Reviews of Physiology, Biochemistry and Pharmacology (Reviews of Physiology, Biochemistry and Pharmacology, 180) 3030834298, 9783030834296

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Stine Helene Falsig Pedersen Editor

Reviews of Physiology, Biochemistry and Pharmacology 180

Reviews of Physiology, Biochemistry and Pharmacology Volume 180

Editor-in-Chief Stine Helene Falsig Pedersen, Department of Biology, University of Copenhagen, Copenhagen, Denmark Series Editors Diane L. Barber, Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA, USA Emmanuelle Cordat, Department of Physiology, University of Alberta, Edmonton, Canada Mayumi Kajimura, Department of Biochemistry, Keio University, Tokyo, Japan Jens G. Leipziger, Department of Biomedicine, Aarhus University, Aarhus, Denmark Martha E. O’Donnell, Department of Physiology and Membrane Biology, University of California Davis School of Medicine, Davis, USA Luis A. Pardo, Max Planck Institute for Experimental Medicine, G€ ottingen, Germany Nicole Schmitt, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark Christian Stock, Department of Gastroenterology, Hannover Medical School, Hannover, Germany

The highly successful Reviews of Physiology, Biochemistry and Pharmacology continue to offer high-quality, in-depth reviews covering the full range of modern physiology, biochemistry and pharmacology. Leading researchers are specially invited to provide a complete understanding of the key topics in these archetypal multidisciplinary fields. In a form immediately useful to scientists, this periodical aims to filter, highlight and review the latest developments in these rapidly advancing fields. 2019 Impact Factor: 4.700, 5-Year Impact Factor: 6.000 2019 Eigenfaktor Score: 0.00067, Article Influence Score: 1.570

More information about this series at http://www.springer.com/series/112

Stine Helene Falsig Pedersen Editor

Reviews of Physiology, Biochemistry and Pharmacology

Editor Stine Helene Falsig Pedersen Department of Biology University of Copenhagen Copenhagen, Denmark

ISSN 0303-4240 ISSN 1617-5786 (electronic) Reviews of Physiology, Biochemistry and Pharmacology ISBN 978-3-030-83429-6 ISBN 978-3-030-83430-2 (eBook) https://doi.org/10.1007/978-3-030-83430-2 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are solely and exclusively licensed 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

Acknowledgements

Contributions to this volume have partly been personally invited, with kind support of the series editors D.L. Barber, E. Cordat, M. Kajimura, J. Leipziger, M.E. O’Donnell, L. Pardo, N. Schmitt, C. Stock.

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Contents

Role of Histone Deacetylases in Monocyte Function in Health and Chronic Inflammatory Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . Rosa Marı´a Tordera and Marı´a Corte´s-Erice Neuronal Nitric Oxide Synthase (nNOS) in Neutrophils: An Insight . . . Rashmi Saini, Zaffar Azam, Leena Sapra, and Rupesh K. Srivastava When Glycosylation Meets Blood Cells: A Glance of the Aberrant Glycosylation in Hematological Malignancies . . . . . . . . . . . . . . . . . . . . Huining Su, Mimi Wang, Xingchen Pang, Feng Guan, Xiang Li, and Ying Cheng The Placenta as a Target for Alcohol During Pregnancy: The Close Relation with IGFs Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . Irene Martı´n-Estal, Inma Castilla-Corta´zar, and Fabiola Castorena-Torres

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Rev Physiol Biochem Pharmacol (2021) 180: 1–48 https://doi.org/10.1007/112_2021_59 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 Published online: 12 May 2021

Role of Histone Deacetylases in Monocyte Function in Health and Chronic Inflammatory Diseases Rosa María Tordera and María Cortés-Erice Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Role of Class I HDACs in Monocyte Function in Healthy and Pathological States . . . . . . . . 2.1 Role of HDAC1 in Monocyte Function in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . 2.2 Role of HDAC2 in Monocyte Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Role of HDAC3 in Monocyte Function in Healthy and Disease States . . . . . . . . . . . . . . 2.4 Role of HDAC8 in Monocyte Function in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . 3 Role of Class II HDACs in Monocyte Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Regulation of Class IIa HDAC Transcriptional Activity by Nucleocytoplasmic Shuttling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Role of Class IIa HDACs in Inflammatory Processes, Host Defense, and Cancer . . . 3.3 Role of the Class IIb HDAC6 in Macrophage Migration in Inflammatory Processes, Host Defense, and Tumor Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Role of Class III HDAC in Monocyte Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Role of SIRT1 in Monocyte Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Role of SIRT6 in Monocyte Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Role of Other Class III HDACs in Monocyte Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Role of Class IV in Monocyte Function in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 5 5 9 15 18 19 21 22 24 25 25 29 34 35 36 38

Abstract Histone deacetylases (HDACs) are a family of 18 members that participate in the epigenetic regulation of gene expression. In addition to histones, some HDACs also deacetylate transcription factors and specific cytoplasmic proteins. Monocytes, as part of the innate immune system, maintain tissue homeostasis and help fight infections and cancer. In these cells, HDACs are involved in multiple processes including proliferation, migration, differentiation, inflammatory response, infections, and tumorigenesis. Here, a systematic description of the role that most HDACs play in these functions is reviewed. Specifically, some HDACs induce a R. M. Tordera (*) and M. Cortés-Erice Department of Pharmacology and Toxicology, Universidad de Navarra, School of Pharmacy and Nutrition, Pamplona, Spain e-mail: [email protected]

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pro-inflammatory response and play major roles in host defense. Conversely, other HDACs reprogram monocytes and macrophages towards an immunosuppressive phenotype. The right balance between both types helps monocytes to respond correctly to the different physiological/pathological stimuli. However, aberrant expressions or activities of specific HDACs are associated with autoimmune diseases along with other chronic inflammatory diseases, infections, or cancer. This paper critically reviews the interesting and extensive knowledge regarding the role of some HDACs in these pathologies. It also shows that as yet, very little progress has been made toward the goal of finding effective HDAC-targeted therapies. However, given their obvious potential, we conclude that it is worth the effort to develop monocyte-specific drugs that selectively target HDAC subtypes with the aim of finding effective treatments for diseases in which our innate immune system is involved. Keywords Autoimmune diseases · Cancer · Epigenetics · HDACs inhibitors · Immunosuppression · Inflammation · Macrophage reprogramming · Viral infection

Abbreviations BMDMs COPD COX-2 CSE FDA FOXO HAT HDAC HIF-1α HIV-1 HSPCs IAV IL JEV KLF2 LOX-1 LPMCs LPS LXR MCP-1 MEF2 MIP-1α MMP9 NES

Bone marrow–derived macrophage cells Chronic obstructive pulmonary disease Cyclooxygenase 2 Cigarette smoke extract Food and Drug Administration Forkhead box Histone acetyltransferase Histone deacetylase Hypoxia-inducible factor 1 alpha Human immunodeficiency virus 1 Human stem and progenitor cells Influenza A virus Interleukin Japanese encephalitis virus Krüppel-like factor 2 Lectin-like oxidized low-density lipoprotein receptor-1 Intestinal lamina propia mononuclear cells Lipopolysaccharide Liver X receptor Monocyte chemoattractant protein Myocyte enhancer factor 2 Macrophage inflammatory protein alpha Matrix metalloproteinase-9 Nuclear export sequence

Role of Histone Deacetylases in Monocyte Function in Health and Chronic. . .

NF-ĸB NLS Nrf2 PBMCs PI3Kδ SAHA SIRT TLR TNFα

3

Nuclear factor kappa B Nuclear localization sequence Nuclear factor erythroid 2-related factor 2 Peripheral blood mononuclear cells Phosphoinositide-3-kinase-δ Suberoylanilide hydroxamic acid Sirtuin Toll-like receptor Tumoral necrosis factor alpha

1 Introduction Epigenetics is defined as the study of stable and heritable modifications of chromatin that occur without changes in DNA sequence and that influence the phenotypic features of living organisms (Eccleston et al. 2007). Among the different epigenetic mechanisms, histone modification has been the focus of much investigation. Chromatin can exist in a condensed state, heterochromatin, which avoids gene transcription, or in an open active state, euchromatin, which allows the genes to be transcribed. Histone modifications affecting chromatin occur mainly in the N-terminal of histones 3 and 4. These modifications include acetylation, methylation, phosphorylation, ubiquitination, SUMOylation, and ADP-ribosylation (Zhang et al. 2015). Of these, histone (de)acetylation of lysine residues is one of the most widely studied modifications, a process carried out by histone acetyltransferases (HAT) and histone deacetylases (HDAC). HATs enzymes acetylate the lysine residues of histones. Histone acetylation unwinds the DNA-histone conformation, allowing the transcription factors to interact with DNA and facilitating gene expression. Conversely, HDACs remove acetyl groups from the lysine residues of the N-terminal tail of histones. Histone deacetylation results in a more compact chromatin state and, therefore, promotes gene silencing. Drugs that inhibit HDACs produce an increase in histone acetylation within hours and thereby help to maintain DNA in a more open and transcriptionally active state (Zhao et al. 2018). Moreover, in addition to histones, HDACs can also deacetylate transcription factors modulating their ability to activate specific promoters. Further, some HDACs can shuttle between the nucleus and the cytoplasm and also regulate the function of specific cytoplasmic proteins (Narita et al. 2019). On the other hand, HDACs not only function as gene transcription repressors as one would expect, but they can also act as gene transcription activators depending on their influence over specific promoters. For instance, in some cases HDACs activate transcription when they inactivate specific genes that are repressors of the expression of other targeted genes. HDACs belong to an evolutionarily conserved family of 18 members divided into four classes (De Ruijter et al. 2003). Class I (HDAC1, 2, 3, and 8), IIa (HDAC4, 5, 7, and 9), IIb (HDAC6 and 10), and IV (HDAC11) are

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similar as they require Zn2+ as a cofactor. In contrast, class III HDACs, also called sirtuins (SIRT1–7), depend on nicotinamide adenine dinucleotide (NAD+) as a cofactor. HDACs regulate multiple processes in monocytes including proliferation, maturation and differentiation, inflammatory response, bacterial or viral infection, and tumorigenesis (Daskalaki et al. 2018). Monocytes, as white blood cells that develop from the myeloid progenitor cell line in the bone marrow, help fight bacterial or viral infections. Further, when a disturbance in tissue homeostasis occurs in any part of the body, monocytes are capable of migrating to the affected tissue and differentiating to macrophages. Interestingly, macrophages have been classically classified into M1 and M2 subtypes. M1 macrophages enhance the immune response by secretion of pro-inflammatory cytokines and are crucial to protect the host against different types of threats such as viral infections or tumor growth. Conversely, M2 macrophages have anti-inflammatory immunoregulatory functions. Yet, these characteristics make them undesirable during cancer progression as they will permit tumor growth (Sica et al. 2015; Wang et al. 2019). While some HDACs promote monocyte maturation and differentiation, others prevent these processes. In general, relevant HDACs exert a pro-inflammatory function in monocytes, by inducing expression and secretion of pro-inflammatory cytokines. Upregulation of inflammatory genes in monocytes plays a major role in host defense against pathogens but also in the development of autoimmune and chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, or atherosclerosis (Das Gupta et al. 2016; Tabas and Bornfeldt 2016). Therefore, selective inhibition of pro-inflammatory HDACs offers great potential in these immune-mediated inflammatory diseases (Schötterl et al. 2015; Angiolilli et al. 2017). Conversely, other HDACs subtypes play an anti-inflammatory role in monocytes and therefore, their activation could have therapeutic potential in chronic inflammatory diseases (Cosio et al. 2004; Winnik et al. 2012; Ji et al. 2019). Furthermore, HDACs are attractive epigenetic targets for cancer therapy. Aberrant expression of some HDACs favors tumorigenesis because it leads to chromatin condensation and silencing at specific sites where promoters of tumor suppressor genes are located (Hui Ng and Bird 2000). For instance, in myeloid leukemia, increased HDAC activity is almost invariably observed in all blast cells. In contrast, pan HDAC inhibitors, which have been approved by the Food and Drug Administration (FDA) for cancer therapy, induce the expression of tumor suppressor genes and favor pro-apoptotic and anti-proliferative activity in tumor cells. Here, we review relevant studies with the aim of describing the role of specific HDACs in monocyte function both in health and in response to pathological threats. Further, we aim to provide a wealth of targets for pharmacologic intervention in different diseases including autoimmune-inflammatory diseases, infections, and cancer.

Role of Histone Deacetylases in Monocyte Function in Health and Chronic. . .

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2 Role of Class I HDACs in Monocyte Function in Healthy and Pathological States Class I HDACs are made up of HDAC1, 2, 3, and 8 subtypes and are found almost exclusively in the nucleus of the cells with the exception of HDAC3 that can shuttle between the nucleus and the cytoplasm (Thiagalingam et al. 2003).

2.1

Role of HDAC1 in Monocyte Function in Health and Disease

HDAC1 regulates key functions in monocytes such as differentiation and activation of inflammatory processes, immunosuppression, and cancer progression. In addition to histone deacetylation, HDAC1 also deacetylates several transcription factors. Of these, the nuclear factor kappa B (NF-κB), which binds to the promoter region of inflammatory genes, plays a primary role in monocyte function.

2.1.1

HDAC1 Promotes Monocyte Differentiation and Inflammatory Response in Monocytes

HDAC1 deacetylation of histone 3 at lysines 9 and 14 (H3K9/14ac) promotes monocyte differentiation from progenitor stem cells (Ewelina et al. 2017). Moreover, several studies show that HDAC1 activates osteoclastogenesis from the monocyte/ macrophage lineage. In contrast, osteoclastogenesis is prevented by the class I HDAC inhibitor drugs NW-21, which preferentially target both HDAC1 and HDAC2, and MS-275 which shows selectivity for HDAC1 (Algate et al. 2020). Many studies highlight a key role of HDAC1 in triggering inflammatory processes in monocytes (Algate et al. 2020). For instance, in in vitro experimental models based on cytokine-stimulated monocyte cultures, HDAC1 upregulation triggers the expression of the inflammatory cytokines IL-1β and TNFα as well as the expression of the monocyte chemoattractant protein MCP-1 and the macrophage inflammatory protein 1α (MIP-1α) (Algate et al. 2020). Moreover, the expression of these inflammatory proteins is prevented by treatment with the HDAC1/2 inhibitor NW-21. In particular, HDAC1 is found to be upregulated in rheumatoid arthritis and participates in mechanisms mediating inflammation in this disease. In rheumatoid arthritis, monocytes, together with dendritic and T-cells, are recruited to inflammatory sites, become activated in synovial tissue, and trigger the expression of inflammatory cytokines leading to tissue destruction. In the synovial tissue, recruited monocytes differentiate into macrophages and activate surrounding resident cells. They also differentiate into osteoclasts, which will be the main cause of bone destruction.

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In the synovial cells of rheumatoid arthritis patients, the transcription factor NF-κB is highly activated and induces the expression of TNFα, MCP-1, and MIP-1α. In the infiltrated monocytes, highly expressed HDAC1 will activate NF-κB by deacetylating its p65 subunit at lysine 122 (Kawabata et al. 2010). In contrast, the class I HDAC inhibitor MS-275 has shown a relevant anti-inflammatory and anti-rheumatic action (Kawabata et al. 2010) by inhibiting the NF-κB pathway (Cantley et al. 2011). In addition, MS-275 upregulates the expression of the cyclindependent kinase (CDK) inhibitor p21, considered to be a key checkpoint of cell cycle control. This effect will also contribute to a reduction in the inflammation by exerting an anti-proliferative activity in the arthritic synovial fibroblasts (Cantley et al. 2011).

2.1.2

HDAC1 Maintains Latent Infection of the HIV-1 Virus in Monocytes

HDAC1 maintains the HIV-1 pro-viral DNA transcriptionally inactive in quiescent cells. Specifically, HDAC1 deacetylates histones in specific DNA sites of expression of several transcription factors that promote HIV-1 viral replication. These include NF-κB, AP-4, YY1, and LSF1. In contrast, the selective HDAC1 inhibitor ITF2357 is able to reverse the repressive effect of the HDAC1 enzyme on these transcription factors and induce HIV-1 replication from latently infected monocytes. In addition, ITF2357 has been shown to reduce the expression of HIV-1 entry receptors suggesting the potential therapeutic value of HDAC1 inhibitors to cure HIV-1 latent infection (Matalon et al. 2010; Watters et al. 2013).

2.1.3

HDAC1 Favors Growth and Survival of Myeloid Leukemia Cells

HDAC1 promotes aberrant cell cycle progression in myeloid leukemia cells by downregulating the expression of the CDK inhibitors p16, p21, or p56, the wellknown checkpoints of the cell cycle. In contrast, class I HDAC inhibitors upregulate these cell checkpoints and exert an anti-proliferative action and apoptosis of tumor cells with moderate effects on normal cells (Seidel et al. 2014). Further, some selective HDAC1 inhibitors have shown potent oral anti-tumor activity in both myeloid leukemia cell and solid tumor models with no obvious toxicity (Li et al. 2017a).

2.1.4

HDAC1 Plays a Key Role in Macrophage Adaptation to the Intestinal Microenvironment

The intestinal microenvironment is unique for macrophages because they intimately coexist with the enteric microbiota. In this context, macrophages need to develop tolerance to the enteric microbiota to protect the intestine from an exaggerated

Role of Histone Deacetylases in Monocyte Function in Health and Chronic. . .

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Fig. 1 Main roles of HDAC1 in human monocytes. H3K9/14ac deacetylation by HDAC1 induces monocyte differentiation. In addition, HDAC1 binds to some cytokine promoters and induces intestinal macrophage adaptation. HDAC1 upregulation in rheumatoid arthritis induces NF-κB activation and triggers an inflammatory response. Moreover, HDAC1 silences NF-κB and other transcription factor promoters and maintains HIV-1 latent infection in monocytes. Further, HDAC1 silences CDK inhibitor promoters and favors tumorigenesis. Conversely, Class I HDAC inhibitors prevent all these effects showing a potential therapeutic value for the treatment of rheumatoid arthritis, HIV-1 latent infection, and myeloid leukemia. Meaning of the signals and colors: Red means noxious action, blue means beneficial action. Arrow means activation and bar means inhibition

inflammatory response. HDAC1 appears to be upregulated in human intestinal macrophages as compared to peripheral macrophages. Here, HDAC1 upregulation mediates histone deacetylation at cytokine promoters and, in turn, promotes a switch of the inflammatory phenotype of macrophages into an anti-inflammatory and immunosuppressive phenotype (Sun et al. 2019). This is a relevant mechanism by which HDAC1 contributes to macrophage adaptation to the permanent exposure to inflammatory conditions in the intestinal microenvironment. A summary of the main functions regulated by HDAC1 in monocytes is given in Fig. 1. In addition, the most outstanding studies and experimental models used directed to describe the specific roles of HDAC1 in monocyte function both in healthy and disease states are summarized in Table 1. All these studies suggest that selective HDAC1 inhibitors could have a therapeutic value for the treatment of osteoporosis and arthritis. So far, existing drugs that stop bone destruction or improve synovial inflammation show limited efficacy in advanced disease. Yet, given that HDAC1 exerts a protective effect on the

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Table 1 Roles of HDAC1 in monocyte function in healthy and disease states Illness/process Monocyte activation and differentiation

Rheumatoid arthritis (RA)

Latent HIV-1 infection

Main finding HDAC1 deacetylates H3K9/14ac and triggers monocyte differentiation from stem cells HDAC1 upregulation in TNFα-stimulated monocytes triggers the expression of IL-1β and MCP-1 HDAC1 inhibition prevents osteoclast differentiation by supressing the expression of MCP-1 and MIP-1α chemokines HDAC1 upregulation in TNF-stimulated osteoclasts triggers the expression of IL-1, IL-6, TNFα, COX-2, MCP-1, and other cytokines HDAC1/2 inhibition with NW-21 prevents this inflammatory response and suppresses osteoclast formation and bone resorbing activity HDAC1 is highly expressed in the synovium of RA patients and correlates with TNF expression The HDAC1 inhibitor MS-275 upregulates the CDK inhibitor p21 and induces antiproliferative activity The HDAC1 inhibitor MS-275 suppresses inflammation and bone loss in animal models of arthritis HDAC1 suppresses different transcription factors (NF-κB, AP-4, YY1, and LSF1) and maintains HIV-1 in a latent state HDAC1 inhibitor ITF2357 induces HIV-1 replication from latently infected cells

Model used HSPCs THP-1 cells

Reference (Ewelina et al. 2017)

Human monocyte cultures

(Algate et al. 2020)

Osteoclast cell cultures differentiated from human PBMCs

(Cantley et al. 2011)

Osteoclast cell cultures differentiated from human PBMCs

(Cantley et al. 2011; Algate et al. 2020)

Synovial fibroblast cell cultures obtained from fresh synovial tissue of RA patients E11 cell line

(Kawabata et al. 2010)

(Choo et al. 2013)

Collagen antibodyinduced arthritis (CAIA) mouse model of inflammatory arthritis

(Cantley et al. 2011)

U1 and ACH2 cell lines

(Matalon et al. 2010)

(continued)

Role of Histone Deacetylases in Monocyte Function in Health and Chronic. . .

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Table 1 (continued) Illness/process Tumor growth and survival

Tolerance to the intestinal inflammatory microenvironment

Main finding HDAC1 inhibits CDK inhibitors expression (p16, p21, and p56) causing tumor growth and survival HDAC1/2 inhibitors exert pro-apoptotic and antiproliferative effects by upregulating CDK inhibitors HDAC1 upregulation in intestinal inflammatory microenvironment deacetylates cytokine promoters and induces macrophage reprogramming to an anti-inflammatory phenotype

Model used K-562 cell line

Reference (Seidel et al. 2014)

Intestinal myeloid cells from non-inflammatory bowel disease patients activated with muramyl dipeptide Human monocytederived macrophages Mouse bone marrow– derived macrophages

(Sun et al. 2019)

THP-1 is a human monocytic cell line derived from an acute monocytic leukemia patient. E11 is a cell line developed from human rheumatoid arthritis synovial fibroblasts. U1 and ACH2 are chronically infected HIV-1 promonocytic (U1) and lymphoid (ACH2) cloned cell lines. K-562 is a human monocytic cell line derived from a chronic myelogenous leukemia patient

gastrointestinal system by promoting macrophage adaptation, systemic administration of HDAC1 inhibitors might not be recommended. Perhaps, local infiltration of selective HDAC1 inhibitors in the affected joints would be effective to stop osteoclastogenesis and synovial inflammation.

2.2

Role of HDAC2 in Monocyte Function

Located in the nucleus, HDAC2 is a Class I HDAC enzyme that induces an antiinflammatory action in macrophages and monocytes (Ito et al. 2005). Particularly, HDAC2 plays key roles in the regulation of cytokine inflammatory response in lung inflammatory diseases and in host innate antiviral response. HDAC2 inhibits the activity of NF-κB and promotes an anti-inflammatory action in monocytes. In addition, HDAC2 deacetylates histones at the cytokine promoter sites (Ito et al. 2005).

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2.2.1

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HDAC2 Regulates Lung Inflammation Induced by Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a major global health problem caused by chronic inflammation of peripheral airways and the lung parenchyma. HDAC2 activity and expression are markedly reduced in COPD lung airways and alveolar macrophages (Cosio et al. 2004; Ito et al. 2005). The chronic inflammation in COPD is attributed to decreased HDAC2 activity in alveolar macrophages, thought to be mediated by oxidative and nitrative stress. Oxidants, released from inflammatory cells lead to the activation of the phosphoinositide-3-kinase-δ (PI3Kδ) pathway that may reduce HDAC2 activity. Specifically upregulated/activated PI3Kδ in COPD would phosphorylate HDAC2 and promote HDAC2 ubiquitination and degradation by the proteasome pathway (Barnes 2009; Adenuga et al. 2009; Osoata et al. 2009). Consequently, reduction in HDAC2 favors transcription and activation of the NF-κB transcription factor, which enhances the synthesis of the pro-inflammatory cytokines IL-8 and TNFα (Ito et al. 2005). The enhancement of these cytokines promotes prolonged airway and lung inflammation. Moreover, in COPD patients HDAC2 is also decreased in PBMCs and this has been proposed as a potential biomarker of COPD severity (Tan et al. 2016). In these cells, HDAC2 downregulation also favors the transcription and activation of NF-κB and subsequently the expression of inflammatory genes such as TNFα, IL-6, and IL-8. Conversely, increased HDAC2 prevents a cytokine inflammatory response by maintaining NF-κB deacetylated, and subsequently, promoting the translocation of this inactive transcription factor to the cytoplasm. In addition, monocyte-derived macrophages obtained from COPD patients showed a significant correlation between HDAC2 and the nuclear factor erythroid 2-related factor 2 (Nrf2). This factor plays a crucial role in cellular defense against oxidative stress. Thus, reduced HDAC2 activity in COPD may account for reduced Nrf2 stability and impaired antioxidant defenses (Mercado et al. 2011a). Furthermore, HDAC2 can be downregulated by several factors that induce COPD. For instance, oxidative stress as a result of cigarette smoking is an important etiologic factor in the pathogenesis of COPD. In macrophages, cigarette smoke is known to hinder HDAC2 which plays an important role in COPD-associated inflammation. Moreover, acrolein, a reactive electrophile found in cigarette smoke mimics many of the toxic effects of cigarette smoke exposure in the lungs. Particularly, incubation of recombinant HDAC2 with acrolein leads to the formation of an HDAC2-acrolein adduct and consequently to its inactivation (Randall et al. 2016). Finally, activation of the hypoxia-inducible factor 1 alpha (HIF-1α) by hypoxia decreases HDAC2 levels, resulting in amplified inflammation in COPD (Charron et al. 2009).

Role of Histone Deacetylases in Monocyte Function in Health and Chronic. . .

2.2.2

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HDAC2 Contributes to the Anti-inflammatory Action of Corticosteroids

Corticosteroids suppress inflammatory genes in inflammatory lung diseases by recruiting HDAC2 to the NF-κB-activated inflammatory gene complex and keeping it inactive. Thus, HDAC2 is a prerequisite molecule for the anti-inflammatory action of corticosteroids. Indeed, corticosteroid insensitivity, a major therapeutic problem for COPD, has been suggested to be caused by reduced HDAC2 activity and expression (Cosio et al. 2004; Ito et al. 2005). Conversely, recent studies show that inhibition of PI3Kδ restores HDAC2 activity and corticosteroid function in cells from COPD patients (Marwick et al. 2010; To et al. 2010). Thus, increasing HDAC2 activity appears to be a promising approach to reverse this corticosteroid resistance. This might be achieved by existing pharmacological treatments such as macrolide antibiotics, the methylxanthine theophylline, and the tricyclic antidepressant nortriptyline (Barnes 2010, 2013). Firstly, clinical and experimental data indicate that macrolide antibiotics have anti-inflammatory properties independently of their antibacterial effects (Takizawa et al. 1995). Indeed, macrolides reduce airway inflammation (Crosbie and Woodhead 2009) and show beneficial effects on exacerbation of COPD. Specifically, by inhibiting PI3Kδ signaling under oxidative stress macrolides restore HDAC2 activity and consequently steroid sensitivity in COPD patients (Sun et al. 2015). Moreover, macrolides reverse the HDAC2 downregulation induced by cigarette smoke extracts (CSE), which decreases the expression of NF-κB associated inflammatory mediators. Altogether, these results suggest that relieving inflammation with macrolides is a useful therapeutic approach for modulating intracellular nuclear signaling in chronic airway inflammatory diseases such as COPD (Li et al. 2012). Theophylline is an old drug that has long been used for the treatment for asthma and COPD as a bronchodilator. However, more recently, theophylline has been shown to have anti-inflammatory effects at lower plasma concentrations (Ito et al. 2002, 2005). As a bronchodilator, this drug is known to act as a phosphodiesterase inhibitor and an adenosine antagonist. Yet, at low concentrations theophylline potently inhibits PI3Kδ under oxidative stress suggesting that the anti-inflammatory action of this drug is more obvious under conditions of oxidative stress, as in COPD. PI3Kδ inhibition by theophylline would enhance HDAC2 activity which would attenuate airway inflammation and also contribute to restoration of corticosteroid sensitivity (To et al. 2010). The tricyclic antidepressant nortriptyline restores the corticosteroid sensitivity induced by oxidative stress via direct inhibition of PI3Kδ and therefore it is a potential treatment for corticosteroid-insensitive diseases such as COPD and severe asthma (Mercado et al. 2011b). Finally, other drugs known to exert their anti-inflammatory action by upregulation of HDAC2 are the dietary polyphenol curcumin (Meja et al. 2008), the volatile anesthetic isoflurane (Guo et al. 2020), andrographolide (Liao et al.

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2016), and gallic acid (Lee et al. 2015). Specifically, several studies have shown that curcumin maintains HDAC2 activity and expression by interfering with the ubiquitin-proteasome pathway. Thus, curcumin may also have the potential to reverse steroid resistance, which is common in patients with COPD and asthma (Meja et al. 2008).

2.2.3

HDAC2 Contributes to the Anti-inflammatory Action of IL-10

The recruitment of HDAC2 to inflammatory gene promoters represents a mechanism used by IL-10 to suppress inflammatory gene transcription. Remarkably, it has been found that IL-10 recruits HDAC2-dependent deacetylation of histone 4 specifically at the IL8 promoter site. Thus, IL8 transcriptional suppression by IL-10 proceeds through an HDAC2-dependent mechanism. In keeping with this, PBMCs from patients with acute-phase COPD are intrinsically resistant to the ability of IL-10 to suppress IL8 expression because of their reduced intracellular HDAC2 levels. Interestingly, it appears that the mechanisms through which IL-10 suppresses IL8 transcription closely resemble those used by glucocorticoids. Thus, modulating HDAC2 activity might be a novel approach for developing a broader spectrum of anti-inflammatory therapies (Castellucci et al. 2015).

2.2.4

HDAC2 Is a Component of the Host Innate Antiviral Response

In addition, HDAC2 induces innate antiviral response in host cells. In turn, impaired HDAC2 activity has been linked to successful replication and infection of host cells with Influenza A virus (IAV) (Nagesh et al. 2017), rhinovirus (Footitt et al. 2016), and HIV-1 (Lv et al. 2018). The IAV remains one of the most significant human respiratory pathogens. HDAC2, by stimulating the expression of interferon-stimulated genes possesses anti-IAV properties. In turn, IAV downregulates HDAC2 mainly through ubiquitination and proteasome degradation during infection and minimizes its antiviral effect (Nagesh et al. 2017). Rhinovirus infection can trigger significant increases in airway inflammation and can contribute to exacerbations in COPD patients. Rhinovirus infection of monocytes induces oxidative and nitrosative stress leading to reduced HDAC2 activity and upregulation of the inflammatory cytokines IL-6 and IL-8. Indeed, nitrosylation of HDAC2 followed by HDAC2 degradation is a mechanism of reduced HDAC2 activity that correlates inversely with rhinovirus load. Interestingly, reduced HDAC2 may contribute to corticosteroid resistance in stable or exacerbated COPD. Thus, enhancing HDAC2 activity through inhibiting virus-induced oxidation and nitrosylation could have the potential to improve the therapeutic effect of corticosteroids in COPD exacerbations (Footitt et al. 2016). Macrophages are main target cells for HIV-1 replication. Persistently elevated levels of IL-6 in macrophages is a pathological hallmark of HIV-1 infection.

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Fig. 2 Main roles of HDAC2 in monocyte function in healthy and disease states. HDAC2 prevents the inflammatory response induced by inhibiting the NF-κB pathway and by binding IL-6 and IL-8 promoters and repressing the expression of these inflammatory cytokines. COPD, cigarette smoke, and rhinovirus activate PI3Kδ-dependent HDAC2 phosphorylation and subsequent proteasome degradation. HIF-1α, IAV, and acrolein also stimulate HDAC2 proteasome degradation. Further, HDAC2 downregulation by HIV-1 favors cytokine expression and viral replication. On the other hand, corticosteroids suppress inflammatory genes by recruiting HDAC2 to the NF-κB complex. Pharmacological treatments such as macrolides, theophylline, and nortriptyline inhibit the PI3Kδ pathway and upregulate HDAC2. Curcumin maintains HDAC2 activity by interfering with the ubiquitin-proteasome pathway

Blocking IL-6 signaling prevents both HIV-1 replication and inflammation. HDAC2 binds to the IL-6 promoter and inhibits its expression. During early HIV-1 infection, 4 h after infection, HDAC2 binding to the IL-6 promoter is dramatically reduced leading to increased histone 3 acetylation and IL-6 production (Lv et al. 2018). A summary of the main functions regulated by HDAC2 in monocytes in healthy and disease states is given in Fig. 2. In addition, Table 2 summarizes the most outstanding studies and experimental models describing the role of HDAC2 in monocyte function in COPD and in viral infection. In addition, Table 3 summarizes relevant studies and experimental models used showing pharmacological treatments that enhance HDAC2 activity and therefore can restore the anti-inflammatory action of corticosteroids in COPD. All these studies demonstrate that activation of HDAC2 in the lungs appears to be a promising pharmacological strategy in the successful treatment of COPD and also in acute respiratory infections. Given that prolonged corticosteroid treatment could

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Table 2 Role of HDAC2 in the regulation of cytokine inflammatory response in COPD and in the host innate defense against viral infections Illness COPD

Main findings HDAC2 activity and expression are markedly reduced in COPD alveolar macrophages and PBMCs Oxidative/nitrosative stress present in COPD activates the PI3Kδ pathway and reduces HDAC2 activity HIF-1α upregulation induced by COPD decreases HDAC2 levels and amplifies inflammation Cigarette smoke decreases HDAC2 activity and increases inflammatory cytokines Acrolein from cigarette smoke, forms an HDAC2acrolein adduct inactivating HDAC2

Viral infection Influenza A HDAC2 is a component of the virus (IAV) IAV-induced host innate antiviral response Rhinovirus Reduced HDAC2 activity infection inversely correlates with virus load, inflammatory markers, and nitrosative stress HIV-1

HIV-1 replication correlates with reduced recruitment of HDAC2 to the IL-6 promoter, thus enhancing the pro-inflammatory cytokine IL-6

Model used Broncho alveolar macrophages from COPD patients PBMCs from COPD patients U937 and A549 cell lines

Reference (Ito et al. 2005)

(Tan et al. 2016) (Meja et al. 2008; Osoata et al. 2009)

A549 and U937 cell lines and macrophages from lung resection

(Charron et al. 2009)

PBMCs from smokers U937 exposed to cigarette smoke U937 differentiated to macrophage-like cells

(Meja et al. 2008; Li et al. 2012; Sun et al. 2015; Tan et al. 2016) (Randall et al. 2016)

A549 cell line

(Nagesh et al. 2017)

Sputum and bronchoalveolar macrophages from COPD patients and THP-1 cell line Primary macrophage cell cultures from HIV-1 patients

(Footitt et al. 2016)

(Lv et al. 2018)

U937 is a human monocytic cell line derived from a lymphoma patient. THP-1 is a human monocytic cell line derived from an acute monocytic leukemia patient. A549 is a human alveolar epithelial cell line obtained from human lung carcinoma

compromise the host defense, a combined therapy using HDAC2 activators or PI3Kδ inhibitors has been proposed in many of the studies described above. Perhaps, all this knowledge is telling us that a new drug that stimulates HDAC2 activity locally in the lungs could be developed.

