Inflammation leads through PGE / EP 3 signaling to HDAC 5/ MEF 2‐dependent transcription in cardiac myocytes
2018; Springer Nature; Volume: 10; Issue: 7 Linguagem: Inglês
10.15252/emmm.201708536
ISSN1757-4684
AutoresAndrás Dávid Tóth, Richard E. Schell, Magdolna Lévay, Christiane Vettel, Philipp Theis, Clemens Haslinger, Felix Alban, Stefanie Maria Werhahn, Lina Frischbier, Jutta Krebs-Haupenthal, Dominique Thomas, Hermann‐Josef Gröne, Metin Avkiran, Hugo A. Katus, Thomas Wieland, Johannes Backs,
Tópico(s)Signaling Pathways in Disease
ResumoResearch Article15 June 2018Open Access Source DataTransparent process Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2-dependent transcription in cardiac myocytes András D Tóth András D Tóth orcid.org/0000-0003-2746-9370 Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary Search for more papers by this author Richard Schell Richard Schell Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Department of Cardiology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Magdolna Lévay Magdolna Lévay DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Experimental Pharmacology, European Center of Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Christiane Vettel Christiane Vettel DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Experimental Pharmacology, European Center of Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Philipp Theis Philipp Theis Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Clemens Haslinger Clemens Haslinger Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Felix Alban Felix Alban Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Stefanie Werhahn Stefanie Werhahn Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Lina Frischbier Lina Frischbier Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Jutta Krebs-Haupenthal Jutta Krebs-Haupenthal Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Dominique Thomas Dominique Thomas Institute of Clinical Pharmacology, Goethe University Frankfurt, Frankfurt, Germany Search for more papers by this author Hermann-Josef Gröne Hermann-Josef Gröne Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Metin Avkiran Metin Avkiran Cardiovascular Division, King's College London British Heart Foundation Centre of Research Excellence, The Rayne Institute, St Thomas' Hospital, London, UK Search for more papers by this author Hugo A Katus Hugo A Katus Department of Cardiology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Thomas Wieland Thomas Wieland DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Experimental Pharmacology, European Center of Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Johannes Backs Corresponding Author Johannes Backs [email protected] orcid.org/0000-0002-2322-2699 Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author András D Tóth András D Tóth orcid.org/0000-0003-2746-9370 Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary Search for more papers by this author Richard Schell Richard Schell Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Department of Cardiology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Magdolna Lévay Magdolna Lévay DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Experimental Pharmacology, European Center of Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Christiane Vettel Christiane Vettel DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Experimental Pharmacology, European Center of Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Philipp Theis Philipp Theis Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Clemens Haslinger Clemens Haslinger Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Felix Alban Felix Alban Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Stefanie Werhahn Stefanie Werhahn Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Lina Frischbier Lina Frischbier Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Jutta Krebs-Haupenthal Jutta Krebs-Haupenthal Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Dominique Thomas Dominique Thomas Institute of Clinical Pharmacology, Goethe University Frankfurt, Frankfurt, Germany Search for more papers by this author Hermann-Josef Gröne Hermann-Josef Gröne Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany Search for more papers by this author Metin Avkiran Metin Avkiran Cardiovascular