University of Groningen Compartmentalized cAMP Signaling in COPD Zuo, Haoxiao

University of Groningen

Compartmentalized cAMP Signaling in COPD

Zuo, Haoxiao

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Zuo, H. (2019). Compartmentalized cAMP Signaling in COPD: Focus on Phosphodiesterases and A-Kinase Anchoring Proteins. University of Groningen.

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Compartmentalized cAMP Signaling in COPD

The studies described in this thesis were performed within the framework of the Groningen University Institute for Drug Exploration (GUIDE), the Groningen Research Institute for Pharmacy (GRIP) and the Groningen Research Institute for Asthma and COPD (GRIAC), and financially supported by the Ubbo Emmius program of the University of Groningen and by the Institute of Experimental Cardiovascular Research, University of Hamburg, Germany.

Printing of this thesis was fi nancially supported by: University of Groningen

Graduate School of Science (GSS) Faculty of Science and Engineering (FSE)

Paranimfen: Mariska P.M. van den Berg Sophie Bos

Thesis cover: Human Lung Cover design: Hendra Su

Lay-out: Haoxiao Zuo and Fred van der Spek | Ridderprint BV Printing: | www.ridderprint.nl

ISBN (printed): 978-94-034-1786-8

ISBN (electronic version): 978-94-034-1787-5

Copyright © H. Zuo, 2019

All rights reserved. No part of this book may be reproduced in any manner or by any means without permission.

Compartmentalized cAMP

Signaling in COPD

Focus on Phosphodiesterases and A-Kinase Anchoring Proteins

Phd thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus prof. E. Sterken

and in accordance with the decision by the College of Deans. This thesis will be defended in public on

Friday 28 June 2019 at 16.15 hours

by

Haoxiao Zuo

born on 19 January 1989 in Shandong, China

The studies described in this thesis were performed within the framework of the Groningen University Institute for Drug Exploration (GUIDE), the Groningen Research Institute for Pharmacy (GRIP) and the Groningen Research Institute for Asthma and COPD (GRIAC), and financially supported by the Ubbo Emmius program of the University of Groningen and by the Institute of Experimental Cardiovascular Research, University of Hamburg, Germany.

Printing of this thesis was fi nancially supported by: University of Groningen

Graduate School of Science (GSS) Faculty of Science and Engineering (FSE)

Paranimfen: Mariska P.M. van den Berg Sophie Bos

Thesis cover: Human Lung Cover design: Hendra Su

Lay-out: Haoxiao Zuo and Fred van der Spek | Ridderprint BV Printing: | www.ridderprint.nl

ISBN (printed): 978-94-034-1786-8

ISBN (electronic version): 978-94-034-1787-5

Copyright © H. Zuo, 2019

All rights reserved. No part of this book may be reproduced in any manner or by any means without permission.

Compartmentalized cAMP

Signaling in COPD

Focus on Phosphodiesterases and A-Kinase Anchoring Proteins

Phd thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus prof. E. Sterken

and in accordance with the decision by the College of Deans. This thesis will be defended in public on

Friday 28 June 2019 at 16.15 hours

by

Haoxiao Zuo

born on 19 January 1989 in Shandong, China

Supervisors

Prof. M. Schmidt Prof. V.O. Nikolaev

Assessment Committee

Prof. C. Page Prof. G. Folkerts Prof. B.N. Melgert

CHAPTER 1 General Introduction 7

CHAPTER 2 Epac Function and cAMP Scaffolds in the Heart and Lung 17

CHAPTER 3 Phosphodiesterases As Therapeutic Targets for Respiratory Diseases 39

CHAPTER 4 Cigarette Smoke Upregulates PDE3 and PDE4 to Decrease cAMP in Airway Cells 81

CHAPTER 5 PDE8: a Novel Target in Human Airway Smooth Muscle Cells 111

CHAPTER 6 Function of cAMP Scaffolds in Obstructive Lung Disease: Focus on Epithelial-to-Mesenchymal Transition and Oxidative Stress 117

CHAPTER 7 A-Kinase Anchoring Proteins Diminish TGF-β1/Cigarette Smoke-Induced Epithelial-to-Mesenchymal Transition 141

CHAPTER 8 Cigarette Smoke Exposure Alters Phosphodiesterases in Structural Lung Cells 173

CHAPTER 9 General Discussion 185

CHAPTER 10 Nederlandse Samenvatting 199

Acknowledgements 207

Curriculum Vitae 215

Supervisors

Prof. M. Schmidt Prof. V.O. Nikolaev

Assessment Committee

Prof. C. Page Prof. G. Folkerts Prof. B.N. Melgert

CHAPTER 1 General Introduction 7

CHAPTER 2 Epac Function and cAMP Scaffolds in the Heart and Lung 17

CHAPTER 3 Phosphodiesterases As Therapeutic Targets for Respiratory Diseases 39

CHAPTER 4 Cigarette Smoke Upregulates PDE3 and PDE4 to Decrease cAMP in Airway Cells 81

CHAPTER 5 PDE8: a Novel Target in Human Airway Smooth Muscle Cells 111

CHAPTER 6 Function of cAMP Scaffolds in Obstructive Lung Disease: Focus on Epithelial-to-Mesenchymal Transition and Oxidative Stress 117

CHAPTER 7 A-Kinase Anchoring Proteins Diminish TGF-β1/Cigarette Smoke-Induced Epithelial-to-Mesenchymal Transition 141

CHAPTER 8 Cigarette Smoke Exposure Alters Phosphodiesterases in Structural Lung Cells 173

CHAPTER 9 General Discussion 185

CHAPTER 10 Nederlandse Samenvatting 199

Acknowledgements 207

Curriculum Vitae 215

1

General Introduction

Chronic obstructive pulmonary disease (COPD) is one of the major health problems to induce morbidity and mortality. Based on the estimates from the World Health Organization, 65 million people have moderate to severe COPD all over the world. It is predicted that COPD will become the third leading cause of death (~ 8.3 million) and the fifth leading cause of disability by 2030 (Barnes, 2000; Laudette et al., 2018). According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines, COPD is characterized by progressive and not fully reversible airflow limitation. Obstruction of small airways, emphysema, enlargement of air spaces and destruction of lung parenchyma, loss of lung elasticity, closure of small airways, fibrosis, inflammation, mucus hypersecretion, and pulmonary hypertension are the key features of COPD lung tissue (Barnes, 2000; Giembycz and Maurice, 2014; Vogelmeier et al., 2017). Currently, none of the existing medications used to treat COPD have been shown to improve the long-term decline of lung function. Therefore, novel medications are urgently needed for COPD prevention and treatment.

Exposure to cigarette smoke (CS) is considered to be the primary cause of COPD. Therefore, the most effective way to prevent the development of COPD is smoke cessation (Bergeron and Boulet, 2006; Tønnesen, 2013). Additionally, other factors including exposure to indoor pollution from biomass fuels and outdoor air pollution including occupational dusts particularly in developing countries and genetics may also contribute to pathogenesis of COPD (Boswell-Smith and Spina, 2007; Vogelmeier et al., 2017; Wang et al., 2018).

Pathogenesis and pathophysiology of COPD

COPD is characterized by chronic inflammation and airway obstruction, which is not fully reversible (Vogelmeier et al., 2017). The inflammation in COPD most likely occurs in peripheral airways (bronchioles) and lung parenchyma (Barnes, 2014). It has been shown that patients with severe COPD have infiltration of macrophages and CD8+ T cells and an increased number of neutrophils in bronchial-biopsy (Di Stefano et al., 1998; O’Shaughnessy et al., 1997). A dramatic increase of macrophages and neutrophils has been observed in bronchoalveolar lavage fluid and induced sputum (Keatings et al., 1996; Pesci et al., 1998). Moreover, multiple inflammatory mediators, including lipids, chemokines, cytokines and growth factors also play a crucial role during COPD development (Barnes, 2014; Lamela and Vega, 2009). Chronic inflammation is able to induce structural alterations and mucus hypersecretion, thereby further causing narrowing of small airways and decline in lung function. Epithelial cells and macrophages secrete transforming growth factor-β (TGF-β), which triggers fibroblast proliferation and thus contributes to tissue remodeling (Barnes, 2014). The inflammatory cytokines, proteases and growth factors produced by airway smooth muscle cells are associated with remodeling process and induce phenotypic changes of smooth muscle from contractile to proliferative phenotype (Aghasafari et al., 2018; Chung, 2005).