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Table 3 Pharmacological treatments that increase HDAC2 activity and could restore antiinflammatory action of corticosteroids in COPD Pharmacological treatment Corticosteroids

Macrolides (erythromycin, clarithromycin, and azithromycin)

Theophylline

Nortriptyline

Curcumin

Main findings Reduced HDAC2 activity and expression leads to corticosteroid insensitivity in COPD Erythromycin restores the HDAC2 reduction observed in PBMCs of COPD patients Macrolides restore HDAC2 activity via PI3Kδ inhibition Theophylline increased HDAC2 by inhibiting oxidant-activated PI3Kδ Theophylline increases HDAC2 activity and reduces inflammatory response induced by COPD and Haemophilus influenzae Nortriptyline restored HDAC2 activity decreased by oxidative stress and CSE via PI3Kδ inhibition Curcumin maintains HDAC2 activity and reverses steroid insensitivity induced by CSE and oxidative stress

Model used Alveolar macrophages

Reference (Cosio et al. 2004)

PBMCs from COPD patients U937 cell line PBMCs from COPD patients U937 cell line PBMCs from COPD patients

(Sun et al. 2015)

Alveolar macrophages from COPD patients and U937 cell line U937 cell line

AEC II in a rat COPD model U937 cell line

(Kobayashi et al. 2013) (To et al. 2010) (Cosío et al. 2015)

(Mercado et al. 2011b) (Gan et al. 2016) (Meja et al. 2008)

U937 is a human monocytic cell line derived from a lymphoma patient. AEC II is a type II alveolar epithelial cell line

2.3

Role of HDAC3 in Monocyte Function in Healthy and Disease States

The class I HDAC3 enzyme can shuttle between the cell nucleus and the cytoplasm. HDAC3 preferentially deacetylates histone 4 at lysine residues 5, 12, 14, and 16 (H4K5/12/14/16ac) and by doing so HDAC3 affects the accessibility of several transcription factors to the DNA (Johnson et al. 2002; Yang et al. 2002). In addition, HDAC3 deacetylates non-histone proteins. Among these, the HDAC3 regulation of the NF-κB transcription factor is especially relevant for monocyte function (Chen and Greene 2004).

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R. M. Tordera and M. Cortés-Erice

HDAC3 Regulates Monocyte Differentiation

HDAC3 is involved in monocyte differentiation to dendritic cells and to pro-inflammatory M1-like macrophages. At the same time, HDAC3 prevents macrophage polarization into an M2-like anti-inflammatory phenotype. Specifically, deacetylation of lysine residue 16 of histone 4 (H4K16ac) by HDAC3 might be involved in this action. Conversely, treatment with the specific HDAC3 inhibitor apicidin increases H4K16 acetylation and triggers monocyte differentiation into an M2 macrophage-like phenotype (Nicholas et al. 2015). In agreement with this finding, other studies show that HDAC3-deficient macrophages are unable to activate a large part of the inflammatory gene expression program and display an M2-like anti-inflammatory phenotype (Mullican et al. 2011). Altogether, these studies suggest that development of selective HDAC3 inhibitors that specifically modulate histone H4K16 acetylation could open up a new avenue for the monocyte immune therapy of chronic autoimmune-inflammatory diseases based on reprogramming mechanisms.

2.3.2

HDAC3 Keeps NF-κB in an Active State Thus Playing a Key Role in Inflammatory Diseases

HDAC3 ensures that NF-κB is kept in a primarily deacetylated and thus active state. The NF-κB transcription factor plays an important role in chronic inflammatory diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease, or COPD (Neurath et al. 1998; Collins and Cybulsky 2001; Tak and Firestein 2001). In mammalian cells, the NF-κB family of transcription factors consists of five members: p65, RelB, c-Rel, p50, and p52. These proteins are always found in heterodimeric or homo-dimeric complexes. NF-κB formed by the p65/p50 heterodimer is highly frequent and participates in a variety of physiologic processes including differentiation, proliferation, inflammation, and cell survival (Ghosh and Karin 2002). Interestingly, transcriptional activity of the NF-κB heterodimer can be differently modulated by acetylation of seven specific lysine residues of the p65 subunit (Kiernan et al. 2003). HDAC3 is generally an important activator of inflammatory gene expression associated with NF-κB. HDAC3 is able to deacetylate p65 at lysines 122, 123, 314, and 315 and is a positive regulator of NF-κB activity. Particularly deacetylation of lysines 122 and 123 by HDAC3 favors NF-κB binding to DNA to initiate transcription. Thus, selective inhibition of HDAC3 inhibits pro-inflammatory gene expression and appears a promising therapeutic approach for inflammatory diseases (Tak and Firestein 2001). In rheumatoid arthritis, increased HDAC3 participates in monocyte recruitment to sites of inflammation and induces macrophage cytokine production. In contrast, as a result of the inhibition on the NF-κB pathway, the class I and pan HDAC inhibitors, MS-275 and SAHA respectively, suppress downstream transcription of

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inflammatory cytokines. As these drugs effectively inhibit HDAC3, acetylation of the NF-κB p65 subunit at lysines 122 and 123 will be enhanced and subsequently NF-κB will reduce its DNA binding affinity being exported to the cytoplasm. Thus, suppression of the NF-κB HDAC3-dependent pathway appears an interesting pharmacological strategy for rheumatoid arthritis (Choo et al. 2013). Atherosclerosis is a lipid-driven disease that involves chronic inflammation. Monocytes and macrophages accumulate cholesterol by the uptake of oxidized low-density lipoproteins (oxLDL) through Lox-1 receptors, becoming foam cells and acquiring a pro-inflammatory phenotype (Moore et al. 2013). HDAC3 is upregulated in unstable human atherosclerotic plaques (Hoeksema et al. 2014) and correlates with macrophage activation markers. Conversely, macrophage-specific HDAC3 deletion improves lipid handling in atherosclerotic plaques and induces a more stable plaque phenotype (Hoeksema et al. 2014). In addition, selective HDAC3 inhibitors exert antiatherogenic effects in vitro (Van Den Bossche et al. 2014). Therefore, the ideal drug for atherosclerosis therapy would be a selective macrophage-specific HDAC3 inhibitor. To date, such a drug is not currently available.

2.3.3

HDAC3 Promotes Macrophage Adaptation to Enteric Microbiota and Prevents Bowel Inflammatory Disease

HDAC3 is able to prevent cytokine expression (IL-1β and TNFα) in lipopolysaccharide (LPS) activated monocytes. Specifically, in activated pro-inflammatory intestinal monocytes, HDAC3 has been suggested to deacetylate p65 at lysine 310, which in this case would promote nuclear export of the NF-κB transcription factor and subsequent inactivation (Leus et al. 2016). In line with these studies, silencing HDAC3 in human macrophages has been shown to increase the production of IL-1β and IL-8 in response to LPS stimulation (Winkler et al. 2012). In agreement with these findings, within an intestinal inflammatory microenvironment, HDAC3 appears to be upregulated in human intestinal macrophages and co-participates with HDAC1 to promote immunosuppressive effects and macrophage adaptation (Sun et al. 2019). Specifically, it has been observed that macrophage exposure to commensal bacteria upregulates HDAC3 and the antiinflammatory cytokine IL-10, which cooperates to inhibit the synthesis of the IL-12 cytokine through H4 deacetylation at its promoter site (Kobayashi et al. 2012). Thus, HDAC3 participates in the reprogramming of an anti-inflammatory phenotype of intestinal macrophages. Indeed loss of macrophage tolerance to the enteric microbiota plays a central role in the pathogenesis of inflammatory bowel disease.

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HDAC3 Downregulation in Monocytes Mediates Japanese Encephalitis Virus (JEV) Infection

The most important function of peripheral macrophages is to scavenge viral antigens and induce a first line of defense for the host. Yet, sometimes the virus is capable of activating regulatory systems that prevent inflammation-induced destruction of the host. HDAC3 downregulation in monocytes has been shown to contribute to a viral immune evasion strategy occurring in Japanese encephalitis virus (JEV) infection. Decreases in HDAC3 induced by JEV are associated with the inhibition of NF-κB activity and therefore with a decrease of inflammatory responses in macrophages (Adhya et al. 2013). Consequently, the antiviral and inflammatory reaction does not take place in JEV infected macrophages, which would be required for the virus to be killed. JEV takes advantage of this mechanism to “coexist” with the host and enables it to survive. Sadly, this viral immune evasion strategy allows it to spread to the brain before it can be detected by the peripheral immune system in the early stages of the disease (Adhya et al. 2013).

2.3.5

HDAC3 Inhibition Favors Tumor Cell Survival and Growth

The activation status of NF-κB transcription factor plays a critical role in regulating cancer cells. Various stimuli, including DNA damage present in tumor cells activate kinases that promote NF-κB nuclear translocation and transcription of a number of genes that activate cell proliferation and prevent apoptosis. Generally, in tumor cells, it has been suggested that p65 acetylation at lysine 310 results in NF-κB activation that favors tumor cell survival and growth. Importantly, the anti-tumor properties of the pan HDAC inhibitors such as trichostatin A are counteracted by NF-κB activation through p65 acetylation at lysine 310 (Mayo et al. 2003). Therefore, an ideal drug able to deacetylate NF-κB at this specific site would be expected to potentiate the anti-tumor action of non-selective HDAC inhibitors (Dai et al. 2005). As a summary, Fig. 3 shows the main roles that HDAC3 plays in monocyte function in healthy and disease states. In Table 4 we summarize several outstanding studies describing the role of HDAC3 in monocyte function in healthy and disease states including also the main findings and experimental models used.

2.4

Role of HDAC8 in Monocyte Function in Health and Disease

HDAC8 is a class I nuclear HDAC that exerts mainly a pro-inflammatory function through activation of PI3K/Akt signaling (Ha et al. 2017). Selective HDAC8 inhibition by ITF3056 (an analogue compound of givinostat) reduces LPS-induced pro-inflammatory cytokine production in vivo and in vitro in PBMCs and with low

Role of Histone Deacetylases in Monocyte Function in Health and Chronic. . .

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Fig. 3 HDAC3 plays a key role in monocyte function in healthy and disease states. HDAC3 triggers M1 macrophage and dendritic cell differentiation through deacetylation of H4K16ac. Moreover, HDAC3 is a positive regulator of the inflammatory response induced by the NF-κB pathway playing a key role in rheumatoid arthritis and atherosclerosis. Therefore, HDAC3 inhibition offers potential therapeutic value to treat these illnesses by inhibiting the NF-κB dependent inflammatory response and foam cell formation. However, HDAC3 inhibition favors tumor cell survival by upregulation of the acetylated lysine 310 of the p65 NF-κB subunit. Moreover, HDAC3 downregulation by Japanese encephalitis virus favors viral evasion and infection. On the other hand, HDAC3 can also favor macrophage adaptation to enteric microbiota by deacetylation of histones at the IL-12 promoter

levels of toxicity (Li et al. 2015). More recently, the novel HDAC8 inhibitor WK2–16 was found to improve the resolution of sepsis in vivo by reducing hypercytokinemia. In addition, in THP-1 cell lines, this compound reduced MMP9, TNFα, and IL-1β (Jan et al. 2017). In some myeloid leukemia HDAC8 upregulation deacetylates and inactivates p53, leading to persistence of the leukemia and drug resistance. Thus, HDAC8 inhibition in combination with anti-cancer drugs has been suggested as a promising strategy to treat leukemia (Qi et al. 2015; Long et al. 2020).

3 Role of Class II HDACs in Monocyte Function Class II HDACs, divided into two subclasses, class IIa (HDACs 4, 5, 7, and 9) and class IIb (HDAC 6 and 10), shuttle between the nucleus and the cytoplasm, through a mechanism regulated by kinase phosphorylation with the exception of HDAC6,

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Table 4 Role of HDAC3 in monocyte function in healthy and disease states Illness/process Monocyte differentiation

Rheumatoid arthritis (RA)

Atherosclerosis

Activated monocytes Tolerance to enteric microbiota

JEV infection

Myeloid leukemia

Main finding HDAC3 deacetylates H4K16 and promotes monocyte differentiation to M1 macrophages and dendritic cells Inhibition of HDAC3 by apicidin increases H4K16 acetylation and triggers monocyte differentiation to an M2 macrophage-like phenotype The class I HDAC inhibitors MS-275 and SAHA suppress LPS-induced NF-κB p65 nuclear accumulation and cytokine secretion HDAC3 is upregulated in unstable atherosclerotic plaques HDAC3 deletion induces a switch from M1 to M2 macrophages and improves lipid handling inducing a more stable plaque Smoke exposure reduces HDAC3 activity in macrophages, resulting in enhanced cytokine production Ongoing microbial exposure upregulates HDAC3 in macrophages and reprograms these cells into an immunosuppressive phenotype IL-10 mediated IL-12 inhibition through histone deacetylation by HDAC3 maintains the antiinflammatory phenotype of colonic macrophages

HDAC3 downregulation by JEV-infection in macrophages inhibits NF-κB activity The class I HDAC inhibitor MS-275 and pan HDAC inhibitor SAHA promote the accumulation of acetylated p65 at lysine 310 leading to NF-κB activation which promotes the survival of tumor cells

Model used Human monocyte cultures from healthy donors U937 cell line

Reference (Nicholas et al. 2015)

E11 cell line THP-1 cell line RAW264.7 cell line

(Choo et al. 2013)

RNA samples of unstable atherosclerotic plaques Conditional myeloid HDAC3 knockout mice

(Hoeksema et al. 2014)

Alveolar macrophages derived from PBMCs

(Winkler et al. 2012)

Human monocytederived macrophages

(Sun et al. 2019)

Mouse bone marrow– derived macrophage (BMDMs) cells Intestinal lamina propria mononuclear cells (LPMCs) from mice RAW264.7 cell line

(Kobayashi et al. 2012)

U937 cell line HL-60 cell line

(Adhya et al. 2013) (Dai et al. 2005)

U937 is a human monocytic cell line derived from a lymphoma patient. HL-60 is a promyelocytic cell line derived from human leukemia. THP-1 is a human monocytic cell line derived from an acute monocytic leukemia patient. E11 is a cell line developed from human rheumatoid arthritis synovial fibroblasts. RAW264.7 is a macrophage-like cell line derived from Balb/c mice

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which is found predominantly in the cytoplasm (Mathias et al. 2015). Nucleocytoplasmic shuttling induced by phosphorylation is a finely tuned system for regulation of Class II HDAC transcriptional function in the cells.

3.1

Regulation of Class IIa HDAC Transcriptional Activity by Nucleocytoplasmic Shuttling

Class IIa HDACs have two differentiated regions in the C and the N-terminal regions. The C terminus region contains a conserved deacetylase domain and the nuclear export sequence (NES), which binds to chaperon proteins that facilitate cytoplasmic accumulation. The N-terminal region contains a nuclear localization sequence (NLS) and binding sites for transcription factors. Through a mechanism highly conserved across all class IIa HDACs, phosphorylation at specific serine residues initiate 14-3-3 chaperone protein binding (McKinsey et al. 2001) and shuttling to the cytoplasm. For instance, HDAC5 phosphorylation of the serine residues S259 and S498 promotes HDAC5 binding to this chaperone and nuclear export. The calmodulin kinase (CamK) superfamily or the protein kinase D (PKD) is known to phosphorylate HDAC5 at these serine sites (Chang et al. 2008a). The protein kinase C (PKC) can also phosphorylate directly HDAC5 serine residue S259. On the other hand, phosphorylation of HDAC5 in serine residue S279 by protein kinase A (PKA) (Ha et al. 2010) or cyclin-dependent kinase 5 (CDK5) (Taniguchi et al. 2012) is critical for the nuclear localization of HDAC5. Further, the protein phosphatase 2A (PP2A) family is involved in the dephosphorization of Class IIa HDACs promoting its nuclear accumulation (Paroni et al. 2008). In the nucleus, the HDAC IIa family binds to the transcription factor myocyte enhancer factor-2 (MEF2) (Mathias et al. 2015) and represses the expression of many target genes. MEF2 recruits class II HDACs to responsive genes and forms a repressor complex of gene expression (Aude-Garcia et al. 2010). But when HDACs are shuttled to the cytoplasm upon phosphorylation, MEF2 is released and it forms a new complex, which origins transcriptional activation (Aude-Garcia et al. 2010). Thus, by spatially excluding HDACs from the nucleus, MEF2 initiates gene expression. MEF2 is expressed in human primary monocytes and in macrophages. This factor is upregulated during macrophage differentiation and forms complexes with class II HDACs. Particularly relevant is the HDAC4-MEF2 interaction in triggering monocyte differentiation from myeloid progenitor cells and into macrophages (Aude-Garcia et al. 2010). Indeed, a deregulation of this interaction has been often observed in acute myeloid leukemia (Tarumoto et al. 2018).

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Role of Class IIa HDACs in Inflammatory Processes, Host Defense, and Cancer

Class IIa HDACs are involved in monocyte inflammatory processes and macrophage activation. Of these, the key roles for HDAC4 or HDAC5 enzymes in monocyte activation in inflammatory illness such as rheumatoid arthritis or atherogenesis are well documented. HDAC4 is known to exert an anti-inflammatory action in rheumatoid arthritis by forming a complex with the Krüppel-like Factor 2 (KLF2) transcription factor (Kumar et al. 2005). Specifically, KLF2 is a transcription factor that negatively regulates monocyte activation and osteoclast differentiation directing them towards a quiescent state. KLF2 regulates the activity of monocytes, induces the expression of anti-inflammatory genes, and suppresses transcription of NF-κB genes such as IL-1β and TNFα. During inflammation, KLF2 binds to HDAC4 and acts as a potent inhibitor of NF-κB and AP1 transcription factors, as well as hypoxia-related HIF-1α protein. Further, NF-κB downregulates KLF2, so we can say they are reciprocal antagonists (Kumar et al. 2005). Conversely, there is a direct involvement of HDAC5 in the pro-inflammatory capacity of macrophages, in rheumatoid arthritis, via activation of the transcription factor NF-κB. HDAC5 upregulation is associated with a significant increase of TNFα and MCP-1 mediated by activation of the NF-κB pathway. Moreover, HDAC5-dependent activation of NF-κB in rheumatoid arthritis has been associated with pathological osteoclastic activity (Cantley et al. 2011). Consistent with this, HDAC5 knockdown suppresses cytokine production and bone loss in murine models of arthritis (Poralla et al. 2015). In atherogenesis, nuclear HDAC5 has been shown to repress the activity of the transcription factor KLF2 leading to a decrease in the expression of genes involved in lipid handling, which reduces cholesterol efflux and favors foam cell formation (Wang et al. 2010; Kwon et al. 2014). Interestingly, HDAC9, another Class IIa HDAC, also becomes upregulated during macrophage differentiation, contributing to the expression of pro-inflammatory genes (Neele et al. 2015). Thus, HDAC5 inhibition offers potential therapeutic value for the treatment of chronic autoimmune and/or inflammatory diseases such as rheumatoid arthritis and atherosclerosis. On the other hand, HDAC5 plays a central role in host defense in infections. Following bacterial invasion in macrophages, HDAC5 promotes inflammation through NF-κB activation, secreting pro-inflammatory substances. For instance, lung infection with Legionella pneumophila (Schmeck et al. 2008) or Mycoplasma pneumoniae (Zhao et al. 2019) acutely upregulates HDAC5 in human alveolar epithelial cells which triggers inflammation. Yet, after 24 h, Mycoplasma pneumoniae infection in humans has been shown to reduce HDAC5 at protein and mRNA levels in PBMCs. Reduction of HDAC5 in monocytes weakens the production of inflammatory substances and results in one strategy through which the pathogen avoids the host immune response. Thus, restoration of HDAC5 has been

Role of Histone Deacetylases in Monocyte Function in Health and Chronic. . .

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Fig. 4 Nucleocytoplasmic shuttling of HDAC5 regulates monocyte inflammatory response. HDAC5 phosphorylation by different kinases at serine residues S259 and S498 promotes its nuclear export. In contrast, phosphorylation of HDAC5 in serine residue S279 by other kinases or the protein phosphatase 2A (PP2A) is critical for its nuclear localization. Nuclear HDAC5 acts as a positive regulator of the inflammatory response by activation of the NF-κB pathway or repression of the transcription factors MEF2 and KLF2. Nuclear HDAC5 is upregulated in rheumatoid arthritis and atherosclerosis. Further, HDAC5 downregulation in monocytes 24 h post-infection is a strategy used by bacteria to avoid the host immune response

proposed as a therapeutic intervention for Mycoplasma pneumoniae infection (Zhao et al. 2018). As a summary, Fig. 4 shows the main roles that HDAC5 plays in monocyte function in healthy and disease states. Finally, a recent study has revealed that selective inhibition of Class IIa HDACs could be a promising approach to reprogram monocyte/macrophages and activate in them a robust anti-tumor immune response. Tumor-associated macrophages contribute to cancer progression because they can stimulate angiogenesis and promote tumor cell migration and invasion. Therefore, pharmacological strategies directed towards macrophage reprogramming could be beneficial to enhance cancer therapy. A relevant experimental study has shown that the novel Class IIa HDAC inhibitor TMP195 reduces tumor burden and metastases by modulating macrophages towards a type 1 pro-inflammatory and highly phagocytic phenotype. Furthermore, combining TMP195 with chemotherapy regimens or T-cell checkpoint blockade significantly enhances the durability of tumor reduction. Thus, the inhibition of the Class IIa HDAC subfamily appears to be an interesting pharmacological strategy to improve cancer therapy (Guerriero et al. 2017).

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The activity of class IIa HDAC can be modified by phosphorylation at different sites. It is well known that many drugs used in the clinic bind to cell membrane receptors that initiate intracellular signaling that involves kinases enzymes. Therefore, many drugs commonly used could be potentially able to regulate the activity of these HDACs in monocytes and subsequently to affect their inflammatory response. The potential beneficial or negative interaction of some of these drugs in this system could well be worth exploring.

3.3

Role of the Class IIb HDAC6 in Macrophage Migration in Inflammatory Processes, Host Defense, and Tumor Growth

Of the two Class IIb HDACs, HDAC6 and HDAC10, HDAC6 is best characterized in monocytes. HDAC6 is the largest member of the HDAC family with 1,215 amino acid residues. Expressed primarily in the cytoplasm, it has both important cytoplasmic and nuclear functions. Besides histones, HDAC6 has a unique substrate specificity for non-histone proteins, such as α-tubulin, Hsp90, and cortactin. HDAC6 hence plays a key role in microtubule dynamics, cell shape, division, and migration as well as intracellular transport. In summary, HDAC6 has recently been identified as an important regulator of cytoskeletal dynamics (Hubbert et al. 2002). HDAC6 induces the expression of inflammatory cytokines, in addition to other pro-inflammatory factors, such as MCP-1, that promote inflammation in macrophages (Youn et al. 2016). Moreover, HDAC6 induces the expression of type I interferons in macrophages (Zhu et al. 2011) participating in the host defense against viral and bacterial infection. However, HDAC6 has a primary role in macrophage migration in inflammatory processes. Specifically, it has been shown that LPS challenge induces translocation of HDAC6 from the cytosol to the cell periphery, where it deacetylates cortactin and thereby facilitates formation of filopodia, critical for macrophage migration. Further, the correct functioning of microtubules is required for the secretion of the pro-inflammatory cytokine IL-1β. Thus, blocking IL-1β exocytosis caused by HDAC6 inhibition has been suggested as a potential anti-inflammatory strategy (Carta et al. 2006). Recruitment of monocytes/macrophages at sites of infection where they can phagocytose pathogens and necrotic tissue makes a large contribution to infection clearance (Crane et al. 2009). Moreover, sustained infiltration of monocytes/macrophages at sites of inflammation and subsequent differentiation into inflammatory macrophages are also associated with cancer and a number of chronic autoimmuneinflammatory diseases. Thus, targeting macrophage infiltration is a phenomenon that may represent an effective therapeutic strategy for the treatment of cancer, in addition to inflammatory and immune disorders. Specifically HDAC6 has been proposed as a target for antileukemic drugs in acute myeloid leukemia by inhibiting the acetylation of α-tubulin (Hackanson et al. 2012). In addition, HDAC6 inhibition

Role of Histone Deacetylases in Monocyte Function in Health and Chronic. . .

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reduces the suppressive activity of myeloid-derived suppressor cells (MDSCs) (Hashimoto et al. 2020). Currently, several HDAC6 inhibitors are being developed (Valente et al. 2013) or are under clinical investigation for treatment of cancers, such as multiple myeloma and breast cancer (Valente et al. 2011; Tu et al. 2014; Laubach et al. 2017).

4 Role of Class III HDAC in Monocyte Function Class III HDACs, more commonly called sirtuins, are a highly conserved family of NAD+-dependent enzymes. Sirtuins are capable of deacetylating lysines from histone and non-histone proteins. They are essential in many biological functions such as cellular stress resistance, cell motility and survival, genomic stability, inflammation, carcinogenesis, and metabolism. This family includes seven members: some of which are located mainly in the cell nucleus (SIRT6 and 7), while others are mitochondrial (SIRT3, 4, and 5), SIRT1 and 2 can be found both in the nucleus and the cytoplasm (Dali-Youcef et al. 2007; Yanagisawa et al. 2018). While SIRT1 is mainly located in the nucleus, it can shuttle between cytoplasm and nuclei in some cell types (Yanagisawa et al. 2018).

4.1

Role of SIRT1 in Monocyte Function

Many studies show that SIRT1 and SIRT6 play a key role in the regulation of a monocyte inflammatory response and participate in a number of inflammatory and metabolic illnesses as well as in host defense (Salminen et al. 2008; Kauppinen et al. 2013). Some of these relevant studies are documented here.

4.1.1

SIRT1 Supports Anti-inflammatory Responses by Inhibiting the NF-κB and AP-1 Pathways

At the molecular level, SIRT1 promotes transcriptional repression through histone deacetylation. Upon its recruitment to chromatin, SIRT1 directly deacetylates histone 4 lysine 16 (H4K16) and histone 3 at lysine’s 9 and 14 (H3K9/14) (Liu et al. 2011). In addition, SIRT1 inhibits NF-κB-dependent signaling: specifically, SIRT1 deacetylates lysine 310 of the p65 protein in the NF-κB complex and therefore inhibits NF-κB-associated transcription. In addition, SIRT1 favors p65 methylation at lysine’s 314/315 enhancing its ubiquitination and degradation (Yeung et al. 2004). Further, SIRT1 also recruits the de novo induced RelB NF-κB subunit forming a transcription repressor complex (Liu et al. 2011). Thus, SIRT1 activation could be a pharmacological strategy to treat a multitude of NF-κB-driven inflammatory

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disorders. Conversely, activation of the NF-κB pathway has been shown to downregulate SIRT1 expression and activity (Kauppinen et al. 2013). In addition, SIRT1 suppresses the transcriptional activity of AP-1 causing a reduction in cyclooxygenase 2 (COX-2) gene expression and subsequently prostaglandin E2 (PGE2) or protein inflammatory cytokines levels such as IL-6, MIP-1a, and IL-1b (Zhang et al. 2010; Annamanedi and Kalle 2014).

4.1.2

SIRT1 Inhibits Apoptosis Through Deacetylation of the FOXO3 Transcription Factor

The forkhead box (FOXO) class transcription factor family is regulated by a wide range of external stimuli including nutrients, cytokines, and oxidative stress. Among the FOXO family, SIRT1 deacetylates FOXO1, FOXO3, and FOXO4 and represses their transcriptional activity (Motta et al. 2004). In healthy states, FOXO3 is maintained in a deacetylated state largely through the activity of SIRT1. In particular, SIRT1 inhibits the ability of FOXO3 to induce apoptosis (Li et al. 2017b) enhancing cell survival together with resistance to oxidative stress. However, SIRT1-mediated cell survival may also be caused by deacetylation of other transcription factors such as p53 (Vaziri et al. 2001). LPS induces monocyte activation in vitro and in vivo and causes SIRT1 downregulation leading to an increase of FOXO3 acetylation and subsequent apoptosis. Conversely, SIRT1 overexpression in monocyte cultures deacetylates FOXO3 and blocks apoptosis in response to LPS (Li et al. 2017b). In line with these results, resveratrol, the well-known SIRT1 activator, is recognized as an antiapoptosis and anti-inflammatory compound that can have beneficial effects in various chronic inflammatory diseases (Elmali et al. 2007; Lee et al. 2011) such as rheumatoid arthritis and diabetes. Interestingly, in alcoholic hepatitis patients, there is an increase of circulating monocytes and tissue macrophages. SIRT1 upregulation in peripheral blood monocytes of these patients has been proposed as a protective mechanism against the inflammatory response by inhibiting FOXO3 activation and the subsequent apoptotic response (Li et al. 2017b).

4.1.3

SIRT1 Is Regulated in Different Inflammatory and Metabolic Diseases

SIRT1 appears to be highly regulated in rheumatoid arthritis, atherosclerosis, diabetes, and COPD. Several studies have shown that SIRT1 regulates synovial inflammation in rheumatoid arthritis by preventing monocyte differentiation to inflammatory macrophages. SIRT1 suppresses NF-κB-dependent inflammatory signaling by suppressing TNFα, IL-1β, and IL-6 in human articular macrophages (Park et al. 2016). In keeping with this, myeloid deletion of SIRT1 in a mouse model of inflammatory arthritis enhances macrophage M1 polarization and increases the

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expression of pro-inflammatory cytokines via NF-κB activation (Hah et al. 2014). These findings suggest that activators of SIRT1 offer a possible therapeutic strategy for the treatment targeting rheumatoid arthritis synovial inflammation. Activation of monocytes/macrophages and aggregation are critically implicated in the inflammatory process of atherosclerotic plaque formation. SIRT1 activation in monocytes has been recognized as a new regulator of homoeostasis in the human cardiovascular system that prevents stress-facilitated inflammation and cell senescence (Ota et al. 2007). SIRT1 expression levels are repressed in coronary artery disease monocytes compared to those from healthy subjects (Chan et al. 2017). In addition, patients with high cholesterol show low expression of SIRT1 in circulating monocytes (Song et al. 2011). Importantly, SIRT1 deficiency in monocytes/macrophages enhances oxidative stress, inflammation, and foam cell formation, thereby promoting atherosclerosis and vascular senescence (Kitada et al. 2016). For instance, at the preclinical level, a study using myeloid-specific SIRT1 knockout mice has revealed that animals exposed to a high-fat diet displayed high levels of activated macrophages predisposing them to the development of atherosclerosis (Stein et al. 2010). Conversely, a healthy SIRT1 function in macrophages is sufficient to reduce foam cell formation and exert a protective effect against atherogenesis. At the molecular level, the NF-κB signaling pathway and the liver X receptor (LXR) are important for atherosclerotic plaque formation. Specifically, SIRT1 suppresses the expression of Lox1 receptors in macrophages via inhibition of the NF-κB signaling pathway which prevents OxLDL uptake and subsequent formation of foam cells (Stein and Matter 2011; Stein et al. 2010). Moreover, SIRT1-mediated activation of LXR stimulates cholesterol efflux from cells to high-density HDL lipoproteins. Therefore, the activation of SIRT1 in endothelial cells, monocytes/macrophages should be considered as a novel therapeutic target to prevent atherosclerosis. In line with this, SIRT1 upregulation induced by calorie restriction improves metabolic disorders related to cardiovascular disease (Kitada et al. 2016). In line with this recent studies suggest that increased SIRT1 plasma levels correlate with fat mass loss in obese patients (Mariani et al. 2015). In type 2 diabetes patients, decreased SIRT1 levels are found in monocytes and are negatively correlated with fasting plasma glucose plasma (Song et al. 2011) as well as with the inflammatory cytokines IL-6 and TNFα (Li et al. 2016). In addition, SIRT1 expression is decreased in monocytes from patients with insulin resistance (De Kreutzenberg et al. 2010). In contrast, under caloric restriction conditions, SIRT1 expression is upregulated and this is associated with decreased expression of inflammation-related markers in the blood and decreased insulin resistance (Crujeiras et al. 2008). Cigarette smoke mediates alterations in SIRT1 that could be directly linked to the pathogenesis of COPD. Cigarette smoke exposure triggers SIRT1 post-translational covalent modifications which may be irreversible, leading to uncontrolled expression of pro-inflammatory mediators, via activation of the NF-κB pathway in macrophages/lungs of smokers and patients with COPD. Further, SIRT1 reduction leads to acetylation of FOXO3 and the tumor suppressor p53, resulting in lung cell

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senescence and apoptosis. Conversely, the SIRT1 activator resveratrol inhibits cigarette smoke-mediated pro-inflammatory cytokine release (Yang et al. 2007). In contrast, matrix metalloproteinase-9 (MMP9) is a protein involved in the breakdown of extracellular matrix in inflammatory diseases such as COPD (Russell et al. 2002), arthritis (Chang et al. 2008b), cancer metastasis (Van Kempen and Coussens 2002), and also in aging. A study has shown that while SIRT1 reduction under oxidative stress causes MMP9 levels to rise, SIRT1 activation inhibits the increased MMP9 expression. Decreased SIRT1 in the peripheral lungs of COPD patients has been inversely correlated with MMP9 expression. Cigarette smoke exposure also causes a reduction of SIRT1 expression in mice lung tissue with concomitant elevation of MMP9. Interestingly, in this murine model, intranasal treatment of the SIRT1 activator, SRT2172, has been shown to block the increase of MMP9 expression in the lungs. Thus, activation of SIRT1 could be a promising novel strategy for the treatment of COPD in which MMP9 levels are increased (Nakamaru et al. 2009). Further SIRT1 activation plays an important anti-aging role in skin. For instance, natural extracts rich in phlorotannins have been shown to increase SIRT1 activity in epithelial cells and exert a potent inhibitory activity against oxidative stress, inflammation, and senescence. Indeed, phlorotannin rich extracts have been proposed in topical therapeutic formulations against aging (Dutot et al. 2012).

