2a). Although both T and DHT can bind to the AR, T functions as a prohormone in prostate, where it is converted to DHT, a five-fold more potent androgen, by 5α-reductase enzymes (Fig. 7.2b) [15]. Two isoenzymes, 3-oxo-5-alpha-steroid 4-dehydrogenase 1 (SRD5A1) and 2 (SRD5A2), have been identified in both epithelial and stromal cells, but SRD5A2 has been indicated as the main isoenzyme expressed in stromal cells [16]. Androgens not only control fetal and neonatal prostate organogenesis, but they also participate in prostate growth during puberty and regulate the function and homeostasis of the mature adult gland [17, 18]. In fact, the adult prostate remains exquisitely sensitive to withdrawal of circulating androgens and, in response, undergoes tissue atrophy, that can be reversed by their re-administration. To exert their functions, androgens can act directly on the prostatic epithelium via epithelial AR, which was shown to be expressed in the later phases of prostate development, or indirectly via stromal AR-induced secretion of paracrine mediators (Fig. 7.2). Unlike other organs, most of the mesenchymal/stromal-epithelial interactions in prostate are androgen-dependent, but the mechanisms responsible for tissue-specific AR signaling in physiological conditions remain largely unexplored. Nonetheless, the comparison between different androgen-responsive tissues from rodent models (including prostate, kidney, and epididymis) showed minimal overlap among AR-mediated transcription events [19]. Moreover, although several research teams have attempted to clarify which molecules are induced by androgen signaling in the prostate, the reduced similarities between data sets abrogated comparisons (most probably a reflection of the prostate tissue heterogeneity) [20–23]. Additionally, many studies that have examined androgen-mediated gene expression focused on epithelial cells and undervalued the mesenchymal/stromal compartment.

7.4 More for Hormones in the Prostate Prostate morphology and physiology have long been thought to be almost exclusively dependent on hormonal control by androgens. Although androgen-mediated signaling is still considered the central player, it is currently known that other steroid and non-steroid hormones regulate complex gene networks involved in prostate organogenesis and homeostasis. To increase complexity, several studies reported the local production of hormones and hypothesized relevant autocrine and/or paracrine roles beside their conventional endocrine functions.

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7.4.1 Sex Steroid Hormones Estrogens (E) and progesterone (PG) are mostly known for their actions on female reproduction, but they also exhibit important functions on males. The levels of 17βestradiol (E2), the most potent E in circulation, are maintained generally low, with peaks observed only during embryogenesis and aging [24]; while the circulatory levels of PG in males are quite similar to those observed in women (not considering the luteal phase) [25]. With aging, the levels of free circulating T decrease with concomitant increase in the levels of free circulating E2, increasing the E2-to-T ratio. This leads to reactivation of prostate growth and has been associated with malignant transformation [26]. The gonadal production of E in males had long been thought to be restricted to Leydig cells. It was only in the 90s that other structures of the male reproductive tract became recognized as sources of E (in fact, only about 20% are produced by testes) [26–28]. In the prostate, T can be converted into E in a reaction catalyzed by the aromatase enzyme CYP19, which is expressed by stromal cells (Fig. 7.2). Therefore, the E function in the prostate is complex and diverse, involving both endocrine (via pituitary gland to decrease T synthesis in testes) and prostate-specific actions, since E can act independently of the circulatory levels via autocrine and/or paracrine signaling [24]. Like androgens, E and PG can signal through genomic and non-genomic pathways. Classically, E and PG bind to and activate E receptors (ERs) and PG receptors (PGRs), respectively, which act as transcription factors. Unbound receptors localize on the cytoplasm in complexes with heat shock proteins (HSPs). Upon ligand binding, the receptors are released from the complexes and undergo conformational changes that allow their translocation to the nucleus and binding to specific response elements in the DNA, thereby inducing the transcription of target-genes [26, 29]. ERs have two isoforms, ESR1 and ESR2, which are found in higher levels in the prostate than in neighboring structures, such as seminal vesicles and urethra [26]. Differential expression of the receptors is consistent with isoform-specific downstream signaling and distinct actions in the prostate (Table 7.2). Important findings concerning ESR2 signaling have emerged mainly from studies on the prostate. ESR2 is believed to oppose AR signaling on prostatic epithelium to restrain proliferation and inflammation [30]. Prostatic epithelium also expresses ligand-independent ESR2 isoform variants that can act as either constitutive activators, transcription enhancers, or dominant negative regulators of estrogen action. Additionally, E can signal through G protein-coupled estrogen receptors (GPERs) and downstream mitogen-activated protein kinase signaling [26]. PGRs also exist in two isoforms: PGRA and PGRB. Although the expression of PGRs had been reported in stromal cells, particularly in a subset of fibroblasts and smooth muscle cells, their presence in the epithelial compartment is still controversial [31]. In humans, stromal PGR suppresses prostate stromal cell proliferation by inhibiting cell cycle progression, despite isoform-specific regulation of gene transcription has been also described [32].

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Table 7.2 Prostate-specific actions of estrogen receptors isoforms Receptor

Preferential localization

Effects on prostate

References

ESR1

Stroma

(+) Branching morphogenesis (fibroblast ESR1) (+) Stromal cell proliferation (smooth muscle ESR1) (+) Extracellular matrix deposition (smooth muscle ESR1) (+) Stem cell self-renewal (+) Progenitor cell proliferation (+) Prostate squamous metaplasiaa

[26, 30, 151–153]

ESR2

Epithelium

(–) Growth (+) Epithelial cell differentiation (–) Epithelial-mesenchymal transition (+) Apoptosis (–) Inflammation (+) Progenitor cell differentiation (–) Stem cell self-renewal

[26, 30, 154, 155]

a Condition

characterized by the total replacement of the columnar secretory epithelium by layers of stratified squamous cells (reversible following removal of the estrogenic stimulus). It is a direct effect of ESR1 in the prostate, in which ESR1-mediated paracrine signaling is required to elicit estrogen-induced prostatic squamous metaplasia: stromal ESR1 stimulates epithelial proliferation, while epithelial ESR1 mediates epithelial squamous differentiation

7.4.2 Non-steroid Hormones Several non-steroid hormones have also been implicated in prostate development and homeostasis. The prostate is responsive to prolactin (PRL) and oxytocin (OXT), two polypeptide hormones secreted by the central nervous system that are mostly known for their roles during pregnancy and after childbirth [33, 34]. Local production of both PRL and OXT is observed in other tissues, including the prostate, which suggests potential autocrine and/or paracrine actions besides their classical endocrine routes [35]. PRL can be detected in males’ circulation, though at lower levels than in females. It binds to the PRL receptor (PRLR), a member of the class I cytokine receptor superfamily, which is expressed by human prostate cells [35]. The intracellular signaling is then transduced by multiple non-receptor tyrosine kinases that interact with the PRLR’s intracellular domain, for the most common belonging to the tyrosine-protein kinase JAK (JAK)/signal transducer and activator of transcription (STAT) pathway [36]. Similar to other tissues, PRL signaling in the prostate seems to be primarily mediated through the long isoform of the PRLR (despite the presence of a short

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PRLR form is also reported [35]) and its downstream effectors from the canonical PRLR/JAK2/STAT5 pathway. In addition, it may signal through non-canonical pathways involving the AR [33]. Several reports suggest that PRL regulates prostate development, growth, and function [33]. Most studies, nonetheless, used rodent models and lack confirmation in humans. PRL induces growth and differentiation of the prostate epithelium [33, 37]. These effects can be in part explained by a synergistic action with androgens, but also by androgen-independent mechanisms [33]. PRL activates two protein kinase C isoforms, PRKCA and PRKCE, both identified in prostate epithelial cells. PRL-mediated activation of PRKCE is involved in the stimulation of the mitochondrial aspartate aminotransferase (GOT2), a key citrate synthesis regulatory enzyme, and other metabolic entities [34]. Interestingly, these effects are likely to be cell-specific, since responsiveness to PRL varied in rat cells derived from distinct prostate zones [34]. Even slight elevations of PRL serum levels (which tend to occur with aging [38]) were shown to produce significant changes in rats’ prostate epithelium, although no effect was observed regarding sexual behavior [39]. Moreover, it was shown that local overexpression of PRL leads to the expansion of the stem cell subpopulation in rodents’ prostate, which is likely to be involved in malignant transformation [40]. PRL was also shown to increase prostatic uptake and metabolism of T in patients with prostate cancer (PCa) [37]. OXT, oxytocin-associated neurophysin, and oxytocin receptor (OXTR) are present in both epithelial and stromal cells, despite the preferential localization in the epithelial compartment [41]. OXT binds to the OXTR, which belongs to the G-protein coupled receptor (GPCR) superfamily and stimulates multiple signaling pathways [42]. OXT has been suggested to promote prostate growth by stimulating mitosis and inhibiting apoptosis, but the mechanisms are not fully understood. Nonetheless, it has also been implicated in the regulation of prostate steroidogenesis and contractility, being a more effective constrictor than adrenergic agonists. Comprehensive reviews on the roles of OXT in prostate can be found elsewhere [43, 44]. In humans, OXT expression, secretion, and mitogenic activities seem to be induced by complex interactions with androgens and estrogens [45, 46]. These interactions are likely to be maintain in prostatic diseases [47, 48]. The prostate is the major producer and secretor of the thyrotropin-releasing hormone (TRH) and TRH-like peptides among the organs from the male reproductive tract. According to ancient publications, TRH concentrations in rat ventral prostate could exceed those observed in the hypothalamus [49]. Its levels and biosynthesis are hormonally controlled by, for instance, thyroid hormones (TH) and androgens [49–51]—remarkably, this constituted the first evidence that a neuropeptide could be under hormonal control in an extra-hypothalamic site [49]. The existence of a prostate-thyroid axis has been demonstrated in rodent models. Prostatic TRH stimulates the secretion of THs, both triiodothyronine (T3) and its prohormone thyroxine (T4), either directly or via the pituitary gland [52]. TH signaling in the prostate is far from being understood, but TH receptors are expressed by human prostate cells [53]. Proper expression and activity of THs during pre-

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and neonatal periods are determinant for AR status in adult prostate [54]. T4 acts directly on rat prostate gland to increase the release of calcitonin gene-related peptide (CGRP), which is produced by a subpopulation of neuroendocrine cells [55]. Calcitonin levels are also higher in the prostate than in other human organs (apart from those found in the thyroid gland, the main source of circulating calcitonin). These findings suggest that the prostate is the main source of the seminal calcitonin and that this peptide family may play important physiological roles [56]. Locally produced calcitonin may induce the release of the prostatic acid phosphatase (ACPP) from rat prostate explants, possibly by interacting with the prostatic cholinergic system [57]. In contrast to the lack of information regarding physiological roles, calcitonin involvement in prostate carcinogenesis has been widely reported [58]. Conversely, T3 stimulates the activity of prostatic glycosidases, which are important mediators of the glycoprotein metabolism [59]. Prostate hyperplastic conditions have been associated with high levels of T3, that induce cell proliferation and stimulate the expression of prostate-specific antigen (KLK3) [60, 61].

7.5 Prostate Regulation by Growth Factor Signaling In addition to hormones, several growth factors are involved in the regulation of prostate growth, differentiation, and homeostasis. These regulators are mostly locally produced to act via autocrine and/or paracrine signaling (Fig. 7.3). As referred in Sect. 7.3, androgens stimulate stromal cells to synthesize growth and survival factors that are internalized by epithelial cells, where they activate signaling pathways that modulate cell proliferation (Fig. 7.2). The first evidence of such paracrine modulators belongs to the FGF family, but although their assured relevance in prostate physiology, their classification as andromedins is still a focus of major discussion [62]. In fact, no growth factor expressed in the mesenchyme has previously been identified as being a direct target of androgenic regulation (in vitro and in vivo studies are not consistent). In addition to FGFs, a whole range of growth factors has been reported in the prostate, including epidermal growth factors (EGFs), insulin-like growth factors (IGFs), and transforming growth factors (TGFs) [63, 64]. It is worth mentioning that almost all these studies were performed in rodent models, whose prostates exhibit relevant anatomical and histological differences from humans’ [65]. In fact, a study that analyzed human tissues, both fetal and adult (from BPH), reported differential expression of growth factors according to the developmental stage, with relevant differences to the findings in rodents: TGFA, TGFB1, TGFB2, TGFB3, and EGF were observed in human fetal prostate, but not FGF7, or the receptors FGFR2 and EGFR; on the other hand, all growth factors and corresponding receptors were found in human adult prostate, except for FGFR2 [64]. Therefore, extrapolations should be done carefully and efforts should be done to unveil human prostate organogenesis. FGFs are key molecules for organ development in general and comprehensive reviews on their role in prostate development can be found elsewhere [62, 66, 67].

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Fig. 7.3 Simplified schematic representation of the potential growth factor signaling network that regulates prostate homeostasis. Different colors correspond to different signaling pathways; arrows indicate positive regulation and blind-ended arrows indicate negative regulation; squares indicate ligands, rectangles represent receptors, and circles denote binding-proteins. Figures were produced using Servier Medical Art (https://smart.servier.com)

Among its members, FGF7 and FGF10 are particularly important for prostate development. Their highest concentrations are achieved during periods of active prostatic growth, and whilst some level of FGF7 expression is maintained in the organ throughout life, FGF10 expression in growth quiescent adult organ is residual. Both FGF7 and FGF10 act as mitogens for prostate epithelial cell via paracrine signaling (Fig. 7.3). FGFs bind to and activate transmembrane tyrosine kinase FGFRs and the intracellular signaling is transduced mainly by mitogen-activated protein kinases (MAPKs) [68]. FGFR2-mediated signaling is particularly important to maintain epithelial cell proliferation and branching of mice’s developing prostate [66], as well as to prevent stem cell differentiation (Fig. 7.3) [69]. Two isoforms of the FGFR1 were identified in the prostate: FGFR1A is primarily expressed by luminal epithelial cells, whereas FGFR1B, which is believed to have greater affinity to member of the FGF family, is the main variant in smooth muscle cells and basal epithelial cells [70]. Additional members of the FGF signaling have been identified in the prostate, but their roles are not yet clarified (Fig. 7.3). As inferred from the ancient studies that suggested FGF7 as an andromedin, FGF signaling is, at least in part, regulated by androgens [71], but

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FGF7 was also shown to be capable of activating the androgen signaling pathway in epithelial cells [72]. EGF and IGF signaling also have mitogenic effects on the prostate (Fig. 7.3). EGF and related polypeptides, such as TGFA and amphiregulin, are expressed in normal prostate cells and are important regulators of prostate epithelium proliferation during early stages of development and of structural integrity in adult prostate. EGF and TGFA bind to and activate the tyrosine kinase receptor EGFR, which is expressed on the basolateral surfaces of prostate epithelial cells [73]. While EGF is mainly produced by prostate epithelial cells and is secreted to the prostatic fluid at high concentration (the prostatic fluid contains the highest concentration of EGF in the human body), TGFA is secreted in low amounts by prostate stromal cells (it is mainly secreted by tumor cells where it acts via autocrine signaling to promote growth) [73]. Therefore, EGF is believed to be the major EGF-related growth factor in normal adult prostate [74]. When activated, EGFR signals through numerous intracellular pathways [73]. For instance, EGFR is required to interleukin-6 (IL6)mediated activation of MAPK signaling in prostate epithelial cells [75]. IGF signaling is complex; two highly homologous ligands—IGF1 and IGF2— might signal through IGF receptors (IGF1R and IGF2R) and insulin receptor, and their effects are modulated by binding to IGF-binding proteins (IGFBPs) (in fact, most of the free IGF is bounded to IGFBPs). Several members of the signaling have been identified in prostate cells (Fig. 7.3). IGF ligands are produced by prostatic stromal cells (mainly smooth muscle cells) in response to androgen stimulation [76]. Since IGF1R is localized in epithelial cells, IGF signaling works in a paracrine manner to stimulate proliferation and induce basal to luminal differentiation of prostatic epithelial cells [77, 78]. In terms of efficiency, IGF1 was shown to be a more potent mitogen than IGF2 or insulin [63]. The IGF-associated mitogenic effect is primarily mediated by the downstream activation of phosphatidylinositol 3-kinase (PI3K)/RAC-alpha serine/threonine-protein kinase (AKT) and MAPK pathways [79]. In the opposing side of balanced growth regulation is TGFβ signaling (Fig. 7.3). TGFβ is a large gene superfamily that encompass TGFβ, bone morphogenetic proteins (BMPs) and activins. Their effects in the prostate are complex since they can be either inhibitors or stimulators depending on the concentration of the mediators. For instance, TGFB1 inhibits prostate growth, but was shown to promote branching morphogenesis in rat models, which support distinct roles according to prostate zones [80]. The function of TGFβ signaling in the prostate, as well as crosstalk with other signaling pathways was reviewed in detail elsewhere [81–83]. TGFβ ligands bind to the TGFβ receptor 2 (TGFBR2), which then forms heterodimers with TGFBR1. The heterodimeric receptor has serine/threonine kinase activity and activates the downstream intracellular signaling through phosphorylation of protein SMADs. Several studies support the major role of TGFB1, BMP4, and BMP7 ligands in prostate development. BMP signaling through the receptor BMPR1A regulates prostatic epithelial differentiation by controlling the NKX3-1 regulatory gene, one of the earliest markers of prostate development [84]. TGFβ signaling also cooperates with other signaling

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pathways to accomplish its functions. A positive feedback loop involving TGFBR1 and Notch signaling is linked to the homeostasis of prostate basal cells [85].

7.6 Additional Pathways Involved in Prostate Paracrine Signaling 7.6.1 Hedgehog Signaling Pathway Hedgehog signaling pathway regulates complex morphogenic processes during embryonic development of several tissues as well as adult organ homeostasis and regeneration [86, 87]. Three ligands—sonic hedgehog (SHH), Indian hedgehog, and desert hedgehog—bind to the patched (PTC) transmembrane receptor and attenuate the smoothened receptor inhibitor. The intracellular signal transduction positively regulates members of the GLI-Kruppel family of transcription factors, resulting in proliferative stimulus [86, 87]. Hedgehog signaling regulates prostate epithelial proliferation and ductal morphogenesis in a developmental stage- and hormonal environment-dependent way; paracrine signaling promotes epithelial proliferation and budding prenatally, while inhibits these processes postnatally [88]. SHH is the most abundantly expressed and is believed to be the master ligand in the developing prostate, despite functional redundancy among ligands is reported [89]. SHH expression level increases with the onset of ductal budding, peaks during active prostatic bud elongation (when it also re-localizes to sites of active growth of the UGE) and diminishes gradually until residual levels. Both SHH expression level and localization are dependent on the expression of testicular androgens [90]. SHH expression induces the expression of target genes in the adjacent UGM, including members of the Hedgehog signaling, thus establishing an autoregulatory feedback loop [88, 90–92]. Notwithstanding the predominance of paracrine signaling, focal expression of PTC1 and GLI1 in the epithelium of growing prostatic duct tips also support the existence of autocrine signaling in prostate development [93], which is believed to promote the proliferation of progenitor cells at the bud tip [94]. The activity of the Hedgehog signaling pathway in the adult prostate is limited (and less understood), but at least the paracrine mode of action is thought to be preserved (Fig. 7.3). SHH is expressed by basal epithelial cells and binds to receptors in the surrounding stromal stem cells [95]. The association and function of Hedgehog signaling in prostate was recently reviewed by several authors [87, 94, 96].

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7.6.2 NOTCH Signaling Notch signaling pathway mediates cell-cell interactions and is essential to maintain tissue integrity. The mammalian neurogenic locus notch homolog protein family (NOTCH) consists of four highly-conserved transmembrane receptors, NOTCH1-4, which establish physical interactions with ligands that are expressed in the membrane of the neighboring cell. Five canonical ligands are identified in humans: two jagged ligands (JAG1 and JAG2), and three delta-like ligands (DLL1, DLL2, and DLL3). The ligand binding triggers a series of proteolytic cleavages on the receptor and the intracellular domain translocate to the nucleus where it takes part of a transcriptional activation complex that regulates the expression of several target genes [97]. Differential expression of NOTCH signaling components has been shown in the human prostate epithelium, although with some discrepancies among studies. Most reports refer to NOTCH1, whose expression was found in both cultured prostate cells and human prostate tissues, but JAG1, JAG2, DLL1, and NOTCH2 expression was also found in cultured prostate cells [98]. NOTCH signaling is involved in prostate formation, development, and maintenance [99]. Functional studies in rodent models showed that it controls the growth of prostatic progenitor cells, promotes luminal cell differentiation, and downregulates AR activity [100, 101]. NOTCH1 is temporally and spatially regulated in rodent’s prostates during normal development. It is essential for prostatic branching morphogenesis in the developing prostate and for prostatic re-growth in adults [102]. NOTCH1-inducible knockout mice displayed similar prostatic morphological alterations to those with NKX3-1 or RB1 deficiency, showing uncontrolled proliferation of prostatic epithelial cells and impaired differentiation [100]. NOTCH1 knockdown was found to affect multiple signaling cascades, leading to significant mRNA levels increase of proto-oncogene c-Fos (FOS), transcription factor AP-1 (JUN), FGF18, and prostate stem cell antigen [100].

7.6.3 Wnt Signaling Pathway Wnt is a large family of secreted glycoproteins with multiple biological functions. Wnt canonical signaling is catenin beta-1 (CTNNB)-dependent and is triggered by the binding of Wnt ligands to frizzled cell surface receptors (FZD) [103]. Several studies have provided evidence for a central role of canonical Wnt signals in prostate formation [104–106]. A role for canonical Wnt signals in early prostate development has been suggested by the expression of numerous Wnt ligands in both UGM and UGE prior to and during prostate formation [104]. In fact, several Wnt ligands, as well as co-activators of the canonical Wnt pathway, display a sexually dimorphic expression pattern and are specifically detected in male UGS [106]. Furthermore, recent studies have shown that deletion of CTNNB impairs

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prostate specification and bud formation, with only residual levels of the developmental genes NKX3-1 and HOXB13 being detected, suggesting an essential role for canonical Wnt signaling in prostate formation (Fig. 7.1) [105, 107, 108]. Importantly, Wnt signaling regulates many other processes in the developing prostate, including branching morphogenesis, proliferation of epithelial progenitor cells, and luminal cell differentiation [109]. Additionally, Wnt signal might be transduced through non-canonical, CTNNBindependent pathways [110]. The non-canonical protein Wnt-5a (WNT5A) was specifically associated with the regulation of prostate buds’ position and size and it has a negative effect in epithelial proliferation and branching morphogenesis in rodent models [111]. The levels of secreted frizzled-related protein 1 (SFRP1), a Wnt antagonist, were also shown to be rather high in the developing mesenchyme and low in the adult prostate of a mouse model [112]. Indeed, increased SFRP1 expression leads to enhanced epithelial proliferation and decreased expression of secretory proteins, indicating a signal is transduction through a non-canonical pathway [113].

7.7 Is It Reasonable to Talk About a Prostate-Specific Proteome? As reviewed in the prior sections, several signaling pathways were reported as essential for prostate-specific growth and differentiation, since the early steps that guide prostate specification from the UGS. Moreover, the prostate exhibits interesting features, such as its high capacity to store Zn2+ and its tendency to grow with aging. Given that proteins do most of the work in cells, regulating their morphology, function, and metabolism, it is reasonable to ask whether a prostate-specific proteome might exist. According to the Human Protein Atlas database (date of access: 12 July 2018), 73% of all human proteins are expressed in the prostate, and despite the currently unknown existence of prostate exclusive proteins, the expression of 183 genes is higher in the prostate when compared with other tissues. From those, 20 are classified as prostate enriched genes (Table 7.3) and 51 are group enriched genes (most sharing expression with testis and cerebral cortex). Prostate enriched genes are expressed by epithelial cells and most encode for secreted or membranous proteins [114], which is consistent with its dependence on autocrine and/or paracrine signaling.

7.8 So Why does the ‘Prostate-Specific Signaling’ Matter? The prostate gland is associated with major disorders of elderly, such as BPH and carcinoma. Over 80% of men aged more than 80 are likely to harbor BPH [115],

212 Table 7.3 Prostate enriched genes

J. Felgueiras et al. Gene name

Protein name

ACPP

Prostatic acid phosphatase

CHRNA2

Neuronal acetylcholine receptor subunit alpha-2

COL9A1

Collagen alpha-1(IX) chain

KLK2

Kallikrein-2

KLK3

Prostate-specific antigen

KLK4

Kallikrein-4

MSMB

Beta-microseminoprotein

NCAPD3

Condensin-2 complex subunit D3

NEFH

Neurofilament heavy polypeptide

NKX3-1

Homeobox protein Nkx-3.1

OR51E2

Olfactory receptor 51E2

RDH11

Retinol dehydrogenase 11

RFPL2

Ret finger protein-like 2

RLN1

Prorelaxin H1

SLC30A4

Zinc transporter 4

SLC45A3

Solute carrier family 45 member 3

SP8

Transcription factor Sp8

STEAP2

Metalloreductase STEAP2

TGM4

Protein-glutamine gamma-glutamyltransferase 4

TRPM8

Transient receptor potential cation channel subfamily member 8

Data available from https://www.proteinatlas.org/ humanproteome/prostate, version 18 (date of access: 12 July 2018) [114]

whereas PCa is the most prevalent cancer in men worldwide [116]. Indeed, these facts, together with the increased population aging, constitute major health concerns. BPH and PCa are characterized by abnormal growth of the gland. Hence, they might result, at least in part, from the unbalanced action of the signaling pathways involved in prostate development (giving emphasis to the need of a deeper understanding of prostate biology).

7.8.1 Androgen Signaling as Target for Prostate Cancer Therapeutics Similar to normal cells, AR signaling remains essential for growth and survival of PCa cells [117]. This dependency is exploited in androgen deprivation therapy (ADT), the mainstay therapeutic regimen, which encompasses surgical

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(orchidectomy) or chemical castration (by using luteinizing hormone-releasing hormone/gonadotropin-releasing hormone agonists or antagonists). This treatment aims to decrease circulating testicular androgens, depriving AR of its activating ligand and consequently hindering its pro-survival effects. Since adrenal glands also produce reduced amounts of androgens, the ADT is frequently complemented with the administration of antiandrogens [118]. These drugs act by competing with endogenous androgens for binding to the AR and often preclude AR access to the nucleus and to its transcriptional targets. Although, initially ADT is effective, about one third of the patients will relapse in 2–3 years with castration-resistant prostate cancer (CRCP), for which there is still no cure available. Even though CRPC becomes refractory to ADT, most CRPCs still express AR and its target KLK3 which implies that AR signaling is still active [119–121]. The reactivation of the AR signaling can be achieved through multiple mechanisms that may be broadly divided into ligand-dependent and ligand-independent. Liganddependent mechanisms include AR amplification and overexpression (leading to increased sensitivity to low levels of androgens), intra-tumoral steroidogenesis (de novo synthesis of androgens) and increased expression of AR co-factors (which facilitate AR’s transcriptional function). Ligand-independent mechanisms include AR mutations (broaden AR’s ligand-specificity) and constitutively active AR variants [116]. Hence, AR remains the main target in the context of CRPC. Based on improved survival in Phase III clinical trials, two drugs have been approved for CRPC patients’ treatment, abiraterone [122, 123] and enzalutamide [124, 125], which can be used in pre-or post-chemotherapy settings. Abiraterone is a selective and potent inhibitor of steroid 17-alpha-hydroxylase/17, 20 lyase (CYP17A1), an enzyme that is necessary for androgen biosynthesis and enzalutamide, a second-generation antiandrogen that competes with androgens for the binding to AR. Nevertheless, this therapy is not without caveats since not all CRPC patients respond to these drugs and resistance to treatment may develop with time [116]. Importantly, metastatic CRPCs frequently display AR expression heterogeneity, with cells exhibiting different AR expression levels or even AR loss. In fact, PCa with reduced, or even absent, expression of AR is increasingly more common, especially in patients previously treated with abiraterone and enzalutamide [126–128]. Thus, suggesting that at some point CRPCs evolve to become independent of the AR signaling, which poses serious concerns, due to the lack of an effective therapeutic option.

7.8.2 The Complex Crosstalk Between Signaling Pathways in PCa Non-androgenic pathways have also been shown to activate the AR. AR signaling may be activated by the crosstalk with signaling pathways (e.g. MAPK and PI3K/AKT) that are activated because of an extrinsic signal, namely growth factors

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(e.g. EGF and IGF1) or cytokines (IL6 and IL8). Several peptide growth factors were reportedly increased during progression to CRPC and were suggested to regulate AR’s transcriptional activity in androgen-depleted conditions [129]. They often do so by promoting the phosphorylation of AR itself or of its co-factors, increasing AR transcription [130]. On the other hand, AR signaling may also condition the expression of growth factors and a cooperative effect between growth factors and AR can be observed. This is the case of the androgenic induction of EGFR [131]. Cooperation between androgens and EGF promotes proliferation by stimulating the activity of cyclin-dependent kinase 2 (CDK2), which facilitates G1-S transition [132]. Furthermore, IL6 signals through EGFR modulate the expression of androgen-regulated KLK3 gene [133]. The activation of AR by the EGFR-related receptor tyrosine-protein kinase erbB-2 (ERBB2) also seems to be required for optimal transcriptional activity of AR in PCa cells [134]. Indeed, heregulin-mediated ERBB2 activation induced activated CDC42 kinase 1 (TNK2)-driven AR phosphorylation at tyrosine (Tyr) residues 267 and 363, promoting AR´s transcriptional activity independently of ligand stimulation [135]. IGF1/IGF1R is an important growth promoting signaling pathway. In PCa, IGF1 signals via PI3K/AKT and MAPK/RAS GTPase signaling pathways to promote survival by phosphorylation (and consequent inactivation) of Bcl2-associated agonist of cell death (BAD) [136]. Additionally, IGF1 or insulin promote PI3K/AKT-mediated phosphorylation of forkhead box protein O1 (FOXO1), an AR co-repressor, rendering it inactive and consequently enhancing AR´s transcriptional activity [136]. Conversely, upon ligand stimulation, AR can also regulate IGF1/IGF1R signaling by modulating the expression of IGF1 [137], IGF1R [138] in PCa cells and IGFBPs [139] in normal fibroblasts. In normal prostate, TGFβ released by stromal cells inhibits cell growth and promotes apoptosis, by inducing the expression of apoptosis regulator BAX (BAX), cyclin-dependent kinase inhibitor 1B (CDKN1B), IGFBP3 and activating caspase-1 (CASP1). Conversely, TGFBR2 loss or mutation renders PCa cells refractory to the growth inhibitory effect of TGFβ signaling. Indeed, direct interactions between AR and TGFβ signaling mediators, mothers against decapentaplegic homolog 3 (SMAD3) and 4 (SMAD4), have been reported in vivo and in vitro. Nevertheless, there is still controversy on whether SMAD3 has a negative or positive effect on the AR’s transcriptional activity and on the dependency of a SMAD3/SMAD4 complex for a repressive effect [140, 141]. The involvement of AR in the apoptotic effects mediated TGFβ signaling was demonstrated in a castration-resistant PCa cell line in which in the absence of DHT, AR expression reduced the TGFB1/SMAD transcriptional activity, thus preventing TGFB1-induced apoptosis and growth inhibition [78]. In contrast, in an androgen-dependent PCa cell line, the treatment with DHT enhanced TGFβ-induced apoptosis, via CASP1 activation and targeting of the apoptosis regulator Bcl-2 (BCL2) [142]. Concurrently, bounded AR inhibits TGFβ signaling by preventing SMAD3 binding to the SMAD-binding elements [143]. In parallel, FGF/FGFR expression changes also seem to be associated with progression to CRPC. AR may alter the FGF1, FGF2, FGF8 and FGF10 expression patterns’ in both prostate tumor epithelial and stromal cells [78]. Remarkably, AR is

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up-regulated by paracrine FGF10 [144], which can induce the expression of FGF2 and FGFBP, hence facilitating the FGF-induced survival of PCa cells [145]. Moreover, VEGF, an angiogenic cytokine whose expression is induced by hypoxia-inducible factor 1-alpha (HIF1A) in response to low oxygen tension (hypoxia), might be activated by the AR. Specifically, in androgen sensitive tumors, AR promotes angiogenesis by inducing HIF1A which in turn activates VEGF [146]. Nonetheless, in androgen depleted conditions, intracellular reactive oxygen species (ROS) activate a small GTPase, Ras-related protein Ral-A (RALA), which, in turn, promotes vascular endothelial growth factor C (VEGFC) upregulation. As a result, VEGFC increases the expression levels of BAG family molecular chaperone regulator 1 (BAG1), an AR co-activator, thus contributing to increased AR transcriptional activity [147]. Interestingly, pro-inflammatory cytokines, IL6 and IL8, also seem to play a role in AR activation and were found overexpressed in PCa. IL6 can act through the activation of different signaling pathways, namely c-Jun NH2-terminal kinase (JNK)/STAT3, MAPK and PI3K/AKT [148]. IL6 treatment of androgen-dependent PCa cells led to AR transactivation through MAPK/STAT3 signal transduction pathways [133]. Additionally, IL6 may also engage SRC-mediated direct phosphorylation of Tyr-534 of AR, which is a critical residue of AR’s transcriptional activity in the presence of low doses of androgens [149]. Finally, IL-8 also activates AR and confers androgen-independent growth via proto-oncogene tyrosine-protein kinase Src (SRC) and focal adhesion kinase 1 (PTK2) [150]. Altogether, these evidences suggest that despite being a central mediator in prostate biology, AR does not act alone; instead, it takes part of a complex interacting network capable of regulating the most diverse molecular events and biological processes that underlie cancer hallmarks. Therefore, the apparent re-awakening of key developmental signaling pathways and crosstalk during malignant transformation supports the relevance of characterizing, in-depth, such cascades in view of empowering translational medicine in the context of PCa. Acknowledgments The authors would like to acknowledge the support of the Institute for Biomedicine—iBiMED (UID/BIM/04501/2013 and UID/BIM/04501/2019) and POCI-01-0145FEDER-007628), supported by the Portuguese Foundation for Science and Technology (FCT), Compete2020 and FEDER fund, and of the Programa Operacional Competitividade e Internacionalização (POCI), in the component FEDER, and by national funds (OE) through FCT/MCTES, in the scope of the project HyTherCaP (PTDC/MECONC/29030/2017). This work was also financed by FEDER funds through the “Programa Operacional Competitividade e Internacionalização— COMPETE 2020” and by National Funds through the FCT—Fundação para a Ciência e Tecnologia (PTDC/DTPDES/6077/2014). Juliana Felgueiras was supported by an individual grant from FCT of the Portuguese Ministry of Science and Higher Education (SFRH/BD/102981/2014). The HyTherCaP project is also acknowledged for the junior researcher contract of Vânia Camilo.

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Chapter 8

Testicular Signaling: Team Work in Sperm Production Joana Santiago, Daniela Patrício, and Joana Vieira Silva

Abstract The male gonads, testis, have two main functions: testosterone production (steroidogenesis), a fundamental hormone for the development and maintenance of several physiological functions; and sperm production (spermatogenesis), essential for male fertility. The synthesis of both products is mainly regulated by endocrine hormones, synthesized in the hypothalamus and pituitary gland, and paracrine signals. This chapter will explore the signaling pathways involved in testosterone production by Leydig cells. We will also discuss both classical and non-classical pathways of testosterone action in spermatogenesis, and the contribution of follicle stimulating hormone to spermatogenesis maintenance. Finally, the signaling pathways involved in blood-testis-barrier regulation as well as other paracrine signals involved in spermatogenesis control will be explored. Despite these pathways occur in most somatic cells, they have a unique role in regulating the most peculiar and exceptional process in one of the most complex tissue in male body.

J. Santiago and D. Patrício—Contributed equally. J. Santiago · D. Patrício · J. V. Silva (B) Laboratory of Signal Transduction, Department of Medical Sciences, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal e-mail: [email protected] J. Santiago e-mail: [email protected] D. Patrício e-mail: [email protected] D. Patrício Department of Chemistry, CICECO, Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal J. V. Silva i3S - Instituto de Investigação e Inovação em Saúde, University of Porto, Porto, Portugal Laboratory of Cell Biology, Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_8

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Keywords Steroidogenesis · Endocrine signaling · Spermatogenesis · Blood-testis-barrier

Abbreviations 17OH-Allo 17OH-DHP 17OHPreg 17OHProg AA AC AKR1C2/4 AKR1C3 AKT AKT1S1 AR ARE ATP BCL6B BTB CAM cAMP CAR CDC42 CDK1 CLDN CRE CREB CREM CSF1 CXCL12 CXCR4 CYP11A1 CYP17A1 CYP19A1 DAG DHEA DHT DMRT DNA EGF EGFR EGR1

17OH-allopregnanolone 17OH-dihydroprogesterone 17α-hydroxypregnenolone 17α-hydroxyprogesterone Arachidonic acid Adenylyl cyclase Aldo-keto reductase family 1 member C2/4 Aldo-keto reductase family 1 member C3 RAC-alpha serine/threonine-protein kinase Proline-rich AKT1 substrate 1 Androgen receptor Androgen response elements Adenosine triphosphate B cell CLL/lymphoma 6 member B Blood-Testis-Barrier Calmodulin Cyclic adenosine monophosphate Coxsackievirus and adenovirus receptor Cell division control protein 42 homolog Cyclin D1 Claudin cAMP-response element Cyclic AMP-responsive element-binding protein 1 cAMP-responsive element modulators Colony stimulating factor Stromal cell-derived factor 1 C-X-C chemokine receptor type 4 Cholesterol side-chain cleavage enzyme Steroid 17-alpha-hydroxylase/17,20 lyase Aromatase Diacylglycerol Dehydroepiandrosterone Dihydrotestosterone Double sex- and mab-3 related transcription factor Deoxyribonucleic acid Epidermal growth factor Epidermal growth factor receptor Early growth response protein 1

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ER ERBB4 ERK ES ETV5 FAK FDX1 FDXR FGF FSH FSHR GATA-4 GDF9 GDNF GFRA1 GNE GnRH GPRC HSD17B3 HSD3B2 HSP ID4 IGF IL IMM INSL3 IP3 JAM KITLG KO LC LDH LDHA LH LHCGR LHX1 LICH MAPK MMP9 mTOR mTORC1 mTORC2 NF-κB NRG OCLN

Endoplasmic reticulum Receptor tyrosine-protein kinase erbB-4 Mitogen-activated protein kinase Ectoplasmic specialization ETS translocation variant 5 Focal adhesion kinases Ferredoxin-1 Ferredoxin reductase Fibroblast growth factor Follicle stimulating hormone Follicle stimulating hormone receptor Transcription factor GATA-4 Growth/differentiation factor 9 Glial cell-derived neurotrophic factor GDNF family receptor alpha-1 Guanine nucleotide exchange factor Gonadotropin releasing hormone G protein-coupled receptor 17β-hydroxysteroid dehydrogenase type 3 3β-hydroxysteroid dehydrogenase type II Heat shock proteins DNA-binding protein 4 Insulin-like growth factor Interleukins Inner mitochondrial membrane Insulin-like factor 3 Inositol 1,4,5-trisphosphate Junctional adhesion molecules Stem cell factor/kit ligand Knock out Leydig Cell Lactate dehydrogenase L-lactate dehydrogenase A chain Luteinizing Hormone Luteinizing hormone/choriogonadotropin receptor LIM homeobox 1 Cholesteryl ester hydrolase Mitogen-activated protein kinase Matrix metalloproteinase-9 Serine/threonine-protein kinase mTOR Mammalian target of rapamycin complex 1 Mammalian target of rapamycin complex 2 Nuclear factor-kappa B Neuregulin Ocludin

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OPRL1 PDPK1 PDE PGE2 PIK3 PIP2 PLA2 PLC POR PRKA PRKB PRKC RA RAF RAR REC8 RHOX5 RNA RPS6K RoDH RXR SC SCAR SFK SRC SRD5A2 SRF SSC StAR STF1 TGF TJ TNF VEGF VIM γ-GTP

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Nociceptin receptor Phosphoinositide dependent protein kinase 1 Phosphodiesterase Prostaglandin E2 Phosphatidylinositol 3 kinase Phosphatidylinositol 4,5-bisphosphate Phospholipase A2 Phospholipase C P450 oxidoreductase Protein kinase A Protein kinase B Protein kinase C Retinoic acid RAF proto-oncogene serine/threonine-protein kinase Nuclear retinoic receptor Meiotic recombination protein rec8 Homeobox protein Rhox5 Ribonucleic acid Ribosomal protein S6 kinase Retinol dehydrogenase Retinoic X receptor Sertoli Cell Sertoli cells-specific androgen receptor SRC family kinase Proto-oncogene tyrosine-protein kinase Src 5α-reductase type II Serum response factor Spermatogonial stem cells Steroidogenic acute regulatory protein Steroidogenic factor 1 Transforming growth factor Thigh junctions Tumor necrosis factor Vascular endothelial growth factor Vimentin γ-glutamyl peptidase

8.1 Introduction Spermatozoa and androgen production (spermatogenesis and steroidogenesis, respectively), are the two main functions of the male gonad, testis. Both functions are crucial for male fertility maintenance and are tightly regulated by several signaling mechanisms. The huge number of pathways occurring in testis and the complex

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interplay between them is not surprising since the seminiferous epithelium is one of the most complex epithelial tissue in the body. Due to the limitation in using human testis for experimental purposes, most of the experimental work on spermatogenesis and testicular endocrine function was performed in two common rodent models, mice and rat. This chapter describe the main signaling pathways regulating testis function, the key players and outputs.

8.2 Brief Introduction to Testicular Structure and Function 8.2.1 The Testis Testes, the male sexual glands, are ovoid structures housed within the scrotum, a separated compartment with 2–4 °C lower than the body temperature, that have both endocrine and exocrine functions [1]. They are surrounded by two different layers of protective tissue, the outer tunica vaginalis and the tunica albuginea. The tunica albuginea is a dense connective tissue layer that covers the testis and invaginates the testicular tissue, creating septa thus forming the lobules [2]. The testicular parenchyma is composed of 1–3 highly convoluted seminiferous tubules within each lobule, where the production of spermatozoa occurs (spermatogenesis). The seminiferous epithelium is complemented with sustentacular Sertoli cells (SC) and a stratified layer of developing male germ cells—spermatogonia, spermatocytes and spermatids—responsible for 90% of testis volume [3]. Beside the role in the maintenance of the integrity of seminiferous epithelium, SC have an important role in the regulation of germ cell proliferation, differentiation and nutrition [4]. Adjacent to the seminiferous tubules are the interstitial Leydig cells (LC) that may be found either in small groups or alone, mostly located around the blood vessels in a perivascular sheath. The LCs are responsible for testosterone production (steroidogenesis) in the presence of luteinizing hormone (LH) [5]. Small amounts of estrogens and insulin-like factor 3 (INSL3) are also produced by LCs. Two types of LCs were identified: (i) Fetal LCs, responsible for the production of androgens needed for masculinization of the fetus and (ii) adult LCs that are definite and functional LCs in the adult testis, responsible for the testosterone production essential for masculinization and maintenance of spermatogenesis [6].

8.2.2 An Overview of Spermatogenesis Spermatogenesis is a highly regulated process that takes place within the seminiferous tubules and consists in the development of a diploid spermatogonia into a highly specialized haploid male gamete (spermatozoa) through a complex series of divisions. Spermatogenesis usually takes around 64 days in human and can be divided in four

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key phases: (1) self-renewal and differentiation of spermatogonia, (2) meiosis, (3) spermiogenesis, and (4) spermiation. The first phase consists in self-renewal of progenitor A dark and A pale spermatogonia and differentiation of A pale spermatogonia through mitosis producing type B spermatogonia. The type B spermatogonia suffer a meiotic division and two primary spermatocytes are produced. The primary spermatocytes divide into two secondary spermatocytes, that then divide into two equal haploid spermatids. Spermatids differentiate into a spermatozoon under a process called spermiogenesis. Spermiogenesis is a complex process where the spermatid suffers a intricate sequence of events, like acrosome and flagellum development; chromatin condensation by the histones substitution by protamines; phagocytosis of excess cytoplasm, known as residual bodies; tail development and reorganization of cellular organelles such as centrioles and mitochondria. Finally, the mature spermatozoa are released from the protective SC into the lumen of the seminiferous tubule in a process called spermiation [7, 8].

8.2.3 The Hypothalamic-Pituitary-Testicular Axis The testicular function is regulated by hormones, growth factors and factors secreted by immune cells that play a pivotal role in management of spermatogenesis and steroidogenesis. In boys, the hypothalamic-pituitary-gonadal axis becomes reactivated around 12 years old. The hypothalamic gonadotropin releasing hormone (GnRH) stimulates gonadotropin secretion (LH/FSH) by the pituitary gland and the pubertal testis development is activated. Follicle stimulating hormone (FSH) bind to its receptors located on SC, responsible to promote spermatogenesis and stimulate activin and inhibin’s secretion. Activins induce FSH biosynthesis [9] and inhibin regulates the secretion of FSH in a negative feedback to the anterior pituitary [10]. LH acts on LH receptors located at LCs to promote testosterone production. Once testosterone is released in the bloodstream and reaches a sufficiently high level, it acts on the pituitary and hypothalamus via a negative feedback loop to suppress GnRH and LH production, which will in turn lead to a passive reduction in testosterone production by LCs [11] (Fig. 8.1).

8.3 Signaling Pathways Regulating Steroidogenesis It is well known that the main product of steroidogenesis is testosterone. Most of testosterone on the male body is produced in testes (~95%) with a small portion being produced in the adrenal cortex and in peripheral organs through conversion of precursor steroids [12]. These steroid hormones regulate several developmental and physiological processes from fetal to adult life [13, 14]. The mechanisms involved in androgens biosynthesis will be described in detail.

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Fig. 8.1 Hypothalamic–pituitary–testicular axis. The gonadotrophin releasing hormone (GnRH) is released by the hypothalamus stimulating the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the pituitary gland. LH acts on LCs stimulating the biosynthesis of testosterone and FSH is recognized by FSH receptors in SCs that has an important role in spermatogenesis. When testosterone is released in the bloodstream inhibits LH secretion from the pituitary—negative feedback. Besides the effect of testosterone, this axis is also regulated by inhibin and activin produced in SCs, that regulate the levels of FSH

8.3.1 Classical Pathway On LCs, activation of steroidogenesis occurs after LH binding to high affinity receptors, called LH/choriogonadotropin receptor (LHCGR), on the cells surface. LH induces a coupling reaction between receptor and adenylyl cyclase (AC) which results in conversion of ATP to cAMP [15]. cAMP is then degraded by a phosphodiesterase (PDE) to 5 AMP or binds to the regulatory subunit of a protein kinase. It will stimulate protein kinases such as protein kinase A and C (PRKA and PRKC) that phosphorylate intracellular proteins which will activate the sequence of events involved in steroid biosynthesis [16]. PRKA phosphorylates proteins such as cholesteryl ester hydrolase (LICH) and transcription factors like steroidogenic factor 1 (STF1), transcription factor GATA-4, and cAMP response-element binding protein (CREB)/cAMP response element modulator that activate genes involved in steroidogenesis, including steroidogenic acute regulatory protein (StAR) [15]. On the other hand, LHstimulated LCs produce insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) that can induce StAR expression without altering the levels of cAMP, through the MAP kinase cascade [15]. Testosterone is an androgen, and like other steroid hormones, is derived from cholesterol. Most of cholesterol supply for testosterone production comes from plasma low-density lipoproteins derived from dietary cholesterol [17]. The first step for testosterone production involves the transport of cholesterol molecules to the inner mitochondrial membrane (IMM) and StAR facilitates the movement of cholesterol across the membranes [18]. Cholesterol is then converted into pregnenolone by cholesterol side-chain cleavage enzyme (CYP11A1), ferredoxin-1 (FDX1) and ferredoxin reductase (FDXR) in the IMM. The enzyme steroid 17-alphahydroxylase/17,20 lyase (CYP17A1) is then activated by 17a-hydroxylase activity

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and P450 oxidoreductase (POR) and converts pregnenolone through the delta 5 pathway to 17α-hydroxypregnenolone (17OHPreg). CYP17A1 thus converts 17OHPreg into dehydroepiandrosterone (DHEA), that is converted into testosterone through the intermediacy of androstenediol or androstenedione by the 17β-hydroxysteroid dehydrogenase type 3 enzyme (HSD17B3/AKR1C3) and 3β-hydroxysteroid dehydrogenase type II enzyme (HSD3B2), respectively (Fig. 8.2) [17]. A small amount of testosterone and/or androstenedione may be converted to estrogens by aromatase (CYP19A1). Recent immunohistochemical findings suggest that the LC expresses small amounts of 5α-reductase type II (SRD5A2) to convert testosterone into dihydrotestosterone (DHT). Compared to testosterone, DHT is more effective since it has an higher affinity for the androgen receptor (AR) [19].

Fig. 8.2 Androgen synthesis pathways in Leydig cell. The classic pathway for testosterone biosynthesis in the LC (black) begins when LH binds the LH receptor and AC is activated, converting ATP into cAMP. Cholesterol is transported to the inner mitochondrial membrane by StAR. At the inner mitochondrial membrane cholesterol is cleaved to pregnenolone by CYP11A1 and its redox partners adrenodoxin/adrenodoxin reductase. Pregnelone is then converted by CYP17A1 to 17OHPreg and DHEA in the ER, for the second reaction is required CYB5. DHEA is then converted either through the intermediacy of androstenediol or androstenedione to testosterone by the enzymes HSD3B2 and AKR1C3/HSD17B3. Backdoor pathway (grey) for DHT is mainly ensured by SRD5A1 and HSD3B2. HSD3B2 produce 17OHProg from 17OHPreg or from Pregnenolone through Prog. SRD5A1 will convert 17OHP to 17OH-DHP, and HSD3B2 activity of AKR1C2/4 will yield 17OH-Allo, it is converted to androsterone by CYP17. Further conversion of androsterone by AKR1C3/HSD17B3 leads to androstanediol and by AKR1C2/4 or RoDH finally to DHT

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8.3.2 Backdoor Pathway An alternative pathway for DHT production was first described in Tammar wallaby testis, now known has the backdoor pathway. It diverges from the common precursors of the classic pathway of testosterone biosynthesis with DHT being produced without the testosterone as an intermediate [20]. Firstly, cholesterol is transformed by CYP11A1 into pregnenolone that is further converted by 3a-HSD into progesterone. Progesterone is then 5α-reduced by SRD5A1 in 17OH-dihydroprogesterone (17OH-DHP) that lately is 3α-reduced by aldo-keto reductase family 1 member C2/4 (AKR1C2/4) to 17OH-allopregnanolone (17OH-Allo). 17OH-Allo is an excellent substrate for CYP17A1 to form androsterone. Androsterone is further converted into androstanediol by AKR1C3 that is then oxidized into DHT by AKR1C2/4 or RoDH. Alternatively, androsterone may be first oxidized to androstenedione before being converted to DHT [7] (Fig. 8.2). The production of DHT through the “backdoor pathway” in the human testis was confirmed, since patients with POR deficiency revealed steroid metabolites needed for this pathway (like 17OH-Allo) in an urine steroid analysis. [21]. Another proof for a role of the backdoor pathway in human sexual development came from 46, XY patients suffering from moderate to severe undervirilization. Those patients have mutations in the genes AKR1C2/4, but the classical pathway is not compromised. Since these patients showed an incomplete masculinization, with abnormal differentiation and development of the testis despite testosterone being produced by the classical pathway, it is clear that one pathway cannot compensate the other [22].

