University of Groningen
Compartmentalized cAMP Signaling in COPD
Zuo, Haoxiao
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Publication date: 2019
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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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General Discussion
Research on cyclic nucleotides was initiated in early 1953 by Earl Sutherland (Berthet et al., 1957). Over the last decades, it has been demonstrated that cAMP plays a crucial role including but not limited to the airway smooth muscle (ASM) contraction and proliferation, cell differentiation, apoptosis, cancer migration, secretion of inflammatory mediators, deposition of extracellular matrix, and the maintenance of the endothelial and epithelial barrier (Beavo and Brunton, 2002; Billington et al., 2013; Feinstein et al., 2012; Zhang et al., 2016).
Compartmentalization – meanwhile widely accepted as a key feature of cAMP signaling - allows extracellular signals to propagate into the cells along defined and specific pathways within cellular network (Baillie, 2009; Han et al., 2015; Poppinga et al., 2014). As shown in Fig. 1, stimulation of Gs protein-coupled receptors, such as
the β2-adrenoreceptors (β2-AR) and distinct prostanoid receptors, leads to the
activation of adenylyl cyclases (ACs) which catalyze the synthesis of cAMP from adenosine triphosphate. cAMP is able to exert such diverse signaling properties by activating multiple effectors, which include: protein kinase A (PKA) (Taylor et al., 1992), the exchange proteins directly activated by cAMP (Epacs) (de Rooij et al., 1998), cyclic nucleotide-gated ion channels (Biel and Michalakis, 2009; Kaupp and Seifert, 2002) and the most recently defined novel class of three-pass transmembrane popeye domain containing proteins which bind cAMP with a high affinity (Schindler and Brand, 2016). The intracellular concentration of cAMP is spatially and temporally controlled by phosphodiesterases (PDEs) – a superfamily of enzymes which hydrolyze cAMP and thereby terminate its signaling properties (Fig. 1)
(Omori and Kotera, 2007; Zuo et al., 2019a). A-kinase anchoring proteins (AKAPs) which are a group of structurally diverse proteins localized at specific subcellular sites play a critical role in maintaining subcellular cAMP compartmentalization by generation of spatially discrete signaling complexes that create local gradients of cAMP (Fig. 1) (Beene and Scott, 2007; Poppinga et al., 2014; Skroblin et al., 2010;
Zuo et al., 2019b).
The objective of this thesis was to investigate the role of cAMP compartmentalization in COPD, mainly focusing on phosphodiesterases (PDEs) and A-kinase anchoring proteins (AKAPs) during cigarette smoke (CS) exposure. The studies described in this thesis emphasized the importance of PDEs and AKAPs as potential drug targets in CS-induced COPD.
Visualization of cAMP dynamics in the airway
It is challenging to monitor intracellular cAMP levels and dynamics using standard biochemical techniques. Therefore, biophysical methods including Förster resonance energy transfer (FRET)-based biosensors have been developed to facilitate its real-time measurements. FRET allows visualization of cAMP fluctuations in living cells with high temporal and spatial resolution (Adams et al., 1991; DiPilato et al., 2004; Nikolaev et al., 2004; Sprenger et al., 2015; Violin et al., 2008). Limited studies have applied FRET to structural cells of the lung, including human airway epithelial cells (Schmid et al., 2006, 2015), smooth muscle cells (Billington and Hall, 2011) and
9
Research on cyclic nucleotides was initiated in early 1953 by Earl Sutherland (Berthet et al., 1957). Over the last decades, it has been demonstrated that cAMP plays a crucial role including but not limited to the airway smooth muscle (ASM) contraction and proliferation, cell differentiation, apoptosis, cancer migration, secretion of inflammatory mediators, deposition of extracellular matrix, and the maintenance of the endothelial and epithelial barrier (Beavo and Brunton, 2002; Billington et al., 2013; Feinstein et al., 2012; Zhang et al., 2016).
Compartmentalization – meanwhile widely accepted as a key feature of cAMP signaling - allows extracellular signals to propagate into the cells along defined and specific pathways within cellular network (Baillie, 2009; Han et al., 2015; Poppinga et al., 2014). As shown in Fig. 1, stimulation of Gs protein-coupled receptors, such as
the β2-adrenoreceptors (β2-AR) and distinct prostanoid receptors, leads to the
activation of adenylyl cyclases (ACs) which catalyze the synthesis of cAMP from adenosine triphosphate. cAMP is able to exert such diverse signaling properties by activating multiple effectors, which include: protein kinase A (PKA) (Taylor et al., 1992), the exchange proteins directly activated by cAMP (Epacs) (de Rooij et al., 1998), cyclic nucleotide-gated ion channels (Biel and Michalakis, 2009; Kaupp and Seifert, 2002) and the most recently defined novel class of three-pass transmembrane popeye domain containing proteins which bind cAMP with a high affinity (Schindler and Brand, 2016). The intracellular concentration of cAMP is spatially and temporally controlled by phosphodiesterases (PDEs) – a superfamily of enzymes which hydrolyze cAMP and thereby terminate its signaling properties (Fig. 1)
(Omori and Kotera, 2007; Zuo et al., 2019a). A-kinase anchoring proteins (AKAPs) which are a group of structurally diverse proteins localized at specific subcellular sites play a critical role in maintaining subcellular cAMP compartmentalization by generation of spatially discrete signaling complexes that create local gradients of cAMP (Fig. 1) (Beene and Scott, 2007; Poppinga et al., 2014; Skroblin et al., 2010;
Zuo et al., 2019b).
The objective of this thesis was to investigate the role of cAMP compartmentalization in COPD, mainly focusing on phosphodiesterases (PDEs) and A-kinase anchoring proteins (AKAPs) during cigarette smoke (CS) exposure. The studies described in this thesis emphasized the importance of PDEs and AKAPs as potential drug targets in CS-induced COPD.