4.1.4

SIRT1 Activation Favors Phagocytosis

SIRT1 activates LC3-associated phagocytosis and by doing so, SIRT1 activation favors the host defense. For instance, SIRT1 enhances the antimicrobial response against Mycobacterium tuberculosis infection (Paik et al. 2019) or reduces infection in peritoneal macrophages (Zhang et al. 2010). On the other hand, SIRT1 activation in Müller glial cells enhances its phagocytosis function and prevents the development of choroidal neovascularization (Ishida et al. 2017).

4.1.5

SIRT1 Favors Endotoxin Tolerance in Macrophages Exposed to Sepsis

Endotoxin tolerance in macrophages is an example of epigenome reprogramming following the acute systemic inflammation associated with sepsis (West and Heagy 2002). It involves repression of acute pro-inflammatory genes and activation of antiinflammatory genes. In particular, in epigenetic reprogramming, endotoxin activates TLR4 signaling which rapidly accumulates nuclear SIRT1 at the promoters of TNFα and IL-1. In addition SIRT1 deacetylates nucleosomal H4K16 and promotes termination of NF-κB-dependent transcription (Liu et al. 2011). Furthermore, in the cytoplasm, SIRT1 enhances autophagy in monocytes/macrophages and exerts a protective effect against the cellular stresses induced by sepsis. Conversely, SIRT1 inhibition leads to autophagy dysfunction, indicated by p62 accumulation and this

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effect is connected with NF-κB signaling activation. All these effects mediated by SIRT1 generate endotoxin tolerance in macrophages (Takeda-Watanabe et al. 2012).

4.1.6

Role of SIRT1 in Aging Related Disease

Pro-inflammatory mechanisms gain increasing importance in the course of senescence. Particularly, aging and various age-related diseases are associated with reductions in SIRT1 activity. In contrast, SIRT1 activation may be a viable target for treatment or prevention of these diseases. For instance, resveratrol has been shown to decrease MMP9 in the cerebrospinal fluid of Alzheimer disease patients and modulates neuro-inflammation and induces adaptive immunity (Moussa et al. 2017). In addition, resveratrol can effectively inhibit lung cancer progression by suppressing the activation of tumor-associated macrophages. Similarly, SIRT1 activation induced by caloric restriction has been shown to delay cancer progression. Further, bone cell senescence is associated with decreased SIRT1 expression (Zhang et al. 2013) and SIRT1 activation plays an important anti-aging role in skin. For instance, natural extracts rich in phlorotannins have been shown to increase SIRT1 activity in epithelial cells and exert potent inhibitory activity against oxidative stress, inflammation, and skin senescence. Indeed, phlorotannin rich extracts have been proposed in topical therapeutic formulations against aging (Dutot et al. 2012). A summary of the main functions regulated by SIRT1 in monocytes is provided in Fig. 5. In Table 5 we summarize several outstanding studies and experimental models describing the role of SIRT1 in monocyte function in several disease states.

4.2

Role of SIRT6 in Monocyte Function

The nuclear sirtuin SIRT6 also exerts a protective anti-inflammatory action against inflammatory, metabolic diseases and infections and might be a potential therapeutic target (Fig. 6).

4.2.1

SIRT6 Regulates Inflammatory and Metabolic Diseases

In general, SIRT6 promotes an anti-inflammatory action by inhibiting the NF-κB pathway. Firstly, SIRT6 interacts with the p65 subunit of NF-κB and inactivates this transcription factor (Fig. 6). In addition, SIRT6 deacetylates H3K9 at the promoter sites of NF-ĸB target genes and reduces inflammatory cytokine expression (Kawahara et al. 2009; Woo et al. 2018). SIRT6 is essential for the development of arthritis and its overexpression exerts a positive effect in collagen-induced arthritic mice (Lee et al. 2013). Particularly, SIRT6 deficiency in macrophages causes more inflammation in rheumatoid arthritis by increasing proliferation, polarization to M1, and infiltration to the synovium. In

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Fig. 5 SIRT1 plays a key role in monocyte function in healthy and disease states. SIRT1 exerts an anti-inflammatory action by inhibition of NF-κB signaling and the AP1 transcription factor. SIRT1 activation inhibits M1 differentiation, inflammatory response, foam cell formation, insulin resistance, osteoporosis, and MMP9 expression. Therefore, SIRT1 offers potential therapeutic value for the treatment of chronic autoimmune and/or inflammatory diseases including rheumatoid arthritis, atherosclerosis, type 2 diabetes, and COPD. In addition, SIRT1 activation exerts an anti-apoptotic and anti-aging effect and enhances autophagy by deacetylation of the FOXO3 transcription factor. Further, SIRT1 activation exerts LC3-dependent phagocytosis and induces antimicrobial action and slows cancer progression. Finally, SIRT1 upregulation induced by septic shock induces endotoxin tolerance by macrophage reprogramming into an anti-inflammatory phenotype

addition to NF-ĸB, SIRT6 is capable of interacting with some FOXO family transcription factors, which are increased in arthritic joints. FOXO1 regulates pro-inflammatory genes and participates in macrophage chemotaxis and migration, contributing to RA development (Woo et al. 2018). SIRT6 deacetylates FOXO1 (Song et al. 2016) and promotes its cytosolic degradation (Fig. 6). SIRT6 can be protective in age-related metabolic diseases, such as obesity, hypercholesterolemia, and diabetes (Moschen et al. 2013). In these diseases SIRT6 downregulation and enhanced NF-ƙB activity and ROS formation have been observed (Kim et al. 2017). SIRT6 regulates the activity of insulin, insulin growth factor 1 (IGF-1), and HIF-α, critical molecules that regulate glucose metabolism (Zhong et al. 2010; Lombard and Miller 2012). Interestingly the administration of sitagliptin, a DPP-4 inhibitor prevents SIRT6 downregulation, which could avert the vascular alterations associated with diabetes. Further, in diabetic nephropathy, where M1 macrophages are activated mainly due to the effect of high glucose levels, SIRT6

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Table 5 Role of SIRT1 in monocyte function in healthy and disease states Illness/process Rheumatoid arthritis (RA)

Atherosclerosis

Alcoholic hepatitis

Diabetes

COPD

Main finding SIRT1 inhibits monocyte to macrophage differentiation by suppressing PU.1 phosphorylation and NF-κB inflammatory signaling Myeloid cell-specific deletion of SIRT1 exacerbates inflammatory arthritis via the hyperactivation of NF-κB signaling SIRT1 expression levels were repressed in monocytes of atherosclerosis patients Monocytic SIRT1 expression in atherosclerosis patients correlated positively with HDL lipoprotein levels SIRT1 prevents from atherosclerosis by inhibiting NF-κB pathway and enhancing liver-X-receptor High expression of SIRT1 in circulating monocytes of alcoholic hepatitis patients prevents apoptosis induced by FOXO3 and subsequent inflammatory response SIRT1 is decreased in high glucose conditions and increased under conditions of calorie restriction SIRT1 is decreased in peripheral lungs of smokers and COPD patients Cigarette smoke decreases levels of SIRT1 associated with NF-kB activation and release of proinflammatory chemokines (IL-8 and TNFα) SIRT1 downregulation is linked to increase in MMP9 expression in COPD

Model used Monocytes from synovial fluid of RA patients THP-1 cell line Mouse bone marrow– derived monocytes Mouse bone marrow– derived monocytes from WT and SIRT1 knockout mice

Reference (Park et al. 2016)

PBMCs from atherosclerosis patients and healthy controls

(Chan et al. 2017)

PBMCs from atherosclerosis patients and healthy controls

(Breitenstein et al. 2013)

U937 cell line

(Zhang et al. 2013)

PBMCs from alcoholic hepatitis patients and healthy controls

(Li et al. 2017a)

Circulating leukocytes from 52 healthy controls and 113 type 2 diabetes mellitus patients Lung samples of nonsmokers, smokers, and patients with COPD MonoMac6 cell line MonoMac6 cell line exposed to CSE

(Song et al. 2011)

Lung tissue and PBMCs from COPD patients

(Nakamaru et al. 2009)

(Hah et al. 2014)

(Rajendrasozhan et al. 2008)

(Yang et al. 2007)

(continued)

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Table 5 (continued) Illness/process

Endotoxin tolerance

Phagocytosis

Cell senescence

Main finding SIRT1 knockdown leads to an increase in MMP9 expression in human monocytes

Model used U937 cell line and sputum macrophages

Cigarette smoke exposure caused reduction of SIRT1 expression in lung tissue of A/J mice, with concomitant elevation of MMP9 Therapeutic intranasal treatment with the SIRT1 activator SRT2172 for 3 days after cigarette smoke exposure significantly inhibited MMP9 expression and also increased SIRT1 activity Activation of SIRT1 by TLR4 inhibits the NF-kB pathway and limits gene transcription of pro-inflammatory cytokine SIRT1 inhibits AP-1 transcriptional activity and subsequent COX-2 expression leading to improvement of phagocytosis and tumoricidal functions Celecoxib inhibits macrophage phagocytosis by activating SIRT1

Murine model of acute cigarette smoke exposure

Sirtuin 1 activation in Müller glial cells suppressed the development of choroidal neovascularization Phlorotannins showed inhibitory activity against oxidative stress, inflammation, and senescence through SIRT1 activation

Reference

Murine model of acute cigarette smoke exposure

Circulating leukocytes from subjects with septic shock and healthy controls THP-1 cell line Murine peritoneal macrophages activated by thioglycollate

Mouse macrophages, RAW 264.7, cells exposed to Staphyloccous aureus bacteria Primary Müller glial cells

U937 cells differentiated into macrophages

(Liu et al. 2011)

(Zhang et al. 2010)

(Annamanedi and Kalle 2014)

(Ishida et al. 2017)

(Dutot et al. 2012)

THP-1 is a human monocytic cell line derived from an acute monocytic leukemia patient. MonoMac6 is a human monocyte-macrophage cell line, which was established from peripheral blood of a patient with monoblastic leukemia. U937 is a human monocytic cell line derived from a lymphoma patient

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Fig. 6 Anti-inflammatory action of SIRT6 in monocytes. SIRT6 exerts an anti-inflammatory action by inhibition of NF-κB signaling. In addition, SIRT6 inhibits macrophage migration by deacetylation of the FOXO1 transcription factor. Thus, SIRT6 activation offers potential therapeutic value for the treatment of chronic autoimmune and/or inflammatory diseases including rheumatoid arthritis, atherosclerosis, and type 2 diabetes. In addition, SIRT6 deacetylates H3K9 at the promoter sites of NF-ĸB target genes and reduces inflammatory cytokine expression. Further, SIRT6 activation induces an antioxidant action by activation of the Nrf2 transcription factor. Finally, SIRT6 activation by sepsis induces macrophage adaptation and tolerance

is capable of polarizing macrophages to M2, causing alleviation in the disease (Ji et al. 2019). Moreover, fisetin and luteolin, two flavonoid compounds, upregulate SIRT6 and exert an antioxidant effect improving diabetes (Kim et al. 2017) (Fig. 6). SIRT6 is an anti-atherosclerotic enzyme and could be a potential therapeutic target. It is known that SIRT6 is reduced in atherosclerotic patients. Recently, Jin et al. (2020) observed that leukocyte adhesion molecules could be upregulated by cholesterol crystals, creating endothelial dysfunction. SIRT6 inhibits monocyte adhesion to endothelium (Xu et al. 2016; Liu et al. 2016) by inhibiting the expression of adhesion molecules, an effect mediated by SIRT6 deacetylation of H3K9 at NF-ƙB target genes promoter (Kawahara et al. 2009). In contrast, SIRT6 depletion causes macrophage accumulation and destabilizes atherosclerotic plaques (Liu et al. 2016). In line with these observations, overexpression of SIRT6 activates Nrf2, which enhances antioxidant enzymes and produces an alleviation of endothelial dysfunction (Jin et al. 2020) (Fig. 6).

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R. M. Tordera and M. Cortés-Erice

SIRT6 Promotes Successful Progression of Sepsis

SIRT6, besides SIRT1, plays a key role in the successful progression of sepsis preventing an exacerbated inflammatory response and facilitating the recovery of normal homeostasis (Liu et al. 2011, 2015; Qin et al. 2019). In the initial phase of sepsis, TLR4 activation decreases SIRT6 in monocytic cell lines. The acuteinflammatory phase is mainly glycolytic because of an upregulation of HIF-1α. In the adaption phase, SIRT6 inactivates NF-ĸB and normal homeostasis is recovered (Liu et al. 2011, 2015) (Fig. 6). All these studies show the potential therapeutic value of SIRT1 and SIRT6 activation for chronic inflammatory and metabolic illness as well as for host defense. In addition to the existing natural extracts rich in polyphenols, the development of more potent and selective SIRT1 and SIRT6 activators is a very interesting field of research for treating diabetes, atherosclerosis, and/or age-related disorders.

4.3

Role of Other Class III HDACs in Monocyte Function

In addition to SIRT1 and SIRT6, other sirtuins regulate monocytes and in general exert a beneficial effect in these cells. Sirtuin deficiency could contribute to the development of inflammatory and metabolic diseases, infections, and cancer. SIRT2 is located predominantly in the cytoplasm but it can also interact with nuclear proteins playing a role in the transcriptional activity of genes that participate in cell proliferation, differentiation, and survival (Eskandarian et al. 2013). In the cytoplasm, SIRT2 participates in microtubule organization through deacetylation of α-tubulin. It seems that a high degree of tubulin acetylation is essential for monocyte migration to the sites of inflammation or infection with pathogens as well as for cytokine secretion from the vesicles. In line with this, SIRT2 inhibitors alter microtubule organization, affecting monocyte motility and inhibiting IL-1 secretion (Carta et al. 2006). Further, in rheumatoid arthritis SIRT2 deacetylates p65 at lysine 310, resulting in lower expression NF-ĸβ-dependent genes such as cytokines and MCP-1. Thus, SIRT2 deficiency causes more severe joint damage. Furthermore, SIRT2 participates in bacterial infection with Listeria Monocytogenes. This bacterium induces SIRT2 translocation to the monocyte nucleus. There, SIRT2 deacetylates H3 on lysine 18 (H3K18) and represses the expression of genes that participate in host defense (Eskandarian et al. 2013). The sirtuins SIRT3, 4, and 5 are located in the mitochondria. Of these, SIRT3 carries out an important protective role against mitochondria damage, oxidative stress, and autophagy. SIRT3 is essential in antimicrobial host defense against Mycobacterium tuberculosis (Kim et al. 2019). Indeed, SIRT3 is reduced in the peripheral immune cells of patients with tuberculosis infection, which causes an exacerbated increase of ROS and an upregulation of pro-inflammatory cytokines. Therefore, SIRT3 activation could be a potential target to reduce the excessive

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inflammation and oxidation that characterize tuberculosis infection (Kim et al. 2019). In addition, SIRT3 can have a protective function in nephropathies, because it mediates the polarization of macrophages from M1-like macrophages to M2. The anti-inflammatory M2-like macrophages prevent the formation of renal calcium oxalate crystals. SIRT3 deacetylates FOXO3 and causes its translocation to the nucleus where it facilitates the expression of some anti-inflammatory genes such as IL-10, interleukin that helps in M2 polarization (Xi et al. 2019). Similarly, SIRT3 upregulation participates in macrophage adaptation to endotoxin tolerance following septic shock (Liu et al. 2015; Qin et al. 2017). Finally, another important role for SIRT3 is in inflammation and wound healing. If levels of SIRT3 are decreased, as occurs in diabetic mice macrophages, there will be a pro-inflammatory environment where wound repair is impaired (Boniakowski et al. 2019). On the other hand, the mitochondrial sirtuin SIRT4 breaks immune tolerance in monocytes and rescues TNFα expression. SIRT4 negatively regulates SIRT3 and 1 activity, which is important for the maintenance of the normal homeostasis of the mitochondria (Tao et al. 2018). The last sirtuin, SIRT7, plays different roles in several kinds of cancers. It is known that it decreases with aging, and this downregulation can be seen in age-related leukemia. SIRT7 could act as a tumor suppressor in myeloid leukemia. It also might be a biomarker for assessing treatment outcome or disease progression (Kaiser et al. 2020). However, it has been reported that SIRT7 acts mainly as an oncogene in other cancers, including breast, bladder, or colorectal cancers.

5 Role of Class IV in Monocyte Function in Health and Disease HDAC11 is the only class IV HDAC, and of all the HDACs, the most recently identified and the smallest. Although little is known about its biological activity, evidence has shown that it can play a role in the inflammatory response in monocytes. HDAC11 physically interacts with other HDAC enzymes regulating cytokine release (Liu et al. 2020). In addition, some studies have observed that HDAC11 has a main function as a fatty acid deacetylase (Kutil et al. 2018; Cao et al. 2019). HDAC11 deacetylates H3 at the IL-10 promoter. In line with this, S. aureus infections in prosthetic joints reduce HDAC11, causing an increase of the antiinflammatory IL-10 and facilitating the maintenance of the infection (Heim et al. 2020). Further, HDAC11 has been shown to be decreased in monocytes in systemic lupus erythematosus patients (Tak Leung et al. 2015). In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), HDAC11 knockdown reduces the clinical severity in the post-acute phase of the disease because of decreased peripheral macrophage activation. In addition, HDAC11 knockdown reduces the infiltration of peripheral monocytes and myeloid dendritic cells into the MS brain (Sun et al. 2018).

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As other HDACs, inhibition/activation of HDAC11 could be of potential therapeutic interest. Interestingly, a natural anti-inflammatory antioxidant, garcinol, shows inhibitory activity toward HDAC11 (Son et al. 2020).

6 Conclusions and Perspectives This review shows that some HDAC subtypes in monocyte cells are potential targets for pharmacologic intervention in different autoimmune-inflammatory diseases, infections, and cancer. Firstly, some HDAC subtypes in monocyte cells have potential therapeutic value for autoimmune-inflammatory diseases. Specifically, it seems that HDACs are promising pharmacological targets to address rheumatoid arthritis, atherosclerosis, COPD, and diabetes for which existing drugs show limited effects on illness progression. In general, pharmacological treatments that inhibit the activity of pro-inflammatory HDACs or enhance the activity of the anti-inflammatory HDCAs show beneficial effects in mouse disease models. In particular, selective HDAC1, HDAC3, HDAC5, and HDAC6 inhibitors could have therapeutic value as modulators of the monocyte inflammatory response. However, both Class I HDACs, HDAC1 and HDAC3, are known to be crucial in reprogramming intestinal macrophages to adapt to chronic microbial exposure. Thus, systemic administration of HDAC1 or HDAC3 inhibitors could trigger important gastrointestinal side effects such as bowel inflammation. In the case of arthritis, local infiltrations of these inhibitors in the inflamed arthritic joints could be an alternative to avoid these side effects. In the case of atherosclerosis, the development of HDAC3 inhibitors that specifically target foam cell macrophages could also minimize side effects. On the other hand, drugs that selectively activate HDAC2, SIRT1, SIRT3, or SIRT6 which are considered to be anti-inflammatory HDACs, seem promising options to improve the treatment of COPD, type II diabetes, and inflammatory nephropathies. Furthermore, existing pharmacological treatments known to enhance the activity of antiinflammatory HDACs show beneficial effects on the immune response of monocytes. Furthermore, unlike class I HDACs, the therapeutic value of class IIa HDACs in autoimmune-inflammatory diseases has been explored very little. Importantly, the fact that class IIa HDACs can reversibly shuttle between the nucleus and the cytoplasm upon phosphorylation at specific serine residues seems a therapeutic advantage over the nuclear HDACs. For instance, cytoplasmic shuttling of HDAC5 could have a rapid and transient anti-inflammatory action in activated macrophages. In contrast, nuclear shuttling of HDAC5 may help to maintain host defense. Thus, drugs able to phosphorylate HDAC5 at specific serine residues should be developed in order to study their potential anti-inflammatory activity. In this context, the effect of pharmacological treatments known to activate membrane receptors coupled to kinases able to phosphorylate HDAC5 in the pro-inflammatory phenotype of monocytes should be also studied.

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Secondly, some HDAC subtypes are potential targets for pharmacological intervention in different viral or bacterial infections. For instance, the antiviral role of HDAC1, HDAC2, HDAC3, and SIRT1 in monocytes could be an opportunity for developing alternative antiviral strategies. Of particular interest currently are viral infections with a global presence, but with limited response to existing drugs or for which universal vaccines are lacking. The potential emergence of new genetic variants of existing viruses is a further concern. Sadly, actual respiratory viruses meet these criteria and, for some individuals, illness severity can be dramatically high. We suggest that pharmacological strategies aimed at maintaining high levels of activity of specific HDACs that combine both antiviral and anti-inflammatory roles, such HDAC2 and SIRT1, should be implemented. Further, this review highlights the protective effect of the sirtuins SIRT1, SIRT3, and SIRT6 in circulating monocytes against the pathological acute systemic inflammation associated with septic shock. Thirdly, some HDAC subtypes are potential targets for pharmacological intervention in different types of cancer. Specifically, in addition to class I HDAC inhibitors, with limited anti-tumor effectiveness, targeting class II HDACs is an emerging strategy that stimulates tumor associated-macrophage immune responses. Interestingly, macrophage polarization toward an M1 pro-inflammatory phenotype is showing promising results in cancer therapy. In summary, this review shows that the HDAC superfamily is a nice set of tools that help monocytes to exert innate immunity. It consists of 18 enzymes in total, with different cellular mechanisms of action. They share, however, many similar physiological functions and thus it is possible to classify them into three groups. A first group of HDACs stimulates the immune response and activates pro-inflammatory mechanisms. The opposing group does exactly the opposite by reprogramming monocytes toward an immunosuppressive phenotype and preventing an exacerbated cytokine storm that could be fatal. A third group shuttles between both of the other groups adapting monocytes to the physiological/pathological stimuli of the moment. We could wonder about the need for up to 18 different enzymes when, perhaps, just 2 or 3 could be sufficient. Perhaps, having such a complete set of enzymes coparticipating in the regulation of our innate immunity is one of the explanations why we are still here and have not disappeared due to some catastrophic pathogen. Still, this set is not perfect. Even if our monocytes have learnt to use each HDAC at the right time and in the necessary proportion, on some occasions, a pathogen such as a virus or a tumor cell manages to evade control, takes advantage of a multiple failure, and starts to propagate. On other occasions, a misuse of these HDACs leads to the development of a chronic inflammatory disease. This review shows that, despite all the interesting and extensive knowledge regarding the role of some HDACs in monocyte function, surprisingly, very little progress has been made toward the goal of finding effective HDAC-targeted therapies. Perhaps, the side effects and limited effectiveness of the existing pan HDAC inhibitors have led to past research as being seen as somewhat disappointing. However, we believe that it is very much worth the effort to continue exploring the pharmacology of each HDAC at preclinical and clinical levels. The development of monocyte-specific drugs that selectively target HDAC subtypes would open a

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wide avenue for pharmacological research. This research could help to find effective treatments for the three big health problems, in which our innate immune system is involved: chronic autoimmune-inflammatory illness, infections, and cancer. Acknowledgements We would like to thank the Departamento de Salud (Gobierno de Navarra, Spain) for funding a project focused on the role of different HDACs on peripheral monocytes in depressed patients. Thanks to Ministerio de Ciencia, Innovación y Universidades (Gobierno de España) for supporting to M.C.E (FPU17/05039) with a fellowship. In addition, thanks to all the patients and volunteers that are collaborating in our research, and specially, to Ms Sandra Lizaso for all her technical support in all the monocyte studies. The authors declare no competing interests. Thanks to O.L.F for inspiring me the initial idea of this review during the first weeks of the Covid19 pandemic.

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Rev Physiol Biochem Pharmacol (2021) 180: 49–84 https://doi.org/10.1007/112_2021_61 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 Published online: 12 June 2021

Neuronal Nitric Oxide Synthase (nNOS) in Neutrophils: An Insight Rashmi Saini, Zaffar Azam, Leena Sapra, and Rupesh K. Srivastava

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Structural Biology of nNOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 nNOS: The Constitutive Contributor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 The nNOS Gene: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Alternative Splice Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 The Promoter Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Regulation of the Catalytic Machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Substrate and Cofactor Availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Feedback Inhibition by NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Post-Translational Modification: Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Protein–Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Expanding the Connections Through the PDZ Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Coupling to Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Reciprocal Regulation by Caveolin and Calmodulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Modulation by Molecular Chaperon: Hsp90 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 PIN, NOSIP: Inhibitory Potential Towards nNOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Intracellular Compartmentalization of nNOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Trafficking to the Nuclear Compartment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 nNOS Associated with Plasma Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Cytoplasmic nNOS: Association with Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

R. Saini (*) Department of Zoology, Gargi College, University of Delhi, Delhi, India e-mail: [email protected] Z. Azam Department of Zoology, Dr. Harisingh Gour Central University, Sagar, MP, India Department of Biotechnology, All India Institute of Medical Sciences (AIIMS), New Delhi, India L. Sapra and R. K. Srivastava (*) Department of Biotechnology, All India Institute of Medical Sciences (AIIMS), New Delhi, India e-mail: [email protected]

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6.4 Localization in Mitochondria and Endoplasmic Reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Sequestration in Primary Granules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Clinical Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract NO (nitric oxide) is an important regulator of neutrophil functions and has a key role in diverse pathophysiological conditions. NO production by nitric oxide synthases (NOS) is under tight control at transcriptional, translational, and posttranslational levels including interactions with heterologous proteins owing to its potent chemical reactivity and high diffusibility; this limits toxicity to other cellular components and promotes signaling specificity. The protein–protein interactions govern the activity and spatial distribution of NOS isoform to regulatory proteins and to their intended targets. In comparison with the vast literature available for endothelial, macrophages, and neuronal cells, demonstrating neuronal NOS (nNOS) interaction with other proteins through the PDZ domain, neutrophil nNOS, however, remains unexplored. Neutrophil’s key role in both physiological and pathological conditions necessitates the need for further studies in delineating the NOS mediated NO modulations in signaling pathways operational in them. nNOS has been linked to depression, schizophrenia, and Parkinson’s disease, suggesting the importance of exploring nNOS/NO-mediated neutrophil physiology in relation to such neuronal disorders. The review thus presents the scenario of neutrophil nNOS from the genetics to the functional level, including protein–protein interactions governing its intracellular sequestration in diverse cell types, besides speculating possible regulation in neutrophils and also addressing their clinical implications. Keywords Intracellular compartmentalization · Neuronal NOS · Neutrophils · Protein interactions · Subcellular trafficking

Abbreviations ADP BH4 CaM CaMKII cGMP CtBP DHRs eNOS FAD FMN Hsp90 IFN

Adenosine diphosphate Tetrahydrobiopterin Calmodulin Ca2/calmodulin-dependent protein kinase II Cyclic guanosine monophosphate C-terminal-binding protein Disks-large homology repeats Endothelial nitric oxide synthase Flavin adenine dinucleotide Flavin mononucleotide Heat-shock protein 90 Interferon

Neuronal Nitric Oxide Synthase (nNOS) in Neutrophils: An Insight

iNOS LPS NADPH NFκB NF-Kb NLS NMDA nNOS NO NOSIP PDZ PIN PKA PKC PKG PMN PSD sGC TNF

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Inducible nitric oxide synthase Lipopolysaccharide Reduced nicotinamide adenine dinucleotide phosphate Nuclear factor kappa B Nuclear factor kappa-light-chain-enhancer of activated B cells Nuclear localization signal N-methyl-D-aspartate Neuronal nitric oxide synthase Nitric oxide NOS interacting protein PSD-95/disc-large/ZO-1 Protein inhibitor of NOS Protein kinase A Protein kinase C Protein kinase G Polymorphonuclear leukocytes Post synaptic density Soluble guanylyl cyclase Tumor necrosis factor

1 Introduction Neutrophils have a complex, highly integrated, and sophisticated functional repertoire, which enables them to act as a first line defense in the host by opposing invasion of microbes. Neutrophils constitute about 50–60% of the circulating leukocytes that contribute significantly to the amount of NO in circulation, i.e. at a rate of 10–100 nm/5 min/106 cells (Sethi and Dikshit 2000). There has been lot of ambiguities regarding the presence of different NOS isoforms in neutrophils. Occurrence of both iNOS and nNOS isoforms has been demonstrated, and a report has also been cited for the presence of eNOS in neutrophils (Salvemini et al. 1989). Earlier, nNOS was thought to be present only in neurons, where it was first isolated, however, it has also been located in neutrophils, endometrium, skeletal muscle, pancreatic islets, bones as well as in respiratory and gastrointestinal epithelia (Saini et al. 2006; Cameron and Campbell 1984; Rothe et al. 2005; Ort et al. 2000; Van’t Hof et al. 2004; Sherman et al. 1998; Lu et al. 2006). nNOS is constitutively expressed and functions in a Ca2+/calmodulin-dependent manner (Singh et al. 2005). Human neutrophils according to Greenberg et al. (1998) lack a functional NOS system despite its constitutive expression. Inhibition imposed on enzymatic activity could be a consequence of interactions with negative modulatory elements like calmodulin which is discussed in the following section. Several factors regulate the expression of this constitutive contributor. Human neutrophils incubated in vitro with 17β-estradiol (10
8 mol/L) resulted in an increase in nNOS protein

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expression through AP-1, Sp-1 transcription factors (Molero et al. 2002). 17β-estradiol treated neutrophils also reduced ADP-induced platelet activation (Duran et al. 2000). Moreover augmented cGMP levels have also been observed in platelets co-incubated with 17-β estradiol treated neutrophils. nNOS derived NO is found to be an important modulator during early neurogenesis independent of sGC/cGMP pathway and more through regulation of intracellular calcium homeostasis (Arnhold et al. 2002). NO alters gene expression profiles, regulates growth, differentiation, and acts as an anti-proliferative agent through p21 (Ishida et al. 1997). It also mediates the action of growth factors and controls the balance between proliferation and differentiation in neuronal, cardiomyocyte, adipocyte, osteoblast, and endothelial cell cultures (Enikolopov et al. 1999; Zhao et al. 2019). Most of the investigations regarding nNOS have been directed towards the central and peripheral nervous system, where its role as a neurotransmitter is well established in normal physiological as well as in various diseased conditions. Presence and participation of nNOS in different activities carried out by the immune-competent cells (Glial cells) have gained curiosity in the last few years in the research field with respect to neurobiology of nNOS. This has also been reviewed by Luo and Zhu (2011), but the importance of nNOS in neutrophils is yet to be established. Our lab research has been focused on investigating the NO mediated response of neutrophils in various pathophysiological conditions such as Parkinson’s disease (Barthwal et al. 1999; Singh et al. 2005), schizophrenia (Srivastava et al. 2001), and depression (Srivastava et al. 2002). Previous studies have revealed that nNOS in neutrophils is found to be a fulllength PDZ (PSD-95/Disc large/ZO-1) domain containing protein (Saini et al. 2006). PDZ domains are protein interacting domains that play crucial role in targeting of proteins to its effective sites in membranes and assembly of these proteins into supramolecular complexes. Though association of nNOS with caveolin-1 protein has been demonstrated in cytoplasmic as well as nuclear compartments in neutrophils (Saini et al. 2006), its interaction with other PDZ containing proteins still needs to be explored in these cells. Investigations have demonstrated that NO content increases with neutrophil maturation and seems to play a crucial role in maturation of neutrophils (Kumar et al. 2008). NO generating ability with molecular/biochemical characteristics of nNOS has also been characterized in bone marrow neutrophil precursor cells (Kumar et al. 2010) and an attempt was made to delineate the involvement of NO/nNOS in differentiation of neutrophils. This might provide an effective therapeutic intervention in future for treatment of neutropenic ailments, during which patients fall easy prey to frequent infections. Neutrophil nNOS remained to be explored from more than a few aspects as knowledge regarding these cells is still limited when compared to the vast array of literature reporting investigations in other cells/cell lines. Thus, in the present manuscript we have attempted to include evidences from those to provide the potential patterns and pathways involved in the regulation of NOS synthesis and modulation that can be operational in neutrophils, highlighting the aspects that remained to be investigated in these granulocytes. Besides providing insight into the genetic and catalytic modulation of nNOS in these cells, the present review emphasizes the relevance of compartmentalization of nNOS isoform at intracellular

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Fig. 1 Diagrammatic representation of structural biology of neutrophil nNOS, its expressional regulation, regulation of the catalytic machinery, as well as its intracellular compartmentalization and protein–protein interactions of neutrophil nNOS determining the microenvironment, activity, and its contribution as a trafficking protein for NO signaling specificity. PDZ PSD-95/disc-large/ ZO-1, ARG L-arginine, HAEM Haem, NADPH reduced nicotinamide adenine dinucleotide phosphate, FMN flavin mononucleotide, BH4 tetrahydrobiopterin, CaM calmodulin, M mitochondria, N nucleus, ER endoplasmic reticulum, AG azurophilic granules, PIN Protein Inhibitor of NOS, Hsp Heat shock protein, NOSIP NOS interacting protein

sites and its association with other interacting proteins in the functioning of neutrophils (Fig. 1).