Division, King's College London British Heart Foundation Centre of Research Excellence, The Rayne Institute, St Thomas' Hospital, London, UK Search for more papers by this author Hugo A Katus Hugo A Katus Department of Cardiology, Heidelberg University, Heidelberg, Germany Search for more papers by this author Thomas Wieland Thomas Wieland DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Experimental Pharmacology, European Center of Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Johannes Backs Corresponding Author Johannes Backs [email protected] orcid.org/0000-0002-2322-2699 Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany Search for more papers by this author Author Information András D Tóth1,2,3,‡, Richard Schell1,2,4,‡, Magdolna Lévay2,5, Christiane Vettel2,5, Philipp Theis1,2, Clemens Haslinger1,2, Felix Alban1,2, Stefanie Werhahn1,2, Lina Frischbier1,2, Jutta Krebs-Haupenthal1,2, Dominique Thomas6, Hermann-Josef Gröne7, Metin Avkiran8, Hugo A Katus4, Thomas Wieland2,5,‡ and Johannes Backs *,1,2,‡ 1Department of Molecular Cardiology and Epigenetics, Heidelberg University, Heidelberg, Germany 2DZHK (German Centre for Cardiovascular Research), Heidelberg/Mannheim, Germany 3Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary 4Department of Cardiology, Heidelberg University, Heidelberg, Germany 5Experimental Pharmacology, European Center of Angioscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 6Institute of Clinical Pharmacology, Goethe University Frankfurt, Frankfurt, Germany 7Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany 8Cardiovascular Division, King's College London British Heart Foundation Centre of Research Excellence, The Rayne Institute, St Thomas' Hospital, London, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +49 6221 56 35271; E-mail: [email protected] EMBO Mol Med (2018)10:e8536https://doi.org/10.15252/emmm.201708536 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The myocyte enhancer factor 2 (MEF2) regulates transcription in cardiac myocytes and adverse remodeling of adult hearts. Activators of G protein-coupled receptors (GPCRs) have been reported to activate MEF2, but a comprehensive analysis of GPCR activators that regulate MEF2 has to our knowledge not been performed. Here, we tested several GPCR agonists regarding their ability to activate a MEF2 reporter in neonatal rat ventricular myocytes. The inflammatory mediator prostaglandin E2 (PGE2) strongly activated MEF2. Using pharmacological and protein-based inhibitors, we demonstrated that PGE2 regulates MEF2 via the EP3 receptor, the βγ subunit of Gi/o protein and two concomitantly activated downstream pathways. The first consists of Tiam1, Rac1, and its effector p21-activated kinase 2, the second of protein kinase D. Both pathways converge on and inactivate histone deacetylase 5 (HDAC5) and thereby de-repress MEF2. In vivo, endotoxemia in MEF2-reporter mice induced upregulation of PGE2 and MEF2 activation. Our findings provide an unexpected new link between inflammation and cardiac remodeling by de-repression of MEF2 through HDAC5 inactivation, which has potential implications for new strategies to treat inflammatory cardiomyopathies. Synopsis Understanding the link between cardiac inflammation and heart failure is key to develop therapeutic strategies for inflammatory cardiomyopathies. Here, an inflammatory pathway induced by prostaglandin E2 (PGE2) is reported to activate MEF2, well known in the setting of adverse cardiac remodeling. Prostaglandin E2 induces MEF2 via multiple signaling pathways, consisting of EP3 receptor, Gi/o-βγ, Tiam1, Rac1, PAK2 and PKD, converging on HDAC5 by regulating its nucleo-cytoplasmic shuttling. Cardiac inflammation elevates the levels of PGE2 and activates MEF2 in vivo. The presented findings strongly imply a novel yet unrecognized link between cardiac inflammation and adverse remodeling. Introduction Evidence has been provided that sustained stimulation of receptors on the plasma membrane of cardiac myocytes by distinct mediators including endothelin-1 or α-adrenergic agonists is able to trigger the development of pathological structural changes along with alterations of cardiac gene expression (Backs & Olson, 2006a). A pathognomonic change is the reactivation of the dormant embryonic isoforms of genes regulating cardiac