Visualization of cAMP dynamics in the airway
It is challenging to monitor intracellular cAMP levels and dynamics using standard biochemical techniques. Therefore, biophysical methods including Förster resonance energy transfer (FRET)-based biosensors have been developed to facilitate its real-time measurements. FRET allows visualization of cAMP fluctuations in living cells with high temporal and spatial resolution (Adams et al., 1991; DiPilato et al., 2004; Nikolaev et al., 2004; Sprenger et al., 2015; Violin et al., 2008). Limited studies have applied FRET to structural cells of the lung, including human airway epithelial cells (Schmid et al., 2006, 2015), smooth muscle cells (Billington and Hall, 2011) and
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endothelial cells (Yañez-Mó et al., 2008). However, real-time monitoring of cAMP levels in the lung tissue using FRET-based biosensors has not been reported so far. Precision cut lung slices (PCLS), which has been recognized as a reliable model to study airway responsiveness and drug toxicity (Schlepütz et al., 2012), retain the complex micro-composition and environment of the airways. In Chapter 4, we report
that PCLS from ubiquitously expressing transgenic Epac1-camps FRET reporter mice represent a useful model to monitor airway cAMP levels in real time, indicating that the combination between FRET measurements and PCLS offers the unique opportunity to study the airway as a whole structural unit (Zuo et al., 2018). Moreover, using this novel approach to monitor cAMP, it could be shown that ex vivo CS extract exposure largely mimics cellular cAMP dynamics with distinct functional responses in lung preparations from live mice pre-exposed to CS (Chapter 4).
Figure 1. Schematic overview of cyclic nucleotide compartmentalization in the lung. PDEs dynamically control cAMP and cGMP signals in different subcellular microdomains. Consequently, the activities of downstream effectors, such as PKA, Epacs and PKG, are modulated. PDEs are presented in a highly spatio-temporal dynamics, meaning individual PDEs are most likely to be recruited to specific locations at specific time points based on different stimulations/activations. A-kinase anchoring proteins (AKAPs) are a group of scaffolding proteins with the ability to associate with PKA via a short α-helical structure. It is known that some PDEs and AKAPs are highly expressed, for instance, in mitochondria (AKAP1) or at the plasma membrane (AKAP5 and AKAP12) (Cong et al., 2001; Merrill & Strack, 2014; Tao & Malbon, 2008). However, the molecular link between AKAPs and PDEs has not yet been studied in specific compartments in the lung. PDEs, phosphodiesterases; PKA, cAMP-dependent protein kinase; Epacs, exchange proteins directly activated by cAMP; PKG, cGMP-dependent protein kinase G; AKAPs, A-kinase anchoring proteins; EP, E prostanoid receptors; β2-AR, β2-adrenoceptor; AC,
adenylyl cyclase; pGC, particulate guanylate cyclase; sGC, soluble guanylyl cyclase; NO, nitric oxide; IP3R, inositol trisphosphate receptor; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase.
The impact of CS exposure on the function of phosphodiesterases
The superfamily of PDEs is composed of 11 families with a distinct substrate specificity, molecular structure and subcellular localization (Zuo et al., 2019a).
Chapter 3 summarizes the regulation of several PDEs (PDE3, PDE4, PDE5, PDE7
and PDE8) and the roles of selective inhibitors in chronic pulmonary diseases (COPD and asthma) (Fig. 1). In addition, the combination of different PDE inhibitors is
described as well, thereby providing a more comprehensive review of up-to-date research. In Chapter 4, the effects of CS on airway-specific regulation of PDE3 and
PDE4 are elucidated. Using acute CS exposure in vivo and ex vivo models, we demonstrate that CS increases the expression and activity of PDE4. This upregulation correlates an increase in both PDE4D mRNA and protein. Our findings are in line with previous reports that the activity of PDE4 is increased in prenatal cigarette exposed mice (Singh et al., 2009, 2003). Also, an increase in PDE4A and PDE4B was observed in CS exposure models (in vivo, ex vivo), strongly implicating that PDE4 subtypes may be effective drug targets to, for example, improve cilia motility of ciliated cells (Milara et al., 2012), inhibit proinflammatory cytokines secretion (Ariga et al., 2004; Jin and Conti, 2002; Ma et al., 2014) and diminish cell proliferation (Fig. 2) (Selige et al., 2011). Later on, to test if cAMP changes induced
by upregulation of PDE4 expression are correlated with physiological responses of epithelial cells, ciliary beating frequency (CBF) is used to be indicative for epithelial function. In Chapter 4, we find that inhibition of PDE4 fully reverses CBF
downregulation induced by ex vivo CS extract exposure, which intriguingly matches completely previous studies (Milara et al., 2012). Similar to PDE4, we show that PDE3 activity but not expression is upregulated by exposure to CS. It is widely accepted that PDE3 plays a crucial role in modulating airway contractility. Therefore, the potential relaxation of PDE3 and PDE4 inhibitors is measured. Intriguingly, only airway relaxation to cilostamide is increased in ex vivo PCLS exposed to CS extract (Chapter 4). Thus, our findings indicate that whereas both PDE3 and PDE4 are
involved in CS induced cAMP dynamics, the physiological outcomes are rather distinct. Currently, the selective PDE4 inhibitor roflumilast is used as an add-on treatment for patients with severe COPD associated with bronchitis and a history of frequent exacerbations, however unwanted side effects including nausea and vomiting still limit the oral administration of PDE4 inhibitors (Giembycz and Maurice, 2014; Vogelmeier et al., 2017). Our findings in Chapter 4, which are consistent with
previous studies, highlight a better therapeutic benefit of the combined inhibition of PDE3 and PDE4 compared to inhibition of PDE3 or PDE4 alone (BinMahfouz et al., 2015; Giembycz and Newton, 2011; Milara et al., 2011).
Chapter 5 demonstrates the transcript, protein and functional presence of PDE8 in
human ASM cells. PDE8, as another cAMP-specific PDE, exhibits a higher-affinity and lower Km (≈0.04 - 0.15 µM) for cAMP compared to other PDE isoforms, thus
acting as a potential drug target to shape low-level intracellular cAMP signals (Fisher, Smith, Pillar, St Denis, & Cheng, 1998; Hayashi et al., 1998; Soderling, Bayuga, & Beavo, 1998; Vang et al., 2010; Yan, Wang, Cai, & Ke, 2009). Most intriguingly, as
9
endothelial cells (Yañez-Mó et al., 2008). However, real-time monitoring of cAMP levels in the lung tissue using FRET-based biosensors has not been reported so far. Precision cut lung slices (PCLS), which has been recognized as a reliable model to study airway responsiveness and drug toxicity (Schlepütz et al., 2012), retain the complex micro-composition and environment of the airways. In Chapter 4, we report
that PCLS from ubiquitously expressing transgenic Epac1-camps FRET reporter mice represent a useful model to monitor airway cAMP levels in real time, indicating that the combination between FRET measurements and PCLS offers the unique opportunity to study the airway as a whole structural unit (Zuo et al., 2018). Moreover, using this novel approach to monitor cAMP, it could be shown that ex vivo CS extract exposure largely mimics cellular cAMP dynamics with distinct functional responses in lung preparations from live mice pre-exposed to CS (Chapter 4).