8.4 Endocrine Signaling in Sertoli Cells and Spermatogenesis Spermatogenesis is highly dependent upon the interaction between somatic SCs and germ cells. SCs physically support the developing germ cells during their maturation, supplying nutrients (lactate) and growth factors (stem cell factor, TGFα and TGFβ, IGF1, FGF and EGF) as well as hormones (Mullerian-inhibiting substance and inhibin) that regulate the development of the male reproductive structures or feedback to regulate the hormonal signals [23]. They also provide a specialized and safe environment within the seminiferous tubules, protecting germ cells from immune attack. Indeed, adjacent SCs form tight junctions (TJs) with each other avoiding the crossing of molecules larger than 1000 Daltons to the inside of the tubule [24]. The TJs between all SCs within the tubule form the Blood-Testis-Barrier (BTB) [25]. Since within the seminiferous tubules only SCs have receptors for both FSH and testosterone, this type of cell is the main target of the hormonal signal that regulate spermatogenesis, transducing signals from hormones into the production of factors required for germ cells maturation [26]. Additionally, synergistic actions likely occur within the SC. The main signaling pathways activated by FSH and testosterone are described in this section.

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8.4.1 FSH Signaling The number of SC determine the spermatogenic potential. SC proliferate during the fetal and early neonatal period and then immediately before puberty [27]. At puberty, proliferation stops, and the cells acquire a differentiated phenotype, capable of support spermatogenesis. Disturbances in SC proliferation usually leads to small testis and diminished sperm output [27]. FSH constitute an important endocrine controller in the post-natal development of testis. In fact, this hormone sustains SC mitotic activity and promotes their differentiation together with other endocrine, paracrine and autocrine factors, such as interleukin-1β (IL1β), IGF1 or glial cellderived neurotrophic factor (GDNF) [28]. The importance of FSH to male fertility was revealed by the FSHβ and FSH receptor (FSHR) KO models [29]. The animals showed decreased testicular volume, but spermatogenesis was not completely absent, so the mice were fertile [30, 31]. Both KO mice showed a reduction in the number of spermatogonia, spermatocytes and spermatids suggesting that FSH acts to increase the number of spermatogonia and the entry of these cells into meiosis. In addition to pre-meiotic effects, FSH also seems to play an important role in the normal completion of spermiogenesis. In FSHR KO mice, ejaculated spermatozoa showed alterations in DNA condensation and an increase in morphological abnormalities [32, 33]. These studies also indicate that per se, FSH is not necessary for spermatogenesis but appears to be required for adequate viability and morphology of spermatozoa, in synergy with intra-testicular testosterone. In adults, FSH stimulates mitotic and meiotic DNA synthesis in spermatogonia and preleptotene spermatocytes and acts as a survival factor for these premeiotic germ cells by acting on SCs [3]. By stimulating these steps, FSH indirectly promotes the meiotic steps and regulates spermatogenesis.

8.4.1.1

FSH—FSH Receptor Binding

FSH is a member of the glycoprotein hormone family that acts by binding to the FSHR, a G protein-coupled receptor (GPRC) on the surface of SCs [26]. In adult human and rat, serum FSH levels remain relatively stable, while the expression of FSH receptor on SCs varies in a cyclical and stage-specific manner [34]. When FSH interacts with the receptor it undergoes a conformational change acquiring a rigid structure. After binding to the receptor, a structural alteration in the membrane domain of the receptor also occurs allowing guanine nucleotide exchange in associated Gs proteins. Ligand binding can also result in dimerization of FSHR through contacts confined to the cytoplasmic domains, contributing to the signaling. FSHR causes receptor coupled G proteins to activate AC and increase intracellular cAMP levels [34]. FSH binding to its receptor can activate at least 5 known signaling pathways in SCs, described in Fig. 8.3 being expected some crosstalk between them to reach the final desired consequence.

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MAP Kinase Pathway

The first action of FSH on testis occurs postnatally, when SCs acquire FSHR expression. FSH seems to stimulate prenatal and prepubertal proliferation of SCs fixing the final cell number. Interestingly, FSH requires the insulin/IGF signaling pathways to mediate its effects on pubertal SC proliferation [35]. Crepieux and colleagues showed that, during puberty, the MAP kinase pathway is activated in response to FSH in cultured rat SCs [36]. MAP kinase and ERK kinase (MAPK) were activated mainly through cAMP interactions with guanine nucleotide exchange factors (GNEs) and activation of Ras-like G proteins (Fig. 8.3). ERK is capable of activating transcription factors such as serum response factor SRF, c-Jun and Creb. Since FSH only activates MAPK in cultured SCs of rats with 5–11 days old, and not in mature SCs, it was proposed that this FSH-regulated pathway may not be involved in spermatogenesis maintenance in adult rats [37]. On the other hand, this study suggested that MAP kinase pathway stimulates SC proliferation, which occurs in the first 15 days after birth. The induction of cyclin D1 (CDK1) and transcription factor E2F, two cell-cycle promoters, also support the mitogenic effects of FSH mediated by MAP kinase signaling during puberty [36].

Fig. 8.3 Signaling pathways activated by FSH in Sertoli cell. Briefly, FSH binding to FSH receptor (FSHR) leads to adenylyl cyclase (AC) activation by receptor coupled G proteins (Gs) and increase in cellular cAMP levels. The increase in cAMP levels results in the activation of PRKA that phosphorylate several proteins in the cell and regulates the expression and activity of transcription factor such as CREB. cAMP elevation also causes an influx of Ca2+ into SCs, resulting in the phosphorylation of cytoskeleton proteins, like VIM, and the regulation of CREB, by calmodulin and CaM kinases. During puberty, FSH also acts activating the MAP kinase cascade and ERK kinase, particularly by the activation of Ras-like G protein. ERK activates transcription factors including CREM, SRF and c-Jun. The cAMP elevation also cause activation of PIK3 and then PDK1 and AKT in SCs. Finally, FSH also mediated the induction of PLA2 and consequent release of arachidonic acid (AA). The metabolism of AA leads to the production of eicosanoids, as PGE2, that can act as intracellular or extracellular messenger

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cAMP-PRKA Pathway

In adult rats, pre-spermatogonia (gonocyte) maturation by FSH acts as an important antiapoptotic survival factor for spermatogonia, spermatocytes and spermatids [38]. Increased intracellular cAMP levels induced by FSH binding to its receptor results in the release of the catalytic subunit of PRKA from repressing subunits allowing the phosphorylation of target proteins [39]. One of those targets is a class of transcription factors that binds to cAMP response elements (CREs), in particular CREB transcription factor that is rapidly activated after being phosphorylated on Ser133 by PRKA (Fig. 8.3) [39]. The importance of CREB phosphorylation for fertility was identified using in vivo studies. A mutant CREB isoform that could not be phosphorylated at Ser133 was overexpressed in rat SCs, resulting in disrupted spermatogenesis in more than 40% of the seminiferous tubules due to spermatocytes apoptosis and loss of at least 75% of round spermatids [40]. Once phosphorylated, CREB activates transcription of several genes, including nociceptin, involved in spermatocytes meiosis (see Sect. 6.2.1). Additionally, FSH signaling through PRKA pathway has been associated with the expression of several cyclic AMP-responsive element modulators (CREM) isoforms that are expressed in spermatocytes and spermatids and are crucial for survival [41, 42]. FSH also acts via cAMP and PRKA to block RAS activation of RAF1 and thus, ERK phosphorylation, a target of the non-classical signaling pathway (see Sect. 8.4.2.3) in mature SCs [24, 36]. Additionally, SCs stimulation by FSH results in the release of NF-κB from its cytoplasmic anchoring partner IκB due to PRKA phosphorylation of IκB that marks it for degradation. Free NF-κB is then translocated to the nucleus, where it acts as a transcription factor, similar to CREB [37].

8.4.1.4

Calcium Pathway

Some evidences exist that FSH supports meiosis and spermiogenesis by regulating the adhesion complexes between germ cells and SCs [43]. In particular, Ca2+ seems to play and important role in BTB maintenance, mostly at the TJ dynamics level [44]. After stimulation of SC with FSH, the levels of cAMP rise which also causes an influx of Ca2+ into SCs within few seconds of stimulation [45–47]. The Ca2+ influx through the plasma membrane occurs via either voltage gated or voltage independent calcium channels [46] and results in the activation of calmodulin (CAM) and CaM kinases that may affect cytoskeletal structures, particularly vimentin (VIM), of SCs and the phosphorylation levels of transcription factors like CREB (Fig. 8.3) [44, 48]. The phosphorylation of VIM and consequent rearrangement of the cytoskeleton can be involved in the secretion of specific proteins in response to hormones and might contribute to the compartmentation and mechanical integration of processes. The influx of Ca2+ is also triggered by testosterone (see Sect. 8.4.2.3), so FSH and androgens seems to cooperate to spermatogenesis regulation.

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Phosphatidylinositol 3 Kinase (PIK3) Pathway

Few studies suggested the role of FSH and cAMP in the activation of PIK3 signaling pathway. FSH seems to activate PIK3 and then, phosphoinositide dependent protein kinase 1 (PDPK1) and PRKB in SCs via FSH-mediated increase in cAMP levels (Fig. 8.3) [49]. The involvement of this cascade in metabolic processes required to support germ cells was determined using PIK3 inhibitors. The inhibition of this pathway results in the reduction of the capacity of FSH to induce the activity of lactate dehydrogenase (LDH) required to produce lactate for germ cells and γ-glutamyl peptidase (γ-GTP), a transporter of amino acids through the plasma membrane. PIK3 is also described as being involved in the uptake of glucose, that is converted to lactate to support germ cells energy needs, and transferrin secretions, essential for spermatogenesis [49]. In addition, FSH also regulates the proliferation of SCs through the PIK3/AKT/mTOR pathway. Studies performed in 8-day-old rat SC cultures showed that FSH increases p-AKT, p-mTOR, and p-p70S6K (RPS6K) levels, as well as p-PRAS40 (AKT1S1) levels, probably contributing to improving mTOR signaling [50]. Since mTOR has been involved in the proliferation of several cell types, this study suggests that, the combined signal of this cascade and MAP kinase cascade (see Sect. 8.4.1.2) may be responsible for SC proliferation and thus, spermatogenesis quantity determination.

8.4.1.6

Phospholipase A2 (PLA2) Pathway

In immature testis, FSH stimulation leads to the release of arachidonic acid (AA) through the activation of PLA2, with consequent metabolism by the cyclooxygenase pathway. AA is a second messenger that is metabolized to eicosanoids such as prostaglandin E2 (PGE2 ), which can act as intracellular or extracellular messengers. Consequently, androgen aromatization is stimulated in SCs possibly affecting germ cells via their G-protein coupled eicosanoid receptor [51]. On the other hand, eicosanoids could play an important role in modulating the functions of somatic cells of the seminiferous epithelium, in both autocrine and paracrine ways.

8.4.1.7

FSH-Inducible Genes in Sertoli Cells

The AR levels increase with FSH stimulation, so FSH also have an important role in the regulation of androgen responsiveness of SCs [52, 53]. Several genes involved in spermatogenesis support are found to be regulated by FSH-inducible cAMP and CREB in SCs. These genes include LDH, stem cell factor, required for spermatogonia survival and expansion, as well as aromatase, plasminogen activator and IGF1 [26]. Other FSH-inducible genes comprise doublesex- and mab- 3 related transcription factor (Dmrt) and transferrin and vascular endothelial growth factor (Vegf ). Microarray analysis showed that more than 300 genes are altered with FSH in SCs, most of them related to germ cell survival or development [54].

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8.4.2 Testosterone Signaling As explored in Sect. 8.3, testosterone is produced by LCs in the interstitial space of the testis, in response to LH signaling, and mediate its effect by binding to AR. As a result of local production, the levels of testosterone in testis are more that 30-fold greater than in plasma in men and near 100-fold greater in rodents [55]. In contrast to FSH, the presence of testosterone in testis is essential for normal spermatogenesis. Indeed, spermatogenesis requires high levels of testosterone (>70 ng/ml in rat testis) and does not proceed if testosterone concentration falls above 20 ng/ml [56]. The absence of either testosterone or AR result in spermatogenic arrest at meiosis stage, resulting in infertility [57, 58]. The development of a mice model with the loss of AR in SCs (SCARKO mice) allowed the establishment of the precise effects of testosterone action in SCs. In the absence of SCs-specific AR (SCAR), spermatogenesis does not progress beyond the pachytene or diplotene stages of meiosis [58, 59]. Additionally, high intra-testicular testosterone is essential for the transition of type A to type B spermatogonia, earlier in spermatogenesis [60]. Testosterone signaling in SCs can result directly in changes in gene expression (classical pathway) or testosterone can activate kinases that are responsible for the regulation of spermatogenesis (non-classical pathway) [61]. Both pathways are active in SC and both contribute to the maintenance of normal spermatogenesis (Fig. 8.4).

8.4.2.1

Androgen Receptors—The Targets of Testosterone in Testicular Cells

In testis, AR are only present in LCs, peritubular and SCs [62]. Despite requiring androgens for maturation and survival, germ cells do not express AR [63]. Thus, SCs are the major cellular target and translator of testosterone signal in the seminiferous tubules [64]. Lack of testosterone or AR affects processes required for spermatogenesis, such as regulation of BTB and spermatid differentiation [58, 65, 66].

8.4.2.2

Classical Testosterone Signaling Pathway

Testosterone can act via two distinct pathways: the classical and the non-classical [24, 61]. In the classical pathway, testosterone produced by the LCs diffuses through the plasma membrane of SCs and binds to AR that are sequestered by heat shock proteins (HSP) in the cytoplasm. The AR undergoes a conformational change allowing their release from HSP and then their translocation to the nucleus. In the nucleus, the AR binds to specific DNA sequences called androgen response elements (AREs) in gene promotor regions, recruits co-regulatory proteins and ultimately regulates gene transcription (Fig. 8.4).

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Fig. 8.4 Testosterone signaling pathways in Sertoli cell. In the classical testosterone signaling pathway, testosterone (T) diffuses through the membrane and binds to the androgen receptor (AR) in cytoplasm. The AR undergoes an alteration in conformation allowing the release from the heat shock proteins (HSP) and is translocated to the nucleus where it binds to specific DNA sequences called androgen response elements (AREs). The binding to the AREs recruits the co-activator complex that regulates the transcription of several genes. Testosterone also acts through the nonclassical pathway, inducing the MAP kinase cascade or Ca2+ influx. Testosterone bind to the AR and recruit and activate SRC. The activation of SRC leads to the activation of the EGF receptor (EGFR) and thus the activation of the MAP kinase cascade though Ras, which results in the activation of Raf, MEK and ERK. ERK activates p90rsk kinase that phosphorylate CREB, resulting in the induction of CREB-regulated genes. Testosterone also interacts with a membrane receptor similar to G-protein coupled receptor (GPCR) that activates phospholipase C (PLC). PLC cleaves PIP2 into IP3 and diacylglycerol (DAG). The decrease in PIP2 levels results in the inhibition of K+ ATP channels and thus, membrane depolarization, leading to Ca2+ entry to the SC through L-type Ca2+ channels. Ca2+ influx results in CaM kinase stimulation and translocation to the nucleus where phosphorylates CREB

The activation of the classical pathway requires at least 30–45 min to initiate gene expression and hours to produce new proteins [61]. To identify the transcripts that result from classical pathway, microarray studies were performed in animal models in the presence or absence of testosterone. Despite several genes in the testis being regulated by testosterone signaling, SC-specific genes represent a small group [59]. Although a study reported that 65% of AR-regulated genes are linked to a conserved ARE, only the Rhox5 homeobox transcription factor encoding gene was described as induced in SCs by AR binding to ARE promoter elements [67]. In fact, there is no evidences that the AR-regulated gene expression in SCs are critical for the completion of spermatogenesis and that testosterone acts via gene regulatory mechanisms to support this process [68].

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Non-classical Testosterone Signaling Pathway

A series of findings support the idea that testosterone can act in SCs though pathways other than the classical pathway to support spermatogenesis: (i) intra-testicular testosterone levels are greater than the necessary to regulate transcription via AREs; (ii) few genes are regulated by testosterone or through AREs in SCs; (iii) many genes regulated by testosterone are inhibited in the presence of AR [24]. Several studies have confirmed that testosterone can act through non-classical mechanisms to rapidly activate kinases and signaling pathways capable of regulating the expression of genes without AREs or independent of AR-promoter interaction. The main non-classical testosterone signaling pathways and their role are described in Fig. 8.4.

Testosterone-Mediated Activation of SRC, MAP Kinase Cascade and CREB Testosterone activates several kinases in SCs resulting in the activation of MAP kinase cascade, that are known to regulate spermatogenesis. Stimulation of SCs with testosterone levels similar to those found in testis leads to AR re-localization to the plasma membrane and association of the proline rich region of AR (amino acids 352–359) with the SH3 domain of SRC tyrosine kinase (Proto-oncogene tyrosineprotein kinase Src). This interaction results in the activation of SRC that phosphorylates and stimulates the EGF receptor (EGFR) via an intracellular pathway [69]. The stimulation of EGFR is required to the activation of the MAP kinase cascade (RAF/MEK/ERK) that culminates in the phosphorylation of the CREB transcription factor by p90Rsk kinase [61, 69]. One result of this testosterone-mediated MAP kinase cascade is the induction of CREB-regulated genes in SCs, specifically CREB, LDHA and EGR1 that do not contain AREs [70]. The induced CREB and activation of this pathway by ERK phosphorylation occurs within 1 min and can be sustained for at least 12 h [70]. Several studies suggest that SRC and ERK kinases activation by non-classical testosterone signaling leads to the alteration of processes crucial for spermatogenesis maintenance. The inhibition of these kinases resulted in reduced germ cell attachment to SCs below the basal levels, in adult rats [69]. Additionally, studies performed with AR-defective SCs infected with adenovirus constructs expressing wild type AR or mutant AR that selectively activate classical or non-classical pathway showed that testosterone can act though the activation of SRC and ERK kinases, that are activated in the non-classical pathway, to facilitate Sertoli-germ cells attachment [24]. Testosterone signaling may be, thus, responsible for the remodeling of Sertoligerm cells adhesion during elongation of round spermatids, in rat. The lack of SRC and ERK activation may be responsible for the sloughing off and loss of spermatids that occur in these stages, in the absence of testosterone [69]. The poor cell adhesion that causes the premature detachment of round spermatids from SCs prevents the conversion of round spermatid to elongated spermatids [66, 71]. Since germ cells do not complete meiosis, fully mature spermatozoa are not released to the lumen of the seminiferous tubules and the germ cells are phagocytized by the SCs [66].

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SRC activation by non-classical testosterone signaling is also involved in the regulation of mature sperm release since the treatment of seminiferous tubules sections with SRC inhibitors results in a decrease of 45% in sperm release [24]. These results are supported by studies that showed that activated SRC increase near the Sertoli-elongated spermatids adhesion complex—ectoplasmic specialization (ES)— structurally associated with cell adhesion regulatory proteins [72, 73]. SRC is also responsible for the phosphorylation of focal adhesion kinases (FAK), β-catenin and N-cadherin proteins, participating in the formation of adhesion complexes between SCs and mature elongated spermatids [74]. Phosphorylated N-cadherin diffuses away from β-catenin and the linkage with actin is disrupted, allowing mature sperm release [74]. The activation of SRC kinase and MAP kinase cascade have the potential to regulate the expression of many more genes than the classical pathway and act in SCs in a fast way. Thus, the nonclassical pathway seems to be an essential complement required to maintain spermatogenesis.

Testosterone-Mediated Ca2+ Influx Pathway The testosterone-mediated Ca2+ influx pathway results in the immediate influx of Ca2+ into SCs within 20–40 s. through L-Type calcium channels [75, 76]. Despite the fact that SCs express at least four types of voltage sensitive calcium channels in the plasma membrane, the L-type channels are recognized as the major conveyers of testosterone-induces Ca2+ influx [75, 76]. In the presence of testosterone, an unidentified Gq type G protein coupled receptor is activated and activates phospholipase C (PLC). PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane to produce inositol 1,4,5-trisphosphate (IP3 ) and diacylglycerol (DAG). The reduction in PIP2 levels, an inhibitor of ATP-mediated activation of K+ ATP channels, leads to the closing of these channels which results in an increase in membrane resistance and depolarization of the cell. Consequently, voltage dependent L-type Ca2+ channels open and allow the influx of Ca2+ , which has an impact in several cellular processes [77, 78].

8.5 Regulation of the Blood-Testis-Barrier Sperm production is highly dependent on a specialized structure called BTB. The BTB comprises a physical barrier formed by the SC junctional complex (TJs, gap junctions, ectoplasmic specialization and desmosome-like junctions), a physiological barrier made up SC transporters, controlling the passage of substances from and to the lumen, and an immunological barrier. This structure creates two compartments within the seminiferous tubules, the basal (also called “interstitial”) and adluminal. Blood vessels, interstitial cells and earlier germ cells (spermatogonia and preleptotene spermatocytes) are located at the basal compartment. Later meiotic (leptotene, zygotene and pachytene spermatocytes) and post-meiotic (round and

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elongating spermatids) germ cells are confined in the adluminal compartment, apart from the external environment and blood supply [79]. During normal spermatogenesis, the BTB transiently “open” and “close” to allow immature germ cells to cross into the protected environment. Three hypotheses for germ cell translocation have been proposed, including (i) the transient formation of an intermediate compartment enclosing early leptotene spermatocytes [80], (ii) the progressive opening of TJs above migrating germ cells that close once migration is completed [81] and (iii) the proliferation of TJs for assembly by an endocytic pathway (reviewed by [82]). Several TJ proteins have been described in SCs, comprising members of claudin (CLDN) family (CLDN11, CLDN3, CLDN5, CLDN12, CLDN13, CLDN1 and occluding (OCLN)), junctional adhesion molecules (JAM) family, tricellulin and coxsackievirus and adenovirus receptor (CAR). These proteins are associated to actin cytoskeleton and provide links to other junctional types, such as adherent or gap junctions, in the BTB (for more information see [25, 83]). From all integral TJ proteins, only CLDN11 seems to be critical for spermatogenesis since CLDN11 KO mice are infertile [84, 85] but the KO of CLDN3, CAR and JAM-A did not show any testicular phenotype [79]. The absence of claudin results in loss of TJ integrity and disruption of BTB. The BTB is regulated by the combined effect of both endocrine and paracrine factors, described below.

8.5.1 Regulation of BTB Assembly The testosterone signaling (discussed in Sect. 8.4.2) is the main responsible for the correct assembly and integrity of the BTB. In fact, testosterone signaling up-regulates three TJ components—OCLN, CLDN11 and CLDN13—supporting the hypothesis that testosterone and AR are required to reform TJs after pre-meiotic cells pass through the BTB [65, 86]. Additionally, the integrity of the TJs of the BTB are disturbed, either due to the reduced levels of BTB TJ-associated proteins and their localization [65, 86, 87]. Cultured SCs stimulated with testosterone showed high rates of integral membrane adhesion proteins endocytosis and then recycled to the membrane, suggesting that testosterone may be involved in the cyclical remodulation of the BTB after the passage of the leptotene spermatocytes though the barrier [88]. An important regulatory mechanism in normal testis, besides testosterone signaling, to balance the activin action and prevent an uncontrolled opening of the BTB is retinoic acid (RA) signaling, which is emerging as a key regulator of spermatogenesis [89–91]. RA stimulates the BTB in vivo through the induction of SC transepithelial electrical resistance and CLDN11 expression an localization at TJs [90]. The compromised integrity of BTB results in the exposure of post meiotic germ cells to autoimmune attack and cytotoxic factors, ultimately leading to infertility.

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8.5.2 Regulation of the BTB Disassembly for Spermatocyte Translocation In general, BTB function is enhanced by testosterone and RA [79]. Additionally, local paracrine and autocrine factors, such as cytokines, are essential to regulate the translocation of the spermatocytes through the BTB [79]. Several cytokines are known to be produced in testis, including tumor necrosis factor alpha (TNFα), members of the transforming growth factor beta (TGFβ) superfamily (TGFβ3, TGFβ2, GDF9 and activins A/B), interleukins (IL-1α and IL-6) and interferons [74, 92, 93]. TGFβ3 was identified in rat testis immediately prior to germ cell translocation [94, 95]. This factor suppresses SC TJs by almost 50%, however, the remaining TJs were independent of TGFβ3, suggesting that other factors are involved in the disassembly of TJs [94]. The role of activin A in BTB dynamics was also studied in vitro and in vivo. In testis, activins are produced by all cell types, including germ cells, and a marked peak of production happens upon germ cell translocation, similar to what occurs with RA. In the presence of activin A, the TJs function is completely ablated and their formation is prevented, in vitro, probably due to the reduction in CLDN11 expression [96]. Additionally, activin A seems to stimulate the de-differentiation of SCs in vitro and, in vivo, the excess of activin action cause the loss of BTB function [96]. Together, these factors seem to regulate the remodeling of the BTB during spermatocyte translocation, particularly promoting the TJ disassembling. Additionally, RA and activin A signaling seem to cooperate in the regulation of TJs disassembly and assembly, avoiding the unnecessary opening of the BTB. Despite the growing knowledge on the multiple effects produced by autocrine and paracrine factors, it is still poor understood how signals are propagated and how they are coordinated within SCs to achieve a correct BTB restructuring. The best pathway associated to BTB disassembly is the p38 MAP kinase cascade [92]. In SC, signaling molecules, such as TGFβ3 and TNFα stimulate activators such as small G proteins (Rac, Cdc42) that activate MAPK kinases kinases (MEKK1-4 and TGF-β activated kinase 1) [92, 95]. In turn, these kinases activate MAPK kinases (MKK3, 4 and 6) that phosphorylate and activate p38 MAPK [92]. In testis, it is known that p38 MAPK is implicated in BTB disassembly. In fact, TGFβ3, TNFα and environmental toxicants such as cadmium increase the phosphorylated levels of p38 MAPK resulting in the loss of BTB integrity [92]. The activation of p38 MAPK in SCs is highly dependent upon CDC42 since p38 MAPK phosphorylation is induced by CDC42 overexpression [95]. mTORC1 and mTORC2 also promote BTB remodeling and BTB integrity, respectively. It is through the antagonistic effects of these two mTOR signaling proteins that the dynamic of the BTB during the transport of preleptotene spermatocytes across the immunological barrier can be rapidly modulated. In vitro studies revealed that mTOR binds to its binding partner Raptor creating the mTORC1 signaling complex. mTORC1 works with its downstream signaling protein ribosomal protein S6 (RPS6) and the p-AKT1/2 signaling molecule involving both the Arp2/3 complex and its

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upstream activator N-WASP, and MMP9, thus affecting actin organization and the stability of adhesion protein complexes at the BTB, resulting in BTB disruption [33]. Recently, the mTORC1/RPS6 complex was also indicated as a regulator of spermatogenesis and BTB by modulating the organization of the actin- and microtubule-based cytoskeletons [97].

8.6 Spermatogenesis Control by Paracrine Signals The complex process of spermatogenesis is regulated by a combined effect of signaling pathways within a well-structured and organized tissue. In this section, we will describe the main signaling pathways involved in self-renewal and differentiation of spermatogonia, regulation of spermatocyte meiosis, spermiogenesis and spermiation.

8.6.1 Spermatogonia Renewal and Differentiation Spermatogonial stem cells (SSC) and spermatogonia reside within a specialized testicular microenvironment known as the “niche”, where the balance between SSC renewal and differentiation is regulated. Half of SSCs, known as daughter cells, move out of the niche and subsequently differentiate, while the other half stay in the niche and self-renewal. Is the proliferation of spermatogonia clones and their differentiation which determine the constant production of spermatozoa during adulthood [3, 98]. As described earlier in this chapter, the suppression of androgens and FSH generally results in block of spermatogonia development being clear that these hormones have a supportive effect in this process. However, since spermatogonia lack receptors for both FSH and androgens, the action of these hormones must be indirect, via SCs and/or other testicular somatic cells. SCs secrete GDNF upon FSH stimulation, that acts on receptors on undifferentiated spermatogonia regulating spermatogonial self-renewal and differentiation [99]. SCs also control the SSC niche through fibroblast growth factor 2 (FGF2) production and expression of ETV5. Other somatic cells within the testis are involved in this phase of spermatogenesis, such as peritubular myoid cells and LCs that secrete colony stimulating factor (CSF1), important for SSC self-renewal in mice [100]. Despite the involvement of other transcription factors and signaling pathways in spermatogonia self-renewal and differentiation, in this chapter we will focus on the contribution of the most known signaling pathways to this process (Fig. 8.5).

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Fig. 8.5 Signaling pathways regulating SSC self-renewal. The binding of GDNF to GFRA1 receptor coupled to RET activate PIK3/AKT and SFK intracellular signaling cascades, regulating transcription factors involved in SSC self-renewal such as BCL6B, EVT5, LHX1, ID4. FGF2 is also involved in self-renewal of a niche of SSC prone to differentiation via MAP2K1 activation. The activation of this kinase results in the upregulation of genes like Evt5, Bcl6b and Lhx1. The SCsecreted CXCL12 acts via CXCR4 receptor present on SSCs and acts via an unidentified signaling pathway to regulate self-renewal

8.6.1.1

GDNF Signaling Regulates SSC Self-renewal

The GDNF is a soluble factor secreted by SC that is involved in self-renewal of SSC and inhibits their differentiation [101]. Overexpression of GDNF leads to an accumulation of undifferentiated spermatogonia including SSCs, and conversely, ablation of GDNF by gene targeting results in the depletion of spermatogonia [101]. In vitro, the addition of GDNF to cultured SSCs promotes their proliferation further demonstrating that GDNF is a crucial factor for self-renewal and maintenance of mouse SSCs [102, 103]. GDNF signaling acts via a receptor complex containing GDNF family receptor alpha-1 (GFRA1) and RET tyrosine kinase, both present on undifferentiated spermatogonia [103, 104] to promote spermatogonia self-renewal. The silencing of Gfra1 by RNA interference results in a switch from proliferation of mouse SSCs to differentiation into spermatogonia type A, an initial stage of spermatogenesis [105]. The binding of GDNF to the receptor results in the activation of the PIK3/AKT pathway or the SRC family kinase (SFK) pathway (Fig. 8.5) [99]. These pathways lead to the transcription of genes crucial for SSC self-renewal like transcription factor B cell CLL/lymphoma 6 member B (Bcl6b), ETS variant 5 (Etv5), DNA-binding protein 4 (Id4) and LIM homeobox 1 (Lhx1) [106].

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FGF2-MAP2K1/AKT Signaling Regulates SSC Self-renewal

Fibroblast growth factor 2 (FGF2) is expressed and secreted by SCs and stimulates SSC self-renewal. In vitro experiments showed that supplementation with FGF2 and GDNF results in long-term self-renewing of SSCs [102]. Although both factors expanded a large subset of undifferentiated spermatogonia, the FGF2 expands a differentiation-prone subset in mouse testis [107]. FGF2 induces both MAPK1/3 (ERK2/1) and AKT phosphorylation in germ line stem cells, and cells expressing activated MAP2K1 (MEK1) not only induced MAPK1/3 phosphorylation but also proliferated without FGF2, indicating that MAP2K1 can substitute FGF2 [99]. The activation of MAP2K1 results in the upregulation of Evt5, Bcl6b and Lhx1 genes that allows the expansion of SSCs [99]. Since FGF2-depleted testes exhibited increased levels of GDNF and were enriched for SSCs [108], SSC self-renewal in vivo seems to result from the balance between FGF2 and GDNF signaling.

8.6.1.3

CXCL12-CXCR4 Signaling Regulates SSC Self-renewal

The chemokine Stromal cell-derived factor 1 (CXCL12) is expressed and secreted by SCs and binds to the C-X-C chemokine receptor type 4 (CXCR4), a GPCR, on SSCs to regulate self-renewal and maintenance [109]. The disruption in CXCL12-CXCR4 signaling in primary cultures of undifferentiated mouse spermatogonia results in SSCs loss with a reduction in Fgf2 transcript [110], suggesting that CXCL12, FGF and GDNF work together to regulate SSC self-renewal. The presence of CXCL12 and its receptor was identified in human testis [111] suggesting that this signaling pathway may be involved in human SSC regulation. However, the intracellular mechanisms of CXCL12-CXCR4 needs further investigation.

8.6.1.4

PIK3/AKT and Retinoic Acid Signaling Pathways Regulate Spermatogonia Differentiation

In the neonatal period, migration and proliferation of primordial germ cells and prespermatogonia (gonocytes) is a crucial step in the establishment of spermatogenesis. Besides, the commitment of spermatogonia to differentiation and entry to meiosis provide the pulses of spermatozoa production along the seminiferous tubule. For that, the action of stem cell factor/kit ligand (KITLG) produced by SCs, on c-Kit receptor, located on spermatogonia, is required [112]. The action of KITLG on c-Kit induces the PIK3 signaling pathway in spermatogonia resulting in cell proliferation. In vivo studies confirm that the failure of binding of KITLG to c-Kit diminishes activation of AKT, leading to a decrease of proliferation and an increase of apoptosis of SSCs and eventually results in an arrest of spermatogenesis [112]. The RA signaling pathway is also emerging as critical regulator of spermatogonial differentiation. RA stimulates spermatogonia differentiation by activating the PIK3/AKT/mTOR signaling pathway to induce the translation of the c-Kit and other

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proteins involved in spermatogonia differentiation [99]. The acquirement of a c-kit protein on the surface of spermatogonia is a key marker of differentiation and is essential for their development and entry in meiosis [113].

8.6.2 Regulation of Meiosis Meiosis begins with the differentiation of type B spermatogonia into preleptotene spermatocyte, which start DNA synthesis. As discussed in Sect. 8.4.2. The absence of either testosterone or AR results in spermatogenic arrest at meiosis stage. Additionally, other signaling pathways are involved in the progression from pachytene to diplotene spermatocyte, especially nociceptin–nociceptin receptor (OPRL1) signaling and RA signaling.

8.6.2.1

Nociceptin-OPRL1 Signaling

Nociceptin is secreted by SCs in response to FSH and is involved in spermatocyte meiosis [114]. The binding of nociceptin to their receptor, OPRL1, exclusively expressed on the plasma membrane of spermatocytes, induces and maintains meiotic recombination protein REC8 phosphorylation, which is required for normal meiotic chromosomal dynamics in spermatocytes, during meiosis [115, 116]. Despite the mechanisms by which nociceptin stimulates REC8 remains unclear, nociceptin/OPRL1 plays a crucial role in the progression of meiosis.

8.6.2.2

Retinoic Acid/Neuregulin Signaling

The entry in meiosis and spermatocytes production requires the RA pathway [117]. In juvenile rat testis, RA synthetized by SCs in response to FSH positively regulates the spermatocytes commitment to meiosis and also regulates the progression of the early stages of meiotic prophase through the stimulation of RA-inducible Stra8 gene [118]. In the absence of Stra8, preleptotene spermatocyte are formed and replicate their DNA, however, they not entry into the meiosis prophase [118]. Additionally, RA and FSH can act on SCs to promote the expression of Neuregulin (NRG) 1 and 3. NRG1, a member of the EGF family, acts on its receptor ERBB4 on the surface of germ cells inducing meiosis in spermatocytes [119]. However, the mechanisms by which RA promotes NRG1 and 3 expression in SCs remain to be elucidated. RA also induces Rec8 activation, a meiosis-specific component of the cohesion complex suggesting that this molecule acts through distinct pathways to initiate meiosis.

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8.6.3 Regulation of Spermiogenesis and Spermiation The meiotic division of a spermatocyte culminates in the generation of four round, haploid spermatids that undergo extensive morphological changes to become spermatozoa. The development of the sperm tail, the nuclear changes involving the condensation of the DNA, the cessation of transcription and the delay in translation as well as the repositioning of the cell nucleus, organelles and cytoplasm require a tight control [120]. Both spermiogenesis and spermiation are well known targets of androgen action in testis. In fact, androgen insufficiency also causes a failure in round spermatid to attach to SCs and enter in the elongation phase of spermiogenesis and mature spermatids fail to be released at the end of spermiation, as discusses early in Sect. 8.4.2.3. On the other hand, administration of FSH to men undergoing gonadotropin suppression can support spermiogenesis, and acute suppression on FSH alone causes spermiation failure in rats, suggesting that both testosterone and FSH co-operate to promote spermiation. Additionally, spermiogenesis and spermiation appears to be regulated by the RA signaling pathway [121]. SC-secreted RA, metabolized from retinol (vitamin A) acts by binding to the nuclear retinoic receptor (RARα, β and γ) and retinoic X receptor (RXRα, β and γ). Particularly, the binding of RA to a RARα/RARβ heterodimer expressed in SCs seems to be essential for spermiation [121, 122]. The receptor heterodimerizes to control the expression of RA responsive genes. Deletion of genes encoding RARα (Rara) from SCs causes anomalies in spermiogenesis and spermiation. Interestingly, the expression of Rara rescues the spermiogenesis and spermiation defects seen in Rara null mice, suggesting that germline expression of RARα is also important for these processes [121]. RARα/RARβ may co-operate with AR signaling in SCs to regulate spermatogenesis and/or regulate the expression of adhesion junction components.

8.7 Conclusion The testis functions are mainly regulated by hormones and hormone receptors, but also by growth factors and secreted compounds from somatic cells, such as SCs. The LCs, located in the interstitial space of the testicular parenchyma, are the main responsible for androgen biosynthesis, particularly testosterone and DHT, mediated by the binding of LH to its receptor. LH starts a series of signaling pathways that culminate in the expression of StAR, essential for steroidogenesis. FSH is the main responsible for SCs proliferation and testicular size determination, through the MAP kinase and PIK3/AKT pathways. This hormone also acts, in combination with testosterone, on SCs to induce Ca2+ influx, maintain spermatogonia self-renewal and differentiation as well as meiosis. Retinoic acid signaling also seems to play an important role in testicular signaling, particularly in the maintenance of BTB integrity, spermatocytes meiosis, spermiogenesis and spermiation.

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Acknowledgments This work was supported by FEDER Funds through Competitiveness and Internationalization Operational Program—COMPETE 2020 and by National Funds through FCT— Foundation for Science and Technology under the project PTDB/BBB-BQB/3804/2014. We are thankful to Institute for Biomedicine—iBiMED (UIDB/04501/2020 and POCI-01-0145-FEDER007628) for supporting this project. iBiMED is supported by the Portuguese Foundation for Science and Technology (FCT), Compete2020 and FEDER fund. This work was also support by individual grant from FCT of the Portuguese Ministry of Science and Higher Education to JS (SFRH/BD/136896/2018), DP (SFRH/BD/137487/2018) and JVS (SFRH/BPD/123155/2016).

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Chapter 9

Sperm Signaling Specificity: From Sperm Maturation to Oocyte Recognition Maria João Freitas, Daniela Patrício, and Margarida Fardilha

Abstract The sperm cell is unique in its function. It is the only human cell that must leave the body where it is produced and fulfills its goal in a different organism being, thus, a highly specialized cell. Sperm cells are produced in the testis, acquire motility during the epididymis journey and fertilize the oocyte in the female reproductive system. Moreover, since these cells are virtually transcriptionally silent, they rely exclusively on protein-protein interactions and post translational modifications to control signaling pathways. These sperm cell unique features are reflected in sperm-specific signaling. Several sperm-specific/enriched proteins are responsible for controlling sperm functions such as sperm motility and acrosome reaction. In this chapter, we describe the signaling events that characterize sperm motility and acrosome reaction as well as the unique proteins that control such events.

Abbreviations ADCY ADCY10 AKAP4 AKT ATP

Adenylyl cyclase Adenylyl cyclase 10 A-Kinase anchor protein 4 Protein kinase B Adenosine triphosphate

M. J. Freitas · M. Fardilha (B) Laboratory of Protein Phosphorylation and Proteomics, Faculty of Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium e-mail: [email protected] M. J. Freitas e-mail: [email protected] D. Patrício Laboratory of Signal Transduction, Medical Sciences Department, iBiMED—Institute for Research in Biomedicine, University of Aveiro, Aveiro, Portugal e-mail: [email protected] Department of Chemistry, CICECO, Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_9

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CarSper DAG DNA GABA GAPDH GSK3 HPA IP3 LDH LRP6 NGC OAM PDPK2 PGK1 PIK3C PIP2 PIP3 PLA2 PLC PLC PM PPME1 PPP1 PPP1CC2 PPP1R11 PPP1R2 PPP1R2P3 PPP1R7 PPP2CA PR PRKA PRKC PTMs RyRs SERCA SNARE SOC TEX101 VOCCs ZP

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Cation channel of sperm Diacylglycerol Deoxyribonucleic acid Gamma aminobutyric acid Lactate glyceraldehyde-3-phosphate dehydrogenase Glycogen synthase kinase 3 Human Protein Atlas Inositol trisphosphate Lactate dehydrogenase Low-density lipoprotein receptor-related protein 6 Nucleotide-gated channel Outer acrosomal membrane PDP kinase 2 Phosphoglycerate kinase type 1 Phosphatidylinositol 3-kinase catalytic subunit Phosphatidylinositol 4,5-bisphosphate Phosphatidylinositol 3,4,5 trisphosphate Phospholipase A2 Phospholipase C 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase Plasma membrane Protein phosphatase methylesterase 1 Phosphoprotein phosphatase 1 Phosphoprotein phosphatase 1 catalytic subunit C2 Phosphoprotein phosphatase 1 regulatory subunit 11 Phosphoprotein phosphatase 1 regulatory subunit 2 PPP2CA pseudogene 3 Phosphoprotein phosphatase 1 regulatory subunit 7 Phosphoprotein phosphatase 2 catalytic subunit A Purinergic receptors cAMP-dependent protein kinase catalytic subunit alpha Protein kinase C Post-translational modifications Ryanodine receptors Sarcoplasmic-endoplasmic reticulum Ca2+ ATPase SNAP Receptor Store-operated channels Testis-expressed protein 101 Voltage-dependent calcium channel Zona pellucida

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9.1 Introduction The human male gamete, or spermatozoon, differentiates from the spermatogonia, in a process named spermatogenesis (see Chap. 8). In humans, spermatozoa begin to be release in the early stages of puberty and continues throughout life. This cell is responsible for the safe transport and delivery of the male genetic information to the female gamete, the oocyte [1]. Due to the unique nature of its function, the sperm cell presents exceptional features: (1) it is one of most differentiated cells in the human body; (2) it is the only cell that is produced in one living form but achieves its goal in another; (3) it is an haploid cell, and (4) one of the few cells that is virtually transcriptionally silent [2]. The uniqueness of sperm cells is reflected morphologically and functionally. Morphologically, sperm cells must have an intact flagellum, present an interchangeable membrane fluidity, have high efficiency energy producing organelles and an optimal shape for forward movement. Functionally, sperm cells must undergo maturation in the epididymis and capacitation in the female reproductive system [3]. Since sperm cells are virtually incapable of producing new proteins, sperm functional maturation relies on quick and reversible mechanisms to receive, interpreter and react to external signals. Post-translational modifications (e.g. phosphorylation) and alterations in protein-protein interactions are the main molecular mechanisms that control cellular events in sperm cells. Moreover, it is hypothesized that sperm cells have one of the most unique proteomic composition. Up to 17% of the proteins present in testis are restricted or enriched in this tissue [4]. Dozens of sperm-enriched proteins have been reported in the literature, from enzymes (e.g. PPP1CC2, a phosphatase; PLCZ, a phospholipase and ADCY10, a cyclase) [5–7] to structural proteins (e.g. AKAP4) [8] and receptors (e.g. CatSper) [9]. The uniqueness of human sperm cells is unquestionable. In this chapter we focus on describing the sperm signaling events involved in two essential processes for oocyte fertilization: acquisition of sperm motility in the epididymis and sperm-oocyte interactions in the female reproductive system. Both processes are exclusively present in the sperm cell and require unique morphological and function features.

9.2 The Uniqueness of the Sperm Cell: From Head to Tail Morphologically the sperm cell can be divided in two main structures: the head and the flagellum. Surrounding all sperm structures is the plasma membrane. The human sperm head is spatula-shaped and is mainly constituted by the nucleus and the acrosome. Human sperm nucleus is smaller than somatic cells’, because it only contains half of the genetic information compared to somatic cells and the DNA is highly compacted. The latter results from histones substitution by protamines, a positively charged protein that empowers DNA hypercondensation. Moreover, protamines are responsible for spermatozoa’s genetic silencing, since DNA polymerases

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cannot access the DNA [10–12]. Surrounding the nucleus is the nuclear envelope and contrary to somatic cells, it does not present nuclear pore complex. Furthermore, the sperm nucleus is protected by a rigid structure formed by bonding of structural proteins, the perinuclear theca. Altogether these features result in optimal shaped nucleus for mobility and better protection of the genetic information [13, 14]. The acrosome lies anteriorly to the nucleus and covers half to two thirds of the sperm head. During spermatogenesis, the Golgi complex stores several proteases, hidroglycolases and esterases (some of which are sperm-specific) giving rise to the acrosome. Upon specific signaling the acrosome will fuse with the sperm plasma and release its content [15]. Another features that sets human sperm cells apart from somatic cells is the small amount of cytoplasm and cytoskeleton structures present in the head. Consequently, the sperm is light and easier to propel forward [16]. Figure 9.1 schematizes the ultrastructure of the human spermatozoa. The flagellum is the longest part of the sperm and is a critical structure for motility [17] (see Fig. 9.1). This structure contains the motile apparatus necessary for sperm

Fig. 9.1 Schematic representation of human spermatozoon and flagellum structure. a Human sperm is divided into two parts: head and flagellum. The flagellum is further divided into four structures: connecting piece; midpiece; principal piece and endpiece. A cross-section shows the flagellum structure of the principal piece. b Representation of the on-and-off mechanism that ensures a wave like beating of the sperm flagellum. In this mechanism, half of the microtubule doublets establish a connection with the adjacent microtubule doublet. The other half of the microtubule doublets are inactive. This results in half of the flagellum being pulled towards the end piece and the other half is static. Consequently, the flagellum bends and forms a wave like structure. It is still unknown the how the on-and-off mechanism is controlled

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motility and is divided into four ultrastructures: connecting piece, midpiece, principal piece and end piece. Immediately behind the sperm head is the connecting piece. This structure is flexible and functions as an anchor between the head and the tail. The midpiece houses the mitochondrial sheath, which is organized as individual mitochondria coiled helically around the axoneme. The energy required for sperm motility is produced in the mitochondrial sheath. In humans, the midpiece is a dozen mitochondrial turns in length. Lastly, the principal piece and the end piece generate the flagellar waveform pattern of motility [18–20]. Motility is only achieved by the interplay between the axoneme, fibrous sheath and outer dense fibers that run along the length of the flagellum (approximately 50 μm in human sperm cells). The axoneme extends from the connecting piece being the central structure of the flagellum [19, 21, 22]. It is organized in 9 + 2 microtubule-doublets structure, with a central pair of microtubes and 9 doublets around the central pair, connected by the radial spokes. The latter as responsible for spacing the microtubules doublets around the central microtubule pair. Projecting from the microtubules doublets there are the dynein arms (outer and inner). Dyneins are able to convert energy from ATP hydrolysis into sliding motion of a microtubule doublet in relation to the adjacent. The flagellar beating pattern begins with a dynein from one doublet transiently interacting with the following doublet. In the presence of ATP, axonemal dynein “walks” toward the base of the flagellum, forcing the adjacent microtubule doublet to slide down. Since microtubules are attached to the connecting piece, this movement encounters resistance, leading to the bending of the flagellum. At the end, the dynein detaches from the adjacent microtubule [18–22]. The outer dense fibers are present in the midpiece and extend into the principal piece of the flagellum [18]. This structure is directly above the axoneme microtubules doublets and appear to be responsible for maintaining the passive elastic structure and recoil of the flagellum and to protect the axoneme against shearing forces [23]. Fibrous sheath confers flexibility, shape and plane to the flagellar beat. It also supports and ensures compartmentalization of signaling proteins that regulate motility, capacitation, and hyperactivation [24]. Figure 9.1 illustrates a detail description of the flagellum structure. Another feature that sets apart human sperm cells from somatic cells is the unique plasma membrane composition. In general, membranes contain 70% phospholipids, 25% neutral lipids and 5% glycolipids [25]. The most abundant phospholipid of the sperm plasma membrane is sphingomyelin, which confers rigidity to bilayer membranes. Regarding neutral lipids, human sperm membrane contains very high amounts of cholesterol. Cholesterol is a plasma membrane stabilizer with the particularity of maintaining membrane fluidity. Consequently, cholesterol is vital for sperm permeability and motility [26]. In human sperm plasma membrane, the only glycolipid is seminolipid, which is exclusively present in mammalian sperm and Schwann cells. It is believed that this molecule contributes to restrict lipids to a specific region of the plasma membrane [27, 28]. Although spermatozoa are the smallest cell from the human body they are highly complex perform one of the most complex function: cross the female reproductive tract and deliver their genetic information to an oocyte.