2 Structural Biology of nNOS NO is predominantly synthesized from guanidino nitrogen atom present in L-arginine molecule. In a two-step reaction, NOS enzyme converts L-arginine molecule into NO and L-citrulline that requires cofactors including FMN, FAD,

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Fig. 2 Schematic representation of the structure of neuronal NOS showing the PDZ domain and its splice variants. Domain structure showing the PSD-95/disc-large/ZO-1(PDZ) domain and the binding sites for L-arginine (ARG), Haem (HAEM), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), tetrahydrobiopterin (BH4), and calmodulin (CaM). [Numbers in figure indicate amino acid positions]

NADPH, calmodulin (CaM), and tetrahydrobiopterin (BH4) (Palmer et al. 1988; Fostermann 1994). Among the NOS isoforms, the neuronal NOS has a predicted molecular weight around 160.8 kDa and consists of 1,434 amino acids (Boissel et al. 1998). Monomeric form of nNOS exhibits a bidomain structure in which the central CaM-binding motif separates oxygenase domain (NH2 terminal) from reductase domain (COOH terminal) (Fig. 2). The oxygenase domain which binds the substrate L-arginine comprises an active site, i.e. known to be BH4 binding site, as well as binding site for Zn atom that helps in dimerization of nNOS. Whereas reductase domain that binds the substrate NADPH contains binding sites for FAD and FMN (Hemmens et al. 2000; Sagami et al. 2001; Noguchi et al. 2001). It is active in its dimeric form which requires Heme, BH4, and L-arginine binding (Reif et al. 1999) while its monomeric form is inactive. The dimeric interactions mainly involve the reductase domain which interacts loosely as monomeric tail with the oxygenase domains of two subunits (Panda et al. 2001). Allosteric modulation of nNOS takes place by the transient elevations in the intracellular free Ca2+ levels which are caused due to binding of calmodulin (Alderton et al. 2001). Within the FMN binding domain, there is 40 to 50 amino acids sequence insert that serve as an auto-inhibitory loop (AL), which controls nNOS activity by suppressing electron transfer from the FMN to heme in the absence of Ca2+/calmodulin and disrupting calmodulin binding at diminished levels of Ca2+ concentrations (Daff et al. 1999; Guan and Iyanagi 2003; Roman and Masters 2006). Though the structure of nNOS has been previously reviewed (Zhou and Zhu 2009), it is the presence of the PDZ domain in nNOS

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(absent in iNOS and eNOS), which has caught attention from lot of researchers. The current review emphasizes on structure, compartmentalization of nNOS at intracellular sites and its association with the various interacting proteins via the PDZ domain, emphasizing its significance in the proper functioning of neutrophils. Structure of nNOS with the PDZ domain (a.a:17–99) along with the oxygenase and reductase domains has been diagrammatically represented in Fig. 2. Modular PDZ domains, GLGF (after a conserved Gly-Leu-Gly-Phe sequence found within the domain) and disks-large homology repeats (DHRs), comprise around 80 amino acids (van Huizen et al. 1998; Sheng and Sala 2001). Initially these domains were found as repeated sequences in the Drosophila septate junction protein disks-large (dlg), neuron-specific postsynaptic density protein (PSD-95/SAP-90), and the epithelial tight-junction protein zona occludens-1 (Woods and Bryant 1993). Studies have deciphered its role not only in the protein–protein interactions involving nNOS but also in nNOS trafficking into the mitochondria after processing of this domain (Carreras et al. 2008). nNOS containing the PDZ domain though has been demonstrated to be present in neutrophil (Saini et al. 2006), yet its interacting partners, possibility of such processing in the domain itself and thereby its influence in subcellular trafficking still needs further investigations.

3 Regulation Tissue and development specific expression of nNOS is governed by the complex pattern of alternate splicing events. Previously we reported the presence of nNOS-α having the PDZ domain in neutrophils of rat (Saini et al. 2006). Alternate splicing modifies the stability of mRNA, post-translational modifications, enzyme catalysis and confers novel functions to resulting NOS variants. It is therefore critical to explore the alternate splice variants of nNOS in neutrophils in pathophysiological situations where differential NO generation is monitored and also explore NO in influencing neutrophil differentiation.

3.1

nNOS: The Constitutive Contributor

The constitutive production of NO at basal level by neutrophils is in part attributed to nNOS activity. Constitutive expression of a functional nNOS system with its mRNA and protein has been demonstrated in rat neutrophils (Greenberg et al. 1998), but human studies indicate towards a distinct regulatory mechanism, which affects or controls the translation and thereby constitutive functionality of nNOS in polymorphonuclear leukocytes (PMNs) despite its continued expression as deciphered by the mRNA levels. The constitutive generation of NO has been implicated to maintain homeostatic vascular tone or prevent adherence of PMNs to the vascular endothelium (Sandoo et al. 2010). Absolute and relative mRNA and protein levels in rat

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neutrophils are significantly less than that found in cortex and cerebellum; but the amount of protein per unit length of mRNA is greater presumably due to increased translational rate or decreased protein degradation. Unlike rat PMN, the human PMN does not express constitutive nNOS protein. The inability of the human PMN to constitutively express nNOS protein despite the presence of nNOS mRNA suggests that human nNOS mRNA may be vestigial in nature (Greenberg et al. 1998). Neutrophils isolated from premenopausal women show increased level of circulating estrogen as well as estrogen receptors concurrently causing an over-expression of nNOS in them during the ovulatory phase. Neutrophils derived from men also showed the presence of ERα and ERβ but an increase in ERα was observed only after 17β-estradiol incubation in vitro (10
8 mol/L) and an increase in nNOS protein expression prevented by mithramycin (10
6 mol/L), curcumin (10
6 mol/L), thereby suggests the involvement of AP-1,Sp-1 transcription factors (Molero et al. 2002). Furthermore, 17β-estradiol incubation reduces ADP induced platelet activation (Duran et al. 2000), corresponding to increased level of cGMP in platelets, suggesting a NO connection. However, to prevent excessive NO generation, various stimuli such as lipopolysaccharide (LPS) triggering the switch to the induced iNOS expression pose an inhibitory effect on constitutive nNOS expression. Using Biopredsi algorithm the target sequence of nNOS mRNA was determined (Dieter et al. 2005). The presence and role of nNOS and its enhancement during the intervertebral disk’s regeneration through increased expression of nNOS mRNA content have been well demonstrated (Vitor et al. 2017). Although it plays a key role in clearance of bacteria especially in case of staphylococcal induced arthritis, it has also been known to contribute in articular damage and bone loss (Van’t Hof et al. 2004). Production of NO at inflammatory sites reduces pain through suppression of neutrophil’s migration and it also enhances the sGC/cGMP/PKG analgesic pathway in nociceptive neurons. All the above facts indicate that the effects of NO are multifaceted, varied, and antagonistic as well (Fernando et al. 2019). Thus, it is relevant to investigate and understand the intrinsic regulatory mechanisms that underlie this constitutive activity.

3.2

The nNOS Gene: An Overview

The human nNOS gene encoding for a protein of 160 kDa is assigned to 12q24.2 region of chromosome 12 spanning a region of 240 kb comprising of 29 exons and 28 introns. The full-length ORF (open reading frame) spans 4,302 bp and encodes for 1,434 amino acids (Boissel et al. 1998). nNOS mRNA transcripts have been isolated from noradrenergic, non-cholinergic nerves, skeletal muscle, macula densa, pancreatic beta cells, adrenal medulla, pituitary gland, and distal nephron of kidneys. The translation initiation and the termination sites are localized in exon 2 and 29, respectively. Whereas the transcription start site shows tissue and cell specific variations resulting from alternate promoter usage facilitating extricate regulatory mechanisms for tissue and development specific gene expression.

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Alternative Splice Variants

nNOS is found to be expressed in various human tissues where it displays a complex transcriptional regulatory mechanism with the occurrence of nine alternative first exons (1a-1i) which results in development of nNOS transcripts with distinct 50 -untranslated regions. The 50 ends of the mRNA species from various tissues show different first exons alternately spliced to a common second exon. Eight different exon 1 have been demonstrated that correspond to the 5-prime flanking/promoter regions accounting for different regulatory mechanisms spanning for about 200 kb upstream of exon 2. In human colon, skeletal muscle, brain, and the TGW-nu-I neuroblastoma cells, the expression patterns of the first exon were investigated by quantitative RT-PCR that revealed that one of the crucial and most abundant first exons of nNOS is exon 1c. Several splice variants of nNOS have been reported (as depicted in Fig. 2), among which nNOSα, a full-length protein containing exon-2 accounts for majority of the enzymatic activity, has the PDZ domain, while nNOSβ and nNOSγ lack the PDZ domain (Alderton et al. 2001). nNOSγ lacks catalytic activity. It’s worth investigating the splice variant(s) present in neutrophils to ascertain its regulatory mechanism and elements in comparison with the available information in literature. Earlier study by Greenberg et al. (1998) utilizing primers did not verify presence of the PDZ domain in nNOS expressed in rat neutrophils. It was reported that rat circulating neutrophils constitutively expressed nNOS mRNA and nNOS protein. Constitutive nNOS mRNA was also found in human neutrophils but nNOS protein was not expressed in them. However, based on the data from western blotting and RT-PCR experiments, nNOS isoform in rat neutrophils appeared to be a full-length PDZ domain containing protein (Saini et al. 2006). The PDZ domains are protein interacting domains that play crucial role in trafficking of proteins to particular compartments localized in the membrane and help in the assembly of these proteins into supramolecular complexes.

3.4

The Promoter Region

The 50 prime flanking regions in human neurons bear potential transcription factor binding sites for TEF-1/MCBF, AP-2, NRF-1. Ets, CREB/ATF/c-fos, NF-1, and Nuclear factor kappa-light-chain-enhancer of the activated B cells (NF-kBb). A 89-nucleotide alternatively spliced exon acts as a potent repressor of translational machinery that inhibits translation of native nNOS open reading frame. This spliced exon is found to be localized in the 50 -untranslated region between the exon 1 variants and exon 2 that covers the translational initiation codon. Furthermore, RNase protection and RT-PCR assays specified that nNOS mRNAs containing this exon are expressed in a promoter-specific as well as in tissue-restricted manner. Mutational analysis and a secondary structure prediction identified the functional cis-element that forms a putative stem-loop within this novel exon. Interaction

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between specific cytoplasmic RNA binding with this motif was proved by RNA electrophoretic mobility shift assay techniques. Hence, a unique splicing event within the 5’-UTR region introduces a translational control element. This represents a newer model for translational control of a mammalian mRNA (Newton et al. 2003).

4 Regulation of the Catalytic Machinery Activity of NOS isoforms is governed by different regulatory mechanisms which results in differential participation of these isoforms in diverse physiological activities. Catalytic process is determined by availability of cofactors, NO mediated protein interactions, feedback regulation, and post-translational modifications. The kinetic parameters of the three isoform differ significantly causing variation in their steady state of NO synthesis as stated by Stuehr et al. (2004).

4.1

Substrate and Cofactor Availability

The apparent Km and Vmax for cNOS in human neutrophils are 1,454  160 μM and 0.11  0.02 μM/min/mg of protein, respectively (Riesco et al. 1993) and for nNOS from rat peritoneal neutrophils Km is 22 μM and Vmax is 485 nM/min/mg protein (Yui et al. 1991). The human neutrophils act as a reservoir of nNOS substrate, L-arginine, which is considered to be at an estimated intracellular concentration of 5.22  0.46 nmol/108 cells (Rysz et al. 2003). Apart from synthesis and uptake, recycling from citrulline also contributes in enhancing L-arginine and thereby helps in sustaining the production of NO. It is found to be conditionally a vital amino acid that plays crucial role during growth but becomes limited under numerous pathological conditions and thus affects NO generation (Rysz et al. 2003). Under such pathological conditions, NOS is known to generate superoxide instead of NO and likely to perform functions other than the catalytic activity. Under physiological conditions, regeneration of L-arginine from the end product is very sensitive to citrulline concentration in plasma (0.1–0.5) mM. L-arginine recycling in the endothelial cells and macrophages correlates to the need of L-arginine availability to support their function. On the other hand, arginase limits the levels of intracellular L-arginine thereby keeping generation of NO in control. Recycling of L-arginine in neutrophils still remains unexplored; however, there is report of no reduction in the availability of L-arginine following phagocytosis in neutrophils, suggesting the maintenance of active inducible NOS following phagocytosis that was well supported by the augmented nitrite/NO generation (Nagarkoti et al. 2019). BH4 is one of the most vital cofactors essential for regulating homodimeric conformation in all the three isoforms of NOS (Klatt et al. 1995; Gorren et al. 1996). BH4 serves as a donor of electrons to the heme group in NOS reactions but is

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neither involved in the direct substrate oxidation nor its regeneration. BH4 mediated homodimerization reduces the susceptibility of NOS to Protein kinase C (PKC) mediated phosphorylation as seen in low temperature SDS-PAGE studies with stabilized and unstabilized nNOS dimers (Okada 1998). Purified nNOS is reversibly inhibited in vitro by PKC mediated phosphorylation events. PKC-dependent phosphorylation in HEK293 cells results in reversible suppression of nNOS activity that further acts as a regulatory mechanism to control superoxide generation in the absence of adequate BH4, whereas in cerebellar slices, mesencephalic and striatal neurons and pinealocytes culture, it is known to activate nNOS (Okada 1998). Studies have demonstrated that there is increase in superoxide anion and hydrogen peroxide generation by nNOS due to its uncoupling resulting due to loss of BH4 (Katusic et al. 2008). It was identified that BH4 could be oxidized by potent oxidant peroxynitrite, which is generated by the reaction between NO and superoxide anion (Milstien and Katusic 1999). The chemical antagonism between peroxynitrite and BH4 suggests that uncoupling of nNOS might be related with enhanced production of peroxynitrite. Another cofactor in enzyme calmodulin has been suggested to be involved in inter-subunit electron transfer in nNOS (Panda et al. 2001), which is also a central regulator of NADPH oxidase activity in PMNs. CaM binding is known to displace the auto-inhibitory loop (AL) leading to enzyme activation. Studies on neutrophil nNOS demonstrated the association of nNOS with caveolin-1 protein, which is known to negatively modulate its activity. Increase in CaM concentration positively correlated with increase in enzyme activity that suggests a “CaM-caveolin cycle” of activation or inactivation of nNOS operational in these cells too (Saini et al. 2006).

4.2

Feedback Inhibition by NO

Due to higher stability of ferrous nitrosyl (Fe2+-NO) complexes, any enzyme that forms a reduced-ferrous-heme intermediate has the potential to be suppressed by NO (Cooper 1999). nNOS is no exception and during turnover it can form inhibitory nitrosyl species. Up to 95% of nNOS enzyme in its stable state can be in the tighter Fe2+-NO form (Abu-Soud et al. 1995). Unlike iNOS, nNOS appears to be able to react with NO within the enzyme active site, which has been explained in unified model proposed by Stuehr’s group (Santolini et al. 2001). It has also been discovered that NO generated at the completion of enzymatic reaction remains in distal pocket and rebinds to heme iron, thus suppress the enzymatic activity (auto-inhibition). Although the crystal structure of oxygenase domain is identical in all three isoforms of NOS enzyme but the extent of auto-inhibition by NO is different which is as follows: nNOS > iNOS > eNOS (Li et al. 2004).

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Post-Translational Modification: Phosphorylation

Various studies have reported that nNOS activity is also modulated by phosphorylation. Protein Kinase C (PKC), PKA, and Ca2/Calmodulin-dependent Protein Kinase II (CaMKII) have been found to modulate activity of nNOS through phosphorylation. It has been reported that nNOS is phosphorylated at serine amino acid present at 847th position, a potential CaMKII phosphorylation site (Hayashi et al. 1999). This CaMKII mediated phosphorylation of nNOS at ser-847 position partly inhibits the binding of Ca2+-CaM and suppresses nNOS mediated enzymatic activity (Komeima et al. 2000). PSD95, which binds nNOS to N-methyl-D-aspartate (NMDA) receptor, may promote Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of nNOS at serine residue (Rameau et al. 2007). LPS/IFN-γ induced inhibition of nNOS activity has been reported to be mediated by tyrosine kinase, causing tyrosine phosphorylation (Colasanti et al. 1999; Palomba et al. 2004). The regulatory role for BH4 in phosphorylation of nNOS has also been proposed. BH4 mediated homodimerization reduces susceptibility to PKC mediated phosphorylation as seen in low temperature SDS-PAGE studies with unstabilized and stabilized nNOS dimers. PKC-dependent phosphorylation results in reversible inhibition of nNOS activity in HEK293 cells whereas in cerebellar slices, mesencephalic, striatal neurons and pinealocytes in culture it is known to activate nNOS. Purified nNOS has been shown to be inhibited by in vitro phosphorylation events mediated by PKC. Whereas in vivo studies with HEK293 cells stably expressing nNOS reveal that nNOS can produce superoxide in absence of adequate BH4 and that phosphorylation-dependent inhibition acts as a regulatory mechanism to keep generation of superoxide in check. Thus, investigations should be directed towards involvement of such post-translational mechanisms of nNOS regulation in neutrophils as well as generation of superoxide in absence of adequate amounts of BH4.

5 Protein–Protein Interactions Interactions of all major isoforms of NOS with different heterologous proteins have emerged as a mechanism by which the functional activity, spatial distribution, and proximity of the NOS isoforms to the regulatory proteins and the intended targets are administered. Dimerization, which is required for the functional activity of NOS isozymes exhibits various distinguishing features among all these proteins. This may serve as an important regulated process and can be used as target for therapeutic intervention (Kone et al. 2003). As compared to other two isoforms of NOS, nNOS seems to be lacking acylation and hence uses the other target mechanisms (Mungrue and Bredt 2004). Rather, nNOS is recruited to the membranes mainly PSD or the sarcolemma via protein–protein interactions involving its unique N-terminal PDZ domain, which is found to be lacking in the other two NOS isoforms (Govers and

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Oess 2004). A wide array of proteins that range from the membrane receptors to scaffolding proteins have also been displayed to function as nNOS-interacting proteins (Kone et al. 2003) and confer vital consequences including the enzyme activation, inhibition, and trafficking as detailed in Table 1. Although previous reviews highlighted the significance of protein–protein interaction that involves nNOS and their functional importance in neurons (Zhou and Zhu 2009) and other tissues/cell types (Nedvetsky et al. 2002; Kone et al. 2003; Oess et al. 2006), neutrophils are largely unexplored in this area. The present review thus summarizes the interacting domains involved in the interaction associated with nNOS and the consequences of the interaction on its activity in different cell/tissues, addressing also the possibilities and effectiveness of such interactions in influencing functionality of neutrophils.

5.1

Expanding the Connections Through the PDZ Domain

Among other isoforms of NOS, the PDZ domain is exclusive to nNOS isoform. Proteins that contain the PDZ domains typically localize to specialized cell to cell contacts and in the ternary complexes where they connect the components of signal transduction pathways (Kone et al. 2003). Several proteins bearing the PDZ domains have direct interactions with N-terminal of nNOS and have been reported not only to influence the functional activity of enzyme in the muscle and brain but also their subcellular distribution (Brenman et al. 1996a, b; Hashida-Okumura et al. 1999). The PDZ domain of nNOS found at the NH2 terminus is involved in the development of active nNOS dimers and is responsible for its interaction with several proteins in specific regions of cell. NO signaling gets altered by anchoring nNOS to specified targets in this manner (Kone et al. 2003). Though the PDZ domains have a common 3-D structure, but mostly it differs in their binding specificities. Earlier studies have demonstrated that the PDZ domain containing proteins typically associate with target proteins without disturbing the ligand function and overall structure due to their ability to bind short extreme C-terminal sequences (Saini et al. 2006; Sheng and Sala 2001; Hung and Sheng 2002; Trejo 2005). Detection of the PDZ domain encoding nNOS-α splice variant in neutrophils (Saini et al. 2006) calls for extensive explorations to characterize its interacting partners and their functional significance. Interaction of the PDZ domain with carboxy-terminal tail is crucial for K+ channels, PSD-95 and for the NMDA receptor clustering, depicting the physiological significance of proper subcellular location of these proteins (Brenman et al. 1996a, b; Cao et al. 2005; Cui et al. 2007). In the skeletal muscles, the PDZ domain of syntrophin mediates the association of nNOS with the dystrophin complex (Segalat et al. 2005). NIDD, nNOS-interacting DHHC domain containing protein is known to enhance nNOS enzymatic activity by targeting nNOS enzyme to synaptic plasma membrane in the PDZ domain-dependent manner (Saitoh et al. 2004). A receptor tyrosine phosphatase like protein related with the neuronal and

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Table 1 Protein–protein interaction involving nNOS

Protein partners Tissue/cell line A. Interactions through the PDZ domain CAPON Murine brain

Domains of interacting protein involved in interaction COOH terminal domain+ (GDAV)

Consequences of interaction on enzyme activity/ trafficking Disruption of PSD-95–NOS1 complexes and coupling to NO targets Activation triggered via coupling to NMDA receptors Increased activity by enhanced N-methyl-Daspartate (NMDA)stimulated Ca2+ loading Proximity with intended target (Sarcolemma)

PSD-95

Rat neurons

Second PDZ motif

PSD-93

Mice neurons

Second PDZ motif

α1Syntrophin

Mice skeletal muscle, brain

PDZ domain (amino acids 59–166)

NIDD

Rat brain

5-HT2B receptor

Mouse cardiomyocytes, carcinoid cells, serotonergic cell line HEK

COOH terminal domain+ (EDIV) COOH terminal PDZ group 1 motif+VSYI (D/E)

Targeting the synaptic plasma membrane Triggers intracellular cGMP accumulation

COOH terminal sequence+ (GEEV) COOH terminal sequence (ETSV)

Unknown

α1A-adrenergic receptors Plasma membrane calcium/calmodulindependent calcium ATPase (PMCA-4b)

HEK, embryonic kidney and neuro-2a neuroblastoma cells

Phosphofructokinase-M

Rat brain, skeletal muscle

COOH terminal domain+ (GDAV)

Reduces the local calcium concentration, leading to reduced activity of nNOS Help disposition of cytosolic NOS

Reference (Jaffrey et al. 1998)

(Brenman et al. 1996a, b) (Brenman et al. 1996a, b)

(Brenman et al. 1996a, b; HashidaOkumura et al. 1999) (Saitoh et al. 2004) (Manivet et al. 2000)

(Pupo and Minneman 2002) Schuh et al. (2001)

(Firestein and Bredt 1999) (continued)

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Table 1 (continued)

Protein partners CtBP

Tissue/cell line Rat brain, kidney, COS-7

Islet cell autoantigen 512

Rat insulinoma INS-1 cells, pancreatic β cells

Vac14/ArPIKfyve

HEK 293-T cells

Domains of interacting protein involved in interaction Carboxy-terminal sequence+ (DXL) Cytoplasmic domain+ (T-AV)

Internal motif (G-D-H-L-D)

Consequences of interaction on enzyme activity/ trafficking Regulates nuclear protein localization Links secretory granules to the signaling pathway involving NO Unknown

B. Interactions through segment connecting PDZ domain and oxygenase domain PIN Rat kidney, brain PIN binding Destabilizes domain homodimer (PINB, a.a: formation 161–245) C. Interactions through oxygenase domain Caveolin-1 BAEC, Caveolin scaf- Scaffolding and BLMVEC, BRE, folding negative allosteCOS-7, rat domain ric effector neutrophils (a.a: 82–101) and (a.a: 135– 178) Caveolin-3 Rat skeletal Caveolin scaf- Scaffolding and muscle folding trafficking domain (a.a: 65–84) and (a. a: 109–130) NOSIP HUVEC Not identified Redistribution of enzyme from plasma membrane to intracellular compartments MembraneInterference with Bradykinin B2 receptors HEK cells proximal flavin to heme subdomain electron transfer (C-terminal intracellular domain 4) (a.a: 310–329)

Reference (Riefler and Firestein 2001) (Ort et al. 2000)

(Lemaire and McPherson 2006) (Fan et al. 1998; Hemmens et al. 1998) (GarciaCardena, et al. 1996; Saini et al. 2006) (Venema et al. 1997)

(Dedio et al. 2001)

(Marrero et al. 1999)

(continued)

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Table 1 (continued) Domains of interacting Consequences of protein interaction on involved in enzyme activity/ Protein partners Tissue/cell line interaction trafficking D. Interaction through linker region between the oxygenase and reductase domain Calmodulin BAEC, COS-7, Latch domain Induction of rat brain (tight junction intra-molecular between electron transfer helixes 2 and occurs due to 6) interruption in binding with caveolin-1 E. Interaction through amino terminus Hsp-90 Canine cerebral M domain Increases the arteries, binding to BLMVEC, calmodulin PCAEC, rat smooth muscle cells

Reference (Vorherr et al. 1993)

(Song et al. 2001)

+ PDZ domain [human nNOS (a.a: 17–96), rat nNOS (a.a: 15–98)] bind to domains encoding the canonic C-terminal sequence G(DE)-X-V/L/I-COOH, the interaction can also be mediated by their association with the other PDZ domains HUVEC human umbilical vein endothelial cells, BRE bovine retinal endothelial cells, BAEC bovine aortic endothelial cells, PCAEC porcine coronary artery endothelial cells, BLMVEC bovine lung microvascular endothelial cells, HEK human embryonic kidney cells

endocrine secretary granules, islet cell autoantigen 512 of type 1 diabetes binds to the PDZ domain of nNOS isoform by its cytoplasmic domain (Kone et al. 2003; Ort et al. 2000). Another protein CAPON, the COOH-terminal PDZ ligand of nNOS isoform, competes with PSD-95 for interaction with nNOS isoform in brain (Jaffrey et al. 1998). Over-expression of CAPON leads to disruption of PSD-95-nNOS complexes in transfected cells that further interrupts the proximity of nNOS to NMDA-mediated influx of Ca2+, thereby restricting generation of NO (Kone et al. 2003). CAPON has now been reported to function as an adapter protein that links nNOS to specific targets such as synapsins and dexras-1 (Jaffrey et al. 2002). The physiological relevance of these interactions is designated by the alterations in the subcellular localization of CAPON and nNOS. Receptor tyrosine phosphatase like protein, calcium/CaM-dependent ATPase, the COOH-terminal-binding protein (CtBP), and phosphofructokinase-M (Schuh et al. 2001; Riefler and Firestein 2001; Firestein and Bredt 1999) are also found to be important PDZ interacting proteins. The consequences of the interaction of these protein partners on nNOS activity/trafficking and the domains of the protein partners involved in the interactions are elaborated in Table 1. These PDZ mediated interactions are studied in various tissue/cell types but in neutrophils this aspect has not been addressed so far. Since nNOS isoform in neutrophils possesses the PDZ domain, it has been found to

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be linked with other proteins that contain the PDZ domain to regulate several essential functions. Thus, to get an insight into the role of the PDZ domain operational in neutrophils, further investigations to identify the PDZ mediated nNOS protein interactions in these cells need to be done.

5.2

Coupling to Receptors

Interactions of nNOS with various receptors that are found to be involved in signal transduction have been demonstrated. It has been found that serotonin 5-HT2B receptor interacts with nNOS to modulate its activation via COOH-terminal PDZ domain (Manivet et al. 2000; Chanrion et al. 2007). Further, interaction between bradykinin B2 and nNOS has been confirmed via immunoprecipitation experiments in HEK cells, those were stably transfected with human nNOS (Kone et al. 2003; Marrero et al. 1999). In correlation to this study, eNOS and nNOS isoforms were also known to interact with a synthetic peptide derived from the inhibitory sequence of the bradykinin B2 receptor (a.a. residues 310–329). Moreover, interaction between this peptide and nNOS blocked the transfer of electrons from flavin to heme group (Marrero et al. 1999). Interaction between α1A-adrenergic receptors with the PDZ domain of the nNOS enzyme has also been documented (Kone et al. 2003; Pupo and Minneman 2002), but the functional relevance of this interaction is unknown. In human neutrophils, adrenergic receptors almost entirely inhibit the production of superoxide; generated by at least 50,000 formyl peptide receptors. In previous studies (Mueller et al. 1991), it has been reported that the speed and potency of the β-adrenergic receptor system in human neutrophils is remarkable: 500 β-adrenergic receptors are able to inhibit cell response stimulated by up to 105 formyl peptide receptors within seconds of addition of agonists to β-adrenergic receptors. The role of the pathway is to limit the extent of inflammatory response of the cells when they are stimulated to participate in host defense. It would be interesting to investigate nNOS coupling to adrenergic receptors in the neutrophils too and delineate its significance in physiology of these cells.

5.3

Reciprocal Regulation by Caveolin and Calmodulin

Neutrophils are known to be phagocytic cell and to serve their immune response function it is found to be involved in both intracellular and extracellular vesicular transport. Caveolin protein that majorly constitutes the transport vesicles might be involved in trafficking of NOS into and out of the various compartments. Previously, neutrophils were observed to be deprived of caveolin protein in plasmalemmal caveolae (Sengelov et al. 1998). Though confocal and electron microscopic explorations in our lab (Saini et al. 2006) documented caveolin-1-nNOS association presumably accounting for its reduced activity (Yan et al. 1994) as reported earlier.

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NOS becomes active when there is a dimeric interaction between the reductase and oxygenase domain and calmodulin binding occurs between these two domains, which further triggers the electron transfer between the heme and flavin groups. It has been proposed that CaM activates the reductase domain towards reduction of artificial electron acceptors independently; both the mechanisms are supposed to occur from large-scale conformational changes after CaM binding, having negligible influence on the redox properties of the enzyme’s cofactors mainly flavin (Daff 2003). Auto-inhibitory loop (AL) is known to block the electron flow from flavin to heme group in absence of CaM. Binding of CaM displaces the auto-inhibitory loop (AL) leading to enzyme activation. Caveolin inflicts an inhibitory effect on all NOS isoforms (Felley-Bosco et al. 2000; Felley-Bosco et al. 2002), antagonized by Ca2+/ CaM and by the phosphorylation events proposing the role of “caveolin cycle” in the activation or inactivation and re-localization of the enzyme (Michel et al. 1997). Caveolin serves a key role in vesicular trafficking of NOS to the effector sites within cell in an inactivated state to circumvent uncalled production of NO in the cytosolic region. Studies have also proposed a model for the reciprocal regulation of nNOS in endothelial cells where binding of Ca2+-calmodulin complex to nNOS leads to the activation of enzyme and disrupts the inhibitory nNOS-caveolin complex. nNOS is constitutively expressed and requires calmodulin binding from elevated intracellular calcium to synthesize NO. The expression of iNOS is induced by cytokines and tends to produce NO at a greater rate than the other two NOS isoforms. Inhibitory complex is formed between nNOS and caveolin which may cause the latency of NO signal until extracellular, i.e. calcium-mobilizing stimuli disrupts nNOS-caveolin complex and activates the nNOS enzyme. Several studies reported that NO exhibits cytotoxic as well as signaling properties, thus the reciprocal modulation of nNOS enzyme by calmodulin and caveolin may signify a novel mechanism for the intensive control of NO production. In myocardium, extravasation of neutrophils to inflammatory sites under hypoxic conditions is observed that may involve the suppression of caveolin proteins for perpetuation of NO (Navarro-Lerida et al. 2004) or a concomitant reduction of caveolin proteins, as mediated by NO for caveolin-3 and due to LPS/IFN-γ for caveolin-1 and caveolin-2 is also probable. Moreover, CaM and intracellular levels of calcium are of enormous relevance and might play a key role in dissociation of NOS from the inactivating caveolin complex to begin catalysis as we have demonstrated in nuclear and cytosolic compartments of neutrophils (Saini et al. 2006).

5.4

Modulation by Molecular Chaperon: Hsp90

Heat-shock protein 90 (Hsp90) is a well-known molecular chaperone that is found to be localized in the cytosol where it serves a key role in folding of proteins and in maturation events, but it has been highly recognized as an integral component involved in signaling networks. It has also been reported that nNOS-hsp90 heterocomplex results in enhanced production of NO (Kone et al. 2003; Song et al. 2001).

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Several studies were conducted to explore whether hsp90 regulates activity of nNOS by altering CaM binding and it has been confirmed that there was enhancement in nNOS activity (EC (50) of 24.16.4 nM) in the presence of hsp90 in a dosedependent manner. The CaM-nNOS dose response curve was shifted to left in the presence of hsp90, with elevation of its maximal activity. Thus, hsp90 directly enhances nNOS catalytic activity mediated by CaM-binding enhancement (Song et al. 2001). In oxygen deprived conditions, neutrophils exhibit prolonged survival as compared to the other cells. The survival of these granulocytes might possibly be due to interaction of NO, Hsp90, and PI3K-Akt mediated signaling pathways which are active participants of their anti-apoptotic defense mechanism (Saini and Singh 2019). Marked O2
.generation has been observed at diminished levels of L-arginine and hsp90 causes augmentation in synthesis of NO by nNOS enzyme as compared to conditions where normal levels of L-arginine are observed. Intracellular levels of L-arginine have also been reported to result in generation of significant amount of O2
. from nNOS. A study reported that addition of hsp90 prevented this O2
. production and thus led to enhanced nNOS activity (Song et al. 2002). Studies on purified rat nNOS have reported that hsp90 not only monitors the generation of NO from nNOS but also that of O2.
. Hsp90 inhibits O2
production from purified rat nNOS (Song et al. 2001). Such uncoupling phenomenon of nNOS and subsequent production of O2.
can add up to respiratory burst in neutrophils.

5.5

PIN, NOSIP: Inhibitory Potential Towards nNOS

Protein inhibitor of NOS (PIN), an 89-amino acid protein that interacts with PIN binding domain (PINB) of nNOS and destabilizes its dimerization (Fan et al. 1998; Hemmens et al. 1998) is speculated to control its activity despite constitutive presence. It is claimed that PIN neither suppresses activity of nNOS nor promotes its monomerization (Zhou and Zhu 2009), but it inhibits NOS as well as NADPH oxidase functional activity of nNOS in time-dependent manner (Xia et al. 2006). The documentation of PIN as light chain of dynein and myosin (Straub et al. 1998) has led to the suggestion of a different role played by PIN as one of the axonal transport proteins for nNOS rather than modulator of nNOS (Xia et al. 2006; Zhou and Zhu 2009). Presence of PIN and its association if any with nNOS in neutrophils still remains to be explored. NOS Interacting Protein (NOSIP) has been previously known to inhibit eNOS (Dedio et al. 2001) but investigations have revealed that it also has inhibitory potential towards nNOS (Dreyer et al. 2004). It has been observed that subcellular compartmentalization of NOSIP in the hippocampal neurons differs with neuronal activity: after NMDA stimulation it alters in favor of cytosol compartment and when neuronal activity is silenced NOSIP is observed towards the nuclear compartment. The findings that subcellular targeting may regulate the nNOS activity is provided by

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the study in differentiated form of Pheocytochroma (PC12) cells which demonstrated that NMDA promotes the trafficking of nNOS from subcellular sites to the plasma membrane (Oess et al. 2006; Arundine et al. 2003).