Figure 1. Schematic overview of cyclic nucleotide compartmentalization in the lung. PDEs dynamically control cAMP and cGMP signals in different subcellular microdomains. Consequently, the activities of downstream effectors, such as PKA, Epacs and PKG, are modulated. PDEs are presented in a highly spatio-temporal dynamics, meaning individual PDEs are most likely to be recruited to specific locations at specific time points based on different stimulations/activations. A-kinase anchoring proteins (AKAPs) are a group of scaffolding proteins with the ability to associate with PKA via a short α-helical structure. It is known that some PDEs and AKAPs are highly expressed, for instance, in mitochondria (AKAP1) or at the plasma membrane (AKAP5 and AKAP12) (Cong et al., 2001; Merrill & Strack, 2014; Tao & Malbon, 2008). However, the molecular link between AKAPs and PDEs has not yet been studied in specific compartments in the lung. PDEs, phosphodiesterases; PKA, cAMP-dependent protein kinase; Epacs, exchange proteins directly activated by cAMP; PKG, cGMP-dependent protein kinase G; AKAPs, A-kinase anchoring proteins; EP, E prostanoid receptors; β2-AR, β2-adrenoceptor; AC,
adenylyl cyclase; pGC, particulate guanylate cyclase; sGC, soluble guanylyl cyclase; NO, nitric oxide; IP3R, inositol trisphosphate receptor; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase.
The impact of CS exposure on the function of phosphodiesterases
The superfamily of PDEs is composed of 11 families with a distinct substrate specificity, molecular structure and subcellular localization (Zuo et al., 2019a).
Chapter 3 summarizes the regulation of several PDEs (PDE3, PDE4, PDE5, PDE7
and PDE8) and the roles of selective inhibitors in chronic pulmonary diseases (COPD and asthma) (Fig. 1). In addition, the combination of different PDE inhibitors is
described as well, thereby providing a more comprehensive review of up-to-date research. In Chapter 4, the effects of CS on airway-specific regulation of PDE3 and
PDE4 are elucidated. Using acute CS exposure in vivo and ex vivo models, we demonstrate that CS increases the expression and activity of PDE4. This upregulation correlates an increase in both PDE4D mRNA and protein. Our findings are in line with previous reports that the activity of PDE4 is increased in prenatal cigarette exposed mice (Singh et al., 2009, 2003). Also, an increase in PDE4A and PDE4B was observed in CS exposure models (in vivo, ex vivo), strongly implicating that PDE4 subtypes may be effective drug targets to, for example, improve cilia motility of ciliated cells (Milara et al., 2012), inhibit proinflammatory cytokines secretion (Ariga et al., 2004; Jin and Conti, 2002; Ma et al., 2014) and diminish cell proliferation (Fig. 2) (Selige et al., 2011). Later on, to test if cAMP changes induced
by upregulation of PDE4 expression are correlated with physiological responses of epithelial cells, ciliary beating frequency (CBF) is used to be indicative for epithelial function. In Chapter 4, we find that inhibition of PDE4 fully reverses CBF
downregulation induced by ex vivo CS extract exposure, which intriguingly matches completely previous studies (Milara et al., 2012). Similar to PDE4, we show that PDE3 activity but not expression is upregulated by exposure to CS. It is widely accepted that PDE3 plays a crucial role in modulating airway contractility. Therefore, the potential relaxation of PDE3 and PDE4 inhibitors is measured. Intriguingly, only airway relaxation to cilostamide is increased in ex vivo PCLS exposed to CS extract (Chapter 4). Thus, our findings indicate that whereas both PDE3 and PDE4 are
involved in CS induced cAMP dynamics, the physiological outcomes are rather distinct. Currently, the selective PDE4 inhibitor roflumilast is used as an add-on treatment for patients with severe COPD associated with bronchitis and a history of frequent exacerbations, however unwanted side effects including nausea and vomiting still limit the oral administration of PDE4 inhibitors (Giembycz and Maurice, 2014; Vogelmeier et al., 2017). Our findings in Chapter 4, which are consistent with
previous studies, highlight a better therapeutic benefit of the combined inhibition of PDE3 and PDE4 compared to inhibition of PDE3 or PDE4 alone (BinMahfouz et al., 2015; Giembycz and Newton, 2011; Milara et al., 2011).
Chapter 5 demonstrates the transcript, protein and functional presence of PDE8 in
human ASM cells. PDE8, as another cAMP-specific PDE, exhibits a higher-affinity and lower Km (≈0.04 - 0.15 µM) for cAMP compared to other PDE isoforms, thus
acting as a potential drug target to shape low-level intracellular cAMP signals (Fisher, Smith, Pillar, St Denis, & Cheng, 1998; Hayashi et al., 1998; Soderling, Bayuga, & Beavo, 1998; Vang et al., 2010; Yan, Wang, Cai, & Ke, 2009). Most intriguingly, as
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Chapter 9
shown in Fig. 1, it reveals that PDE8 inhibition selectively increases cAMP levels
which is generated by β2-AR, but has no effect when Prostaglandin E2 receptor
(EP)2 or EP4 are activated, indicating a functional β2-AR-AC6-PDE8 signalosome
expresses in caveolae of ASM cells (Johnstone et al., 2018). Additionally, Johnstone and colleagues demonstrate for the first time that inhibition of PDE8, together with β2
-AR stimulation by isoproterenol, profoundly reduces the serum-induced human ASM cells proliferation compared to isoproterenol alone (Johnstone et al., 2018), thereby indicating a potential pharmaceutical benefit of PDE8 in ASM cells. In Chapter 8, we
find that the gene expression of PDE8A in basal epithelium and PDE8A and PDE8B in bronchial epithelium are downregulated by chronic cigarette smoke exposure. Therefore, the effect of PDE8 in COPD, especially regarding the cigarette smoke exposure, needs more investigation in the future.