1

Chronic obstructive pulmonary disease (COPD)

Chronic obstructive pulmonary disease (COPD) is one of the major health problems to induce morbidity and mortality. Based on the estimates from the World Health Organization, 65 million people have moderate to severe COPD all over the world. It is predicted that COPD will become the third leading cause of death (~ 8.3 million) and the fifth leading cause of disability by 2030 (Barnes, 2000; Laudette et al., 2018). According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines, COPD is characterized by progressive and not fully reversible airflow limitation. Obstruction of small airways, emphysema, enlargement of air spaces and destruction of lung parenchyma, loss of lung elasticity, closure of small airways, fibrosis, inflammation, mucus hypersecretion, and pulmonary hypertension are the key features of COPD lung tissue (Barnes, 2000; Giembycz and Maurice, 2014; Vogelmeier et al., 2017). Currently, none of the existing medications used to treat COPD have been shown to improve the long-term decline of lung function. Therefore, novel medications are urgently needed for COPD prevention and treatment.

Exposure to cigarette smoke (CS) is considered to be the primary cause of COPD. Therefore, the most effective way to prevent the development of COPD is smoke cessation (Bergeron and Boulet, 2006; Tønnesen, 2013). Additionally, other factors including exposure to indoor pollution from biomass fuels and outdoor air pollution including occupational dusts particularly in developing countries and genetics may also contribute to pathogenesis of COPD (Boswell-Smith and Spina, 2007; Vogelmeier et al., 2017; Wang et al., 2018).

Pathogenesis and pathophysiology of COPD

COPD is characterized by chronic inflammation and airway obstruction, which is not fully reversible (Vogelmeier et al., 2017). The inflammation in COPD most likely occurs in peripheral airways (bronchioles) and lung parenchyma (Barnes, 2014). It has been shown that patients with severe COPD have infiltration of macrophages and CD8+ T cells and an increased number of neutrophils in bronchial-biopsy (Di Stefano et al., 1998; O’Shaughnessy et al., 1997). A dramatic increase of macrophages and neutrophils has been observed in bronchoalveolar lavage fluid and induced sputum (Keatings et al., 1996; Pesci et al., 1998). Moreover, multiple inflammatory mediators, including lipids, chemokines, cytokines and growth factors also play a crucial role during COPD development (Barnes, 2014; Lamela and Vega, 2009). Chronic inflammation is able to induce structural alterations and mucus hypersecretion, thereby further causing narrowing of small airways and decline in lung function. Epithelial cells and macrophages secrete transforming growth factor-β (TGF-β), which triggers fibroblast proliferation and thus contributes to tissue remodeling (Barnes, 2014). The inflammatory cytokines, proteases and growth factors produced by airway smooth muscle cells are associated with remodeling process and induce phenotypic changes of smooth muscle from contractile to proliferative phenotype (Aghasafari et al., 2018; Chung, 2005).

As cigarette smoke can induce oxidative stress, there is accumulating evidence that oxidative stress is involved in COPD (Kirkham and Barnes, 2013; Wang et al., 2018). Except for cigarette smoke, exogenous sources of reactive oxygen species (ROS) such as air pollutants, or endogenously released ROS from leukocytes and macrophages can also induce oxidative stress and break the balance between oxidant and antioxidant (Kirkham and Barnes, 2013). The activated immune cells, for instance neutrophils and macrophages, are able to release ROS during the inflammatory process(Meijer et al., 2013). Endogenously released ROS reacts with lipid, protein, DNA, RNA and mitochondrial DNA, thereby, leading to epithelium injury and contributing to COPD development (Boukhenouna et al., 2018).

Fibrosis, which is a key feature in chronic pulmonary diseases, plays a vital role in COPD pathogenesis. It has been shown that epithelial-to-mesenchymal transition (EMT), which was triggered either by environmental stresses such as oxidative stress (Rhyu et al., 2005) or by extracellular mediators such as TGF-β1 (Hackett et al., 2009), contributed to pulmonary fibrosis (Jolly et al., 2018; Rout-Pitt et al., 2018; Sakuma, 2017). EMT was first identified in the 1980s by Greenburg and Hay (Greenburg and Hay, 1986). EMT is a process in which epithelial cells gradually lose epithelial proteins including E-cadherin, ZO-1, which are responsible for cell-cell contact, and undergo transition to a more mesenchymal phenotype as they gain mesenchymal markers such as N-cadherin, vimentin and fibronectin (Nieto, 2011; Zuo et al., 2019b). Transcription factors involved during EMT process are Snail, Slug, Zeb and Twist (Baulida, 2017; Yang et al., 2013). In 2010, Sohal and colleagues demonstrated for the first time that the fibroblast protein marker S100A4 was significantly increased in cells within reticular basement membrane clefts of smokers and COPD patients compared with never-smoking control subjects by immunohistochemistry, indicating an active EMT process in the large airway of CODP patients which was highly correlated with cigarette smoke exposure (Sohal et al., 2010). However, the role of cAMP scaffold in TGF-β1/cigarette smoke-induced EMT is still unclear.

Cyclic AMP targeted therapies in COPD

According to GOLD guidelines, the aim of therapy in COPD is to relieve symptoms, to reduce the frequency and severity of exacerbations and to improve health status and exercise performance (Vogelmeier et al., 2017). Unfortunately, no existing COPD medication has been conclusively shown to modify the long-term clinical outcomes. Cyclic AMP (cAMP) has been proven to be a promising target in COPD treatment due to its excellent performance on bronchodilation and anti-inflammation which is mediated by cAMP/ cAMP-dependent protein kinase A (PKA) and exchange proteins activated by cAMP (Epacs) (Dekkers et al., 2013; Oldenburger et al., 2012b; Roscioni et al., 2011b, 2011a; Schmidt et al., 2013). Nowadays, medicines used for COPD treatment relies mainly on bronchodilator therapy (β2-agonists, anticholinergics and theophylline), and on PDE4 inhibitors used in concert with either corticosteroid or bronchodilator treatment especially in COPD patients with a high risk of

exacerbations (Giembycz and Maurice, 2014; Maji et al., 2018; Vogelmeier et al., 2017; Wang et al., 2018).

Figure 1. Compartmentalised cAMP signaling. Two distinct cAMP pools are shown in the schematic. One cAMP pool is generated by an AC anchored at the plasma membrane and activated by a GPCR exposed to the extracellular stimulus; the other one is generated by an internalized GPCR in the cytoplasm. PDEs, as key actors in limiting the spread of cyclic nucleotides, are responsible for cAMP hydrolysis and hence compartmentalize the cyclic nucleotide signal. The AKAP family, which binds to the regulatory subunits of PKA and targets PKA to discreet sites/macromolecular complexes, is also indicated in the schematic. GPCR, G-protein coupled receptor; AC, adenylyl cyclase;PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; AKAPs, A-kinase anchoring proteins;

cAMP compartmentalization

The first evidence for a compartmentalized cAMP signaling has been provided in the heart more than 40 years ago. Hayes et al. and Buxton et al. demonstrated differences in heart contractility when comparing hearts perfused with different agonists to activate the cAMP cascade. The force of heart contraction was enhanced with β1-adrenoceptor agonist isoproterenol, whereas there was no change in heart contractility when activating prostaglandin E1 receptor with PGE1, even though cAMP was elevated and soluble PKA activity was also increased in both cases (Buxton and Brunton, 1983; Hayes et al., 1979). These findings provided functional evidence for the selectivity of cAMP action, indicating a compartmentalized cAMP signaling.