6 Intracellular Compartmentalization of nNOS Intracellular localization of NOS might modulate its enzymatic activity due to the microenvironment, availability of cofactors, substrates in optimal concentrations, and interacting proteins. The cell is endowed with means in form of compartments to seize NO, the signal transducer to the precise locations for its effective and utmost proposed use. This perhaps provides the option of regulating the enzyme at the activity level following the genetic checkpoints. Trafficking of the enzyme to such intracellular locales via protein–protein associations and its significant implications inside them is attaining importance lately, which is summarized in Table 2. Table 2 Compartmentalization of nNOS at distinct subcellular locales in different cells/tissues Subcellular organelles Mitochondria

Primary granules Plasma membrane Cytoplasm

Cytosolic vesicles Nucleus

Endoplasmic reticulum

Type of cells/tissues Turtle retina, human skeletal muscles, rat pancreatic β-cells and neutrophils, mice brain, muscle, heart, kidney, and liver Rat neutrophils and eosinophils, human eosinophils, and bone marrow neutrophil precursor cells Teleost brain, rat neutrophils Rat brain, eosinophils, neutrophils, bone marrow neutrophil precursor cells, MDCK cells, COS-7, turtle retina, human mast-cell lines (HMC-1, LAD 2), eosinophils, and skeletal muscles Rat kidney, teleost brain, and pancreatic-β cells Rat cerebral cortical astrocytes, eosinophils, neutrophils, bone marrow neutrophil precursor cells, human eosinophils, MDCK cells, COS-7 and pancreatic β-cells Rat brain and neutrophils, human skeletal muscles, rabbit heart, mice heart, turtle retina, teleost brain

References (Frandsen et al. 1996; Stamler and Meissner 2001; Lajoix et al. 2001; Schild et al. 2005; Saini et al. 2006) (Saini et al. 2006; Saluja et al. 2010; Kumar et al. 2010) (Holmqvist and Ekstrom 1997; Saini et al. 2006) (Frandsen et al. 1996; Riefler and Firestein 2001; Gilchrist et al. 2004; Saini et al. 2006; Saluja et al. 2010; Kumar et al. 2010; Stamler and Meissner 2001) (Vodovotz et al. 1996; Holmqvist and Ekstrom 1997; Lajoix et al. 2001 (Bentz et al. 1997; Riefler and Firestein 2001; Lajoix et al. 2001; Yuan et al. 2004; Saini et al. 2006; Saluja et al. 2010; Kumar et al. 2010) (Xu et al. 1999; Saini et al. 2006; Stamler and Meissner 2001; Michel et al. 1997; Holmqvist and Ekstrom 1997)

MDCK Madin-Darby canine kidney cells, HMC-1 & LAD 2 Leukaemia-derived human mast-cell lines

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Unlike most other endogenous messengers that are deposited in vesicles, processed on demand and/or secreted in a regulated fashion, NO is a highly active molecule that readily diffuses through cell membranes and thus cannot be stored inside the producing cell. Rather, its signaling capacity must be controlled at the level of biosynthesis and local availability. The importance of temporal and spatial control of NO production is previously reviewed by Oess et al. (2006) emphasizing that NOS belong to most tightly controlled enzymes, being regulated at transcriptional and translational levels, through co-translational and post-translational modifications, by substrate availability and not least via specific sorting to subcellular compartments, where they are in close proximity to their target proteins. Previous study from our lab has also explored various subcellular compartments where nNOS is localized in neutrophils (Saini et al. 2006), which is diagrammatically represented in Fig. 3, however the correlation with its function in distinct compartments is still to

Fig. 3 Diagrammatic representation of a neutrophil showing compartmentalized distribution of nNOS. Distinct subcellular compartments such as cytoplasm, nucleus, azurophilic granules, specific granules, and plasma membrane are shown to be associated with nNOS in these cells. NOS in the endoplasmic reticulum and mitochondria are also shown

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be investigated. Neutrophils are vastly ignored in this aspect, but it is essential to inspect the situation as posed by other cellular systems before evaluating the possibilities in neutrophils.

6.1

Trafficking to the Nuclear Compartment

nNOS has been demonstrated to be sequestered in distinct cytoplasmic compartments and even in the nucleus of peripheral neutrophils (Saini et al. 2006) as well as in bone marrow neutrophil precursor cells (Kumar et al. 2010) where a correlation with functionality needs to be deciphered. Interestingly, report on the nuclear localization of CaM and BH4 biosynthesizing enzymes, GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase are of considerable relevance to the availability of a redox sensitive cofactors to support NO synthesis in the nuclear compartment (Bachs et al. 1992; Elzaouk et al. 2004) Earlier studies have evidenced nuclear nNOS in different cell/tissues as elaborated in Table 2. Studies from our lab have demonstrated colocalization of nNOS with caveolin and its activity modulation by CaM in nucleus of neutrophils, suggesting intracellular trafficking of nNOS by NLS bearing caveolin in non-functional state but ready to be activated state by CaM via seemingly “caveolin-cycle” operational in these cells (Saini et al. 2006). It has been demonstrated that NO by augmenting free radical generation mediates neutrophil extracellular traps (NET) release at the site of inflammation/infection (Patel et al. 2010) and nuclear NOS might be involved in disrupting and extricating the genetic material to form extracellular traps for extracellular bacterial killing (Brinkmann et al. 2004). However, the intricate mechanism resulting in the dissociation of nNOS with caveolin, nuclear localization of CaM and BH4 biosynthetic enzymes is yet to be explored in neutrophils to decipher clearly the role of NO in nuclear compartment. nNOS in the nuclear compartment has also been reported in eosinophils though its correlation with functionality is still to be investigated (Saluja et al. 2010). Nuclear nNOS acts in protein trafficking like, employing its PDZ domain for nucleo-cytoplasmic shuttling of carboxy terminal binding protein (CtBP) (Riefler and Firestein 2001). Though unprecedented, based on the existing studies it cannot be ruled out that nNOS might even perform functions other than NO synthesis such as protein activity modulation by physical interactions and their localization in the specific cellular compartment that may not necessarily require enzymatically active NOS. Though several studies have demonstrated nNOS to be present in the nucleus, but they have remained speculative to define the role of nuclear NOS (Xu et al. 1999; Lajoix et al. 2001; Giordano et al. 2002; Yuan et al. 2004; Gilchrist et al. 2004). S-Nitrosylated GAPDH has been found to initiate apoptotic cell death in neurons, indicating that S-nitrosylation is a ubiquitous post-translational modification to regulate protein functions and their translocation (Hara et al. 2005). NO mediated S-nitrosylation, ubiquitination, oxidation, protein phosphorylation, activation of phosphatases or altered protein interactions might activate or inhibit transcription

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by modulating transcription factors and signaling cascade (Turpaev et al. 2005; Hess et al. 2005). Even initiation of transcription process by RNA polymerase I and II in conjunction with actin and myosin I (Philimonenko et al. 2004) seems to be modulated by cGMP-dependent protein kinase by dephosphorylating myosin light chain (Surks et al. 1999), which further signifies the presence of intra-nuclear NOS. Further studies are however required to define the exact role of NOS/NO in the nuclear compartment of neutrophils.

6.2

nNOS Associated with Plasma Membrane

Plasma membrane contains proteins involved in receptor signaling and it links extracellular and intracellular processes. It becomes enriched with several other proteins from intracellular reservoirs when the cell is in activated state. nNOS associated with plasma membrane seems to play vital role as plasma membrane limits the ingested material in compartments which are formed by local projections of the plasma membrane which extend along the object, surround, fuse, and produce phagocytic vacuoles. In neutrophils, the professional phagocytes and nNOS presence in the membrane fractions ensures its proximity for prospective interaction with components of the NADPH oxidase complex in modulating its assembly thereby directly governing respiratory burst associated with these cells. This also potentiates its enclosure in the phagolysosomal vesicles to deliver NO for microbicidal actions or function in trafficking of cargo proteins employing protein–protein interactions.

6.3

Cytoplasmic nNOS: Association with Cytoskeleton

Our defense against microbes depends largely on the ability of neutrophils to migrate from the blood stream to sites of infection and its cytoskeleton is involved in chemotaxis and degranulation. In an actively migrating cell such as neutrophil, structures involved in cell locomotion (actin filaments) and in intracellular transport of granules (microtubules) have immense significance and both the azurophilic and specific granules employ these vehicles. Granule movement is organized by the microtubules, which radiate from the center of the cell while actin network depolymerization facilitates granules to fuse with plasma membrane. Thus, the cytoskeletal network evokes trafficking of NOS to sites of action in the phagosomes or organelles for localizing NOS activity as per need; while NO critically inspects the changes in the cytoskeletal parameters ensuring a mutual effect on each other. There are considerable evidences that NO and cyclic GMP act as endogenous mediators of the chemotactic response of neutrophils (Armstrong 2001), yet there is a vast hiatus of ignorance as to how these responses work. The isolation of both actin and myosin from neutrophils (Stossel and Pollard 1973; Tatsumi et al. 1973) has raised the possibility that the interaction between these proteins is the driving force for

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movement within phagocytes and it is seemingly controlled in similar ways as muscle contraction. Undoubtedly these cells are powered with such mechanism which facilitates them to traverse between endothelial cells and undergo dramatic deformation passing through pores 3–4 times smaller than the cell diameter (Malech et al. 1977). Almost certainly cytoplasmic actin-myosin microfilaments as well as microtubules play a role in mediating the cytoplasmic movements necessary for chemotaxis and phagocytosis; microfilaments provide the motor mechanism while microtubules act as cytoskeletal scaffolding. Continuous reorganization of the actin cytoskeleton is required for efficient chemotaxis. In unstimulated, randomly moving cells, F-actin is localized in newly extended pseudopods. F-actin depolymerization has been found to be very sensitive to peroxynitrite. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade under the peroxynitrite mediated oxidative stress for some pathophysiological conditions. NO has been shown to inhibit neutrophil chemotaxis through peroxynitrite, which is produced under conditions of high NO and superoxide NO-dependent mechanism involving ADP-ribosylation of actin (Forslund et al. 2000). Additionally, Tiago et al. (2006) have also demonstrated that peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation. Actin-binding proteins, mainly cofilin, coronin, and filopodin probably have a function in regulating the amount of F-actin and the reorganization of filaments. These are localized to extended pseudopods during chemotaxis (Gerisch et al. 1995; Kreitmeier et al. 1995; van Es and Devreotes 1999). The question arises that by what mechanisms these proteins become localized to the leading edge, when cells are exposed to a gradient. Thus, possibility of nNOS acting in a non-catalytic manner such as a carrier protein cannot be ruled out in such transport of actin binding proteins, as it has been shown to transport CtBP (Riefler and Firestein 2001). Importance of cofilin has been proposed in phagocyte functions through dephosphorylation and translocation to the plasma membrane regions. It has also been demonstrated that NO caused translocation of cofilin to the cell periphery, though dephosphorylation of cofilin was not detected (Adachi et al. 2000). Moreover, investigation has also demonstrated the actin-destabilizing protein cofilin’s NO-dependent effects in primary neuronal cultures and that cofilin phosphorylation is involved in nitric oxide/cGMP-mediated nociception (Zulauf et al. 2009). Additionally, differentiation of neutrophils involves considerable cytoskeletal rearrangements in the form of shape change, constitution of granules and segmentation of nucleus, also likely to be monitored by NO. Recently, NO generating ability and the molecular/biochemical characteristics of nNOS isoforms in bone marrow neutrophil precursor cells demonstrated that expression of nNOS was predominantly present in all the stages of neutrophil maturation (Kumar et al. 2010), seemingly NO has vital importance in the cytoskeletal rearrangements during differentiation. This is a vastly uncovered area of research in neutrophils that seeks much attention.

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Localization in Mitochondria and Endoplasmic Reticulum

In a terminally differentiated cell like neutrophil, where minimal synthesis of new compounds takes place, cytoplasm is scarce in mitochondria, endoplasmic reticulum, and golgi. nNOS was found to be associated with mitochondria and endoplasmic reticulum in neutrophils (Saini et al. 2006). Most remarkably, nanomolar concentrations of NO can inhibit mitochondrial respiration, so even a small amount of NO in the mitochondrial matrix may regulate ATP synthesis. Therefore, the idea that mitochondria themselves are capable of NO production is an important concept in several physiological and pathological mechanisms (Lacza et al. 2006). Besides effects of NO on mitochondrial enzymes and the stimulation of mitochondrial H2O2 production, a NO-dependent increase in mitochondrial biogenesis in several tissues has been reported (Schild et al. 2005). The importance of NO in the mitochondrial compartment has been recognized in the apoptotic execution of these cells. Study by Carreras et al. (2008) demonstrated nNOS trafficking into the mitochondria after processing of the PDZ domain. Studies related to the PDZ domain in neutrophil nNOS and its role in nNOS trafficking however need to be investigated in neutrophils. The majority of nNOS immune reactivity in neurons is associated with rough endoplasmic reticulum and within specialized electron-dense synaptic membrane structures (Michel and Feron 1997). Although synthesis of the NOS proteins involves this organelle, it is less clear what could be the function of nNOS in the endoplasmic reticulum of neutrophils where minimal synthesis of proteins takes place.

6.5

Sequestration in Primary Granules

Neutrophils depict not only spatial distribution but also compartmentalization of a wide range of hydrolytic enzymes involved in non-oxygen dependent microbicidal activity and degradation of substances. Two major populations, primary or azurophilic and specific granules are employed where azurophilic granules are predominantly located in the outer region of the cell, whereas specific granules are placed more centrally. These partitioning of granules show different degranulation dynamics, specific granules being mobilized selectively, albeit to a varying extent, in response to most soluble stimuli. This is primarily due to different Ca2+ requirements for exocytosis, specific granules being more sensitive to a rise in ([Ca2+]i) and consequently released before the azurophils (Lew et al. 1986). Interestingly, reports demonstrated the localization of NOS in azurophilic granules and its absence in specific granules (Saini et al. 2006). Additionally, nNOS has also been found to be localized in eosinophilic granules, where its correlation with functionality still needs to be explored (Saluja et al. 2010). Neutrophils are known to accomplish vital task to

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remove microbes by segregating them intracellularly in sealed structures by phagocytosis, the regulated exocytosis of granules enables the neutrophil to deliver its arsenal of potentially cytotoxic granule proteins in a targeted manner, thus preventing widespread damage to host tissue in most situations.

7 Clinical Implications The potential importance of neutrophil is exemplified in several pathologies and the significance of neutrophil nNOS is emphasized in neuronal disorders (Gatto et al. 2000). Activated neutrophils infiltrating the central nervous system suppress potential autoimmune T cell response to myelin and adjuvant antigens depending on the orchestrated IFN-γ secretion from the target T cells and NOS catalysis among neutrophils (Zehntner et al. 2005). A thorough understanding of differential operation of nNOS in neutrophils could possibly provide us with peripheral markers in neurological disorders such as Parkinson’s disease, where an up-regulated nNOS expression and nitrite load was documented (Barthwal et al. 1999; Gatto et al. 2000). Gatto et al. found markedly increased nNOS expression and activity in activated neutrophils from peripheral circulation of Parkinson’s disease patients. This could entail strengthening promoter activity by exposure to environmental mutagens causing prevalence to Parkinsonism or plasma factors; or it could be an adaptive mechanism secondary to decreased dopaminergic activity. Overall, a generalized increase in NO and peroxynitrite formation contributes to excitotoxicity, mitochondrial damage, apoptotic neurodegeneration in Parkinson’s disease (Barthwal et al. 1999; Gatto et al. 2000). Conversely, decrease in neutrophil total nitrite content is evident in patients of depression (Srivastava et al. 2002), also extending to the case of schizophrenic patients in which with augmented dopaminergic activity is activated (Srivastava et al. 2001). Studies from our lab on depression patients have demonstrated decreased nNOS activity in neutrophils concurrent with reduction in the expression of β-adrenergic receptors (Srivastava et al. 2002). Schizophrenic patients with augmented dopaminergic activity too exhibit attenuated nitrite content in neutrophils. Thus, exploring NO mediated intonation of neutrophil physiology, in relation to such neuronal disorders is of grave importance.

8 Conclusion nNOS bears the potential to interact with a wide array of proteins in the intracellular environment, which opens a new era of nNOS biology to be explored. It also emphasizes on the plausible involvement of nNOS itself as a signaling, docking/ scaffolding, regulatory protein in relaying signals apart from its conventional purpose of generating NO in neutrophils. Besides localization of nNOS into target specific intracellular compartments by means of protein–protein interactions also

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delivers NO to local target molecules and exerts a control over their functionality. Inhibition of nNOS directly may disturb physiological functions and thus interfering with nNOS-specific pathways involving uncoupling of nNOS with its protein partner would thus be a better alternative and may have vital clinical implications. In comparison with the vast array of literature available for endothelial, macrophages, and neuronal cells, neutrophil nNOS has remained unexplored on various aspects, thus this review highlights the need to look into the loopholes that call for further investigations in these cells. Acknowledgements This work was financially supported by projects: DST-SERB (EMR/2016/ 007158), Govt. of India and intramural project from All India Institute of Medical Sciences (AIIMS), New Delhi-India, sanctioned to RKS. RS acknowledges the Department of Zoology, Gargi College, University of Delhi, Delhi-India for providing infrastructural facilities. RKS, ZA, and LS acknowledge the Department of Biotechnology, AIIMS, New Delhi-India for providing infrastructural facilities. ZA also thanks Dr. Harisingh Gour Central University, Sagar (MP) for providing infrastructural facilities. ZA and LS thank UGC for research fellowships. Conflicts of Interest All the authors state that no conflict of interest exists regarding the described work.

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Rev Physiol Biochem Pharmacol (2021) 180: 85–118 https://doi.org/10.1007/112_2021_60 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 Published online: 25 May 2021

When Glycosylation Meets Blood Cells: A Glance of the Aberrant Glycosylation in Hematological Malignancies Huining Su, Mimi Wang, Xingchen Pang, Feng Guan, Xiang Li, and Ying Cheng

Contents 1 2 3 4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycosylation in Hematological Malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycosylation in Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Acute Myeloid Leukemia (AML) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Chronic Myeloid Leukemia (CML) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Acute Lymphoblastic Leukemia (ALL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Chronic Lymphocytic Leukemia (CLL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Glycosylation in Lymphomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Hodgkin’s Lymphoma (HL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Non-Hodgkin’s Lymphoma (Non-HL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Glycosylation in Multiple Myeloma (MM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Glycosylation in Other Blood Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Glycosylation in Myeloproliferative Neoplasms (MPNs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Glycosylation in Myelodysplastic Syndrome/Myeloproliferative Neoplasms (MDS/MPNs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Glycosylation in Chronic Neutrophilic Leukemia (CNL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

H. Su, M. Wang, and Y. Cheng (*) Center for Mitochondrial Biology and Medicine, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, China e-mail: [email protected] X. Pang, F. Guan, and X. Li (*) Key Laboratory of Resource Biology and Biotechnology Western China, College of Life Science, Northwest University, Xi’an, China e-mail: [email protected]

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Abstract Among neoplasia-associated epigenetic alterations, changes in cellular glycosylation have recently received attention as a key component of hematological malignancy progression. Alterations in glycosylation appear to not only directly impact cell growth and survival, but also alter the adhesion of tumor cells and their interactions with the microenvironment, facilitating cancer-induced immunomodulation and eventual metastasis. Changes in glycosylation arise from altered expression of glycosyltransferases, enzymes that catalyze the transfer of saccharide moieties to a wide range of acceptor substrates, such as proteins, lipids, and other saccharides in the endoplasmic reticulum (ER) and Golgi apparatus. Novel glycan structures in hematological malignancies represent new targets for the diagnosis and treatment of blood diseases. This review summarizes studies of the aberrant expression of glycans commonly found in hematological malignancies and their potential mechanisms and defines the specific roles of glycans as drivers or passengers in the development of hematological malignancies. Keywords Glycans · Glycosyltransferase · Hematological malignancies · Lectin · N-glycosylation · O-glycosylation

Abbreviations 2DG 9-OAcSGs Ac5GalNTGc aCML ADC AFP ALG9 ALL ALL AML asialo GM2 Asn B-ALL B-CLL BCR BL CALR CD138 CD62L CD82 CEA Cer CLL

2-Deoxy-D glucose 9-O-acetylated sialoglycoconjugates Peracetyl N-thioglycolyl-D-galactosamine Atypical chronic myeloid leukemia Antibody-drug conjugate α-Fetoprotein α-1,2-Mannosyltransferase Acute lymphoblastic leukemia Acute lymphoblastic leukemia Acute myeloid leukemia Gangliotriaosylceramide Asparagine B cell type acute lymphoblastic leukemia B cell phenotype chronic lymphocytic leukemia B cell antigen receptor Burkitt lymphoma Calreticulin Cell surface proteoglycan syndecan-1 Lymphocyte homing receptor L-selectin Kai1 Carcinoembryonic antigen Ceramide Chronic lymphocytic leukemia

When Glycosylation Meets Blood Cells: A Glance of the Aberrant. . .

CML CMML CNL CSF3R DLBCL EMT ET FL FLT3 FNG Fuc GAG Gal GalNAc GCS Gg3 Glc GlcA GlcNAc GM3 GPI GSL HCLL HER2 HEV HGF HL HNRNPH1 HTLV-1 IdoA IFNα Ig VH IKKβ ITD JMML LGALS3 LSCs mAb Man MDR MGAT3 MGL MM

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Chronic myeloid leukemia Chronic myelomonocytic leukemia Chronic neutrophilic leukemia Colony-stimulating factor-3 receptor Clinical common large B cell lymphoma Epithelial mesenchymal transition Essential thrombocytosis Follicular lymphoma FMS-related tyrosine kinase 3 Fringe glycosyltransferase Fucose Glycosaminoglycan Galactose β-D-N-acetylgalactosamine Glucose ceramide synthase Gangliotriaosylceramide Glucose Glucuronic acid β-D-N-acetylglucosamine Sialosyllactosylceramide Glycosyl phosphatidylinositol Glycosphingolipid Hematopoietic cell L-selectin ligand Human epidermal growth factor receptor 2 High endothelial venule Hepatocyte growth factor Hodgkin’s lymphoma Heterogeneous nuclear ribonucleoprotein H1 T cell leukemia virus type 1 Iduronic acid Interferon alpha Immunoglobulin variable heavy chain genes IκB kinase subunit β Internal tandem duplications Juvenile myelomonocytic leukemia Galectin-3 Leukemic stem cells Monoclonal antibody Mannose Multidrug resistance Mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase 3 Macrophage galactose-type lectin Multiple myeloma

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MPL/TpoR MPN MRP1 MTOG NEU3 Neu5Ac non-HL Pgp PMF POFUT1 POGLUT1/Hclp46 PSA PSGL1/CD162 PV Ser sIgs SNHG3 SRGN ST3GAL1/4/5/6 ST8SIA4/6 T-ALL T-CLL Thr TKIs TLRs TPO TrfRs V regions Xyl

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TPO receptor Myeloproliferative neoplasm MDR-associated protein 1 Mucin-type O-glycosylation Membrane type- and ganglioside-specific sialidase 5-N-acetylneuraminic acid Non-Hodgkin’s lymphoma P-glycoprotein Primary myelofibrosis O-fucosyltransferase 1 O-glucosyltransferase 1 Prostate specific antigen P-selectin glycoprotein ligand 1 Polycythemia vera Serine Surface immunoglobulins Small nucleolar RNA host gene 3 Serglycin α-2,3-Sialyltransferase 1/4/5/6 α-2,8-Polysialytransferase 4/6 T cell type acute lymphoblastic leukemia T cell phenotype chronic lymphocytic leukemia Threonine Tyrosine kinase inhibitors Toll-like receptors Thrombopoietin Transferrin receptors Variable regions Xylose

1 Introduction Carbohydrates, together with lipids, proteins, and nucleic acids, are the four major organic compounds in cells. Each has its own important functions in the development of organisms. Among them, the carbohydrates are not only important energy sources and structural components of cells, but can also be employed to modify lipids and proteins via the covalent addition of monosaccharides or even whole oligosaccharides (glycans). This enzyme-catalyzed process of glycosidic linkage of saccharides to other saccharides, proteins, or lipids is called glycosylation (Pinho and Reis 2015). Glycosylation has a fundamental impact on a diverse range of biological processes. Glycosylated lipids or proteins mediate not only processes such as cell development and differentiation, but also biological events such as

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transport, signaling, folding, and adhesion. Therefore, it is not hard to imagine that minor alterations in glycosylation will lead to a variety of physiological and pathological changes. Aberrant alteration of glycosylation is a hallmark of cancer (Abroun et al. 2008; Moremen et al. 2012; Pinho and Reis 2015). It has been well documented that aberrant glycans and glycosylation are associated with malignant transformation and the development of cancers, especially in the early stages of cancer development (Marth and Grewal 2008; Moremen et al. 2012). Well-known proteins involved in tumorigenesis and epithelial mesenchymal transition (EMT), such as p53, IκB kinase subunit β (IKKβ), c-Myc, and Snail are all regulated by glycosylation (Yang et al. 2006; Keiko et al. 2009; Chou et al. 1995; Yoon et al. 2014). In addition, glycans, glycoproteins, and glycolipids can be secreted or can leak into serum when cancer cells undergo necrosis and apoptosis. These tumor-derived molecules detected in serum, such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), and prostate specific antigen (PSA), can be used as markers for cancer diagnosis and prognosis (Dhanisha et al. 2018; Chandler et al. 2013; Kirwan et al. 2015; Eichhorn et al. 2005; Reis et al. 2010; Silva 2015). At such, the mechanisms by which these abnormal glycosylation products are generated can be investigated to help to reveal the molecular basis of cancers (Ju and Cummings 2002). During the past decades, remarkable progress has been achieved in our understanding of the role of glycosylation in hematological malignancies. Here we briefly summarize the recent advances in our knowledge of the relationship between aberrant glycosylation and the development of hematological malignancies.

2 Glycosylation Glycosylation is a complex synergistic process involving enzymes, organelles, and other factors that are necessary for the generation of carbohydrate-associated posttranslational modifications (Stowell et al. 2015). In mammals, the major glycans contain 10 monosaccharide building blocks, glucose (Glc), galactose (Gal), fucose (Fuc), mannose (Man), xylose (Xyl), glucuronic acid (GlcA), iduronic acid (IdoA), β-D-N-acetylglucosamine(GlcNAc), β-D-N-acetylgalactosamine (GalNAc), and 5-N-acetylneuraminic acid (Neu5Ac), all of which can be originally derived from Glc in every cell (Stowell et al. 2015). As shown in Fig. 1, according to the conserved core structures, glycosylation is roughly divided into N-glycosylation, O-glycosylation, glycosaminoglycan (GAG), glycosphingolipid (GSL), and glycosyl phosphatidylinositol (GPI)-linked proteins (Pang et al. 2018). N-glycosylation is the most common type of glycosylation. The structures of N-glycosylation, that is, N-glycans, which usually have a common conserved pentasaccharide core structure, are linked to asparagine (Asn) residues of proteins, specifically a subset residing in the Asn-X-serine (Ser)/threonine (Thr) (N-X-S/T) motif, in which X can be any amino acid except proline (Pro). The most

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Fig. 1 Glycosylation and hematological malignancies

common abnormal N-glycosylation processes found in hematological malignancies are roughly divided into three categories including sialylation, fucosylation, and bisecting GlcNAc (Pang et al. 2018). O-glycosylation is another common type of glycosylation essential for the biosynthesis of mucins, and it usually occurs on the Ser and Thr residues of glycoproteins. The most common type of protein Oglycosylation is O-GalNAc modification, which involves α linkage of GalNAc to the hydroxyl group of Ser or Thr by an O-glycosidic bond. Other types of covalent modifications of Ser/Thr residues can involve Glc, Fuc, and GlcNAc. Additionally, O-linked glycans usually have much simpler oligosaccharide structures than Nlinked glycans (Pang et al. 2018). GAGs are a class of highly sulfated and long unbranched polysaccharides with a repeating disaccharide structure. GAGs can be mainly categorized into four subclasses: heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratin sulfate. GAG chains are usually covalently attached to Ser or Thr residues of proteins. The GAG chains are indispensable functional parts. They are produced through various biosynthetic pathways and are usually highly sulfated, thus having the ability to bind cytokines, chemokines, or growth factors. Through binding, GAG-modified proteins can change cell growth and differentiation, thereby contributing to the regulation of embryogenesis, angiogenesis, and homeostasis (Pang et al. 2018). In addition to N-glycans and O-glycans, GSLs are composed of glycans attached to sphingolipid ceramide (Cer). The core structure of GSLs includes a monosaccharide, usually Glu or Gal, that is directly

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attached to Cer molecules. The GSL core glycan structure can be extended by other monosaccharides. Since GSLs on the cell membranes are involved in the regulation of retention, quiescence, mobilization, and homing of hematopoietic stem/progenitor cells, they are important for hematological oncogenesis and ontogenesis (Ogretmen and Hannun 2004; Ratajczak and Adamiak 2015). Glycosylation is usually catalyzed by glycosyltransferases and glycosidases with distinct substrate specificities. Glycosyltransferases are in charge of “adding” sugar blocks, while glycosidases are in charge of “removing” sugar blocks. Abnormalities in glycosyltransferases and glycosidases are closely related to the development of cancers (Pinho and Reis 2015; Stowell et al. 2015). Glycans can be recognized by lectins, such as selectins, galectins, and calreticulin (CALR), which can modulate activity and signaling in cells, and thus contribute to the regulation of cell physiological and pathological processes. Lectins are glycan-binding proteins that are typically highly selective for specific glycan structures. Aberrant glycosylation alters the abundance of ligands of endogenous lectins, and thereby affects multiple cellular mechanisms involving the corresponding glycans (Pinho and Reis 2015; Stowell et al. 2015).

3 Glycosylation in Hematological Malignancies Glycan aberrations can be found in most hematological malignancies. Increased glycosylation in hematological malignancies is triggered by overexpression of glycoproteins that carry certain specific glycans and altered expression of glycosyltransferases and glycosidases (Marth and Grewal 2008; Pinho and Reis 2015). These glycoproteins are involved in signaling pathways related to cell proliferation, adhesion inhibition and immune escape. Because changes in glycosylation occur in the early stage of cancer development, the detection of tumor associated-glycosylation markers is an effective strategy that can improve the clinical diagnosis and treatments. Here, we will outline and discuss glycosylation abnormalities in different hematological malignancies, including leukemia, lymphoma, myeloma, and other types of malignancies.

4 Glycosylation in Leukemia Leukemia is a broad term encompassing several hematological malignancies with increased numbers of leukocytes in blood or bone marrow. Once the hematopoietic stem cells in bone marrow transform into leukemic cells, they will grow and survive better than normal cells, and subsequently suppress the development of normal cells. The type of leukemia depends on the type of blood cells involved and the rate of leukemia progression.

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Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. The survival rates of AML for both adults and children are very poor, with the overall five-year survival rate is 27–65% (Almeida and Ramos 2016). AML is a heterogeneous blood cancer that results from abnormal proliferation of white blood cells that are initiated and maintained by leukemic stem cells (LSCs) featuring aberrations such as translocations of t(6;9)(p22;q34), t(8;21), t(15;17), or inv (16) (Grimwade et al. 1998; Rowley and Potter 1976). In addition, aberrations of chromosomes 5 and 7 or abnormalities of 11q23 in AML are involved in the poor response to chemotherapy (Schoch et al. 2005). Aberrant glycosylation, such as abnormalities in N-glycosylation, O-glycosylation, and GAG modifications, can occur in AML (Table 1). Aberrant glycosylation is involved in changes in proliferation-related signaling molecules during tumor formation. NOTCH signaling plays crucial roles in the growth of AML cells. The activation of NOTCH signaling induces overexpression of glycosyltransferases including protein O-fucosyltransferase 1 (POFUT1), Fringe (FNG) glycosyltransferase and protein O-glucosyltransferase 1 (POGLUT1, also known as hCLP46), which further increases the glycosylation levels of O-Fuc and O-Glu in NOTCH (Ma et al. 2011; Chu et al. 2013; Yao et al. 2011; Wang et al. 2018). Glycosylation leads to the increased sensitivity of NOTCH to ligands, further enhancing the NOTCH activity in a positive feedback manner (Wang et al. 2018). FMS-related tyrosine kinase 3 (FLT3), a receptor tyrosine kinase that mediates the signaling involved in the proliferation of hematopoietic stem and progenitor cells during development, has been established as a molecular marker on leukemic blasts of patients with AML (Reilly 2003). FLT3/ITD (internal tandem duplications) mutations have been found in approximately 30% of AML patients (SchmidtArras et al. 2005). FLT3 needs to undergo posttranslational modification after translation to serve as a functional cell surface receptor. FLT3 is first glycosylated in the ER to form an immature protein, undergoes final glycosylation in the Golgi complex to become a mature receptor, and then translocates to the cell surface (Williams et al. 2012). FLT3/ITD protein exists predominantly in an immature, underglycosylated 130-kDa form in AML, whereas the wild-type FLT3 protein is a mature, completely glycosylated 150-kDa molecule (Schmidt-Arras et al. 2005). Because they induce cell apoptosis through inhibition of glycosylation of FLT3, fluvastatin and 2-Deoxy-D-glucose (2DG) are potential drugs that can prolong the survival of AML patients in the clinic (Williams et al. 2012; Larrue et al. 2015). In addition to fluvastatin and 2DG, an Fc-optimized antibody termed 4G8SDIEM that directly targets FLT3 was developed to treat AML. This type of antibody was designed by defined modifications of the glycosylation pattern or the amino acid sequence of the human immunoglobulin G1 Fc part to enhance its ability to induce cellular cytotoxicity against FLT3-expressing cell lines as well as blasts of AML patients (Hofmann et al. 2012). In addition, several flavonoid derivatives have the potential to be further optimized as FLT3 inhibitors and provide valuable chemical information for the development of new AML drugs (Yen et al. 2021).