Figure 2. The effect of cigarette smoke (CS) on the cAMP compartmentalization in the airway structural cells. CS exposure upregulates expression and activity of both PDE3 and PDE4, which regulate real-time cAMP dynamics. Also, CS exposure stimulates TGF-β1 release, which activates epithelial-to-mesenchymal transition (EMT) process. A-kinase anchoring proteins (AKAPs), especially Ezrin, AKAP95 and Yotiao, are able to modulate the TGF-β1/CS-induced EMT. Additionally, chronic CS exposure alters the gene and protein expressions of PDEs in the nasal/ bronchial brushings and also in the whole lung tissue.
Epithelial-to-mesenchymal transition (EMT), which allows polarized epithelial cells to undergo multiple biochemical changes and gradually gain mesenchymal cell phenotypes, is also observed during the progression of COPD (Bartis et al., 2014; Jansen et al., 2018; Jolly et al., 2017; Nieto, 2011). In Chapter 7, we show that
pre-incubation of in human bronchial epithelial BEAS-2B cells with the PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram before addition of TGF-β1 is able to significantly diminish the protein upregulation of mesenchymal marker collagen Ӏ,
indicating that cAMP stimulation via PDE3 and PDE4 inhibition is able to partly inhibit TGF-β1-induced EMT. And this finding is also in line with previous observations from Kolosionek et al. that PDE4 inhibition reverses TGF-β1-induced EMT in alveolar type 2 epithelial A549 cells (Kolosionek et al., 2009).
Chapter 8 aims to answer if the profiles of PDEs were changed in human epithelial
cells which were obtained from nasal and bronchial brushes and in the whole lung tissues after CS exposure (Fig. 2). We show that the gene expression of PDE1B,
PDE4A, PDE7A, and PDE8A is significantly decreased in the nasal epithelium of current smokers compared to never-smokers, whereas PDE9A and PDE10A are significantly increased. In bronchial epithelium from current smokers the gene expression of PDE1A, PDE3A, PDE4D, PDE5A, PDE6D, PDE7A, PDE7B, PDE8A, PDE8B, and PDE11A are significant downregulated. PDE6A, PDE6B and PDE10A gene expression is upregulated in the current smokers compared to never-smokers. In lung tissue, only 4 genes are changed, with a decrease of PDE1A and PDE11A and an increase of PDE4D and PDE6A in current smokers versus never-smokers. In the protein expression, PDE3A tends to decrease in current smokers compared to never-smokers and ex-smokers and PDE4D tends to be increased in ex-smoker and current smokers compared to never-smokers. Thus, the data indicate that PDE superfamily represent promising targets for further development of precise and effective therapeutics in COPD.
The impact of AKAPs in cAMP compartmentalization in EMT
Epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells gradually lose their epithelial phenotype and undergo transition to mesenchymal typical characteristics (Kalluri and Neilson, 2003; Kim et al., 2006; Zeisberg and Neilson, 2009). It has been reported that EMT is observed on the reticular basement membrane in large airways from endobronchial biopsies of smokers, and the findings positively correlated with the subjects’ smoking history (Sohal et al., 2010, 2010; Wang et al., 2013). AKAPs are able to 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; Wong and Scott, 2004). In this thesis, we investigated the role of AKAPs in TGF-β1/ CS-induced EMT. In Chapter 7, the collagen I upregulation induced by TGF-β1 is
diminished upon disruption of AKAP-PKA interactions by the dominant-interfering peptide st-Ht31. Gene and protein expressions of Ezrin, Yotiao and AKAP95 are significantly changed by TGF-β1, indicating that Ezrin, Yotiao and AKAP95 are involved in TGF-β1-induced EMT. Further investigations specifically focusing on Ezrin, AKAP95 and Yotiao show that knockdown of Ezrin, AKAP95 or Yotiao individually using targeted siRNA decreases TGF-β1-induced collagen Ӏ upregulation. We achieved a more pronounced inhibition of collagen Ӏ upregulation by a simultaneous reduction of Ezrin, AKAP95 and Yotiao expression. Importantly, focusing on the functional outcome we show that the combined silencing of Ezrin, AKAP95 and Yotiao inhibited TGF-β1-induced cell migration, measured by wound
9
shown in Fig. 1, it reveals that PDE8 inhibition selectively increases cAMP levels
which is generated by β2-AR, but has no effect when Prostaglandin E2 receptor
(EP)2 or EP4 are activated, indicating a functional β2-AR-AC6-PDE8 signalosome
expresses in caveolae of ASM cells (Johnstone et al., 2018). Additionally, Johnstone and colleagues demonstrate for the first time that inhibition of PDE8, together with β2
-AR stimulation by isoproterenol, profoundly reduces the serum-induced human ASM cells proliferation compared to isoproterenol alone (Johnstone et al., 2018), thereby indicating a potential pharmaceutical benefit of PDE8 in ASM cells. In Chapter 8, we
find that the gene expression of PDE8A in basal epithelium and PDE8A and PDE8B in bronchial epithelium are downregulated by chronic cigarette smoke exposure. Therefore, the effect of PDE8 in COPD, especially regarding the cigarette smoke exposure, needs more investigation in the future.
Figure 2. The effect of cigarette smoke (CS) on the cAMP compartmentalization in the airway structural cells. CS exposure upregulates expression and activity of both PDE3 and PDE4, which regulate real-time cAMP dynamics. Also, CS exposure stimulates TGF-β1 release, which activates epithelial-to-mesenchymal transition (EMT) process. A-kinase anchoring proteins (AKAPs), especially Ezrin, AKAP95 and Yotiao, are able to modulate the TGF-β1/CS-induced EMT. Additionally, chronic CS exposure alters the gene and protein expressions of PDEs in the nasal/ bronchial brushings and also in the whole lung tissue.
Epithelial-to-mesenchymal transition (EMT), which allows polarized epithelial cells to undergo multiple biochemical changes and gradually gain mesenchymal cell phenotypes, is also observed during the progression of COPD (Bartis et al., 2014; Jansen et al., 2018; Jolly et al., 2017; Nieto, 2011). In Chapter 7, we show that
pre-incubation of in human bronchial epithelial BEAS-2B cells with the PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram before addition of TGF-β1 is able to significantly diminish the protein upregulation of mesenchymal marker collagen Ӏ,
indicating that cAMP stimulation via PDE3 and PDE4 inhibition is able to partly inhibit TGF-β1-induced EMT. And this finding is also in line with previous observations from Kolosionek et al. that PDE4 inhibition reverses TGF-β1-induced EMT in alveolar type 2 epithelial A549 cells (Kolosionek et al., 2009).