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9.3 Sperm-Specific Proteome The Human Protein Atlas (HPA) reported that testis shows the largest number of tissue-enriched genes (1079 genes). Furthermore, testis presents around 2.5 times more enriched genes than cerebral cortex, the second tissue with the largest number of tissue-enriched genes. This reflects the specificity of testis/sperm proteome and consequently the uniqueness of sperm function [4]. Since gene expression does not occur on spermatozoa, alteration of function is coordinated by activation and/or inhibition of several signaling pathways and posttranslational modifications. Thus, identifying and understanding sperm proteome is key to understand sperm physiology. The study on sperm proteins was first initiated by Miescher with the discovery of salmon sperm protamine [29]. This field has advanced enormously in the last century and the studies performed so far have resulted in the identification of more than 600 sperm proteins involved in several cellular pathways [30]. In 2011, Gilany et al. compiled and characterize the normospermic sperm proteome. The results suggest that the most prominent biological function of the sperm proteome is associated with catabolic processes, including proteins responsible for energy production required for sperm movement. The study also reported that 3.6% of proteins are linked to spermatogenesis and 0.9% to spermiogenesis [31]. The most intriguing conclusion of this study is that approximately 30% of the proteins had no biological function associated, which reveals the necessity of a deeper study of the sperm proteome. In an effort to stablish a transcriptome-proteome correlation, Wang et al. identified the sperm proteomic profile and compared the identified proteome with published sperm transcriptome data. Little overlap between proteome and transcriptome was found, hinting that the transcriptomic profile of sperm cells has little influence on sperm physiology [32]. Moreover, this attests the importance of sperm studies at the protein level. From the 4675 proteins identified, 233 were testis-specific proteins. Germ cell-specific enzymes, lactate glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and phosphoglycerate kinase type 1 (PGK1) were identified. Over represented proteins were found to be associated with energy metabolism (glycolysis, gluconeogenesis, etc.), signal transduction and cytoskeleton [32]. So far, more than 50 studies have attempted to decipher the human sperm proteome using high throughput methods. They focus not only into reveal normospermic human sperm proteome [32–36] but also depicted the proteomes of different stages of sperm maturation (e.g. ejaculated and capacitated); the proteome of males with distinct fertility issues (e.g. asthenozoospermic; normal but In Vitro Fertilization failure) [37–44]; specific subcellular sperm component (e.g. head or flagellum) [30, 45]; posttranslational protein modifications (phosphoproteome, nitrosylations, glycosylation) [46–48] and finally sperm proteome of males with associated pathologies (diabetes, obesity, epididymitis) [49–51].

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Throughout the years, several sperm-specific proteins have been identified in an effort to reveal sperm-specific functions. We highlight the CatSper channel, responsible for the increase of Ca2+ in flagellum upon progesterone binding and essential for sperm hyperactivated motility [9]; ADCY10, a soluble isoform of adenylyl cyclase that does not interact with guanosine 5 -tiphosphaye and requires HCO3 − and Ca2+ for its activation [52] and PPP1CC2, a PPP1 testis/sperm-specific isoform indispensable for spermatogenesis and sperm motility [5, 53]. As previously stated, post-translational modifications are key mechanism for sperm physiology. The numerous possible post-translation modifications (PTMs) increase protein complexity and modulate protein functions on spermatozoa. Protein N-linked glycosylation and protein phosphorylation may be the most common PTMs in the human sperm cell [54]. In fact, the human sperm includes various glycosyltransferases, kinases and phosphatases. The importance of PTMs on sperm physiology is reflected in the fact that the primary indicator of a successful sperm capacitation is the increase of general tyrosine phosphorylation in human sperm cells. In 2016 an in-depth study on PTMs in sperm cells, identified 6069 modified sites, specifically 1791 lysine acetylation sites, 3009 phosphorylation sites, 543 n-linked glycosylation sites and 726 protein N-terminal acetylation site, resulting on 2132 proteins presenting PTMs [55].

9.4 Signaling Pathways in Sperm Motility In the human body, the ability of progressive motility is exclusive to sperm cells. Thus, this cells present protein profiles that are key to motility itself and to its regulation. In the next section we will focuses on describing the unique features of the signaling pathways involved in two types of human sperm motility: primary and hyperactivated sperm motility.

9.4.1 Sperm Motility Acquisition in the Epididymis: The Central Role of PPP1 Spermatogenesis results in morphological complete sperm but it remains functionally immature. To acquire progressive motility, or primary motility, in the epididymis, several signaling pathways must be activated/inhibited [56]. It has been proposed that primary regulation of flagellar beating, that happens in the epididymis, occurs through reversible phosphorylation of axonemal proteins. Therefore, modulation of both kinases and phosphatases are in order. In 1996 Smith et al. revealed the important role of phosphoprotein phosphatases (PPP) in the regulation of spermatozoa motility through the epididymis. The group showed, for the first time, that inhibition of phosphoprotein phosphatase 1 (PPP1) activity resulted in the initiation and stimulation

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of motility [5]. Several studies have demonstrated that PPP1CC2, a testis/spermenriched isoform of PPP1, is key in regulating sperm motility. In the spermatozoa, PPP1CC2 is localized along the entire flagellum, consistent with a role in sperm motility. PPP1CC2 may be also found in the posterior and equatorial regions of the spermatozoa head, suggesting a role in the acrosomal reaction [5, 53, 57]. In human spermatozoa several mechanisms that control PPP1 activity have been uncover [58]. It is now known that in the epididymis caput (immotile spermatozoa), PPP2CA is demethylated and phosphorylated and consequently active, which in turn dephosphorylates GSK3 at serine residues, rendering GSK3 active. GSK3 phosphorylates PPP1R2 at Thr73 which inhibits its interaction with PPP1 resulting in an active PPP1 [59–64]. Besides PPP1R2, other two PPP1 regulatory subunits keep PPP1 activity inhibited in sperm, PPP1R7 and PPP1R11. In caput spermatozoa, bovine sperm PPP1R7 and PPP1CC2 do not directly interact. Instead, PPP1R7 is associated with a 17 kDa protein (p17) [65], resulting in free and active PPP1. Consequently, PPP1 dephosphorylates key proteins for sperm motility, resulting in immotile spermatozoa. In cauda epididymis, Protein phosphatase methylesterase 1 (PPME1) activity decreases and methylation of PPP2CA increases, rendering an inactive PPP2CA. Consequently, GSK3 serine phosphorylation increases leading to its inhibition. GSK3 might be also inhibited through Wnt binding to low-density lipoprotein receptor-related protein 6 (LRP6) receptor. Moreover, ATP binds to purinergic receptors (PR), resulting in calcium influx. This calcium influx activates adenylyl cyclase (ADCY), which produces Cyclic adenosine monophosphate (cAMP) that latter will activate Protein kinase B (AKT) that phosphorylates GSK3 at serine residues inactivating it. Due to GSK3 inhibition, PPP1R2 is no longer phosphorylated and can bind and inhibit PPP1. Moreover, PPP1 might bind to PPPP1R2P3 in a complex with PPP1R7, actin and PPP1R11 [58, 66]. PPP1R2P3, a PPP1R2 pseudogene 3, has a unique feature of Thr73 being replaced by Pro avoiding GSK3 phosphorylation. PPP1R2P3 protein is able to bind directly to PPP1 and the inactive PPP1inhibitor complex formed cannot be activated by GSK3 phosphorylation. This rises the hypothesis that PPP1R2P3 is only present in caudal motile sperm, representing a constitutively inhibitor of PPP1, independent of GSK3 phosphorylation, and therefore responsible for the unidirectional process of spermatozoa motility acquisition along the epididymis [60]. Thus, PPP1 activity is inhibited and Ser/Thr phosphorylation of key residues increases leading to motile sperm. Figure 9.2 depicts the signaling pathways involved in sperm motility acquisition in human sperm cells. After epididymal maturation, spermatozoa are stored in the distal part of the caudal epididymis where they remain quiescent.

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Fig. 9.2 Schematic representation of human spermatozoa motility. In the epididymis motility is partial controlled by PPP1 inhibitions centered signaling pathways. In the female reproductive system, activation of tyrosine kinases is central for motility. P, phosphorylation; arrows, activation; stop arrow, inhibition

9.4.2 Hyperactivated Motility in the Female Reproductive System: The Central Role of PKRA Upon ejaculation, the sperm cell is functionally and morphologically prepared to survive the female reproductive system. Yet, the latter imposes several obstacles to sperm cell progression (e.g. acidity of the vagina and protective coating around the oocyte). To overcome these hurdles, the sperm cell must suffer sperm capacitation. One of the first phenomena of capacitation is the acquisition of hyperactivated motility. This type of motility is characterized by high amplitude and asymmetric flagellar bends that allows sperm to overcome dense mucus, detach from the oviductal epithelium, and penetrate the egg’s protective vestments [56, 67]. Hyperactivated sperm motility is triggered during ejaculation when spermatozoa are mixed with fluids from accessory glands and stimulated upon entrance in the female reproductive system environment. In the sperm cell, specifically in the flagellum, the three major alterations that must occur so hyperactivated motility is prompted are: (1) increased concentration of HCO3 − , (2) Ca2+ , and (3) cholesterol efflux. The rationale behind this necessity is the fact that most motility promoter proteins respond to an increase of these two ions [68, 69]. The female reproductive system is rich in bicarbonate (HCO3 − ) and presents an alkaline environment. The activation of the sperm-specific Na+ /HCO3 − cotransporter mediates the influx of HCO3 − . As a result, there is an increase in internal sperm pH and hyperpolarization. This sperm-specific isoform of Na+ /HCO3 − responds to cAMP, electrical potential and phosphorylation. Yet, HCO3 − influx is insufficient to promote a complete hyperpolarization of the plasma membrane. An influx

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of Na+ by the sperm-specific Na+ /HCO3 − cotransporter, an influx of K+ mediated by the calcium-activated potassium channels and the ion transporter Na,K-ATPase ensure a full hyperpolarization. In sperm cytoplasm, HCO3 − promotes phospholipids exchange factors that result in exposure of cholesterol to albumin, the main cholesterol receptor in the female reproductive system. Consequently, cholesterol is removed from the plasma membrane and membrane fluidity and permeability to ions increases. Interestingly, this efflux of cholesterol further activates the sperm-specific Na+ /HCO3 − cotransporter promoting a positive feedback loop [69–73]. The intracellular increase of Ca2+ is crucial for hyperactivated motility promotion. Several mechanisms have been described that induce the influx of this ion. First, the Ca2+ -ATPase pump, that ensures low basal Ca2+ intracellular concentrations is inhibited and activation of voltage-dependent calcium channel (VOCCs) and of spermspecific channel (CatSper) promote Ca2+ influx [9, 74, 75]. It has been described that progesterone promotes the influx of Ca2+ through CatSper channel and has a dose-dependent effect on sperm flagellar beat frequency and hyperactivated motility [76]. CatSper was identified in 2001 by Ren et al. and rapidly its function on sperm motility was revealed [9]. In human sperm cells, CatSper responds to progesterone, is pH sensitive, is exclusively localized to the flagellum and null mice for CatSper1 are infertile [9, 77, 78]. The increase of Ca2+ activates PIK3C (Phosphatidylinositol 3-kinase catalytic subunit; Ca+ dependent), that forms PIP3 from PIP2. PIP3 will bind and consequently activate the 3-phosphoinositide-dependent protein kinase (PDPK1) that phosphorylates the AKT on Thr308. Consequently, Ser473 becomes exposed and may be phosphorylated by PDP kinase 2 (PDPK2), activating AKT. In turn AKT phosphorylates tyrosine residues on several key proteins for sperm motility [79]. In the meantime, HCO3 − together with Ca2+ activates soluble ADCY. Note that ADCY instead of responding to guanosine-5’-triphosphate responds to HCO3 − and Ca2+ increase [69]. Activation of ADCY increases the production of cAMP and protein kinase A (PKA/PRKA) is activated. Consequently, PRKA phosphorylates multiple proteins during hyperactivated motility, specially proteins related with tyrosine kinase activation [80, 81]. PRKA is typically composed of 2 regulatory subunits and 2 catalytic subunits. Interestingly, sperm contains an unique catalytic subunit, termed Cα2. Upon elimination of sperm specific PRKA subunit or ADCY sperm cannot acquire motility [68, 82, 83]. In order to achieve high levels of protein phosphorylation, protein phosphatases must be inhibited. Several Ser/Thr phosphatases, like PPP1CC2 or PPP2A are kept inhibited by tyrosine phosphorylation mediated by the Src family protein tyrosine kinases [84]. Figure 9.2 represents the signaling pathways involved in hyperactivated motility in human sperm.

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9.5 Boy Meets Girl: Signaling Pathways in Acrosome Reaction As a result of a successful hyperactivated motility, the sperm cells reach the vicinities of the oocyte. For oocyte fertilization, sperm must suffer acrosome reaction. In the next section we will describe the sperm signaling events triggered by the oocyte and/or cellular components surrounding the oocyte: acrosome reaction. In early 1950s Dan and colleagues observed for the first time the acrosome reaction in sea urchin spermatozoa. Acrosome reaction was then described as: “Response of the acrosome to the chemical stimulation of the dissolved jelly, which is manifested by an almost instantaneous local breakdown of the acrosome membrane so that the acrosome substance is exposed at the tip of the sperm head as a relatively labile mass” [85]. What is the purpose of the acrosome reaction? Where does the acrosome reaction occurs? What type of proteins are released from the acrosome? Which is the duration of the acrosome reaction? What are the sperm signaling events that coordinate acrosome reaction? These are some of the questions that in the last decades, the scientific community have been focused in order to understand the acrosome reaction. Although all the questions are pertinent, we will focus our attention on describing the signaling events behind human acrosome reaction. Acrosome reaction is a calcium-dependent exocytic event based on fusion between the acrosomal outer membrane and the apposed sperm plasma membrane (see Fig. 9.3). The fusion patched formed allows dispersion of acrosomal enzymes, crucial to oocyte surrounding cells and extracellular matrixes penetration [86]. Moreover, acrosome reactions exposes and reallocate sperm intracellular proteins which are essential for oocyte fertilization. Upon reaching the mature oocyte, the sperm cell encounters an oocyte surrounded by a narrow layer of follicular cells (corona radiata) followed by cumulus cells embedded in an extracellular matrix rich in hyaluronan and proteoglycans (cumulus oophorus). Continuously to the cumulus oophorus towards the oocyte there is a mesh of fine filaments in a glycol-protein matrix, the zona pellucida. The main components of this structure are 4 isoforms of a glycoproteins, the zona pellucida glycoproteins (ZP1-ZP4) [87–89]. The cumulus cells produce and release progesterone, a key player not only in acrosome reaction but also in hyperactivated motility (see Sect. 9.4.2) [90]. Apparently, the release of progesterone is promoted through interaction between cumulus cells and the testis-expressed protein 101 (TEX101), a glycosylphosphatidylinositolanchored membrane protein which is released from the sperm surface [91]. Progesterone binds to plasma membrane receptors (contrary to somatic cells in which progesterone acts through nuclear receptors) and promotes increase Ca2+ within the acrosome and plasma membrane depolarization [75, 77]. Interestingly the sperm-specific receptor described as progesterone responsive and that promotes Ca2+ influx, the CatSper, is restricted to the sperm flagellum [9] (see Sect. 9.4.2). The involvement of tyrosine kinase pathway and gamma aminobutyric acid (GABA)

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Fig. 9.3 Schematic representation of human acrosome reaction. Acrosome reaction is activated by both progesterone and zona-pellucida proteins. The activated signaling pathways result on increase of Ca2+ between the plasma membrane and the outer acrosomal membrane. As a result, F-actin depolymerizes and solubilizes. Since actin forms a physical barrier between plasma membrane and outer acrosomal membrane, its solubilization allows the fusing between both membranes. Activation of dynamin stabilizes the fusing pores (points of contact between both membranes), which results in prolonged exocytotic event (acrosome reaction). P in light circle, progesterone; P in dark circle, phosphorylation; OAM, outer acrosomal membrane; TKR, tyrosine kinase receptor; ZP, zona-pellucida

a like receptor have been suggested as mediators of Ca2+ influx due to progesterone action in the acrosome. Regarding membrane depolarization, progesterone promotes the efflux of Cl− through a GABAa/Cl− channel and influx of Na+ through voltage-gated Na+ channels [3, 92–96]. Similarly to progesterone, ZP proteins are key for the increase of Ca2+ and pH within the acrosome. Several receptors for ZP have been proposed, from receptors coupled to Gi proteins, glycine receptor/Cl− channel, tyrosine kinase receptors to VOCCs [92, 97–99]. Another peculiarity of ZP proteins relies on the fact that they are partially responsible for species-specific fertilization. The combination and role of each ZP isoform is species-specific resulting in species ZP-ZP receptor incompatibility [100].

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Currently, there is still some debate on whether progesterone or zona pellucida proteins are the trigger for acrosome reaction and where does the acrosome reactions occurs: in the cumulus oophorous or in zona pellucida. The most supported hypothesis is a priming phenomenon. More specifically, progesterone primes spermatozoa for acrosome reaction upon ZP proteins binding [101, 102]. This type of sequential events is a common mechanism in sperm maturation, since epididymal maturation is a requirement for sperm capacitation and acrosome reaction only occurs in capacitated spermatozoa [103]. A successful acrosome reaction requires an alkalization and increase of Ca2+ in the intra-acrosomal environment. In the acrosome, alkalization partially relies in the influx of HCO3 − . It appears that a Na+ /HCO3 − cotransporter is the main responsible for the increase of HCO3 − within the acrosome [69]. Alternatively, the presence of Na+ /Cl− /HCO3 − cotransporter and Cl− /HCO3 − exchangers has been proposed in sperm cells. Yet, their involvement in acrosome reaction is debatable [104–107]. Several efforts have been made to uncover the signaling events responsible for the increase in intracellular Ca2+ . The increase of sperm intracellular Ca2+ during sperm capacitation is promoted by: inhibition of the Ca2+ /ATPase pump; activation of VOOCs, low-voltage T-type channels and CatSper; action of GTP-binding protein signaling pathway and calcium store depletion pathway [9, 86, 108–112]. The latter imposes that sperm cells contain Ca2+ intracellular storages. Currently, it is accepted that the acrosome region is rich in Ca2+ and possible a storage location of this ion. Moreover, the fact that sperm cells express sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA); ryanodine receptors (RyRs) and respond to phospholipase C signaling, all involved in either Ca2+ storage, reinforces the hypothesis the Ca2+ is stored in human sperm cells [113]. Ultimately, Ca2+ is directly involved in the fusion of the plasma membrane and outer plasma membrane of the acrosome. The increase of intracellular Ca2+ results in activation of PRKA, PRKC, phospholipase C and A and depolarization of actin. Note that although Ca2+ exerts it function in the free form, it can be associated with calmodulin in human sperm to activate specific enzymes (for example calcineurin) [114, 115].

9.5.1 Signaling Pathways Acrosome reaction signaling can be divided in two types: progesterone activated and zona pellucida activated. The cross-talk between both signaling mechanisms is significant and, as already stated previously, progesterone seems to have a priming effect for zona pellucida induced acrosome reaction. Note that animal models are still a significant source of information and results are extrapolated for human sperm mechanism.

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Zona Pellucida Induced-Acrosome Reaction

Zona pellucida proteins activate two type of pathways: pertussis toxin-independent pathways and pertussis toxin-depended pathways. In pertussis toxin-independent pathways, zona pellucida proteins bind to tyrosine kinase receptors, which results in phosphorylation of Ca2+ transporter and entry of Ca2+ . Also, PLCG1 is phosphorylated and upon increase of Ca2+ activated. Consequently, PLCG1 is translocated and coupled to the plasma membrane. Another pertussis toxin-independent mechanism results from activation of a poorly cation selective channel by ZP that results in depolarization of the plasma membrane and consequently opening of VOCCs (mainly T-type) and increase in intracellular Ca2+ . In pertussis toxin-dependent pathway zona pellucid proteins activate Gi receptors which (1) promote the influx of Na+ through the Na+ /H+ exchanger and increase of intracellular pH; (2) activate adenylate cyclase, leading to cAMP synthesis, and (3) induce PLC1beta1 activation and translocation to the equatorial region. Both PLCB1 and PLCC hydrolyse PIP2 in the membrane or in association with actin, leading to the generation of IP3 and DAG. The increase in pH results in IP3 binding to its receptor in the outer acrosomal membrane. This leads to the release of Ca2+ from acrosomal Ca2+ stores. The increase of cAMP also leads to the influx of Ca2+ from acrosomal stores, either directly by acting on the nucleotide-gated channel (NGC) or indirectly via activation of PRKA. The decrease of Ca2+ within the acrosome (intracellular stores) results in SOC (Store-operated channels) activation in the plasma membrane and a sustained enhancement of Ca2+ . The production of DAG activates and translocate PRKC to the plasma membrane, which in turn stimulates influx of Ca2+ from extracellular medium (channel/pump unknown). Even more, DAG results in a PLA2 stimulation, directly or via PRKC and PLA2 hydrolyses lipids in the phospholipids resulting in plasma membrane vesiculation (formation of PM and OAM hybrid vesicles). In response to the increase in Ca2+ (around 500 nM) and pH, F-actin depolymerizes to form soluble monomeric actin (G-protein). Since actin provides a physical barrier between plasma membrane and outer acrosomal membrane, its depolarization allows PM and OAM to come closer. Actin depolarization together with vesiculation leads to acrosomal content release at the site of sperm-oocyte binding [75, 102, 116].

9.5.1.2

Progesterone Induced-Acrosome Reaction

In sperm cells, progesterone induces Cl− efflux via a GABA like receptor (the activity is tyrosine kinase dependent). Moreover, progesterone promotes Na+ influx probably through a voltage-operated channel (either T or L type). The synergetic Cl− and Na+ fluxes can explain the depolarizing effect of progesterone. Besides promoting ion fluxes, progesterone stimulates protein Gi receptors which activate both PLC and PLA2. Reid and colleagues defend that progesterone signaling is fundamental for the formation of hybrid vesicles and vesiculation. Upon activation of PLC, IP3 is formed and activates downstream kinases that phosphorylate dynamin. Alongside this, SNARE (SNAP receptor) proteins within the outer acrosomal membrane would

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assemble (unknow trigger) and associate with dynamin (protein involved in membrane manipulation in exocytosis events) and position and stabilize the fusion pores of the hybrid vesicles thus leading to the prolonged exocytotic event that characterizes the sperm acrosome reaction [117]. Figure 9.3 represents the signaling pathways involved in acrosome reaction.

9.6 Concluding Remarks Sperm cells are unique: they have the ability to move forward; they fulfil their goal in another organism; and they are morphologically highly specialized. Most signaling pathways described in somatic cells have also been shown (or at least hinted) to be present in sperm. Thus, the distinct features of sperm cells do not arise for exclusive signaling pathways but from exclusive players on ubiquitous signaling pathways. The activation of PRKA and PPP1, the importance of progesterone as a primary messenger and the presence of a plethora of Ca2+ channels is common to most human cells. Yet, in sperm, all these events present sperm-specific features due to sperm-specific isoforms of key proteins. Identification and functional characterization of sperm-specific protein isoforms opens avenues not only to fully understand sperm physiology, but also to take a step towards diagnosis and development of new therapies for idiopathic male infertility. Acknowledgments This work was financed by FEDER funds through the “Programa Operacional Competitividade e Internacionalização—COMPETE 2020” and by National Funds through the FCT—Fundação para a Ciência e Tecnologia (PTDB/BBB-BQB/3804/2014). We are thankful to Institute for Biomedicine—iBiMED (UID/BIM/04501/2013, POCI-01-0145-FEDER-007628 and UID/BIM/04501/2019) for supporting this project. iBiMED is supported by the Portuguese Foundation for Science and Technology (FCT), Compete2020 and FEDER fund. This work was also supported by an individual grant from FCT of the Portuguese Ministry of Science and Higher Education to D.P. (SFRH/BD/137487/2018).

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Chapter 10

Hormone Signaling Pathways in the Postnatal Mammary Gland Fátima L. Monteiro, Inês Direito, and Luisa A. Helguero

Abstract The mammary gland is an organ that in female mammals develops after birth and specializes to produce milk to feed the offspring. The mammary gland undergoes continued remodeling in response to hormonal cues that direct its development and function throughout sexual development and reproductive age. This gland can undergo numerous cycles of proliferation, differentiation and apoptosis after each pregnancy. This is accomplished by mammary stem cells (MaSCs) capable of sustaining continuous remodeling of the mammary tissue. The biological processes that regulate mammary morphogenesis and remodeling are regulated by steroid and peptide hormones which tightly control gene expression and epigenetic changes. This chapter aims to give a general overview of the main hormones and signaling pathways that control the female mammary gland differentiation leading to a fully functional milk producing gland. For this purpose, a summary of findings from commonly used cell and animal models, as well as humans was selected to explain the effects exerted by the main hormones implicated in mammary gland biology. Keywords Mammary gland · Morphogenesis · Differentiation steroid hormones · Growth factors · Peptide hormones · Epigenetics · Mammary stem cells

F. L. Monteiro and I. Direito—Contributed equally. F. L. Monteiro · I. Direito · L. A. Helguero (B) Hormones and Cancer Research Group, iBiMED—Institute of Biomedicine, University of Aveiro, Aveiro, Portugal e-mail: [email protected] F. L. Monteiro e-mail: [email protected] I. Direito e-mail: [email protected] © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_10

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Abbreviations ATF4 ATF6 ATG AR AREG BCL-2 BEC1 BiP BM BMI1 BMP4 BTC CCND1 CDK6 CHOP CSN1S1 CXCR4 CXCL12 DNMTs EGF EGFR EZH2 EIF5 ELF5 EMT EPGN EPR ER Esr FGFs FGFRs FOXP3 FTO GH GR GREs HB-EGF HDMTs HMTs HOXA1 HOXB3 HOXD10

Activating transcription factor 4 Activating transcription factor 6 Autophagy protein Androgen receptor Amphiregulin Bcl2-associated agonist of cell death Beclin-1 EnR stressor sensor-binding immunoglobulin protein Basement membrane Polycomb complex protein BMI-1 Bone morphogenetic protein 4 Betacellulin Cyclin D1 Cyclin-dependent kinase-6 DNA damage-inducible transcript 3 protein Alpha-S1-casein C-X-C chemokine receptor type 4 Stromal cell-derived factor 1 DNA methyltransferases Epidermal growth factor Epidermal growth factor receptor Histone-lysine N-methyltransferase EZH2 Eukaryotic translation initiation factor 5A-1 ETS-related transcription factor Elf-5 Epithelial mesenchymal transition Epigen Epiregulin Estrogen receptor Estrogen receptor gene Fibroblast growth factors FGF receptors Forkhead box protein P3 Alpha-ketoglutarate-dependent dioxygenase FTO Growth hormone Glucocorticoid receptor Glucocorticoid responsive elements Heparin binding-EGF Demethylases Histone methyltransferases Homeobox protein Hox-A1 Homeobox protein Hox-B3 Homeobox protein Hox-D10

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hPTMs hPTMs IRE1α IGF IGF1 IGFBPs IGFRs IGFs IR INS JNK JARID1B KDMs LEF1 MAP1LC3B MAPK MaSCs ncRNAS NFE2L2 NRG NOTCH3 PARP PPARγ PERK PI3K PKC PR PRCs PRL PRLHR PRLRs PTHrP PYGO2 RANKL REnR RSPO1 RTKs SLC2A1 STAT5A SREBP1 TAL1 TCF3/4 TDGF1 TEBs TGFα

Histone post-translational modifications Histones post-translational modifications Inositol-requiring enzyme 1α Insulin -like growth factor Insulin-like growth factor I High affinity binding proteins Insulin growth factor-receptors Insulin growth factors Insulin receptor Insulin Mitogen-activated protein kinase 8 Lysine-specific demethylase 5B K demethylases Lymphoid enhancer-binding factor 1 Microtubule-associated proteins 1A/1B light chain 3B Mitogen-activated protein kinases Mammary stem cells Non-coding RNAs Nuclear factor erythroid 2-related factor 2 Neuroregulins Neurogenic locus notch homolog protein 3 Poly [ADP-ribose] polymerase Peroxisome proliferator-activated receptor gamma Protein kinase RNA-like endoplasmic reticulum kinase Phosphatidylinositol 3-kinase Protein kinase C Progesterone receptor POLYCOMB REPRESSIVE COMPLEXES Prolactin Prolactin-releasing peptide receptor PRL receptors Parathyroid hormone related peptide Pygopus homolog 2 Tumor necrosis factor ligand superfamily member 11 Rough endoplasmic reticulum R-spondin-1 Receptor tyrosine kinases Facilitated glucose transporter member 1 Signal transducer and activator of transcription 5A Sterol regulatory element-binding protein 1 T-cell acute lymphocytic leukemia protein 1 Transcription factor E2-alpha/4 Teratocarcinoma-derived growth factor 1 Terminal end buds Transforming growth factor alpha

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Transforming growth factor beta-1 Tight junctions Cellular tumor antigen p53 Transcription factor SOX-4 Transcription factor SOX17 Unfolded Protein Response Vimentin X-box-binding protein 1 Zinc finger E-box-binding homeobox 1/2

10.1 General Overview of the Mammary Gland Development and Function The mature mammary gland structure resembles that of a tree and consists of an extensive branched ductal network adorned by a group of alveoli embedded within the subcutaneous fatty tissue (Fig. 10.1). In humans, groups of alveoli organize into lobules with a lactiferous duct that drains into a principal duct that opens in the nipple [1, 2]. Ducts and alveoli are composed of luminal epithelial cells and basal myoepithelial cells, which are separated from the surrounding stroma by a basement membrane (BM) [3]. The luminal epithelial cells line both ducts and alveoli as a single layer of epithelia that forms a central lumen, while the basal myoepithelial cells are found beneath the luminal cells in direct contact with the BM. Luminal cells can also contact BM if there are microscopic gaps in the myoepithelium, which occurs more frequently in the alveoli than in the ducts [3]. The alveolar luminal cells produce and secrete milk [4, 5], while the surrounding myoepithelial cells contract to expel the milk from the alveoli through the lactiferous ducts toward the nipple, where it is secreted in response to suckling [1, 6, 7]. The BM is rich in growth factors and extracellular matrix proteins like collagens, laminin, glycoproteins and proteoglycans which play a fundamental role in mammary morphogenesis and function [8]. The mammary gland stroma consists of adipose and connective fibrous tissue and a variety of cellular types including fibroblasts, immune and endothelial cells [9, 10], which participate in the paracrine regulation leading to a mature mammary gland [11]. Most of the mammary gland development occurs after birth and can be divided into embryonic, pubertal, pregnancy, lactation and involution [1, 12, 13]. These stages are defined by the tissue morphology and differential patterns of gene expression [1, 4, 14]. Each stage is under the control of steroid and peptide hormones as well as local factors via autocrine and paracrine interactions, which activate a variety of signaling pathways [1, 12, 13, 15–17]. The embryonic stage begins at mid-gestation when a surface ectodermal thickening migrates into the surrounding stroma to form an elementary mammary bud. This initial process is exclusively dependent on stromal-epithelial interactions stimulated

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Fig. 10.1 The mature mammary gland structure. Mature mammary gland consists of an extensive branched ductal network adorned by a group of alveoli embedded within the subcutaneous fatty tissue. In humans, groups of alveoli organize into lobules with a lactiferous duct that drains into a principal duct that openings in the nipple. Ducts and alveoli are composed of luminal epithelial cells and basal myoepithelial cells, which are separated from the surrounding stroma by a basement membrane. The alveolar luminal cells produce and secrete milk, while the surrounding myoepithelial cells contract to expel the milk from the alveoli through the lactiferous ducts toward the nipple. The mammary gland stroma consists of adipose and connective fibrous tissue and a variety of cellular types including fibroblasts, immune and endothelial cells in blood vessels

by parathyroid hormone related peptide (PTHrP) and WNT signaling molecules [13, 17]. At birth, the mammary bud invades the surrounding adipose tissue to form primitive branched ductal structures contained in a rudimentary mammary gland that is arrested at this stage until puberty [1, 18]. Although the initial stages of mammary gland development are independent of systemic cues, the cells are already responsive to hormonal stimuli and depend on reciprocal signaling between the epithelium and the surrounding stroma [1, 18, 19]. Throughout puberty, estrogens, glucocorticoids and growth hormone (GH) are responsible for ductal elongation throughout each menstrual cycle [20]. Terminal end buds (TEBs) are specialized structures at the tips of the elongating ducts, composed of mammary progenitor cells called the ‘body cells’ and ‘cap cells’ [11, 21], which, as the duct elongates, differentiate into luminal and myoepithelial cells, respectively [11, 22]. TEBs generate the lobular portions of the gland that elongate and bifurcate to completely fill the mammary fat pad, creating branches at regular intervals and forming a tree-like ductal structure. On branching, mammary epithelial cells display a significant alteration of their interaction with the extracellular matrix and epithelial mesenchymal transition (EMT) occurs [1, 23]. This process is characterized, amongst other things, by disruption of epithelial architecture, loss of apico-basal polarity, an increase in mesenchymal morphology

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and secretion of extracellular proteases, which degrade extracellular matrix components and promote invasiveness and resistance to apoptosis [23, 24]. In pregnancy, systemic levels of estrogens (such as 17β-estradiol), progesterone and placental lactogen/prolactin (PRL) increase. The estrogen surge stimulates ductal elongation, while additional ductal side branching is under the control of progesterone [14]. Firstly, progesterone induces proliferation and maturation of the side branches culminating in the amplification of alveolar progenitor cells to form the alveoli clusters. In late pregnancy and after parturition, the pituitary hormone PRL acts in synergy with insulin growth factors (IGFs) and glucocorticoids, to induce specific gene expression leading to ductal terminal differentiation and lactogenesis [25–27] to achieve a fully differentiated gland capable of milk production and secretion during lactation [28, 29]. The involution process begins with weaning, with the subsequent accumulation of milk in the mammary gland [1, 30, 31]. Involution starts right after weaning and is characterized by a transient destruction of the BM by matrix metalloproteases (MMPs) and anoikis of mammary epithelial cells (apoptosis due to detachment from the BM) [8, 30, 32]. The mammary adipose tissue redevelops and the mammary tissue remodels to yield a state that is morphologically, but not genetically, similar to the gland before pregnancy [30, 33]. Following menopause, estrogen and progesterone serum plasma levels abruptly decrease while testosterone levels remain basically the same which results in a regression of the mammary epithelial and stromal compartments and an increase in adipose tissue [34]. The proportion of epithelial cell populations changes with age showing decreased myoepithelial cell numbers and increased luminal CD49f/CK19/MUC1/CD227 progenitors, as well as c-kit+ progenitors [35].

10.2 Epigenetic Control of Mammary Epithelial Cell Differentiation Several decades ago, DeOme and his colleagues observed that following transplantation of epithelium isolated from several different regions of the mammary gland, these transplants were able to reconstitute the normal mammary ductal tree [36]. Presently, the idea widely accepted is that of a mammary stem/progenitor cell hierarchy, where MaSCs localized throughout the mammary tree generate early bipotent progenitors which in turn can differentiate into either luminal or basal committed progenitors (Fig. 10.2) [37]. The luminal progenitors can give rise to cells that are either ductal cells—with or without estrogen receptor (ER) expression, or alveolar progenitors that maintain the capacity to originate fully differentiated alveolar cells. Additionally, the basal progenitor cells can give rise to the myoepithelial lineage [37–42], although some evidences suggest that basal progenitor cells can also give rise to luminal progenitor cells [38, 43]. Accordingly, it is increasingly recognized that the hierarchal differentiation model is not rigid, instead it constitutes a flexible process [38, 44, 45]. Tight gene expression regulation is critical to induce MaSC

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Fig. 10.2 Epigenetic control of the mammary cell hierarchy. Mammary stem cells (MaSCs) originate bipotent progenitors that can commit to both myoepithelial and luminal/alveolar lineages through the respective progenitors. The luminal progenitors can either generate luminal cells expressing or not estrogen receptor (ER), or alveolar progenitors, which can in turn fully differentiate into alveolar cells. Additionally, the basal progenitor cells can give rise to myoepithelial cells. Some evidences also suggest that basal progenitor cells can also give rise to luminal progenitors (dashed arrow). Epigenetic regulators that positively (green) or negatively (red) influence specific cell fate decisions are represented. Detailed information on these can be found in the main text. MaSC—mammary stem cells; ER—Estrogen receptor

lineage commitment and functional differentiation of daughter cells during normal mammary gland development [38]. The regulation of stem-cell or cell-type specific commitment is under strict epigenetic control [46, 47]. Epigenetic mechanisms can be divided into four main categories: DNA methylation, histones post-translational modifications (hPTMs), substitution of histone variants and isoforms in the nucleosome, and non-coding RNAs (ncRNAS) [47–49]. In the next section we summarize the main epigenetic mechanisms governing mammary gland differentiation (Fig. 10.2).

10.2.1 DNA Methylation The methylation of large stretches of CpG dinucleotide (CpG islands) is associated with transcriptional repression and it is carried out by DNA methyltransferases (DNMTs 1, 3a and 3b) [47]. DNA methylation regulates MaSCs self-renewal and maintenance providing cellular memory during repeated cell divisions [48]. DNMT1 expression increases with mammary epithelial cell differentiation which correlates with increased CpG island methylation of developmental and non-lineage-specific genes [38]. The differences in methylation patterns from MaSC and progenitor to differentiated cells are mainly related with the expression of stem cell maintenance genes (e.g. Homeobox protein Hox-A1—HOXA1), which are hypomethylated in stem/progenitor cells, while the cell-specific genes are hypermethylated [50].

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DNMT1 activity has been described to regulate the maintenance of MaSCs [38, 46, 48] and TEB development during puberty, and to promote stem/progenitor cells expansion during pregnancy [48]. During lactation, specific genes coding for milk components such as β, γ and κ-casein, become hypomethylated [51]. Interestingly, parity seems to leave a long-term epigenetic memory conducted by transcription co-regulator STAT5A which modifies the mammary gland response to a new pregnancy, producing milk faster and in higher quantity [52].

10.2.2 Histone Post-translational Modifications The combination of histone post-translational modifications (hPTMs) enriched in specific DNA regulatory sequences determines if transcription is activated or repressed, known as the histone code [53]. Combinations of hPTMs at both promoters and enhancers differ between basal progenitor, luminal progenitor, and mature cells [43, 54]. Histone lysine (K) methylations are the most studied hPTM in the mammary gland development and MaSC hierarchization processes [38, 47]. The active mark H3K4me3 is found in slightly higher amount in basal cells, while the repressive mark H3K27me3 coverage is higher in mature luminal cells [43, 50]. Examples of genes marked by H3K27me3 in mature luminal cells are Transcription factor SOX-4 (SOX4), Transcription factor E2-alpha/4 (TCF3/4), Homeobox protein Hox-B3 (HOXB3) and Zinc finger E-box-binding homeobox 1/2 (ZEB1/2), known regulators of stem cells and EMT [50]. Promoters containing both active and repressive marks (bivalent marks) are often found in progenitor cells to allow expression of lineage-specific genes upon differentiation. Mature luminal cells have specific gene promoters enriched in bivalent marks, which suggests that these marks are not restricted to lineage-specific genes but also to proliferative genes, to allow the cells to rapidly respond to environmental cues [38, 43, 50]. In basal progenitor cells, the genes containing bivalent marks regulate stem cell properties (e.g. Transcription factor SOX17 (SOX17), Homeobox protein Hox-D10 (HOXD10), T-cell acute lymphocytic leukemia protein 1 (TAL1)). Accordingly, in luminal progenitor cells, bivalent marks are found enriched in genes regulating epithelial tissue morphogenesis and development (e.g. Bone morphogenetic protein 4 (BMP4), Transforming growth factor beta-1 (TGFB1)). While, in mature luminal cells, bivalent marks are located in genes related with cell migration, proliferation and receptor tyrosine kinases (e.g. Vimentin (VIM), Cyclin-dependent kinase-6 (CDK6), Epidermal growth factor receptor (EGFR)) [43]. Histone methylation is regulated by both histone methyltransferases (HMTs) and demethylases (HDMTs). Epigenetic maintenance of the H3K27me3 repressive chromatin mark is preserved by polycomb repressive complexes (PRCs) which are required for the maintenance of stem cells, as well as to silence lineage-specific transcription factors until the proper cues stimulate differentiation. There are two PRCs, PRC1 and PRC2 [38]. Polycomb complex protein BMI-1 (BMI1), a member of the PCGF paralog group of the PRC1, has been shown to repress alveolar

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differentiation and induce MaSC self-renewal [46, 55, 56]. MaSC self-renewal is also dependent on the histone methylation reader Pygopus homolog 2 (PYGO2), a co-factor of WNT/β-Catenin pathway, expressed in the basal epithelium [38, 57, 58]. PYGO2 recruits WDR5-histone methyltransferase complex to facilitate enrichment of activating H3K4me3 mark at WNT/β-Catenin target genes allowing MaSCs expansion [57]. Moreover, WNT/β-Catenin signaling mediated by PYGO2 facilitates a bivalent chromatin configuration at Neurogenic locus notch homolog protein 3 (NOTCH3) locus maintaining its expression low and preventing MaSC/basal progenitor cells to undergo luminal cell differentiation [58]. Furthermore, Histone-lysine N-methyltransferase EZH2 (EZH2), a component of PRC2, is involved in maintaining luminal progenitor cells during puberty and lobuloalveolar differentiation during pregnancy [39, 59]. Progesterone induces EZH2 expression thus driving luminal and alveolar expansion during pregnancy [38, 46, 59]. K demethylases (KDMs), particularly, Lysine-specific demethylase 5B (JARID1B), a protein from KDM1 JmjC domain family, has been suggested to promote ductal elongation and side branching during puberty, by repression of basal-specific genes and induction of luminal lineage-specific gene expression (ETSrelated transcription factor Elf-5 (ELF5), Estrogen receptor (ESR1), Progesterone receptor (PGR), Prolactin-releasing peptide receptor (PRLHR) and Signal transducer and activator of transcription 5A (STAT5A)) [38, 60–62]. JARID1B usually acts by removing the active marks di and trimethylation of H3K4, however in some specific circumstances JARID1B can directly bind these histone marks enhancing their active role in transcription [38].

10.2.3 Histone Variants and Non-canonical Isoforms Histones are amongst the most highly conserved proteins in both sequence and structure. Each histone class consists of different variants and isoforms which are equally conserved across species. While histone variants are present as single copies and are thought to be constitutively expressed, histone isoforms are usually found clustered in repeated arrays and their transcription is normally replication-dependent [49, 63]. Histone variants and isoforms for all subtypes of core histones, except H4, have been described. Replacement of canonical histone by its non-canonical variants or isoforms contribute to distinct nucleosomal architectures, which can influence gene expression as well as DNA replication, recombination and repair, among other [64]. Histone variants have specialized functions and their expression patterns change during development and differentiation, with alterations found in cancer [63, 65]. However, their involvement in the mammary gland development has been poorly studied. mRNAs and proteins from several histone variants as well as isoforms were found to be differentially expressed between the stem cell-like and differentiated stages of the mouse mammary epithelial cell line HC11. HIST2H2AC isoform expression is maximum in mouse MaSC-like stage and decreased along with lactogenic differentiation [28]. EGF induces HIST2H2AC expression in undifferentiated cells which is

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necessary to allow proliferation and EMT. This may be due to HIST2H2AC repressive effects over Cadherin-1 (CDH1) gene transcription via ZEB1 upregulation [66]. The histone variant H2AFX is involved in DNA damage response and increases during pregnancy. This may be due to promoter demethylation of H2AFX in basal myoepithelial cells in response to paracrine stimulation by luminal neighboring cells in response to PRL during late pregnancy [67, 68].

10.2.4 Non-coding RNAs There are three main classes of small non-coding RNAs. In most cases, these molecules have complex and precise patterns of expression during differentiation, development and tissue specificity, being miRNAs the most well studied in the mammary gland context [44]. miRNAs control cell growth, differentiation, proliferation, metabolism, apoptosis and stem cell biology by targeting one or multiple pathways simultaneously. For instance, elements of the miRNA-200 family, particularly miRNAs 200a and b, were found to be downregulated in HC11 MaSC-like cells when compared to a differentiated state. These miRNAs were shown to inhibit EMT by downregulating ZEB1/2 and SUZ12 genes and inducing CDH1 expression [69]. Similarly, miRNA-205 was also reported to target ZEB1/2 genes inhibiting EMT [70]. Conversely, miRNA-22 was found to be highly expressed in mammary progenitor cells, where it targets Tet family enzymes leading to inhibition of DNA demethylation of genes responsible for the expansion of the MaSC pool such as ZEB1/2 and BMI1 [71]. Members of miRNA-29 family (miRNA-29a, b and c) regulate DNA methylation levels by inhibiting DNMT3a and DNMT3b in dairy cow mammary gland development. Even though global DNA methylation seem to be affected by miRNA29 action, this is particularly important for lactation-related genes like Alpha-S1casein (CSN1S1), Eukaryotic translation initiation factor 5A-1 (EIF5), Peroxisome proliferator-activated receptor gamma (PPARγ), Sterol regulatory element-binding protein 1 (SREBP1) and Solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1) and secretion of other milk components (triglycerides, lactose and lactoprotein) [72]. Exosomes containing miRNAs are secreted by mammary gland epithelial cells together with other milk components. These may induce epigenetic changes in the offspring, which are intended to only last during the breastfeeding period. The most abundant miRNA found in both human and cow’s milk is miRNA-148a. This microRNA is known to repress DNMT1 activity leading to active expression of developmental genes such as Insulin (INS), Forkhead box protein P3 (FOXP3), Insulin-like growth factor I (IGF1), Nuclear factor erythroid 2-related factor 2 (NFE2L2) and Alpha-ketoglutarate-dependent dioxygenase FTO (FTO) which have a critical role in adipogenesis and feeding control. Moreover, miRNA-125b, also found in milk, suppresses Cellular tumor antigen p53 (TP53) expression promoting cell proliferation and anabolic metabolism [73].

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10.3 Steroid Hormone Signaling in the Mammary Gland Steroid hormones with functions in the mammary gland include estrogens, progestins androgens and glucocorticoids. Steroids exert their actions through intracellular receptors which function as ligand-activated transcription factors. These receptors are members of the nuclear receptor superfamily and share a protein structure which can be divided into modular domains with specific functions (Fig. 10.3) [74].

10.3.1 Estrogens These group of molecules, of which 17β-estradiol is the most active, can bind and activate two estrogen receptor subtypes: ERα and ERβ. These two proteins bind 17β-estradiol with similar affinity, but show differential binding to other natural and synthetic ligands [75–77]. Moreover, ERα and ERβ exhibit distinct cellular and tissue distribution patterns and display unique structural and functional features [75, 78] which allow them to regulate different set of genes [79, 80]. In the breast, ERα is predominantly expressed in luminal cells [81] but also in cells within the supporting stroma [82]. In humans, ERα is detectable in the breast epithelium of fetus since the 30th week of gestation and apparently it is functional for the first 2–3 months of life [83]. Still, analyzing female Esr1−/− mice no mammary gland development is usually observed until puberty [84], when ER signaling is activated as a result of plasma estrogen increase. ERα mediates the rapid growth and expansion of the ducts to originate a functional gland being a critical regulator of mammary gland development [1, 12, 85]. Although 17β-estradiol stimulates mammary luminal cell proliferation, ERα and markers of proliferation such as ki67 or BrdU do not colocalize, which indicates that proliferation occurs in ER-negative luminal cells [86, 87]. 17β-estradiol induces

Fig. 10.3 Functional structure of nuclear receptors. Nuclear receptors are formed by functionally distinct domains. Amino-terminal (NTD) A/B domains encodes a conserved amino acid sequence, the activation function 1 (AF1), that is responsible for regulating the transcriptional activity by protein-protein interactions with transcription coregulators independently of ligand-binding. The central C region containing the DNA-binding domain (DBD) is responsible for the transcription of target genes. The flexible hinge, or D domain contains a nuclear localization signal that directs the receptor towards the nucleus. The multifunctional carboxyl-terminal E domain, also known as the ligand-binding domain (LBD), is a globular region that contains a hormone-binding site, a dimerization interface and a ligand-dependent co-regulator activation function (AF2). AF1 and AF2 can function independently or synergize mediating full transcriptional activation of nuclear receptors. Finally, the F domain is localized at the extreme carboxyl-terminus

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mouse mammary luminal cell proliferation through amphiregulin (AREG) signaling in a paracrine manner. 17β-estradiol induces AREG expression and release by epithelial ERα-positive cells [88, 89]. AREG in turn, activates proliferation of neighboring ERα-negative cells either directly or indirectly by inducing growth factor (i.e. Fibroblast growth factors (FGFs) secretion from stromal cells (Fig. 10.4a) [84, 90]. This crosstalk between epithelial and stromal compartments is tightly regulated by negative feedback responses mainly through the action of TGFβ1. In the adult mouse mammary gland, 17β-estradiol contributes to maintain differentiated ERα-positive cells and also the MaSC pool through paracrine signaling [91]. In humans, systemic 17β-estradiol and progesterone levels decrease with menopause but local mammary 17β-estradiol levels actually increase due to a higher aromatase activity in adipose stromal cells [92]. This may be a plausible explanation for the increased number of ERα-positive proliferative epithelial cells after menopause [93], although the overall mammary epithelial cell proliferation is reduced.