As one of the most important second messengers, cAMP localizes in well-organized intracellular signaling microdomains. As shown in Fig 1, cAMP is synthesized from adenosine triphosphate, following activation of adenylyl cyclases (ACs) (Omori and Kotera, 2007). Subsequently, cAMP binds to specific intracellular effector proteins, such as cyclic nucleotide-gated ion channels, PKA and Epacs (Oldenburger et al., 2012a; Omori and Kotera, 2007). In addition, PDEs, as key actors in limiting the spread of cyclic nucleotides, are responsible for cAMP and cGMP hydrolysis and

1

oxidative stress is involved in COPD (Kirkham and Barnes, 2013; Wang et al., 2018). Except for cigarette smoke, exogenous sources of reactive oxygen species (ROS) such as air pollutants, or endogenously released ROS from leukocytes and macrophages can also induce oxidative stress and break the balance between oxidant and antioxidant (Kirkham and Barnes, 2013). The activated immune cells, for instance neutrophils and macrophages, are able to release ROS during the inflammatory process(Meijer et al., 2013). Endogenously released ROS reacts with lipid, protein, DNA, RNA and mitochondrial DNA, thereby, leading to epithelium injury and contributing to COPD development (Boukhenouna et al., 2018).

Fibrosis, which is a key feature in chronic pulmonary diseases, plays a vital role in COPD pathogenesis. It has been shown that epithelial-to-mesenchymal transition (EMT), which was triggered either by environmental stresses such as oxidative stress (Rhyu et al., 2005) or by extracellular mediators such as TGF-β1 (Hackett et al., 2009), contributed to pulmonary fibrosis (Jolly et al., 2018; Rout-Pitt et al., 2018; Sakuma, 2017). EMT was first identified in the 1980s by Greenburg and Hay (Greenburg and Hay, 1986). EMT is a process in which epithelial cells gradually lose epithelial proteins including E-cadherin, ZO-1, which are responsible for cell-cell contact, and undergo transition to a more mesenchymal phenotype as they gain mesenchymal markers such as N-cadherin, vimentin and fibronectin (Nieto, 2011; Zuo et al., 2019b). Transcription factors involved during EMT process are Snail, Slug, Zeb and Twist (Baulida, 2017; Yang et al., 2013). In 2010, Sohal and colleagues demonstrated for the first time that the fibroblast protein marker S100A4 was significantly increased in cells within reticular basement membrane clefts of smokers and COPD patients compared with never-smoking control subjects by immunohistochemistry, indicating an active EMT process in the large airway of CODP patients which was highly correlated with cigarette smoke exposure (Sohal et al., 2010). However, the role of cAMP scaffold in TGF-β1/cigarette smoke-induced EMT is still unclear.

Cyclic AMP targeted therapies in COPD

According to GOLD guidelines, the aim of therapy in COPD is to relieve symptoms, to reduce the frequency and severity of exacerbations and to improve health status and exercise performance (Vogelmeier et al., 2017). Unfortunately, no existing COPD medication has been conclusively shown to modify the long-term clinical outcomes. Cyclic AMP (cAMP) has been proven to be a promising target in COPD treatment due to its excellent performance on bronchodilation and anti-inflammation which is mediated by cAMP/ cAMP-dependent protein kinase A (PKA) and exchange proteins activated by cAMP (Epacs) (Dekkers et al., 2013; Oldenburger et al., 2012b; Roscioni et al., 2011b, 2011a; Schmidt et al., 2013). Nowadays, medicines used for COPD treatment relies mainly on bronchodilator therapy (β2-agonists, anticholinergics and theophylline), and on PDE4 inhibitors used in concert with either corticosteroid or bronchodilator treatment especially in COPD patients with a high risk of

exacerbations (Giembycz and Maurice, 2014; Maji et al., 2018; Vogelmeier et al., 2017; Wang et al., 2018).

Figure 1. Compartmentalised cAMP signaling. Two distinct cAMP pools are shown in the schematic. One cAMP pool is generated by an AC anchored at the plasma membrane and activated by a GPCR exposed to the extracellular stimulus; the other one is generated by an internalized GPCR in the cytoplasm. PDEs, as key actors in limiting the spread of cyclic nucleotides, are responsible for cAMP hydrolysis and hence compartmentalize the cyclic nucleotide signal. The AKAP family, which binds to the regulatory subunits of PKA and targets PKA to discreet sites/macromolecular complexes, is also indicated in the schematic. GPCR, G-protein coupled receptor; AC, adenylyl cyclase;PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; AKAPs, A-kinase anchoring proteins;

cAMP compartmentalization

The first evidence for a compartmentalized cAMP signaling has been provided in the heart more than 40 years ago. Hayes et al. and Buxton et al. demonstrated differences in heart contractility when comparing hearts perfused with different agonists to activate the cAMP cascade. The force of heart contraction was enhanced with β1-adrenoceptor agonist isoproterenol, whereas there was no change in heart contractility when activating prostaglandin E1 receptor with PGE1, even though cAMP was elevated and soluble PKA activity was also increased in both cases (Buxton and Brunton, 1983; Hayes et al., 1979). These findings provided functional evidence for the selectivity of cAMP action, indicating a compartmentalized cAMP signaling.

As one of the most important second messengers, cAMP localizes in well-organized intracellular signaling microdomains. As shown in Fig 1, cAMP is synthesized from adenosine triphosphate, following activation of adenylyl cyclases (ACs) (Omori and Kotera, 2007). Subsequently, cAMP binds to specific intracellular effector proteins, such as cyclic nucleotide-gated ion channels, PKA and Epacs (Oldenburger et al., 2012a; Omori and Kotera, 2007). In addition, PDEs, as key actors in limiting the spread of cyclic nucleotides, are responsible for cAMP and cGMP hydrolysis and

hence compartmentalize the cyclic nucleotide signal. The superfamily of PDEs is composed of 11 families with a distinct substrate specificity, molecular structure and subcellular localization (Omori and Kotera, 2007; Zuo et al., 2019a). Each PDE family has at least one (e.g. Pde5a) and often multiple coding genes, resulting in the mammalian PDE superfamily being composed of more than 21 genes (Omori and Kotera, 2007; Page and Spina, 2012). Moreover, most PDE encoding genes have distinct promoters, and multiple transcriptional products which are generated by alternative splicing, resulting in nearly 100 different PDE messenger RNAs (Conti and Beavo, 2007; Otero et al., 2014). In addition, the communication between β2-adrenoreceptor, cAMP effectors, PDEs and other downstream targets are coordinated by A-kinase anchoring proteins (AKAPs) (shown in Fig 1) (Beene and Scott, 2007; Carnegie et al., 2009; Han et al., 2015; Poppinga et al., 2014). Members of the AKAPs family bind to the regulatory subunits of PKA and target PKA to discreet sites/macromolecular complexes, thereby playing a central role in the regulation of cAMP compartmentalization (Beene and Scott, 2007).

Scope of the thesis

The main objective of this thesis is to explore the role of compartmentalized cAMP signaling in the pathogenesis of COPD, focusing on PDE subfamilies and AKAPs. Using in vitro, ex vivo and in vivo approaches, we investigate the potential therapeutic targets in cAMP signaling pathways and link it with the current therapies available on the market.

In chapter 2, we provide an overview of Epac function and cAMP scaffolds in the heart and the lung. We highlight recent studies in heart and lung pertaining to cAMP compartmentalization, which provides more insights in understanding the role of cAMP scaffolds in different organs.

In chapter 3, we review the regulation of several PDEs (PDE3, PDE4, PDE5, PDE7 and PDE8) and demonstrate the roles of their selective inhibitors in chronic pulmonary diseases (COPD and asthma). In addition, the combination of different PDE inhibitors is also described, thereby providing a more comprehensive overview of the up-to-date research findings.

In chapter 4, we describe a new method to monitor cAMP dynamics in the airway by combining Förster resonance energy transfer (FRET) and precision cut lung slices (PCLS). Using this novel setup, the effect of cigarette smoke on cAMP hydrolyzing enzymes, PDE3 and PDE4, is studied. We show that cigarette smoke upregulates the activity and expression of both PDE3 and PDE4, which in turn, induces changes of intracellular cAMP dynamics. Moreover, these findings from PCLS are further confirmed using human bronchial epithelial cells and airway smooth muscle cells transfected with the cAMP biosensor adenovirus, indicating the different strategies in epithelial cells and airway smooth muscle cells with cAMP hydrolysis. In addition, functional changes of PDE3 and PDE4 after cigarette smoke exposure are examined by airway contractility and ciliary beating frequency (CBF) test. This study provides

strong evidence of the underlying changes induced by cigarette smoke regarding PDE3 and PDE4, providing increased impetus towards the development of improved dual PDE3/4 inhibitors for clinical use in smoke-related airway diseases.