Chapter 8 aims to answer if the profiles of PDEs were changed in human epithelial
cells which were obtained from nasal and bronchial brushes and in the whole lung tissues after CS exposure (Fig. 2). We show that the gene expression of PDE1B,
PDE4A, PDE7A, and PDE8A is significantly decreased in the nasal epithelium of current smokers compared to never-smokers, whereas PDE9A and PDE10A are significantly increased. In bronchial epithelium from current smokers the gene expression of PDE1A, PDE3A, PDE4D, PDE5A, PDE6D, PDE7A, PDE7B, PDE8A, PDE8B, and PDE11A are significant downregulated. PDE6A, PDE6B and PDE10A gene expression is upregulated in the current smokers compared to never-smokers. In lung tissue, only 4 genes are changed, with a decrease of PDE1A and PDE11A and an increase of PDE4D and PDE6A in current smokers versus never-smokers. In the protein expression, PDE3A tends to decrease in current smokers compared to never-smokers and ex-smokers and PDE4D tends to be increased in ex-smoker and current smokers compared to never-smokers. Thus, the data indicate that PDE superfamily represent promising targets for further development of precise and effective therapeutics in COPD.
The impact of AKAPs in cAMP compartmentalization in EMT
Epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells gradually lose their epithelial phenotype and undergo transition to mesenchymal typical characteristics (Kalluri and Neilson, 2003; Kim et al., 2006; Zeisberg and Neilson, 2009). It has been reported that EMT is observed on the reticular basement membrane in large airways from endobronchial biopsies of smokers, and the findings positively correlated with the subjects’ smoking history (Sohal et al., 2010, 2010; Wang et al., 2013). AKAPs are able to 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; Wong and Scott, 2004). In this thesis, we investigated the role of AKAPs in TGF-β1/ CS-induced EMT. In Chapter 7, the collagen I upregulation induced by TGF-β1 is
diminished upon disruption of AKAP-PKA interactions by the dominant-interfering peptide st-Ht31. Gene and protein expressions of Ezrin, Yotiao and AKAP95 are significantly changed by TGF-β1, indicating that Ezrin, Yotiao and AKAP95 are involved in TGF-β1-induced EMT. Further investigations specifically focusing on Ezrin, AKAP95 and Yotiao show that knockdown of Ezrin, AKAP95 or Yotiao individually using targeted siRNA decreases TGF-β1-induced collagen Ӏ upregulation. We achieved a more pronounced inhibition of collagen Ӏ upregulation by a simultaneous reduction of Ezrin, AKAP95 and Yotiao expression. Importantly, focusing on the functional outcome we show that the combined silencing of Ezrin, AKAP95 and Yotiao inhibited TGF-β1-induced cell migration, measured by wound
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healing assay, xCELLigence and Incucyte (Chapter 7). In line with our findings,
several research groups have shown that Ezrin overexpression was associated with enhanced tumor aggressiveness, while knockdown of Ezrin expression reduced the proliferation, migration, and invasion of cancer cells (Huang et al., 2010; Li et al., 2008, 2012; Saito et al., 2013). AKAP95, also known as AKAP8, is an AKAP that has been primarily identified to reside in the nucleus, which leaves no surprise that AKAP95 is involved in DNA replication and the expression levels of several proteins that regulate the cell cycle (Han et al., 2015; Skroblin et al., 2010). In Chapter 7, we
show that knockdown of AKAP95 decreases TGF-β1-induced collagen Ӏ production. This may be explained by the fact that silencing of AKAP95 inhibits TGF-β1-induced cell proliferation in BEAS-2B cells. In addition, it has been demonstrated previously that knockdown Yotiao by short hairpin RNA inhibits tumor growth in mice, which is partly due to the fact that Yotiao knockdown induces an increase of epithelial marker E-cadherin and a decrease of mesenchymal markers N-cadherin and vimentin (Hu et al., 2016). Thus, Ezrin, AKAP95 and Yotiao are potential drug targets to inhibit TGF-β1/ CS-induced cell migration. Additionally, in Chapter 7, co-silencing of Ezrin, AKAP95 and Yotiao is associated with β2-AR but not PDE3 or PDE4 in decreasing
TGF-β1-induced collagen Ӏ upregulation, indicating that Ezrin, AKAP95 and Yotiao could have an add-on effect on β2-agonist. Also, as one of the most important risk
factors for COPD, CS exposure is able to activate TGF-β1 signaling, inducing EMT process (Fig. 2).
Future perspective
The studies described in this thesis show that cAMP compartmentalization, mainly focusing on PDEs and AKAPs, plays a vital role in the pathophysiological processes of COPD. cAMP, as one of the most ubiquitous second messengers, controls a wide range of physiological functions by modulating signaling cascades in a spatio-temporal manner (Musheshe et al., 2018b). Therefore, comprehensive understanding of the fluctuations (generation and degradation) of cAMP and its potential functions within certain compartments will most likely help in the screening of novel pharmaceutical targets which have higher efficiency and less side effects (shown in Figure 1). Recently, it has been demonstrated by Johnstone and colleagues that β2
-AR/-AC6/-PDE8 are mainly expressed in caveolae of human ASM cells, emphasizing the microdomain-specific cAMP modulation and pointing at the future direction of studies in cAMP compartmentalization in the lung. However, of note is that monitoring intracellular cAMP dynamics using standard biochemical methods is extremely difficult (Sprenger and Nikolaev, 2013). So far, several FRET-based biosensors have been developed to achieve real-time visualization of cAMP with high spatial and temporal resolutions targeting specific subcellular microdomains, including but not limited to sodium-potassium ATPase (Bastug-Özel et al., 2018), the sarcoplasmic/endoplasmic reticulum calcium ATPase (Sprenger et al., 2015), AKAP79 (Musheshe et al., 2018a) and mitochondria (Monterisi et al., 2017) in cardiovascular studies. Therefore, designing and using microdomain-specific targeted
cAMP biosensors to study the local cAMP dynamics will be more helpful to reveal microdomain-specific cAMP dynamics in the lung.
In addition, there are no reports using cGMP FRET biosensors to investigate the intracellular cGMP levels in either lung structural cells or tissues so far. Thus, it is conceivable that monitoring microdomain-specific intracellular cAMP and cGMP levels and more importantly, their crosstalk modulated by PDE2 and PDE3, will provide more evidence to help design novel drugs targeting cyclic nucleotides with higher efficiency and with fewer side effects.