growth, myocardial contractility and Ca2+-handling (Backs & Olson, 2006a). In particular, the reactivation of fetal cardiac gene programs is suggested to lead to the progression of cardiac remodeling and heart failure. As those mediators play crucial roles in the activation of fetal gene programs, it seems a promising approach to identify and describe the distinct upstream signaling pathways, which may reveal new targets for therapeutic interventions. The myocyte enhancer factor 2 (MEF2) transcription factors belong to the family of MADS (MCM1, agamous, deficiens, SRF) proteins (Potthoff & Olson, 2007). In mammals, four different MEF2 proteins exist, named MEF2A, B, C, and D. They bind a common DNA sequence and drive the expression of specific target genes (Potthoff & Olson, 2007). The MEF2 proteins have a crucial role in the proper cardiac development. Due to severe cardiac abnormalities, MEF2A- and MEF2C-null mice exhibit pre- and perinatal lethality, respectively (Lin et al, 1997; Naya et al, 2002). In addition to their critical role in the embryonic development, the MEF2 isoforms and their splicing variants also govern the stress response in the adult heart by reactivation of fetal cardiac gene programs (van Oort et al, 2006; Kim et al, 2008; Gao et al, 2016). Whereas MEF2 transgenic mice develop ventricular dilation and contractile dysfunction, mice lacking the MEF2D gene are protected against fetal gene activation, fibrosis, and cardiac hypertrophy (van Oort et al, 2006; Kim et al, 2008). Therefore, the activity of MEF2 is tightly controlled. Whereas phosphorylation by mitogen-activated protein (MAP) kinases and acetylation by the histone acetyltransferase p300 enhance the transcriptional activity of MEF2 (Potthoff & Olson, 2007; Wales et al, 2014; Wei et al, 2017), class IIa histone deacetylases (HDACs; HDACs 4, 5, 7, and 9) physically interact with MEF2 and recruit other repressive epigenetic factors (Backs & Olson, 2006a; Lehmann et al, 2014). A protective role of class IIa HDACs in heart failure has been suggested. For example, HDAC5 and HDAC9 prevent cardiac hypertrophy (Zhang et al, 2002; Chang et al, 2004) and HDAC4 is required for the maintenance of cardiac function during physiological exercise (Lehmann et al, 2018). Class IIa HDACs can be phosphorylated by several kinases, including the Ca2+/Calmodulin-dependent protein kinase II (CaMKII), protein kinase D (PKD), or G protein-coupled receptor kinase 5 (Backs & Olson, 2006a; Kreusser et al, 2014; Lehmann et al, 2014; Weeks & Avkiran, 2015). In turn, phosphorylated HDACs are guided by 14-3-3 chaperones from the nucleus to the cytoplasm and MEF2 is released from the inhibition and can activate downstream genes (Backs & Olson, 2006a). On the other hand, there are pathways which protect from the harmful hyper-activation of MEF2 (Lehmann et al, 2014). For instance, activation of protein kinase A (PKA) has been shown to inhibit MEF2 activity and moreover to counteract CaMKII-mediated activation of MEF2 (Backs et al, 2011; Weeks et al, 2017; Lehmann et al, 2018). The upstream activation signal of the HDAC-MEF2 axis often originates from G protein-coupled receptors (GPCRs). The divergent pathways resulting from GPCRs could have antagonistic effects on MEF2 activation; therefore, it is difficult to predict the net effect on MEF2 activation by different mediators. The aim of this study was to identify new mediators that signal via GPCRs in cardiac myocytes and which were not known to activate the HDAC-MEF2 axis before. Furthermore, we sought to explore the detailed resulting downstream signaling pathway. We found that prostaglandin E2 (PGE2), which is one of the main inflammatory mediators, strongly activates MEF2. PGE2 binds to the Gi/o-coupled EP3 receptor, which activates divergent, parallel operating signaling pathways; one involving Tiam1-, Rac1-, and p21-activated kinases (PAK) and another involving PKD and HDAC5. We found that full MEF2 activation depends on participation of both pathways and that MEF2 is activated in vivo in inflamed hearts. Results PGE2 activates MEF2 through the EP3 receptor To identify unknown GPCR-dependent signaling pathways that regulate MEF2 activity, we conducted a screening experiment using neonatal rat ventricular myocytes (NRVMs). NRVMs were infected with an adenovirus harboring a