ALL

CML

Disease types Leukemia AML

Glycosaminoglycan

O-glycosylation

Glycosaminoglycan

N-glycosylation Glucosamine and Galactosamine Sialylation

CD43 CD45 9-O-acetylated sialoglycoprotein

Glycoproteins in granulocytes cell surface NKG2D Galectin-3

GP Ib, IIb, IIIa, and IIIb IFNα

FLT3-ITD P-gp

Cell differentiation and proliferation regulation Involve in NK-mediated lysis Cell proliferation regulation and antiapoptosis Drug resistance Cell adhesion Cell adhesion Cell survival and antiapoptosis Escape from host defenses

SRGN HNRNPH1 Galectin-3

Underglycosylation Unknowna

Cell proliferation and antiapoptosis Unknowna Leukemia microenvironment niche Poor survival outcome Immune escape Progression, bone marrow retention, and chemo-resistance Cell proliferation regulation Antiapoptosis Drug resistance Unknowna Cell proliferation regulation

NOTCH

O-glycosylation

Galectin-9 CD162

Biological impacts Bone marrow homing Cell adhesion and trafficking Cell proliferation regulation

Related proteins CD82

Glycosylation types/ levels N-glycosylation

Table 1 Aberrant glycosylation associated with different types of leukemia

(continued)

(Mazurov et al. 2012) (Mazurov et al. 2012) (Bandyopadhyay et al. 2005; Ghosh et al. 2007)

(Cebo et al. 2006) (Nakayama et al. 2014; YamamotoSugitani et al. 2011)

(Cyopick et al. 1993)

(Larrue et al. 2015; Williams et al. 2012) (Del Poeta et al. 1996; Turzanski et al. 2005) (Clezardin et al. 1985) (Labdon et al. 1984)

(Chu et al. 2013; Ma et al. 2011; Wang et al. 2018; Yao et al. 2011) (Niemann et al. 2007; Peng et al. 2020) (Balkhi et al. 2006) (Cheng et al. 2013; Gao et al. 2017; Ruvolo 2019; Ruvolo et al. 2018) (Goncalves Silva et al. 2017) (Erbani et al. 2020)

References (Marjon et al. 2016)

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a

HCLL CD9 CD95 DC-SIGN L-SIGN TrfRs P-selectin p53 c-Myc Akt B cell antigen receptor CD79a μ-constant region of IgM

N-glycosylation

Igα and Igβ CD62L

Galectin-1

Underglycosylation Immature mannosylation N-glycosylation Unknowna

Glycosaminoglycan

Underglycosylation Unknowna O-GlcNAcylation

GM3

Related proteins NOTCH

Glycosphingolipid

Glycosylation types/ levels

BCR signaling activation Cell trafficking and proliferation regulation Drug resistance Cell proliferation and invasion Immune escape

Cell proliferation, apoptosis, and differentiation Cell adhesion Cell adhesion IFNγ resistance Immunosuppressive responses Immunosuppressive responses Unknowna Cell adhesion Cell proliferation regulation Cell proliferation regulation Cell proliferation regulation B cell hemostasis and transduction Cell proliferation regulation BCR signaling activation

Biological impacts Cell proliferation regulation

(Croci et al. 2013)

(Kriss et al. 2012) (Lafouresse et al. 2015)

(Sackstein and Dimitroff 2000) (Komada and Sakurai 1994) (Dorrie et al. 2002) (Gijzen et al. 2008) (Gijzen et al. 2008) (Petrini et al. 1989) (Pluchart et al. 2021) (Shi et al. 2010) (Shi et al. 2010) (Shi et al. 2010) (Shi et al. 2010; Wu et al. 2017) (Vuillier 2005) (Krysov et al. 2010)

(Tringali et al. 2009)

References (Chu et al. 2013; Ma et al. 2011; Wang et al. 2010)

Means not be mentioned in the current research progress. Underglycosylation means the glycosylation is absent or half-baked

CLL

Disease types

Table 1 (continued)

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Changes in glycosylation of signaling molecules associated with cell migration and bone marrow homing also contribute to the development of AML. CD82 (also known as Kai1) is considered a metastasis suppressor in solid tumors (Dong et al. 1995). The glycosylation level of CD82 affects the molecular structure of N-cadherin by modulating N-glycosylation and the adhesion and trafficking of AML cells, and thus controls the progression of disease (Marjon et al. 2016). Serglycin (SRGN) is the major cell-associated proteoglycan of hematopoietic cells and the level of SRGN in plasma from patients with AML was reported to be significantly higher than that in plasma from patients with acute lymphoblastic leukemia (ALL) or Philadelphia chromosome-negative chronic myeloproliferative leukemia (CML) (Niemann et al. 2007). An abnormal expression level of SRGN can be used as a marker for AML diagnosis (Niemann et al. 2007). A recent study revealed the underlying mechanism of SRGN in AML. The expression of SRGN can be modulated by small nucleolar RNA host gene 3 (SNHG3), a recently identified long noncoding RNA. Increased expression of SRGN stimulates cell proliferation by enhancing Ki67 expression and inhibits cell apoptosis by reducing caspase 3 expression in AML patients (Peng et al. 2020). Galectins belong to a family of lectins which have high affinities for β-galactosides (Ruvolo 2019). Fifteen galectins have been identified and they are involved in cell differentiation, inflammation, adhesion, migration, and apoptosis (Ruvolo 2019). Previous studies have shown that higher galectin-3 (LGALS3) expression in bone marrow is an independent prognostic factor for poor survival in AML patients (Gao et al. 2017; Cheng et al. 2013; Ruvolo 2019). Galectin-3 is related to the regulation of mesenchymal stromal cell homeostasis, and cell localization and cell survival in the leukemia microenvironment niche for AML (Ruvolo et al. 2018). In addition, another member of the galectin family, galectin-9, is believed to be involved in the immune escape of human myeloid leukemia cells (Goncalves Silva et al. 2017). Because of the important role of galectins in the development of AML, the novel galectin inhibitor GCS-100 may be a potential drug for AML therapy (Ruvolo et al. 2016). E-selectin receptor CD162 was reported that it is associated with AML chemo-resistance (Erbani et al. 2020). Actually, the alterations in cell surface glycosylation associated with oncogenesis enhance AML blast binding to E-selectin and enable promotion of pro-survival signaling through AKT/NF-κB pathways. Absence or therapeutic blockade of E-selectin using small molecule mimetic GMI-1271/Uproleselan effectively inhibits this niche-mediated pro-survival signaling and dampens AML blast regeneration (Barbier et al. 2020). Changes in glycosylation also affect molecules associated with the drug resistance in AML. Cer is an apoptosis inducer in response to chemotherapy in AML (Haimovitz-Friedman et al. 1997; Hannun and Obeid 1997). P-glycoprotein (P-gp) can be targeted to reverse resistance to Cer-induced apoptosis (Turzanski et al. 2005) and has already been used as a classical marker for overcoming multidrug resistance (MDR) in AML (Del Poeta et al. 1996). Specific inhibition of glycosylation with the P-gp inhibitor chlamycin caused a decrease in the activity of glucose ceramide synthase (GCS) (Turzanski et al. 2005). Therefore, it is speculated that the underlying mechanism of MDR in AML is caused by increased enzyme activity of GCS, which is involved in the synthesis of P-gp. Low expression of glycosyltransferase

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ALG9 is a predicted poor prognosis of AML patients and lncRNAs target ALG9, which plays a post-transcriptional regulatory role providing a novel therapeutic target for AML chemo-resistance (Yu et al. 2020). In addition, proteomic analysis showed that heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1), an RNA-binding protein, was overexpressed and modified with O-GlcNAc in AML patients with chromosome 11q23 ectopic translocation, although the association between HNRNPH1 and glycosylation modification in AML remains to be further investigated (Balkhi et al. 2006). Although many glycosylation aberrations have been found in AML patients, to the best of our knowledge, glycosylation changes have not been reported in relatively rare subtypes of AML, such as acute megakaryoblastic leukemia, which is associated with a high risk of Down’s syndrome children.

4.2

Chronic Myeloid Leukemia (CML)

CML is caused by a chromosomal translocation between the long arms of chromosomes 9 and 22 that forms the fusion gene Bcr-abl1 (PeterC.Nowell 2002; Stagno et al. 2016). The Bcr-abl1 gene encodes the oncoprotein BCR-ABL1, which has constitutively increased tyrosine kinase activity, resulting in aggression and proliferation advantage of hematopoietic stem cells (Ren 2005; Giallongo et al. 2011; Stella et al. 2013; Manzella et al. 2016; Preyer et al. 2011; Massimino et al. 2014). As shown in Table 1, glycosylation aberrations have been reported in CML. Currently, most of the studies on glycosylation modification in CML focus on Nglycosylation. Abnormal modification of glycoprotein components on the surface of macromolecules of CML platelets was the first to be reported. Platelets from CML patients show elevated galactosyl and GalNAc modifications of macromolecular surface glycoprotein constituents (Vainer and Bussel 1976). Later, a study showed abnormalities in the terminal sialic acid residue of platelet membrane glycoprotein IIIb and the penultimate Gal/GalNAc residues of GP Ib, IIb, IIIa, and IIIb (Clezardin et al. 1985). Subsequently, leukocyte interferon alpha (IFNα) isolated from CML patients showed various degrees of glycosylation, such as glucosamine and galactosamine modifications (Labdon et al. 1984). At present, sialylation of glycoproteins seems to be a major feature of immature granulocytes in CML. Granulocytes in CML patients exhibit markedly increased sialylation of glycoproteins on the cell surface, which may be due to an increased activity of sialyltransferases and altered activity of other glycosyltransferases and sialidases (Cyopick et al. 1993). Aberrant glycosylation of CML granulocytes may reduce the binding of hematopoietic growth factors, resulting in aberrant differentiation and proliferation of myeloid-lineage cells (Cyopick et al. 1993). These abnormal sialylation events may be caused by overexpression of the sialyltransferases ST3GAL4 and ST6GAL1 (Nasirikenari et al. 2014; Zhou et al. 2017). Recently, it was reported that a synthetic GalNAc analog, peracetyl N-thioglycolyl-D-galactosamine (Ac5GalNTGc) can regulate

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immune activation in the CML K562 cell line by inhibiting mucin-type O-glycosylation (MTOG) of CD43 (Dwivedi et al. 2018). In addition, the disruption of the immune system through glycosylation of the NK cell receptor is another potential mechanism underlying the formation of CML. Glycosylation modification of NK cell surface receptors, particularly NKG2D, results in the failure of these structures to induce their normal effect of NK-mediated lysis (Cebo et al. 2006). In addition, β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase 3 (MGAT3) is the key enzyme for the biosynthesis of N-glycans. MGAT3 catalyzes the addition of a bisecting GlcNAc residue to the L-Man of the mannosyl core of N-glycans (Taniguchi et al. 1999). The activity of MGAT3 is obviously elevated in patients with CML in blast crisis (Yoshimura et al. 1995). Overexpression of MGAT3 in K562 leukemia cells results in an increase in bisecting GlcNAc modifications and a decrease in external sialic acid modifications, which changes the binding to effector cells, leading to K562 cell resistance to NK cell cytotoxicity (Yoshimura et al. 1996). Glycosylation modification is not only crucial for the molecules associated with cell proliferation in the development of CML, but also reflects MDR to tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, in the treatment of CML (Stagno et al. 2016). Acquired resistance to TKIs is frequently associated with poor clinical outcomes in CML patients. The most common causes of resistance can be categorized as BCR-ABL1-dependent and BCR-ABL1-independent. BCR-ABL1-dependent resistance is due to point mutations in the BCR-ABL1 kinase domain that interfere with the binding with TKIs, and BCR-ABL1-independent resistance is mediated by activation of alternative survival pathways under the effective inhibition of TKIs (Ma et al. 2014b; Patel et al. 2017). Another potential mechanism of MDR to TKIs may be the changes in the glycosylation of CML cells. Elevated expression of α-2, 3-sialyltransferase 4 (ST3GAL4) was found in CML cells with imatinib resistance (Taniguchi et al. 1999). In addition, CML cells from patients and cell lines with MDR showed overexpression of α-2, 8-polysialytransferase 4 (ST8SIA4) and ST3GAL4, and insufficient expression of α-2, 3-sialyltransferase 1 (ST3GAL1), ST3GAL5 and α-2, 8-polysialytransferase 6 (ST8SIA6) (Zhang et al. 2015; Li et al. 2016). Furthermore, expression of galectin3, which was mentioned in the AML section, induced by the leukemia microenvironment can promote cell proliferation and MDR to TKI in CML cells by inducing accumulation of the antiapoptotic protein Mcl-1 and suppression of the bovine SERPINA1-fetal bovine serum albumin complex (Yamamoto-Sugitani et al. 2011; Nakayama et al. 2014).

4.3

Acute Lymphoblastic Leukemia (ALL)

ALL is a type of blood and bone marrow cancer caused by the neoplasm of immature lymphoid progenitors (Mullighan 2012). ALL is the most common cancer and the most frequent cause of cancer-related death among children and teenagers (Hunger

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and Mullighan 2015). Based on the type of lymphoid linage, ALL is divided into different types, B cell type (B-ALL) and T cell type (T-ALL); most childhood cases of ALL are B-ALL. More than 75% of B-ALL cases show aneuploidy or recurring gross chromosomal rearrangements, such as ETV6-RUNX1, TCF3-PBX1, and BCR-ABL1, and other mutations, such as mutations in SH2B3 and CDKN2A/ CDKN2B, as we reported before (Cheng et al. 2016). T-ALL cases always exhibit mutations in the oncogenic transcription factors TAL1 and TLX1 (Mullighan 2012; Hunger and Mullighan 2015). In addition to the genetic changes in ALL, the aberrant glycosylation involved in the development of ALL has drawn attention (Table 1). Currently, the types of aberrant glycosylation involved in ALL are O-glycosylation and N-glycosylation, which affect the processes of cell adhesion, proliferation, antiapoptosis, and MDR. C-type lectins like DC-SIGN and L-SIGN are a diverse group of proteins involved in many human physiological and pathological processes. Aberrant glycosylation of blast cells can alter their interaction with C-type lectins and induce an immunosuppressive response. Recent evidence has shown that leukemic blasts from B-ALL and T-ALL patients have increased binding with C-type lectins thereby affecting their immunological elimination (Gijzen et al. 2008). In addition, increased binding of B-ALL peripheral blood cells to DC-SIGN and L-SIGN is correlated with poor prognosis (Gijzen et al. 2008). Macrophage galactose-type lectin (MGL) is another C-type lectin. A recent study found that MGL can recognize terminal GalNAc-containing structures, such as the specific Tn antigen expressed on T cell leukemia cells (Pirro et al. 2019). Hematopoietic cell L-selectin ligand (HCLL) is modified by N-glycosylation with sialylated and fucosylated structures. These structures act as a complete membrane glycoprotein that is expressed in normal hematopoietic cells. However, HCLL from the blasts of ALL patients shows highly sialofucosylated structures on complex-type N-glycans and exhibits higher activity than HCLL from normal cells (Sackstein and Dimitroff 2000). It was also reported that as a marker of platelet activation, P-selectin may be a new relevant marker, which is easier to be analyzed in clinical practice (Pluchart et al. 2021). Besides lectin receptors and lectin ligands on ALL blasts, another study found that transferrin receptors (TrfRs) are highly expressed on blasts from T-ALL patients; however, molecular weight of TrfRs was decreased due to the absence of certain types of glycosylation, although the types of glycosylation were not investigated (Petrini et al. 1989). In addition to the aberrant glycosylation found on ALL blasts, abnormal glycosylation was found on red cells from ALL patients as well. Anemia is a major feature in pediatric B-ALL and T-ALL. The glycosylation in red cells from patients is elevated approximately three folds compared with that in red cells from healthy people (Ghosh et al. 2005). Another ribonucleoprotein, HNRNPH1, which was mentioned in the AML section, has been recently identified in cases of B-ALL (Ohki et al. 2019). Although there is no evidence to show that the glycosylation of HNRNPH1 is aberrant in these B-ALL patients, changes in glycosylation of HNRNPH1 may promote the development of B-ALL given the role of such modification in AML, in which HNRNPH1 is

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overexpressed and modified with O-GlcNAc in AML patients. Further investigation may be needed to provide important insights into this molecule. The abnormal glycoprotein antigens CD9, CD43, CD45, and CD95 are all overexpressed in ALL blasts, which is characteristic of ALL (Komada and Sakurai 1994; Blixt et al. 2012; Dorrie et al. 2002). Heavy O-glycosylation of CD43 and CD45 on the cell surface can help the blasts to reduce cell adhesion, prevent inappropriate cell contact, and facilitate T cell leukemia virus type 1 (HTLV-1) particles to assembly into large, highly infectious structures on the surface of T cells (Mazurov et al. 2012). A study showed that the lectin affinity of the carbohydrate moiety of the CD9 antigen in ALL reflects the presence of N-linked oligosaccharide chains having groups with GlcNAc residues, a Man core and a terminal D-Gal, although the detailed effects of aberrant CD9 glycosylation on the development of ALL are still unknown (Komada and Sakurai 1994). It was reported that 9-O-acetylated sialoglycoprotein is overexpressed in children with ALL, and sialylation deficiency played a prominent role in promoting the survival of lymphoblasts in ALL (Bandyopadhyay et al. 2005; Ghosh et al. 2007). With changes in 9-O-acetylated sialoglycoconjugates (9-OAcSGs), 9-OAcSA-specific IgG2 is incapable to activate Fc-glycosylation-sensitive effector in ALL, which results in escape from host defenses (Bandyopadhyay et al. 2005). Furthermore, 9-O-acetylated sialoglycoprotein also decreases the activity of the apoptotic protein caspase 3, which protects blasts from apoptosis (Ghosh et al. 2007). This may be another reason for the survival of ALL blasts. Several glycosyltransferases have been reported to be involved in the development of ALL. The neuraminidase NEU3 (a membrane type- and ganglioside-specific sialidase) regulates the levels of gangliosides and Cers, which are associated with proliferation and apoptosis, by regulating PKC/ERK/p38 MAPK signaling (Tringali et al. 2009). The enzyme POGLUT1, also known as hCLP46, is overexpressed in T-ALL (Wang et al. 2010). Its overexpression enhances activation of NOTCH signaling and regulates cell proliferation in a cell type-dependent manner, similar to that observed in AML (Ma et al. 2011; Chu et al. 2013). Glycosylation is also involved in the MDR of ALL. When B-lineage leukemia cell lines are treated with neuraminidase and mannosidase inhibitors, resistance to IFNγ treatment is reversed. This result demonstrates that high N-linked glycosylation of CD95 is associated with IFNγ resistance in B-ALL (Dorrie et al. 2002). Therefore, glycosyltransferase inhibitors can be used to modulate the sensitivity of B-ALL blasts to IFNγ and induce cell apoptosis by inhibiting the glycosylation of CD95. The expression level of ST6GAL1, a class of sialyltransferases, is positively correlated with increased risk of pediatric ALL (Mondal et al. 2010). Increased expression of ST6GAL1 is associated with MDR in leukemia cells by regulation of P-gp and MDR-associated protein 1 (MRP1) through PI3K/Akt signaling (Ma et al. 2014a). Recently, a new recombinant human lactoferrin carrying humanized glycosylation (rhLf-h-glycan) has been reported to be a potential therapeutic drug for ALL (Nakamura-Bencomo et al. 2021).

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Chronic Lymphocytic Leukemia (CLL)

Chronic lymphocytic leukemia (CLL), the most frequent leukemia in developed countries, is a disease with clonal expansion of mature-appearing lymphocytes in blood, bone marrow, and secondary lymphoid tissues. CLL is one of the most common type of leukemia in adults, especially in older people, with a median diagnosis age of 72 years (Hallek et al. 2018; Scarfo et al. 2016). More than 95% of cases are B cell phenotype (B-CLL), which is characterized by clonal proliferation and accumulation of mature CD19-positive B cells with co-expressing CD5 and CD23, and few cases are T cell phenotype (T-CLL) (Scarfo et al. 2016; Boelens et al. 2009). Compared with normal circulating cells, CLL cells express high levels of OGlcNAcylated proteins, such as p53, c-Myc, and Akt (Table 1). High levels of glycosylation of proteins change the intracellular signaling processes in CLL cells (Shi et al. 2010). O-GlcNAcylation can increase the downstream signaling of Tolllike receptors (TLRs) after cytokine stimulation in CLL cells. However, high baseline O-GlcNAc levels lower the response to the stimulation and result in the resistance to TLR agonists, chemotherapeutic agents, B cell receptor (BCR) crosslinking agents, and mitogens. In addition, O-GlcNAc levels are associated with the grades of tumor aggression in CLL. CLL with aggressive clinical behavior showed less O-GlcNAcylation than indolent cells, which showed relatively highly O-GlcNAcylation (Shi et al. 2010). In addition, O-GlcNAcylation is very important for B cell hemostasis and transduction of BCR-mediated activation signals. Lacking of O-GlcNAc transferase enhances apoptosis of mature B cells and perturbs B cell homeostasis, resulting in severe defects in the activation of BCR signaling (Wu et al. 2017). N-glycosylation aberrations found in CLL are mainly involved in BCR signaling (Krysov et al. 2010). BCR signaling plays the most important physiological and pathological role in B cell survival and proliferation. Aberrant activation of BCR signaling leads to many B cell malignancies including CLL (Burger and Chiorazzi 2013). BCRs comprise surface immunoglobulins (sIgs) and CD79a/b. Surface IgM has an important influence on the clinical behavior of CLL. Compared with the mature complex glycan modifications of the μ-constant region of IgM of normal cells, the μ chain of CLL blasts bears immature mannosylated modifications, which should be restricted to the ER and absent on the cell surface (Krysov et al. 2010; Vuillier 2005). Glycosylation and folding defects of the μ chain and CD79a chain, but not the CD79b chain, have been reported in CLL patients. The defects were associated with low expression levels of BCR, which is a characteristic of B-CLL (Vuillier 2005). Moreover, the BCR of CLL blasts carries high levels of mannosylation in the heavy chain constant region but not variable region (Sedlik et al. 2016). Aberrant expression of molecules involved in BCR signaling also changes the glycosylation of the BCR and affects the activation of BCR signaling. TCL1 is expressed in approximately 90% of human CLL patients and its overexpression

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leads to the activation of BCR signaling (Herling et al. 2009; Kriss et al. 2012). Overexpression of TCL1 increases the expression of IgM and alters the N-glycosylation of Igα and Igβ, which contributes to the hyperactivation of BCR signaling in CLL (Kriss et al. 2012). Lectins are also involved in the development of CLL (Table 1). The lymphocyte homing receptor L-selectin (CD62L) is a crucial molecule mediating the binding of B-CLL cells to high endothelial venule (HEV) walls of lymph nodes (Lafouresse et al. 2015). Lymph nodes are sites of malignant hyperplasia, and their enlargement is related to poor prognosis. Aberrant glycosylation of L-selectin accounts for the aggressive cell growth and MDR of CLL (Lafouresse et al. 2015). Galectin-1 is a β-galactoside-binding lectin and is secreted by myeloid cells to establish the appropriate microenvironment to facilitate CLL blast proliferation and invasiveness by inhibiting T cell-mediated immunity (Croci et al. 2013). Given the role of aberrant glycosylation in the pathogenesis of CLL, targeting certain glycoproteins is an effective therapeutic strategy. Several drugs including glycol-engineered monoclonal antibodies (obinutuzumab and rituximab), BCR signaling inhibitors (ibrutinib, idelalisib, duvelisib, and acalabrutinib), and a BCL-2 inhibitor (venetoclax) have been used in the clinic (Yelvington 2018; Zinzani et al. 2019; Awan et al. 2019; Brander et al. 2019; Patel et al. 2019). Recently, antibodydrug conjugates (ADCs) that combine the specificity of monoclonal antibodies (mAbs) recognizing the aberrant glycosylation in tumors with anticancer drugs with powerful cytotoxicity have been introduced, and they may be able to target the aberrant glycosylation of the Tn antigen in CLL and thus become a new therapeutic strategy (Aller et al. 1996; Sedlik et al. 2016).

5 Glycosylation in Lymphomas Lymphomas can be divided into Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (non-HL). More than 90% of cases are non-HL, which further classified as B cell type, T cell type, and NK cell type (Mugnaini and Ghosh 2016). Diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), and Burkitt lymphoma (BL) are all types of non-HL. Less than 10% of lymphoma cases are HL, which is divided into classical HL and non-classical HL (Mugnaini and Ghosh 2016). Here, we summarize current advances in the understanding of aberrant glycosylation in HL and non-HL. In our knowledge, signaling molecules related to abnormal glycosylation including Gg3, Gb3, Ig VH genes, IgG, IgM, sIg, galectin-3, c-Myc, PSGL-1, galectin-1 and mucin1, have been reported in lymphoma progression (Table 2). Aberrant glycosylation is associated with cell adhesion, malignant growth, and the innate immune system.

a

N-glycosylation

Non-HL

Mannosylation and oligomannose Glycosaminoglycan Glycosphingolipid

O-glycosylation

Glycosylation types/levels Glycosphingolipid

HL

Means not be mentioned in the current research progress

Disease types Malignant lymphomas

Galectin-3 Gb3

IgM/IgG V regions

Leukemia microenvironment niche Antiapoptotic Unknowna

Unknowna Unknowna Immune escape Poor survival outcome Unknowna Drug resistance Tumor clones and subclones accumulation BCR signaling activation Cell transformation

GM3 Gg3 Galectin-1 SRGN CD45 Ig heavy-chain V regions BCR c-Myc

Biological impacts Unknowna

Related proteins Asialo GM2

Table 2 Aberrant glycosylation associated with malignant lymphomas

(Koning et al. 2019) (Chou et al. 1995; Kamemura et al. 2002) (Coelho et al. 2010; Radcliffe et al. 2007) (Clark et al. 2012) (Sugawara et al. 2017; Wiels et al. 1984)

(Zhu et al. 2002) (Clark et al. 2012) (Odabashian et al. 2020)

References (Hakomori 1984; Kniep et al. 1983) (Kojima and Hakomori 1989) (Kojima and Hakomori 1989) (Juszczynski et al. 2007)

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Hodgkin’s Lymphoma (HL)

Studies of the role of glycosylation modification in the development of HL are rare. Limited studies have reported that GSLs, such as gangliotriaosylceramide (asialo GM2), sialosyllactosylceramide (GM3), gangliotriaosylceramide (Gg3), and galectin-1, seem to play critical roles in HL progression. Asialo GM2 is a GSL and serves as a marker for cell lines derived from patients with HL, and the aberrant expression of GM2 results from the accumulation of incomplete GSL synthesis (Kniep et al. 1983; Hakomori 1984). A highly specific interaction between Gg3 and GM3 has also been reported in HL cell (Kojima and Hakomori 1989). Galectin-1 is overexpressed in HL and facilitates immune privilege in classical HL. High Galectin-1 is correlated with poor outcome and the event-free survival (Juszczynski et al. 2007). Therefore, galectin-1 reflects tumor burden and can serve as a predictive biomarker for relapsed classical HL (Ouyang et al. 2013; Kamper et al. 2011).

5.2

Non-Hodgkin’s Lymphoma (Non-HL)

Studies have shown that N-glycosylation changes at specific sites in the immunoglobulin variable heavy chain (Ig VH) genes are very common in FL and in a subset of DLBCLs (Zhu et al. 2002; Odabashian et al. 2020). In addition, the variable regions (V regions) of IgM/IgG of primary lymphoma cells are highly modified by mannosylation, and oligomannose residues are located in the antigen-binding site, possibly precluding conventional antigen binding to create a unique microenvironment to support the growth of tumor cells and escape from the BCR-mediated immune system (Coelho et al. 2010; Radcliffe et al. 2007). Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B cell lymphoma also be demonstrated acquired N-linked glycosylation motifs in the BCRs (Koning et al. 2019). Another antigen-independent BCR activation pathway has also been reported. Lectin can directly bind to the V region, which usually binds with cognate antigen in sIg from FL cells, through N-linked oligomannose glycans within the antigen-binding domain to activate BCR signaling. This antigen-independent binding will provide a constitutive activation signaling to facilitate FL cell survival and proliferation (Amin et al. 2015; Linley et al. 2015). Galectin-3 is overexpressed in DLBCL, which is the most common type of non-HL (Clark et al. 2012). Galectin-3 is the only antiapoptotic member of the galectin family. It is secreted by DLBCL cells and binds with a subset of highly glycosylated CD45 with N-acetyllactosamine sequences on the cell surface through C2GnT-1 glycosyltransferase. The binding of galectin-3 with CD45 inhibits the phosphatase activity of CD45, leading to resistance to chemotherapeutic agents in DLBCL cells (Clark et al. 2012). The oncoprotein c-Myc is overexpressed in many tumors including lymphoma. c-Myc can be dynamically modified by either glycosylation or phosphorylation at

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the Thr58 site (Kamemura et al. 2002; Chou et al. 1995). The Thr58 site is located in the malignant transformation domain of c-Myc, where mutations are frequently found in BL (Chou et al. 1995). It has been reported that increased O-GlcNAc glycosylation of Thr58 in c-Myc can inhibit the activity of c-Myc and decrease transformation of cells (Kamemura et al. 2002; Chou et al. 1995). In addition, elevated levels of the GSL Gb3 were found in BL and the abnormal elevation was caused by high enzyme activity of UDP-Gal:LacCer α-galactosyltransferase, which is involved in the synthesis of Gb3 (Sugawara et al. 2017; Wiels et al. 1984).

6 Glycosylation in Multiple Myeloma (MM) MM is a neoplastic disorder characterized by the clonal proliferation of malignant transformed plasma cells derived from B cells (Caraccio et al. 2020). Plasma cell clones expand in the bone marrow and produce excessive monoclonal immunoglobulins. Patients with MM usually suffer from tumor-induced bone pain and fractures, anemia, renal failure, and immunodeficiency. Occasionally, the malignant transformed plasma cells infiltrate multiple organs and produce other symptoms (Caraccio et al. 2020). As shown in Table 3, in MM cells, glycosaminoglycans such as the secretoryvesicle proteoglycan SRGN and the cell surface proteoglycan syndecan-1 (CD138) are highly expressed. MM cells constitutively secrete SRGN to facilitate cell adhesion to collagen type I by increasing the expression of collagen-degrading enzymes, such as the matrix metalloproteinases MMP-2 and MMP-9 (Skliris et al. 2013). Syndecan-1 is a heparan sulfate proteoglycan, which is expressed on the surface of MM cells and actively sheds from the cell surface. Syndecan-1 regulates apoptosis and inhibits the growth of MM cells, resulting in reduced osteoclastogenesis and enhanced osteoblastogenesis (Dhodapkar et al. 1998). Furthermore, syndecan-1 promotes activation of PI3K/PKB and RAS/MAPK signaling to enhance cell Table 3 Aberrant glycosylation associated with multiple myeloma Disease types Multiple myeloma

Glycosylation types/levels Glycosaminoglycan

Related proteins SRGN CD138

Biological impacts Cell adhesion Cell proliferation and apoptosis regulation

CD162

Cell adhesion

Galectin-1 Galectin-9

Cell invasion Cell proliferation regulation

References (Skliris et al. 2013) (Derksen et al. 2002; Dhodapkar et al. 1998) (Davenpeck et al. 2000; Florena et al. 2005) (Abroun et al. 2008) (Kobayashi et al. 2010)

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survival and proliferation in MM cells by binding to hepatocyte growth factor (HGF) (Derksen et al. 2002). Therefore, glycosaminoglycans play crucial roles in MM cell survival, proliferation, and metastasis. In addition to glycosaminoglycans, altered glycosyltransferases are associated with malignant transformation and metastasis in MM progression. It has been reported that sialyltransferase ST3 β-galactoside α2,3-sialyltransferase 6 (ST3GAL6) is highly expressed in MM cells and patients. It is a key regulator of selectin ligand synthesis and the expression is positively correlated with the homing and engraftment of MM cells (Glavey et al. 2014). The activity of MGAT3, which is elevated in CML patients, was also elevated in patients with MM (Yoshimura et al. 1995). Lectins are also involved in the pathogenesis of MM (Table 3). Galectin-1 is overexpressed in MM cells and promotes cell survival. Galectin-1 also activates ERK signaling and contributes to the invasive ability in CD45RA negative but not CD45RA positive MM cells by affecting their adhesion to the extracellular matrix and cellular aggregation (Abroun et al. 2008). Galectin-9 has an antiproliferative effect on MM cell lines and patient-derived myeloma cells by inhibiting the JNK and p38 MAPK signaling pathways. Therefore, galectin-9 can be used as a new therapeutic option to treat MM (Kobayashi et al. 2010). P-selectin glycoprotein ligand 1 (PSGL-1, CD162), the major P-selectin ligand, is constantly expressed in MM cells and a marker of lymphoma cells with plasmacytoid differentiation (Florena et al. 2005; Davenpeck et al. 2000). Furthermore, PSGL-1 can be used as a therapeutic target for treatment of MM. Immunotherapy with an anti-PSGL-1 mAb induced complement-mediated lysis of MM cells in vivo (Tripodo et al. 2009).

7 Glycosylation in Other Blood Cancers As shown in Table 4, aberrant glycosylation alterations are involved in the development of other blood cancers.

7.1

Glycosylation in Myeloproliferative Neoplasms (MPNs)

MPN are a group of clonal hematopoietic diseases involving excessive bone marrow cell production, comprising essential thrombocytosis (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Most MPN patients show somatically acquired mutations on Jak2, Calf, and Mpl genes, which result in hyperactivation of JAK-STAT signaling (Nangalia and Green 2017). An immature and highly mannosylated form of thrombopoietin (TPO) receptor (MPL/TpoR), which is due to mutation of calreticulin (CALR), has been reported (Pang et al. 2018; Chachoua et al. 2016). The pathogenic CALR mutation interacts with extracellular N-glycosylation residues of MPL through a C-terminal glycan-binding site, leading to

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Table 4 Aberrant glycosylation associated with other hematological malignancies Disease types Other hematological malignancies

Glycosylation types/levels N-glycosylation

Related proteins MPL

Biological impacts Cell growth

Galactosylation

β4GALT1

MDS/MPN

O-glycosylation

CSF3R

Increased TPO plasma level Cell proliferation

CNL

O-glycosylation

CSF3R

Cell trafficking

N-glycosylation

CSF3R

Ligand-independent cellular expansion

MPN

References (Araki et al. 2016; Elf et al. 2016) (Di Buduo et al. 2021) (Fleischman et al. 2013; Maxson et al. 2014, 2016) (Maxson et al. 2014; Price et al. 2020) (Spiciarich et al. 2018)

TPO-independent activation of MPL and downstream JAK2-STAT5 activation, promoting cell growth and resulting in the development of MPN (Pang et al. 2018; Araki et al. 2016; Elf et al. 2016). The sialylated derivatives of the glycan structure β4-N-acetyllactosamine expression is significantly increased in the platelets isolated from MPNs (Di Buduo et al. 2021). Up to now, there are some clinical drugs for the treatment of MPN, such as ruxolitinib and hydroxyurea; however, there have been no reports of the drug targeting aberrant glycosylation occurred in MPN, since there are very few studies focusing on the role of glycosylation changes in the pathogenesis of MPN.