In chapter 5, we discuss the scientific and therapeutic value of a recently published research paper in American Journal of Respiratory Cell and Molecular Biology “PDE8 is expressed in human airway smooth muscle and selectively regulates cAMP signaling by β2-AR-AC6”. PDE8, an IBMX insensitive PDE, can be inhibited by PF-04957325, which is a highly potent and selective PDE8 inhibitor developed by Pfizer. The role of PDE8 was demonstrated by Johnstone et al. for the first time in human airway smooth muscle cells. We highlight the findings on the transcript, protein and functional presence of PDE8 and β2-AR-AC6-PDE8 signalosome which is expressed in caveolae.

In chapter 6, we review the recent knowledge about the role of cAMP scaffolds and oxidative stress in EMT process. How cAMP scaffolds (PDEs and AKAPs) and their distinguished signalosomes in different subcellular compartments may contribute to COPD is described here.

In chapter 7, we investigate the role of cAMP compartments during TGF-β1/ cigarette smoke induced EMT by modulating intracellular AKAPs. The contribution of PKA-AKAP complexes to EMT process is studied using the peptide st-Ht31, which inhibits the interaction between RII subunits of PKA and AKAP. Among more than 50 members, specific attention focuses on Ezrin, AKAP95 and Yotiao. The role of Ezrin, AKAP95 and Yotiao on EMT process is studied by small interfering RNA.

In chapter 8, the nasal and bronchial brushes and lung tissues from never-smokers, ex-smokers and current smokers are used to study the effect of cigarette smoke on PDE3 and PDE4 protein expression. In addition, human mRNA is isolated from bronchial and nasal brushings of never-smokers and healthy smokers.

In chapter 9, we provide a summary of our work, give future perspective of cAMP studies, and also describe the challenges.

1

composed of 11 families with a distinct substrate specificity, molecular structure and subcellular localization (Omori and Kotera, 2007; Zuo et al., 2019a). Each PDE family has at least one (e.g. Pde5a) and often multiple coding genes, resulting in the mammalian PDE superfamily being composed of more than 21 genes (Omori and Kotera, 2007; Page and Spina, 2012). Moreover, most PDE encoding genes have distinct promoters, and multiple transcriptional products which are generated by alternative splicing, resulting in nearly 100 different PDE messenger RNAs (Conti and Beavo, 2007; Otero et al., 2014). In addition, the communication between β2-adrenoreceptor, cAMP effectors, PDEs and other downstream targets are coordinated by A-kinase anchoring proteins (AKAPs) (shown in Fig 1) (Beene and Scott, 2007; Carnegie et al., 2009; Han et al., 2015; Poppinga et al., 2014). Members of the AKAPs family bind to the regulatory subunits of PKA and target PKA to discreet sites/macromolecular complexes, thereby playing a central role in the regulation of cAMP compartmentalization (Beene and Scott, 2007).

Scope of the thesis

The main objective of this thesis is to explore the role of compartmentalized cAMP signaling in the pathogenesis of COPD, focusing on PDE subfamilies and AKAPs. Using in vitro, ex vivo and in vivo approaches, we investigate the potential therapeutic targets in cAMP signaling pathways and link it with the current therapies available on the market.

In chapter 2, we provide an overview of Epac function and cAMP scaffolds in the heart and the lung. We highlight recent studies in heart and lung pertaining to cAMP compartmentalization, which provides more insights in understanding the role of cAMP scaffolds in different organs.

In chapter 3, we review the regulation of several PDEs (PDE3, PDE4, PDE5, PDE7 and PDE8) and demonstrate the roles of their selective inhibitors in chronic pulmonary diseases (COPD and asthma). In addition, the combination of different PDE inhibitors is also described, thereby providing a more comprehensive overview of the up-to-date research findings.

In chapter 4, we describe a new method to monitor cAMP dynamics in the airway by combining Förster resonance energy transfer (FRET) and precision cut lung slices (PCLS). Using this novel setup, the effect of cigarette smoke on cAMP hydrolyzing enzymes, PDE3 and PDE4, is studied. We show that cigarette smoke upregulates the activity and expression of both PDE3 and PDE4, which in turn, induces changes of intracellular cAMP dynamics. Moreover, these findings from PCLS are further confirmed using human bronchial epithelial cells and airway smooth muscle cells transfected with the cAMP biosensor adenovirus, indicating the different strategies in epithelial cells and airway smooth muscle cells with cAMP hydrolysis. In addition, functional changes of PDE3 and PDE4 after cigarette smoke exposure are examined by airway contractility and ciliary beating frequency (CBF) test. This study provides

strong evidence of the underlying changes induced by cigarette smoke regarding PDE3 and PDE4, providing increased impetus towards the development of improved dual PDE3/4 inhibitors for clinical use in smoke-related airway diseases.

In chapter 5, we discuss the scientific and therapeutic value of a recently published research paper in American Journal of Respiratory Cell and Molecular Biology “PDE8 is expressed in human airway smooth muscle and selectively regulates cAMP signaling by β2-AR-AC6”. PDE8, an IBMX insensitive PDE, can be inhibited by PF-04957325, which is a highly potent and selective PDE8 inhibitor developed by Pfizer. The role of PDE8 was demonstrated by Johnstone et al. for the first time in human airway smooth muscle cells. We highlight the findings on the transcript, protein and functional presence of PDE8 and β2-AR-AC6-PDE8 signalosome which is expressed in caveolae.

In chapter 6, we review the recent knowledge about the role of cAMP scaffolds and oxidative stress in EMT process. How cAMP scaffolds (PDEs and AKAPs) and their distinguished signalosomes in different subcellular compartments may contribute to COPD is described here.

In chapter 7, we investigate the role of cAMP compartments during TGF-β1/ cigarette smoke induced EMT by modulating intracellular AKAPs. The contribution of PKA-AKAP complexes to EMT process is studied using the peptide st-Ht31, which inhibits the interaction between RII subunits of PKA and AKAP. Among more than 50 members, specific attention focuses on Ezrin, AKAP95 and Yotiao. The role of Ezrin, AKAP95 and Yotiao on EMT process is studied by small interfering RNA.

In chapter 8, the nasal and bronchial brushes and lung tissues from never-smokers, ex-smokers and current smokers are used to study the effect of cigarette smoke on PDE3 and PDE4 protein expression. In addition, human mRNA is isolated from bronchial and nasal brushings of never-smokers and healthy smokers.

In chapter 9, we provide a summary of our work, give future perspective of cAMP studies, and also describe the challenges.

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Conti, M., Beavo, J., 2007. Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu. Rev. Biochem. 76, 481–511.

Dekkers, B.G.J., Racké, K., Schmidt, M., 2013. Distinct PKA and Epac compartmentalization in airway function and plasticity. Pharmacol. Ther. 137, 248–265.

Di Stefano, A., Capelli, A., Lusuardi, M., Balbo, P., Vecchio, C., Maestrelli, P., Mapp, C.E., Fabbri, L.M., Donner, C.F., Saetta, M., 1998. Severity of airflow limitation is associated with severity of airway inflammation in smokers. Am. J. Respir. Crit. Care Med. 158, 1277–1285.

Giembycz, M.A., Maurice, D.H., 2014. Cyclic nucleotide-based therapeutics for chronic obstructive pulmonary disease. Curr. Opin. Pharmacol., Respiratory • Musculoskeletal 16, 89–107.

Greenburg, G., Hay, E.D., 1986. Cytodifferentiation and tissue phenotype change during transformation of embryonic lens epithelium to mesenchyme-like cells in vitro. Dev. Biol. 115, 363–379.