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Fig. 10.4 Signaling pathways and hormone action during mammary gland development. Development of mammary gland occurs in distinct stages divided into embryonic, pubertal, adult/early pregnancy, pregnancy, lactation, involution and aging. These stages are defined by the tissue morphology and differential patterns of gene expression. Each stage is under the control of steroid and peptide hormones as well as local factors via autocrine and paracrine interactions, which activate a variety of signaling pathways. a Estrogen Receptor α (ERα) mediates the rapid growth and expansion of the ducts to originate a functional gland, being a critical regulator of mammary gland development. Estrogens induce luminal cell proliferation through amphiregulin (AREG) signaling in a paracrine manner. In epithelial ERα+ cells, E2 induces AREG expression and its release from the cell membrane by metalloproteinase ADAM17. AREG in turn, directly promotes proliferation of neighboring ERα- cells by binding to their epidermal growth factor receptor (EGFR). Additionally, AREG also stimulates luminal cell proliferation in an indirect fashion by binding to EGFR in stromal cells. This results in secretion of fibroblast growth factors (FGFs) which will bind to fibroblast growth factors receptor 2 (FGFR2) in mammary epithelial cell to activate downsteam signalling, which in turn promotes cell proliferation. Pituitary growth hormone (GH) induces IGF-1 production both in the liver and in the mammary stromal cells. Then, IGF-1 acts on IGF-1 receptors (IGFR) of the mammary epithelial cells, to promote cell proliferation which results in ductal elongation. b During early pregnancy, progesterone (P) promotes the expansion of both luminal and basal epithelial progenitor cell populations. Progesterone receptor negative (PR− ) luminal progenitor cells and basal progenitor cells proliferate in a paracrine fashion, in response to factors released by non-proliferative ERα+ /PR+ cells, by a mechanism mediated by RANKL and WNT4. RANKL and WNT4 expression is directly induced by progesterone in PR+ luminal cells. RANKL binds to its receptor—RANK—in progenitor cells, leading to the activation of NF-κB-CCND1 pathway. P also indirectly induces the expression of RSPO1 in luminal progenitor cells. Both WNT4 and RSPO1 act together in progenitor cells promoting their expansion. c The pituitary hormone prolactin (PRL) acts directly on the mammary epithelium by binding to prolactin receptor (PRLR). Upon PRL binding, PRLR becomes activated and can trigger JAK/STAT pathway which is important for alveologenesis and lactogenesis. Negative regulation of JAK/STAT signaling pathway occurs through a negative feedback loop. The interaction of JAK2 and STAT5 is inhibited by members of the suppressors of cytokine signaling (SOCS) family, whose expression is, in turn, activated by PRL. Glucocorticoids (G) action, trough protein-protein interaction with phosphorylated STAT5, in combination with PRL, Insulin (INS) signaling is necessary for the expression of milk genes like whey acidic protein (WAP) and beta casein (CSN2) and its release in secretory granules

The role of ERβ in mammary development remains controversial due to lack of reliable antibodies used for its investigation. Although ERβ was shown to be the most expressed ER isoform in the human mammary epithelium being both mRNA and protein levels detected [94, 95], this has recently been challenged in a study comparing human RNA-Seq data with immunohistochemical characterization of several antibodies [96]. This later study concluded that ERβ protein is not expressed at detection levels in human breast [96]. However, studies using Esr2−/− mice showed that while this isoform is not necessary for ductal elongation [97, 98], it is necessary to induce epithelial differentiation during lactation, where Esr2−/− glands showed reduced membrane E-cadherin and increased proliferation [99]. Since the Esr2−/− used for this study was a complete knock-out, it can be argued that the effects observed are due to loss of paracrine signaling. But in vitro studies using the mouse mammary epithelial cell line HC11 support a direct regulation of epithelial differentiation by ERβ [100]. In these studies, loss of E-cadherin and increased proliferation was observed in cells treated with siRNA to ERβ [100, 101].

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Since ERβ is not expressed in human cell lines, most functional studies have been carried out using breast cancer cells overexpressing this protein [102]. In opposition to ERα which promotes cell proliferation, ERβ, in certain experimental conditions, can counterbalance ERα effects and inhibit proliferation and survival [103, 104]. This can be explained by the fact that ERα mediates a stimulatory effect of 17β-estradiol on cyclin D1 (CCND1) expression, which is essential for cell cycle progression, whereas ERβ has an inhibitory effect [105]. However, the effects mediated by ERβ when it is naturally expressed by the cells, as found in mouse mammary epithelial (HC11) and cancer cells (MC-L2), are more complex, and ERβ effects can change from anti-proliferative to proliferative if Mitogen-activated protein kinases (MAPK) or Phosphatidylinositol 3-kinase (PI3K) signaling are active [103]. ERβ expression is necessary to support mammary epithelial cell E-cadherin expression and regulates integrin expression [106]. Thus, although ERα and ERβ bind the same hormone and have common target genes, they also appear to have distinct expression programs [107, 108], where ERα expression is necessary for ductal epithelial cell proliferation and ERβ may be more involved in maintaining the epithelial phenotype. Weather ERβ is expressed differently in human and mouse mammary gland needs to be clarified.

10.3.2 Progesterone In the adult mammary gland, progesterone is the most potent mitogenic stimulus and is responsible for changes in the ductal tree that are necessary for a lactation-competent gland [1, 12, 85]. Although in humans PR is initially detected at 2–3 months after birth [83], progesterone effects are only evident after sexual maturity, peaking during the luteal phase of menstrual cycle and pregnancy, when the ratio of progesterone/estrogen is increased [109]. PR is expressed mainly as two isoforms, PR-A and PR-B, that arise from the same gene located in chromosome 11q22.1 [110]. Expression profiling studies revealed that PR-A and PR-B control distinct as well as overlapping sets of target genes [111]. PR is co-expressed with ERα in both epithelial and stromal cells [112, 113], which is expectable since PR is one of the major target genes upon 17β-estradiol stimulation. In the past years several authors have shown that ERs and PRs collaborate through protein-protein interactions to regulate gene expression in breast cancer cells [114–117]. Weather these mechanisms are also at play in the normal epithelial response has not been investigated. Transplantation studies have shown that epithelial PR is necessary for sidebranching and alveologenesis [118]. Both PR-A and PR-B isoforms are expressed in the mammary gland of virgin and pregnant mice and PR-A levels seems to be higher than PR-B [119]. However, characterization studies of Pgr-a−/− and Pgr-b−/− mice demonstrate that only PR-B is required for mammary development [119, 120]. During pregnancy, progesterone, in combination with PRL, promotes alveolar differentiation. First, progesterone promotes the expansion of both luminal and basal epithelial progenitor cell populations via WNT signaling [121]. At a later stage, progesterone plus PRL induce terminal epithelial/alveolar differentiation [122, 123].

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Studies with ovariectomized mice demonstrated that progesterone promotes the proliferation of mammary luminal cells by two distinct mechanisms: (i) a subset of epithelial ERα-positive/PR-positive cells proliferate in an autocrine way by a CCND1-dependent mechanism and (ii) a larger number of PR-negative neighboring cells proliferate in a paracrine fashion, in response to factors released by nonproliferative ERα-positive/PR-positive cells by a mechanism mediated by Tumor necrosis factor ligand superfamily member 11 (RANKL) or by WNT4 (Fig. 10.4b) [20, 124]. RANKL is a TNF family member that is induced by progesterone in PRpositive luminal cells, it binds to its receptor (RANK) localized in PR-negative luminal and basal progenitors leading to the activation of IKKα/IκBα-NF-κB-CCND1 pathway [20, 125]. Additionally, WNT4, which is also secreted by luminal PRpositive cells, can induce proliferation in PR-negative progenitors via the agonist of WNT/β-catenin pathway, to mediate progesterone-induced side branching [126]. Progesterone directly stimulates WNT4 expression in PR-positive luminal cells and indirectly induces R-spondin-1 (RSPO1) expression in PR− luminal progenitor cells. Both WNT4 and RSPO1 act together on basal cells promoting their expansion via WNT/β-catenin signaling [121, 124]. Finally, progesterone also plays a critical role in maintaining and generating MaSC and progenitor cell populations that are required for alveolar development, through a PR-dependent signaling axis involving C-XC chemokine receptor type 4 (CXCR4)/Stromal cell-derived factor 1 (CXCL12) [127, 128].

10.3.3 Androgens There is increasing evidence that independently of genetic sex, androgens inhibit mammary cell proliferation [129–132]. The androgen receptor (AR) gene is in chromosome Xq11-12 and comprises 8 exons coding a 110 kDa protein. The exact mechanism by which androgens inhibit the stimulatory effects of estrogens on the mammary gland is not completely understood. Androgens could act by decreasing mammary epithelial cell ER expression [133] or by inhibiting ER activity trough binding of AR NH2-terminus to the ER ligand-binding domain [134]. Additionally, androgens may favor 17β-estradiol metabolization into estrone by changing 17β-hydroxysteroid dehydrogenase activity [135]. During embryonic development of the mammary gland, AR expressed by mesenchymal cells of mammary buds regulates mammary anlagen regression in males in response to testosterone produced by embryonic testes; while in females, the rudimentary mammary gland continues to develop [130]. After birth AR is expressed by epithelial and stromal cells [136, 137]. During pregnancy and lactation, androgens and steroidogenic enzymes that transform the steroid precursors into androgens are differently expressed at different stages, which suggest that androgens may have a role in functional differentiation of the mammary gland [130] and in milk production [138, 139]. Although most findings support a suppressive role of androgens in mammary cell proliferation and function, some contradictory experiments using female rats and the HC11 cells, which are AR-positive, point to a stimulatory effect

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mediated by AR [140, 141] however, it is important to note that this can be the result of heterogeneity between species.

10.3.4 Glucocorticoids The involvement of glucocorticoids in mammary gland growth differentiation and lactation has been discussed over the years due to their roles in a variety of processes, such as energy homeostasis glucose metabolism and organ development [142]. Glucocorticoid signaling is mediated by the glucocorticoid receptor (GR). Glucocorticoids are essential lactogenic hormones. However, the signaling pathways by which they regulate milk protein expression synergistically with PRL and INS (Fig. 10.4c) are not completely clear and appear to differ depending on the milk gene [142]. Several mechanisms have been proposed such as GR binding to glucocorticoid responsive-elements (GREs) within promoter regions [143], protein-protein interactions with Stat5 [144], potentiation of PRL signaling as well as acting as a permissive hormone by inducing the expression of PRL receptors (PRLRs) during periparturient period so that PRL can exert its lactogenic effects [145, 146]. Mammary GR levels change with the reproductive state. In pregnant rats, GR expression gradually increases and remains constant until late pregnancy, reaching a peak at parturition. During lactation, GR expression returns to pre-partum levels and remains relatively high until involution when a rapid decline is observed [147]. These changes in GR expression are related with glucocorticoids signaling necessary for secretory cell differentiation and lactation. In fact, studies using mice with GR that are impaired in the DNA binding-dependent function (GRdim ), demonstrate that direct GR binding to GREs is necessary for epithelial proliferation and ductal development of the mammary gland of virgin females [148]. On the other hand, lactating GRdim mice have normally differentiated mammary glands and normal milk protein synthesis since this process seems to be mediated by protein-protein interaction of phosphorylated STAT5 with GR [148]. Glucocorticoids are also involved in the formation of ultrastructural components, such as rough endoplasmic reticulum (REnR) and tight junctions (TJs), that are necessary to support milk synthesis and secretion [149–153]. In fact, when mammary epithelial cells are treated with hydrocortisone in the presence of insulin their REnR and Golgi apparatus increase in size but their ultrastructural differentiation (translocation of REnR, Golgi apparatus and nucleus to a more basal position) is only completed when PRL is added to the culture medium [149]. The combined action of these three hormones also induces the formation of secretory protein granules within the cytoplasm [149]. TJs closure is essential, in alveolar cells, to the transition from the colostral phase to the mature milk phase of lactation and failure of this process results in precocious involution of the gland [153]. TJ closure is a process triggered by progesterone withdrawal [154]. This process is only induced when alveolar cells are co-treated with PRL and dexamethasone and occurs in parallel with high β-casein expression [151].

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Early studies showed that injection of glucocorticoids in rats delayed involution, highlighting the role of glucocorticoids as mammary secretory cell survival factors [155]. Indeed, dexamethasone acts systemically and completely inhibits involution in all mammary glands by repressing the expression of extracellular matrix protease stromelysin-1 (MMP3), which is necessary for cell death induced by ECM degradation [156].

10.4 Peptide Hormone Signaling in the Mammary Gland The role of peptide hormones in the mammary gland development has long been known [13, 157, 158]. In this section we summarize the actions mediated by the main peptide hormones throughout mammary development with focus on those that activate receptor tyrosine kinases (RTKs) to mediate gene expression changes.

10.4.1 Insulin—Insulin-Like Growth Factor System All three structural homolog ligands of the Insulin—insulin-like growth factor (IGF) system (insulin, IGF type I and II), play important roles in the mammary gland development process [159]. INS plays a central role in glucose uptake by most of the tissues in the body [159]. In the mammary gland its role has long been linked to milk fat synthesis and secretion during lactogenesis [160–165]. It has been difficult to study the effects of INS in the mammary gland development in vivo since INS manipulation interferes with the metabolism of the entire organism and there is crosstalk between INS and IGF signaling pathways [159, 166]. Therefore, the mechanisms of INS action were revealed only a decade ago [167]. Even though INS can also activate IGF type I receptor (IGF-1R), it binds preferentially to insulin receptor (IR) [159, 166]. There are two isoforms of IR, A and B, and they are both present in pregnant and lactating mammary glands [168]. Following insulin biding and activation of IR α-subunits, its intracellular β-subunits auto-phosphorylate activating mainly the PI3K/ISR2/AKT signaling pathway [159, 167–169]. Isoform B of IR (IR-B), which is the most specific for insulin binding [159], was found upregulated in the lactating mammary gland [168, 169]. Consequently IR-B has been highlighted as the main responsible for INS action during lactation [168], where it plays an important role in the regulation of lactogenic protein synthesis and secretion (Fig. 10.4c) [159, 166]. Recently, INS has also been implicated in the regulation of the transition from proliferation to differentiation of the mammary gland during the second half of pregnancy in mice [166]. IGF-I and IGF-II regulate proliferation, survival and differentiation, particularly during puberty and pregnancy [170]. They are structural homologs of pro-insulin but in contrary, they can be produced in most tissues of the body [171]. IGF-I and

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IGF-II have different affinities for Insulin growth factor-receptors (IGFRs) which may explain the distinct effects that these growth factors have during mammary gland development [159, 172]. Both IGF-I and IGF-II bind IGF-1R, high affinity binding proteins (IGFBPs) [171], and the hybrid receptor IGF-1R/IR-A. IGF-II also activates IR-A, and IGF-I the hybrid receptor IGF1R/IR-B [159]. In adulthood, IGF-I influences mammary gland ductal branching [27, 173] in both paracrine [174] and endocrine manner (Fig. 10.4a) [175]. Pituitary GH induces IGF-I production both in the liver, the main source of circulating IGFs [170], and in the mammary stromal cells [1, 173, 176]. Then, IGF-I acts on IGF-I receptors of the mammary epithelial cells, leading to IRS/PI3K/AKT and Ras/Raf/MAPK signaling pathway activation [170] to promote TEB formation and ductal elongation [170, 173]. IGF-I also acts during pregnancy in alveologenesis and milk protein synthesis [27]. Additionally, IGF-II expression induced by PRL results in ductal branching and alveologenesis during early-pregnancy in mice [26]. Depending on the context, IGFBPs can modulate IGFs by either inhibiting or enhancing their action thereby controlling their bioavailability [170]. IGFBPs can also act on their own, independently of IGF [177, 178]. There are six IGFBPs (IGFBP-1–IGFBP-6) and they are all expressed in the mammary gland [177]. Their expression pattern changes during puberty, pregnancy, lactation and involution stages [172, 179]. IGFBPs are particularly implicated in the involution process [172], when they inhibit the survival effect of IGFs [177] and, stimulate cell death and extracellular matrix remodeling [178]. Particularly, IGFBP-5, which is synthesized by the secretory epithelial cells, is overexpressed during involution [177–181].

10.4.2 Epidermal Growth Factors Family Ligands There are several epidermal growth factor (EGF) family members [182] and most of them are present in the postnatal mammary gland with unique expression patterns across its development. These include EGF, transforming growth factor alpha (TGFα), AREG, heparin binding-EGF (HB-EGF), betacellulin (BTC), epiregulin (EPR), epigen (EPGN), neuroregulins (NRG1–NRG4), and Teratocarcinomaderived growth factor 1 (TDGF1) [88, 183]. All EGF family members are synthesized as membrane-bound precursors that are cleaved by proteases (e.g. ADAM17) to release the active ligands. They all share a conserved EGF-like domain composed of three disulphide-loop structures which can in turn, directly or indirectly, activate members of the ERBB receptor tyrosine kinase family. Once bound to a ligand, the ERBB receptors can form homo- or hetero-dimers with other family members. There are four members of the ERBB receptor family and they are all expressed throughout postnatal mammary gland development [88]. These are the epidermal growth factor receptor (EGFR, also known as ERBB1 or HER-1), ERBB2 (HER-2/neu), ERBB3 (HER-3) and ERBB4 (HER-4) [182, 184]. While all EGFR-family members are expressed during pregnancy and lactation, during puberty ERBB4 is not expressed [185].

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EGF, TGF-α, and AREG bind exclusively to EGFR. While, NRG-1 and NRG2 can bind both ERBB3 and ERBB4, NRG-3 and NRG-4 uniquely bind ERBB4. HB-EGF and BTC can bind both EGFR and ERBB4, while EPGN appears to be a broad-spectrum ligand. Additionally, cripto-1 does not directly bind to any of the EGFR-family members but can indirectly activate ERBB4; and ERBB2, does not directly bind any ligand [182, 184, 186]. Even when not binding specific receptors the different ligands can still activate other EGFR-family members by inducing heterodimerization. Thus, multiple signaling pathways can be activated by the EGFR-family members including the Ras/MAPK, PI3K/AKT, PLCγ1/PKC, STAT and Par6-atypical Protein kinase C (PKC) pathways [182, 186]. In the mammary gland, the effects of EGFR and its exclusive ligands (EGF, AREG and TGF-α) are the most well studied. EGFR can be expressed in multiple sites across the mammary gland including cap cells of TEBs, adipocytes, myoepithelial and luminal epithelial cells and stromal fibroblasts. However, its role in ductal growth during puberty is mainly attributed to stromal EGFR [19, 187] and ERBB2. Ductal growth during puberty mediated by EGFR might be induced by any of its specific ligands, indicating overlapping and/or compensatory mechanisms [188]. Studies suggest that exogenous EGF may be sufficient to activate stromal EGFR and promote ductal growth. While circulating EGF is capable of rescuing normal ductal morphogenesis in ovariectomized mice [189], exogenously added TGFα had similar effects in Esr1−/− mice promoting ductal migration and side branching [190]. Additionally, several studies have highlighted the role of AREG in the mammary gland development during puberty, mediating 17β-estradiol (Fig. 10.4a) and progesterone response via a paracrine mechanism. AREG was also shown to mediate progesterone-induced TEB formation and ductal elongation during pre-puberty and puberty via a similar mechanism [191]. Hence, EGFR is a necessary component of the stromal-epithelial signaling interactions required for ductal growth [19]. During pregnancy and lactation EGFR signaling is activated by EGF and TGFα which act together in lobuloalveolar development and lactogenesis [186, 188]. According to their expression patterns, TGFα seems to be more important during pregnancy, whereas EGF seems to be more important during lactation [88, 192, 193]. Despite not having any known ligand, ERBB2 is highly expressed during puberty and can heterodimerize with ERBB3 to promote ductal elongation and TEB differentiation [194]. Furthermore, during pregnancy and lactation NRGs from basal myoepithelial cells binds to epithelial ERBB3 which forms a heterodimer with ERBB4. Both ERBB3 and ERBB4 expression are induce by PRL. The heterodimer then activates STAT5 and PI3K/AKT signaling pathways promoting lobuloalveolar development [195]. Interestingly, NRG1 has long been related with alveolar terminal differentiation [190]. Finally, cripto-1 is synthesized by mammary epithelial cells and may play an autocrine role in ductal growth during puberty [183, 193], in ductal branching and progesterone-mediated alveologenesis during pregnancy, and in the mammary gland remodeling during involution [196].

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10.4.3 Fibroblast Growth Factor Family FGFs are produced by stromal fibroblasts and adipocytes to promote ductal elongation [11]. FGFs bind to FGF receptors (FGFRs) [197] which are receptor tyrosine kinases located in the mammary epithelial cells. This leads to RAS/MAPK and PI3K/AKT pathways activation and stimulation of cell proliferation. There are at least 22 members of the FGF ligand family [184, 198] but only a subset (e.g., FGF2, 4, 7, 10) have been implicated in the mammary gland development [11]. In mice, FGF2 is predominantly expressed in the stroma and possibly in myoepithelial cells during puberty [199] when it induces cell proliferation and ductal elongation via FGFR2 and epithelial branching via other unidentified receptor, potentially FGFR1 (Fig. 10.4a) [200]. Similarly, in bovine mammary epithelial cells, FGF2 protects cells from endoplasmic reticulum (EnR) stress (see below) and also induces cell proliferation by activating CCND1 expression via AKT and Mitogen-activated protein kinase 8 (JNK) signaling pathways [201]. Throughout post-puberty development, FGF4 expression increases and activates the Bcl2-associated agonist of cell death (BCL-2) pathway to inhibit apoptosis within TEBs. Although FGF4 appears to not affect the mammary gland during pregnancy, its re-expression inhibits apoptosis and retards involution [202]. FGF7 is mainly synthetized in the mammary stroma [199], and it targets FGFR2b in putative MaSCs/progenitor cells. This activates MAPK signaling to promote ductal elongation. However, FGF7 was shown to inhibit ductal branching, even when TGFα an inducer of ductal side branching, was concomitantly added [203]. FGF10 and its receptor FGFR2b were first identified in mice as being involved in the embryonic stage by regulating WNT10b and Lymphoid enhancer-binding factor 1 (LEF1) expression [204, 205]. Stromal FGF10 is also important during all postnatal developmental stages [206], although the phase in which it is better studied is during puberty. Even though it is clear that FGF10 plays an important role maintaining mammary progenitor cells and in ductal elongation during puberty, its specific actions regarding ductal branching are controversial [200, 203, 205, 206]. While in a study from 2007, FGF10 was shown to have similar effects as FGF7 [203], in more recent studies FGF10/FGFR2b were contradictorily implicated in TEBs formation and ductal branching [200, 205, 206]. Furthermore, FGF10 seems to also inhibit apoptosis via MAPK/ERK pathway [206]. During pregnancy, FGF10 expression was only observed during a short period of time [206] however it may be sufficient to critically influence lobuloalveolar development through FGFR2b binding [207].

10.4.4 Prolactin The pituitary hormone PRL acts directly on the mammary epithelium by binding to PRLR [5, 15, 208], a member of the class I cytokine receptor superfamily. Upon PRL binding, PRLR becomes activated and can trigger multiple signaling pathways

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including the JAK/STAT pathway, which is the most studied in the mammary gland development [1, 13, 15, 209, 210]. Upon PRL binding, PRLR dimerizes and the associated JAK molecules phosphorylate each other. Once activated, JAK phosphorylates PRLR phosphotyrosine residues enabling cytoplasmic STAT binding and phosphorylation by JAK. Phosphorylated STATs dissociate from the receptor, dimerize and translocate into the nucleus where it regulates transcription of target genes. Many studies have highlighted the specific role of PRL in mammary gland morphogenesis, mainly using Prlr and Prl−/− mice [5, 15, 211–213]. According to these studies, blocking PRL signaling regulates the mammary gland development in three stages [12, 208]. The first stage is at the end of puberty, where PRL contributes to ductal side branching and lobule budding giving rise to a fully mature virgin gland [213, 214]. This is, mainly, due to PRL systemic role in the mammary gland development [12]. The second moment is throughout pregnancy, mostly in late stages, when PRL is necessary to induce ductal expansion and terminal differentiation of the lobuloalveolar structures [5, 15, 212]. The third stage is following delivery, when PRL stimulates alveolar epithelial cells to produce and secrete milk during lactation [208, 212]. The activation of JAK/STAT signaling pathway by PRL/PRLR has been directly linked to the alveologenesis process (Fig. 10.4c) [12, 214], since Jak2 and STAT5A/B null mammary epithelium failed to form alveoli during pregnancy [215, 216]. The JAK/STAT signaling pathway also has an important role in lactogenesis, because it activates the expression of milk protein genes like beta casein (CSN2) [210, 217, 218] and whey acidic protein (WAP) [219]. Interestingly, placental lactogen can also bind and activate PRLR inducing the same response as PRL itself [13, 214]. The JAK/STAT signaling pathway is positively influenced by specific integrins in response to signals from the extracellular matrix during lactation [220, 221]. Negative regulation of JAK/STAT signaling pathway occurs through a negative feedback loop. The interaction of JAK2 and STAT5 is inhibited by members of the suppressors of cytokine signaling (SOCS) family, whose expression is, in turn, activated by PRL (Fig. 10.4c) [4, 222]. During both pregnancy and lactogenesis PRL also exerts endocrine functions [5, 12, 208] in the ovary, where it induces progesterone and 17β-estradiol biosynthesis, which, as seen above, have an active role in mammary gland morphogenesis [12, 208]. Thus, progesterone and PRL regulate each other’s expression, and both work together to favor the proliferation and differentiation of alveolar cells to generate a lactation-competent gland during pregnancy [1, 4, 223].

10.4.5 Transforming Growth Factor Beta All three isoforms of the cytokine TGFβ (TGFβ1, TGFβ2, TGFβ3) are expressed in the epithelium of the mammary gland. TGFβ is secreted as an inactive complex that is activated by proteases (e.g. MMP-9) [224]. The activated form of TGFβ then binds to heteromeric complexes composed of type I (TGFβR1) and type II (TGFβR2) serine/threonine kinase receptors, activating one of the two pathways, canonical or non-canonical. The canonical pathway, also known as SMAD-dependent consists on

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the phosphorylation of cytoplasmic SMAD1 and SMAD2 by TGFβR1. Activated SMADs form a complex with SMAD4 which is then translocated into the nucleus where they regulate transcription. The non-canonical or SMAD-independent signaling cascades include TGFβ-induced JNK/p38, ERK/MAPK and PI3K/AKT [225]. TGFβ is usually associated with an anti-proliferative response to stromal and/or systemic stimuli, being its activity highly regulated throughout the normal mammary gland development [224]. All three TGFβ isoforms as well as TGFβRI and TGFβRII are expressed during all phases of mammary gland development [226, 227]. Moreover, TGFβRI and TGFβRII have been localized in both epithelial and stromal compartments [226]. During pubertal ductal growth, TGFβ1 and TGFβ3 have overlapping expression patterns. The highest levels of expression for TGFβ1 and TGFβ3 were found in the TEB’s body and cap cells, respectively [227]. In puberty, TGFβ1 has been the most extensively studied. TGFβ1 generally acts as negative regulator of ductal elongation and branching by inhibiting epithelial proliferation [228, 229] maybe through WNT5A [230]. Concomitantly, stromal TGFβR2 deletion leads to aberrant ductal elongation and branching [226, 231]. However, other studies have shown that TGFβ1 can have opposite effects depending on its concentration [232], target cells [233] and way of action [234]. While high levels (picomolar) of TGFβ1 inhibit ductal branching, low concentrations (femtomolar) stimulate it [232]. During puberty active TGFβ1 activity seems to be, at least in part, regulated by ovarian hormones [229]. In the high proliferative phase, TGFβ1 expression induced by 17βestradiol activates proliferation of ER-negative cells and inhibits it in ER-positive cells [233]. Another study showed that during puberty autocrine TGFβ1 inhibits ductal growth, but paracrine/endocrine TGFβ1stimulates it [234]. During pregnancy mainly TGFβ2 but also TGFβ3 showed very high expression in ducts and alveoli, while TGFβ1 expression in these structures was very low [227]. It is postulated that early in pregnancy during branching morphogenesis TGFβ might induce apoptosis specifically in the luminal side of the TEB helping create the lumen [224]. TGFβs also inhibit milk protein secretion before lactation where the expression of all three isoforms is reduced [235]. Following weaning, TGFβ3 expression increases early in the involution process due to its activation of STAT3 activity [236]. TGFβ1 peaks at later stages of involution where it is thought to promote apoptosis and extracellular matrix remodeling [237].

10.5 The Unfolded Protein Response and Autophagy in Mammary Gland Development and Homeostasis Protein quality control mechanisms are important for mammary gland development and function, especially during lactation when cells have increased protein production demands and an excessive load accumulation of unfolded/misfolded/aggregated proteins that could impair basic cell survival functions. So, it is not surprising that mechanisms, such as autophagy and the Unfolded Protein Response (UPR), are necessary for normal mammary gland development and tissue homeostasis [238–242].

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Autophagy, as a self-degradative process, is important for energy balance at critical times in development. By clearing unfolded/misfolded/aggregated proteins and removing damaged organelles, this mechanism enables cells to recover resources and maintain homeostasis [243]. On the other hand, UPR is a stress response that is activated to protect cells from detrimental conditions that compromise EnR function (protein folding, post-translational modifications, lipid and steroid synthesis and calcium signaling) [244]. UPR is initiated when the Chap. 1 EnR stressor sensorbinding immunoglobulin protein (BiP) frees itself from the EnR-resident proteins and associates with misfolded proteins. This results in activation of the three UPR branches: inositol-requiring enzyme 1α (IRE1α) arm, protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm and activating transcription factor 6 (ATF6) arm [245]. As a result of this activation mRNA transcription and protein translation rates decrease, degradation of damaged proteins is enhanced as well as the induction of genes involved in proteostasis and lipid biosynthesis control [246–248]. It is now known that during alveologenesis, autophagy is induced in the cells localized in the center of the developing alveoli as a survival mechanism prior to apoptosis induction necessary for the formation of hollow lumens [249–252]. It has been shown that upon losing their contact with ECM, these inner mammary epithelial cells activate the canonical PERK pathway, which regulates the transcription of multiple autophagy genes, such as Autophagy protein 5 (ATG5), beclin-1 (BEC1), Atg8 and Microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), as an attempt to deal with the stress conditions [250]. These findings were further supported by complementary immunohistochemical studies made ex vivo on murine mammary glands isolated at the lactation period where activated PERK was found to be highly expressed in cells of luminal epithelium while LC3 was only detected in the detached cells, suggesting that activation of PERK promotes autophagy as a survival mechanism of mammary epithelial cells during lactation [250]. Additionally, recent studies using 3D cultures of bovine mammary epithelial cells demonstrate that during mammogenesis estrogens and progesterone are involved in the induction of autophagy [251, 252]. It was shown that 17β-estradiol increases the expression of total LC3 by increasing the level of LC3-I protein as well as by accelerating the formation of LC3-II (the membrane-bound form) [251]. In fact, both synergistic genomic regulation by 17β-estradiol and progesterone of autophagy-related genes such as ATG3, ATG5 and BEC1, as well as the ability of these hormones to regulate signaling pathways such as PI3K/AKT/mTOR; AMPK/mTOR seems to be in the origin of autophagy induction at the earlier time points of acini formation [252]. During lactation, the required increase in protein and lipid synthesis for milk production induces activation of the IRE-1α/X-box-binding protein 1 (XBP-1) and PERK branches of UPR [29, 238, 239, 242]. On the other hand, ATF6 branch doesn’t seem to be involved in mammary gland developmental since its levels don’t change significantly across the developmental stages [239]. Activation of IRE-1α/XBP-1 and PERK pathways leads to an expansion of EnR capacity which is needed to support alveolar expansion and the secretory phenotype, as demonstrated by the fact that XBP-1 deletion is sufficient to impair lobuloalveolar development during early lactation due to reduced epithelial cell proliferation [238] and to reduced lactogenic

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protein mRNA levels in response to dexamethasone-PRL-INS stimulation [239]. On the other hand, PERK branch is necessary to allow lactogenic protein synthesis, since tissue-specific PERK deletion in the mouse mammary epithelium reduces levels of the lipogenic genes and cyclic AMP-dependent transcription factor (ATF4) knockdown reduces INS and GR mRNA levels [242]. PERK branch, in coordination with autophagy, is also involved in the mammary alveolar involution process through a positive feedback loop mechanism where ATF4 enhances DNA damage-inducible transcript 3 protein (CHOP) transcriptional activity leading to G0/G1 arrest [253] and to the expression of apoptosis markers such as active caspases and cleaved Poly [ADP-ribose] polymerase (PARP) [254]. This results in reduced cell proliferation and differentiation and increased response to lactogenic hormones [240, 241]. On the other hand, during the early stages of involution in mice (24 h upon forced weaning), LC3 is highly expressed in the cells that remain within the alveoli and actively reabsorb the apoptotic cells from the lumens, suggesting that autophagy is induced during the process of efferocytosis [255].

10.6 Conclusion The mammary gland developmental stages are temporarily separated and occur over the lifespan of mammals. The mammary gland is composed of many specialized cell types which during reproductive age are under continuous hormonal stimulation. These hormonal cues activate several signaling pathways that converge in the regulation of epigenetic marks and consequently determine gene expression patterns and epithelial cell fate. Hormonal regulation begins at puberty, with the increase of circulating ovarian hormones that in conjunction with peptide mediators establish the communication between the stroma and epithelium. This crosstalk leads to a fully mature mammary ductal tree capable of responding to lactogenic hormones released during pregnancy and lactation. Unlike many other organs, the mammary gland is easily accessible and develops after birth. This, together with its regenerative properties make the mammary gland an excellent model to study how signal transduction pathways regulate adult stem cell differentiation, epithelial stability and mesenchymal transition, cell survival mechanisms and response to metabolic stress. Acknowledgments We apologize to authors whose work was not cited due to space limitations. We are thankful to Institute for Biomedicine—iBiMED (UID/BIM/04501/2013, POCI-01-0145FEDER-007628 and UID/BIM/04501/2019) for supporting this project. iBiMED is supported by the Portuguese Foundation for Science and Technology (FCT), Compete2020 and FEDER fund. FLM and ID thank FCT for their fellowship (SFRH/BD/123821/2016 and SFRH/BD/117818/2016).

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Chapter 11

Oogenesis Signaling from Development to Environmental Plasticity and Aging Bruno Marques, Ricardo Matos, and Rui Gonçalo Martinho

Abstract Oogenesis is a unique process of cell division and differentiation, where a mature oocyte is formed from a germ cell precursor. In this chapter, the complex regulatory network responsible for oocyte differentiation and maturation in Drosophila melanogaster and human female gametogenesis is discussed. There is a focus in the hormonal and non-hormonal signaling pathways responsible for the communication between the oocyte and the surrounding somatic cells, with a special emphasis to starvation-dependent and age-dependent loss of female fertility. Keywords Gametogenesis · Oogenesis · Signaling · Oocyte

Abbreviations 20E Baf Dilps Dpp DSBs Ec FSC

20-Hydroxyecdysone Barrier to autointegration factor Drosophila insulin-like peptides Decapentaplegic Double-stranded breaks Ecdysone Follicle somatic stem cells

B. Marques · R. Matos · R. G. Martinho (B) Center for Biomedical Research (CBMR), Universidade do Algarve, Faro, Portugal e-mail: [email protected] B. Marques e-mail: [email protected] R. Matos e-mail: [email protected] R. G. Martinho Department of Medical Sciences, Institute for Biomedicine (iBiMED), Universidade de Aveiro, Aveiro, Portugal Instituto de Medicina Molecular, Universidade de Lisboa, Lisbon, Portugal © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_11

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Follicle-stimulating hormone Gonadotropin-releasing hormone Germ-line stem cells Insulin-like growth factor 1 Insulin receptor The Juvenile Hormone Luteinizing hormone Nucleosomal histone kinase-1 Phosphatidylinositide-dependent kinase 1 Phosphoinositide kinase-3 Phosphatidylinositol-4,5-bisphosphate Phosphatidylinositol-3,4,5-trisphosphate Synaptonemal complex Superoxide dismutase Target of Rapamycin Tuberin/tuberous sclerosis complex Yolk proteins

11.1 Oogenesis Overview Gametogenesis is a unique process of cell division and differentiation, where a diploid cell gives rise to one or more mature haploid gametes, capable of fertilization and forming the ultimate totipotent cell: the zygote. Male and female gametes are generated from germ-line cells, supporting the distinction between an immortal germ cell lineage and a “disposable” soma and explaining the Weisman barrier hypothesis that states that hereditary information necessarily moves from the germline towards the soma. Although male and female gametogenesis have meiosis and differentiation as central points of gamete formation, differentiation of the sperm only occurs after meiotic-completion and reduction of ploidy [1]. Oocyte differentiation, in contrast, occurs during meiosis raising interesting questions regarding the way these two genetic programs are coordinated. An apparent solution to this conundrum is the fact that in most studied multicellular organisms there is an extended meiotic arrest during the diplotene stage of prophase I, which provides a window of opportunity for oocyte growth and maturation [2].

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11.1.1 Drosophila Melanogaster Drosophila melanogaster has two ovaries, each composed of 16–20 independent ovarioles that contain their own stem cell population [3]. Egg chambers are connected by somatic stalk cells and they can be linearly found within each ovariole at various stages of oocyte development (Fig. 11.1) [4]. Oogenesis takes approximately one week to be completed. Mostly based in size and morphological changes, the developing egg chambers can be divided in 14 stages of development [4]. Each Drosophila ovariole contains two main stem cell populations, the germ-line stem cells (GSC) and follicle somatic stem cells (FSC), which are harbored in an anterior structure named the germarium [5, 6]. The germarium is divided in four regions. At the anterior tip of the germarium, in region 1, 2–3 GSCs divide asymmetrically to produce another stem cell and a daughter cell that will soon differentiate into a cystoblast [5, 6]. The new formed cystoblast undergoes four mitotic divisions with incomplete cytokinesis forming a 16-cell cyst, where cells are interconnected by the ring canals and a cytoskeleton structure known as fusome [5, 6]. The synaptonemal complex (SC) assembly between the paired homologous chromosomes is the first cytological evidence of meiosis beginning [3]. In region 2A of the germarium, up to four nuclei of the developing cyst show detectable assembly of SC proteins, by region 2B only two are typically positive for SC assembly [7]. In region 3 only one nucleus, the single pro-oocyte, remains in meiosis, whereas the other 15 cells of the 16-cell cyst will endoreplicate their own genome at later stages of egg chamber development and give rise to the supporting polyploid nurse cells. These supporting cells are necessary for oocyte growth and maturation, as they synthesize RNA’s and proteins that are actively transported into the oocyte [8].

Fig. 11.1 In Drosophila melanogaster (fruit fly), oogenesis starts with an asymmetrical division of the germline stem cells, resulting in daughter stem cell and a daughter cell that will differentiate into a cystoblast. The newly formed cystoblast will undergo four mitotic divisions with incomplete cytokinesis forming a 16-cell cyst. One of the cysts will remain in meiosis and become the prooocyte, whereas the remaining 15 cysts will endoreplicate their own genome, giving rise to the supporting nurse cells. Similar to humans, the developing oocyte also arrests at the diplotene stage of prophase I, with its nucleus forming a highly condensed structure, known as the karyosome. The transcriptionally quiescent oocyte transiently reactivates gene expression before meiotic progression into metaphase I, where it is arrested again until egg activation

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Homologous chromosomes pairing is essential for meiotic recombination and normal meiosis. In Drosophila, pairing of the centromeres starts during the premeiotic mitotic divisions (region 1 of the germarium), but they only become fully paired at the eight-cell cyst stage [9, 10]. After initiation of SC assembly between the paired homologous chromosomes, programmed double-stranded breaks (DSBs) are generated in region 2A of the germarium [7]. A subset of these DSBs give rise to crossing-overs that physically link the homologous chromosomes together until the onset of anaphase at latter stages of oogenesis. In region 3 of the germarium most DSBs are already repaired [7]. Once the 16-cell cyst buds off from the germarium, an individualized egg chamber is formed, where the oocyte and the flanking nurse cells are surrounded by somatic follicle cells (Fig. 11.1). During stages 2–3 egg chambers, the mid-prophase I oocyte chromatin is reorganized into a compacted structure known as karyosome. This relies among other factors in the phosphorylation of protein Barrier to autointegration factor (Baf) by Nucleosomal histone kinase-1 (Nhk-1), which helps to release the oocyte chromatin from its nuclear envelope attachments [11]. Loss of nhk-1 is associated with an abnormal loading of condensins into the karyosome, a delay in disassembly of euchromatic SC and dispersed chromosomes capable of nucleating abnormal assembly of meiotic spindles [12, 13]. This strongly suggests that karyosome formation is essential for normal chromosome segregation during meiosis [7, 10]. Drosophila oogenesis has a prolonged meiotic arrest at the diplotene stage of prophase I, being the condensed oocyte karyosome transcriptionally quiescent between stage 5 and 9. Before progression into metaphase I (during stage 13), there is however a transient opening of the oocyte karyosome and reactivation of gene expression during late diplotene I-arrest (stages 9–10) [14, 15]. The functional relevance of such transcriptional reactivation for meiotic progression and female fertility is still poorly understood, however Histone demethylase Kdm5/Lid not only avoids precocious reactivation of oocyte transcription [15], but is also important for the correct levels of Histone H3K4 trimethylation, karyosome architecture, SC maintenance, centromere pairing, meiotic progression, and female fertility [15, 16], which suggests some degree of functional interdependence. Drosophila oocyte growth mostly relies in the transport of RNA’s, proteins, and other cytoplasmic components from the supporting nurse cells into the developing oocyte. Such cytoskeleton-based transport can be divided in two phases [17–19], a slow-initial phase that occurs between stage 2 until stage 10A, and a rapid phase that occurs between stage 10B and 12 (nurse cell dumping), where there is an actin-myosin-based contraction of the nurse cells and their cytoplasm is rapidly transported into the oocyte through the connecting ring canals. Once oocyte growth stops and the migrating follicle cells cap the oocyte, closing the connecting ring canals, meiosis progresses into metaphase I, where it is arrested again until egg activation. Drosophila egg activation occurs independently of fertilization, being induced by the physically stimulated passing of the egg in the oviduct and rehydration [20]. During egg activation, there is considerable change of the oocyte transcriptome and proteome [21, 22], which prepares the female gamete for fertilization and one of the most dramatic cellular transitions in biology: the oocyte-to-zygote transition.

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11.1.2 Homo Sapiens Human oocyte development starts at around 6 weeks of gestation when primordial germ cells migrate from the yolk sac to the genital ridges, where they proliferate by mitosis and give rise to approximately 7 million oogonia at 20 weeks of gestation [23–25]. As oogonia proliferate, a subset of cells progressively exit proliferation and enter prophase I of meiosis, starting their differentiation into oocytes (Fig. 11.2). During fetal female gametogenesis, oocytes advance through various stages of prophase I: leptotene, zygotene, pachytene, and finally their development is transiently arrested at the diplotene stage of prophase I [26]. In humans, the diplotene I arrest of the quiescent primary oocyte can last several years, or even decades, from fetal development until the onset of ovulatory cycles at puberty, when the dormant oocytes are progressively reactivated during the entire women’s fertile period [25]. Concurrent with oocyte differentiation, a layer of somatic cells that support oocyte development, progressively maturate in a process called folliculogenesis [27]. This begins in the fourth month of gestation until birth, when the primordial follicles containing primary oocytes arrested at the diplotene stage of prophase I are fully formed [27, 28]. These follicles are surrounded by flattened granulosa cells, also arrested in the G0 phase of the cell cycle, which produce several growth factors and cytokines crucial the viability and maturation of the developing oocyte. The mechanisms underlying follicular quiescence and maintenance of oocyte viability are still poorly understood [11, 29]. After birth, only approximately 1 million oocytes survive the process of follicle degeneration, commonly known as atresia [30]. Since the process of atresia continues during growth and adulthood, at puberty there are only

Fig. 11.2 In Homo sapiens (human), oogenesis starts at around 6 weeks of gestation when primordial germ cells start proliferating by mitosis giving rise to approximately 7 million oogonia, which will subsequently divide asymmetrically to give rise to primary oocytes. The developing primordial follicles arrest for several years in a dormant state. After puberty, there is a constant reactivation of the primordial follicles that last until the onset of menopause. During late preovulatory stage, the oocyte is released from the prophase I-arrest, progresses into meiosis II and transiently arrests in metaphase II until fertilization

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around 4,00,000 primary oocytes and from these only approximately 500 oocytes will fully mature and ovulate [25]. Follicle growth initiation and maturation results from a crosstalk between the developing oocyte and the supporting somatic cells [31]. After activation of the primordial follicles, there is an irreversible initiation of oocyte growth and proliferation of the supporting cuboidal granulosa cells, forming the primary follicle. These morphological changes are associated with significant changes of the primary follicle transcriptome [32]. As granulosa cells continuously proliferate, theca cells are recruited to the growing secondary follicles, surrounding the follicles basal lamina [32–34]. Subsequently, capillary vessels form within the thecal layers, blood begins to circulate to and from the follicle, and the fully-grown oocyte becomes surrounded by a zona pellucida. Formation of the antral follicles (also known as tertiary follicles) correlates with the appearance of a fluid-filled cavity, the antrum, adjacent to the oocyte, which dramatically increases follicle size. Whereas most of the growing late tertiary follicles die by atresia due to competition for growth-inducing folliclestimulating hormone (FSH), the single surviving one, the dominant follicle, grows rapidly and becomes preovulatory. During late preovulatory stage, the oocyte is finally released from the prophase I-arrest, progresses into meiosis II and transiently arrests in metaphase II until fertilization [35]. Meiotic resumption results from an endocrine cascade that induces a peak of luteinizing hormone (LH) secretion leading to ovulation. After ovulation, the corpus luteum, which is formed from the remaining terminally differentiated theca and granulosa cells, maintains the endometrium by secreting progesterone and minor amounts of estrogen [36, 37].

11.2 Hormonal Regulation of Oogenesis 11.2.1 Drosophila Melanogaster In Drosophila melanogaster, hormonal and other signaling pathways are crucial for development and maturation of the oocyte. The Juvenile Hormone (JH) and Ecdysone (Ec) are key hormones for oogenesis [38, 39]. In adult females, the ovaries follicles cells are a key source of ecdysone [39]. Its active form, 20-Hydroxyecdysone (20E), results from the activity of a 22-hydroxylase named Shade, that converts Ec into 20E. Shade is expressed in the ovaries and in the fat body [40]. During early third instar larvae development, ecdysone signaling plays an important function for gonadal morphogenesis, where the developing larvae ovaries need sufficient time to produce adequate number of precursors for both somatic and germline populations [41]. This function relies in the repression of a precocious expression of ecdysone-target gene Broad. Interestingly, at mid/late third instar larvae development, ecdysone stops repressing Broad expression and actually stimulates the expression of this gene in the soma, which is important for stem cell niche formation and later on to primordial germ cells differentiation [41].

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During stage 8 of oogenesis there is an important developmental checkpoint of oocyte development, where the developing oocyte engages in an irreversible growth and maturation or enters apoptosis. Ecdysone (Ec) and Juvenile hormones (JH) are both crucial for vitellogenic development and oocyte maturation. In the absence of Ec or JH, oogenesis is severely abnormal, without vitellogenic stages in the ovary [42–44]. JH has the capacity to induce the expression of distinct vitellogenin genes in the fat body and stimulating the establishment of significant intercellular spaces between follicle cells, enabling vitellogenin uptake in the developing oocyte [45]. Development of vitellogenic egg chambers relies among others in the expression and oocyte accumulation of three major yolk proteins (YP) [42]. YP expression is switched-off at stage 11 when synthesis of the chorion in the follicle cells begins [40, 46]. The decision to engage in vitellogenic development is strongly dependent on the nutritional status of the animal, as starvation induces apoptosis of stage 8/9 egg chambers [47, 48]. Interestingly, whereas nutritional shortage increases the levels of active ecdysone (20E) in the ovaries, injection of high levels of 20E in well-feed animals is sufficient to induce apoptosis in developing egg chambers [43, 48–50]. Injection of an analogue of JH (JHA; Methoprene) suppresses the apoptotic effect of starvation during oogenesis [48]. The different signaling pathways responses to starvation will be discussed in more detail bellow (see Sect. 11.4).

11.2.2 Homo Sapiens The continuous cross-talk between the developing oocyte and the supporting somatic cells ensures successful gametogenesis [20, 51]. For example, in humans the gonadotropin-releasing hormone (GnRH) is a hormone released by the hypothalamus that regulates the expression of two hormones important for ovarian development: follicle stimulating hormone (FSH) and luteinizing hormone (LH) [52]. The release of GnRH is controlled by the levels of estradiol produced by the ovarian follicles [53]. Low levels of estradiol at the beginning of follicle development, produce a feedback loop at the hypothalamus repressing the release of GnRH. With ovarian follicle development the levels of estradiol rise and when they reach their maximum this prompts the release of GnRH, and consequently, the production of FSH and LH [54]. FSH plays an important role in the communication between the oocyte and the surrounding granulosa cells [55, 56], being implicated in follicle maturation and oocyte growth, as granulosa cells are responsible for macromolecular synthesis that fulfill the metabolic needs of the growing oocyte [57]. FSH activates several signaling pathways in granulosa cells, including ERK1/2, cAMP/protein kinase A/CREB, and PI3K/AKT. FSH-induced follicle differentiation greatly relies on Insulin-like growth factor 1 (IGF-1) signaling [58, 59]. Likewise, and although FSH stimulates AKT, simultaneous activation of IGF-1 receptor is necessary for full AKT phosphorylation in granulosa cells [60], confirming the synergistic effects of these two pathways

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in ovarian follicle maturation. IGF-1 signaling also enables the production of LH receptors both in granulosa and theca cells making them sensible to the levels of LH. Increased levels of LH stimulate the production of progesterone and some androgens in these cells. When the concentration of LH reaches its peak, follicular cells stop proliferating and the oocyte resumes meiosis until metaphase II-arrest and is ready for ovulation [61].

11.3 Non-hormonal Regulation of Oogenesis 11.3.1 PI3K/PTEN/AKT Signaling Pathway PI3K/PTEN/AKT signaling pathway has important roles in cell proliferation, survival, motility, cytoskeleton rearrangements, metabolism, and aging [62]. At the membrane, activation of PI3K is associated with phosphorylation and conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5trisphosphate (PIP3), which helps to recruit and activate the serine-threonine kinase Akt (also known as protein kinase B) and phosphatidylinositide-dependent kinase 1 (PDK1). Phosphatase and tensin homolog ten (PTEN) is a negative regulator of PI3K function, since converts PIP3 back to PIP2 [63]. Akt1 activation by PDK1-dependent phosphorylation, regulates distinct downstream targets responsible for the multiple functions of this signaling cascade [64]. Akt1 promotes cell survival and proliferation, by inhibiting proapoptotic proteins, like p53 and forkhead, activating pro-survival proteins like BCL2, and stabilizing Myc and cyclin D. Akt-dependent activation of mammalian target of rapamycin (mTOR) pathway integrates inputs from growth factors, nutrients availability, energy levels, oxygen, and stress, stimulating autophagy and synthesis of proteins and lipids.