7.2

Glycosylation in Myelodysplastic Syndrome/ Myeloproliferative Neoplasms (MDS/MPNs)

MDS/MPNs are rare and distinct group of myeloid neoplasms with overlapping clinical, laboratory, and morphologic features (Tanaka and Bejar 2019). There are three different MDS/MPNs overlap conditions based on the clinical features, including chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), and juvenile myelomonocytic leukemia (JMML) (Tanaka and Bejar 2019). Unlike classical CML, which is driven by the BCR-ABL1 fusion gene, aCML is a BCR-ABL1 negative MDS/MPN, that is characterized by leukocytosis, granulocytic dysplasia, and poor outcomes (Sadigh et al. 2020). At present, the only glycosylation modifications known to be involved in the development of aCML are modifications affecting threonine (Thr) linked O-glycosylation at the T618I and T640N sites of

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colony-stimulating factor-3 receptor (CSF3R). These aberrations prevent the Oglycosylation of CRF3R and increased receptor dimerization, resulting in ligandindependent activation of CRF3R downstream signaling pathways, such as the JAK and SRC kinase signaling pathways, in aCML patients (Fleischman et al. 2013; Maxson et al. 2014, 2016). To the best of our knowledge, there is no report on the aberrant glycosylation in CMML and JMML.

7.3

Glycosylation in Chronic Neutrophilic Leukemia (CNL)

CNL displays very similar clinical and hematological characteristics to aCML. CNL is diagnosed based on the expansion of neutrophils in both bone marrow and blood, while aCML is characterized by granulocytic dysplasia and an increased number of neutrophil precursors (Maxson et al. 2013). The CSF3R T618I mutation, which was mentioned above, is also a hallmark of CNL and leads to ligand-independent activation due to loss of O-glycosylation of the receptor (Maxson et al. 2014). A recent study demonstrated that the CSF3R T618I mutation produces a protein that can still be glycosylated but that undergoes enhanced spontaneous internalization and degradation which results in a marked decrease in its surface expression (Price et al. 2020). Thus, we can speculate that the underlying mechanism of CSF3R T618I mutation in CNL may be the induction of oncogenic signals through aberrant trafficking and constitutive phosphorylation of the O-glycosylated receptor. In addition, mutation on N610 site of CSF3R was reported. This mutation prevented membrane-proximal N-glycosylation of CSF3R, thereby driving the ligandindependent cellular expansion (Spiciarich et al. 2018). The underlying mechanism is still unknown and needs to be further explored.

8 Conclusion and Perspectives Glycosylation is one of the most vulnerable cellular processes. Any small pathologic change or metabolic stress will affect glycosylation and result in aberrant glycan and glycoprotein synthesis. Overexpression of glycoproteins, altered expression of sugar donors, and altered enzyme activity of glycosyltransferases and glycosidases are key mechanisms underlying the synthesis of aberrant glycans and glycoprotein forms in the development of hematological malignancies. As summarized in Tables 1, 2, 3, and 4, various glycosylation abnormalities are often found in hematological malignancies. Although some aberrant glycosylation corresponds to different hematological malignancies, it is worth noting that aberrant glycosylation in the same protein could be involved in different hematological malignancies. For example, the abnormalities of O-glycosylation of NOTCH occur in both AML and ALL (Ma et al. 2011; Yao et al. 2011; Chu et al. 2013; Wang et al. 2018). Another example, the

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aberrant O-glycosylation of CD45 promotes cell adhesion in ALL, but the Nglycosylation mutation of CD45 occurred in non-HL and plays an important role in drug resistance (Clark et al. 2012; Mazurov et al. 2012). Therefore, microheterogeneity and structural complexity of glycans pose significant analytical challenges. As detection and analysis methods become more accurate, it will be necessary to deeply investigate the glycosylation changes in different types of hematological malignancies to discover aberrant glycosylation patterns and explore the underlying mechanisms. This knowledge will expand the utility of aberrant glycosylation as diagnostic markers and therapeutic targets for hematological malignancies. Acknowledgement The authors thank Dr. Huadong Liu for critical reading and English editing of the manuscript. This work was supported by the National Natural Science Foundation of China (31971053, 81770123), China Postdoctoral Science Foundation, the Scientific and Technical Foundation of Shaanxi Province (2020JM015) and the Fundamental Research Funds for the Central Universities from Xi’an Jiaotong University. Declaration of Competing Interest: The authors declare no competing financial interests. Author Contributions: Y. C. and X. L. conceived and designed the article frame. H. S., M. W., X. P., F. G., X. L., and Y. C. wrote the paper.

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Rev Physiol Biochem Pharmacol (2021) 180: 119–154 https://doi.org/10.1007/112_2021_58 © Springer Nature Switzerland AG 2021 Published online: 23 June 2021

The Placenta as a Target for Alcohol During Pregnancy: The Close Relation with IGFs Signaling Pathway Irene Martín-Estal, Inma Castilla-Cortázar, and Fabiola Castorena-Torres

Contents 1 2 3 4 5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Placenta: A Key Organ during Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Feto-Placental Alterations Due to Alcohol Consumption Throughout Pregnancy . . . . . . . . Placental Ethanol Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insulin-Like Growth Factors (IGFs) as a Main Target for Ethanol Consumption . . . . . . . . . 5.1 IGFs Main Functions during Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Ethanol Molecular Alterations in Trophoblast Invasion and Migration . . . . . . . . . . . . . . . . . . . 7 Mitochondria as a Target Generator of ROS Due to Ethanol Metabolism . . . . . . . . . . . . . . . . 8 Ethanol Alterations in the IGFs Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Alcohol is one of the most consumed drugs in the world, even during pregnancy. Its use is a risk factor for developing adverse outcomes, e.g. fetal death, miscarriage, fetal growth restriction, and premature birth, also resulting in fetal alcohol spectrum disorders. Ethanol metabolism induces an oxidative environment that promotes the oxidation of lipids and proteins, triggers DNA damage, and advocates mitochondrial dysfunction, all of them leading to apoptosis and cellular injury. Several organs are altered due to this harmful behavior, the brain being one of the most affected. Throughout pregnancy, the human placenta is one of the most important organs for women’s health and fetal development, as it secretes numerous hormones necessary for a suitable intrauterine environment. However, our understanding of the human placenta is very limited and even more restricted is the Inma Castilla-Cortázar and Fabiola Castorena-Torres contributed equally to this work. I. Martín-Estal and F. Castorena-Torres (*) Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Monterrey, NL, Mexico e-mail: [email protected] I. Castilla-Cortázar Fundacion de Investigacion HM Hospitales, Madrid, Spain

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knowledge of the impact of toxic substances in its development and fetal growth. So, could ethanol consumption during this period have wounding effects in the placenta, compromising proper fetal organ development? Several studies have demonstrated that alcohol impairs various signaling cascades within G protein-coupled receptors and tyrosine kinase receptors, mainly through its action on insulin and insulin-like growth factor 1 (IGF-1) signaling pathway. This last cascade is involved in cell proliferation, migration, and differentiation and in placentation. This review tries to examine the current knowledge and gaps in our existing understanding of the ethanol effects in insulin/IGFs signaling pathway, which can explain the mechanism to elucidate the adverse actions of ethanol in the maternal–fetal interface of mammals. Keywords Alcohol consumption · Fetal growth restriction · IGF-1 · Oxidative stress · Placenta

Abbreviations AAH ADH ALDH ALS ARBD CCM3 CNS CTCF CYP450 CYP2E1 Dnmt ERKs ETC EtOH FAS FASD FGR GH GHRH GPCRs H2O2 ICR IGF-1 IGF-2 IGF1R IGF2R IGFBPs INSR

Aspartyl-asparaginyl β-hydroxylase Alcohol dehydrogenase Aldehyde dehydrogenase Acid-labile subunit Alcohol-related birth defects Cerebral cavernous malformation protein 3 Central nervous system CCCTC site binding factor Cytochrome P450 system Cytochrome P450 2E1 DNA methyltransferase Extracellular signal-regulated kinases Electron transport chain Ethanol Fetal alcohol syndrome Fetal alcohol spectrum disorders Fetal growth restriction Growth hormone Growth hormone-releasing hormone G protein-coupled receptors Hydrogen peroxide Imprinting control region Insulin-like growth factor 1 Insulin-like growth factor 2 IGF-1 receptor IGF-2 receptor IGF-1 binding proteins Insulin receptor

The Placenta as a Target for Alcohol During Pregnancy: The Close Relation. . .

IRSs JAK JNKs MAPKs ND-PAE NO PAS PDGF PI3K PKA PKB PKC PTEN PTP-1b RNS ROS RTKs STATs TGFβ VEGFR1 VEGFR2

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Insulin receptor substrates Janus kinases c-Jun N-terminal kinases Mitogen-activated protein kinases Neurobehavioral disorder associated with prenatal alcohol Nitric oxide Placental associated syndromes Platelet-derived growth factor Phosphatidylinositol-3-kinase Protein kinase A Protein kinase B Protein kinase C Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase Protein-tyrosine phosphatase 1b Reactive nitrogen species Reactive oxygen species Tyrosine kinase receptors Signal transducer and activator of transcription proteins Transforming growth factor β Vascular endothelial growth factor receptor 1 Vascular endothelial growth factor receptor 2

1 Introduction To date, alcohol continues to be the most consumed drug in the world, even during pregnancy: approximately 2.3 billion people drink spirituous beverages worldwide (WHO 2018). This harmful behavior has several consequences on the health of women and children. For this reason, its consumption is a vast health and economic problem worldwide, due to the high costs to attend its medical illness and dependence. Alcohol use during pregnancy is a risk factor to develop adverse outcomes, such as fetal death, miscarriage, fetal growth restriction (FGR), low birth weight, and premature birth (Lee et al. 2020; Lees et al. 2020). Also, alcohol consumption during this period can result in a great variety of lifelong conditions known as fetal alcohol spectrum disorders (FASD) that have numerous physical, cognitive, and behavioral defects in newborns and children (Hamilton et al. 2020; National Institute on Alcohol Abuse and Alcoholism 2018; O’Leary et al. 2010). FASD is not a clinically used name and encompasses other several terms with diagnostic criteria, depending on the nature and harshness of the damage: fetal alcohol syndrome (FAS, the most severe form of FASD) and partial FAS, both with and without confirmed maternal alcohol exposure, respectively; alcohol-related birth defects (ARBD); and neurobehavioral disorder associated with prenatal alcohol exposure (ND-PAE)

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(National Institute on Alcohol Abuse and Alcoholism 2018; Ehrhart et al. 2018). The observed physical, cognitive, and behavioral defects found in FASD patients vary in severity and differ by the timing, dose, and duration of alcohol exposure, nutritional status, genetic polymorphisms, maternal characteristics (gravity, parity, body mass index), and additional exposures (smoking/drugs) (National Institute on Alcohol Abuse and Alcoholism 2018; Meyer-Leu et al. 2011; Currie et al. 2020). Health damages derived from alcohol consumption occur through four main mechanisms: (1) development of alcohol dependence; (2) intoxication and psychoactive effects of alcohol after drinking; (3) alcohol toxic effects in various organs and tissues in consumer’s body; and (4) alcohol metabolism as a significant source of free radicals that lead, in turn, to oxidative damage in all cells and tissues (Ehrhart et al. 2018). Alcohol’s damaging effects are related to its strength and frequency of consumption. For this reason, several animal and human studies have been conducted to evaluate the different effects of moderate (1 drink or 2 drinks per day in females and males, respectively) and binge drinking (4 drink or 5 drinks per occasion or at least 1 day in the past month in females and males, respectively) during pregnancy, as well as their association with harmful neonatal outcomes. Results have shown that both moderate and binge drinking are associated with reduced fetal growth, neonatal asphyxia, and poor neurodevelopment (Meyer-Leu et al. 2011; Currie et al. 2020; Flak et al. 2014), as well as behavioral problems and physical deformities (Denny et al. 2017; Hoyme et al. 2016), including FAS and other FASD conditions (Flak et al. 2014). These results imply that there is no safe amount of alcohol to consume while pregnant. However, the genetic predisposition of each individual to alcohol metabolism and adverse effects is another risk issue that should be analyzed, in order to provide better results. Of note, timing of alcohol consumption, especially if this use occurs during periods of development of specific fetal organ systems, is another risk factor. For example, alcohol exposure during the first trimester of pregnancy in humans can affect facial and skull bones, resulting in the characteristic facial abnormalities observed in FAS children. Conversely, alcohol use during the second and third trimesters alters fetal growth and brain development, since both are periods where a prompt brain development occurs (Day and Richardson 2004; Aros et al. 2011). Throughout pregnancy, the human placenta is an extremely important and complex organ for both mother and fetal health. One of its main functions is the synthesis of hormones and other mediators, all crucial for gestational success (Guttmacher et al. 2014). One of these placental-produced hormones is the insulin-like growth factor 1 (IGF-1), an anabolic and pleiotropic hormone involved in cell survival and proliferation, an adequate placentation and proper intrauterine and postnatal growth. This hormone, together with insulin-like growth factor 2 (IGF-2, both belonging to the IGFs family), regulates cell growth and differentiation and stimulates placental and fetal growth and development (Martín-Estal et al. 2019; Fowden and Forhead 2004; Fowden 2003). Although there are several experimental studies conducted in the placenta in order to understand the harmful effects produced by alcohol use during pregnancy in this organ, its morphology and function, there are very few

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experimental studies carried out to comprehend the effects that ethanol has on the IGF-1 signaling pathway in the placenta, as this is an extremely important cascade for placental development and function. Several studies in animal models of alcohol consumption have provided data suggesting that alcohol, like other drugs of abuse, alters numerous molecular cascades, mainly through its action on the IGF-1 signaling pathway (Hu et al. 2020; Dean et al. 2017; Tong et al. 2013; de la Monte et al. 2012). Moreover, these alterations could lead to several cellular and molecular alterations, e.g. trophoblast motility and invasion, resulting in impairments in placental morphology (especially in the syncytiotrophoblast layer) and function, hence altering fetal growth and development (de la Monte et al. 2006; Cantarini et al. 2006; Gundogan et al. 2008, 2015; Holbrook et al. 2019; Tong et al. 2017). The aim of the present review is to examine the current knowledge about the effects of ethanol (EtOH) in the insulin/IGFs signaling pathway in placenta, which can explain the mechanism to elucidate EtOH adverse actions in the maternal–fetal interface of mammals.

2 The Placenta: A Key Organ during Pregnancy Throughout pregnancy, the human placenta is an extremely important organ for both mother and fetal health. During fetal development, the placenta functions as a symbiosis between renal, respiratory, hepatic, gastrointestinal, endocrine, and immune fetal systems. This organ is the major determinant of intrauterine growth, because it serves as a protective barrier against external and internal insults: it can prevent infections, the dispersion of maternal diseases, and fetal transport of certain xenobiotics that could be harmful for fetal development (Creeth and John 2020). Furthermore, the placenta allows the communication between mother and fetus (Martín-Estal et al. 2019). As above mentioned, the synthesis of hormones and other mediators, a core placental function, is crucial for gestational success (Guttmacher et al. 2014). Also, the placenta releases these hormones into both maternal and fetal circulations, being their synthesis and secretion responsive to environmental changes. A dysregulated placental hormone secretion is associated with FGR, among other abnormalities (Creeth and John 2020; McNamara and Kay 2011). In fact, placental hormones are important during pregnancy, since they play different roles in the establishment and maintenance of pregnancy and fetal development. For example, placental progesterone and lactogen influence maternal metabolism to support glucose delivery to the fetus; glucocorticoids control organ development and maturation; IGF-1 and IGF-2 stimulate placental and fetal growth and development (Martín-Estal et al. 2019; Fowden and Forhead 2004; Fowden 2003; Creeth and John 2020). As can be revised elsewhere, the placenta is a complex organ conformed by several stratums, such as the cytotrophoblast and the syncytiotrophoblast layers.

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This last layer is the main source of placental hormones and it contains the enzymatic machinery to produce them for endocrine, paracrine, and/or autocrine purposes, guiding the gestational course (Costa 2016). Additionally, the placenta is involved in nutrient transport and metabolism of several molecules that will be released into maternal and/or fetal circulations, e.g. respiratory gases (O2, CO2), carbohydrates (glucose), amino acids, lipids, water, inorganic ions (potassium, magnesium, calcium and phosphate), minerals, and vitamins (Martín-Estal et al. 2019). The placenta is a dynamic organ, it can adjust its transport capacity in response to fetal nutrient demand signals and maternal signals for nutrient availability, thus affecting maternal metabolism to ensure nutrient supply for fetal growth and guaranteeing an optimal pregnancy outcome (Sferruzzi-Perri et al. 2006). Noteworthy, impairments in respiratory gases supply, specially oxygen, could lead to hypoxia, thus resulting in pregnancy complications, e.g. FGR and preeclampsia. This hypoxic environment, a common pregnancy alteration as a result of several exogenous insults, such as alcohol consumption, smoking, anemia, cord occlusion, and/or poor placental vascularity, could be activated in the placenta due to the diminution in oxygen supply and the reduced intervillous pO2 that take place in FGR and preeclampsia pregnancies (Creeth and John 2020; Higgins et al. 2016; Giussani and Davidge 2013; Zhang et al. 2015; Hutter et al. 2010). All these abovementioned placental functions are potential targets for EtOH. Consequently, alcohol use throughout pregnancy can impair placental morphogenesis, transport, and hormone secretion, leading to placental abnormalities that promote poor fetal outcomes.

3 Feto-Placental Alterations Due to Alcohol Consumption Throughout Pregnancy EtOH’s effects can be translated into maternal, placental, and/or fetal abnormalities (Fig. 1). As aforementioned, the placenta is an essential organ for an appropriate pregnancy outcome, due to its endocrine and protective functions. For this reason, the placenta possibly could play a crucial role in EtOH-related effects throughout pregnancy. Reports in human cohorts reveal that EtOH consumption throughout pregnancy can result in an increased risk to develop placental-associated syndromes (PAS), such as placental abruption, responsible for up to a third of all perinatal deaths due to the disruption of gestation length and fetal growth; placenta previa, a condition that can restrict fetal growth and produce preterm delivery and perinatal mortality; placenta accreta, coupled to premature birth and disproportionate vaginal bleeding during delivery; placental hemorrhage and early stillbirth (Ohira et al. 2019; Carter et al. 2016; Aliyu et al. 2011; Salihu et al. 2011; Odendaal et al. 2020) (Fig. 1).

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Fig. 1 Maternal and placental effects due to ethanol use during pregnancy: alterations in the hypothalamic-pituitary-adrenal/gonadal/thyroid axis. Ethanol consumption throughout pregnancy can lead to several placental and fetal growth and development abnormalities. Also, ethanol use during this period can result in several alterations in maternal axes, e.g. hypothalamic-pituitaryadrenal axis, hypothalamic-pituitary-gonadal axis, and hypothalamic-pituitary-thyroid axis

Several studies have been conducted in vitro cultures of human and animal placentas and in animal models of prenatal alcohol exposure, proving the harmful effects of EtOH on placental size, structure, and function (Gundogan et al. 2008, 2015; Carter et al. 2016; Clave et al. 2014; Lui et al. 2014; Carter et al. 2012; Kwan et al. 2020; Ventureira et al. 2019). Epigenetic changes are known to play a crucial role in placentation and fetal development, e.g. environmental factors could deregulate and change the genomic imprinting resulting in placental defects (Kappil et al. 2015). Epigenetic modifications to DNA (methylation) or histones (methylation, acetylation, phosphorylation, ubiquitination, sumoylation) may act as repressive or active marks, which alter the chromatin conformation, stability, and therefore gene transcription (Bowman and Poirier 2015). Intrauterine alcohol exposure can cause fetal alcohol spectrum disorders including behavioral disabilities, brain development, facial deformities, and growth retardation in the offspring that are clearly associated with epigenetic changes (Lussier et al. 2017).

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Haycock and Ramsay studied in a mouse model the effects of alcohol exposure in utero and showed that ethanol-exposed midgestation embryos and placentas exhibited severe growth retardation compared to controls. H19 imprinting control region (ICR) hypomethylation was observed in alcohol-exposed placentas, although the overall methylation status of the fetus remained unchanged and paternal alleles were significantly less methylated in ethanol-exposed placentas. Data showing a relationship between placental weight and ethanol treatment suggested that this feature was partially dependent on DNA methylation at the CCCTC site binding factor (CTCF) at the paternal allele in placentas (Haycock and Ramsay 2009). Also, in early stage fish embryos, exposure to ethanol appears to have a lasting effect on the expression of DNA methyltransferase (Dnmt) enzymes, e.g. Dnmt1 reduces its expression, while Dnmt3a and Dnmt3b expression levels remained unchanged (Dasmahapatra and Khan 2015). On the other hand, it is known that IGF-2 participates in the development of the placenta and the fetus. Downing et al. (2011) studied the state of methylation and expression of the Igf2 locus both in the embryo and in the placenta after alcohol exposure in utero. Exposed mice showed hypomethylation and reduced expression at the Igf2 locus in embryos at 4 CpG sites; however, no changes were observed in placentas. After methyl supplementation during pregnancy, methylation levels returned to normal, and a decrease in mortality due to ethanol exposure was observed, suggesting that epigenetic changes are reversible in nature, and methyl supplementation at the appropriate time can improve the health of the offspring (Downing et al. 2011). Nevertheless, to date it is not clear how alcohol consumption might influence the genetic imprint on placental and embryonic development, particularly in the periconceptional/preimplantation period, and its relation with IGF-1 and/or IGF-2. For this reason, this mechanism needs to be further studied (Lussier et al. 2017). Due to its nature, EtOH enters maternal circulation, diffuses through the placenta, and distributes promptly into the fetal compartment, where it accumulates, triggering the fetus to have similar alcohol levels as seen in the mother. As a result of this accumulation in the fetal compartment and the minimal capacity of the placenta to metabolize EtOH, this molecule interferes with placental transport of nutrients, oxygen, and waste products (Gundogan et al. 2008, 2015; Kwan et al. 2020) (Fig. 1). This prenatal alcohol exposure decreases the levels of several proteins involved in vascular remodeling, such as vascular endothelial growth factor receptor 1 and 2 (VEGFR1 and VEGFR2), annexin 4 and cerebral cavernous malformation protein 3 (CCM3) (Holbrook et al. 2019; Ventureira et al. 2019; Savage et al. 2020). Also, EtOH exposure during pregnancy impairs nitric oxide (NO) synthesis, a key regulator of angiogenesis and vasodilation (Reynolds et al. 2005; Redmer et al. 2005) and/or dysregulates the thromboxane–prostacyclin balance (Lui et al. 2014). These results suggest that EtOH use throughout pregnancy leads to vasoconstriction of placental and umbilical cord vessels, resulting in hypoxia and fetal malnutrition (Holbrook et al. 2019; Lui et al. 2014; Lo et al. 2017). A clinical study has disclosed that gestational alcohol exposure is associated with uteroplacental malperfusion, producing preterm delivery and reduced mean birth weights, this leading to FGR

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(Tai et al. 2016). Consequently, this impairment in placental transport function leads to an increase in oxidative stress that compromises placentation as it alters trophoblast cell motility (Gundogan et al. 2008; Kay et al. 2000; Siler-Khodr et al. 2000; Gundogan 2013). In this way, several studies in rats and humans demonstrate that EtOH impairs biotin, vitamin B6, leucine, omega-3 polyunsaturated fatty acids, and adenosine transport from mother to fetus across the placenta, all of them essential for proper placental and fetal growth and development. Moreover, prenatal alcohol exposure was related to an increase in iron deficiency anemia, condition that exacerbates alcohol-related growth restriction. This development of iron deficiency anemia could be due to an alteration in placental iron transport due to alcohol consumption during pregnancy (Carter et al. 2012; Kwan et al. 2020). During pregnancy, several endocrine and metabolic changes occur to accommodate the growing embryo, ensuring a suitable development. These changes result from physiological alterations at the interface between mother and fetus, known as the feto-placental unit. This boundary is a key site of protein and steroid hormone production and secretion (Creeth and John 2020). Alcohol use throughout gestation can disrupt the normal hormonal interactions between mother and fetus, impairing natural homeostasis and hence, leading to poor pregnancy outcomes. For example, alcohol exposure during this period can alter the hypothalamic-pituitary-adrenal axis (which regulates responses to stress), leading to an excessive activation and glucocorticoid release, similar to that observed in stress circumstances (Burgess et al. 2019; Weinberg et al. 2008; Wieczorek et al. 2015) (Fig. 1). Moreover, EtOH use during pregnancy can impair the hypothalamic-pituitary-gonadal axis, which controls reproductive functions; and the hypothalamic-pituitary-thyroid axis, involved in the metabolism of nearly all tissues, resulting in deprived pregnancy outcomes, adverse neurodevelopment, and behavioral alterations (Weinberg et al. 2008; Frias 2002; Wilcoxon et al. 2005; Donald et al. 2018) (Fig. 1). Similarly, EtOH alters the endocrine function of the placenta, e.g. it disturbs the GnRH/GnRH-receptor system and exacerbates cytokine production, thus increasing the risk of pregnancy loss, fetal damage, and intrauterine infections (Holbrook et al. 2019) (Fig. 1). As shown in diverse studies in rodents and humans, the most critical phase for an appropriate placental development is the establishment of the maternal–fetal interface, where invasive trophoblasts must invade maternal spiral arteries, disrupting the media and replacing the endothelial cells, allowing the continuous nutrient supply between mother and fetus (Gundogan et al. 2008, 2015; Kalisch-Smith et al. 2019). These studies disclose that EtOH consumption during pregnancy impairs placental morphology, and hence fetal development, at three levels: (1) modifying the morphogenesis of the labyrinthine zone in the placenta; (2) suppressing the invasive trophoblastic precursor cells; and (3) inhibiting the trophoblastic cell adhesion and motility, all of them crucial stages for placental growth and development (Gundogan et al. 2008, 2015; Coll et al. 2018) (Fig. 1). Additionally, this alteration in placental morphology leads to a decrease in placental blood flow, resulting in vasoconstriction of the placenta and umbilical vessels, diminished nutrient transfer to the fetus, and

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hypoxia (Gundogan et al. 2015; Ohira et al. 2019; Bosco and Diaz 2012; Mesquita 2010). As a consequence, this harmful behavior can promote fetal adverse outcomes, such as fetal death, FGR, reduced fetal weight, neonatal depression, FASD, and impairments in neurodevelopment (cerebral palsy, central nervous system abnormalities, etc.); plus, pregnancy adverse outcomes, such as miscarriage, stillbirth, premature birth, and fetal distress during labor (Tai et al. 2016; Shankar et al. 2007) (Fig. 1). Despite all these promising results on EtOH-related effects during pregnancy, few studies have focused on the placenta as a target organ of ethanol toxicity, especially on the IGF-1 signaling pathway, giving more attention to other organs such as fetal brain and liver. Consequently, more emphasis should be placed on the study of this organ as a main target for EtOH’s effects.

4 Placental Ethanol Metabolism Ethanol diffuses through the placenta and distributes quickly into the fetal compartment, where this molecule accumulates due to its slow elimination rate, resulting in similar blood alcohol concentrations in mother and fetus (Gupta et al. 2016a). The great majority of ingested EtOH is metabolized, with the remainder part excreted in urine, breath, and sweat. This molecule can be metabolized via oxidative and non-oxidative routes (Zelner and Koren 2013). Regarding the oxidative processes, EtOH is metabolized to acetaldehyde, mainly in the liver, through two key pathways: 90% approximately by alcohol dehydrogenase (ADH) and 10% by cytochrome P450 2E1 (CYP2E1), an isoform of the cytochrome P450 system (CYP450), a superfamily of enzymes involved in the metabolism of endogenous and exogenous compounds, e.g. steroids, fatty acids, alcohol, drugs, and chemicals (Cho et al. 2017; Wilson and Matschinsky 2020; Zakhari 2006) (Fig. 2). Then, acetaldehyde is converted to acetate by aldehyde dehydrogenase (ALDH) and enters the Krebs cycle to produce water and carbon dioxide. Acetaldehyde is also toxic by itself. This molecule can contribute to tissue damage by binding enzymes, proteins, and microtubules, also forming adducts with DNA and neurotransmitters such as serotonin, leading to neurodegeneration (Lui et al. 2014), and dopamine, contributing to alcohol dependence (Wilson and Matschinsky 2020; Manzo-Avalos and Saavedra-Molina 2010). This ethanol-derived product can accumulate in fetal blood at concentrations similar to those found in maternal blood, being freely present in the placenta, amniotic fluid, and fetal liver. Herein, this metabolite can reduce placental growth and decrease offspring size (Lui et al. 2014). To date, there is controversy about acetaldehyde levels observed in the fetus, as it cannot be explained by the action of fetal ADH and fetal ALDH, because the activities of both enzymes are relatively low, and there is scarce and novel information on whether this molecule can cross the placental barrier due to its

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Fig. 2 Ethanol oxidative metabolism in placenta (modified from Zakhari S. 2006). Ethanol (EtOH) can be metabolized to acetaldehyde via three pathways: catalase, alcohol dehydrogenase (ADH), and cytochrome P450 2E1 (CYP2E1). Then, acetaldehyde is metabolized into acetate by acetaldehyde dehydrogenase (ALDH), which enters the Krebs cycle to produce CO2 and H2O. In the reaction catalyzed by CYP2E1, several reactive oxygen species (ROS) are produced, thus producing DNA damage and the oxidation of lipids, proteins, and metabolites. This oxidative environment activates the intrinsic apoptotic pathway, leading to poor pregnancy outcomes, e.g. neurodegeneration, hypoxia, mitochondrial dysfunction, and placental alterations. The thickness of the arrows represents the predominance of the reaction

permeability, its miscibility with water, and its solubility in lipoid solvents, e.g. body fluids, tissues, and membranes. Several differences in EtOH metabolism have been observed, according to the gender. Women have a higher EtOH bioavailability and augmented rates of hepatic EtOH metabolism than men, due to their lower expression levels of gastric ADH and higher relative liver volumes, respectively (Zelner and Koren 2013; Baraona et al. 2001). Moreover, there are numerous ADH and CYP450 enzyme isoforms expressed broadly in the body and located in the cytoplasm and endoplasmic reticulum that can account for the diverse genotoxic EtOH effects observed in different human subjects (Table 1) (Shankar et al. 2007; Zelner and Koren 2013; Estonius et al. 1996; Nishimura et al. 2003; Green and Stoler 2007; Zanger and Schwab 2013; Li et al. 2016). All of these isoforms vary in how efficiently break down or oxidize alcohol and in their tissue distribution. CYP2E1, as well as CYP2B6 (another isoform less important for EtOH metabolism), located in the

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Table 1 Tissue distribution and reaction site of alcohol dehydrogenase (ADH), cytochrome P450 (CYP), and catalase in human body Enzyme Alcohol dehydrogenase (ADH)

Cytochrome P450 (CYP)

Isoforms I II III IV V 1A2 2A6, 13 2B6

2C9 2D6, 7 2E1

Catalase

3A4 1 2 3

Tissue distribution Liver, kidney, lung, stomach Liver Brain, stomach Stomach Liver Liver Liver, lung Liver, brain, kidney, heart, placenta, lung Liver Brain, liver Liver, placenta, brain, kidney, testis, ovaries, gastrointestinal tract, heart Liver, intestine Most tissues

Reaction site Cytoplasm

References Estonius et al. (1996), Green and Stoler (2007)

Endoplasmic reticulum Mitochondria (CYP2E1)

Bansal et al. (2010), Nishimura et al. (2003), Robin et al. (2002), Zanger and Schwab (2013)

Peroxisomes

Zelner and Koren (2013)

Mitochondria

endoplasmic reticulum and in the mitochondria (Bansal et al. 2010; Robin et al. 2002), is mainly expressed in the placenta, being the main enzyme responsible for EtOH metabolism after chronic alcohol use (Cho et al. 2017; Nishimura et al. 2003; Zanger and Schwab 2013). Throughout pregnancy, CYP2E1 is active from week 16 and ADH is active from week 26 of gestation (Ehrhart et al. 2018), suggesting that during high alcohol intake, EtOH placental metabolism is mostly due to the CYP2E1 activity as a result of ADH saturation (Quertemont 2004) and also aggravated by the decreased activity of ALDH. Noticeably, in the placenta, mainly in the first trimester, CYP2E1 metabolizes EtOH attributable to its higher affinity for this molecule, in contrast to the low affinity presented by ADH. In addition to these two main EtOH oxidative pathways, catalase can metabolize EtOH into acetaldehyde in the presence of a hydrogen peroxide (H2O2)-generating system, such as the enzyme complex NADPH oxidase or the enzyme xanthine oxidase (Zelner and Koren 2013; Mohammed et al. 2020) (Fig. 2). This is a minor pathway of alcohol oxidation, except in the fasted state or in restricted circumstances, where an increase in H2O2 production is observed. As CYP2E1 is the core system responsible for EtOH metabolism throughout pregnancy, reactive oxygen species (ROS), such as hydroxyl radical, hydroxyethyl

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radical and hydroperoxyl radical, are produced as a consequence of this chemical reaction due to its remarkably high NADPH oxidase activity (Koop 2006). These side products can oxidize lipids, proteins, and other metabolites, trigger DNA damage, produce mitochondrial dysfunction, and disrupt neuronal cell–cell adhesion (Ramachandran et al. 2001; de La Monte and Wands 2001). Animal and in vitro studies have shown that EtOH metabolism related products activate the intrinsic apoptotic pathway, e.g. increase cytochrome c and caspase 3 levels (Ramachandran et al. 2001) and promote mitochondrial damage (de La Monte and Wands 2001), resulting in placental vasoconstriction and impairments in placental metabolism and hormonal function (Gupta et al. 2016a). These alterations can result in placental abruption, fetal hypoxia, and malnutrition, producing in turn FGR or stillbirth (Carter et al. 2016; Aliyu et al. 2008, 2011; Salihu et al. 2011). Besides, this increase in ROS generation by CYP2E1 can compromise the antioxidant capability of the cell, decreasing both enzymatic (superoxide dismutase, catalase, and/or glutathione peroxidase levels) and non-enzymatic (e.g., tocopherol and melatonin) antioxidant cellular mechanisms (Gupta et al. 2016a). For this reason, both ROS and, currently, acetaldehyde-protein adducts (compounds that have longer half-life than free acetaldehyde and remain high in blood after alcohol exposure) could be used as indirect biomarkers of alcohol abuse. To date, several biomarkers, e.g. fatty acid ethyl esters, ethyl glucuronide, ethyl sulfate, and phosphatidyl ethanol, have been suggested as the most suitable to detect prenatal alcohol exposure (Bager et al. 2017). During pregnancy, the growing fetus is really vulnerable to alcohol exposure, due to the amniotic accumulation of EtOH, fetal immature antioxidant system, fetal lower liver levels, and activity of ethanol metabolizing enzymes (ADH and CYP2E1), and hence, reduced EtOH elimination (Ehrhart et al. 2018; Gupta et al. 2016a). As a consequence, fetal tissues and organs, particularly the developing brain, liver and skeleton, are extremely susceptible to the oxidative stress produced as a result of EtOH metabolism. Numerous studies have focused their attention on fetal brain as one of the key targets for EtOH effects during pregnancy, as it has the greatest oxygen rate of all body tissues; its richness in polyunsaturated fatty acids and oxidizable neurotransmitters that can act as targets for ROS; and its lower levels of antioxidant and EtOH degrading enzymes (Ehrhart et al. 2018). However, a modest question arises: if the placenta is the major organ necessary for an appropriate fetal growth and development, could EtOH and its derived products impair placental functions and metabolism, thus leading to poor fetal outcomes, such as FGR? It is important to notice that not only local EtOH metabolism in the placenta is a crucial factor for the related consequences of alcohol use during pregnancy. EtOHrelated metabolites, such as acetaldehyde and acetate, can be generated elsewhere in the body, contributing in this way to fetal and placental damage throughout this important period of development (Carter et al. 2016; Aliyu et al. 2008, 2011; Salihu et al. 2011). On the other hand, EtOH is also metabolized, to a lesser extent, by non-oxidative pathways where it conjugates with several molecules, such as fatty acids,

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phospholipids, sulfate, or glucuronic acid to form fatty acid ethyl esters, phosphatidyl ethanol, ethyl sulfate, and ethyl glucuronide, respectively (Zelner and Koren 2013). Fatty acid ethyl ester synthases catalyze the reaction to form fatty acid ethyl esters, compounds involved in EtOH-induced organ injuries, especially in heart, an organ lacking EtOH oxidative metabolism (Quertemont 2004). As aforementioned, in this non-oxidative pathway, EtOH can also be metabolized to phosphatidyl ethanol by phospholipase D, predominantly at high EtOH levels. This product can disrupt phosphatidic acid generation, thus altering cell signaling. Recent studies suggest that EtOH consumption increases the levels of CYP2E1 and other P450 isoforms such as CYP2A3, CYP1A1/2, and CYP4B; hinting that these enzymes could be used as potential novel biomarkers for liver injury (Cho et al. 2017). In spite of these promising biomarkers, to date there is no specific biomarker to detect placental alterations related to EtOH consumption.