Hackett, T.-L., Warner, S.M., Stefanowicz, D., Shaheen, F., Pechkovsky, D.V., Murray, L.A., Argentieri, R., Kicic, A., Stick, S.M., Bai, T.R., Knight, D.A., 2009. Induction of epithelial-mesenchymal transition in primary airway epithelial cells from patients with asthma by transforming growth factor-beta1. Am. J. Respir. Crit. Care Med. 180, 122–133.

Han, B., Poppinga, W.J., Schmidt, M., 2015. Scaffolding during the cell cycle by A-kinase anchoring proteins. Pflugers Arch. 467, 2401–2411.

Hayes, J.S., Brunton, L.L., Brown, J.H., Reese, J.B., Mayer, S.E., 1979. Hormonally specific expression of cardiac protein kinase activity. Proc. Natl. Acad. Sci. U. S. A. 76, 1570–1574.

Jolly, M.K., Ward, C., Eapen, M.S., Myers, S., Hallgren, O., Levine, H., Sohal, S.S., 2018. Epithelial-mesenchymal transition, a spectrum of states: Role in lung development, homeostasis, and disease. Dev. Dyn. Off. Publ. Am. Assoc. Anat. 247, 346–358.

Keatings, V.M., Collins, P.D., Scott, D.M., Barnes, P.J., 1996. Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am. J. Respir. Crit. Care Med. 153, 530–534.

Kirkham, P.A., Barnes, P.J., 2013. Oxidative Stress in COPD. Chest 144, 266–273.

Lamela, J., Vega, F., 2009. Immunologic aspects of chronic obstructive pulmonary disease. N. Engl. J. Med. 361, 1024.

Laudette, M., Zuo, H., Lezoualc’h, F., Schmidt, M., 2018. Epac Function and cAMP Scaffolds in the Heart and Lung. J. Cardiovasc. Dev. Dis. 5.

Maji, K.J., Dikshit, A.K., Arora, M., Deshpande, A., 2018. Estimating premature mortality attributable to PM2.5 exposure and benefit of air pollution control policies in China for 2020. Sci. Total Environ. 612, 683–693.

Meijer, M., Rijkers, G.T., van Overveld, F.J., 2013. Neutrophils and emerging targets for treatment in chronic obstructive pulmonary disease. Expert Rev. Clin. Immunol. 9, 1055–1068.

Nieto, M.A., 2011. The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu. Rev. Cell Dev. Biol. 27, 347–376.

Oldenburger, A., Maarsingh, H., Schmidt, M., 2012a. Multiple facets of cAMP signalling and physiological impact: cAMP compartmentalization in the lung. Pharm. Basel Switz. 5, 1291–1331.

Oldenburger, A., Roscioni, S.S., Jansen, E., Menzen, M.H., Halayko, A.J., Timens, W., Meurs, H., Maarsingh, H., Schmidt, M., 2012b. Anti-inflammatory role of the cAMP effectors Epac and PKA: implications in chronic obstructive pulmonary disease. PloS One 7, e31574.

Omori, K., Kotera, J., 2007. Overview of PDEs and their regulation. Circ. Res. 100, 309–327.

O’Shaughnessy, T.C., Ansari, T.W., Barnes, N.C., Jeffery, P.K., 1997. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am. J. Respir. Crit. Care Med. 155, 852–857.

Otero, C., Peñaloza, J.P., Rodas, P.I., Fernández-Ramires, R., Velasquez, L., Jung, J.E., 2014. Temporal and spatial regulation of cAMP signaling in disease: role of cyclic nucleotide phosphodiesterases. Fundam. Clin. Pharmacol. 28, 593–607.

Page, C.P., Spina, D., 2012. Selective PDE inhibitors as novel treatments for respiratory diseases. Curr. Opin. Pharmacol. 12, 275–286.

Pesci, A., Balbi, B., Majori, M., Cacciani, G., Bertacco, S., Alciato, P., Donner, C.F., 1998. Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease. Eur. Respir. J. 12, 380–386.

Poppinga, W.J., Muñoz-Llancao, P., González-Billault, C., Schmidt, M., 2014. A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases. Br. J. Pharmacol. 171, 5603–5623.

Rhyu, D.Y., Yang, Y., Ha, H., Lee, G.T., Song, J.S., Uh, S., Lee, H.B., 2005. Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J. Am. Soc. Nephrol. JASN 16, 667–675.

Roscioni, S.S., Dekkers, B.G.J., Prins, A.G., Menzen, M.H., Meurs, H., Schmidt, M., Maarsingh, H., 2011a. cAMP inhibits modulation of airway smooth muscle phenotype via the exchange protein activated by cAMP (Epac) and protein kinase A. Br. J. Pharmacol. 162, 193–209.

Roscioni, S.S., Prins, A.G., Elzinga, C.R.S., Menzen, M.H., Dekkers, B.G.J., Halayko, A.J., Meurs, H., Maarsingh, H., Schmidt, M., 2011b. Protein kinase A and the exchange protein directly activated by cAMP (Epac) modulate phenotype plasticity in human airway smooth muscle. Br. J. Pharmacol. 164, 958–969.

Rout-Pitt, N., Farrow, N., Parsons, D., Donnelley, M., 2018. Epithelial mesenchymal transition (EMT): a universal process in lung diseases with implications for cystic fibrosis pathophysiology. Respir. Res. 19, 136.

Sakuma, Y., 2017. Epithelial-to-mesenchymal transition and its role in EGFR-mutant lung adenocarcinoma and idiopathic pulmonary fibrosis. Pathol. Int. 67, 379–388.

Schmidt, M., Dekker, F.J., Maarsingh, H., 2013. Exchange Protein Directly Activated by cAMP (epac): A Multidomain cAMP Mediator in the Regulation of Diverse Biological Functions. Pharmacol. Rev. 65, 670–709.

Sohal, S.S., Reid, D., Soltani, A., Ward, C., Weston, S., Muller, H.K., Wood-Baker, R., Walters, E.H., 2010. Reticular basement membrane fragmentation and potential epithelial mesenchymal transition is exaggerated in the airways of smokers with chronic obstructive pulmonary disease. Respirology 15, 930– 938.

Tønnesen, P., 2013. Smoking cessation and COPD. Eur. Respir. Rev. 22, 37–43.

Vogelmeier, C.F., Criner, G.J., Martinez, F.J., Anzueto, A., Barnes, P.J., Bourbeau, J., Celli, B.R., Chen, R., Decramer, M., Fabbri, L.M., Frith, P., Halpin, D.M.G., López Varela, M.V., Nishimura, M., Roche, N., Rodriguez-Roisin, R., Sin, D.D., Singh, D., Stockley, R., Vestbo, J., Wedzicha, J.A., Agustí, A., 2017. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Lung Disease 2017 Report. GOLD Executive Summary. Am. J. Respir. Crit. Care Med. 195, 557–582. Wang, C., Xu, J., Yang, L., Xu, Y., Zhang, Xiangyan, Bai, C., Kang, J., Ran, P., Shen, H., Wen, F., Huang, K.,

Yao, W., Sun, T., Shan, G., Yang, T., Lin, Y., Wu, S., Zhu, J., Wang, R., Shi, Z., Zhao, J., Ye, X., Song, Y., Wang, Q., Zhou, Y., Ding, L., Yang, T., Chen, Y., Guo, Y., Xiao, F., Lu, Y., Peng, X.,

1

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Conti, M., Beavo, J., 2007. Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Annu. Rev. Biochem. 76, 481–511.

Dekkers, B.G.J., Racké, K., Schmidt, M., 2013. Distinct PKA and Epac compartmentalization in airway function and plasticity. Pharmacol. Ther. 137, 248–265.

Di Stefano, A., Capelli, A., Lusuardi, M., Balbo, P., Vecchio, C., Maestrelli, P., Mapp, C.E., Fabbri, L.M., Donner, C.F., Saetta, M., 1998. Severity of airflow limitation is associated with severity of airway inflammation in smokers. Am. J. Respir. Crit. Care Med. 158, 1277–1285.