Moreover, the compartmentalized cAMP signaling may also offer answers to yet unresolved questions underlying the distinct stages of the EMT process that is linked to a diverse subset of lung responses, including pulmonary fibrosis (Jolly et al., 2018). Recent evidence also indicates that cAMP scaffolds maintained by a diverse subset of receptors, PDEs, PKA, Epac and members of the AKAP superfamily bear the potential to target distinct aspects of the EMT process, with the EMT process being closely related to factors such as TGF-β1, tumor necrosis factor alpha and/or IL-13 (Chen et al., 2014; Hu et al., 2016; Jansen et al., 2016; Jia et al., 2018; Milara et al., 2014). To date only a very limited number of drugs are available to target lung fibrosis, therefore future studies with a special focus on the distinct role of cAMP compartmentalization will new therapeutic targets and hence novel drugs in the treatment of lung fibrosis (Gourdie et al., 2016; Kalluri, 2016; Mora et al., 2017).
Main conclusions
We show that FRET which allows real time monitoring of cAMP in intact cells and tissues could be combined with precision cut lung slices (PCLS) which mimic the airway micro-environment, offering the unique opportunity to study the airway as a whole structural unit (Chapter 4).
CS causes an increase in capacity for PDE3 and PDE4 activities in mouse PLCS and cultured human ASM and epithelial cells that likely underpins alterations of intracellular cAMP levels resulting from CS exposure (Chapter 4). CS alters the PDE
expression profile, increasing PDE4 mRNA, protein and activity, whereas though the activity of PDE3 is increased this isoform is refractory to CS-induced change in expression (Chapter 4).
CS extract exposure alters the PDE activity profile in a cell type specific manner, with cilostamide sensitive PDE3 activity primarily increased in human ASM cells, whereas rolipram-sensitive PDE4 activity is increased in both human ASM and epithelial cells (Chapter 4). In ex vivo PCLS exposed to CS extract, rolipram reversed
downregulation of ciliary beating frequency, whereas only cilostamide significantly increased airway relaxation of methacholine pre-contracted airways.
9
healing assay, xCELLigence and Incucyte (Chapter 7). In line with our findings,
several research groups have shown that Ezrin overexpression was associated with enhanced tumor aggressiveness, while knockdown of Ezrin expression reduced the proliferation, migration, and invasion of cancer cells (Huang et al., 2010; Li et al., 2008, 2012; Saito et al., 2013). AKAP95, also known as AKAP8, is an AKAP that has been primarily identified to reside in the nucleus, which leaves no surprise that AKAP95 is involved in DNA replication and the expression levels of several proteins that regulate the cell cycle (Han et al., 2015; Skroblin et al., 2010). In Chapter 7, we
show that knockdown of AKAP95 decreases TGF-β1-induced collagen Ӏ production. This may be explained by the fact that silencing of AKAP95 inhibits TGF-β1-induced cell proliferation in BEAS-2B cells. In addition, it has been demonstrated previously that knockdown Yotiao by short hairpin RNA inhibits tumor growth in mice, which is partly due to the fact that Yotiao knockdown induces an increase of epithelial marker E-cadherin and a decrease of mesenchymal markers N-cadherin and vimentin (Hu et al., 2016). Thus, Ezrin, AKAP95 and Yotiao are potential drug targets to inhibit TGF-β1/ CS-induced cell migration. Additionally, in Chapter 7, co-silencing of Ezrin, AKAP95 and Yotiao is associated with β2-AR but not PDE3 or PDE4 in decreasing
TGF-β1-induced collagen Ӏ upregulation, indicating that Ezrin, AKAP95 and Yotiao could have an add-on effect on β2-agonist. Also, as one of the most important risk
factors for COPD, CS exposure is able to activate TGF-β1 signaling, inducing EMT process (Fig. 2).
Future perspective
The studies described in this thesis show that cAMP compartmentalization, mainly focusing on PDEs and AKAPs, plays a vital role in the pathophysiological processes of COPD. cAMP, as one of the most ubiquitous second messengers, controls a wide range of physiological functions by modulating signaling cascades in a spatio-temporal manner (Musheshe et al., 2018b). Therefore, comprehensive understanding of the fluctuations (generation and degradation) of cAMP and its potential functions within certain compartments will most likely help in the screening of novel pharmaceutical targets which have higher efficiency and less side effects (shown in Figure 1). Recently, it has been demonstrated by Johnstone and colleagues that β2
-AR/-AC6/-PDE8 are mainly expressed in caveolae of human ASM cells, emphasizing the microdomain-specific cAMP modulation and pointing at the future direction of studies in cAMP compartmentalization in the lung. However, of note is that monitoring intracellular cAMP dynamics using standard biochemical methods is extremely difficult (Sprenger and Nikolaev, 2013). So far, several FRET-based biosensors have been developed to achieve real-time visualization of cAMP with high spatial and temporal resolutions targeting specific subcellular microdomains, including but not limited to sodium-potassium ATPase (Bastug-Özel et al., 2018), the sarcoplasmic/endoplasmic reticulum calcium ATPase (Sprenger et al., 2015), AKAP79 (Musheshe et al., 2018a) and mitochondria (Monterisi et al., 2017) in cardiovascular studies. Therefore, designing and using microdomain-specific targeted
cAMP biosensors to study the local cAMP dynamics will be more helpful to reveal microdomain-specific cAMP dynamics in the lung.
In addition, there are no reports using cGMP FRET biosensors to investigate the intracellular cGMP levels in either lung structural cells or tissues so far. Thus, it is conceivable that monitoring microdomain-specific intracellular cAMP and cGMP levels and more importantly, their crosstalk modulated by PDE2 and PDE3, will provide more evidence to help design novel drugs targeting cyclic nucleotides with higher efficiency and with fewer side effects.
Moreover, the compartmentalized cAMP signaling may also offer answers to yet unresolved questions underlying the distinct stages of the EMT process that is linked to a diverse subset of lung responses, including pulmonary fibrosis (Jolly et al., 2018). Recent evidence also indicates that cAMP scaffolds maintained by a diverse subset of receptors, PDEs, PKA, Epac and members of the AKAP superfamily bear the potential to target distinct aspects of the EMT process, with the EMT process being closely related to factors such as TGF-β1, tumor necrosis factor alpha and/or IL-13 (Chen et al., 2014; Hu et al., 2016; Jansen et al., 2016; Jia et al., 2018; Milara et al., 2014). To date only a very limited number of drugs are available to target lung fibrosis, therefore future studies with a special focus on the distinct role of cAMP compartmentalization will new therapeutic targets and hence novel drugs in the treatment of lung fibrosis (Gourdie et al., 2016; Kalluri, 2016; Mora et al., 2017).