11.3.2 Mammals Mouse Foxo3 transcription factor is expressed at high levels in the dormant primordial follicles, being its expression sharply decreased after follicle activation [65–67]. AKT-dependent phosphorylation of the transcription factor FOXO negatively regulates its function, since promotes its translocation from the nucleus into the cytoplasm, after binding with 14-3-3 proteins, and targets it for degradation [68]. Consistent with the hypothesis that inhibition of PI3K signaling is crucial for maintenance of follicle dormancy due to its repression of Foxo3 transcription factor, depletion of mouse Pten or Foxo3 produce similar phenotypes, including primordial follicles overactivation, premature ovarian follicle depletion, and secondary infertility [66, 67, 69]. Human female fertility relies in a continuous activation of a pool of dormant follicles that will grow and maturate during the next twelve months, until they become

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responsive to the monthly female hormonal cycle, are release from the prophase I meiotic arrest, progress into metaphase II-arrest, and ovulate [70]. Depletion of the ovarian pool of primordial follicles is associated with the onset of menopause. In mouse, activation of the dormant primordial follicles relies in the stimulation of the PI3K signaling pathway and AKT activation, which inhibits Foxo3 [67, 71]. Since primordial follicles are not yet sensitive to FSH, their activation relies in an intrinsic signaling crosstalk between the oocytes and the surrounding granulosa cells, with Kit being expressed on the surface of the oocytes and the respective ligand on the granulosa cells [72]. Besides the inhibition of Foxo3, AKT also inhibits tuberin/tuberous sclerosis complex (TSC), which is a negative regulator of mTOR [73–75]. Activation of mTOR pathway has a profound effect in oocyte metabolism and growth, being absolutely crucial for its growth and maturation.

11.4 Starvation and Fertility 11.4.1 Drosophila Melanogaster Drosophila female fertility is highly dependent on nutrition and protein availability [76], being the size of the ovary and egg laying significantly reduced under starvation. On a rich diet, a single Drosophila melanogaster female lays on average more than 60 eggs per day at its peak (4–7 days after pupae eclosion) and its ovaries contain all stages of oocyte development within their ovarioles [76, 77]. In contrast, under protein-poor diet ovaries morphology is dramatically changed, with a reduced number of vitellogenic stages, retention of mature stage 14 eggs, and a 60-fold reduction in egg laying [77, 78]. Proliferation of the germ-line and follicle stem cells is decreased in the germarium and apoptosis is frequently seen in the boundary between regions 2a and 2b of the germarium and at stage 8 egg chambers [77, 78]. No significant cell death was observed within the germ-line stem cells or the somatic follicle cells from stages 1 through 7 egg chambers. When switched from a nutritional poor to rich food egg laying is increased within two days, clearly showing that egg production and laying responds rapidly to changes in diet [76–78]. Several nutrient sensitive signaling pathways respond to starvation [79, 80]. Insulin/insulin-like signaling is stimulated by protein feeding and during oogenesis it acts in two distinct developmental checkpoints, where program cell death is known to occur upon starvation: in the region 2A of the germarium and stage 8 of oogenesis [79]. In Drosophila, there are seven insulin-like peptides (Dilps) that interact with the insulin receptor (InR) [81]. Both InR and the insulin receptor substrate chico are required for proliferation of follicle cells and respective mutants are sterile with no egg chambers at vitellogenic stages [77, 80, 81]. A primary pathway for nutrient sensing is the insulin-mediated phosphoinositide kinase-3 (PI3K) pathway. This pathway mediates nutrient responses in the germarium, negatively regulating

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cell death. Mutations in positive regulators of this pathway result in a reduction of body size and female sterility [82, 83]. Target of Rapamycin (Tor) protein kinase is a downstream target of the PI3K pathway that plays a central role in coupling growth with nutrition, being a key target of insulin signaling [84]. Tor responds cell autonomously to levels of cellular amino acids, ATP and oxygen [85]. Tor negatively regulates insulin/insulin-like signaling through feedback between the kinase S6K and insulin substrate (IRS) proteins, and its activity is also required to activate AKT [74, 86]. The negative regulation of S6K kinase seems to correlate with the cellular response to Dilp signaling when nutrient levels are high, whereas AKT activation presumably blocks the potentially harmful effects of inappropriate insulin-like signaling when cells are critically starved for amino acids, glucose or oxygen [87]. Starvation represses the expression of several of Dilps, depletes the insulin-like signaling second messenger PIP3, and inhibits the activity of the TOR target S6K [88, 89]. Homozygous Tor mutants are reduced in size compared to wild-type and have increased cell death in the germarium [90, 91]. As mentioned before, activation of AKT is associated with phosphorylation of Foxo transcription factor, its translocation into the cytoplasm and subsequent degradation activation [92]. On a poor protein diet, Dilp secretion is blocked, which leads to reduced insulin signaling, inactivation of AKT, and translocation of the unphosphorylated Foxo into the nucleus, where it acts as a growth inhibitor by regulating the expression of multiple target genes [92, 93]. Upon a poor protein diet, foxo mutant flies no longer arrest oogenesis at stage 7/8, which indicates that this transcription factor is crucial for the regulation of oocyte growth upon starvation [94]. Interestingly, mutations in the insulin pathway also allow Foxo to induce the expression of multiple genes that promote longevity, stress resistance, fat storage, and growth attenuation [95, 96].

11.4.2 Mammals How early-life nutrition can affect human oocyte growth, maturation, and overhaul development of the mother and offspring is poorly understood. Data collected from historical observations have given important information on the effects that maternal nutrient intake can have on reproductive performance and development. Offspring whose mothers were exposed to starving conditions present reduced ovarian size [97], decreased number of primordial follicles [98], an abnormal timing of menarche [99], an early onset of menopause [100], and low ovulatory rates [99]. Maternal starvation can also lead to an increase in offspring primary infertility [101]. Rodent models showed that offspring of a nutritional starvation population tend to exhibit delayed reproductive maturity and puberty [102], as undernutrition significantly reduced primordial and secondary follicle numbers in adult offspring [103]. Insulin signaling in mammals is required to regulate the blood glucose levels, being

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essential for the regulation of energy storage, glucose metabolism, cell growth, survival/aging and reproduction [104, 105]. Insulin is secreted from mammalian pancreatic β cells in response to glucose. Other nutrients, such as free fatty acids and amino acids, can also augment glucose-induced insulin secretion [106]. Starvation is associated with an abnormal maturation of the oocytes. This is most likely because reduced insulin signaling is associated with a decrease of the number of gonadotropin receptors and sensitivity/binding capacity of LH to its receptor [107] and because PI3K/AKT and mTOR signaling pathways are most likely impaired in the ovaries, being these two pathways essential for primordial follicles activation, and oocyte survival, growth and maturation [69, 73, 108].

11.5 Aging and Loss of Fertility 11.5.1 Drosophila Melanogaster Aging in Drosophila melanogaster is associated with a significant reduction of egg laying and female fertility [109]. Maintenance of the germ-line stem cells (GSCs) is dependent on Decapentaplegic (Dpp) signaling and the cell adhesion molecule E-Cadherin within the stem cell niche, being their age-dependent decline associated with aging and loss of GSCs [110–112]. The significant increase in cystoblasts and egg chamber apoptosis coupled with a reduced stem cell proliferation also contributes to the reduced fertility [113]. The higher levels of apoptosis in the ovaries result from increased oxidative stress, as overexpression of superoxide dismutase (SOD), an enzyme that helps to remove oxygen radical species, delays GSCs loss and increases their proliferation rates [110, 114]. Finally, the reduced food intake that occurs with age is also likely to have a significant impact in female fertility, reinforcing egg chamber degeneration and cell death [77, 113]. Age-dependent endocrinal misregulation of the ovaries likely contributes to reduced Drosophila female fertility. Yet, since lower levels of juvenile hormone (JH) and ecdysone promote adult longevity, whereas these two hormones are instead important for vitellogenesis during oogenesis [43, 115–117], an age-dependent reduction of these two hormones is not likely to be directly related to a decline in Drosophila female fertility. Nonetheless, a tissue-specific reduction of these two hormones receptors within the ovaries and the fat body might potentially contribute to the observed oogenesis defects. Importantly, female age can also influence offspring fitness, as older females show a detectable decrease in offspring viability and longevity [109, 118, 119]. The link between aging and fertility has been an object of study for several years in Drosophila melanogaster, revealing the impact that age can have on the normal process of oogenesis, female fertility and offspring viability. There are however various aspects that are not yet fully understood, namely the way the environment and distinct genetic backgrounds can modulate fertility and offspring fitness of older females.

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11.5.2 Humans/Mammals Nowadays it is common for women to delay their decision of having a child, which carries a risk since fertility rates sharply decline with age [120]. This decline is due to several factors being the progressive loss of ovarian primordial follicles from birth to menopause perhaps the most relevant, as there is only a limited number of available primordial follicles that were formed during in fetal development and dictate the fertile life of the women [121]. Every day some primordial follicles are recruited from this dormant pool and begin their growth and maturation, which is essential for maintenance of the monthly hormonal cycle and fertility. Interestingly, the number of recruited primordial follicles increases with age [122], which is likely to speed up the depletion of the available ovarian follicle pool. The recruitment of the primordial follicles will continue until the onset of menopause. With age, the menstrual cycle tends to get shorter as the there is a reduced number of follicles ready to progress and form the ovulatory follicle [123]. Moreover, there is an associated decrease in the quality of oocytes and increased risk for complications during oocyte maturation and development, which can ultimately lead to abnormal chromosome segregation and loss of oocyte viability [123]. The reduction of the number of follicles also has an impact on hormonal regulation, especially in the increased levels of FSH due to reduced secretion of inhibin B [124]. With the onset of menopause there are lower levels of estradiol, which results in increased levels of gonadotropins until 2–3 years after the menopause [124]. The large majority of studies investigating the effect of aging in fertility mostly focused on fully grown oocytes. Yet, the signaling cascades associated with the hormonal and non-hormonal regulation of the early stages of oogenesis are crucially important for the formation and maturation of a viable oocyte [125]. A recent transcriptomics study of oocytes from young and aged mice found that expression of genes related to mitochondria and chromosome dynamics were comparatively higher in young mice [126], whereas oocytes from aged mice show an increased expression of genes involved in immune and inflammatory processes, clearly suggesting that the microenvironment surrounding the developing oocyte is likely to have a significant impact in the correct maturation of the oocyte and age-depend loss of female fertility [125, 126]. In conclusion, a better understanding of the communication between the oocyte and the surrounding somatic cells during early folliculogenesis, including the signaling networks that regulate these processes is of crucial importance for the development of better strategies in terms of reproduction at later stages of fertile life and a delay of precocious menopause, which is known to have a significant in the health and quality of living of a woman.

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Acknowledgments Rui G. Martinho is supported by Portuguese national funding through Fundação para a Ciência e a Tecnologia [FCT grant refs. PTDC/BEX-BID/0395/2014, PTDC/BIABID/28441/2017, UID/BIM/04773/2013, UID/BIM/04773/2019, UID/BIM/04501/2013, and UID/BIM/04501/2019]. Bruno Marques is supported by Portuguese national funding through Fundação para a Ciência e a Tecnologia, ref. PD/BD/128342/2017 within the scope of the ProRegeM PhD program (Ref. PD/00117/2012, CRM:0027030).

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Chapter 12

Key Signaling Pathways in the Cardiovascular System Fábio Trindade, Inês Falcão-Pires, Andreas Kavazis, Adelino Leite-Moreira, Daniel Moreira-Gonçalves, and Rita Nogueira-Ferreira

Abstract The activity of the heart and vessels is permanently modulated in response to electrical, mechanical and chemical signals to maintain cardiovascular system homeostasis. Some effects are rapidly manifested (e.g. contraction after an electrical stimulus), while others are observed at long-term (e.g. hypertrophy resulting from gene expression modulation). In any case, an orchestrated set of events follows from receptor to intracellular messengers and effectors via complex signaling routes. These include neurohumoral signaling targeting G protein-coupled receptors (such as adrenaline, angiotensin II and endothelin-1 receptors), growth factor pathways initiated at tyrosine (including insulin, vascular endothelial growth factor and fibroblast F. Trindade (B) · I. Falcão-Pires · A. Leite-Moreira · D. Moreira-Gonçalves · R. Nogueira-Ferreira (B) Department of Surgery and Physiology, Cardiovascular R&D Center, Faculty of Medicine of the University of Porto, Porto, Portugal e-mail: [email protected] R. Nogueira-Ferreira e-mail: [email protected] I. Falcão-Pires e-mail: [email protected] A. Leite-Moreira e-mail: [email protected] D. Moreira-Gonçalves e-mail: [email protected] F. Trindade Department of Medical Sciences, iBiMED–Institute of Biomedicine, University of Aveiro, Aveiro, Portugal A. Kavazis School of Kinesiology, Auburn University, Auburn, AL, USA e-mail: [email protected] A. Leite-Moreira Department of Cardiothoracic Surgery, Centro Hospitalar Universitário São João, Porto, Portugal D. Moreira-Gonçalves Faculty of Sport, CIAFEL, University of Porto, Porto, Portugal © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_12

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growth factor) or serine/threonine kinase receptors (transforming growth factor-β) or even direct intracellular/nuclear pathways (triggered by calcium, nitric oxide or thyroid hormones). Herein, the signaling pathways taking place in cardiomyocytes, endothelial cells, vascular smooth muscle cells and fibroblasts, mainly involved in the regulation of cardiac contraction, vasorelaxation, mechanotransduction, cell survival and hypertrophy are described. Finally, the role of extracellular matrix in cardiac remodeling and fibrosis is reviewed. Keywords Cardiomyocytes · Contraction · Fibrosis · Hypertrophy · Intracellular signaling · Vascular cells

Abbreviations AC AKAP ALK Ang II ANP β-AR BNP Ca2+ CaM CaMKII cAMP cGMP CREB DAG ECC ECM ECs EGF eNOS ERK ET-1 ETA ETB ETC FAK FGF FGFR FoxO GC GPCRs

Adenylate cyclase A-kinase anchor protein Activin receptor-like kinases Angiotensin II Atrial natriuretic peptide β-adrenergic receptors B-type natriuretic peptide Calcium Calmodulin Ca2+ /CaM-dependent protein kinase II Cyclic adenosine monophosphate Cyclic guanosine monophosphate cAMP-responsive element-binding protein Diacylglycerol Excitation-contraction coupling Extracellular matrix Endothelial cells Epidermal growth factor Nitric oxide synthase, endothelial Extracellular signal-regulated protein kinase Endothelin-1 Endothelin receptor A Endothelin receptor B Excitation-transcription coupling Focal adhesion kinase Fibroblast growth factor Fibroblast growth factor receptor Forkhead box protein O Guanylate cyclase G protein-coupled receptors

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GSK3β HB-EGF HDAC HIF-1α IGF IP3 IRS JNK LTCC MLC MLCK MLCP MMP MyBP-C NCX NFAT NF-κB NO NRG1 PDGF PDGFR PI-3K PIP2 PIP3 PKA PKC PKG PLB PLC RAAS RyR SERCA SR STAT T3 T4 TGF-β TH TR TβR VEGF VEGFR VSMCs

Glycogen synthase kinase-3β Heparin-binding EGF-like growth factor Histone deacetylase Hypoxia-inducible factor-1α Insulin-like growth factor Inositol 1,4,5-trisphosphate Insulin receptor substrate c-Jun N terminal kinase Voltage-dependent L-type calcium channels Myosin light chain Myosin light chain kinase Myosin light chain phosphatase Matrix metalloproteinase Myosin-binding protein C Sodium/calcium exchanger Nuclear factor of activated T-cells Nuclear factor NF-kappa-B Nitric oxide Neuregulin 1 Platelet-derived growth factor PDGF receptor Phosphatidylinositol 3-kinase Phosphatidylinositol 4,5-bisphosphate Phosphatidylinositol 3,4,5-trisphosphate Protein kinase A Protein kinase C Protein kinase G Phospholamban Phospholipase C Renin-angiotensin-aldosterone system Ryanodine receptor Sarcoplasmic/endoplasmic reticulum calcium ATPase Sarcoplasmic reticulum Signal transducer and activator of transcription 3,5,3 -triiodothyronine 3,5,3 ,5 -tetraiodothyronine Transforming growth factor-β Thyroid hormone TH receptors TGF-β receptors Vascular endothelial growth factor VEGF receptor Vascular smooth muscle cells

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12.1 Introduction The heart is at the center of the cardiovascular system and has the essential function of pumping blood for the entire body via the blood vessels allowing oxygen and nutrients distribution to the tissues and removal of carbon dioxide. In addition to cardiomyocytes (i.e., contracting cells), the heart comprises other cell types, namely endothelial cells (ECs), fibroblasts, vascular smooth muscle cells (VSMCs), immune cells and progenitor cells. Although cardiomyocytes represent only approximately 30% of the total cell number, they account for 70–80% of the heart’s mass [1–4]. Interactions amongst the different cell types, as well as the extracellular matrix (ECM), modulate cardiovascular structure and function. Comprehending how different electrical, mechanical and chemical signals interact at the molecular and cellular levels to modulate cardiovascular structure and function is essential for better understanding the cardiovascular adaptations and alterations under physiological and pathological conditions. In this chapter, the main signaling pathways activated in the heart in order to cope with the demands imposed to the cardiovascular system are described.

12.2 Excitation-Contraction Coupling and Active Relaxation The conversion of an electrical stimulus (action potential) into cardiac mechanical action (contraction of the heart) requires tight control and is commonly known as excitation-contraction coupling (ECC) [5, 6]. When the membrane potential reaches the activation threshold, voltage-dependent calcium (Ca2+ ) channels, mainly the Ltype (LTCC), become activated and allow Ca2+ to enter the cardiomyocyte, generating an inward Ca2+ flux. The Ca2+ itself triggers Ca2+ release from the sarcoplasmic reticulum (SR), through ryanodine receptors (RyR), leading to an increase in cytosolic Ca2+ concentration (calcium-induced calcium release, CICR, Fig. 12.1). Then, Ca2+ reaches the sarcomere, where it binds to troponin C, promoting sarcomere contraction (systole). Briefly, Ca2+ -troponin C connection moves the troponin-tropomyosin complex away from actin, exposing a myosin binding site. Using ATP, myosin binds to actin forming cross-bridges, pulling actin towards the sarcomere center. It is the subsequent sarcomere shortening that translates into contraction on a tissue/organ level. Right after generation of active force, relaxation is imperative (diastole). This process requires a reduction on the cytosolic Ca2+ levels (active relaxation), which is achieved primarily through reuptake of Ca2+ by sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) and through sodium/calcium exchanger (NCX)-mediated sarcolemmal extrusion. The sarcolemmal Ca2+ ATPase and mitochondrial Ca2+ ATPase are also responsible for reducing intracellular Ca2+ levels, although at much slower rates (‘slow systems’) [5–8].

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Fig. 12.1 Main signaling pathways involved in excitation-contraction coupling (ECC) and the role of calcium signaling in the cardiovascular system. Arrows depict activation and T-shaped lines inhibition. The contraction-relaxation cycle is regulated by several signaling pathways, promoting inotropy (dotted lines) and/or lusitropy (long dashed lines) (see text for explanation). β-AR, β-adrenergic receptor; AC, adenylate cyclase; Ach, acetylcholine; Adr, adrenaline; Bad, Bcl2 antagonist of cell death; Bcl-xL, B-cell lymphoma extra-large; Ca2+ , calcium; CaM, calmodulin; CaMK, calmoldulin kinase; cAMP, cyclic adenosine monophosphate; CAMTA2, calmodulin-binding transcriptional activator 2; cGMP, cyclic guanosine monophosphate; CICR, calcium-induced calcium release; CytC, cytochrome C; DAG, diacylglycerol; ET-1, endothelin-1; ETA, endothelin receptor A; ETS, electron transport system; GPCR, G protein-coupled receptor; HDACs, histone deacetylases; LTCC, voltage-dependent L-type calcium channels; M2R, muscarinic receptor M2; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; MEF2, myocyte-specific enhancer factor 2; mPTP, mitochondrial permeability transition pore; MyBP-C, myosin binding protein C; NCX, sodium/calcium exchanger; NE, norepinephrine; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor NF-kappa-B; NO, nitric oxide; PDE, phosphodiesterase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PLB, phospholamban; PLC, phospholipase C; RyR, ryanodine receptor; SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; sGC, soluble guanylate cyclase; SRF, serum response factor; TnC, troponin C; TnI, troponin I. Some graphical elements were adapted from Servier Medical Art (https:// smart.servier.com)

12.2.1 Regulation of Excitation-Contraction Coupling by β-Adrenergic Signaling The cardiac muscle is continuously contracting to allow proper perfusion of the entire organism and adjusting its performance to accomplish the requirements of

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the daily demands (e.g. exercise or postural changes). Cardiac contraction is under the control of both autonomic and endocrine mechanisms. Regarding the autonomic regulation of the heart, there are two interacting systems—sympathetic and parasympathetic systems—that exert antagonistic effects on the heart. Sympathetic stimulation increases heart (chronotropism) rate and myocardial contractility (inotropism) through the release of norepinephrine to β1 adrenergic receptors (β1 -ARs), expressed in the sinoatrial node, atrioventricular node, and on atrial and ventricular cardiomyocytes. The parasympathetic system exerts opposite effects through the release of acetylcholine to muscarinic receptors (M2 R) that are abundant in nodal and atrial tissue, but sparse in the ventricles [9, 10]. In addition to the autonomic regulation, the heart is also under the control of endocrine hormones released by the adrenal medulla to the bloodstream (80% is epinephrine and 20% is norepinephrine), particularly important to the initiation of the “fight-or-flight” response. Circulating norepinephrine can increase chronotropism and contractility through interaction with β1 -ARs. At low concentrations, epinephrine is β2 -selective, inducing vasodilation, while at high concentrations it also stimulates α1 , α2 , and β1 receptors, producing vasoconstriction (mediated by α1 and α2 receptors), and increases heart rate and contractility (mediated by β1 receptor) [11]. Figure 12.1 summarizes the molecular mediators of the cardiac effects of β1 -ARs (heart’s most prevalent AR receptor) and β2 -ARs stimulation by catecholamines (inotropism, chronotropism and lusitropism). β-ARs are G protein-coupled receptors (GPCRs) that couple primarily to stimulatory G (Gs) proteins (β1 -ARs and β2 -ARs) and G inhibitory (Gi) protein (β2 -ARs). Activation of β1 -ARs activates the enzyme adenylate cyclase (AC) that converts ATP into cyclic adenosine monophosphate (cAMP), which in turn binds to and activates the enzyme cAMP-dependent protein kinase (protein kinase A, PKA), which affects multiple pathways. The inotropic effects of PKA result from phosphorylation-activation of LTCC and RyR, leading to increased cytosolic Ca2+ concentration. In turn, PKA-mediated lusitropy (relaxation) is achieved, for instance, through phosphorylation of phospholamban (PLB), troponin I and myosin-binding protein-C (MyBP-C). The former event leads to PLB inactivation, rescuing SERCA activity and, thus, promoting Ca2+ reuptake. The latter two translate, essentially, in decreased Ca2+ myofilamentary sensitivity [5, 7, 8, 12– 14]. Additionally, β2 -ARs couple to the inhibitory pertussis toxin-sensitive Gi protein. Once activated, the Gi protein inhibits AC, blocking cAMP synthesis and mitigating the inotropic effect. PKA can also phosphorylate the β-ARs and others GPCRs in the heart, leading to G-protein uncoupling and receptor desensitisation [8, 14]. Owing to the pleiotropic activity of the mediators cAMP and PKA, ECC is further regulated in terms of cardiomyocyte architecture. Cardiomyocytes bear highly specialized microdomains, the so-called dyads, which adjoint t-tubules (150–300 nm deep invaginations of the sarcolemma) to the SR cisternae. The dyads concentrate several key regulatory elements of the ECC, namely the GPCRs, LTCCs, RyRs, cAMP, PKA, phosphodiesterases and protein phosphatases, which together are responsible for the specificity of the β-adrenergic signaling in the contractile apparatus [6, 12]. The compartmentalization of cAMP/PKA activity is also achieved through the action

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of A-kinase-anchoring proteins (AKAPs). These proteins tether PKA to specific protein complexes. For instance, AKAP15 attaches PKA to LTCC and muscle-specific AKAP targets both PKA and the phosphodiesterase PDE4D3 (a cAMP-degrading enzyme) to RyR2. This kind of regulation is essential to prevent diffusion of cAMP and PKA to (extra)-cytosolic compartments and underscores the specificity of β-AR signaling in ECC [12].

12.2.2 Regulation of Excitation-Contraction Coupling by Other Signaling Mediators Apart from β-AR-mediated cascades, there are other signaling pathways involved in the regulation of ECC. The control of the heart’s ECC (especially heart rate and cardiac output) is also engaged by muscarinic receptors, activated by acetylcholine released from parasympathetic neurons (Fig. 12.1). In this pathway, acetylcholine mediates the activation of the muscarinic receptor M2 , which, in turn, activates Gi with two main effects. In one hand, the Gi subunit α directly inactivates AC, arresting the cAMP-PKA axis. On the other hand, the Gi subunits βγ lead to G protein-activated inward rectifying K+ (GIRK) channels opening. Subsequent K+ influx results in membrane hyperpolarization favoring relaxation. Beyond the effects on the cardiac muscle, muscarinic receptors also regulate vascular tone. For instance, activation of the M3 and M5 receptors by acetylcholine promotes vasodilation of most vessels (with intact endothelium). Vasodilation is accomplished by Gq-mediated activation of phospholipase C (PLC) in ECs, which promotes IP3 formation and Ca2+ release from endoplasmic reticulum (ER) stores. Ca2+ -activated calmodulin (CaM) then activates endothelial nitric oxide synthase (eNOS, see Sect. 12.4), leading to nitric oxide (NO) synthesis [10, 15]. In addition to muscarinic receptors, β3 -ARs are also responsible for eNOS stimulation [16]. Regardless of the pathway, the modulation of ECC by ECs paracrine factors, such as NO, is undeniable (Fig. 12.1). For instance, NO has a concentrationdependent inotropic effect. When NO concentration is low, it stimulates Ca2+ entry and SR Ca2+ release through nitrosylation of LTCC and RyR2. On the contrary, when NO concentration is high, the soluble guanylate cyclase is activated, promoting cyclic guanosine monophosphate (cGMP) synthesis. cGMP, in turn, activates phosphodiesterase II, which degrades cAMP, leading to a reduction in Ca2+ influx through LTCC. cGMP-activated protein kinase G (PKG) should also be accounted for, as this kinase directly inhibits LTCC activity and phosphorylates troponin I, leading to decreased myofilament Ca2+ sensitivity (lusitropic effect). ECs also release endothelin-1 (ET-1) (Sect. 12.6.1) that exerts positive inotropic effects (Fig. 12.1). Briefly, ET-1-mediated activation of ET-1 receptor A (ETA ) results in protein kinase C (PKC) activation, which stimulates the activity of the LTCC, NCX and of the Na+ /H+ exchanger, leading to a net increase of Ca2+ influx. ET-1 may also affect myofilament Ca2+ sensitivity through PKC/D-mediated phosphorylation of troponin I and MyBP-C [7, 17].

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12.3 Calcium as a Key Signaling Molecule in the Cardiovascular System Ca2+ governs a myriad of cellular processes in the cardiomyocytes (Fig. 12.1). A very tight temporal and spatial control is observed in Ca2+ signaling, ranging from shortterm, milliseconds-long effects taking place in dyads and sarcomeres (ECC) to longterm, hours/days-long transcriptional effects occurring in the nucleus (commonly referred to as excitation-transcription coupling, ETC) [18, 19]. The Ca2+ signaling effects can be mediated by Ca2+ itself and by Ca2+ -binding proteins, such as CaM, Ca2+ /CaM-dependent protein kinase II (CaMKII), calcineurin and calpains. CaM displays no enzymatic activity, but upon binding to Ca2+ , a shift on its conformation occurs, exposing protein interaction domains. Calcium-activated CaM can activate CaMKII and calcineurin. CaMKII is a serine/threonine (Ser/Thr) kinase with four different isoforms, δ being the predominant one in the cardiac muscle. Additionally, CaMKIIδ has two more isoforms, the cytoplasmic-predominant δC and the nucleuscompartmentalized δB. Calcineurin, also known as protein phosphatase 2B, is a Ser/Thr phosphatase composed of two subunits: the CaM-binding catalytic subunit, CnA, and the Ca2+ -binding regulatory subunit, CnB. Finally, calpains are cysteine proteases, activated upon a substantial increase of Ca2+ concentration [20, 21]. In this section, we will focus our attention on the key regulatory role of Ca2+ and Ca2+ regulated proteins in: (i) ECC; (ii) mitochondria bioenergetics and cell death and (iii) ETC. The signaling effects of Ca2+ on ECC are the fastest and ultimately rely on its concentration. For instance, when the cytosolic concentration of Ca2+ approaches 100 nM, CaM becomes activated and, in turn, activates CaMKIIδ. CaMKIIδC phosphorylates LTCC, increasing the channel opening probability (Ca2+ dependent facilitation). However, when Ca2+ concentration reaches the micromolar range, CaM, which is constitutively attached to LTCC, becomes Ca2+ -saturated, leading to the inhibition of this channel [18, 20, 22]. The RyR2 is also a substrate for CaMKII and its phosphorylation can either increase or decrease the channel opening probability. Besides, the apo-form (Ca2+ free) of CaM partially activates RyR2, while active Ca2+ -bound CaM lowers its activity [18, 20, 22]. CaMKII is also known to phosphorylate SERCA and PLB, leading to increased SR Ca2+ reuptake [20, 22]. Of note, β-adrenergic signaling, with well-known effects on ECC, is regulated by Ca2+ signaling. For instance, on the cAMP-PKA axis, Ca2+ inhibits AC and Ca2+ bound CaM activates phosphodiesterases, leading to a net decrease of cAMP. On the PLC-phosphatidylinositol 4,5-bisphosphate (PIP2) axis, Ca2+ is required for the interaction of PLC with its substrate PIP2 and, further downstream, for the activation of the ‘typical’ PKC isoforms (α, β1, β2 and γ). Finally, the ER inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), the central player in IP3-induced Ca2+ release (IICR, a slower reacting system than CICR) is regulated by CaM. When the cytosolic concentration of Ca2+ increases, CaM binds to IP3R, decreasing its affinity to IP3, thereby keeping Ca2+ homeostasis and preventing hypercontractility [22]. Regulation of muscle contractility is also achieved by tuning of myofilamentary Ca2+ sensitivity, which is regulated by CaM-mediated myosin light chain (MLC) kinase

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(MLCK) activation and subsequent phosphorylation of myosin regulatory light chain 2 [18, 22]. The same mechanisms (i.e., IICR and phosphorylation of myosin light chains) are also implicated in α1-AR-induced smooth muscle contraction, required for vasoconstriction [14, 23, 24]. Of all human cell types, cardiomyocytes have the highest mitochondria content, and this is due to the high-energy demand required for the continuous pumping activity of the heart. Besides ECC, Ca2+ is intimately implicated in the regulation of mitochondrial function. Under conditions of higher energetic expenditure (e.g. increased workload), Ca2+ accumulates in the cytosol and enters the mitochondria, through uniporter channels using the electrochemical driving force generated by proton translocation. Intramitochondrial Ca2+ activates Krebs cycle enzymes, namely pyruvate, isocitrate and α-ketoglutarate dehydrogenases, leading to increased production of NADH. Besides, Ca2+ also activates ATP synthase, collectively resulting in increased ATP synthesis and, thus, in higher energy supply [22]. Nevertheless, when there is a sustained increase of Ca2+ in mitochondria in parallel with a cytosolic ATP depletion (which can be further exacerbated by increased oxidative stress), the opening probability of mitochondrial permeability transition pore (mPTP) increases. The opening of mPTP results in a solute exchange between mitochondrial matrix and cytosol, collapse of inner mitochondrial matrix potential, precipitation of Ca2+ phosphates, ATP synthesis blockade and cell death. Furthermore, activation of mPTP may cause excessive release of Ca2+ , leading to hypercontraction, as well as of cytochrome c outflow which can trigger apoptosis through activation of caspases. However, it will be the extent of ATP reserves that will determine if the cell undergoes apoptosis or necrosis [19, 22]. The apoptotic process can also be facilitated by calcineurinmediated dephosphorylation of the pro-apoptotic protein Bad, which translocates to the mitochondria inhibiting the anti-apoptotic protein Bcl-xL. Dephosphorylation of dynamin-related protein 1 (Drp1) by calcineurin also assists the apoptotic process, by promoting Drp1 translocation to the mitochondria. Drp1 oligomerization in mitochondria favors the fission process, which is required for proper clearance of damaged organelles [25]. Sustained Ca2+ signaling also exhibits nuclear effects (ETC), taking place in a longer time frame and commonly being associated with cardiac remodeling (mainly pathological hypertrophy). As mentioned above, an increase of cytosolic Ca2+ triggers CaM, CaMKII, calcineurin and calpains signaling. Indeed, all these proteins show a direct or indirect role in the regulation of gene expression. CaM can directly and indirectly (through CaMKII and calcineurin) regulate the expression of hypertrophy-related genes. Direct regulation involves calmodulin-binding transcriptional activator 2 (CAMTA2)-mediated coactivation of the NKX2-5 homeobox protein, associated with the transcription of hypertrophic genes [21]. Regarding CaMKII, the nuclear effects are a consequence of the phosphorylation of numerous transcription factors, histone deacetylases (HDAC) and histones themselves. However, some of the exact effects are not entirely known, namely for cAMP-responsive elementbinding protein (CREB) and for activating transcription factor 1. Still, it has been shown that CaMKII phosphorylates and activates the serum response factor (SRF) transcription factor, which together with its coactivator myocardin, are involved in the

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preservation of cardiac integrity and hypertrophy. CaMKIIδB also activates the transcription factor heat shock factor 1, promoting increased expression of inducible heat shock protein 70, a well-known chaperone with anti-apoptotic activity. Activation of nuclear factor NF-κB (NF-κB) by CaMKII results in increased chemokine expression, which contributes to maladaptive remodeling through inflammation and fibrosis. The nucleus-specific CaMKIIδ isoform (B) phosphorylates HDAC4, promoting 14-3-3 chaperone binding and consequent nuclear export. If exported, HDAC4 is no longer capable of repressing the myocyte-specific enhancer factor 2 (MEF2) and, thus, the fetal gene program is initiated, with expression of hypertrophic genes. Moreover, whenever HDAC5 oligomerizes with HDAC4, it can be phosphorylated by CaMKII, creating another mechanism for nuclear export of HDACs and consequent activation of MEF2. Finally, CaMKIIδB can directly interfere with chromatin dynamics by phosphorylation of histone 3, thus creating higher accessibility to hypertrophy-response regions of chromatin [18, 19, 21]. The most important effect of calcineurin in ETC is mediated by nuclear factor of activated T-cells (NFAT). Under persistent and high cytosolic Ca2+ concentrations, calcineurin is activated. Calcineurin catalyzes NFAT dephosphorylation, allowing its translocation to the nucleus. Nuclear accumulation of NFAT is also achieved by calcineurin-mediated inhibition of exportin-1. In the active state, NFAT stimulates the transcription of genes related to contraction, Ca2+ handling, metabolism and hypertrophy. Because NFAT interacts weakly with DNA, it cooperates with other transcription factors, such as AP1 (activating protein 1), c-Jun/c-Fos, GATA4 and MEF2. Regulation of NFAT signaling is achieved by inhibitory signals from PKA, c-Jun N-terminal kinase (JNK), p38 MAPK, casein kinases, glycogen synthase kinase 3β (GSK-3β) and dualspecificity Tyr phosphorylation-regulated kinase 1A, which re-phosphorylate NFAT [19, 21, 22, 25]. Finally, calpain effects on ETC are mainly the result of PKCα and calcineurin cleavages. The former leads to the release of PKC’s catalytic fragment that enters to the nucleus, leading to HDAC5 phosphorylation and nuclear export, de-repressing MEF2-mediated gene transcription. The latter drives to the elimination of calcineurin’s auto-inhibitory domain, which allows its nuclear translocation and contributes to the accumulation of dephosphorylated NFAT and, thus, to an increase in NFAT-mediated gene transcription [21].

12.4 Nitric Oxide Role in the Cardiovascular System NO is perhaps the most important and the best-characterized vasodilator agent in the cardiovascular system [26, 27]. NO is synthesized in ECs, from L-Arg, by eNOS. This enzyme is basally found attached to caveolin and can be detached and activated by Ca2+ -activated CaM. Increased shear stress (due to increased blood flow) or the activity of NO agonists (such as acetylcholine, ATP, ADP, substance P, thrombin and bradykinin) are two leading mechanisms of increasing cytosolic Ca2+ levels. When shear stress increases, Ca2+ -activated K+ channels are activated, promoting K+ efflux

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and Ca2+ influx. In turn, NO agonists promote Ca2+ release from ER, leading to depletion of intracellular stores and activation of plasma membrane Ca2+ channels, together resulting in a net rise of intracellular Ca2+ levels. Following CaM activation, eNOS is released and synthesizes NO which diffuses freely from endothelium to VSMCs where its vasodilator properties are manifested [26, 28]. Inside VSMCs, NO binds and activates soluble guanylate cyclase (GC) that generates cGMP from GTP. cGMP turns PKG on, which mediates most of NO’s vasorelaxant properties. For instance, PKG phosphorylates IP3R and PLB, mitigating IICR and promoting Ca2+ reuptake, respectively. PKG may also directly phosphorylate LTCC and Ca2+ ATPase, dropping Ca2+ influx and enhancing Ca2+ efflux [29, 30]. NO signaling may also drive VSMCs hyperpolarization, through cGMP or SHP-1 Tyr phosphatase-mediated voltage-gated K+ channel dephosphorylation, PKG-mediated Ca2+ -activated K+ channel phosphorylation and directly through NO-mediated activation of ATP-sensitive K+ channels, all leading to K+ efflux and turning membrane potential even more negative [27, 29, 30]. The NO effect is also observed on MLC Ca2+ sensitivity. PKG phosphorylates MLC phosphatase (MLCP), increasing its activity and, thus, lowering contractility by reducing MLC’s sensitivity to Ca2+ . Another mechanism is based on RhoA phosphorylation, which prevents its interaction with Rho kinase (ROCK) and, consequently, its activation. By remaining inactive, phosphorylation of MLCP’s myosin-binding site is prevented, MLCP activity is maintained and so the hypocontractile state of MLCs [29, 30]. Apart from modulating contractility, NO also regulates mitochondrial function in cardiomyocytes. In fact, through a cGMP-dependent mechanism, NO triggers the expression of peroxisome proliferator-activated receptor gamma coactivator 1α, leading to increased mitochondrial biogenesis and boosting oxidative phosphorylation [31].

12.5 Mechanotransduction Mechanotransduction corresponds to the cellular mechanism by which mechanical forces are converted into intracellular biochemical signals and generate suitable responses altering the structure and/or function of the cell [4, 32–34]. This mechanism affects the regulation of cardiac performance as well as the proliferation, growth, differentiation and survival of cardiac and vascular cells [34, 35]. Several signaling molecules and structural proteins play relevant roles in converting mechanical stress and strain into biochemical signals, including membrane-associated receptors such as integrins, adaptor proteins, such as talin, vinculin, FAK (focal adhesion kinase) and melusin and ion channels (stretch-sensitive channels) [36]. Moreover, sarcomere related protein complexes, specifically those involving titin, can also have a role in cardiomyocyte mechanotransduction [37] (Fig. 12.2).

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Fig. 12.2 Cardiomyocyte signaling pathways involved in integrin- and titin-mediated mechanotransduction (see text for explanation). β1-AR, β1-adrenergic receptor; AC, adenylate cyclase; Adr, adrenaline; Ang II, angiotensin II; ANP, atrial natriuretic peptide; AT1R, angiotensin II receptor; BNP, B-type natriuretic peptide; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; DAG, diacylglycerol; IQGAP1, IQ motif-containing GTPase activating protein 1; ECM, extracellular matrix; ERK1/2, extracellular signal-regulated protein kinases 1 and 2; ET-1, endothelin-1; ETA, endothelin receptor A; FAK, focal adhesion kinase; FHL, four-and-a-half LIM domain protein; GC-CR, guanylate cyclase-coupled receptor; GPCR, G protein-coupled receptor; MLP, muscle LIM protein; MuRF, muscle RING-finger protein; Nbr1, neighbor-of-BRCA1-gene-1 protein; NE, norepinephrine; NFAT, nuclear factor of activated T cells; NO, nitric oxide; NPRA, natriuretic peptide receptor A; PEVK, proline-glutamic acid-valine-lysine rich region; pGC, particulate guanylate cyclase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PLC, phospholipase C; sGC, soluble guanylate cyclase; T-Cap, titin-Cap; us, unique sequence of cardiac N2-B region. Some graphical elements were adapted from Servier Medical Art (https://smart.servier.com)

12.5.1 Integrins-Mediated Signaling Integrins are expressed in all cellular components of the cardiovascular system, including the cardiomyocytes, non-muscle cardiac cells and vascular cells [35]. Integrins are transmembrane receptors that link the ECM to the cytoskeleton at focal adhesion sites. Integrins are also located in the intercalated discs that connect cardiomyocytes [32, 33, 38]. These receptors are heterodimeric, presenting an α-subunit that confers ECM specificity and a β-subunit that interacts with the cytosol in cardiomyocytes [33, 38]. The most common integrin heterodimers in the cardiomyocytes are α1β1, α5β1 and α7β1, which are predominantly collagen, fibronectin and

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laminin-binding receptors, respectively [32]. Binding of ECM ligands to integrins activates intracellular signaling pathways through a process known as outside-in signaling. Integrins lack enzymatic activity and thus require the interaction with adaptor proteins for the propagation of the signals from the focal adhesion sites. In the cytosol, integrins connect and aggregate with a variety of adaptor proteins (with structural, scaffolding or catalytic functions) such as FAK, integrin-linked kinase (ILK), vinculin, talin, melusin, particularly interesting new cysteine-histidine rich protein (PINCH), paxillin, parvin, α-actinin and actin [32, 34]. These proteins then activate signaling pathways involving mediators such as Rho GTPases, phosphatidylinositol 3-kinase (PI-3K), PKC, Akt, JNK, extracellular signal-regulated protein kinase (ERK), p38 and NF-κB, thus regulating cardiomyocyte hypertrophy, survival and cytoskeletal organization [32, 35]. FAK has a major role in integrin-mediated mechanotransduction (Fig. 12.2). This tyrosine kinase is expressed in costameres (in the striated muscle, the equivalent to focal adhesion) and intercalated discs of cardiomyocytes. FAK colocalizes with integrins through its association with the proteins paxillin and talin. FAK is activated by mechanical stretch/hemodynamic loading, as well as by hypertrophic agonists such as ET-1 and angiotensin II (Ang II). FAK activation (Sect. 12.7) potentiates its interaction with Src family tyrosine kinases and also affects the activity of Rhofamily GTPases that can modulate the organization of actin’s cytoskeleton. FAK can also activate the proteins Ras, ERK1/2 and Akt, leading to hypertrophy [32, 34]. Another protein involved in mechanotransduction is melusin (Fig. 12.2). Melusin is a muscle-specific protein localized at the costameres that bind to the β1-integrin cytosolic domain [32]. Melusin overexpression was shown to induce mild cardiac hypertrophy with no apparent structural and functional alterations. Interestingly, after prolonged pressure overload, melusin overexpressing mice showed beneficial left ventricle remodeling with preserved contractile function. Melusin overexpression was related with an increase in phosphorylation of Akt, GSK3β, and ERK1/2, after pressure overload, which are mediators involved in anti-apoptotic signaling pathways [39]. Additionally, melusin was shown to form a complex with the mitogen-activated protein kinases (MAPKs), the c-Raf, MEK1/2, ERK1/2, FAK and IQGAP1 (IQ motif-containing GTPase activating protein 1). Furthermore, FAK and IQGAP1 are required for melusin-dependent ERK1/2 activation and are essential to melusininduced cardiomyocyte hypertrophy and survival [40].

12.5.2 Stretch-Activated Channels Stretch-activated channels also have a crucial role in mechanotransduction, by converting mechanical forces into ion fluxes through the cell membrane. These channels play several roles in the cardiovascular system, including regulation of blood pressure, vasoreactivity, and cardiac arrhythmias as well as the maladaptive remodeling related to cardiac hypertrophy and heart failure [41].

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Transient receptor potential channels, TRPC1 and TRPC6, are widely expressed in cardiomyocytes and VSMCs and have been implicated in muscle mechanotransduction. TRPC1 is a non-selective “store-operated ion channel” (SOC) involved in Ca2+ entry following Ca2+ depletion of the ER. TRPC6 is a “receptor-operated channel” (ROC), directly activated by diacylglycerol (DAG), independently of PKC [42, 43]. Stimulation of AT1 R by mechanical stress (i.e. tension in the membrane) or by its ligand Ang II, leads to PIP2 hydrolysis and the resulting DAG directly activates TRPC channels. Activation of calcineurin by Ca2+ -CaM dephosphorylates NFAT which then translocates into the nucleus, leading to the expression of prohypertrophic genes including TRPC6 [43]. Other Gq/11-coupled receptors such as the ETA receptor for ET-1 can substitute for AT1R, whereas Gs-coupled receptors such as the β2-AR and tyrosine kinase receptors do not associate mechanical stimuli with TRPC6 activation. TRPC1 may also be implicated in regulating cardiac hypertrophy [42, 43].

12.5.3 Titin-Mediated Mechanotransduction The high molecular weight sarcomeric protein titin can interact with several other proteins that have a role in mechanotransduction- and hypertrophy-associated pathways (Fig. 12.2) [44]. Titin runs from the Z-disk to the M band (center of the sarcomere). Two main titin isoforms are described in mammalian heart: N2B (stiffer) and N2BA (compliant), which are co-expressed at the sarcomere level [45–47]. Three titin regions are principally involved in protein-protein interactions: the Z-disk region, the I band segment and the M band region [45, 48]. T-Cap binds to the NH2-terminal domain at the Z-disk of titin and complexes with MLP (muscle LIM protein). This complex (MLP/T-Cap/titin) is associated with the activation of the calcineurin/NFAT signaling cascade, through the interaction with calsarcin which is linked to hypertrophic signaling. In the I band segment, the N2-Bus (unique sequence) domain binds FHL (four-and-a-half LIM protein) 1 and FHL2, which are transcriptional coactivators. FHL1 binding to titin is associated with the MAPK signaling cascade (Raf1/MEK1/2/ERK2). Additionally, ERK2 can phosphorylate titin in the N2Bus sequence, possibly modulating titin stiffness. The third region for titin-related hypertrophic signaling is the titin kinase domain, located in the M band periphery. This domain can interact with ubiquitin-associated zinc-finger protein neighbor of BRCA1 gene 1 (Nbr1) forming a complex with p62/SQSTM1 and muscle-specific ubiquitin E3 ligases MuRF1 and MuRF2, which can repress transcription and inhibit hypertrophy [45, 46, 48]. In addition to the interactions with several signaling proteins, the presence of diverse phosphorylation sites in the Z-disc, I band, and M band segments demonstrate the key role of titin in cardiac signaling. Phosphorylation by PKA (activated in response to β-adrenergic stimulation by catecholamines), PKG (activated by NO or natriuretic peptides), PKC and ERK 2 (both activated by ET-1 or Ang II) was described and has a role in the modulation of titin stiffness [45] (for detail, see articles [49, 50]).

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12.6 Neurohumoral and Growth Factors Signaling In this section, the most relevant signal transduction mechanisms activated by neurohumoral factors (ET-1, Ang II, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)) and by growth factors/hormonal cues (insulin, insulin-like growth factor-1 (IGF-1), other growth factors and thyroid hormone (TH)) in the cardiovascular system will be described (Fig. 12.3). Their role in the regulation of cardiovascular structure and function will be highlighted.

12.6.1 Endothelin-1 Signaling ET-1 is the predominant endothelin in the human cardiovascular system [51]. ECs primarily synthesize ET-1, but VSMCs, fibroblasts and cardiomyocytes also produce this potent vasoconstrictor [52, 53]. ET-1 regulates vascular remodeling, angiogenesis, ECM synthesis, cardiac contractility and hypertrophy [23, 54]. ET-1 is upregulated by mediators such as Ang II and transforming growth factor-β (TGF-β) and downregulated by vasodilators as NO and natriuretic peptides [54]. ET-1 signals through two GPCRs, endothelin receptor A (ETA ) and endothelin receptor B (ETB ). ETA and ETB receptors are both present in VSMCs and mediate contraction and proliferation by interacting with its ligand ET-1. Both receptors share the Gq/11 signaling pathway, inducing an increase in cytosolic Ca2+ and muscle contraction. ET-1 interaction with receptors will activate PLC that will lead to an increase in cytosolic Ca2+ and to muscle contraction, as described above [52]. ETB receptor is the principal receptor in ECs, and ligand interaction will activate PKC that, in turn, activates eNOS, phospholipase A2 and prostaglandin G/H synthase 2 (a.k.a. cyclooxygenase2), leading to the production of the vasodilators NO and prostacyclin. ETB is also responsible for the clearance of circulating ET-1 and is also involved in the process of angiogenesis [52–54]. Both ETA and ETB receptors are present in fibroblasts, and ligand interaction with the receptors is associated with fibrosis [54]. In cardiomyocytes, the ETA receptors are the prevalent ones, and its activation promotes cardiac remodeling and hypertrophy via Gq/11 signaling pathway [1, 23, 55].