Giembycz, M.A., Maurice, D.H., 2014. Cyclic nucleotide-based therapeutics for chronic obstructive pulmonary disease. Curr. Opin. Pharmacol., Respiratory • Musculoskeletal 16, 89–107.

Greenburg, G., Hay, E.D., 1986. Cytodifferentiation and tissue phenotype change during transformation of embryonic lens epithelium to mesenchyme-like cells in vitro. Dev. Biol. 115, 363–379.

Hackett, T.-L., Warner, S.M., Stefanowicz, D., Shaheen, F., Pechkovsky, D.V., Murray, L.A., Argentieri, R., Kicic, A., Stick, S.M., Bai, T.R., Knight, D.A., 2009. Induction of epithelial-mesenchymal transition in primary airway epithelial cells from patients with asthma by transforming growth factor-beta1. Am. J. Respir. Crit. Care Med. 180, 122–133.

Han, B., Poppinga, W.J., Schmidt, M., 2015. Scaffolding during the cell cycle by A-kinase anchoring proteins. Pflugers Arch. 467, 2401–2411.

Hayes, J.S., Brunton, L.L., Brown, J.H., Reese, J.B., Mayer, S.E., 1979. Hormonally specific expression of cardiac protein kinase activity. Proc. Natl. Acad. Sci. U. S. A. 76, 1570–1574.

Jolly, M.K., Ward, C., Eapen, M.S., Myers, S., Hallgren, O., Levine, H., Sohal, S.S., 2018. Epithelial-mesenchymal transition, a spectrum of states: Role in lung development, homeostasis, and disease. Dev. Dyn. Off. Publ. Am. Assoc. Anat. 247, 346–358.

Keatings, V.M., Collins, P.D., Scott, D.M., Barnes, P.J., 1996. Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am. J. Respir. Crit. Care Med. 153, 530–534.

Kirkham, P.A., Barnes, P.J., 2013. Oxidative Stress in COPD. Chest 144, 266–273.

Lamela, J., Vega, F., 2009. Immunologic aspects of chronic obstructive pulmonary disease. N. Engl. J. Med. 361, 1024.

Laudette, M., Zuo, H., Lezoualc’h, F., Schmidt, M., 2018. Epac Function and cAMP Scaffolds in the Heart and Lung. J. Cardiovasc. Dev. Dis. 5.

Maji, K.J., Dikshit, A.K., Arora, M., Deshpande, A., 2018. Estimating premature mortality attributable to PM2.5 exposure and benefit of air pollution control policies in China for 2020. Sci. Total Environ. 612, 683–693.

Meijer, M., Rijkers, G.T., van Overveld, F.J., 2013. Neutrophils and emerging targets for treatment in chronic obstructive pulmonary disease. Expert Rev. Clin. Immunol. 9, 1055–1068.

Nieto, M.A., 2011. The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu. Rev. Cell Dev. Biol. 27, 347–376.

Oldenburger, A., Maarsingh, H., Schmidt, M., 2012a. Multiple facets of cAMP signalling and physiological impact: cAMP compartmentalization in the lung. Pharm. Basel Switz. 5, 1291–1331.

Oldenburger, A., Roscioni, S.S., Jansen, E., Menzen, M.H., Halayko, A.J., Timens, W., Meurs, H., Maarsingh, H., Schmidt, M., 2012b. Anti-inflammatory role of the cAMP effectors Epac and PKA: implications in chronic obstructive pulmonary disease. PloS One 7, e31574.

Omori, K., Kotera, J., 2007. Overview of PDEs and their regulation. Circ. Res. 100, 309–327.

O’Shaughnessy, T.C., Ansari, T.W., Barnes, N.C., Jeffery, P.K., 1997. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am. J. Respir. Crit. Care Med. 155, 852–857.

Otero, C., Peñaloza, J.P., Rodas, P.I., Fernández-Ramires, R., Velasquez, L., Jung, J.E., 2014. Temporal and spatial regulation of cAMP signaling in disease: role of cyclic nucleotide phosphodiesterases. Fundam. Clin. Pharmacol. 28, 593–607.

Page, C.P., Spina, D., 2012. Selective PDE inhibitors as novel treatments for respiratory diseases. Curr. Opin. Pharmacol. 12, 275–286.

Pesci, A., Balbi, B., Majori, M., Cacciani, G., Bertacco, S., Alciato, P., Donner, C.F., 1998. Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease. Eur. Respir. J. 12, 380–386.

Poppinga, W.J., Muñoz-Llancao, P., González-Billault, C., Schmidt, M., 2014. A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases. Br. J. Pharmacol. 171, 5603–5623.

Rhyu, D.Y., Yang, Y., Ha, H., Lee, G.T., Song, J.S., Uh, S., Lee, H.B., 2005. Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J. Am. Soc. Nephrol. JASN 16, 667–675.

Roscioni, S.S., Dekkers, B.G.J., Prins, A.G., Menzen, M.H., Meurs, H., Schmidt, M., Maarsingh, H., 2011a. cAMP inhibits modulation of airway smooth muscle phenotype via the exchange protein activated by cAMP (Epac) and protein kinase A. Br. J. Pharmacol. 162, 193–209.

Roscioni, S.S., Prins, A.G., Elzinga, C.R.S., Menzen, M.H., Dekkers, B.G.J., Halayko, A.J., Meurs, H., Maarsingh, H., Schmidt, M., 2011b. Protein kinase A and the exchange protein directly activated by cAMP (Epac) modulate phenotype plasticity in human airway smooth muscle. Br. J. Pharmacol. 164, 958–969.

Rout-Pitt, N., Farrow, N., Parsons, D., Donnelley, M., 2018. Epithelial mesenchymal transition (EMT): a universal process in lung diseases with implications for cystic fibrosis pathophysiology. Respir. Res. 19, 136.

Sakuma, Y., 2017. Epithelial-to-mesenchymal transition and its role in EGFR-mutant lung adenocarcinoma and idiopathic pulmonary fibrosis. Pathol. Int. 67, 379–388.

Schmidt, M., Dekker, F.J., Maarsingh, H., 2013. Exchange Protein Directly Activated by cAMP (epac): A Multidomain cAMP Mediator in the Regulation of Diverse Biological Functions. Pharmacol. Rev. 65, 670–709.

Sohal, S.S., Reid, D., Soltani, A., Ward, C., Weston, S., Muller, H.K., Wood-Baker, R., Walters, E.H., 2010. Reticular basement membrane fragmentation and potential epithelial mesenchymal transition is exaggerated in the airways of smokers with chronic obstructive pulmonary disease. Respirology 15, 930– 938.

Tønnesen, P., 2013. Smoking cessation and COPD. Eur. Respir. Rev. 22, 37–43.

Vogelmeier, C.F., Criner, G.J., Martinez, F.J., Anzueto, A., Barnes, P.J., Bourbeau, J., Celli, B.R., Chen, R., Decramer, M., Fabbri, L.M., Frith, P., Halpin, D.M.G., López Varela, M.V., Nishimura, M., Roche, N., Rodriguez-Roisin, R., Sin, D.D., Singh, D., Stockley, R., Vestbo, J., Wedzicha, J.A., Agustí, A., 2017. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Lung Disease 2017 Report. GOLD Executive Summary. Am. J. Respir. Crit. Care Med. 195, 557–582. Wang, C., Xu, J., Yang, L., Xu, Y., Zhang, Xiangyan, Bai, C., Kang, J., Ran, P., Shen, H., Wen, F., Huang, K.,

Yao, W., Sun, T., Shan, G., Yang, T., Lin, Y., Wu, S., Zhu, J., Wang, R., Shi, Z., Zhao, J., Ye, X., Song, Y., Wang, Q., Zhou, Y., Ding, L., Yang, T., Chen, Y., Guo, Y., Xiao, F., Lu, Y., Peng, X.,

Zhang, B., Xiao, D., Chen, C.-S., Wang, Z., Zhang, H., Bu, X., Zhang, Xiaolei, An, L., Zhang, S., Cao, Z., Zhan, Q., Yang, Y., Cao, B., Dai, H., Liang, L., He, J., China Pulmonary Health Study Group, 2018. Prevalence and risk factors of chronic obstructive pulmonary disease in China (the China Pulmonary Health [CPH] study): a national cross-sectional study. Lancet Lond. Engl. 391, 1706–1717. Yang, Z.-C., Yi, M.-J., Ran, N., Wang, C., Fu, P., Feng, X.-Y., Xu, L., Qu, Z.-H., 2013. Transforming growth

factor-β1 induces bronchial epithelial cells to mesenchymal transition by activating the Snail pathway and promotes airway remodeling in asthma. Mol. Med. Rep. 8, 1663–1668.