MEF2-reporter (3xMEF2-Luc), which responds to endogenous MEF2. Thereafter, the cells were stimulated with different GPCR agonists for 24 h in serum-free medium. Similar to previous reports, endothelin-1 (100 nM) activated MEF2 to a high extent (Backs et al, 2011). On the other hand, the β-adrenergic receptor agonist isoproterenol (1 μM) significantly decreased the basal level of MEF2 activity. We identified several mediators, which slightly elevated the activity of MEF2, such as sphingosine-1-phosphate (1 μM) or lysophosphatidic acid (10 μM). Strikingly, we observed a 20-fold activation by two related compounds, namely prostaglandin E1 (PGE1; 10 μM) and prostaglandin E2 (PGE2; 10 μM; Fig 1A). In contrast, analogs of other prostaglandins (fluprostenol and treprostinil, agonists of prostaglandin F and prostacyclin receptors, respectively) had no significant effect. In the following experiments, we focused on PGE2 because of its higher abundance in the heart in vivo (Herman et al, 1987). The PGE2 effect on MEF2 activity was concentration-dependent (Fig EV1A). In good agreement with the ability to activate MEF2, PGE2 triggered the expression of known specific MEF2 target genes (Potthoff & Olson, 2007; Lehmann et al, 2018), such as Nur77, Myomaxin, or Adamts1 (Fig 1B). MEF2 activation is commonly associated with the induction of hypertrophic gene programs. Likewise, PGE2 increased the mRNA levels of the hypertrophy marker BNP (Fig 1B) and induced cellular hypertrophy of NRVMs, which correlated with the induction of MEF2 activity (Fig 1C). In addition, we observed a similar extent of protein synthesis induction after PGE2 stimulation as with the well-known hypertrophic α-adrenoceptor agonist phenylephrine (Fig EV1B). Next, we aimed to determine the receptor involved in PGE2-mediated MEF2 activation. The four different isoforms of PGE2 receptors (EP1, EP2, EP3, EP4) show a different degree of G protein coupling and expression patterns (Woodward et al, 2011). Each of the four types is expressed in cardiac myocytes, but EP3 and EP4 receptors are the most abundant isoforms (Di Benedetto et al, 2008). The EP3 receptor antagonist L798106 inhibited PGE2-dependent MEF2 activation (Figs 1D and EV1C), while neither the EP4 receptor antagonist L-161,982 (2 μM) nor the EP1/EP2 receptor antagonist AH6809 (10 μM) led to an alteration of MEF2 activity, suggesting the EP3 receptor as the underlying receptor to activate MEF2 by PGE2 in NRVMs. Figure 1. Screening the effect of different GPCR agonists on MEF2 activity Neonatal rat ventricular myocytes (NRVMs) were infected with the 3xMEF2-Luc reporter, serum starved for 20 h, and stimulated for 24 h with different agonists: 100 nM endothelin-1 (ET1), 1 μM sphingosine-1-phosphate (S1P), 10 μM lysophosphatidic acid (LPA), 1 μM WIN55,212-2 (WIN55, cannabinoid receptor agonist), 1 μM isoproterenol (ISO, β-adrenergic receptor agonist), 100 nM angiotensin II (AngII), 10 μM prostaglandin E1 (PGE1), 10 μM prostaglandin E2 (PGE2) or 100 nM prostanoid F receptor agonist fluprostenol (Flupro), 100 nM treprostinil (Trepro, prostacyclin receptor agonist). PGE2 induces the expression of MEF2 target genes. NRVMs were serum starved for 24 h, then were stimulated with 1 μM PGE2 for 2 h, and mRNA levels of the indicated genes were determined. Correlation between MEF2 activation and NRVM hypertrophy. MEF2 activity was measured as in (A) upon increasing concentration of PGE2. Cell size of NRVMs was determined after 48-h stimulation with the indicated concentrations of PGE2. Left: Representative images of α-actinin stained NRVMs stimulated with DMSO, 300 nM PGE2, or 1 nM ET1. Scale bar is 10 μm. PGE2 activates MEF2 via EP3 receptor. NRVMs were infected with the 3xMEF2-Luc reporter and serum starved for 20 h. The cells were stimulated with DMSO or 1 μM PGE2 for 24 h in the presence or absence of different EP receptor antagonists: AH6809 (10 μM, EP1- and EP2-antagonist) or 798106 (200 nM, EP3-antagonist) or L161,982 (2 μM, EP4-antagonist). Data information: Values are mean ± s.e.m. In (A), the experiment was performed in triplicates, and similar results were obtained in three different experiments. Student's two-tailed t-test, *P < 0.05 vs. control (ET1, P = 0.0013; S1P, P = 0.0144; LPA, P = 0.026; WIN55, P = 0.6205; ISO = 0.0399; AngII, P = 0.5812; PGE1, P = 0.001; PGE2, P < 0.0001; Flupro, P = 0.0846; Trepro, P = 0.1958). In (B), n = 5, technical replicates, Student's two-tailed t-test, *P < 0.05 vs. control (Nur77, P = 0.0148; Myomaxin, P = 0.0417; Adamts1, P = 0.0112; BNP, P = 0.0065). In (C), NRVM cell size was determined from a minimum of 100 cells of three technical replicates upon different concentrations of PGE2, and MEF2 activity was parallel assessed from six technical replicates. Correlation was statistically analyzed by calculating Pearson correlation coefficient (Pearson r = 0.9626, P = 0.0021). In (D), n = 3, independent experiments, *represents significant interaction between the two treatments (P < 0.05, two-way ANOVA, AH6809, P = 0.1092; L798106, P = 0.0002; L161,982, P = 0.5579). Source data are available online for this figure. Source Data for Figure 1 [emmm201708536-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. PGE2 activates MEF2 via the Gi/o protein-coupled EP3 receptor Concentration dependency of the PGE2-triggered MEF2 activity. MEF2 activity was assessed in 3xMEF2-Luc-infected NRVMs upon 24-h stimulation with increasing concentration of PGE2. PGE2 induces protein synthesis in NRVMs. After serum starvation, NRVMs were treated with media containing 1 μCi/ml [3H]-leucine and 100 μM PE or 10 μM PGE2 for 24 h. Thereafter, the cellular [3H]-content was detected. Concentration-dependent effect of L798106 on the PGE2-induced MEF2 activation. MEF2 activity was determined in 3xMEF2-Luc-expressing NRVMs pretreated with increasing concentration of L798106 for 20 min and treated with DMSO or 1 μM PGE2 for 24 h. The PGE2-induced response was calculated by subtracting the MEF2-luciferase activity measured in vehicle-treated cells from the PGE2-induced response in the case of all 798106 concentrations. L798106 inhibits the Gi/o protein-coupled receptor of PGE2. To prove that 798106 inhibits EP3 receptor, a cAMP signal-decreasing receptor, we assessed the phosphorylation of phospholamban at a target site (Ser-16) of protein kinase A, a well-known cAMP-regulated kinase. NRVMs were pretreated with 200 nM L798106 for 20 min, then with 100 nM PGE2 for 2 min. The β-adrenergic receptor agonist isoproterenol (ISO, 10 nM) was used to induce a cAMP signal. Phosphorylation of phospholamban was detected by immunoblot. In the presence of the EP3 receptor inhibitor, PGE2 elevated the ISO-induced phosphorylation of phospholamban, showing that L798106 inhibits the cAMP-decreasing effect of PGE2. Data information: Values are mean ± s.e.m. In (A) and (C), n = 4, technical replicates; curve was fitted and half maximal inhibitory concentration (IC50 = 27 nM) was determined using GraphPad Prism. In (B), n = 9, independent experiments. *P < 0.05 vs. control, Student's two-tailed unpaired t-test (PE, P = 0.0008; PGE2, P = 0.0005). The exact n and P-values can also be found in the Source Data Excel file for Fig EV1. Source data are available online for this figure. Download figure Download PowerPoint PGE2 activates MEF2 through Gi/o proteins The EP3 receptor is generally considered to couple to pertussis toxin (PTX)-sensitive Gi/o proteins, thereby decreasing intracellular cAMP levels. In accordance, EP3 receptor inhibition by L798106 increased the isoproterenol-induced phosphorylation of phospholamban, a known cAMP-regulated protein (Cuello et al, 2007; Fig EV1D). However, the EP3 receptor has several splice variants, which differ in the G protein-coupling properties (Woodward et al, 2011). Therefore, we examined which heterotrimeric G protein types are essential for the PGE2-mediated MEF2 activation by a pharmacological and molecular approach (Fig 2A). Regulator of G protein signaling (RGS) proteins are GTPase activating proteins (GAPs) and specifically block different classes of G proteins. RGS16 is a GAP for Gi/o and Gq/11 proteins, RGS2 suppresses Gq/11, while RGS-LSCII inhibits the G12/13 signaling pathway (Vettel et al, 2012). Like RGS2, p63∆N, a specific scavenger of activated Gαq/11 proteins did not affect the PGE2-induced activity of MEF2. In contrast, the Gi/o and Gq/11 inhibitor RGS16 (Fig 2B), as well as pretreatment with pertussis toxin (PTX, 100 ng/ml) attenuated the PGE2 response (Fig 2C). We did not observe any significant effect of RGS-LSCII overexpression in this setting (Fig 2B). These data suggest that neither Gq/11- nor