12.6.2 Angiotensin II Signaling Ang II is part of the renin-angiotensin-aldosterone system (RAAS). This mediator is converted from Ang I by angiotensin converting enzyme. Ang I results from the enzymatic cleavage of angiotensinogen by renin. However, in the human heart, the majority of Ang I is converted to Ang II by chymase. Acute stimulation with Ang II modulates blood pressure by regulating salt/water homeostasis and vasoconstriction, while chronic stimulation promotes hyperplasia and hypertrophy of VSMCs

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Fig. 12.3 Major pathways associated with neurohumoral (adrenaline, norepinephrine, ET-1, Ang II, ANP and BNP; left side of the image) and hormonal (insulin, IGF-1, other growth factors and thyroid hormone; right side of the image) signals in the cardiomyocyte (see text for explanation). β-AR, β-adrenergic receptor; 4E-BP1, eukaryotic translation initiation factor 4E binding protein 1; AC, adenylate cyclase; Adr, adrenaline; Ang II, angiotensin II; ANP, atrial natriuretic peptide; AT1R, angiotensin II receptor; BNP, B-type natriuretic peptide; Ca2+ , calcium; CaM, calmodulin; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CREB, cAMP-responsive element-binding protein; DAG, diacylglycerol; EGF, epidermal growth factor; ERK1/2, extracellular signal-regulated protein kinases 1 and 2; ET-1, endothelin-1; ETA, endothelin receptor A; FoxO1, forkhead box protein O 1; GC, guanylate cyclase; GC-CR, guanylate cyclase-coupled receptor; Glc, glucose; GPCR, G protein-coupled receptor; GSK3, glycogen synthase kinase 3; IGF-1, insulin-like growth factor-1; IP3, inositol 1,4,5-trisphosphate; IRS, insulin receptor substrate; LTCC, voltage-dependent L-type calcium channels; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; mTOR, mammalian target of rapamycin; NE, norepinephrine; NFAT, nuclear factor of activated T cells; NPRA, natriuretic peptide receptor A; PDK-1, 3-phosphoinositide-dependent protein kinase 1; PI-3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PLC, phospholipase C; ROC, receptor-operated channel; RSTK, receptor serine/threonine kinase; RTK, receptor tyrosine kinase; RyR, ryanodine receptor; S6K, 70 kDa ribosomal protein S6 kinase; SOC, store-operated channel; TAK-1, TGF-β activated kinase-1; TGF-β, transforming growth factor-β; THT, thyroid hormone transporter. Some graphical elements were adapted from Servier Medical Art (https://smart.servier.com)

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as well as cardiac hypertrophy and remodeling [56]. The Ang II receptors (AT1 R, AT2 R) are GPCRs. Most of the effects of Ang II are mediated by AT1 Rs, expressed in vascular smooth muscle, endothelium and heart, and are primarily coupled to a Gq/11 protein. Ligand binding to these receptors activates PLC resulting in IP3 and DAG production, increasing Ca2+ cytosolic levels, which causes phosphorylation of MLC by MLCK and vascular/cardiac contraction [56–58]. Additionally, Ang II binding to AT1 Rs coupled to Gq/11 protein can activate other pathways involving PKC, reactive oxygen species (ROS) production and tyrosine kinases (such as Src) that activate metalloprotease ADAM-17, cleaving proheparin-binding EGF-like growth factor (proHB-EGF) to HB-EGF. HB-EGF binds, phosphorylates and consequently activates EGFR/ERK1/2 pathway, leading to protein synthesis, cell growth and hypertrophy. Gαq-mediated ROS might also comprise platelet derived growth factor (PDGF) receptor (PDGFR) transactivation, leading to growth and vascular remodeling. Furthermore, AT1 Rs are also coupled to Gα12/13, whose activation stimulates ROCK, which causes vascular contraction and hypertrophy. AT1 Rs activation also stimulates G protein-independent signaling pathways such as β-arrestin, which results in uncoupling of G protein signaling and activation of p42/44 MAPK and Akt/PKB/eNOS pathways, leading to a reduction in cardiac apoptosis. Another G-protein-independent signaling pathway activated by Ang II is the Src-JAK/STAT (Janus kinase/signal transducer and activator of transcription). Ang II activates JAK2 via both G-protein-independent and dependent mechanisms, leading to the proliferation of VSMCs and cardiac hypertrophy and remodeling [56–58].

12.6.3 Atrial Natriuretic Peptide and B-Type Natriuretic Peptide Signaling ANP and BNP are synthesized in the heart and released into circulation mainly in response to myocardial stretch. Although ANP is mostly expressed in the atria, the ventricles are responsible for a higher release of this peptide in the failing heart. BNP is mainly released from the ventricles, although it is expressed in both atria and ventricles [59, 60]. These peptides display diuretic, natriuretic and hypotensive effects. Additionally, they have a cardioprotective role, reducing cardiac hypertrophy and fibrosis and also enhancing angiogenesis and cardiomyocyte viability [59]. ANP and BNP bind to the receptors NPRA and NPRC in target cells (such as cardiomyocytes, fibroblasts and VSMCs). NPRA is a GC-coupled receptor, and its activation leads to cGMP synthesis and, thus, PKG activation. In vascular and cardiac cells, ANP and BNP effects are mediated by PKG phosphorylation of downstream targets such as PLB and LTCCs, leading to a decrease in cytosolic Ca2+ levels. The NPRC lacks GC activity. NPRC is involved in peptide clearance and is also coupled to AC through a Gi protein. Thus, it is associated with AC inhibition, resulting in a decrease of intracellular cAMP concentration. Additionally, NPRC can activate cGMP signaling indirectly through activation of eNOS [59, 61–63].

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12.6.4 Thyroid Hormones Signaling The thyroid secretes two main hormones, namely the 3,5,3 ,5 -tetraiodothyronine (T4) and the 3,5,3 -triiodothyronine (T3). Despite being released in smaller amounts, T3 is more active than T4, showing higher affinity to TH receptors (TR). T4, in turn, must be converted to T3 by deiodinases in target cells [64, 65]. THs target TR inside the cell, after being carried across the membrane by specific transporters such as monocarboxylate transporters. From the three known TR isoforms (α1, β1, and β2), TRα1 and TRβ1 predominate in cardiomyocytes. TRs can be present either in the cytosol or in the nucleus and once activated by TH trigger gene transcription through binding to TH response elements in the promoter region of specific genes. In cardiomyocytes, the nuclear effects of T3/T4 are translated into an increased expression of α-myosin heavy chain, voltage-gated K+ channels, Na+ /K+ ATPase, β1 -AR and guanine nucleotide regulatory proteins and into a decreased expression of β-myosin heavy chain, Na+ /Ca2+ exchanger, PLB, and ACs V and VI. Together, this leads to increased cardiac inotropy, chronotropy and lusitropy [65–67]. Other nuclear effects of TH comprise the positive regulation of matrix metalloproteinases (MMPs) expression and the repression of pro-α1 (I) collagen gene on cardiac fibroblasts, explaining its anti-fibrotic effects. Furthermore, TR-mediated gene expression regulation favors efficient energy utilization in cardiomyocytes, by stimulating glycolysis, lipolysis and mitochondrial biogenesis [68]. However, T3/T4 signaling is not limited to nuclear effects. In fact, T3/T4 can activate intracellular signaling pathways in a cytosolic TR-dependent or independent manner. For instance, in cardiomyocytes, T3 binds to cytosolic TRα1 which promotes the activation of the PI-3K/Akt/mTOR axis that, in turn, is responsible for increased protein synthesis and subsequent cardiomyocyte hypertrophy [68, 69]. In ECs and VSMCs, T3-mediated activation of TRα1, followed by PI-3K/Akt activation, leads to augmented eNOS activity and expression of hypoxia-inducible factor-1α (HIF-1α), promoting vasodilation and angiogenesis, respectively [65, 68, 69]. TR-independent THs effects include the activation of ion transporters and the activation of integrin αVβ3. The former leads to the regulation of Na+ , K+ and Ca2+ currents, while the latter has been associated with angiogenesis. Integrin αVβ3 pro-angiogenic effect has been attributed to MAPK-ERK1/2 signaling, and to PKD-activated HDAC5, which upregulates the expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and HIF-1α [66, 68, 69]. T3/T4 also confer protection against apoptosis, either through activation of PI-3K/Akt/mTOR, PKC, ERK1/2 signaling pathways, inhibition of p38 MAPK pathway or by nuclear effects, such as upregulation of p53-regulating miRNAs or heat shock proteins 70 and 27 [65, 68, 69].

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12.6.5 Insulin and Insulin-Like Growth Factor Signaling The skeletal muscle, adipose tissue and the liver are traditionally recognized as the principal targets of insulin. However, insulin receptors are also expressed in the heart muscle. Indeed, the principal insulin receptor isoform (A) in the cardiomyocytes is also the most sensitive to insulin (~2-fold compared to B isoform). Insulin receptors are also highly abundant in ECs [70, 71]. This suggests that insulin signaling plays an important role in the regulation of heart and vessel’s activity. In fact, insulin has been implicated in the regulation of heart metabolism, myocardial perfusion/vasorelaxation, hypertrophy, autophagy and survival [70–72]. The IGF is also a chief regulator of the cardiovascular system. IGF promotes cardiac effects similar to insulin, which can be explained, at least in part, by the homology and cross-reactivity between their receptors [73, 74]. Insulin and IGF receptors belong to the same family, the receptor tyrosine kinase family [72, 74]. Upon ligand binding, a reshape of these receptors occurs, promoting trans-autophosphorylation of specific tyrosine residues on the intracellular β subunit. Once the complex becomes activated, insulin receptor substrate (IRS) proteins or Shc (SH2 domain-containing transforming protein) are recruited to the vicinity of its intracellular domain and are themselves phosphorylated. In turn, IRS proteins (IRS-1 and IRS-2) and Shc create Src homology 2 (SH2)-domain containing docking sites for several proteins, such as the regulatory p85 subunit of PI-3K, Nck (Non-catalytic region of tyrosine kinase) and the Grb-2 (GFR-bound protein 2) [70, 72–75]. IRSand Shc-docked proteins mediate the roles of insulin/IGF in the cells through the activation of several signaling branches. One of the classical pathways activated by insulin and IGF is the PI3K/Akt/mTOR. In this cascade, the regulatory p85 subunit of PI-3K binds to IRS, leading to the activation of the catalytic p110 subunit. p110 catalyzes the conversion of PIP2 to phosphatidylinositol 3,4,5-trisphosphate (PIP3). Then, PIP3 activates the 3-phosphoinositide-dependent protein kinase 1 (PDK-1). This kinase activates atypical PKC isoforms as well as Akt (PKB). Two main Akt isoforms exist in the heart: Akt1, mediating physiologic hypertrophy, and Akt2, regulating glucose uptake and metabolism. The former phosphorylates mTOR, leading to phosphorylation of 70 kDa ribosomal protein S6 kinase and of eukaryotic translation initiation factor 4E binding protein 1, favoring translation and increasing protein synthesis. Hypertrophy is also favored by Akt1-mediated inhibition of GSK3β (thus preventing inhibition of the eukaryotic translation initiation factor 2) and of CCAAT/enhancer binding protein-β (C/EBPβ), leading to increased CBP/p300-interacting transactivator 4-mediated cardiomyocyte growth. Akt2, in turn, promotes glucose uptake and utilization by stimulating GLUT4 translocation to the membrane and by inhibiting forkhead box protein O (FoxO)1 (FoxO1), which suppresses glucokinase gene expression [71–73, 76, 77]. Akt signaling also promotes cell survival by inhibiting the pro-apoptotic Bad, pro-caspase 9, GSK3β, NF-κB and FoxO and stimulating the transcription factor CREB [73]. Finally, Akt-mediated eNOS phosphorylation enhances its activity, leading to an increase in NO production and thereby promoting vasorelaxation [71].

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Another critical pathway activated by insulin/IGF signaling is the MEK/ERK pathway. Here, Grb2 forms a complex with Sos, which acts as a nucleotide exchange factor, promoting the release of GDP and replacement by GTP on p21-Ras GTPase. In turn, Ras activates the MAPK cascade by stimulating the MAPKKK Raf kinase. This kinase then phosphorylates a MAPKK (such as MEK) that will phosphorylate a MAPK (such as ERK). Finally, ERK will regulate the activity of transcription factors (namely the p60TCF /Elk-1 and CREB, this last through p90rsk-mediated phosphorylation). The MEK/ERK signaling will ultimately translate into cell growth, mitogenesis and differentiation [70–72, 74, 78]. Overall, IGF activates pro-hypertrophic signaling in the myocardium. Of note, insulin/IGF receptors also dock regulatory proteins such as Syp (SH2containing protein tyrosine phosphatase, SH-PTP2), a protein Tyr phosphatase that blunt insulin/IGF signaling through receptor dephosphorylation. Additional negative modulators of insulin/IGF signaling comprise the PIP3 and dual-specificity protein phosphatase PTEN and the SH2 domain-containing inositol 5-phosphatase-2 (SHIP2) which halt Akt activation by reconverting PIP3 back to PIP2 [72]. The crosstalk between insulin and IGF signaling is particularly important in tackling heart failure, which is associated with insulin resistance. Hyperinsulinemic states (e.g. type 2 diabetes), metabolic disturbances (e.g. due to obesity) and even mechanical stress (e.g. pressure overload) are all known to activate p38 MAPK pathway [76]. This kinase blocks IRS activity by favoring Ser/Thr rather than Tyr phosphorylation and by promoting IRS ubiquitination and degradation. Consequently, Akt phosphorylation is prevented and FoxO1 nuclear localization is perpetuated, further desensitizing insulin signaling [71, 76]. In this regard, IGF-mediated signaling may compensate insulin pathway shut down. Indeed, small clinical trials have shown that recombinant IGF helps improving insulin sensitivity [79]. Besides, in a cardiomyocyte-restricted IGF transgenic model, the increased levels of IGF mitigated insulin resistance caused by a diet rich in fats [73, 80].

12.6.6 Growth Factors Growth factors, such as IGF-1 and neuregulin 1 (NRG1), are involved in the activation of pathways underlying growth and regeneration of the cardiac muscle and vessels. Angiogenesis, apoptosis suppression, cardiomyocyte proliferation and stem cell recruitment are among the biological processes under its influence [81]. Growth factors also drive ECM remodeling (Sect. 12.7). These can target tyrosine kinase receptors (such as IGF-1, FGF, VEGF and epidermal growth factor (EGF)) or Ser/Thr kinase receptors (TGF-β) [82]. Their effects result from the activation of the PI-3K/Akt, MEK/ERK and PLCγ1 pathways [82, 83]. Several FGF classes have been shown to display pathophysiological roles in the heart, namely FGF1, 2, 8–10, 15, 16, 21, and 23 [83–85]. FGFs bind and activate cell surface receptors (FGF receptors, FGFR), promoting the phosphorylation of the intracytoplasmic tyrosine kinase domains, thus activating FGF receptor substrate

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2α (FRS2α) and/or PLCγ1. While the former activates the Ras-MAPK (e.g. p38 MAPK and JNK), STAT signaling routes or PI-3K/Akt pathways, the latter triggers Ca2+ -dependent signaling pathways [82, 83, 85, 86]. Overall, FGFs appear to be cardioprotective. For instance, FGF2 (basic FGF) may exert proangiogenic and cardioprotective effects after ischemia/reperfusion injury, through activation of the ERK and inhibition of the p38 MAPK pathways [86, 87]. FGF1 was also associated to cardioprotection following ischemia/reperfusion [82, 84]. In turn, FGF2 and FGF16 compete for the same receptor (FGFR1c), but the former induces, and the latter prevents hypertrophy [83]. The above FGFs require the use of heparin/heparan sulfate as a cofactor. Contrariwise, FGF21 and FGF23 use, respectively, βKlotho and αKlotho as cofactors for FGFR activation. FGF21 activates FGFR1c, triggering the antiapoptotic PI-3K/Akt1/BAD (Bcl2 antagonist of cell death) and AMPK-Akt/GSK3β pathways and the inhibition of the pro-inflammatory NF-κB pathway and of the profibrotic MMP9. Moreover, FGF21 stimulates the ERK1/2/CREB/Sirtuin 1 pathway leading to increased expression of mitochondrial β-oxidation enzymes and antioxidant proteins (e.g. catalase and peroxiredoxin 5). Altogether, these routes directly or indirectly prevent or mitigate cardiac hypertrophy [83, 88, 89]. Unlike FGF21, FGF23 stimulates hypertrophy, through the activation of PLCγ and subsequently calcineurin/NFAT axis [83, 85]. Regarding VEGF, its signaling is initiated by binding to one of three classes of VEGF receptors (VEGFR), namely VEGFR1 (VEGFA, B and placental growth factor), VEGFR2 (VEGFA, C, D and E) and VEGFR3 (VEGFC and D) [90]. The VEGFR activation mechanism is similar to other receptor tyrosine kinases and involves receptor dimerization [91]. VEGF signaling may also be magnified by coreceptors such as heparan sulfate glycoproteins, neuropilins, integrins and ephrin B2 [91, 92]. A multitude of effects is triggered by specific VEGF-VEGFR interactions, namely ECs survival, proliferation, migration, tubulogenesis, angiogenesis, lymphangiogenesis and regulation of vascular permeability [91, 93]. VEGFR1 activation, for instance, promotes ECs migration and actin reorganization through activation of the receptor for activated kinase 1. Cell proliferation and tubulogenesis are stimulated through recruitment and activation of PI-3K. VEGFR1 also targets PLCγ leading to an increase of cytosolic Ca2+ and NFAT-based gene transcription. Grb-2 and SH2-domain-containing tyrosine phosphatase 2 are also attracted by VEGFR1 [91, 92]. VEGFR2, in turn, stimulates cell migration through SH2 domain-based recruitment of T-cell specific adaptor molecule, SH2 domain-containing protein B (Shb), which favors interaction with FAK, and through the Nck adaptor protein 1-Fyn tyrosine kinase complex. This complex promotes actin remodeling through the p21activated protein kinase 2-cell division cycle 42(Cdc42)-p38 MAPK axis [91, 92]. VEGF binding to VEGFR2 may also drive to increase cell survival and regulates vascular permeability. Recruited Shb activates PI-3K/Akt axis, through PDK1. Akt can then block apoptosis by interaction with BAD and caspase 9. Besides, Akt enhances eNOS expression and, thus, NO synthesis. It is through NO-based cGMP pathway activation that vascular permeability can be increased. VEGF-based activation of ROCK leads to phosphorylation of Rho GTPases (e.g. Rho, Rac and Cdc42). The resulting regulation of actin dynamics may also exert a role in vascular permeability

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[91, 93]. Finally, VEGFR3 activation is linked to cell survival and lymphangiogenesis. The former is achieved by interaction with adaptor protein C10 regulator of kinases that subsequently activates the JNK pathway. The latter is accomplished through the recruitment of Shc/Grb2 adaptor proteins and activation of the ERK1/2 and PI-3K/Akt pathways [91, 92]. Fibroblasts, SMCs and ECs are the main targets of PDGF signaling. Apart from its role in developmental stages, this growth factor participates in wound healing and angiogenesis (mobilizing mural cells). Moreover, it is also associated with atherosclerotic lesions, probably by promoting SMCs migration to injured intima [94–96]. The PDGF family is composed of 4 elements (A, B, C and D), but these are only biologically active as dimers (AA, AB, BB, CC and DD). In turn, PDGFRs can be present in membranes as homo- or heterodimers of α and β subunits. AA dimers can only bind to α-containing receptor dimers, while the remaining can bind to αα, αβ or ββ receptors [94, 95]. PDGFR activation occurs in the same way of other tyrosine receptor kinases. Pathways lying downstream of PDGFR are responsible for actin reorganization, motility, growth and inhibition of apoptosis (this last through the PI3K/Akt/BAD). PDGFR can also recruit PLCγ, generating IP3. This secondary messenger can activate PKC and increase cytosolic Ca2+ influx, modeling actin dynamics and promoting chemotaxis. Another well-characterized axis in PDGF signaling is the Grb2/Sos1-driven Ras activation, which will activate the Raf1/MEK/ERK1/2 pathway. This will result in cell growth, proliferation and differentiation. The Src family of protein kinases may also be docked to activate PDGFR and may promote induction of c-myc, required for a mitogenic response [94, 97]. The family of EGFs is particularly important during cardiac developmental stages. Nonetheless, in the postnatal period, EGF-like factors are still required for cardiomyocyte growth and survival. EGF, HB-EGF and NRG1 are considered the three most important mediators for adult cardiomyocytes activity [98]. These bind to different EGF receptors (EGFR/ErbB/HER). EGF binds exclusively to EGFR/ErbB1/HER1; HB-EGF has an affinity to both EGFR and ErbB4/HER4, and NRG1 recognizes either ErbB3/HER3 or ErbB4/HER4. EGFR belongs to the tyrosine kinase receptor family [98]. The two major pathways activated by EGF and NRG1 in the heart comprise the Grb2/Sos or Grb2/Shc complex-induced Raf/MKK1/ERK1/2 pathway and the PI-3K/PDK1/Akt pathway. The former is known to promote adaptive hypertrophy, while the latter is pro-survival [98–100]. NRG1 has been additionally associated with proangiogenic effects, probably through Akt-driven increase in eNOS expression, as well as in maintaining sarcomere structure, through Src-based activation of FAK [99, 100]. TGF-β can act on different cell types, namely ECs, SMCs, cardiomyocytes and fibroblasts. Its effects are diverse and depend not only on ligand and receptor concentration but also on the cell type, the presence of other growth factors and ECM topology. Indeed, TGF-β can modulate cell growth and differentiation, cell survival and proliferation, actin organization, cell adhesion, ECM deposition, apoptosis and angiogenesis [101–103]. There are two main types of TGF-β receptors (TβR). Class I (TβRI) encompasses seven activin receptor-like kinases (ALK, ALK1–7), while there are five known members of class II (TβRII). A third class can be considered (TβRIII),

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which refers to non-enzymatic co-receptors, such as endoglin and β-glycan, which further regulate TGF-β signaling [101]. Unlike the previous growth factor receptors, which belong to the Tyr kinase family, TβR are Ser/Thr kinase receptors that show a distinct activation mechanism. Upon TGF-β binding, TβR tether as heterotetramers composed by a TβRII dimer and two TβRI monomers, where the first phosphorylates and activates the second. Depending on the assembled receptors, different Smad proteins are activated by phosphorylation, and these lead to different cellular responses. For instance, activation of the TβRI/ALK5 receptor complex favors Smad2 and Smad3 phosphorylation. These then interact with Smad4, yielding a complex that translocates to the nucleus, where it regulates gene transcription [101, 103, 104]. The effects of Smad2/Smad3 signaling are dependent on the cell type involved. For instance, on ECs this pathway inhibits cell proliferation and migration [102]. Smad3 is particularly known to promote the acquisition of the myofibroblast phenotype as well as the synthesis and integrity of ECM, inducing the expression of ECM proteins and inhibitors of their proteases (e.g. tissue inhibitors of metalloproteinases, plasminogen activator inhibitor-1). In ECs, TGF-β is also responsible for the activation of the ALK1 receptor, translating into the phosphorylation and activation of the proteins Smad1, 5 and 8. The presence of the co-receptor endoglin enhances this pathway. Smad1/Smad5 also forms a complex with Smad4 that travels to the nucleus to regulate gene transcription. However, unlike the ALK5-triggered pathway, ALK1 activation has an angiogenic effect, achieved by the proliferation and migration of ECs [101–104]. In cardiomyocytes, TGF-β promotes cell growth, through increased synthesis of structural proteins such as β-myosin heavy chain and skeletal α-actin. However, this effect has been attributed to a Smad-independent pathway, involving the activation of the TGF-β activated kinase-1 and subsequent p38 MAPK activation [102, 104, 105]. The complexity of TGF-β signaling is also proven by the existence of inhibitory Smads, such as Smad6 (Smad1, 5 and 8 inhibitor) and Smad7 (Smad2 and 3 inhibitor), which compete for the interaction with Smad4 and of negative regulators of Smads’ transcriptional activity (e.g. c-Ski and SnoN) [101, 103].

12.7 Signaling Pathways Implicated in Extracellular Matrix Remodeling and Cardiac Fibrosis In the cardiovascular tissue, ECM not only serves as a scaffold for the cellular fraction but also plays a role in the transmission of chemical, electrical and mechanical signals and in mediating cellular responses through specific signaling pathways [106, 107]. ECM is mainly composed of fibrillary collagens, being composed by around 85% of thicker and stiffer type I collagen and 10% of thinner and more compliant type III collagen, in addition to basement membrane-specific type IV and V collagens. Besides, ECM also contains glycosaminoglycans, glycoproteins, proteoglycans and, importantly, it stores latent growth factors and proteases that, upon injury, may be released and trigger signaling cascades, remarkably on cardiac fibroblasts [106, 108].

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Fibroblasts play a key role in cardiac homeostasis, representing around two-thirds of all cardiac cellular entities. In fact, cardiac fibroblasts are central players in the setting of cardiac injury. Upon acute or chronic injuries, such as myocardial infarction, or under pressure- or volume-overload states, fibroblasts are stimulated to undergo differentiation into myofibroblasts. These are hybrid cells that share features of both fibroblasts and cardiomyocytes. Myofibroblasts are capable of synthesizing ECM proteins (collagen I and III, laminin and fibronectin), promoting fibrosis, and present contractile activity (conferred by increased expression of α-smooth muscle actin, α-SMA). Fibroblasts can also release cytokines, growth factors, MMPs and their inhibitors that together modulate ECM turnover [106, 107, 109]. Several factors can stimulate fibroblasts activation, such as Ang II, ET-1, aldosterone, PDGFs, integrins, galectins, tenascin C and cytokines [106, 107, 110, 111]. In the cardiovascular system, Ang II (Sect. 12.6.2) is an important mediator associated with ECM remodeling. Prolonged Ang II activity is associated with fibrosis, through stimulation of cardiac fibroblasts. The activation of the AT1 R is responsible for the initiation of multiple signaling pathways in fibroblasts, namely the PLC, PKC and MAPK pathways, which result in increased fibroblast proliferation, ECM synthesis (e.g. collagen I), ROS production (through induction of NADPH oxidase), tumor necrosis factor (TNF)-α (favoring inflammation), TGF-β1 synthesis (which further induces NAPDH oxidase expression) and secretion. Besides, Ang II signaling stimulates the expression of fibroblast integrins and FAK activity, leading to fibroblast adhesion. AT1 R-related cascades are counterbalanced by AT2 R, whose activation is associated with increased expression of phosphatases that inhibit MAPK signaling and with increased NO synthase expression [106, 107, 110]. Aldosterone, another RAAS element, is also capable of inducing fibroblast proliferation and promotes cardiac fibrosis. One mechanism explaining such activity is the ROS- or mineralocorticoid receptor-mediated activation of TNF-receptor associated factor 3 interacting protein 2, which lies upstream of the IkB kinase/NF-κB and of the JNK/activatorprotein 1 pathway. Activation of these pathways translates into an increased expression of connective tissue growth factor, collagen I and III, MMP2 and of the collagen cross-linking enzyme, lysyl oxidase, all contributing to ECM buildup and stiffening [106, 112]. ET-1 (Sect. 12.6.1) has also been shown to exert pro-fibrotic effects by, for instance, stimulating collagen synthesis, by down-regulating collagenases and by increasing fibroblast survival [106, 113]. Besides, ET-1 induces expression of αSMA, through the PI-3K/Akt/rac pathway, which is a crucial step in fibroblast-tomyofibroblast differentiation [107]. Additionally, four signaling axes play an undeniable role in fibrogenesis: TGFβ/Smad-dependent and Smad-independent pathways, Wnt/β-catenin signaling and integrin/FAK/RhoA pathway. TGF-β can be released upon ECM strain-causing cardiac injury or through secretion from a broad range of cells, such as fibroblasts, platelets, macrophages, vascular cells and even cardiomyocytes [106, 111, 114]. Myofibroblast transdifferentiation, ECM synthesis and increased expression of protease inhibitors (e.g. TIMP2) and integrins are well-known effects mediated by TGF-β that result from Smad3 signaling and, simultaneously, from the activation

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of MAPK pathways (p38, ERK and JNK). Besides, the Smad-independent NFAT pathway has also been shown to promote fibroblast transformation [106, 107, 111, 114]. The Smad-independent activation of the FAK is also a hallmark of TGF-β activity in fibroblasts. FAK is responsible for the activation of the TGF-β activated kinase (TAK) 1 and subsequent JNK activation that leads to increased ET-1 expression. This response further perpetuates the signaling cascades resulting in fibrosis, as discussed above [107]. TGF-β1 may also activate the PI-3K/Akt axis, through FAK-driven Src activation. Moreover, cardiac fibrosis is regulated by Wnt proteins, whose secretion can be triggered by TGF-β [106]. Wnt along with GSK3β regulate the cytosolic levels of β-catenin, a transcriptional coactivator of fibrogenesis-related genes. In the absence of fibrotic cues, GSK3β mediates β-catenin phosphorylation, which leads to its ubiquitination and degradation. However, when Wnt proteins (e.g. Wnt3a) are present, these proteins bind and activate Frizzled receptors, leading to polymerization of Disheveled protein and consequent sequestration of GSK3β. Consequently, cytosolic β-catenin accumulates and translocates to the nucleus promoting the expression of fibrosis-related genes [109, 111]. The activation of the fibrogenesis gene program can also be achieved through the integration of ECM signals at focal adhesions by integrins. When activated, integrins promote FAK autophosphorylation and Src recruitment. Activated Src, in turn, leads to phosphorylation and activation of the FAK-Src complex. This complex regulates both guanine nucleotide exchange factors and GTPases-activating proteins leading to activation of the RhoA GTPase. One of the main consequences of the RhoA activation is the induction of αSMA stress fibers (F-actin) assembly from actin monomers (G-actin). Under normal conditions, the myocardin-related transcription factor (MRTF) is bound to G-actin (repressed state). However, activation of the integrin signaling through mechanical stimuli leads to increased stress fibers polymerization and subsequent reduction of the free G-actin. Hence, MRTF is released and can move to the nucleus, where it can activate SRF-mediated transcription of fibrosis-related genes, such as α-SMA [111]. Therefore, a diverse set of stimuli, such as RAAS mediators, Wnt, TGF-β, oxidative stress and ECM deformation can induce cardiac fibrosis.

12.8 Summary In this chapter, the central signaling pathways governing the activity of the cardiovascular system were reviewed. Different types of stimuli, i.e. electrical, mechanical and chemical, are transduced through multiple cascades, conveying to multiple effects. These encompass faster changes in contractility and slower genetic adaptations, translating into hypertrophy or angiogenesis, among others. Herein, we emphasized the role of the G protein-coupled receptors-activated signaling cascades, responding promptly to variations in neurohumoral factors by fine-tuning the ECC through the multiple actions of PKA. We have also discussed the signaling pathways initiated by hormones/growth factors at tyrosine (e.g. insulin) and serine/threonine kinase receptors (e.g. TGF-β). The former is generally associated to survival and regenerative

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processes, particularly through the PI-3K/Akt/mTOR axis, and the latter to several responses which are cell type-dependent. We also approached the unique roles of Ca2+ and NO inside cardiovascular cells, as important modulators of contractility, vasorelaxation, hypertrophy and mitochondrial bioenergetics. Finally, the main signaling pathways involved in mechanotransduction and the role of ECM remodeling in the cardiovascular system were reviewed. Acknowledgments Thanks are due to the Portuguese Foundation for Science and Technology (FCT), European Union, QREN, FEDER and COMPETE for the financial support for the UnIC (UID/IC/00051/2019), iBiMED (UIDB/04501/2020) and CIAFEL (UIDB/00617/2020) research units and the research projects DOCnet (NORTE-01-0145-FEDER-000003) and NETDIAMOND (POCI-01-0145-FEDER-016385) and the post-graduation student (grant number SFRH/BD/111633/2015 to F.T.).

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Chapter 13

Growth Factor Signaling in the Maintenance of Adult Lung Homeostasis Henrique Araújo-Silva, Jorge Correia-Pinto, and Rute S. Moura

Abstract The lung needs to maintain its integrity and functionality throughout lifespan since it is critical for survival. At steady-state, lung cell turnover is typically very low, and resident progenitor cells are in a quiescent state. The activation or inhibition of different signaling pathways in epithelial and/or mesenchymal cells of the adult lung tissue is crucial to maintaining the quiescence of progenitor’s niches. Interestingly, growth factors that have been reported as essential for lung development are also key to preserve adult tissue. With this chapter, we aim to describe the current knowledge regarding the molecular players that contribute to maintaining the homeostasis of the adult lung, specifically, SHH, FGF, WNT, Retinoic Acid, TGFβ, BMP, VEGF, and PDGF. Keywords Homeostasis · Quiescence · SHH · FGF · WNT · RA · TGFβ-BMP · VEGF · PDGF

H. Araújo-Silva · J. Correia-Pinto · R. S. Moura (B) Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal e-mail: [email protected] H. Araújo-Silva e-mail: [email protected] J. Correia-Pinto e-mail: [email protected] ICVS/3B’s - PT Government Associate Laboratory, University of Minho, 4710-057 Braga/Guimarães, Portugal J. Correia-Pinto Department of Pediatric Surgery, Hospital de Braga, 4710-243 Braga, Portugal © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_13

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Abbreviations ABCA3 AEC AM ASMC BC BMP CYP26A1 DHH FGF FGFR GLI GREM2 IHH LIF LRP NRP PDGF PDGFR PTCH RA RALDH1 RAR SFRP1 SHH SMO SOX2 SP SPRY2 TGFβ VEGF VEGFR WNT

ATP Binding Cassette Subfamily A Member 3 Alveolar epithelial cells Alveolar macrophages Airway smooth muscle cells Basal cells Bone Morphogenetic Protein Cytochrome P450 26A1 Desert Hedgehog Fibroblast Growth Factor Fibroblast Growth Factor Receptor Glioma-associated oncogene Gremlin 2 Indian Hedgehog Lipofibroblast Lipoprotein receptor-related protein Neuropilin Platelet Derived Growth Factor Platelet Derived Growth Factor Receptor Patched Retinoic Acid Retinaldehyde dehydrogenase 1 Retinoic Acid Receptor Secreted frizzled-related protein 1 Sonic Hedgehog Smoothened SRY (sex determining region Y)-box 2 Surfactant protein Sprouty homolog 2 Transforming Growth Factor β Vascular Endothelial Growth Factor Vascular Endothelial Growth Factor Receptor Wingless-related Integration Site

13.1 Lung Overview The respiratory system can be divided into two anatomical regions: the upper respiratory tract (nose, pharynx, and larynx) and the lower respiratory tract (trachea, bronchi, and lungs) [1]. Additionally, it can also be distinguished in two distinct functional areas. The conducting zone that conducts the air into the lungs starts in the nose and ends in the bronchioles; through this path, the air is warmed, filtered, and

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moistened. The respiratory zone includes the respiratory bronchioles, alveolar ducts, and alveoli, and corresponds to the region where gas exchange occurs [2]. The lung has a complex internal structure [3] composed of two related and highly branched tubular systems: the conducting system and the vascular system [4]. To provide efficient gas exchange, the two systems need to be in close contact over a large surface area to assure a continuous renovation of air and blood; this cooperation results in effective oxygen uptake and carbon dioxide removal from the blood flow [5–7]. To fulfill the multiple respiratory functions, different types of cells/glands populate the respiratory tract. Mucoserous glands are present in both trachea and bronchi and are composed of serous and mucous cells. The mucinous secretions of these cells contribute to the mucous bilayer that covers the bronchial epithelium. The tracheal and bronchial mucosa is formed by a pseudostratified ciliated columnar epithelium intermingled with goblet cells over a basement membrane matrix that supports the epithelium (Fig. 13.1A). Besides goblet cells and ciliated cells, basal cells (BC) and neuroendocrine cells are also major cellular constituents of the pulmonary epithelium [5]. Goblet cells are mainly responsible for mucin secretion, which is the major component of the mucus [8]. Ciliated cells are principally involved in propelling foreign particles or organisms by a mucociliary clearance mechanism [5]. Basal cells can act, under specific conditions, as progenitor cells of the columnar airway epithelium. Furthermore, they play an important role in the junctional adhesion of the airways since they promote the attachment of the columnar epithelium to the basal lamina, and in the inflammatory response by upregulating the expression of immune cells receptors [9]. Neuroendocrine cells appear in low numbers and scattered throughout the lung parenchyma and present features of both neural and endocrine cells [10].

Fig. 13.1 Schematic representation of the adult lung and region-specific cell types. Illustration of the cell populations residing in the trachea (A), proximal conducting region (B), and distal respiratory region (C). This figure was produced, in part, using Servier Medical Art (www.servier. com/Powerpoint-image-bank)

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Bronchioles are completely lined with epithelial cells, mainly ciliated columnar cells, and club cells. Club cells comprise the major cell type present in the distal airways and contribute to cell renewal in the normal human conducting airway epithelium [11]. Furthermore, bronchioles are surrounded by a circular layer of smooth muscle cells involved in the regulation of the bronchomotor tone of the airways (Fig. 13.1B). On its turn, alveoli are primarily composed of alveolar epithelial cells (AEC) type 1 and type 2 interconnected by tight junctions (Fig. 13.1C). Type 1 cells are specialized in gas exchange, whereas type 2 cells synthesize and secrete pulmonary surfactant that prevents alveolar collapse [5]. AEC1 are flat (extremely thin) and cover more than 95% of the gas exchange surface. AEC2 are cuboidal and are considered epithelial stem cells since they have the capacity of cell renewal and, upon injury, serve as progenitors for AEC1, thus maintaining lung homeostasis [12]. Additionally, mesenchymal-derived cells such as fibroblasts, myofibroblasts, and pericytes can also be found in the alveolar wall; these cells are responsible for the maintenance and metabolism of proteoglycans and collagen (rigid structural component) and elastic fibers (non-rigid structural component) present in the alveolar walls [5]. Alveolar macrophages (AM) colonize the airways in the first days after birth and are replenished throughout the entire life due to its high ability of self-renewal. Besides participating in the adult lung immunological response, these cells participate in the clearance of apoptotic cells and cellular debris in both health and in disease context [13]. The alveolar wall and the interstitial space also exhibit neutrophils, lymphocytes, eosinophils, mast cells, and plasma cells, although in less amount [5]. The vascular system is a highly branched network lined by endothelial cells that supply nutrients and oxygen to the tissues. Endothelial cells are connected by loose junctions allowing easier passage of macromolecules and fluids into the interstitial compartment. The pulmonary endothelium acts not only as a barrier that regulates water, gas, and solutes transport, but it also regulates locally the amount of vasoactive compounds, thereby modulating the composition of arterial blood. The endothelium clears several molecules, including norepinephrine, serotonin, prostaglandins (E and F), adenine nucleotides, and hormones. At the same time, it is also able to modify and release drugs and metabolites accumulated and certain prostaglandins [5]. Proper respiratory function depends on the appropriate interaction between all types of cellular components that contribute to maintaining adult pulmonary homeostasis. This crosstalk is achieved through numerous signals; among them, growth factors stand out as critical for this process.

13.2 Growth Factors Growth factors are secreted proteins that regulate several cellular processes as, for instance, cell proliferation, differentiation, migration, and survival. These signaling molecules may act in an autocrine and/or paracrine fashion and rely on the presence of specific transmembrane receptors, which activate intracellular signal transduction cascades that convey the appropriate cellular responses. Growth factors are essential for embryonic development and also for adult tissue homeostasis.

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13.2.1 Sonic Hedgehog The Hedgehog (HH) signaling pathway is crucial for embryogenesis, namely cell fate specification and differentiation, stem cell renewal, and tissue homeostasis in the adult [14, 15]. Canonical HH signaling comprises three ligands: Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH) that display different spatiotemporal expression patterns. Succinctly, the ligands recognize a specific transmembrane receptor, Patched (PTCH), that reliefs its suppressor effect on the activity of Smoothened (SMO); in this scenario, SMO proteins facilitate the activation of GLI transcription factors that, subsequently, activate the transcription of HH target genes [15]. During early lung development, the components of HH signaling are differentially expressed in the epithelium and mesenchyme of the developing lung, thus regulating epithelial-mesenchymal interactions and, consequently, lung branching and mesenchymal proliferation [16]. In the adult lung, SHH is expressed in the club epithelial cells in the proximal airway, with dispersed expression in the ciliated epithelium and the alveolar type 2 epithelial cells [17]. Conversely, GLI1-positive cells are present in the interstitial space around the large airways and vessels in the normal adult mouse lung, and virtually absent from the alveolar septa. GLI1-positive cells are typically fibroblasts and do not contribute to the smooth muscle lineage [17, 18]. It has been shown that SHH paracrine signaling actively contributes to maintaining proximal epithelial and mesenchymal quiescence in the adult lung; normal homeostasis requires an active SHH pathway to prevent mesenchymal growth [17]. On the other hand, low SHH signaling in distal airways contributes to alveolar regeneration [17]. In the disease context, SHH signaling may be reactivated upon injury, promoting mesenchymal expansion and, consequently, impairing epithelial cells. In fact, in mouse models of acute or chronic lung injury, it has been reported that SHH signaling is impaired, thus triggering anomalous repair and regeneration of the lung [17–19].

13.2.2 Fibroblast Growth Factor Fibroblast growth factors (FGFs) are a family of secreted proteins that have numerous biological roles during embryonic development and also in the adult organism. FGFs bind to specific tyrosine kinase receptors (FGFR1–4) that display distinct ligand specificity. Upon ligand binding, a signal transduction cascade is activated, ultimately promoting the expression of FGF-target genes as, for instance, spry2, involved in cell growth, migration, and differentiation [20]. Of all 22 members, the most significantly expressed FGFs, in the adult mouse lung, are FGF1, 2, 7, 10, and 18 [21]. However, only FGF7 and 10 have been described as playing a role in lung homeostasis until now. FGF7 is expressed in a mesenchymal alveolar niche cell and promotes alveolar type 2 cell self-renewal and alveolar growth [22]. In the adult lung, FGF10 is expressed in the intercartilage

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mesenchyme of the upper conducting airways where basal cells normally reside, and in the lipofibroblasts (LIFs) adjacent to alveolar type 2 cells [23]. FGF10 positive cells are mediators of epithelial stem cell growth and are inhibited by TGFβ signaling [24]. In the upper conducting airways, mesenchymal FGF10 expression is a consequence of Hippo signaling downregulation; the presence of an active FGF10-FGFR2b axis in this cell population contributes to maintaining BC stem cell state by reducing its proliferation. Conversely, Hippo activation in airway epithelial cells inhibits FGF10 expression in airway smooth muscle cells (ASMC) and, as a result, conducting airway epithelial cells retain its dormant state [25]. Regarding the receptors, FGFR1–4 are widely expressed in the adult lung [21]. FGFR1 and 2 are required for normal tracheal homeostasis [26, 27]. It has been demonstrated that there is an FGFR1-SPRY2 axis that negatively regulates BC proliferation by inhibiting intracellular signaling cascades [26]. Furthermore, it has been shown that FGFR2 is expressed in the airway BC and at the apical surface of secretory cells. In this case, FGFR2 signaling is needed for BC asymmetric self-renewal and differentiation by sustaining SOX2 expression [27]. It seems that FGFR1 and FGFR2 signaling display different roles in adult lung homeostasis.

13.2.3 WNT Canonical WNT/β-catenin signaling pathway is required for proper embryo development and adult tissue homeostasis. There are 19 WNT secreted ligands that bind to specific transmembrane receptors (Frizzled and LRPs) on the adjacent cells. In these circumstances, β-catenin is stabilized, accumulates in the cytoplasm, and is then translocated to the nucleus to elicit the transcription of WNT target genes as, for example, axin2. In the absence of WNT ligands, β-catenin is targeted for destruction in the cytoplasm [28]. WNT/β-catenin machinery is expressed in the adult lung. For instance, WNT1 is present in the bronchial and alveolar epithelium, and the pulmonary endothelial and smooth muscle cells. WNT-3a is expressed in the ciliated airway epithelial cells and alveolar epithelial type 2 cells [29]. WNT2 is detected in the alveolar region of the lung [22]. Additionally, WNT7b is present in the airway epithelial compartment. In the cartilaginous airways, WNT7b expressed by basal cells induces mesenchymal FGF10 expression assuring basal cell maintenance (please refer to the previous section). In the conducting airways, the absence of WNT7b (due to Hippo activation) impairs FGF10 expression and, consequently, quiescence of the airway epithelial cells is maintained [25]. It has also been described that secreted frizzled-related protein 1 (SFRP1), a WNT signaling inhibitor, can maintain bronchoalveolar stem cells in their undifferentiated state. Finally, it has also been shown in club cells that β-catenin may be involved in regulating differentiation and stem cell pools [30, 31]. AXIN2+ cells are located all over the pulmonary mesenchyme, which suggests that WNT signaling must be relevant for adult lung homeostasis. Recently, an unusual

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population of AEC2-AXIN2 positive has been identified and characterized as displaying stem cell activity. These cells respond to WNT5a secreted by neighboring fibroblasts, activating WNT canonical response and, consequently, repressing transdifferentiation into AEC1 cells and maintaining stem cell phenotype [32, 33].

13.2.4 Retinoic Acid Dietary vitamin A is converted, through sequential enzymatic reactions, first into retinol and then into Retinoic Acid (RA). RA binds to specific nuclear receptors (RAR-RXR) that recognize particular promoter sequences, thus modulating the expression of RA-target genes and, therefore, regulating distinct cellular processes [34]. In the adult, serum retinol levels correlate with a healthy lung function [35]. Moreover, a single-nucleotide polymorphism in RARβ (rs1529672) has been associated with impaired lung function [36]. These studies point towards a role for retinoic acid signaling in the adult lung. It has been shown that RA signaling machinery is expressed in discrete lung cell populations; for instance, RARs and CYP26A1 (a negative regulator of RA signaling) are present in the microvasculature surrounding the alveolar walls, whereas RALDH1 (an enzyme involved in RA intracellular synthesis) is detected in fibroblasts. Additionally, RA stimulates lung microvascular angiogenesis by promoting the expression of vascular endothelial growth factor A (VEGFA) and its receptor VEGFR2, thus contributing to the preservation of microvascular endothelium [37]. Moreover, it has been suggested that, in the adult lung, RA signaling balances lung epithelial cell proliferation vs differentiation. In lung organoids, retinoic acid inhibition, downstream-mediated by Hippo and FGFFGFR2b signaling, triggered proliferation in detriment of differentiation; on the other hand, retinoic acid activation impeded the proliferation of adult distal lung epithelial progenitor cells, likely through the repression of FGF7 and FGF10 expression in fibroblasts [38]. Furthermore, tonic RA signaling actively maintains adult airway smooth muscle homeostasis by repressing the TGFβ pathway [39].

13.2.5 Transforming Growth Factor β—Bone Morphogenetic Protein The transforming growth factor-β (TGFβ) family comprises several ligands such as TGFβ1, TGFβ2, TGFβ3, and bone morphogenetic proteins (BMPs). Briefly, ligands bind and signal through cell surface serine-threonine kinases receptors that regulate the phosphorylation status of SMADs that, ultimately, are the main signaling effectors of the pathway [40]. TGFβ controls numerous cellular events such as cell

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proliferation, differentiation, and it is a potent inducer of epithelial-mesenchymal transitions; for this reason, its levels must be tightly regulated in tissues. In the adult human lung, TGFβ signaling machinery is detected in the bronchial epithelium, alveolar macrophages, mesenchymal, endothelial, and airway smooth muscle cells [41, 42]. Alveolar type 2 epithelial cells are characterized by their ability of self-renewing and differentiating in type 1 cells. It has been shown, in vitro, that TGFβ inhibits AEC2 proliferation [43] and promotes transdifferentiation of AEC2 into AEC1; this means that, in the steady-state lung, this mechanism must be intrinsically inhibited so that AEC2 phenotype is preserved [44]. In fact, TGFβ decreases the expression of AEC2 markers such as SP-A, SP-B, SP-C, fatty acid synthase, and the phospholipid transporter ABCA3 [44, 45]. It has been revealed that, in vitro, BMP signaling inhibits AEC2 transdifferentiation. This finding is not surprising, taking into consideration the recognized crosstalk between members of the TGFβ/BMP superfamily in controlling diverse cellular events [40]. It seems that the equilibrium between TGFβ and BMP signaling contributes to maintaining AECs homeostasis in the normal adult lung. Regarding BMP signaling, it has been reported the existence of a gradient of BMP signaling along the airways. This study revealed that the BMP signaling pathway is active in the trachea, conducting airways and distal bronchioles. However, the expression of pSMAD 1/5/8, a readout of BMP signaling activity, was significantly higher in the trachea when compared to the other two compartments [46]. Probably, basal BMP activity is important for sustaining healthy airways. In fact, it has been shown that BMP4 expression in the mesenchyme adjacent to the mucociliary airway epithelium negatively controls basal cell proliferation, thus contributing to its quiescent state [47]. Likewise, in the alveolar niche, pSMAD1/5/8-dependent signaling is elevated, contributing to maintaining AEC2 quiescence and identity at steadystate [48]. Conversely, Zepp et al. [22] showed that mesenchymal alveolar niche cell lineage expresses GREM2 (a BMP inhibitor) and demonstrated that BMP treatment decreases proliferation and differentiation of AEC2 [22]. These contradictory results may be explained by differences in the cell population analyzed.

13.2.6 Vascular Endothelial Growth Factor Vascular Endothelial Growth Factor (VEGF) family of proteins are not only known to regulate angiogenesis, permeability, and mitogenesis but also to control apoptosis, cell communication, and cell plasticity. VEGF bind to specific tyrosine kinases receptors (VEGFR1–3) and specific co-receptors, namely, to neuropilin (NRP) family members and heparan sulfate proteoglycans. Upon binding, a phosphorylation cascade is responsible for initiating intracellular signaling pathways that mediate VEGF actions [49]. The VEGF signaling machinery is present in the adult lung. VEGFA is abundantly expressed in endothelial cells, lung macrophages, smooth muscle cells, and alveolar epithelial type 2 cells. However, AEC2 are the major source of VEGFA,

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and, in these cells, VEGFA alveolar protein levels are exceptionally elevated [50]. VEGFR1 and VEGFR2 are present in lung endothelial cells; furthermore, they are also expressed in the epithelial compartment and alveolar macrophages (VEGFR1) and AEC2 (VEGFR2) [51]. Despite the elevated levels of the VEGF, the classical actions of this growth factor are virtually absent in the steady-state lung, pointing to a different role in adult lung homeostasis [50]. VEGF acts as a growth and anti-apoptotic factor for both endothelial and alveolar epithelial cells contributing to maintaining the alveolar structure [52, 53]. Moreover, in alveolar macrophages, the VEGF-VEGFR1 signaling axis enhances macrophage clearance of apoptotic cells hence promoting tissue structure maintenance [54].