Zuo, H., Cattani-Cavalieri, I., Musheshe, N., Nikolaev, V.O., Schmidt, M., 2019a. Phosphodiesterases as therapeutic targets for respiratory diseases. Pharmacol. Ther. 197: 225-242.

Zuo, H., Cattani-Cavalieri, I., Valença, S.S., Musheshe, N., Schmidt, M., 2019b. Function of cyclic AMP scaffolds in obstructive lung disease: Focus on epithelial-to-mesenchymal transition and oxidative stress. Br. J. Pharmacol.

2

Epac Function and cAMP Scaffolds

in the Heart and Lung

Marion Laudette

_{, Haoxiao Zuo}

_,

Frank Lezoualc’h

_{and Martina Schmidt}

1 Inserm UMR-1048, Institut des Maladies Métaboliques et Cardiovasculaires, Université Toulouse III,

31432 Toulouse, France;

2 Department of Molecular Pharmacology, University of Groningen, 9713AV Groningen, The Netherlands; 3 Groningen Research Institute for Asthma and COPD

(GRIAC), University Medical Center Groningen, University of Groningen, 9713AV Groningen,

The Netherlands

† These authors contributed equally to this work.

J Cardiovasc Dev Dis. 2018 Mar; 5(1): 9.

Z., Zhan, Q., Yang, Y., Cao, B., Dai, H., Liang, L., He, J., China Pulmonary Health Study Group, 2018. Prevalence and risk factors of chronic obstructive pulmonary disease in China (the China Pulmonary Health [CPH] study): a national cross-sectional study. Lancet Lond. Engl. 391, 1706–1717. Yang, Z.-C., Yi, M.-J., Ran, N., Wang, C., Fu, P., Feng, X.-Y., Xu, L., Qu, Z.-H., 2013. Transforming growth

factor-β1 induces bronchial epithelial cells to mesenchymal transition by activating the Snail pathway and promotes airway remodeling in asthma. Mol. Med. Rep. 8, 1663–1668.

Zuo, H., Cattani-Cavalieri, I., Musheshe, N., Nikolaev, V.O., Schmidt, M., 2019a. Phosphodiesterases as therapeutic targets for respiratory diseases. Pharmacol. Ther. 197: 225-242.

Zuo, H., Cattani-Cavalieri, I., Valença, S.S., Musheshe, N., Schmidt, M., 2019b. Function of cyclic AMP scaffolds in obstructive lung disease: Focus on epithelial-to-mesenchymal transition and oxidative stress. Br. J. Pharmacol.

Abstract

Evidence collected over the last ten years indicates that Epac and cAMP scaffold proteins play a critical role in integrating and transducing multiple signaling pathways at the basis of cardiac and lung physiopathology. Some of the deleterious effects of Epac, such as cardiomyocyte hypertrophy and arrhythmia, initially described in vitro, have been confirmed in genetically modified mice for Epac1 and Epac2. Similar recent findings have been collected in the lung. The following sections will describe how Epac and cAMP signalosomes in different subcellular compartments may contribute to cardiac and lung diseases.

Keywords

cAMP; Epac; compartmentalization; A-kinase anchoring proteins; phosphodiesterases

1. Introduction

In the current manuscript, we aim to highlight the most recent insights into signaling by one of the most ancient second messengers cyclic AMP (cAMP). We focus on novel aspects of cAMP scaffolds maintained by a diverse subset of proteins, among them receptors, exchange proteins, phosphodiesterases, and A-kinase anchoring proteins. We will start with the cardiac system and will then proceed with the lung.

2. Epac in Cardiac Disease

Cyclic AMP (cAMP) is one the most important second messengers in the heart because it regulates many physiological processes, such as cardiac contractility and relaxation. The β-adrenergic receptor (β-AR) belongs to the G protein-coupled receptor (GPCR) superfamily, and is essential for the adaptation of cardiac performance to physiological needs. Upon stimulation of β-AR by noradrenaline (released from cardiac sympathetic nervous endings) and circulating adrenaline, cAMP is produced and activates protein kinase A (PKA), which phosphorylates many of the components involved in the excitation-coupling mechanisms, such as L-type the calcium channel (LTCC), phospholamban (PLB), cardiac myosin binding protein C (cMyBPC), and the ryanodine receptor 2 (RyR2), to modulate their activity (Bers, 2008). Activation of LTCCs produces an inward Ca2+ current (ICa) that activates RyR2 through the mechanism known as Ca2+-induced Ca2+ release (CICR), which raises cytosolic Ca2+ concentration and activates contraction. Whereas PKA-dependent LTCC and RyR2 phosphorylation results in mobilization of Ca2+ available for contraction, PKA-mediated phosphorylation of phospholamban, a peptide inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase promotes increased Ca2+ reuptake in the SR, thereby removing Ca2+ from the cytoplasm and accounting for relaxation (Bers, 2008). In addition, binding of cAMP to hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that carry the pacemaker current, increases heart rate in response to a sympathetic stimulation (chronotropic effect). From the three β-adrenergic subtypes expressed in the mammalian heart, regulation of cardiac function is ascribed to the β1- and β2-adrenergic receptor subtypes (Berthouze et al., 2011).

Although acute stimulation of the β-AR pathway has beneficial effects on heart function, a sustained activation of β-AR contributes to the development of pathological cardiac remodeling by inducing ventricular hypertrophy, fibrosis, and ultimately, arrhythmia and heart failure (HF), one of the most prevalent causes of mortality globally (Bristow, 2011; El-Armouche and Eschenhagen, 2009; von Lueder and Krum, 2015). Toxic effects of sustained β-AR stimulation are consistent with the finding that in HF patients, elevated plasma catecholamine levels correlate with the degree of ventricular dysfunction and mortality (Cohn et al., 1984). However, β-blocker therapy in HF may appear counterintuitive, as catecholamines represent the main trigger of cardiac contractility and relaxation. Indeed, β-blockers restore the

2

Abstract

Evidence collected over the last ten years indicates that Epac and cAMP scaffold proteins play a critical role in integrating and transducing multiple signaling pathways at the basis of cardiac and lung physiopathology. Some of the deleterious effects of Epac, such as cardiomyocyte hypertrophy and arrhythmia, initially described in vitro, have been confirmed in genetically modified mice for Epac1 and Epac2. Similar recent findings have been collected in the lung. The following sections will describe how Epac and cAMP signalosomes in different subcellular compartments may contribute to cardiac and lung diseases.

Keywords

cAMP; Epac; compartmentalization; A-kinase anchoring proteins; phosphodiesterases

1. Introduction

In the current manuscript, we aim to highlight the most recent insights into signaling by one of the most ancient second messengers cyclic AMP (cAMP). We focus on novel aspects of cAMP scaffolds maintained by a diverse subset of proteins, among them receptors, exchange proteins, phosphodiesterases, and A-kinase anchoring proteins. We will start with the cardiac system and will then proceed with the lung.