Main conclusions
We show that FRET which allows real time monitoring of cAMP in intact cells and tissues could be combined with precision cut lung slices (PCLS) which mimic the airway micro-environment, offering the unique opportunity to study the airway as a whole structural unit (Chapter 4).
CS causes an increase in capacity for PDE3 and PDE4 activities in mouse PLCS and cultured human ASM and epithelial cells that likely underpins alterations of intracellular cAMP levels resulting from CS exposure (Chapter 4). CS alters the PDE
expression profile, increasing PDE4 mRNA, protein and activity, whereas though the activity of PDE3 is increased this isoform is refractory to CS-induced change in expression (Chapter 4).
CS extract exposure alters the PDE activity profile in a cell type specific manner, with cilostamide sensitive PDE3 activity primarily increased in human ASM cells, whereas rolipram-sensitive PDE4 activity is increased in both human ASM and epithelial cells (Chapter 4). In ex vivo PCLS exposed to CS extract, rolipram reversed
downregulation of ciliary beating frequency, whereas only cilostamide significantly increased airway relaxation of methacholine pre-contracted airways.
194
Chapter 9
AKAPs, mainly Ezrin, AKAP95 and Yotiao are involved in TGF-β1-induced epithelial-to-mesenchymal transition. A combined inhibition of Ezrin, AKAP95 and Yotiao diminishes TGF-β1-induced cell migration (Chapter 7).
β2-AR is associated with Ezrin, AKAP95 and Yotiao, whereas PDE3 and PDE4 are
not co-localized with Ezrin, AKAP95 or Yotiao in the same cAMP compartment (Chapter 7).
The gene expression of PDE subfamilies is altered by CS exposure in the epithelial cells isolated from nasal brushes and bronchial brushes and in the lung tissue (Chapter 8). PDE4 was the only PDE subfamily changed in the 3 cohorts (Chapter 8).
PDE3A mainly expressed in airway smooth muscle cells, whereas PDE4B and PDE4D could be detected in both airway epithelial and smooth muscle cells, indicating the distinct distribution of PDE3 and PDE4 (Chapter 8).
Reference
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Ariga, M., Neitzert, B., Nakae, S., Mottin, G., Bertrand, C., Pruniaux, M.P., Jin, S.-L.C., Conti, M., 2004. Nonredundant Function of Phosphodiesterases 4D and 4B in Neutrophil Recruitment to the Site of Inflammation. J. Immunol. 173, 7531–7538.
Baillie, G.S., 2009. Compartmentalized signalling: spatial regulation of cAMP by the action of compartmentalized phosphodiesterases. FEBS J. 276, 1790–1799.
Bastug-Özel, Z., Wright, P.T., Kraft, A.E., Pavlovic, D., Howie, J., Froese, A., Fuller, W., Gorelik, J., Shattock, M.J., Nikolaev, V.O., 2018. Heart failure leads to altered β2-adrenoceptor/cAMP dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain: Bastug et al. Phospholemman cAMP microdomain. Cardiovasc. Res.
Beavo, J.A., Brunton, L.L., 2002. Cyclic nucleotide research — still expanding after half a century. Nat. Rev. Mol. Cell Biol. 3, 710–718.
Beene, D.L., Scott, J.D., 2007. A-kinase anchoring proteins take shape. Curr. Opin. Cell Biol. 19, 192–198. Berthet, J., Rall, T.W., Sutherland, E.W., 1957. The relationship of epinephrine and glucagon to liver
phosphorylase. IV. Effect of epinephrine and glucagon on the reactivation of phosphorylase in liver homogenates. J. Biol. Chem. 224, 463–475.
Biel, M., Michalakis, S., 2009. Cyclic nucleotide-gated channels. Handb. Exp. Pharmacol. 111–136.
Billington, C.K., Hall, I.P., 2011. Real time analysis of β(2)-adrenoceptor-mediated signaling kinetics in human primary airway smooth muscle cells reveals both ligand and dose dependent differences. Respir. Res. 12, 89.
Billington, C.K., Ojo, O.O., Penn, R.B., Ito, S., 2013. cAMP Regulation of Airway Smooth Muscle Function. Pulm. Pharmacol. Ther. 26, 112–120.
BinMahfouz, H., Borthakur, B., Yan, D., George, T., Giembycz, M.A., Newton, R., 2015. Superiority of combined phosphodiesterase PDE3/PDE4 inhibition over PDE4 inhibition alone on glucocorticoid- and long-acting β2-adrenoceptor agonist-induced gene expression in human airway epithelial cells. Mol. Pharmacol. 87, 64–76.
Chen, M.-J., Gao, X.-J., Xu, L.-N., Liu, T.-F., Liu, X.-H., Liu, L.-X., 2014. Ezrin is required for epithelial-mesenchymal transition induced by TGF-β1 in A549 cells. Int. J. Oncol. 45, 1515–1522.
de Rooij, J., Zwartkruis, F.J., Verheijen, M.H., Cool, R.H., Nijman, S.M., Wittinghofer, A., Bos, J.L., 1998. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396, 474– 477.
DiPilato, L.M., Cheng, X., Zhang, J., 2004. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc. Natl. Acad. Sci. U. S. A. 101, 16513–16518.
Feinstein, W.P., Zhu, B., Leavesley, S.J., Sayner, S.L., Rich, T.C., 2012. Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells. Am. J. Physiol. Cell Physiol. 302, C839-852.
Giembycz, M.A., Maurice, D.H., 2014. Cyclic nucleotide-based therapeutics for chronic obstructive pulmonary disease. Curr. Opin. Pharmacol. 16, 89–107.
Giembycz, M.A., Newton, R., 2011. Harnessing the clinical efficacy of phosphodiesterase 4 inhibitors in inflammatory lung diseases: dual-selective phosphodiesterase inhibitors and novel combination therapies. Handb. Exp. Pharmacol. 415–446.
Gourdie, R.G., Dimmeler, S., Kohl, P., 2016. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease. Nat. Rev. Drug Discov. 15, 620–638.