13.2.7 Platelet Derived Growth Factor The platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) family contributes to a variety of cellular events, like proliferation, migration, differentiation, and survival in various cell types. Once PDGF(A–D) binds the tyrosine-kinase receptor PDGFR(α-β), it activates intracellular signaling transduction pathways that, ultimately, convey the corresponding effect [55]. In the adult lung, PDGFA is produced by the epithelium. Conversely, PDGFRα is expressed in mesenchymal stromal cells, among them LIFs; it has been revealed that this cell population exists in the vicinity of alveolar type 2 cells and contributes to maintaining their proliferation and differentiation [56]. Concurrently, it has been demonstrated that mesenchymal AXIN2+ cells in the alveolar region express PDGFRα, whereas the ones adjacent to the airways express PDGFRβ. Moreover, WNT2+ positive cells express only PDGFRα. The combination of these signaling molecules contributes to the identity of distal vs proximal lung and favors AEC2 cell stemness [22].

13.3 Conclusion Active crosstalk between epithelial and mesenchymal compartments during lung development is well recognized. In the adult lung, the members of growth factors signaling machinery are differentially expressed in both epithelial and mesenchymal cells, mediating paracrine and autocrine regulatory mechanisms that overall contribute to lung tissue homeostasis. Moreover, the unique combination of growth factors, together with the activation status of each pathway is crucial for the identity and fate of a particular cell type. Table 13.1 compiles the contribution of active growth factor signaling to adult lung homeostasis and clearly illustrates that epitheliummesenchyme interactions are key to maintain a healthy organ. The knowledge of the mechanisms underlying progenitor’s cell quiescence is crucial to better understand and, eventually, manipulate repair and regeneration processes.

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Table 13.1 Growth factor signaling underlying adult lung homeostasis. AEC1, alveolar epithelial type 1 cells; AEC2, alveolar epithelial type 2 cells; ASMC, airway smooth muscle cells; BC, basal cells; LIF, lipofibroblasts; (++), high expression; (+), expression; (–), no expression Growth Factor

Signaling member

Expression site

Function/target tissue and/or cell type

SHH

SHH

Proximal airways: club cells

GLI

Large airways: interstitial space

Prevents mesenchymal growth, maintaining mesenchyme quiescence

FGF7

Mesenchymal alveolar niche

Promotes AEC2 self-renewal and growth

FGF10

Upper airways: mesenchyme (++)

Reduces BC proliferation (via Hippo inhibition)

Conducting airways: mesenchyme (–)

Maintains airway epithelial cells dormant (via Hippo activation)

Basal cells

Inhibit BC proliferation

FGF

FGFR1 FGFR2

WNT

RA

TGFβ-BMP

Sustains BC asymmetrical self-renewal and differentiation

WNT2

Mesenchymal alveolar niche

–

WNT7b

Upper airways: basal cells (++)

Basal cell maintenance (via FGF10)

Conducting airways (–)

Maintains airway epithelial cells dormant

WNT5a

Fibroblast adjacent to AEC2

AXIN2

AEC2 (rare)

Represses AEC2 transdifferentiation into AEC1

Pulmonary mesenchyme

–

CYP26A1

Microvasculature adjacent to alveoli

RALDH1

Fibroblasts

Preserves microvascular endothelium via VEGFA/VEGFR2; balances proliferation/differentiation of distal epithelial cells; maintains ASMC homeostasis (via TGFβ)

TGFβ

ASMC, mesenchymal cells

RARs

AECs BMP4

Promotes AEC2 transdifferentiation Inhibits AEC2 transdifferentiation (continued)

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Table 13.1 (continued) Growth Factor

VEGF

PDGF

Signaling member

Expression site

Function/target tissue and/or cell type

Tracheal mesenchyme (++)

Promotes BC quiescence

Alveolar niche (+)

Promotes AEC2 quiescence + transdifferentiation

VEGFA

Endothelial cells, alveolar macrophages, AEC2

Anti-apoptotic factor for AEC2 and endothelial cells

VEGFR1

Endothelial cells, alveolar macrophages

Enhances clearance of apoptotic cells by macrophages

VEGFR2

Endothelial cells, AEC2

–

PDGFA

Epithelium

–

PDGFRα

Mesenchymal cells adjacent to AEC2 (LIF)

Maintain AEC2 proliferation/differentiation state

Acknowledgments This work has been funded by FEDER through the Competitiveness Factors Operational Programme (COMPETE), by National funds through the Foundation for Science and Technology (FCT) under the scope of the project UID/Multi/50026/2019; and by the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER).

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Chapter 14

The Signaling Pathways Involved in the Regulation of Skeletal Muscle Plasticity Alexandra Moreira-Pais, Francisco Amado, Rui Vitorino, Hans-Joachim Appell Coriolano, José Alberto Duarte, and Rita Ferreira Abstract Signal transduction is essential for skeletal muscle functionality, a highly plastic tissue able to respond to mechanical and chemical stimuli by altering its metabolism, size and myogenic status. Several signaling pathways involving membrane and nuclear receptors, and secondary messengers as Ca2+ , cAMP, inositol triphosphate (IP3) and diacylglycerol (DAG), contribute to such plasticity. The contribution of these transduction systems to muscle remodeling is well characterized; however, their input depends on several issues such as type of muscle (slow-twitch or fast-twitch, postural or locomotor), gender, age, muscle performance, duration and intensity of stimulus exposure, among others. All these issues together with the crosstalk between cell signaling pathways make difficult to correlate a unique signaling pathway or molecular player to a specific effect on muscle remodeling. This chapter overviews the mechanical and chemical transduction pathways, and the molecular players involved in the regulation of skeletal muscle mass, contractile activity and metabolism.

J. A. Duarte and R. Ferreira—Equally supervisors. A. Moreira-Pais · J. A. Duarte Faculty of Sport, CIAFEL, University of Porto, Porto, Portugal e-mail: [email protected] J. A. Duarte e-mail: [email protected] F. Amado · R. Ferreira (B) QOPNA & LAQV, Department of Chemistry, University of Aveiro, Aveiro, Portugal e-mail: [email protected] F. Amado e-mail: [email protected] R. Vitorino Department of Medical Sciences, iBiMED, University of Aveiro, Aveiro, Portugal e-mail: [email protected] H.-J. A. Coriolano Physiology and Anatomy, German Sport University, Cologne, Germany e-mail: [email protected] © Springer Nature Switzerland AG 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_14

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Keywords Mechano-transduction · Neurocrine signaling · Calcium signaling · Endocrine and paracrine players

Abbreviations 4E-BP1 AC Ach ActRIIB ADP ALK AMP AMPK AP1 aPKC AR ASK ATF ATP BCL2L11 BDNF CaM CaMKs cAMP Cn CSQ Cx DAG DHPRs DNA Dvl E1s E2s ECM eIF eNOS ER FAK FAs FGF FNDC5 FoxO Fzd

eukaryotic initiation factor 4E-binding protein adenylate cyclase acetylcholine activin receptor type IIB adenosine diphosphate activin-like kinase adenosine monophosphate 5’-AMP-activated protein kinase activator protein-1 atypical protein kinase C androgen receptor apoptosis signal-regulating kinase activating transcription factor adenosine triphosphate Bcl-2-like protein 11 brain-derived neurotrophic factor calmodulin calmodulin-dependent kinases cyclic adenosine monophosphate calcineurin calsequestrin connexin diacylglycerol dihydropyridine receptors deoxyribonucleic acid Dishevelled ubiquitin-activating enzymes ubiquitin-carrier or conjugating proteins extracellular matrix eukaryotic tranlation initiator factor endotelial nitric oxide synthase estrogen receptor focal adhesion kinase focal adhesions fibroblast growth factor fibronectin type III domain-containing 5 Forkhead box O Frizzled

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GAPDH GDF GDNF GH GHR GLUT4 GSK-3 HSF HSPs IGF-1 IGF-1R IGFBP IκBs IKK IL IL-1R IP3 IR IRS JAK JNK LEF LRP MAFbx MAPK MCD MEF2 MHCIIA MnSOD Mstn mTOR mTORC MuRF1 Myf5 MyoD nAChR NADH NFAT NF-κB • NO NOS NRF2 p70S6K

385

glyceraldehyde 3-phosphate dehydrogenase growth differentiation factor glial cell line-derived neurotrophic factor growth hormone growth hormone receptor glucose transporter type 4 glycogen synthase kinase 3 heat shock factor heat shock proteins insulin-like growth factor 1 insulin-like growth factor 1 receptor insulin-like growth-binding protein inhibitors of nuclear factor kappa-light chain-enhancer of activated B cells inhibitors of nuclear factor kappa-light chain-enhancer of activated B cells kinase interleukin interleukin 1 receptor inositol triphosphate insulin receptor insulin receptor substrate Janus kinase c-Jun N-terminal kinase lymphoid enhancer-binding factor low density lipoprotein-receptor-related proteins muscle atrophy F-box mitogen-activated protein kinase malonyl coenzyme A decarboxylase myocyte enhancer factor 2 type IIA myosin heavy chain manganese superoxide dismutase myostatin mammalian target of rapamycin mammalian target complex muslce-specific RING-finger 1 myogenic factor 5 myoblast determination protein nicotinic acetylcholine receptors nicotinamide adenine dinucleotide nuclear factor of activated T-cells nuclear factor kappa light-chain-enhancer of activated B cells nitric oxide nitric oxide synthases nuclear factor erythroid 2-related factor 2 ribosomal protein S6 kinase beta-1

386

PCP PDK-1 PGC-1 PI3K PIP2 PIP3 PKA PKB PLC PP1 PPAR PTEN RAPTOR RICTOR ROS RPS6 RyR1 S6K1 SERCA SH2 SIRT SMN SR STAT TCF TFAM TGF TNF-α TNF-αR UCP UPP

A. Moreira-Pais et al.

planar cell polarity 3-phosphoinositide-dependent protein kinases-1 peroxisome proliferator-activated receptor gamma coactivator 1 phosphatidylinositol-4,5-biphosphate 3-kinase phosphatidylinositol 4,5-biphosphate phosphatidylinositol-3,4,5 triphosphate cAMP-dependent kinases protein kinase B phospholipase C phosphoprotein phosphatase 1 proliferator-activated receptor phosphatase and tensin homologue deleted on chromosome 10 regulatory-associated protein of mammalian target of rapamycin rapamycin-insensitive companion of mammalian target of rapamycin reactive oxygen species ribosomal protein S6 ryanodine receptor p70 ribosomal S6 kinase sarco/endoplasmic reticulum Ca2+-ATPase Src Homology 2 sirtuin spinal motor neuron sarcoplasmic reticulum signal transducers and activators of transcription T-cell specific transcription factor mitochondrial transcription factor A transforming growth factor tumor necrosis factor alpha tumor necrosis factor alpha receptor uncoupling protein ubiquitin-proteasome pathway

14.1 Introduction The human organism comprises around 640 muscles, which accounts for approximately 40–45% of total body weight. These muscles are not only responsible for voluntary movement and maintenance of body posture but also play a crucial role in the delimitation of anatomical cavities, in vascular protection, in heat generation and in the control of whole-body metabolism [1–3]. Each muscle is formed by tens to hundreds of motor units, which comprises a motor neuron and a bunch of muscle fibers. The heterogeneity of muscle fibers in respect to size, metabolism and

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contractile function is the basis of skeletal muscle plasticity [4]. The term plasticity of muscle was developed during the preparation of an international symposium held at the University of Konstanz in 1979 [5]. Skeletal muscle plasticity refers not only to the integrated response of fibers or motor units to several stimuli but also of fiber-associated structures such as capillaries and nerves [6]. These muscle structures can change their properties in response to distinct stimuli such as innervation, load, hormones and modulation of neural input [7]. Myofibers are classified based on the expression of specific myosin heavy chain isoforms into type I, type IIa, type IId/x, and type IIb fibers. Type I and IIa fibers exhibit oxidative metabolism whereas types IIx and IIb are primarily glycolytic fibers [8, 9]. Oxidative or, with respect to their type of contraction, also named slow-twitch myofibers are rich in mitochondria, are surrounded by more capillaries, and present a low velocity of shortening and a high resistance to fatigue. These myofibers are mostly required for the maintenance of posture and tasks involving endurance. Glycolytic or fast-twitch myofibers fatigue rapidly, being mostly required for movements involving strength and power [2, 10, 11]. The combinations of specific molecular and functional properties seem to reflect the match between energy production and energy consumption to cope the constraints imposed by electrical and mechanical influences [4]. Moreover, in response to overload or unloading and to chemical stimuli, myofibers can be transformed, resembling the so-called fiber type transitions from one type of myofiber to another, with functional and structural repercussions. Multiple signaling pathways are involved in the mediation of skeletal muscle ability to sense chemical and mechanical signals and to convert them into biochemical events, leading to alterations in muscle mass, contractile properties and metabolic status [2, 12]. This chapter focuses on the signaling pathways underlying skeletal muscle plasticity and its functional outcomes.

14.2 Mechanical and Chemical Transduction 14.2.1 Mechanical Signaling Transduction Skeletal muscle mass and metabolism are highly dependent on mechanical stress. Alterations in mechanical stress are responsible for changes in the multiprotein complexes (the costameres) involved in mechano-transduction and mechano-sensing. In this set, focal adhesions (FAs) play a critical role in the integration of mechanical signals into molecular events [12]. FAs are integrin-containing complexes with numerous adapter or anchor proteins, which make the link between the cytoskeleton and the extracellular matrix (ECM). Integrins are the major transmembrane ECM receptors that are associated, via their cytoplasmic domains, with several proteins such as vinculin, talin, paxillin and tensin [13]. Among these, vinculin provides a structural connection between actin filaments and FAs on the membrane [13]. Integrins are also associated to signalling molecules as kinases and phosphatases.

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Fig. 14.1 Signaling pathways involved in (A) mechanical and (B) neurocrine transduction. (A) Focal adhesions, which comprehend integrin-containing complexes and small GTPases (Rac1, RhoA) mediate muscle fiber response to mechanical stress. (B) Neurocrine signaling is mediated by the release of acetylcholine (Ach) from spinal motor neuron and its binding to nicotinic acetylcholine receptors, leading to the influx of Na+ and Ca2+ and the subsequent membrane depolarization, RyR1 opening and Ca2+ release from sarcoplasmic reticulum. Ca2+ binds to troponin C and promotes contraction (Figure was made with Servier Medical Art)

Among these, focal adhesion kinase (FAK) plays a critical role in integrating the mechano-sensing function of costamere complexes, particularly at phosphatidylinositol 4,5-bisphosphate (PIP2)-enriched membranes of FAs, into downstream activation of signalling pathways [14]. Indeed, the activation of this tyrosine kinase leads to the recruitment of regulatory proteins of the small GTPases family (Rac1, RhoA), which are involved in FAs assembly and in the regulation of a variety of cellular events related to the cytoskeletal system, and cell survival [13] (Fig. 14.1). FAK activity is regulated by phosphatase and tensin homologue found on chromosome 10 (PTEN), which also mediate mechanical transduction. PTEN not only dephosphorylates FAK but also phosphatidylinositol-3,4,5 triphosphate (PIP3), antagonistic to phosphatidylinositol-4,5-biphosphate 3-kinase (PI3K), suggesting that PI3K and integrins cooperate. PTEN also acts as a negative regulator of mechanically-induced mitogen-activated protein kinase (MAPK) [15]. Mechanical forces can also lead to the activation of growth factors and hormone receptors even in the absence of the ligand. One of the most well-known examples is the stretch- and contraction-induced glucose uptake. Two mechanisms were proposed to explain mechanical stress-induced glucose transport: (i) the ‘feed-forward mechanism’, a calcium-dependent mechanism, which involves the depolarization of the plasma and transverse (T)-tubule membranes with the subsequent release of sarcoplasmic reticulum (SR) Ca2+ , and the glucose transporter glucose transporter type

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4 (GLUT4) translocation; (ii) the ‘feedback mechanism’, or load-dependent mechanism, relies on the strain put on the muscle or on the force developed by the muscle [16]. During contraction, metabolic by-products accumulate, and reactive oxygen species (ROS) are produced. ROS might act as secondary messenger in the regulation of stretch-induced glucose uptake, a signalling cascade that seems to involve p38 MAPK [17]. Taken together, several signalling cascades can be considered downstream effectors of mechano-transduction in skeletal muscle, including PI3K/Akt (or protein kinase B (PKB)), MAPKs, Ca2+ via calmodulin (CaM) and calcineurin (Cn), and 5 -AMP-activated protein kinase (AMPK). These signalling cascades are explored in more detail in Sect. 14.3.

14.2.2 Chemical Signalling Transduction 14.2.2.1

Neurocrine Signalling

The flow of information between the nerve and the muscle is mediated by acetylcholine (ACh). This neurotransmitter is released from the axon terminal of the spinal motor neuron (SMN), diffuses across the synaptic cleft and binds to nicotinic acetylcholine receptors (nAChR) located in the muscle cell membrane [2, 20]. To ensure efficient neuromuscular signal transmission, dense nAChR clusters must be compactly formed, to which SMN- and muscle-derived molecules contribute. Among SMN-derived molecules involved in the regulation of nAChR clustering and neuron motor junction formation are neuregulin-1 and ACh, and among muscle-derived regulators are laminins, fibroblast growth factors (FGFs), collagens, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), Wnts, and transforming growth factor (TGF)-β [21]. The activation of nAChR prompts the influx of the cations Na+ and Ca2+ , causing membrane depolarization. Consequently, voltage-gated Na+ channels located at the fiber membrane are activated, thereby generating an action potential [20]. The depolarization of the membrane reaches the transverse (T)-tubular membrane, and L-type voltage-gated calcium channels in the T-tubules are activated. These channels undergo a conformational change that enables their interaction with a skeletal muscle-specific SR calcium-release channel, the ryanodine receptor (RyR)1. RyR1 opens and releases Ca2+ from SR. Ca2+ then binds to troponin C on actin filaments and initiates contraction [2] (Fig. 14.1). The changes in cytoplasmic Ca2+ content promoted by nAChR activation not only determine muscle contraction but also activate signalling pathways that regulate muscle plasticity. Muscle fiber type transitions from one type of myofiber to another is accompanied by differences in Ca2+ -mediated regulation, which reflect distinct calcium sequestering and buffering systems. For instance, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) 2A is the main SERCA isoform in slow-twitch fibers whereas SERCA1A is the isoform expressed by fast-twitch fibers [20]. Two isoforms of calsequestrin (CSQ), the main calcium buffering system, are found in

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slow-twitch fibers, CSQ1 and CSQ2, whereas only CSQ1 is expressed by fast-twitch ones. The buffering capacity of both isoforms is different through the regulation of RyR activity. CSQ1 inhibits the activity of RyR1 whereas CSQ2 increases the open probability of both RyR1 and RyR2 [22].

14.2.2.2

Endocrine Signalling

Hormones are important regulators of muscle mass and metabolism. Growth hormone (GH), insulin-like growth factor 1 (IGF-1) and insulin are some examples of hormones with a key role in the regulation of muscle mass [23]. Among the main effects of GH on skeletal muscle are enhanced amino acid uptake to support protein synthesis [24], and the fusion of myoblasts with nascent myotubes [25]. IGF-1 has been associated with skeletal muscle hypertrophy [26]. Similar to all tissues of the body, skeletal muscles express GH receptor (GHR), which dimerizes upon GH binding with the concomitant activation of intracellular signalling cascades involving the Janus kinase (JAK) and the signal transducers and activators of transcription (STAT) pathway (Fig. 14.2), likewise to most cytokine receptors [23, 27]. GHR activation also stimulates tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins IRS-1 and IRS-2, followed by PI3K and/or MAPK activation. The activation of the IGF-1 receptor (IGF-1R) also induces the phosphorylation of the IRS proteins, highlighting a cross-talk between GH and IGF-1 signalling [27] (Fig. 14.2). Some years ago, the somatomedin hypothesis was raised based on the assumption that the anabolic action of GH is indirect, being mediated by circulating IGF-1, mainly derived from the liver [24]. However, the effects of GH can be independent of the circulating form of IGF-1, and therefore, an alternative viewpoint to the original somatomedin hypothesis was proposed [27]. Skeletal muscle is the primary target of insulin-stimulated glucose uptake in the postprandial state and a major tissue for glucose utilization [28, 29]. Unlike IGF1 that circulates bound to IGF-binding protein (IGFBP)3, insulin circulates in an unbound form [24]. When this hormone reaches the fiber membrane, insulin binds to its receptor (IR), a α2β2 heterodimeric transmembrane protein with intrinsic tyrosine kinase activity, promoting its autophosphorylation and the activation of receptor tyrosine kinase. Consequently, IRS-1 is recruited and activated, then interacting with PI3K via Src Homology 2 (SH2) domains. This kinase phosphorylates PIP2 that is converted into PIP3, leading to the activation of the 3-phosphoinositide-dependent protein kinases-1 (PDK-1), which in turn activates PKB and the atypical protein kinase C λ and ζ (aPKC λ/ζ). Both these kinases are involved in the regulation of GLUT4-rich vesicles translocation to the sarcolemma through the remodelling of the sub-membrane actin mesh or the activation of Rab-GTPase-activating proteins TBC1D4 (also known as AS160) and TBC1D1, allowing for glucose uptake [28, 30]. PKB phosphorylates, and subsequently, inhibits glycogen synthase kinase 3 (GSK3), allowing phosphoprotein phosphatase 1 (PP1) dephosphorylation, thus activating glycogen synthesis. PKB also induces protein synthesis via mammalian target of rapamycin (mTOR) and downstream players [30] (Fig. 14.2). These are some of the

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Fig. 14.2 Overview of the major signaling pathways involved in skeletal muscle plasticity. Upon stimulation, epinephrine is released and binds to the β2-adrenergic receptor coupled to stimulatory G proteins, resulting in the activation of AC. Then, ATP is converted into cAMP, which modulates Ca2+ release from the SR, increasing the contractile force. Moreover, cAMP activates PKA that phosphorylates the Na+ /K+ pump, culminating in the restoration of muscle excitability. In the canonical pathway, Wnt ligands bind to the Fzd receptors, leading to Dvl recruitment, which inhibits GSK3β. β-catenin is translocated to the nucleus, inducing Wnt target genes expression. The noncanonical pathways include the Wnt/PCP and Wnt/Ca2+ signals, leading to the activation of Rho and Rac, and to the stimulation of trimeric G proteins, respectively. IP3 and DAG promote the intracellular increase of Ca2+ concentration. Skeletal muscle mass is also regulated by sex steroids through their receptors. AR and ERs enhance IGF-1 expression and Akt/mTOR signaling, thus increasing protein synthesis. IGF-1 can activate the IRS1/PI3K/Akt signaling. On the one hand, Akt blocks the repression of mTOR, leading to protein synthesis. GH binds to GHR, leading to its dimerization and activation of intracellular cascades involving the JAK/STAT pathway. In response to cytokines such as TNF-α and IL-1, occurs the activation of the IKK complex that phosphorylates the NF-κB-bound IκBαs, which are targeted for ubiquitination, followed by degradation by the proteasome. ROS can also upregulate the NF-κB pathway and activate the p38 MAPK signaling, that has as target the p53, leading to enhancement of MAFbx. Additionally, ROS can activate JNK through the ASK1 pathway, leading to oxidative-stress mediated apoptosis. In addition, ROS can inhibit calcineurin. Myostatin binds to the ActRIIB, activating the ALK-4 or ALK-5, which in turn, phosphorylates and activates Smad2 and Smad3, which may translocate to the nucleus and activate the transcription of target genes. AMPK activates eNOS, thus increasing the endogenous production of • NO, which in turn, increases PGC-1α expression. In addition, • NO can activate AMPK, leading to a positive feedback mechanism. SIRT1 can activate PGC-1α, FoxO and upregulate the eNOS activity (Figure was made with Servier Medical Art)

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hundreds of molecules known to be involved in the insulin-signalling network, being probably the best characterized signalling players. Skeletal muscle fibers are responsive to catecholamines, which are well known for their role in the body’s adaptive response to a variety of stressors. The main catecholamines, adrenaline (or epinephrine) and noradrenaline (or norepinephrine), are synthetized from tyrosine at the sympathetic nervous fiber extremities (in the case of noradrenaline) and at chromaffin cells of the adrenal medulla (for both adrenaline and noradrenaline) [31]. The contribution of the neural and hormonal components to the regulation of catecholamine-mediated processes depends on the stimulus [32, 33]. For instance, the release of adrenaline from the adrenal medulla may be important to counteract proteolysis in fast-twitch skeletal muscles during long-term fasting. This anti-proteolytic effect on the ubiquitin proteasome and lysosomal systems seems to involve the activation of the cyclic adenosine monophosphate (cAMP)-Aktdependent pathway [34]. Norepinephrine increases glucose uptake in skeletal muscle without an increase in plasma insulin concentration, whereas epinephrine decreases glucose transport possibly by changing GLUT4 activity [29]. These hormones act in skeletal muscle through beta-adrenergic signalling that involves β2-adrenergic receptors coupled to stimulatory G proteins. Once activated, the Gαs subunit is released from its complex with Gγβs and then interacts and activates adenylate cyclase (AC). This enzyme converts ATP into cAMP, which modulates Ca2+ release from the SR, increasing the contractile force [35]. This effect seems to involve the phosphorylation of L-type voltage-dependent Ca2+ channels. cAMP induces the activation of several cAMP-dependent kinases (such as PKA) that, in turn, phosphorylate serine residues in several protein targets, among which the Na+ /K+ pump. The activation of this pump is important in membrane hyperpolarization and in the restoration of muscle excitability. Glycogenolysis is also stimulated by PKA, supporting the energetic needs imposed by muscle contraction [33, 35] (Fig. 14.2). Skeletal muscle mass is regulated by sex steroids in a hormone-specific way, being responsible for the sexual dimorphism underlying muscle plasticity. Both androgens and estrogens, promote the growth and maintenance of muscle mass and strength and exert beneficial metabolic effects. The effects of these hormones are additive and, eventually, synergistic to the effects of anabolic factors as IGF-1 and of mechanical loading [36, 37]. However, there are differences between the effects of androgens and estrogens on skeletal muscle. Testosterone or dihydrotestosterone are more potent than estrogens in the regulation of muscle homeostasis in both males and females. Differences are explained, at least in part, by a sex-specific receptors expression profile [37]. The receptors for these hormones are expressed in the different cell types of skeletal muscle. The androgen receptor (AR) is expressed in satellite cells, myocytes, and fibroblasts. The AR expression in muscle fibers is upregulated by androgens. The deletion of AR causes a decrease of muscle mass in males but not in females [38]. Estrogen receptors (ER), ERα and ERβ, are expressed in satellite cells, myofibers and endothelial cells of both, females and males [39]; however, their expression pattern differs between sexes [40]. ERβ deletion increases male skeletal muscle fatigue properties, but it has no effect in females. ERα, but not ERβ, deletion was shown to decrease the mass and contractile properties of some muscles [37].

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AR and ERs receptors function as transcription factors, regulating gene expression by their direct binding to specific deoxyribonucleic acid (DNA) target sequences. However, non-genomic effects involving the activation of signalling pathways were also reported [41]. Testosterone induces the IGF-1/Akt pathway by upregulating the expression of IGF-1 and Akt/mTOR signaling with the concomitant increase of protein synthesis (Fig. 14.2). Estradiol also activates Akt in myocytes, which among several effects promotes the translocation of GLUT4 to the plasma membrane [37]. Testosterone down-regulates the expression of Forkhead box O (FoxO) with the consequent preservation of muscle proteins [42].

14.2.2.3

Paracrine and Autocrine Signalling

Skeletal muscle is nowadays recognized as an endocrine organ. This organ expresses and releases cytokines and other peptides known as “myokines” that exert paracrine, autocrine, or endocrine effects [43]. Among the myokines studied are myostatin, interleukin (IL)-6, IL-8, IL-15, FGF 12 and irisin. Myostatin, also known as growth differentiation factor (GDF)-8, is a TGF-β ligand that by acting in an autocrine or paracrine manner, negatively regulates the skeletal muscle growth during postnatal development and the skeletal muscle mass in adulthood [44, 45]. Myostatin binds predominantly to the activin receptor type IIB (ActRIIB), activating through phosphorylation the activin type I receptor, activin-like kinase-4 or -5 (ALK-4 or ALK-5) [46, 47]. Consequently, the phosphorylation and activation of the transcription factors Smad2 and Smad3 occur. These transcription factors form a heterodimeric complex with Smad4, which may translocate to the nucleus, activating the transcription of target genes by interacting with DNA and nuclear factors [46] (Fig. 14.2). It has been proposed that myostatin can inhibit the myogenic process through the downregulation of the myogenic differentiation factors myoblast determination protein (MyoD), myogenic factor 5 (Myf5) and myogenin, leading to a decrease in myoblast proliferation [48]. In addition, both activation and self-renewal of satellite cells, are also inhibited by myostatin [46]. This mediator silences the Akt/mTOR signaling by dephosphorylation of serine 473 of Akt, decreasing protein synthesis [46]. Consequently, myostatin enhances protein degradation through activation of the FoxO family of transcription factors, which in turn stimulates the ubiquitin-proteasome mediators, including muscle atrophy F-box (MAFbx)/atrogin-1 [46] (Fig. 14.2). Thus, myostatin is associated with the muscle wasting process [49]. In addition, FoxO upregulates myostatin, leading to a mechanism of positive feedback [46]. Contrarily, this pathway is also controlled by a mechanism of negative feedback through the inhibitory protein Smad7 [46]. IL-6 is secreted by muscle fibers during contraction, enhancing glucose uptake and fatty acid oxidation [43]. IL-6 production and release is also regulated by substrate availability, working as an energy sensor. This myokine regulates myogenesis by promoting myoblast proliferation and/or differentiation. It acts by binding to its membrane-bound receptor (IL-6R; CD126), activating the JAK/STAT pathway. The JAK/STAT1/STAT3 pathway has been directly associated with the proliferation of

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satellite cells and the inhibition of their precocious differentiation [50]. Another myokine upregulated by muscle contraction is irisin. This myokine results from the cleavage of the membrane protein fibronectin type III domain-containing 5 (FNDC5). Irisin was shown to stimulate glucose uptake by muscle fibers through the activation of AMPKα2, a mechanism likely involving p38 MAPK-GLUT4 translocation [51]. An irisin-related increase of fatty acid oxidation also seems to involve AMPK signaling. This myokine was also reported to stimulate mitochondrial biogenesis by regulating the expression of peroxisome proliferator-activated receptor alpha (PPARα) and mitochondrial transcription factor A (TFAM) and of uncoupling protein 3 (UCP3), with the consequent increase in thermogenesis [52]. These are some examples of the complex array of molecular mediators involved in the regulation of skeletal muscle plasticity. In the next section, an in-deep analysis of the intracellular events activated by these molecular players is done, being aware that there is a cross-talk between their effects, which makes it difficult to associate a signaling event to a specific molecular mediator.

14.3 Intracellular Signaling Pathways 14.3.1 Akt/mTOR Pathway The Akt/mTOR pathway promotes protein synthesis and also inhibits protein degradation through the activation of IRS1/PI3K/Akt signaling by the IGF-1 [53]. On the one hand, Akt blocks the repression of mTOR, which leads to protein synthesis by two different multiprotein complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [54]. Basically, the mTORC1, in the presence of the regulatory-associated protein of mTOR (RAPTOR), phosphorylates the p70 ribosomal S6 kinase 1 (S6K1), activating it, and the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), inhibiting it, thus improving ribosomal translation efficiency through their downstream targets—the ribosomal protein S6 (RPS6) and the eukaryotic translation initiator factor (eIF)4E, respectively [47, 55]. Additionally, this complex also increases the protein translation capacity by promoting the synthesis of nucleotides required for DNA replication and ribosome biogenesis [55]. On the other hand, Akt phosphorylates and suppresses the transcription factors of the FoxO family, preventing their translocation to the nucleus and the expression of the two muscle-specific ubiquitin-protein ligases (E3s)—MAFbx/atrogin-1 and muscle-specific RING-finger 1 (MuRF1)—and autophagy genes, thus inhibiting protein degradation [54]. Furthermore, Akt is also responsible for the phosphorylation and inhibition of the GSK3β, releasing the eIF2B, which promotes protein synthesis [47]. Furthermore, Akt-inhibited GSK3β results in dephosphorylated nuclear factor of activated T-cells (NFAT)c1, which translocates to the nucleus, enhancing the myoblast differentiation and fiber-type switching to the slow/oxidative phenotype [47]. The activity of this anabolic pathway is controlled by negative and positive

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feedback loops [54]. The former occurs through inhibition of IRS1, inducing its degradation and altering its cell localization. The latter involves mTORC2 that in the presence of rapamycin-insensitive companion of mTOR (RICTOR) phosphorylates Akt, resulting in its maximum activation [47, 54]. The upregulation of this pathway results in muscle hypertrophy [56] (Fig. 14.2). However, when the Akt/mTOR signaling decreases, for instance, during fasting and/or catabolic diseases, protein synthesis diminishes whereas protein degradation through the FoxO-mediated expression of the atrogene program and fiber atrophy increase [53]. In addition, myostatin, an autocrine inhibitor of normal muscle growth, or its homologue activin A may also inhibit this pathway [53].

14.3.2 ROS-Mediated Signaling The activity of muscle requires low levels of ROS, which are maintained in skeletal muscle under basal conditions [57]. Indeed, ROS and also reactive nitrogen species are generated in response to the contractile activity through the enhanced formation of superoxide and nitric oxide by the skeletal muscle fibers [58]. In normal physiology, ROS mediate adaptive processes in response to physiological stresses by changing gene expression. In this regard, elevated ROS generation during enhanced contractile activity upregulates protective enzymes and stress proteins through several transcription factors, including the prototypical nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB), the activator protein-1 (AP1), the heat shock factor (HSF)1 and the nuclear factor erythroid 2-related factor 2 (NRF2). This upregulation seems to be modulated by hydrogen peroxide (H2 O2 ) [57, 58]. These molecules enhance the expression of antioxidant enzymes, such as the manganese superoxide dismutase (MnSOD), the catalase and also of cytoprotective proteins for ROS named heat shock proteins (HSPs) [57, 58]. In spite of some uncertainty about which redoxmediated processes can regulate other adaptive responses to muscle contraction, it has been suggested that ROS may be involved in the stimulation of the expression of genes related to catabolism and mitochondrial biogenesis [58]. Thus, ROS can activate the p38 MAPK pathway that has as targets p53, NF-κB and activating transcription factor (ATF)2, and in addition, p38 signaling may enhance the expression of the muscle-specific E3 ligase MAFbx in myotubes, leading to muscle protein breakdown [59]. Furthermore, ROS can activate the c-Jun N-terminal kinase (JNK) through the apoptosis signal-regulating kinase (ASK)1 pathway thereby resulting in oxidative-stress mediated apoptosis. In addition, ROS can inhibit Cn, which is involved in muscle hypertrophy and fiber phenotype transformation (Fig. 14.2). It has also been suggested that ROS can activate caspase-3, which promotes degradation of intact actin-myosin complexes [59]. It is also hypothesized that ROS may influence the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) expression; however, more data collection is necessary in this field [60]. ROS can also stimulate the production of pro-inflammatory cytokines, including the

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tumor necrosis factor alpha (TNF-α) and IL-1β through the activation of NF-κB signaling [61]. In addition, ROS may impair the functioning of the RyR1, resulting in leaking of Ca2+ from the SR [61]. Finally, ROS may be involved in the modulation of skeletal muscle glucose uptake during contraction in an AMPK-independent manner [62]. In the cell, the ROS levels are maintained by a production and clearance balance [63]. Excessive ROS stimulation can be deleterious to cells, causing direct and irreversible oxidative damage to lipids, DNA and proteins, disruption of vital redox-dependent signaling processes, inflammation and insulin resistance, resulting, eventually, in cell viability impairment [58, 63, 64].

14.3.3 Ca2+ /Calmodulin-Dependent Pathways The cellular functions are modulated by intracellular secondary messengers, such as Ca2+ [65]. In skeletal muscle, Ca2+ plays a crucial role in the contraction-relaxation cycle [66]. CaM is the ubiquitous intracellular Ca2+ receptor that has been associated with the regulation of, for instance, glycogen phosphorylase and creatine kinase, which are involved in glycogen metabolism and of the myosin light chain kinase that modulates actin-myosin interactions in skeletal muscle, and also, with the activation of the skeletal muscle SR phosphorylating system involved in the release of Ca2+ from the SR [65, 67]. There are two Ca2+ /CaM-dependent signaling pathways— one controlled by Cn and the other by Ca2+ /CaM-dependent kinases (CaMKs) [68]. Regarding Cn, it has been associated with the regulation of the expression of slow fiber type-related myofibrillar proteins and mitochondrial gene expression, and it regulates the phosphorylation state of the transcription factor NFAT [69, 70]. In this way, this mediator can translocate to the nucleus, and in collaboration with the myocyte enhancer factor 2 (MEF2) and other regulatory proteins, activates the slowtype muscle proteins [69] (Fig. 14.2). The CaMKs signaling is also upregulated by slow motor neuron activity, since it intensifies the Cn responses through enhancement of MEF2 [69]. Furthermore, CaMKs activation has been related with the regulation of the oxidative capacity through the stimulation of mitochondrial biogenesis, of type IIa myosin heavy chain (MHCIIa) expression and of Ca2+ re-uptake into the SR in slow type muscle fibers [69, 70]. The Ca2+ /CaM complex activates several proteins, such as the CaM-dependent protein kinase II (CaMKII) that is a multifunctional enzyme highly expressed in muscle, and its signaling appears to be involved in the activation of mitochondrial biogenesis, in the regulation of oxidative enzyme expression and in the muscle hypertrophic response [65, 66]. Activation of CaMKII leads to the phosphorylation of serum response factor and its binding to serum response elements on the genes promoters, including actin gene [68]. In addition, it has been suggested that CaMKII may signal through other transcription factors to activate slower and more oxidative muscle genes or genes related to muscle growth [68]. Indeed, in slowtwitch muscle cells, CaMKII has as target the SERCA, whereas in fast-twitch muscle cells CaMKII may be associated with the RyRs, dihydropyridine receptors (DHPRs) and related proteins, including triadin [71]. Nevertheless, the current knowledge of

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the physiological role of CaMKs holoenzymes in the whole muscle is rather poor [70]. In the field of exercise training, CaMKIV has been associated with the enhancement of PGC-1α expression and mitochondrial biogenesis [68]. Nevertheless, it seems that PGC-1α directly interacts with the MEF2, activating selective slow-twitch muscle genes and, simultaneously, it is a target for the Cn signaling [69]. Additionally, in SR membrane the CaMKII isoform CaMKIIβM exists in complex with glycolytic enzymes, such as glycogen debranching enzyme and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Moreover, in response to Ca2+ signaling, CaMKIIβM can directly phosphorylate GAPDH, increasing its activity, and so, modulating the local levels of nicotinamide adenine dinucleotide (NADH) and ATP, and therefore, Ca2+ transport [65].

14.3.4 Ion Channels In skeletal muscle, the modulation of ions flux is the most direct mechanism for signal transduction [72]. Mechanical loads may influence the activity of several channels that are presented in the skeletal muscle membranes, such as Ca2+ channels, voltagegated Na+ channels, Ca2+ -activated K+ channels and K+ /Na+ -permeable channels; however, the molecular identities of several mechanically sensitive ion channels are unknown [72]. As already mentioned, the nAchR activation causes an influx of Na+ and Ca2+ , depolarizing the muscle cell membrane, thus activating the voltage-gated Na+ channels [20]. Influx of Na+ ions depolarizes further the membrane, initiating the rising phase of the action potential [73]. The action potential runs along the membrane and into the T-tubules and causes the activation of the L-type Ca2+ channels that reside there [20]. Thus, Ca2+ enters down its concentration gradient, leading to the opening of RyRs on the SR [20, 74]. In this way, a further increase in the cytosolic levels of Ca2+ occurs, which binds to troponin C on actin filaments in order to initiate the contraction process [20, 75]. Voltage-gated Ca2+ channels are also involved in the regulation of slow Ca2+ conductance, which increases the cytosolic concentration, regulating the force of the contraction in response to high-frequency trains of nerve impulses [75]. In the skeletal muscle, the NaV 1.4, the CaV 1.1 and the RyRs1 are the principal channels and receptor, respectively [20, 73, 74]. It has been reported that mutations on these channels can cause congenital disorders, such as hypokalemic periodic paralysis, malignant hyperthermia and paramyotonia congenital [73, 76]. Additionally, the Ca2+ -activated K+ channels, which are a heterogeneous family, are activated by high intracellular Ca2+ levels [77, 78]. This family includes the large conductance (BK ) and the small conductance (SK) channels [78]. The SK channels compromise three types (SK1-3), wherein SK3 is the one expressed in myotubes and muscle fibers [77]. In myofibers of developing muscles these channels are normally present, but in adult muscle they are down-regulated, being upregulated following denervation [77]. Thus, it seems that an increased expression of these channels may be involved in denervation and myotonic muscular dystrophy [79].

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14.3.5 UPP Pathway The ubiquitin-proteasome pathway (UPP) is involved in the degradation of the majority of intracellular proteins [80]. In this pathway, firstly the polyubiquitination of targeted proteins occurs. This process implies the sequential action of the ubiquitinactivating enzymes (E1s) and ubiquitin-carrier or conjugating proteins (E2s) that prepare ubiquitin for conjugation, followed by E3s action, which recognizes a specific protein substrate and catalyzes the transfer of activated ubiquitin to it [80]. Subsequently, the ubiquitinated proteins are recognized by the 26S proteasome, which degrades it to small peptides [80]. The UPP is upregulated by two signaling mediators—the NF-κB and p38 MAPK—and inhibited by Akt signaling [47]. Upregulation of the muscle-specific E3 ligases requires the FoxO family of transcription factors, which, when phosphorylated by Akt, is excluded from the nucleus and when dephosphorylated translocate to the nucleus, thus upregulating MuRF1 and MAFbx [47]. In skeletal muscle, an increase of UPP-induced proteolysis is associated with muscle atrophy. This muscle wasting process is related to an enhanced expression of the muscle-specific E3 ligases—MAFbx and MuRF1—and with the inhibition of the Akt/mTOR pathway and the activation of the NF-κB system [80]. It seems that the MuRF1 induces proteolysis of myosin proteins through the direct attack of thick filament from the sarcomere, resulting in muscle atrophy [47]. Regarding MAFbx, it has been suggested that MyoD and Cn are substrates for this muscle-specific E3 ligase; however, it is not known which protein is ubiquitinated under atrophy conditions [47]. Additionally, MAFbx has been shown to be an E3 ligase for eIF3F, suggesting that MAFbx activity leads to muscle atrophy by downregulation of protein synthesis [47]. However, it seems that the UPP is necessary for the normal function of the muscle [81]. In fact, this pathway may be essential for the myogenesis process. This seems to be explained by the synthesis and degradation of myogenic proteins. So, an adaptive proteolysis may play a fundamental role in myoblast differentiation [81]. The proteasome-mediated protein degradation may also be important during exercise. It has been hypothesized that proteolysis following resistance and endurance exercise may be an adaptive response, eliminating damaged proteins and helping myofilament restructuration and muscle growth [81].

14.3.6 NF-κB Pathway Among the dimers of the NF-κB/Rel family, the p50/p65 heterodimer can be found in almost all cell types and seems to be responsible for the NF-κB activity in skeletal muscle [82, 83]. Activation of NF-κB can be stimulated by several factors related to numerous biological processes, normally associated with inflammation [84]. Activation of this pathway also increases inflammation, and so, this signaling requires

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a strict physiological regulation in order to prevent tissue damage [84]. NF-κB proteins are present in the cytoplasm bound to inhibitory proteins collectively known as inhibitors of NF-κB (IκBs), such as IκBα [82, 85]. However, there occurs in response to some stimuli the activation of the IκB kinase (IKK) complex that phosphorylates, at serine 32 and serine 36, the NF-κB-bound IκBαs, which are (the IκBαs) targeted for ubiquitination, followed by degradation through the proteasome [83]. In this way, the NF-κB free dimers translocate to the nucleus in order to regulate the transcriptional activation of hundreds of target genes (Fig. 14.2), including cytokines, chemokines, stress response proteins and several enzymes, such as those related to protein degradation by the UPP [83–85]. The NF-κB signaling can be activated by several stimuli, such as circulating pro-inflammatory cytokines, namely TNF-α (through TNF-α receptor (TNF-αR)1 and TNF-αR2; but primarily TNF-αR1) and IL1 (through IL-1 receptor (IL-1R)), and physical and chemical stresses [85]. Chronic activation of this pathway is related to several pathological conditions, namely muscle wasting and insulin resistance [83]. Indeed, enhanced NF-κB activity may result in muscle wasting through three mechanisms: (i) NF-κB may increase the expression of inflammatory mediators, which can promote muscle wasting directly or indirectly; (ii) NF-κB may impair the myogenic program, which is involved in the regeneration of atrophied skeletal muscle fibers; and (iii) NF-κB may upregulate the expression of several proteins involved in the UPP, which in turn, are responsible for the degradation of specific muscle proteins [84]. It has also been shown that an unusual exercise bout enhances the NF-κB activity, since muscle contractions increase SR calcium release, enhance ROS accumulation and activate MAPK, and all of these processes can activate the NF-κB signaling [83].

14.3.7 Wnt Pathways Wnt ligands might trigger different signaling pathways in skeletal muscle with distinct outcomes. Wnt signaling was shown to activate stem cells in adult muscles, to modulate the differentiation of presynaptic and postsynaptic components, particularly through the regulation of acetylcholine receptors clustering in the sarcolemma, and to play a key role in skeletal muscle fibrosis [86, 87]. These different outcomes may result from the activation of the canonical or β-catenin-dependent, or of the noncanonical or β-catenin-independent pathways. In the canonical pathway, Wnt ligands bind to the seven-transmembrane Frizzled (Fzd) receptors, an interaction that requires the co-receptors low density lipoprotein (LDL)-receptor-related proteins 5/6 (LRP5/6). The scaffold protein Dishevelled (Dvl) is then recruited, promoting the dissociation of the β-catenin destruction complex by sequential phosphorylation reactions that inhibit GSK3β. So, β-catenin accumulates in the cytoplasm and then translocates to the nucleus where it recognizes the T-cell specific transcription factor (TCF) and the lymphoid enhancer-binding factor (LEF), inducing the expression of Wnt target genes [86, 88].

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The noncanonical pathways include the planar cell polarity (Wnt/PCP) and Wnt/Ca2+ signals. In the first case, upon binding of Wnt ligand to its receptor Fzd, Dvl is activated, leading to the activation of small GTPase proteins such as Rho and Rac. Then, transcription factors of the signaling pathway of JNK are activated. In the case of Wnt/Ca2+ pathway, the activation of Dvl following Wnt ligand interaction with its receptor Fzd leads to the activation of trimeric G proteins and subsequent activation of phospholipase C (PLC) with the subsequent production of diacylglycerol (DAG) and inositol triphosphate (IP3). These secondary messengers promote the intracellular increase of Ca2+ concentration and the activation of Ca2+ /Cn dependent proteins [86, 87] (Fig. 14.2).

14.3.8 AMPK/eNOS Axis The AMPK is a ubiquitously expressed multisubstrate serine/threonine protein kinase [89]. This enzyme functions as an intracellular energy sensor, and so, it is activated in response to cellular stresses that result in ATP depletion (i.e. elevation of the adenosine monophosphate (AMP)/ATP and adenosine diphosphate (ADP)/ATP ratios) [89–91]. In this way, energy consuming anabolic processes, including protein synthesis are inhibited (possibly through diminution of the mTOR signal transduction pathway, and consequently, of S6K1 and 4E-BP1) and catabolic energy producing processes, namely glycolysis, fatty acid oxidation and protein degradation are stimulated to increase the potential for ATP production [89, 92, 93]. The rapid contraction of the muscle represents a large energetic challenge to the muscle fibers, since it implies a huge increase in energy turnover (>100-fold), leading to ATP consumption, which results in AMPK activation [89, 90]. In skeletal muscle, this kinase is also related with glucose uptake modulation through the regulation of GLUT4 trafficking [93, 94]. The metabolic changes performed by AMPK activation can be either acute or chronic through direct phosphorylation of metabolic enzymes or control of the gene expression, respectively [90]. The nitric oxide synthases (NOS) family is composed by endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) isoforms, all expressed in skeletal muscle of mammals. The NOS family is responsible for the production of nitric oxide (• NO) in acute and adaptational conditions of normal muscle functioning [95, 96]. Like the AMPK, • NO regulates the contraction process and it has been suggested to mediate the activation of the satellite cells, thus contributing to muscle repair [95, 97]. Actually, these two molecules are related to each other, since AMPK phosphorylated at the α1 subunit phosphorylates the eNOS, activating it, and thereby increasing the endogenous production of • NO, which in turn, enhances the expression of the PGC-1α [95, 98]. This molecule is an important mediator of energy metabolism, mostly through the regulation of mitochondrial biogenesis [95]. Additionally, it has been proposed that endogenous • NO can, in a positive feedback mechanism, activate AMPK, which in turn enhances NOS activity and • NO production [99]. A partial impairment of the eNOS expression in skeletal muscle has been correlated with

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the pathogenesis of chronic metabolic disease states, including obesity and insulin resistance. And again, like for AMPK, inhibition of NOS results in impaired skeletal muscle glucose uptake and GLUT4 translocation [99]. In addition, • NO interacts with mediators of the Akt/mTOR pathway, impacting on protein synthesis in skeletal muscle [95] (Fig. 14.2).