5 Insulin-Like Growth Factors (IGFs) as a Main Target for Ethanol Consumption IGFs are a family of growth factors involved in cell survival and proliferation. Two isoforms belong to this family, IGF-1 and IGF-2, both with different biological activity: although IGF-2 is expressed predominantly in early embryonic and fetal life, IGF-1 is the main factor responsible for an appropriate intrauterine and postnatal growth and development (Sferruzzi-Perri et al. 2006; Clemmons 2019; Puche and Castilla-Cortázar 2012). IGF-1 is an anabolic hormone produced in several tissues, especially in the liver (approximately 75% of circulating IGF-1), but virtually every tissue is able to secrete IGF-1 for autocrine and/or paracrine purposes (Clemmons 2019; Martín-Estal et al. 2016). The secretion of IGF-1 is stimulated by growth hormone (GH), forming the GH/ IGF-1 axis, where GH secretion is stimulated by growth hormone-releasing hormone (GHRH) and inhibited by somatostatin. This GH/IGF-1 axis is regulated by negative feedback mechanisms induced by IGF-1 itself: IGF-1 can inhibit GH gene expression by stimulating the secretion of somatostatin, which inhibits in turn GH secretion (Puche and Castilla-Cortázar 2012). IGF-1 is a pleiotropic hormone with several functions: proliferative, mitochondrial protection (Pérez et al. 2008), cell survival (Vincent and Feldman 2002), tissue growth and development (Fowden and Forhead 2013), anti-inflammatory and antioxidant (García-Fernández et al. 2003, 2005), antifibrogenic (Muguerza et al. 2001), and anti-aging (Puche et al. 2008; García-Fernández et al. 2008). In spite of these several physiological roles of IGF-1, its activities must be strictly controlled by its association with binding proteins (IGFBPs 1-6 with major affinity for IGF-1). These IGFBPs are produced by a variety of biological tissues and found in several biological fluids, such as follicular liquid, amniotic liquid, vitreous humor,

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lymph, plasma, seminal fluid, cerebrospinal fluid, and gastrointestinal secretions (Martín-Estal et al. 2016). In circulation, IGF-1 is mostly found forming ternary complexes with IGFBP-3 and acid-labile subunit (ALS), which serves as a reservoir for IGF-1 and also increases its half-life. These IGFBPs can be associated with cell membranes or extracellular matrix, allowing them to maintain a local pool of IGF-1 (Martín-Estal et al. 2016; Allard and Duan 2018). Recent experimental studies show that chronic exposure to high levels of EtOH during pregnancy reduces fetal weight and maternal and fetal plasma IGF-1 and IGFBP-3 levels; increases IGFBP-1 (an inhibitory protein for IGF-1) and IGF-2 levels; inhibits insulin, IGF-1 and IGF-2 placental gene expression and/or secretion; and reduces insulin and IGF-1 receptor tyrosine kinase activities, thus altering the bioavailability of IGF-1 and its downstream signaling transduction (Aros et al. 2011; Gundogan et al. 2008; Gårdebjer et al. 2014; Röjdmark and Aquilonius 2000; Röjdmark and Brismar 2001; Dal Maso et al. 2007; Dobson et al. 2014; Kumar et al. 2002; de la Monte et al. 2005; Soscia et al. 2006; Gatford et al. 2007; de la Monte et al. 2000; Hallak et al. 2001; Seiler et al. 2000, 2001). The observed increase in IGF-2 and IGF2R mRNA expression levels in these studies could be due to a compensatory mechanism established in response to the impairment in IGF-1 signaling pathway due to insulin receptor substrates (IRSs) depletion (Gundogan et al. 2008). For this reason, as an altered placental and fetal growth and development can be associated with an increased perinatal morbidity and mortality and a larger risk to develop several diseases in adult life, it is crucial to understand the role of IGF-1 and the molecular mediators within its signaling pathway during pregnancy, in order to identify the mechanisms underlying this altered fetal growth.

5.1

IGFs Main Functions during Pregnancy

IGFs (both IGF-1 and IGF-2) play a crucial role in modulating fetal growth via their actions on mother and/or the placenta, especially IGF-1, whose maternal levels correlate positively with fetal growth and birth weight (Sferruzzi-Perri et al. 2011). Before birth, IGF-1 is synthesized by numerous fetal tissues, independently of GH (Sferruzzi-Perri et al. 2006, 2011; Hiden et al. 2009). Maternal IGF-1 levels increase in the first trimester of pregnancy and continue to rise throughout this period of development (Chellakooty et al. 2004; Yang et al. 2013). After birth, circulating IGF-1 concentrations rise due to the commencement of GH-dependent hepatic synthesis of IGF-1 and also the production of this hormone by a large number of other tissues, e.g. uterus, skeletal muscle, and adipose tissue (Sferruzzi-Perri et al. 2011; Lof et al. 2005). IGF-1 influences maternal tissue growth and metabolism, hence modulating nutrient supply and bioavailability for fetal growth. For example, IGF-1 stimulates glucose and amino acid uptake in human trophoblasts in vitro (Sferruzzi-Perri et al. 2011; Roos et al. 2009). Moreover, IGF-1 regulates placental morphogenesis and

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hormone secretion, either into the umbilical or into the maternal circulation, being both processes indispensable for an appropriate intrauterine growth (Sferruzzi-Perri et al. 2011). This could be possible due to the localization of IGF-1 and insulin receptors in the syncytiotrophoblast layer of the placenta, bathed in maternal blood (Sferruzzi-Perri et al. 2006; Hiden et al. 2006, 2009). In addition, IGF-1 has a major role in fetal and placental growth and differentiation (Hiden et al. 2009; Forbes and Westwood 2010), being one of the foremost regulators of intrauterine and normal body growth. For example, in the central nervous system (CNS), IGF-1, together with IGF-2 and insulin, regulates neuronal survival, energy metabolism, and plasticity, all of them critical to maintain cognitive and motor functions. In this fashion, IGF-1 can protect against CNS insults, e.g. EtOH-related neuronal impairments as observed in FASD conditions, enhancing both neuronal generation and growth and white matter production (Cao et al. 2003; Ye et al. 2000; McGough et al. 2009). For this reason, insulin and IGF-1 signaling pathways are main targets of EtOH-mediated neurotoxicity in the immature CNS. The inhibition of one or both pathways due to EtOH exposure could lead to neuronal loss (de la Monte et al. 2000, 2001; Hallak et al. 2001). Furthermore, IGF-1 is a crucial regulator of placental resource allocation to fetal growth both developmentally and in response to external and environmental insults, due to its effect on maternal tissue growth and metabolism, placental morphogenesis, transport and hormone secretion, thus modulating nutrient availability for fetal growth (Sferruzzi-Perri et al. 2011, 2017). IGF-1 and its signaling pathway change aligned with the alterations in placental structure and function, and these changes can be beneficial or detrimental to resource allocation to the fetus depending on the type, severity, and timing of the challenge during pregnancy (Sferruzzi-Perri et al. 2017). For these reasons, IGF-1-related alterations, for example those derived from a chronic EtOH use during pregnancy, could lead to poor pregnancy outcomes that would lead to increased mortality and morbidity and adult diseases (Rehm et al. 2010).

6 Ethanol Molecular Alterations in Trophoblast Invasion and Migration During pregnancy, implantation encompasses several interrelations between trophoblast cells and the receptive endometrium through numerous embryonic and endometrial-derived factors that control trophoblast cell invasion and migration and syncytia formation, processes necessary for normal placental growth and development. This cross talk is extremely regulated by paracrine and autocrine factors (e.g., cytokines, growth factors, and extracellular matrix components), because an excessive and slight invasion and failure in the spiral artery remodeling and cytotrophoblast–syncytiotrophoblast fusion can result in implantation failure or FGR (Soncin et al. 2015; Gupta et al. 2016b).

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To date, the molecular mechanisms by which EtOH triggers its toxic effects during pregnancy, especially in the placenta, are not entirely known. Several studies using animal and in vitro models of alcohol consumption have provided data suggesting that alcohol, like other drugs of abuse, alters numerous molecular cascades involved in an appropriate placentation, via activation of both tyrosine kinase receptors (RTKs) and G protein-coupled receptors (GPCRs) (Fig. 3). Some of these cascades are Ras/Mitogen-activated protein kinase (MAPK) signaling pathway, which activates several substrates, such as extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38MAPKs, involved in trophoblast migration and invasion; the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB or AKT) signaling cascade; embroiled in cell growth, proliferation and survival; and the Janus kinase (JAK)/signal transducer and activator of transcription protein (STAT), Wnt and transforming growth factor β (TGFβ) signaling pathways and their downstream molecules, implicated in placental development (Soncin et al. 2015; Gupta et al. 2016b; Tomás et al. 2002, 2003; Fattoretti et al. 2003; Kumada et al. 2006; Allansson et al. 2001). Concerning the GPCR signaling cascades, chronic EtOH abuse activates Gαsprotein expression, especially in the mesolimbic regions of the brain (Wand et al. 2001; Ron and Messing 2013), which are involved in drug reward and reinforcement of drug addiction (Fig. 3). As a result, cAMP-dependent signaling is augmented due to adenyl cyclase activity, leading to the activation of protein kinase A (PKA). This last protein phosphorylates several substrates (e.g., JNKs and p38MAPKs) that are responsible, in turn, for placental morphogenesis and learning, memory and behavioral responses to drugs of abuse (Ron and Messing 2013) (Table 2). Along with PKA activation, other second messenger cascades, such as protein kinase C (PKC), are altered due to alcohol consumption, increasing intracellular Ca2+ release, disrupting neuronal cell–cell interaction and cell adhesion (Gupta et al. 2016a; Bearer 2001; Del Castillo-Vaquero et al. 2010), deregulating neurotransmission systems (Zhou et al. 2002; Ikonomidou et al. 2000; Sari and Zhou 2004; Sari and Gozes 2006) and altering growth factor and trophic support (Heaton et al. 2000; Miller et al. 2002; Ge et al. 2004; Li et al. 2004), all of them contributing to cellular damage and apoptosis (Ikonomidou et al. 2000; Olney et al. 2000; de la Monte et al. 2003; Bolnick et al. 2014) (Table 2). Additionally, EtOH consumption alters RTKs signaling pathways, via inactivation of the Tyr activating residues within the receptor and/or impairing PI3K/AKT and Ras/MAPK cascades, resulting in placental apoptosis and abnormal cell proliferation, survival, and growth (Fig. 3). Of note, EtOH’s effects are mainly due to its action on insulin and IGFs signaling pathways, thus altering cell: viability, metabolism, and homeostasis, and hence, normal growth and development (de la Monte and Wands 2010; de la Monte et al. 2012; He et al. 2007; Yeo et al. 2000; Sönmez et al. 2016). This cascade will be discussed in the subsequent paragraphs. However, more studies are needed in order to determine the EtOH wounding effects on the placenta and, therefore, on the fetus.

Fig. 3 Ethanol impairments in insulin/IGFs and GPCRs signaling pathways. Ethanol (EtOH) abuse can impair both Tyr kinase receptor (RTK) signaling pathways (e.g., the insulin/IGF-1 signaling pathway) or G protein-coupled receptor (GPCR). Regarding the first one, insulin-like growth factor 1 (IGF-1) can bind to its putative receptor (IGF1R, a RTK) or to the insulin receptor (INSR), but with low affinity than insulin. The stimulation of this signaling pathway

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begins with the activation of Tyr kinase residues within the receptor that in turn recruits several adapter proteins, e.g. Shc and insulin receptor substrates (IRSs). Shc activates the Ras/MAPK cascade, whereas IRS (isoforms 1-6) activates PI3K/AKT signaling pathway. The latter is a crucial protein involved in several processes such as protein synthesis and growth, neuroprotection, glucose metabolism, cardiovascular homeostasis, cell survival and proliferation, and cell survival gene expression (e.g., AAH gene expression, entangled in cell migration, and trophoblast invasion). EtOH consumption inactivates Tyr activating residues within the receptor, impairs protein kinase B (PKB or AKT) and Ras/MAPK cascades (e.g., ERKs, JNKs, p38MAPKs), resulting in apoptosis, abnormal cell proliferation, survival and growth, and aberrant trophoblast invasion and migration, leading to placental dysfunction and fetal growth restriction (FGR). Regarding the GPCRs signaling pathways, EtOH activates Gαs-proteins, increasing cAMP-dependent signaling that, in turn, stimulates protein kinase A (PKA), which phosphorylates numerous substrates (e.g., JNKs, p38MAPKs) that are responsible for the learning, memory, and behavioral responses to drugs of abuse and trophoblast differentiation and function, among other functions. Also, EtOH exposure activates protein kinase C (PKC), resulting in an increase of Ca2 + intracellular release, disruption of neuronal cell–cell interaction and cell adhesion, deregulation of neurotransmission systems, and alteration of growth factor and trophic support. All these EtOH-related alterations promote, in turn, apoptosis. Additionally, mitochondria are altered by EtOH consumption. The abuse of this molecule decreases the activities and expression of oxidative phosphorylation complexes of the electron transport chain (ETC) and increases the expression of NADH oxidase, resulting in a diminution in ATP synthesis and exacerbating reactive oxygen and nitrogen species (ROS and RNS, respectively) production, both leading to the oxidative modification of mitochondrial macromolecules (lipids and proteins) that impairs mitochondrial membrane permeability and increases mitochondrial sensitivity to toxins. Consequently, EtOH abuse causes mitochondrial dysfunction and promotes apoptosis and/or necrosis. EtOH alterations are represented by yellow rays. Proteins regulated by other proteins and signaling pathways other than IGF-1 are represented in gray circles. AAH: aspartyl-asparaginyl β-hydroxylase; AC: adenyl cyclase; AKT/PKB: protein kinase B; Complex I: NADH oxidase/NADH dehydrogenase; Complex II: succinate dehydrogenase; Complex III: CoQ-cytochrome c reductase; Complex IV: cytochrome c oxidase; Complex V: ATP synthase; CoQ: coenzyme Q; DAG: diacylglycerol; EtOH: ethanol; GPCRs: G protein-coupled receptors; GSK-3β, glycogen-synthase kinase 3β; IGF-1, insulin-like growth factor 1; IGF1R: IGF-1 receptor; IP3: inositol-3-phosphate; IP3R: IP3 receptor; IRS: insulin receptor substrate; O2.-: superoxide radical; PDK1: phosphoinositide-dependent kinase-1; PI3K: phosphoinositol-3-kinase; PIP2: phosphatidylinositol biphosphate; PIP3: phosphatidylinositol triphosphate; PKA: protein kinase A; PKC: protein kinase C; PLCβ: phospholipase Cβ; PTEN: phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase; PTP-1b: protein-tyrosine phosphatase 1b; RNS: reactive nitrogen species; ROS: reactive oxygen species; RTKs: receptor Tyr kinase

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Table 2 Ethanol related alterations in proteins involved in insulin/IGFs and GPCRs signaling pathways Cellular process affected by ethanol consumption Apoptosis

ATP synthesis

Nervous system homeostasis Placental morphogenesis Trophoblast motility and invasion Receptor activation and downstream signaling

Ethanol target protein and its impact on the cell "Caspase 3 and "cytochrome c #Bcl2, "Bax and "Bak ""PKC #Cytochrome c oxidase and #ATP synthase "NADPH oxidase ""PKC ""PKA ##AAH "PTP1-b (negatively regulates RTKs) "PTEN (negatively regulates PI3K)

References Ramachandran et al. (2001) Gundogan et al. (2010) de la Monte et al. (2003), de la Monte and Wands (2010), Ikonomidou et al. (2000), Olney et al. (2000) Chu et al. (2007), Ramachandran et al. (2001), Xu et al. (2005)

Bearer (2001), Ikonomidou et al. (2000), Sari and Zhou (2004), Sari and Gozes (2006), Zhou et al. (2002) Ron and Messing (2013) Cantarini et al. (2006), Carter et al. (2008), de la Monte et al. (2006), Gundogan et al. (2008, 2015), Lawton et al. (2010) de la Monte and Wands (2010), Ewenczyk et al. (2012)

": increased levels; "": over-activation; #: decreased levels; ##: inactivation. AAH aspartylasparaginyl β-hydroxylase, PKA protein kinase A, PKC protein kinase C, PTEN phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase, PTP1-b protein-tyrosine phosphatase 1b

7 Mitochondria as a Target Generator of ROS Due to Ethanol Metabolism Mitochondria, the major energy source of the cell, are closely involved in the generation and defense against ROS, being the electron transport chain (ETC) the main source of ROS within this organ. For this reason, mitochondria are targets of and can contribute to oxidative stress (Hoek et al. 2002). In the placenta, an organ that requires energy for its own metabolism, growth, and morphological remodeling; mitochondria need a substantial oxygen requirement and, as the fetus grows, these demands on placental energetics increase. Noteworthy, placental mitochondria can change its functions developmentally to meet the increasing fetal demands and best support its development, as well as placental growth and function, especially in restricted conditions such as hypoxia (SferruzziPerri et al. 2019). Conversely, there is little information about placental

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mitochondrial dysfunction and its potential impact on fetal and newborn development during pregnancy (Mandò et al. 2014). Alcohol consumption throughout pregnancy affects carbohydrate, lipid, and protein metabolism. As can be revised elsewhere, mitochondria are indispensable for the conversion of acetaldehyde (a derived product from EtOH metabolism) into acetate and the generation of NADH (Manzo-Avalos and Saavedra-Molina 2010; Morén et al. 2014). In this way, during EtOH oxidation there is an increase in the NADH/NAD+ ratio and in ROS formation by mitochondria. Moreover, consistent evidence shows that EtOH consumption decreases the activities and expression of oxidative phosphorylation complexes of the ETC (e.g., cytochrome c oxidase, a target of CYP2E1-mediated alcohol toxicity (Bansal et al. 2012), and ATP synthase) and increases the expression of NADH oxidase (Ramachandran et al. 2001; Chu et al. 2007; de la Monte and Wands 2002), resulting in a diminution in ATP synthesis (Manzo-Avalos and Saavedra-Molina 2010; Chu et al. 2007; Goodlett and Horn 2001; Xu et al. 2005) (Table 2). As a result, these alterations in the oxidative phosphorylation system exacerbate the production of ROS and reactive nitrogen species (RNS), leading to lipid peroxidation and protein carbonylation (Bansal et al. 2012) (Fig. 3). This uncontrolled mitochondrial formation of ROS promotes an oxidative environment, enhanced by a decrease in mitochondrial oxidative defenses (e.g., glutathione) (Bansal et al. 2012) that activates the mitochondrial permeability transition pore, increasing mitochondrial sensitivity to toxins, apoptosis (EtOH consumption decreases Bcl2 levels, an antiapoptotic protein; and increases Bax and Bak levels, both proapoptotic proteins (Gundogan et al. 2010)) and the response to other proapoptotic or damage signals (Hoek et al. 2002) (Table 2). Therefore, EtOH abuse increases CYP2E1 expression, promoting this oxidative environment that causes mitochondrial dysfunction and promotes apoptosis and/or necrosis (Fig. 3). Several experimental studies in animal models have shown that alcohol induces oxidative stress and mitochondrial dysfunction in placenta, as it promotes trophoblast apoptosis and lipid peroxidation, suggesting that mitochondria are the foremost organelle involved in all these processes (Gundogan et al. 2010; Green et al. 2006). This mitochondrial dysfunction has been associated with increased rates of preterm delivery, stillbirth, FGR, and sudden infant death (Mandò et al. 2014; Morén et al. 2014). As diverse experimental studies disclose, IGF-1 signaling pathway is crucial for the maintenance of mitochondrial homeostasis, being involved in its biogenesis, dynamics, and turnover, oxidative phosphorylation, mitochondrial DNA/RNA maintenance, and suppression of ROS production (Riis et al. 2020; Ribeiro et al. 2014; Lyons et al. 2017). Consequently, placental mitochondria are becoming a more relevant target for EtOH use during pregnancy, in order to discover new insights about this metabolite and placental and fetal consequences. However, in spite of these promising results, significant data on mitochondrial toxicity derived from EtOH exposure throughout pregnancy are remarkably missing.

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8 Ethanol Alterations in the IGFs Signaling Pathway The majority of IGF-1 actions are mediated through the union of this molecule to its putative receptor, IGF1R, a tyrosine kinase receptor (RTKs) that is one of the most potent natural activators of the PI3K/AKT and MAPK signaling pathways, both related with cell survival, growth, and proliferation (Annenkov 2009; Chitnis et al. 2008). IGF-1 can also bind to the INSR but with lower affinity, being this signaling a secondary pathway by which IGF-1 mediates some of its metabolic functions. Similarly, insulin can bind to the IGF1R with a lower affinity than it does to the INSR (Belfiore et al. 2017). Both receptors are expressed in almost all placental tissues in the first trimester of pregnancy and at term (Martín-Estal et al. 2016). In addition to these archetypal receptors, naturally occurring hybrid receptors have been described, formed by one αβ-chain from INSR and another αβ-chain from IGF1R. Both insulin and IGF-1 can bind to these hybrid receptors but with lower affinity than their own receptors. IGF-2 receptor (IGF2R) is homologous to the manose-6-phosphate receptor and functions as a scavenger receptor, requisitioning IGF-1 and IGF-2 and targeting them for destruction (Xu et al. 2018). IGF-2 can bind to its own receptor and both IGF1R and INSR, but with lower affinity than their specific ligands. This variability of binding and affinities for the aforementioned receptors is due to the homology presented by IGF-1 that shares >60% homology with IGF-2 and 50% homology with proinsulin structures, and the similarity in structure of IGF1R and insulin receptor (INSR) (60%) (Xu et al. 2018). The stimulation of this signaling pathway begins with the activation of Tyr kinase residues within the receptor that in turn recruits several adapter proteins, e.g. Shc and IRSs. These adapter proteins are responsible for the initiation of Ras/MAPK and PI3K/AKT signaling cascades, respectively (Fig. 3). Regarding these adapter proteins, the most important ones are the IRSs, whose six isoforms have been described to date (IRSs 1-6). IRS1 and IRS2 are extensively distributed, IRS3 is limited to adipocytes and brain, IRS4 is expressed mainly in embryonic tissues, and IRS5 and IRS6 have restrained tissue expression and function in signaling pathways (Taniguchi et al. 2006). Several in vitro studies show that IRS3 and IRS4 impair IRS1 and IRS2 signaling, because they cannot activate MAPK and PI3K proteins (Tsuruzoe et al. 2001). Moreover, numerous experimental studies in rodents show that EtOH exposure during pregnancy reduce IRS1, IRS2, and IRS4 levels in EtOHexposed placentas, thus altering the downstream insulin/IGFs signaling transduction (Gundogan et al. 2008; Kumar et al. 2002). In spite of this different tissue distribution and actions, especially regarding IRS1, IRS2, and IRS3, a reasonable question arises: could one of these IRSs, particularly IRS4, IRS5 and IRS6, be mainly expressed in placenta during pregnancy; and could be this expression altered by EtOH consumption? Up to the present time, no studies have been conducted in placenta to solve this question, so this could be a first line of action to determine and possibly revert the EtOH-related effects in placenta and intrauterine environment during pregnancy.

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To date, several experimental studies have shown that chronic EtOH exposure during pregnancy reduces IGF-1, IGFBP-3, IRS1, IRS2, and IRS4 levels; increases IGFBP-1 and IGF-2 levels; inhibits insulin, IGF-1, and IGF-2 placental gene expression and/or secretion; and reduces insulin and IGF-1 receptor tyrosine kinase activities (Gundogan et al. 2008; Gårdebjer et al. 2014; Kumar et al. 2002; de la Monte et al. 2005; Soscia et al. 2006; Gatford et al. 2007; de la Monte et al. 2000; Hallak et al. 2001; Seiler et al. 2000, 2001). In this sense, EtOH abuse during pregnancy can impair the insulin/IGFs signaling pathway at several levels. Firstly, EtOH can inhibit the phosphorylation and activation of corresponding tyrosine kinase domains within the receptor and their immediate downstream effector molecules, hindering Ras-GTPase activity and signaling through PI3K. This harmful effect leads to reduced activation of the MAPK and PKB/AKT signaling cascades, altering cell proliferation, survival and differentiation; placentation; glucose metabolism; neuroprotection and cardiovascular homeostasis (Fig. 3). Secondarily, EtOH abuse can blight cell survival gene expression. An experimental study in rodents reveals that EtOH exposure during pregnancy alters the expression of aspartyl-asparaginyl β-hydroxylase (AAH), an important enzyme in trophoblast motility and invasion, whose gene is regulated by the insulin/IGFs signaling pathway, thus suggesting the importance of this signaling pathway in placentation (de la Monte et al. 2006; Cantarini et al. 2006; Gundogan et al. 2008, 2015; Carter et al. 2008; Lawton et al. 2010) (Table 2). Ultimately, EtOH consumption through pregnancy can increase the activation of phosphatases that negatively regulate RTKs (protein-tyrosine phosphatase 1b, PTP-1b) and PI3K (phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase, PTEN) (Ewenczyk et al. 2012) (Fig. 3, Table 2). All these results disclose that EtOH use during pregnancy leads to impairments in insulin/IGFs downstream signaling transduction, therefore altering placental morphogenesis and functions that affect intrauterine growth and development. Consequently, EtOH exposure during pregnancy produces an insulin/IGFs resistance state that accounts for most of the abnormalities observed, including CNS malformations, placental dysfunction, and FGR. The latter is a condition where reduced IGF-1 and IGFBP-3 levels have been reported in human studies, suggesting that reduced IGF-1 abundance or bioavailability in the fetus, mother and/or placenta may contribute to this growth restriction (Gundogan et al. 2015; Martín-Estal et al. 2016; Gatford et al. 2007; Woods et al. 2002; Spiroski et al. 2020). Similarly, numerous studies in adults show that EtOH consumption reduces IGF-1 and increases IGFBP-1 circulating serum levels, probably affecting IGF-1 liver production and hence, altering IGF-1 signaling cascade (Kumar et al. 2002). Moreover, acute EtOH exposure during pregnancy promotes malformation and malfunction of placenta-yolk sac tissues in rodents, decreasing the labyrinth zone in the placenta and altering cell membrane’s permeability and fluidity (Haghighi Poodeh et al. 2012). In this way, EtOH consumption modifies biological membrane’s composition, e.g. cholesterol content, producing an altered survival signaling pathway. EtOH use leads to a damaged insulin and IGF-1 binding and signal transduction, reducing the activation of the IRS1/PI3K/AKT pathway and

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preserving the MAPK signaling (Cho et al. 2004; Huo et al. 2003; Peiró et al. 2000; Zhang et al. 2019; Parpal et al. 2001). Additionally, the increase in ROS production due to EtOH exposure augments lipid peroxidation that alters caveolae composition in placental membranes, thus decreasing the activation of insulin/IGFs signaling pathway (Huo et al. 2003; Zhang et al. 2019). To date, the vast majority of experimental studies are conducted in brain, in order to determine EtOH harmful effects in this organ, e.g. CNS malformations, neuronal apoptosis, and neurotransmitters release, among others (Soscia et al. 2006; Ewenczyk et al. 2012). Also, the mechanisms that may lead to FGR in EtOHexposed fetuses are still not well-defined. However, although it is known that the third trimester of pregnancy is the most vulnerable period to EtOH genotoxic effects, there are scarce studies that focus on the placenta. This could be an opportunity area to develop more studies that help us to understand the essential functions that this organ plays throughout gestation. For this reason, it is important to understand first the placental role during pregnancy and the signaling pathways involved in its proper development, since the placenta is the first barrier that protects the intrauterine environment and fetal development against external and internal insults. Second, the role of IGF-1 in pregnancy and in the intrauterine environment because this hormone plays a key role in placental and fetal growth and development. And third, the EtOH-related alterations in signaling pathways, especially in the insulin/IGF-1 pathway, involved in cell growth, proliferation and survival, neuroprotection and placentation.

9 Conclusions and Perspectives Alcohol consumption is a vast health and economic problem worldwide, even during pregnancy, being a risk factor for developing adverse outcomes that result in clinical, cognitive, and behavioral defects. For this reason, it is a public dilemma that governmental authorities need to, firstly, understand the underlying mechanisms of its harmful actions, and secondly, solve due to the high costs to attend its medical illness and dependence. EtOH metabolism, particularly by CYP2E1, produces ROS that generate an oxidative environment responsible for the oxidation of biological molecules, mitochondrial dysfunction, and hypoxia, all of them leading to placental and fetal underdevelopment. Although it is known that several organs are affected by EtOH consumption during pregnancy, especially the brain, scarce studies have focused their hypothesis on the placenta. It is important to notice that the placenta is a key organ during pregnancy that functions as a barrier, protecting the fetus from external and internal insults, ensuring in this way its proper development. In this fashion, how EtOH impairs placental and hence fetal development? Different mechanisms may be responsible for the effects of ethanol on the insulin/IGFs signaling pathway during pregnancy, especially in the feto-placental axis. Nonetheless, limited studies have been conducted to resolve EtOH impairments in such cascade, particularly in the

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placenta. Firstly, ethanol use throughout pregnancy alters several proteins involved in the PI3K/AKT signaling cascade involved in numerous processes such as protein synthesis and growth, neuroprotection, glucose metabolism, cardiovascular homeostasis, cell survival, and proliferation. Secondly, ethanol-related changes observed may modify AAH gene expression, altering cell migration and trophoblast invasion. Thirdly, ethanol consumption impairs several proteins that lead to oxidative stress and apoptosis resulting in an abnormal cell proliferation and migration, causing placental dysfunction and fetal growth restriction. These data suggest that IGF-1 deficiency in the gestational state may be one of the main causes of fetal growth retardation, being the placenta as a target organ of IGF-1 relevant for fetal health and human development. Since both IGF-1 and IGF-2 have several functions depending on the time of development which it is being expressed, it is crucial to determine the participation of each one throughout pregnancy to understand the adverse effects of teratogen insults during this period of growth. For example, in IGF-1 partial deficiency conditions seem to be a compensatory mechanism to minimize the effects due to the lack of this hormone. Further investigations are necessary to determine how alcohol consumption could modify the insulin/IGFs signaling pathway and its impact in the placenta and hence, fetal development. Also, it is necessary to identify biomarkers and differentiate genes that might impact the health of both mother and children. Consequently, this would reduce the clinical, social, economic, and government costs related to ethanol consumption. Acknowledgements The authors would like to express their gratitude to MD. Rodolfo Benavides, MD. Andrea Leal, and MD. Marcela Galindo for their invaluable help. Conflict of Interest The authors declare that they have no conflict of interest.

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