G12/13-, but the PTX-sensitive Gi/o protein activation is required for the PGE2 response. To rule out an involvement of Gs proteins, we inhibited adenylyl cyclase, the effector enzyme of Gs proteins. The adenylyl cyclase inhibitor SQ22536 (100 μM) did not affect MEF2 activity (Fig 2C). In addition, since the adenylyl cyclase inhibition also mimics the effect of Gαi/o, the absence of alteration suggested that not the α, but the βγ subunit of Gi/o is the transducer in the PGE2-mediated MEF2 activation. In accordance, overexpression of Gαt to scavenge the free Gβγ subunits abolished the PGE2 effect (Fig 2D). Similar results were obtained with the overexpression of long isoform of RGS3 (RGS3L), another Gβγ scavenger (Vogt et al, 2007; Fig EV2). Taken together, these data indicate that the PGE2-induced MEF2 activation is mediated by the βγ subunit of Gi/o proteins. Figure 2. PGE2-mediated MEF2 activation is Gi/o-βγ dependent A. Illustration of the target points of the used pharmacological and protein-based inhibitors. B–D. (B, D) NRVMs were infected with the 3xMEF2-Luciferase reporter and with recombinant adenoviruses encoding EGFP (AdGFP), RGS16, RGS2, p63ΔN, RGS-LSCII, or Gαt, as indicated. The cells were serum starved for 20 h and stimulated with DMSO or 1 μM PGE2 for 24 h. (C) NRVMs were infected with the 3xMEF2-Luciferase reporter and pretreated with the adenylyl cyclase inhibitor SQ22536 (100 μM) for 20 min or the Gi/o protein inhibitor pertussis toxin (PTX, 100 ng/ml) for 20 h in serum-starved conditions and treated with DMSO or 1 μM PGE2 for 24 h. Data information: In (B–D), n = 3, independent experiments, *represents significant interaction between the two treatments (P < 0.05, two-way ANOVA, RGS16, P < 0.0001; RGS2, P = 0.1725; p63ΔN, P = 0.0666; RGS-LSCII, P = 0.0818, SQ22536, P = 0.385; PTX, P < 0.0001; Gαt, P < 0.0001), values are mean ± s.e.m. Source data are available online for this figure. Source Data for Figure 2 [emmm201708536-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. The PGE2-induced MEF2 activity and protein synthesis is abolished by overexpression of the βγ-scavenger RGS3LLeft panel: NRVMs were infected on the first day with 3xMEF2-Luc, on the next day with adenovirus encoding EGFP or RGS3L, then were serum starved. 24-h vehicle or 1 μM PGE2 stimuli were used. Overexpression RGS3L prevented the PGE2-induced response. Right panel: After infection with adenovirus encoding EGFP or RGS3L, NRVMs were starved, then treated with vehicle or 1 μM PGE2 in media containing 1 μCi/ml [3H]-leucine for 24 h. Thereafter, [3H]-leucine uptake was assessed. RGS3L counteracted the effect of PGE2. Values are mean + s.e.m., n = 3–4, technical replicates, two-way ANOVA, *means significant interaction between RGS3L overexpression and the effect of PGE2, P < 0.05 (MEF2 activity: P = 0.007, [3H]-leucine uptake: P = 0.0393). The exact n and P-values can also be found in the Source Data Excel file for Fig EV2. Source data are available online for this figure. Download figure Download PowerPoint PGE2 induces HDAC5 hyperphosphorylation and de-repression of MEF2 via PKD Among critical regulators of MEF2 are class IIa HDACs. A well-known mechanism of de-repression of MEF2 activity is HDAC phosphorylation followed by its concomitant nucleo-cytoplasmic shuttling. Therefore, we analyzed the phosphorylation status of HDAC5 by Western blot. We overexpressed FLAG-tagged HDAC5 in NRVMs. After stimulation with PGE2, we observed a high extent of hyperphosphorylation of HDAC5 at Ser-498 (Fig 3A), similarly to endothelin-1 (ET1, 100 nM) or phenylephrine (PE, 100 μM). Since Ser-498 is a specific phosphorylation site of protein kinase D (PKD; Haworth et al, 2011), we examined whether PKD is responsible for the phosphorylation of HDAC5. Indeed, pretreatment with the specific PKD-inhibitor BPKDi (3 μM; Meredith et al, 2010; Monovich et al, 2010) diminished the PGE2-induced HDAC5 phosphorylation. In accordance, phosphorylation of PKD was increased at Ser-744/Ser-748 and Ser-916 sites upon PGE2 treatment in NRVMs (Fig 3B), which was partly (phosphorylation at Ser-744/Ser-748) observed in adult mouse ventricular myocytes as well (Fig EV3). Different mechanisms of PKD activation exist. It is known that PKD can be activated in a protein kinase
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