14.3.9 Sirtuins Sirtuins (SIRT) are NAD-dependent deacetylases that catalyze proteins deacetylation, being involved in the oxidative stress response or in the metabolism regulation. Thus, sirtuins are considered metabolic and stress-sensor proteins [64, 100]. There are seven sirtuins in mammals that can be divided into different classes: SIRT1-3 belong to class I, SIRT4 to class II, SIRT5 to class III and SIRT6–7 to class IV [64]. Furthermore, these sirtuins can be found in the mitochondria (SIRT3, SIRT4 and SIRT5), nucleus (SIRT1, SIRT6 and SIRT7) and in both, cytoplasm and nucleus (SIRT2) [100]. It has been shown that sirtuins may activate FoxO3a gene expression and deacetylate it, activating FoxO DNA binding and increasing the expression of target genes associated with cell cycle arrest (p27), oxidative stress (MnSOD) and apoptosis (Bcl-2-like protein 11; BCL2L11/Bim) [101]. However, the activation of FoxO transcription factors by sirtuins seems to impair the FoxO ability to promote cell apoptosis. Instead, this family can shift the FoxO function towards oxidative stress resistance and DNA repair [101]. The most studied member of the sirtuin family is SIRT1 [102]. The enzymatic activity of SIRT1 is regulated by the free concentration of NAD+ [103]. This sirtuin seems to be involved in muscle differentiation and metabolism, but it also appears to have a negative effect on myogenesis, since it has been demonstrated that during this process SIRT1 may regulate the energy metabolism [104, 105]. So, it has been suggested that the upregulation of SIRT1 may impair the regeneration process during muscle wasting, possibly through PGC-1α [102]. In fact, SIRT1 can deacetylate and activate PGC-1α, which is the principal regulator of mitochondrial biogenesis [103, 104]. During fasting, the SIRT1-dependent deacetylation of PGC1α increases, enhancing mitochondrial oxidative phosphorylation [106]. In fact, both, caloric restriction and physical activity can activate AMPK and FoxO3a, inducing the SIRT1, which results in the activation of FoxO4 and PGC-1α [106]. Additionally, SIRT1 can interact with • NO signaling by regulating eNOS activity through acetylation/deacetylation. Indeed, SIRT1 can upregulate eNOS, inducing the mitochondrial biogenesis and the • NO plays an important role in satellite cells proliferation [103]. However, SIRT1 may also decrease the IGF-1 expression and, consequently, can downregulate its signaling through Akt/mTOR and also inhibit NF-κB, reducing its inflammatory pathway [106] (Fig. 14.2). SIRT2 adapts to the energy state of muscle fibers, and so, its expression is enhanced during low-energy conditions (such as caloric restriction) and is repressed during status of energy excess [64]. In response to oxidative stress, SIRT2 may deacetylate

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and activate FoxO3a, which increases the expression of MnSOD to reduce cellular ROS levels [107]. As SIRT2, SIRT3 is involved in the regulation of many proteins activities, including FoxO3 [106]. In the skeletal muscle, upregulation of SIRT3 occurs in the presence of caloric restriction or fasting, enhancing mitochondrial oxidative phosphorylation. The absence of SIRT3 results in the downregulation of PGC-1α, and consequently, in the alteration of mitochondrial function, in the increase of oxidative stress and activation of JNK, impairing, for instance, the insulin signaling [63, 106]. SIRT4 was suggested to suppress fatty acid oxidation in skeletal muscle through the reduction of the malonyl coenzyme A (CoA) decarboxylase (MCD) activity [108]. Therefore, the downregulation of SIRT4 may increase the mitochondrial fatty acid oxidative function in skeletal muscle [100]. The inhibition of SIRT4 also increases the levels of phosphorylated (activated) AMPK [109]. In the skeletal muscle, inhibition of SIRT5 appears to have no effect on the expression of genes involved in the transcriptional regulation of metabolism, mitochondrial function, fatty acid oxidation, lipogenesis or glucose metabolism [110]. Contrarily, SIRT6 is involved in the downregulation of IGF-1 and its signaling pathway Akt/mTOR [106]. Furthermore, SIRT6 can also inhibit the NF-κB, reducing its inflammatory signaling cascade [106]. Concerning SIRT7, few studies have been made in the skeletal muscle, but it has been hypothesized that this sirtuin may counteract sarcopenia [111].

14.4 Conclusions Several signaling pathways are involved in skeletal muscle remodeling in response to distinct stimuli, such as innervation, load, hormones and neural input, a process known as muscle plasticity. The ability of this organ to sense these signals and to convert them into biochemical events is mediated by multiple signaling pathways. For instance, integrin-containing complexes play a critical role in the integration of mechanical stress and downstream activation of signaling effectors, including Ca2+ via CaM and calcineurin, PI3K/Akt, MAPKs and AMPK. Acetylcholine, via nicotinic acetylcholine receptors, mediates the effect of neural input on muscle fiber contraction. These cells are highly responsive to chemical stimuli such as insulin, growth hormone, catecholamines and sex hormones, particularly testosterone, which is justified by the high muscle expression of these hormones’ receptors and downstream players. Muscle-derived molecules, or myokines, may also contribute to alterations in muscle mass and metabolic status, being of notice the involvement of myostatin, irisin and IL-6. Taken together, skeletal muscle plasticity is overseen by a complex array of molecular players that are responsive to distinct mechanical and chemical signals, which comprehension is far from being completely understood.

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Chapter 15

Adipocyte Specific Signaling David F. Carrageta, Pedro F. Oliveira, Mariana P. Monteiro, and Marco G. Alves

Abstract Adipocytes are the most abundant cells within the adipose tissue and are the cell type responsible for the tissue dynamic metabolic and endocrine activity. Under energy surplus conditions, the adipocyte is able to suffer hypertrophy in order to accommodate energy in form of lipids. Simultaneously, new adipocytes are differentiated through a complex and specific process, known as adipogenesis. While this process seems clear for white adipocytes in white adipose tissue, brown adipocytes and brown adipose tissue have distinct characteristics and function. Brown adipocytes are not related with fat accumulation but rather with thermogenesis, a process defined by a rapidly oxidization of lipids in order to produce heat. Additionally, a class of beige adipocytes, which are inducible thermogenic adipocytes originating from white adipose tissue and phenotypically distinct from both, have been described though how these are originated and which are the main functions are still matters of discussion. Interestingly, the induction of thermogenesis seems to improve insulin resistance, adiposity and hyperlipidemia. Thus, inducing the browning of white adipocytes to beige adipocytes is thought to be promising to improve the common metabolic disorders, such as obesity or metabolic syndrome. This chapter focuses on the specific D. F. Carrageta · P. F. Oliveira · M. G. Alves (B) Laboratory of Cell Biology, Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), Department of Microscopy, University of Porto, 4050-313 Porto, Portugal e-mail: [email protected] D. F. Carrageta e-mail: [email protected] P. F. Oliveira e-mail: [email protected] P. F. Oliveira Faculty of Medicine, Department of Genetics, University of Porto, Porto, Portugal i3S—Instituto de Investigação e Inovação em Saúde, University of Porto, 4200-135 Porto, Portugal M. P. Monteiro Unit for Multidisciplinary Research in Biomedicine (UMIB), Department of Anatomy, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal e-mail: [email protected] © The Author(s) 2020 J. V. Silva et al. (eds.), Tissue-Specific Cell Signaling, https://doi.org/10.1007/978-3-030-44436-5_15

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signaling and regulatory control of adipocyte functions, particularly adipogenesis and adipocyte browning. Emerging insights of these processes are herein discussed, as promising therapeutic targets for obesity and other common metabolic disorders. Keywords Adipocyte · Adipogenesis · Beige adipocyte · Brown adipocyte · Browning · Thermogenesis

Abbreviations ACTRII ADSC AT BAT C/EBPA C/EBPB C/EBPD CAMKII cAMP DLK1 EBF2 EHMT1 EN1 ERK FABP/aP2 FGF FGF1R FZD GLUT GPDH Hh hMADSC IRF4 KLF LEF/TCF LRP5/6 LXR MSC MYF5 MYH11 PAX7 PDGFRA PGC1A PKA

Activin type II receptor Adipose-derived stem cell Adipose tissue Brown adipose tissue CCAAT/enhancer-binding protein alpha CCAAT/enhancer-binding protein beta CCAAT/enhancer-binding protein delta Ca2+ /calmodulin-dependent kinase II Cyclic adenosine monophosphate Delta-like 1 homolog Early B-cell factor 2 Euchromatic histone lysine methyltransferase 1 Engrailed 1 Extracellular signal-regulated kinase Fatty acid binding protein/adipose protein 2 Fibroblast growth factor FGF type 1 receptor Frizzled receptor Glucose transporter Glycerol-3-phosphate dehydrogenase Hedgehog Human multipotent adipose-derived stem cell Interferon regulatory factor 4 Kruppel-like factors Lymphoid-enhancer-binding factor/T-cell-specific transcription factor Low-density lipoprotein-receptor-related protein 5 and 6 co-receptors Liver X receptor Mesenchymal stem cell Myogenic factor 5 Myosin heavy chain 11 Paired-box protein 7 Platelet-derived growth factor receptor A Proliferator-activated receptor gamma coactivator 1-alpha Protein kinase A

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PPARG pRb PRDM16 Pref-1 Ptc RIP140 RyR2 SCA1 SERCA2b Smo SRC2 TACE TGF TNF-A TWIST1 UCP WAT Wnt ZFP516

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Peroxisome proliferator-activated receptor gamma Retinoblastoma protein Positive regulatory domain containing protein 16 Preadipocyte factor 1 Patched receptor Receptor-interacting protein 140 Ryanodine receptor 2 Stem cells antigen 1 Sarco/endoplastmatic reticulum Ca2+ -ATPase 2b Smoothened protein Steroid receptor co-activator 2 Tumor necrosis alpha converting enzyme Transforming growth factor Tumor necrosis factor alpha Twist-related protein 1 Uncoupling protein White adipose tissue Wingless-type MMTV integration site Zinc-finger protein 516

15.1 Introduction The adipose tissue (AT) is a metabolically active organ that acts as the main energy repository in the human body and as an endocrine organ able to synthesize several biologically active molecules that regulate metabolic homeostasis. The AT fulfils several functions, which may vary among fat depots due to its size, distribution, and heterogeneity according to the molecular, morphological, and metabolic profiles [1]. In humans, there are two main types of AT, white AT (WAT) and brown AT (BAT), with relevant differences in morphology and function. In the human body, BAT is mainly located in the supraclavicular, periadrenal, and paravertebral regions [2] and its relative proportion decreases since birth into adulthood [3]. Although BAT is also able to store energy in form of fat, the main activity of the tissue lies in heat production or thermogenesis [4]. In contrast, WAT contribution for thermogenesis is nearly irrelevant, but presents much broader physiological functions. Despite WAT being widely distributed in the human body, 80% of total body fat is located subcutaneously, with the main fat depots in the abdominal and femoral-gluteal regions. The remaining 20% are located around the visceral organs, predominantly in proximity to abdominal visceral AT, such as mesenteric and omental, and around the kidney in the retroperitoneal region [5]. By involving organs and infiltrating tissues, the WAT not only offers mechanical protection, but also plays an important role in the regulation of the body temperature, acting as a thermal insulator [4, 6]. Furthermore, WAT accomplishes multiple other functions, such as immune, endocrine, and regenerative [7, 8].

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The AT is mainly constituted by adipocytes. Adipocytes are very unique cells due to their morphology and functions. Adipocytes are responsible for the dynamic activity of the AT, conferring metabolic and endocrine activity to the tissue. The process of cellular differentiation into adipocytes, or adipogenesis, is a specific and finely regulated process which differs in WAT and BAT. While white adipocytes are the “classic” adipocytes, whose main function is to accumulate energy in form of lipids in large lipid droplets, brown adipocytes are rather distinct. Brown adipocytes are described as being smaller in comparison to white adipocytes, with relatively abundant cytoplasm, numerous small lipid droplets of different sizes and numerous mitochondria that produce heat by fatty acids oxidation [4]. More recently, a third type of adipocytes was identified, being characterized as an intermediate morphology between brown and white adipocytes. These adipocytes also have a thermogenic activity and a high number of mitochondria, thus have been termed as beige or brite adipocytes. In this chapter, the specific signaling and regulatory control of adipocytes will be discussed, particularly white and brown adipogenesis, thermogenesis, and the interchange between the two adipocyte phenotypes with focus on WAT browning and beige adipocytes development.

15.2 White Adipocyte Signaling 15.2.1 White Adipogenesis Adipose tissue mass accumulation is associated with adipocyte cell hypertrophy and increased number of white adipocytes. Whenever there is an energy surplus, excess energy is predominantly accumulated in the form of lipids in white adipocytes, which hypertrophy as the lipid droplets size increase. This WAT buffering activity is suggested as an adaptive response to energy excess, which protects other tissues from lipotoxicity [9]. The maintenance of WAT homeostasis includes simultaneous cell hyperplasia, a phenomena by which adipocyte precursors, or preadipocytes, proliferate and differentiate into mature adipocytes [10]. Thus, this process denominated adipogenesis culminates with the formation of new adipocyte cells and consists of a two phase process (Fig. 15.1). The first step towards the differentiation into the adipocyte lineage consists in the generation of preadipocytes from mesenchymal stem cells (MSCs). The second phase includes the terminal differentiation of preadipocytes into functional mature adipocytes, including morphologic alterations and the formation of lipid droplets. As mature adipocytes are not able to proliferate in vivo, the regeneration and hyperplasia of the WAT is thought to be dependent on the proliferative capacity of a pool of precursor cells, the preadipocytes [11]. In general, the term “preadipocyte” is widely used to describe a progenitor cell that is responsible for the formation of mature adipocytes [12]. A crucial property of preadipocytes is the ability to proliferate and differentiate into mature adipocytes in order to maintain the AT homeostasis

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Fig. 15.1 Schematic illustration of white adipogenesis. White adipogenesis is described as two-step process, starting with the proliferation and commitment of white preadipocytes and culminating with the terminal differentiation to mature white adipocytes. The differentiation process starts with the expression of two CCAAT/enhancer-binding proteins (C/EBP), C/EBPB and C/EBPD, which directly induce the expression of C/EBPA and peroxisome proliferator-activated receptor gamma (PPARG), the most important transcriptional regulators of adipogenesis. Then, C/EBPA and PPARG directly induce self-expression in a positive feedback mechanism, activating several downstream adipocyte-specific genes

throughout the lifespan of a living organism [13]. These cells are also characterized as a committed cell population destined to proliferate and differentiate only into the adipose-lineage, although morphologically undistinguished from its progenitors [14, 15]. Preadipocytes are thought to arise from MSCs and more specifically from human adipose-derived stem cells (ADSCs). In fact, ADSCs were first identified in 2002 as a subpopulation of multipotent self-renewing cells isolated from WAT that are morphologically and phenotypically similar to the MSCs [16]. Then, ADSCs were isolated, cultured, and termed human multipotent adipose-derived stem cells (hMADSCs) [17]. As hMADSCs maintain the capacity to enter the adipose lineage and to differentiate into cells that present characteristics highly similar to native human adipocytes, these are considered as a faithful model to study human AT physiology [18–20]. One of the major focus in adipocyte biology research concerns the identification of distinct cellular intermediates between ASCs and fully functional adipocytes, although there is a lack of specific biomarkers to identify and isolate these cells. However, preadipocytes isolated from different AT present different characteristics in terms of gene expression profiles, proliferation, differentiation, and consequently signaling pathways [21]. Furthermore, adipocytes from different fat depots also present different functional properties, contributions towards energy homeostasis and thus different behaviors during metabolic disease scenarios [22, 23]. However, the molecular mechanisms underlying these AT regional-dependent differences remain unknown. The second and last phase of adipogenesis is the terminal differentiation, where preadipocytes acquire a phenotype of mature adipocytes. Mature white adipocytes are cells specialized in the synthesis, storage, and mobilization of lipids, which are accumulated in the lipid droplet. This lipid droplet occupies the majority of the adipocyte cytoplasm and consequently the nucleus is found at the periphery of the cell [24]. The terminal differentiation consists of a cascade of transcriptional events which culminates in the formation of mature adipocytes. It is thought that the differentiation process starts with the expression of two CCAAT/enhancerbinding proteins (C/EBP), C/EBPB and C/EBPD, which directly induce the

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expression of C/EBPA and peroxisome proliferator-activated receptor gamma (PPARG), the most important transcriptional regulators of adipogenesis [25, 26]. Then, C/EBPA and PPARG directly induce its own expression in a positive feedback mechanism [27] and activate several downstream adipocyte-specific genes that are necessary for adipocyte function, including fatty acid binding protein/adipose protein 2 (FABP/aP2), insulin receptor, glucose transporters (GLUTs), acetyl-coA carboxylase, fatty acid synthase, and glycerol-3-phosphate dehydrogenase (GPDH) [28]. Although the factors that control proliferation, commitment, and differentiation of preadipocytes are still poorly characterized, some important regulatory pathways were already identified (Fig. 15.2) and will be discussed on the subsequent topics.

15.2.2 White Adipogenesis Regulation 15.2.2.1

Peroxisome Proliferator-Activated Receptor Gamma (PPARG)

Inducing the expression of PPARG, the major transcriptional regulator of adipogenesis, is sufficient to induce terminal adipocyte differentiation. In fact, it is thought that it is not possible to induce preadipocytes terminal differentiation in the absence of PPARG [29]. Moreover, all essential signaling pathways in adipogenesis seems to converge on the regulation of PPARG expression or activity [30]. There are two isoforms of PPARG (PPARG1 and PPARG2) that are generated by alternative splicing and both are induced during adipogenesis [31]. The adipogenic activity of both PPARG isoforms differs and PPARG2 was found to be more efficient in promoting terminal differentiation. Both PPARG1 and PPARG2 were observed to induce adipocyte differentiation in Pparg knockout fibroblasts, although at higher efficiency for PPARG2 [32]. Additionally, it was demonstrated that 3T3-L1 cells, a cell line originally established from primary murine embryonic fibroblasts able to differentiate into adipocytes, would undergo adipocyte differentiation in the presence of exogenous PPARG2 but not PPARG1 despite the inhibition of Pparg1 and Pparg2 genes by zin-finger proteins [33]. However, studies in animal knockout models yielded contradictory results, which adds complexity and discussion to the topic. For an instance, PPARG2 knockout mice were shown to have decreased AT size and impaired adipogenesis in one study [34], while other authors observed normal AT size and adipocyte morphology in these mice although insulin resistance was reported [35]. Thus, while PPARG2 is important for adipogenesis in vivo, is hypothesized not to be absolutely required when PPARG1 is normally expressed. Additionally, PPARG is essential for the maintenance of adipocytes differentiated state. Inhibition of PPARG in mature 3T3-L1 adipocytes led to de-differentiation with loss of lipid accumulation and reduced expression of adipocyte specific markers [36]. Moreover, in vivo inducible knockout of Pparg led to apoptosis in differentiated adipocytes and stimulated new adipocytes generation [37]. These studies highlight the complexity in PPARG signaling for adipocytes differentiation and physiology.

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Fig. 15.2 Schematic illustration of white adipogenesis regulation. Several factors regulate adipogenesis, leading to preadipocytes proliferation and inhibiting terminal differentiation. Upon ceasing the proliferative stimuli, preadipocytes commit to the terminal differentiation process. Arrows indicate activation or stimulation in the direction of arrowheads. Crossbars indicate suppression or inhibition. Abbreviations ACTRII—activin type II receptor; C/EBPA—CCAAT/enhancer-binding protein alpha; C/EBPB—CCAAT/enhancer-binding protein beta; C/EBPD—CCAAT/enhancerbinding protein delta; DLK1—delta-like 1 homolog; ERK—extracellular signal-regulated kinase; FGF-1—fibroblast growth factor 1; FGF1R—fibroblast growth factor 1 receptor; FGF-2—fibroblast growth factor 2; FZD—frizzled receptor; HH—hedgehog; LRP5/6—low-density lipoproteinreceptor-related protein 5 and 6 co-receptors; PPARG—peroxisome proliferator-activated receptor gamma; PTC—patched receptor; Smo—smoothened protein; TACE—tumor necrosis alpha converting enzyme; TGF-B—transforming growth factor beta; TGFBR—transforming growth factor beta receptor; TNF-A—tumor necrosis factor alpha; Wnt—wingless-type MMTV integration site; B-Cat—Beta-catenin

15.2.2.2

CCAAT/Enhancer-Binding Protein (C/EBP) Family

The C/EBP family is constituted by important regulators of adipocyte differentiation. Indeed, the C/EBP family participates in a signaling cascade where C/EBPB and C/EBPD induce the expression of C/EBPA and thus terminal differentiation of adipocytes. Although C/EBPB and C/EBPD can compensate each other in case of loss or inhibition, there is evidence that both factors are not absolutely needed for adipogenesis. A study with knockout mice for C/EBPB or C/EBPD shown that

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lack of these factors reduces AT size. In addition, double-knockout for C/EBPB and C/EBPD led to a greater reduction of the AT mass. However, the levels of C/ebpa and Pparg mRNA in adipocytes from double-knockout mice remain normal, which raised the hypothesis that absence of C/EBPB or C/EBPD could result only in abnormal lipogenesis and not adipogenesis inhibition [38]. These findings point out that C/EBPB and C/EBPD may not be essential for adipogenesis in vivo as other signaling pathways can produce similar effects. On the other hand, C/EBPA is known to have a much more important role in adipogenesis. For instance, inducing C/EBPA in vitro is sufficient to trigger differentiation of preadipocytes into mature adipocytes [39]. Moreover, mice where the C/ebpa locus was replaced by C/ebpb present reduced AT size [40], while knockout mice for C/ebpa depict an almost absence of WAT [39]. Besides, the expression of C/EBPA in differentiated adipocytes is also important for insulin sensitivity [41]. In this study, it is proposed that C/EBPA triggers the expression of GLUT4 and insulin-dependent glucose transport. In the absence of C/EBPA, GLUT4 is not expressed and cells are rendered insensitive to insulin. Nonetheless, C/EBPA needs the presence of PPARG to efficiently promote adipogenesis since the expression of C/EBPA in knockout fibroblasts for Pparg cannot induce adipocyte differentiation [42]. In addition, C/EBPB needs PPARG to induce the expression of C/EBPA and start the terminal differentiation pathway [43]. Hence, C/EBPs family members, with especial focus in C/EBPA, are indeed important but PPARG expression is crucial for adipogenesis.

15.2.2.3

Fibroblasts Growth Factors (FGF)

One of the most important signaling pathways regulating adipogenesis concerns the autocrine/paracrine role of fibroblast growth factors (FGF). Undifferentiated hMADSCs with reduced FGF-2 secretion were observed to proliferate more slowly [44]. On the other hand, hMADSCs supplemented with FGF-2 would recover and proliferate much faster illustrating that this signaling pathway is essential for proliferation in these cells. FGF-2 has a high affinity for FGF type 1 receptor (FGF1R), which activates extracellular signal-regulated kinase (ERK) 1/2 pathway, leading to cellular proliferation [45]. In fact, hMADSCs treated with a specific FGF1R inhibitor presented a reduced differentiation potential [44, 46]. These findings suggested that FGF-2 is secreted by hMADSCs in order to regulate their proliferative potential through FGF1R/ERK signaling pathway. Additionally, FGF-2 plays a role in preadipocytes differentiation. FGF-2 effects were reported to be dose-dependent, where FGF-2 can function as either a positive or negative adipogenic factor [47]. FGF-2 at concentrations lower than 2 ng/ml promoted differentiation of hMADSCs, while concentrations higher than 10 ng/ml inhibited the differentiation. These effects are also mediated by the FGF1R/ERK signaling pathway, where high concentrations of FGF-2 are able to sustain ERK phosphorylation and lead PPARG phosphorylation, consequently blocking terminal differentiation. Furthermore, FGF-2 levels in WAT of diet induced obese mice were observed to be lower than those from normal diet mice, indicating that FGF-2 expression levels could have a reverse correlation

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with the fat tissues size. Thus, FGF-2 was hypothesized to play a protective role in obesity and metabolic disease. These findings are further supported by another study conducted in AT of morbidly obese patients who underwent bariatric surgery or lean controls who underwent cholecystectomy [48]. ASCs were obtained from WAT of these individuals and the worse metabolic scenarios were accompanied by a reduced ASCs proliferation rate and decreased FGF-2 secretion. Together, these results suggest that FGF-2 and WAT hyperplasia play a protective role against metabolic syndrome. However, not only FGF-2 but also FGF-1 plays a role in adipogenesis [46]. In fact, FGF-1 seems to be involved in the regulation of FGF-2. FGF-1 treated hMADSCs were reported to have reduced FGF-2 levels by 80%. Moreover, FGF-2 knockout hMADSCs presented decreased proliferation and increased adipogenic genes expression [49]. Thus, while FGF-2 seems to be essential for preadipocytes proliferation, FGF-1 seems to downregulate its secretion, promoting differentiation. For instance, obese individuals also present increased expression of FGF-1 in WAT, which supports the hypothesis of a protective role for FGF-1/2 [50]. Interestingly, in obese individuals were subjected to weigh loss FGF-1 levels were reported to remain high, which may constitute a permanent alteration due to obesity. Nevertheless, further studies are needed to test that hypothesis.

15.2.2.4

Activin A and Transforming Growth Factor Beta (TGF-B) Family

Activin A is a member of the transforming growth factor beta (TGF-B) family and is also secreted by undifferentiated hMADSCs. In fact, activin A was proposed as a biomarker of undifferentiated hMADSCs [51]. Similarly to FGF-2, activin A secretion promotes hMADSCs proliferation and blocks differentiation in an autocrine/paracrine fashion. Activin A effects are mediated via activin type II receptor, which activates Smad2 in order to increase cellular proliferation and inhibit C/EBPB [52]. Thus, while activin A secretion is increased, hMADSCs will proliferate but will not differentiate whereas activin A inhibition leads to hMADSCs differentiation. Interestingly, increased levels of activin A were also found in the WAT of obese individuals [51]. When expressed in high levels, activin A was also reported to act as a profibrotic agent, stimulating the transformation of preadipocytes into myofibroblasts [53, 54]. In fact, AT fibrosis in the context of human obesity limits adipocyte hypertrophy, with beneficial effects on systemic metabolism [55]. Therefore, activin A acts together with FGF-1/2 to constitute a complex and finely regulated signaling pathway in adipogenesis. However, the mechanisms that regulate the increased secretion of FGF-2 and activin A by preadipocytes and subsequent inhibition with terminal differentiation still need to be clarified. There is some evidence that WAT microenvironment is the main responsible for this modulation and that not only preadipocytes and adipocytes regulate adipogenesis. For instance, macrophages isolated from the AT of obese individuals are reported to produce factors that stimulated the expression of FGF-2 and activin A in hMADSCs [51]. Indeed, tumor necrosis factor alpha (TNF-A) is one of the factors secreted by macrophages reported to increase FGF-2

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and activin A expression in hMADSCs [20]. Obesity and AT mass accumulation are linked with inflammation and macrophages infiltration [56]. Thus, macrophages are thought to be essential for adipogenesis and WAT homeostasis. Besides activin A, other members of the TGF-B family are reported to regulate adipogenesis. In fact, TGF-B itself is mostly inhibitory [57]. Studies using 3T3 cell lines (fibroblast cell line) showed that TGF-B increases preadipocyte proliferation, while inhibiting differentiation through Smad3 signaling [58]. However, other studies reported that this inhibitory effect is restricted to the first 40 h due to TFG-B type I and II receptors downregulation during differentiation [58, 59]. The activation of the Smad3 pathway is similar to Smad2, decreasing the expression of PPARG and C/EBPA due to the physical inhibition of C/EBPB and C/EBPD [60]. Besides, Smad3 signaling also involves up-regulation of Wnt signaling pathway factors [61], which will be addressed in further detail in the next section. Bone morphogenetic proteins (BMPs), are another subtype of growth factor members of the TGF-B family, which also regulate ASCs adipogenesis in an autocrine and dose-dependent manner. For instance, BMP4 is probably the most notorious member of the BMP family in terms of proadipogenic effects [57]. BMP4 activates Smad1/5/8 and positively regulates PPARG expression, whereas BMP4 inhibition leads to reduced lipid droplets size [62]. Moreover, BMP4 has a role in preadipocytes proliferation. While in high doses significantly reduces cell proliferation, in low doses reduces the number of apoptotic cells and increases proliferation [63]. Interestingly, BMP4 promotes differentiation of brown preadipocytes into white adipocytes, reducing the expression of brown-specific proteins. In addition, obese individuals with type 2 diabetes mellitus present higher levels of BMP4 [64]. These studies highlight a possible role for BMP in adipocytes physiology and disease mechanisms associated with diabetes and obesity, but further studies are needed to unveil the mechanisms controlled by this pathway.

15.2.2.5

Wingless-Type MMTV Integration Site (Wnt) Family

Wingless-type MMTV integration site (Wnt) family members are highly conserved signaling glycoproteins with autocrine/paracrine action, known to for regulating tissue homeostasis and remodeling [25]. Wnts exhibit two distinct signaling pathways, denominated “canonical” and “noncanonical”. The canonical pathway is linked with the transcriptional factor Beta-catenin. In sum, Wnts binds to frizzled receptors (FZD) and low-density lipoprotein-receptor-related protein-5 or protein-6 coreceptors (LRP5/6) blocking the ubiquitination and proteasomal degradation of Betacatenin, which in turn is hypophosphorylated and translocated to the nucleus to bind lymphoid-enhancer-binding factor/T-cell-specific transcription factor (LEF/TCF) and induce the expression of Wnt target genes [65]. The Wnt canonical signaling pathway has an antiadipogenic activity. In support of this hypothesis, are the repeated observations showing that Wnt pathway blocking leads to spontaneous adipocyte differentiation [66–68]. Furthermore, LRP5/6 activation inhibits terminal differentiation, whereas LRP5/6 inactivation exerts the opposite effect [69–71]. In

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fact, Wnt1 represses terminal differentiation through downregulation of PPARG and C/EBPA [66]. In addition, Wnt10b, previously known as Wnt12, is also described as an important antiadipogenic factor and an adipogenic switch in adipogenesis. Wnt10b is another member of the Wnt family whose gene is clustered with Wnt. Wnt10b is highly expressed in preadipocytes, which also stabilizes Beta-catenin and inhibits terminal differentiation. In addition, Wnt10b inhibition also promoted preadipocyte differentiation [72]. Thus, Wnt canonical signaling pathway is hypothesized to be an important molecular switch regulating adipogenesis, which is suppressed until the pathway is no longer activated. In fact, PPARG and Beta-catenin are mutual antagonists. The activation of PPARG decreases Beta-catenin levels due to its degradation through proteasomal complexes [73]. Therefore, once preadipocytes receive the trigger to initiate the PPARG positive feedback mechanism, become committed to terminal differentiation. On the other hand, the noncanonical pathway is related with intracellular Ca2+ release, which activates the phosphatase calcineurin and the Ca2+ -sensitive kinases Ca2+ /calmodulin-dependent kinase II (CAMKII) and protein kinase C [74]. Contrastingly, the noncanonical pathway can either promote adipogenesis through antagonism of the canonical pathway or inhibit adipogenesis in a similar fashion to the canonical pathway. For instance, Wnt5b increases during adipogenesis and promotes terminal differentiation through inhibition of Beta-catenin [75]. However, Wnt5a is reported to inhibit terminal differentiation by activating Beta-catenin and suppressing the activity of PPARG, thus inducing terminal differentiation, via a pathway unrelated to Ca2+ [76]. Altogether, these findings illustrate the complexity of the signaling intrinsic to adipogenesis and further studies are needed.

15.2.2.6

Delta-Like 1 Homolog (DLK1/Pref-1)

Delta-like 1 homolog (DLK1), also known as preadipocyte factor 1 (Pref-1), is a transmembrane glycoprotein that can be cleaved by tumor necrosis alpha converting enzyme (TACE), originating a biologically active soluble form [77, 78]. DLK1 is highly expressed by proliferating preadipocytes and is suppressed during differentiation [79]. In fact, several studies reported that high DLK1 levels in preadipocytes inhibits differentiation [80–82] while reduced levels enhances differentiation [83]. More recently, DLK1 knockout mice were shown to have enhanced preadipocyte proliferation, which was associated with a reduced membrane-bound form of DLK1 whereas a soluble form of DLK1 had no effect on preadipocyte proliferation but in the terminal differentiation process [84]. Thus, it was hypothesized that the membrane form and soluble form of DLK1 are likely to have distinct functions and targets [85]. Although DLK1 is considered essential for adipogenic regulation, these results remain controversial since DLK1 interaction molecules remain to be identified.

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Hedgehog (Hh) Pathway

Hedgehog (Hh) signaling pathway also plays a role in adipogenesis. Hh binds to the Patched receptor (Ptc), which cease the suppressor effect on smoothened protein (Smo) [86, 87]. Smo activates a signaling cascade that results in the stabilization of the transcription factors Gli1 and Gli2, inducing the transcription of Hh target genes [88]. Moreover, Hh signaling pathway seems to interact with the ERK pathway [89] thus suggesting an association with the aforementioned pathways. In fact, Hh basal levels are needed for human MSCs proliferation [90]. Additionally, Smo inhibition by cyclopamine, thus inhibiting the Hh signaling, decreased human MSC proliferation and led to cell cycle arrest, which was associated with a decrease in cyclin A expression and consequently an increase in the active form of retinoblastoma protein (pRb) expression. Conversely, the ability to differentiate human MSCs was not affected, although another study observed that Hh decreases during the differentiation step and that Smo activation impaired preadipocytes terminal differentiation through downregulation of PPARG and C/EBPA [91]. More recently, Hh was demonstrated to inhibit not only adipocyte differentiation but also lipogenesis in adipocytes in vitro, which in turn improved the metabolic condition of diet-induced obese mice [92].

15.2.2.8

Zinc-Finger Proteins

Zinc-finger proteins are crucial molecules involved in adipogenesis regulation. Kruppel-like factors (KLF) family members are zinc-finger proteins, known for playing numerous roles in the regulation of apoptosis, proliferation, and differentiation [93]. In fact, several members of the KLF family are described as adipogenic regulators. KLF15 is known to promote differentiation and the expression of adipocyterelated genes in preadipocytes, such as GLUT4 [94, 95]. KLF4 stimulates the expression of C/EBPB [96], while KLF5 is induced during terminal differentiation by C/EBPB and C/EBPD, which in turn induces the expression of PPARG [97]. Like KLF5, KLF9 binds directly to the PPARG promoter besides also activating PPARG indirectly by binding to C/EBPA [98]. In addition, KLF6 promotes adipogenesis by inhibiting DLK1 [99]. However, some KLFs depict antiadipogenic effects, such as KLF2 that suppresses PPARG expression [100, 101]. Thus, a shift in KLFs expression during terminal differentiation is hypothesized to occur, with KLF2 downregulation that is replaced by the other proadipogenic family members [97]. Similarly to KLFs, GATA zinc fingers found in the GATA-family transcription factors are also known to modulate adipogenesis. GATA2 and GATA3 are expressed in preadipocytes and both inhibit PPARG and C/EBPs expression. In addition, GATA2 and GATA3 expression decreases during terminal differentiation [102, 103]. There is evidence that GATA3 expression is induced by the Wnt/Beta-catenin pathway [104], however the detailed mechanisms underlying this pathway regulation in preadipocytes are still unknown.

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15.3 Brown Adipocyte Signaling 15.3.1 Brown Adipogenesis Brown adipocytes are the main cellular constituent of BAT. Brown adipocytes are morphologically distinct from white adipocytes, as besides depicting a “brown” color that derives from the large number of mitochondria present in the cell cytoplasm, also present a high number of small lipid droplets. The size of brown adipocytes lipid droplets allows to increase the droplet surface area, which was hypothesized to promote the high rate of metabolite exchange with the mitochondria that is necessary for the thermogenic activity [105]. Brown adipocytes arise from brown preadipocytes, although the pathways underlying brown preadipocytes differentiation are not fully understood. In dedicated BAT depots, brown preadipocytes are known to originate from cells residing in the dermomyotome and express specific transcription factors, including paired-box protein 7 (PAX7), engrailed 1 (EN1), and myogenic factor 5 (MYF5) [106]. Yet, different brown adipocytes subpopulations were described in different BAT depots, which despite not expressing all these factors are still able to differentiate into mature brown adipocytes. Indeed, both brown and white adipocytes from different fat depots were shown to exhibit different gene expression profiles and functional characteristics [107]. In a similar process as depicted by white adipocytes, brown adipocytes can expand in size through lipid accumulation leading to hypertrophy or increase in cell number leading to BAT hyperplasia. However, the main stimulus for brown adipocyte expansion is not energy surplus as for white adipocytes but is instead regulated by the needs to maintain body temperature. In thermoneutral conditions sympathetic stimuli are reduced, thus decreasing brown adipocytes thermogenic activity and increasing lipid accumulation. Several lipid droplets are then combined into a single lipid droplet, leading the brown adipocyte into a hypertrophic state [107]. On the other hand, in a cold environment, thermogenesis is activated and both brown adipocytes cells and BAT sizes decrease. In conditions of persistent cold environment exposure, brown adipogenesis is induced [108, 109]. Therefore, brown adipogenesis and thermogenic activity are highly dependent on the cold stimulus.

15.3.1.1

Brown Adipogenesis Regulation

In general, brown and white adipogenesis share the same signaling cascade, highlighting PPARG and C/EBP family members’ role in adipocytes and lipids metabolism. However, some brown adipocyte-specific transcription factors have been identified (Fig. 15.3). Positive regulatory domain containing protein 16 (PRDM16) is described as a transcriptional factor that acts as the main switch of brown adipogenesis. PRDM16 is not only part of a transcriptional complex with C/EBPB and PPARG [110, 111], but has also the ability to induce uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1A)

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Fig. 15.3 Schematic illustration of brown adipogenesis regulation and thermogenesis. Brown adipogenesis is triggered with the expression of PRDM16, leading to the thermogenic phenotype and the expression of UCP1. Abbreviations: C/EBPB—CCAAT/enhancer-binding protein beta; cAMP— cyclic adenosine monophosphate; EBF2—early B-cell factor 2; EN1—engrailed 1; IRF4—interferon regulatory factor 4; MYF5—myogenic factor 5; PAX7—paired-box protein 7; PGC1A— proliferator-activated receptor gamma coactivator 1 alpha; PKA—protein kinase A; PPARG—peroxisome proliferator-activated receptor gamma; PRDM16—Positive regulatory domain containing protein 16; UCP1—uncoupling protein 1; ZFP516—zinc-finger protein 516

expression, resulting in the consequent thermogenic phenotype of brown adipocytes [112, 113]. Moreover, genetic deletion of euchromatic histone lysine methyltransferase 1 (EHMT1), an essential component of PRDM16 complex, decreases brown adipocytes terminal differentiation [114]. Conversely, PRDM16 deletion in brown preadipocytes was found not to affect brown terminal differentiation, which suggests the existence of compensatory pathways [115]. These pathways could be mediated by other PRDM family members, which can induce brown adipogenesis in vivo. One of these members is PRDM3, a PRDM family member that presents a high homology with PRDM16 and forms a complex with EHMT1 and C/EBPB to induce PPARG expression [116]. Thus, EHMT1 is highlighted as a crucial enzymatic regulator of brown adipogenesis. Additionally, early B-cell factor 2 (EBF2) is a transcription factor essential for brown adipogenesis. EBF2 is highly and specifically expressed in brown preadipocytes and adipocytes [117, 118]. EBF2 suppresses the expression of several transcription factors, ensuring preadipocytes commitment to the brown adipocyte terminal differentiation [119]. Zinc-finger protein 516 (ZFP516) expression is also reported to induce PRDM16 and brown adipogenesis [120, 121].

15.3.2 Thermogenesis Brown adipocytes are able to rapidly exhibit a high oxidative respiration and substrate oxidation capacity due to the high thermogenic potential and UCP1 expression, a protein that acts as a respiration uncoupler. UCP1 is only found in significant

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amounts in the mitochondria of thermogenic adipocytes and is described as identical to mitochondrial ADP/ATP carriers [122]. The mechanism of thermogenesis was first proposed by Mitchell and later developed by Nicholls [123]. Briefly, mitochondrial respiratory chain complexes I, III, and IV act as proton pumps, generating a proton gradient where protons are transported from the mitochondria matrix to the intermembrane space. ATP-synthase consume the protons in order to produce ATP from the phosphorylation of ADP, in an endothermic reaction. However, in the presence of an uncoupler protein such as UCP1, which is an inner mitochondrial membrane protein that acts as a proton channel, protons re-entry the mitochondrial matrix from the intermembrane space, collapsing the proton gradient and activating the respiratory chain to compensate the decreased membrane potential. Since the ATP-synthase is no longer able to consume protons in order to produce ATP from ADP, the energy derived from oxidation is then dissipated as heat. Brown adipocytes are known to produce heat in a process denominated nonshivering thermogenesis. In general, thermogenesis occurs by stimulation of the sympathetic nervous system in response to cold exposure. Whenever there are no requirements to produce extra heat, brown adipocytes remain quiescent and UCP1 is inhibited by purine nucleotides [124]. In fact, inactive brown adipocytes are morphologically similar to white adipocytes, but still preserving the specific genetic and metabolic identity [125]. On the other hand, upon cold stress, norepinephrine is released activating the beta-3 adrenergic receptors and increasing cyclic adenosine monophosphate (cAMP), which results in cAMP-driven protein kinase A (PKA) signaling activation. This signaling pathway culminates in lipolysis, the process where triacylglycerol stored in lipid droplets is hydrolyzed in free fatty acids and glycerol, and increased expression of PGC1A and UCP1 [126, 127]. PGC1A has a crucial role in thermogenesis regulation in brown adipocytes, although interestingly the knockout of PGC1A in brown preadipocytes does not affect brown adipogenesis [128, 129]. PGC1A is highly expressed upon cold stress and PRDM16 induction by cAMP signaling, which results in thermogenic genes activation, such as UCP1 [113]. In addition, cAMP signaling also activates interferon regulatory factor 4 (IRF4), which interacts with PGC1A to form a complex and induces the expression of UCP1 [130]. However, some proteins were identified as inhibitors of PGC1A expression or activity. Receptor-interacting protein 140 (RIP140), together with liver X receptor (LXR) are able to suppress the effects of PGC1A [131, 132]. Steroid receptor co-activator 2 (SRC2) and twist-related protein 1 (TWIST1) also suppress PGC1A effects [133, 134]. Conversely, recent studies have shown that as long as exogenous free fatty acids are available these may directly increase UCP1 expression, thus brown adipocytes might not be fully dependent of brown adipocytes lipolysis [127, 135, 136]. In addition, brown adipocytes are able to perform lipogenesis, where free fatty acids are synthesized from circulating glucose [137], besides the ability to also use acyl-carnitines to sustain thermogenesis [138]. Interestingly, upon cold exposure, brown adipocytes depict both catabolic and anabolic reactions by performing thermogenesis and lipogenesis. Due to this paradoxical activity, it is hypothesized that lipogenesis may have other metabolic implications since several intermediates may also act as signaling agents [107, 139].

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15.4 White Adipose Tissue Browning and Beige Adipocytes 15.4.1 Beige Adipocytes Development Studies with 18 F-fluodeoxyglucose PET-CT imaging allowed to identify several metabolic and thermogenic active adipose depots in the abdominal and subcutaneous regions in adult human individuals. These studies led to the conclusion that WAT was not completely white [140–142]. Indeed, a second type of UCP1-expressing and thermogenic adipocytes were found in WAT. These adipocytes were named brite (brown-like in white) or beige adipocytes. The morphology of beige adipocytes is somewhat intermediary between white and brown adipocytes and can be found within WAT depots mainly upon cold exposure [106]. Thus, when the formation of beige adipocytes is induced, the phenomenon of WAT browning can be observed. Interestingly, each WAT depot has a different susceptibility to browning. While subcutaneous WAT seems to possess a high capacity to undergo a browning process upon cold exposure, the same does not occur in visceral WAT [143]. Even when WAT browning is artificially induced, visceral WAT is not able to reach the browning ability of subcutaneous WAT, which further highlights the molecular differences between fat depots. The origin of beige adipocytes could be explained by two different theories, although not mutually exclusive (Fig. 15.4). The first line of thought hypothesized that beige adipocytes arise from a precursor cell pool through beige adipogenesis [144]. Beige adipocytes are reported to originate from a preadipocyte population expressing platelet-derived growth factor receptor A (PDGFRA) and stem cells antigen 1 (SCA1) or from smooth muscle-like precursors expressing myosin heavy

Fig. 15.4 Schematic illustration of beige adipocytes differentiation. Upon cold exposure, beige adipocytes differentiate from beige preadipocytes or through transdifferentiation of mature white adipocytes. Beige adipocytes express PRDM16 and present a brown phenotype with heat production through thermogenesis. However when the cold stimulus ceases, beige adipocytes are able to return to a white adipocyte phenotype. Abbreviations MYH11—myosin heavy chain 11; A—plateletderived growth factor receptor A; PRDM16—PR domain containing protein 16; SCA1—stem cells antigen 1; UCP1—uncoupling protein 1

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chain 11 (MYH11) [106]. On the other hand, another theory defends that beige adipocytes are originated in conditions of thermogenic needs from pre-existing mature adipocytes that undergo a trans-differentiation process, known as adipocyte browning [145, 146]. The expression of PRDM16 in preadipocytes upon cold exposure triggers the beige-linked genes expression and beige adipocytes development while suppressing WAT-selective genes [112]. In fact, both hypotheses are possible, but the regulation of the mechanisms leading to each specific route are still unclear. In addition, beige adipocytes were shown to share some activity and differentiation regulation with brown adipocytes, although whether there are beige-specific transcription factors remains to be elucidated. In support of both theories, beige adipocyte differentiation can be induced in experimental conditions using two different approaches. The first method consists in animal models exposure to severely low temperatures (4–6 °C), which is thought to be the closest approach to physiological conditions. Low temperature exposure induces UCP1-expressing beige adipocytes in WAT either by beige adipogenesis [147] or through browning of mature white adipocytes [145]. The second method consists in using beta-3 adrenergic agonists to stimulate white adipocytes, which mimics the effects of cold exposure and induces the process of adipocyte browning [148]. Interestingly, treatment with beta-3 adrenergic agonists only functions in mature white adipocytes in vitro as inhibition of this pathway in vivo does not prevents the browning induced by cold stimulus, which suggests that browning induction may be dependent on multiple signaling pathways. Indeed, several other conditions were reported to induce beige adipocytes development, such as physical exercise, cancer cachexia, and peripheral tissue injury [149, 150]. Thus, beige adipocytes development seems to be stimulated not only as a thermogenic need, but also to contribute to energy homeostasis. A distinctive characteristic of beige adipocytes is that the thermogenic ability is inducible and reversible depending on the environmental conditions, as when the cold stress ceases, beige adipocytes are slowly replaced by white adipocytes both in vitro and in vivo [108, 151]. These findings suggest that beige adipocytes are only maintained during the presence of the stimuli and upon its withdrawal, these revert to the white adipocytes’ phenotype. This short-term maintenance and beige-to-white adipocyte conversion or “adipocyte whitening” was attributed to the loss of mitochondrial biogenesis and activity upon the withdrawal of external stimuli [149]. It was hypothesized that adipocyte whitening is linked with mitophagy, a selective form of autophagy responsible for mitochondria degradation. The blockade of the mitophagy pathway prevents adipocyte whitening even upon external stimuli removal and thus leads to the prolongation of the thermogenic activity of beige adipocytes. Moreover, this thermogenic activity was associated with higher energy expenditure and protection against diet-induced obesity [151]. As autophagy pathways are dysregulated in AT of obese and diabetic patients [152] and adipocyte whitening seems to be faster in obese subjects [151], detailed understanding of these pathways could be highly relevant for the development of new pharmacological targets for obesity treatment.

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15.4.2 Unconventional Thermogenesis of Beige Adipocytes UCP1 has been described as the main thermogenic protein in AT thermogenesis. However, it was reported that UCP1 knockout mice exposed to cold still exhibited higher respiration rates in WAT [153]. Moreover, chronic treatment with beta-3 adrenergic receptor agonists in the same animal model also increased the respiration of WAT [154]. Therefore, this implies the existence of UCP1-independent thermogenic pathways. In fact, a specific UCP1-independent thermogenic pathway in beige adipocytes was noticed [155]. This novel and unconventional pathway involves ATP-dependent Ca2+ cycling by sarco/endoplasmatic reticulum Ca2+ -ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). This specific thermogenic pathway is only possible in beige adipocytes due to the high capacity of ATP generation by glycolysis and tricarboxylic acid metabolism. These findings also led the authors to state that this novel pathway could be a potential pharmacological target to improve metabolic health in the elderly population, known to possess a low number of UCP1-positive adipocytes.

15.5 Conclusions and Future Perspectives White, brown, and beige adipocyte differentiation and function are regulated by unique and complex signaling processes. Although some pathways were already identified, the search for factors that regulate adipogenesis just reached the light of the day. While the C/EBP family and PPARG are the main regulators of both white and brown adipogenesis, other transcription factors and signaling proteins that regulate in a direct way and/or upstream/downstream the terminal differentiation signaling cascade are still missing in the big signaling puzzle. In this sense, the new technology of CRISPR-Cas9 may be revolutionary for genetic studies and further extend the knowledge on key adipocyte regulators. Moreover, emerging evidence also highlights the role of miRNAs signaling and epigenetic regulation during adipogenesis and metabolic dysregulation scenarios. Compelling data concerning adipocyte-selective miRNAs that modulate adipocyte proliferation and differentiation [156, 157] point out a considerable new trend in adipocyte biology research. Additionally, the high cellular heterogeneity among adipocytes from different or even within fat depots makes it imperative to characterize their origin and distinct signaling pathways. Nonetheless, WAT browning and inducible thermogenesis is a compelling treatment approach for metabolic diseases such as obesity and type 2 diabetes. A better understanding of the pathways concerning beige adipogenesis and adipocyte browning may point out to promising novel drugs. Given the increasing prevalence and the high health and economic impact of obesity and obesity related disorders, new insights concerning beige adipogenesis and adipocyte browning must be set as a priority in the years to come.

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Acknowledgments This work was supported by the Portuguese Foundation for Science and Technology: PTDC/MEC-MET/32151/2017; David F. Carrageta (SFRH/BD/136779/2018); Marco G. Alves (IFCT2015, PTDC/BIM-MET/4712/2014 and PTDC/MEC-AND/28691/2017); Pedro F. Oliveira (IFCT2015 and PTDC/BBB-BQB/1368/2014); UMIB (Pest-OE/SAL/UI02015/2019); and co-funded by FEDER funds through the POCI/COMPETE 2020. Conflict of Interest None to declare.

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