2. Epac in Cardiac Disease

Cyclic AMP (cAMP) is one the most important second messengers in the heart because it regulates many physiological processes, such as cardiac contractility and relaxation. The β-adrenergic receptor (β-AR) belongs to the G protein-coupled receptor (GPCR) superfamily, and is essential for the adaptation of cardiac performance to physiological needs. Upon stimulation of β-AR by noradrenaline (released from cardiac sympathetic nervous endings) and circulating adrenaline, cAMP is produced and activates protein kinase A (PKA), which phosphorylates many of the components involved in the excitation-coupling mechanisms, such as L-type the calcium channel (LTCC), phospholamban (PLB), cardiac myosin binding protein C (cMyBPC), and the ryanodine receptor 2 (RyR2), to modulate their activity (Bers, 2008). Activation of LTCCs produces an inward Ca2+ current (ICa) that activates RyR2 through the mechanism known as Ca2+-induced Ca2+ release (CICR), which raises cytosolic Ca2+ concentration and activates contraction. Whereas PKA-dependent LTCC and RyR2 phosphorylation results in mobilization of Ca2+ available for contraction, PKA-mediated phosphorylation of phospholamban, a peptide inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase promotes increased Ca2+ reuptake in the SR, thereby removing Ca2+ from the cytoplasm and accounting for relaxation (Bers, 2008). In addition, binding of cAMP to hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that carry the pacemaker current, increases heart rate in response to a sympathetic stimulation (chronotropic effect). From the three β-adrenergic subtypes expressed in the mammalian heart, regulation of cardiac function is ascribed to the β1- and β2-adrenergic receptor subtypes (Berthouze et al., 2011).

Although acute stimulation of the β-AR pathway has beneficial effects on heart function, a sustained activation of β-AR contributes to the development of pathological cardiac remodeling by inducing ventricular hypertrophy, fibrosis, and ultimately, arrhythmia and heart failure (HF), one of the most prevalent causes of mortality globally (Bristow, 2011; El-Armouche and Eschenhagen, 2009; von Lueder and Krum, 2015). Toxic effects of sustained β-AR stimulation are consistent with the finding that in HF patients, elevated plasma catecholamine levels correlate with the degree of ventricular dysfunction and mortality (Cohn et al., 1984). However, β-blocker therapy in HF may appear counterintuitive, as catecholamines represent the main trigger of cardiac contractility and relaxation. Indeed, β-blockers restore the

adrenergic signaling system which is desensitized by high and chronic concentrations of catecholamines (Bristow, 2011). Thus, it is not so much to block the whole adrenergic signaling which seems important, but rather to modulate its different aspects. It is in this context that several research groups are interested in understanding the role of exchange proteins directly activated by cAMP (Epac) proteins in the development of cardiac arrhythmia and HF (El-Armouche and Eschenhagen, 2009).

Evidence collected over the last ten years indicates that Epac proteins play a critical role in integrating and transducing multiple signaling pathways at the basis of cardiac physiopathology. Some of the deleterious effects of Epac, such as cardiomyocyte hypertrophy and arrhythmia, initially described in vitro, have been confirmed in genetically modified mice for Epac1 and Epac2. The following sections will describe how Epac signalosomes in different subcellular compartments of the cardiomyocyte may contribute to cardiac disease.

2.1. Epac Signalosome in Pathological Cardiac Remodeling

Given the importance of the β-AR-cAMP pathway in cardiac pathophysiology, several studies aim to investigate the role of Epac proteins in the development of cardiac remodeling and HF. Remodeling pathological disorder comprises multiple attacks of which the best described are the modification of the geometry of the cardiac cavity associated with cardiomyocyte hypertrophy, fibrosis, and alterations of calcium handling and energy metabolism (Hill and Olson, 2008). In the long term, these changes affect cardiac contractility and favor progression of HF, a process predominantly relying on cardiac signaling in response to the β1-AR subtype.

Among the two Epac isoforms, Epac1 expression was found to be upregulated in various models of cardiac hypertrophy, such as chronic catecholamine infusion and pressure overload induced by thoracic aortic constriction, as well as in the end stages of human HF (Métrich et al., 2008; Ulucan et al., 2007). On the contrary, the anti-hypertrophic action of some hormones and microRNA, including the growth hormone-releasing hormone and microRNA-133, involves Epac1 inhibition (Castaldi et al., 2014; Gesmundo et al., 2017). A more direct evidence of Epac1’s role in the regulation of cardiac remodeling came from the observation that Epac1 overexpression, or its direct activation with the Epac1 preferential agonist, 8-pCPT-2-O-Me-cAMP (8-CPT), increased various markers of cardiomyocyte hypertrophy, such as protein synthesis and hypertrophic genes in primary ventricular myocytes (Métrich et al., 2008; Morel et al., 2005; Schwede et al., 2015). It is hypothesized that in the setting of cardiac remodeling, adaptive autophagy antagonizes Epac1-induced cardiac hypertrophy (Laurent et al., 2015). In vitro studies revealed that the pharmacological inhibition of Epac1 by a tetrahydroquinoline analogue, CE3F4, prevented the induction of cardiomyocyte hypertrophy markers in response to a prolonged β-AR stimulation in rat ventricular myocytes (Bisserier et al., 2014; Courilleau et al., 2012; Laurent et al., 2015). These findings indicate that Epac1 signaling may provide a novel means for the treatment of pathological cardiac

hypertrophy. It is worth mentioning that Epac1 has also been recently identified as a potential mediator of radiation-induced cardiomyocyte hypertrophy, suggesting that this cAMP-sensor is involved in the side effects of anticancer therapy (Monceau et al., 2014).

Compelling evidence indicates that Epac1 signalosome is highly compartmentalized and occurs in several micro subcellular compartments, such as the plasma membrane, sarcoplasm, and the nuclear/perinuclear region of cardiomyocytes (Cazorla et al., 2009; Lezoualc’h et al., 2016; Métrich et al., 2008; Pereira et al., 2015). A macromolecular complex containing the scaffolding protein β-arrestin, Epac1, and Ca2+/calmodulin-dependent protein kinase II (CaMKII), has been reported in the heart (Mangmool et al., 2010). Epac1 constitutively interacts with the β-arrestin in the cytoplasm under basal conditions. Stimulation of β1-AR, but not β2-AR, induces the recruitment of β-arrestin–Epac1 signaling complex at the plasma membrane, whereby it activates a pro-hypertrophic signaling cascade involving the small GTPases Rap2 and Ras, and CaMKII (Berthouze-Duquesnes et al., 2013). This acts as a trigger for histone deacetylase type 4 (HDAC4) nuclear export, which initiates a pro-hypertrophic gene program (Figure 1). Interestingly, Epac1 is prevented from undertaking similar signaling at the β2-AR, as the cAMP-hydrolyzing enzyme, phosphodiesterase (PDE)4D5, impedes the interaction of Epac1 with β-arrestin, and therefore its recruitment to activated β2-AR. Of particular importance, disruption of PDE4D5–β-arrestin complex formation with a cell-permeant peptide promotes binding of Epac1–β-arrestin to β2-AR and, consequently β2-AR signaling switches to a β1-AR-like pro-hypertrophic signaling to increase cardiac myocyte remodeling (Berthouze-Duquesnes et al., 2013). Taken together, these data provide evidence that Epac compartmentalization contributes to the functional differences between cardiac β-AR subtypes.

Besides its sarcolemma distribution, Epac1 is also concentrated in the nuclear/perinuclear region of cardiomyocytes, positioned well to regulate nuclear signaling (Métrich et al., 2008; Pereira et al., 2015). Specifically, it was shown that Epac1 is scaffolded at the nuclear envelope with phospholipase C (PLC)ε and muscle-specific A-kinase anchoring proteins (AKAPs) to regulate the hypertrophic gene program in primary cardiomyocytes (Dodge-Kafka et al., 2005; Lezoualc’h et al., 2016; Zhang et al., 2013) (Figure 1). Interestingly, a detailed analysis of Ca2+ mobilization in different microdomains demonstrated that Epac (probably Epac1) preferentially elevated Ca2+ in the nucleoplasm, correlating with the perinuclear/nuclear localization of Epac1 (Pereira et al., 2012). Additional in vitro studies showed that Epac1, via its downstream effector, the small G protein Rap2, activated PLC to promote the production of inositol 1,4,5-trisphosphate (IP3) (Métrich et al., 2010). Based on this finding, a working hypothesis has been proposed, whereby Epac1 can activate PLC, causing nuclear Ca2+ increase via perinuclear IP3 receptor (IP3-R), which results in the activation of Ca2+-dependent transcription factors involved in cardiac remodeling (Pereira et al., 2012; Ruiz-Hurtado et al., 2013)