Han, B., Poppinga, W.J., Schmidt, M., 2015. Scaffolding during the cell cycle by A-kinase anchoring proteins. Pflugers Arch. 467, 2401–2411.
Hu, Z.-Y., Liu, Y.-P., Xie, L.-Y., Wang, X.-Y., Yang, F., Chen, S.-Y., Li, Z.-G., 2016. AKAP-9 promotes colorectal cancer development by regulating Cdc42 interacting protein 4. Biochim. Biophys. Acta 1862, 1172–1181.
Huang, H.-Y., Li, C.-F., Fang, F.-M., Tsai, J.-W., Li, S.-H., Lee, Y.-T., Wei, H.-M., 2010. Prognostic implication of ezrin overexpression in myxofibrosarcomas. Ann. Surg. Oncol. 17, 3212–3219.
9
AKAPs, mainly Ezrin, AKAP95 and Yotiao are involved in TGF-β1-induced epithelial-to-mesenchymal transition. A combined inhibition of Ezrin, AKAP95 and Yotiao diminishes TGF-β1-induced cell migration (Chapter 7).
β2-AR is associated with Ezrin, AKAP95 and Yotiao, whereas PDE3 and PDE4 are
not co-localized with Ezrin, AKAP95 or Yotiao in the same cAMP compartment (Chapter 7).
The gene expression of PDE subfamilies is altered by CS exposure in the epithelial cells isolated from nasal brushes and bronchial brushes and in the lung tissue (Chapter 8). PDE4 was the only PDE subfamily changed in the 3 cohorts (Chapter 8).
PDE3A mainly expressed in airway smooth muscle cells, whereas PDE4B and PDE4D could be detected in both airway epithelial and smooth muscle cells, indicating the distinct distribution of PDE3 and PDE4 (Chapter 8).
Reference
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Baillie, G.S., 2009. Compartmentalized signalling: spatial regulation of cAMP by the action of compartmentalized phosphodiesterases. FEBS J. 276, 1790–1799.
Bastug-Özel, Z., Wright, P.T., Kraft, A.E., Pavlovic, D., Howie, J., Froese, A., Fuller, W., Gorelik, J., Shattock, M.J., Nikolaev, V.O., 2018. Heart failure leads to altered β2-adrenoceptor/cAMP dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain: Bastug et al. Phospholemman cAMP microdomain. Cardiovasc. Res.
Beavo, J.A., Brunton, L.L., 2002. Cyclic nucleotide research — still expanding after half a century. Nat. Rev. Mol. Cell Biol. 3, 710–718.
Beene, D.L., Scott, J.D., 2007. A-kinase anchoring proteins take shape. Curr. Opin. Cell Biol. 19, 192–198. Berthet, J., Rall, T.W., Sutherland, E.W., 1957. The relationship of epinephrine and glucagon to liver
phosphorylase. IV. Effect of epinephrine and glucagon on the reactivation of phosphorylase in liver homogenates. J. Biol. Chem. 224, 463–475.
Biel, M., Michalakis, S., 2009. Cyclic nucleotide-gated channels. Handb. Exp. Pharmacol. 111–136.
Billington, C.K., Hall, I.P., 2011. Real time analysis of β(2)-adrenoceptor-mediated signaling kinetics in human primary airway smooth muscle cells reveals both ligand and dose dependent differences. Respir. Res. 12, 89.
Billington, C.K., Ojo, O.O., Penn, R.B., Ito, S., 2013. cAMP Regulation of Airway Smooth Muscle Function. Pulm. Pharmacol. Ther. 26, 112–120.
BinMahfouz, H., Borthakur, B., Yan, D., George, T., Giembycz, M.A., Newton, R., 2015. Superiority of combined phosphodiesterase PDE3/PDE4 inhibition over PDE4 inhibition alone on glucocorticoid- and long-acting β2-adrenoceptor agonist-induced gene expression in human airway epithelial cells. Mol. Pharmacol. 87, 64–76.
Chen, M.-J., Gao, X.-J., Xu, L.-N., Liu, T.-F., Liu, X.-H., Liu, L.-X., 2014. Ezrin is required for epithelial-mesenchymal transition induced by TGF-β1 in A549 cells. Int. J. Oncol. 45, 1515–1522.
de Rooij, J., Zwartkruis, F.J., Verheijen, M.H., Cool, R.H., Nijman, S.M., Wittinghofer, A., Bos, J.L., 1998. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396, 474– 477.
DiPilato, L.M., Cheng, X., Zhang, J., 2004. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc. Natl. Acad. Sci. U. S. A. 101, 16513–16518.
Feinstein, W.P., Zhu, B., Leavesley, S.J., Sayner, S.L., Rich, T.C., 2012. Assessment of cellular mechanisms contributing to cAMP compartmentalization in pulmonary microvascular endothelial cells. Am. J. Physiol. Cell Physiol. 302, C839-852.
Giembycz, M.A., Maurice, D.H., 2014. Cyclic nucleotide-based therapeutics for chronic obstructive pulmonary disease. Curr. Opin. Pharmacol. 16, 89–107.
Giembycz, M.A., Newton, R., 2011. Harnessing the clinical efficacy of phosphodiesterase 4 inhibitors in inflammatory lung diseases: dual-selective phosphodiesterase inhibitors and novel combination therapies. Handb. Exp. Pharmacol. 415–446.
Gourdie, R.G., Dimmeler, S., Kohl, P., 2016. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease. Nat. Rev. Drug Discov. 15, 620–638.
Han, B., Poppinga, W.J., Schmidt, M., 2015. Scaffolding during the cell cycle by A-kinase anchoring proteins. Pflugers Arch. 467, 2401–2411.
Hu, Z.-Y., Liu, Y.-P., Xie, L.-Y., Wang, X.-Y., Yang, F., Chen, S.-Y., Li, Z.-G., 2016. AKAP-9 promotes colorectal cancer development by regulating Cdc42 interacting protein 4. Biochim. Biophys. Acta 1862, 1172–1181.
Huang, H.-Y., Li, C.-F., Fang, F.-M., Tsai, J.-W., Li, S.-H., Lee, Y.-T., Wei, H.-M., 2010. Prognostic implication of ezrin overexpression in myxofibrosarcomas. Ann. Surg. Oncol. 17, 3212–3219.
196
Chapter 9
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