Regulation of Cardiac Transcription Factor GATA4 by Post-Translational Modification in Cardiomyocyte Hypertrophy and Heart Failure Yasufumi Katanasaka,1,2,3 PhD, Hidetoshi Suzuki,1, Yoichi Sunagawa,1,2,3 PhD, Koji Hasegawa,2 MD, and Tatsuya Morimoto,1,2,3 MD Summary Heart failure is a leading cause of cardiovascular mortality in industrialized countries. During development and de- terioration of heart failure, cardiomyocytes undergo maladaptive hypertrophy, and changes in the cellular phenotype are accompanied by reinduction of the fetal gene program. Gene expression in cardiomyocytes is regulated by various nu- clear transcription factors, co-activators, and co-repressors. The zinc finger protein GATA4 is one such transcription fac- tor involved in the regulation of cardiomyocyte hypertrophy. In response to hypertrophic stimuli such as those involving the sympathetic nervous and renin-angiotensin systems, changes in protein interaction and/or post-translational modifi- cations of GATA4 cause hypertrophic gene transcription in cardiomyocytes. In this article, we focus on cardiac nuclear signaling molecules, especially GATA4, that are promising as potential targets for heart failure therapy. (Int Heart J 2016; 57: 000-000) Key words: Acetylation, Phosphorylation, Protein interaction, Metylation, SUMOylation ardiac hypertrophy is an adaptive response to pressure taining transcription factors that bind to the specific consensus or volume overload, mutations in sarcomeric pro- DNA sequence 5’-WGATAR-3’. Six members of the GATA teins, or loss of contractile mass due to myocardial family have been identified, of which GATA4, GATA5, and C 1) infarction and is a risk factor for heart failure. The sympathet- GATA6 are expressed in the heart. GATA4 plays essential roles ic nervous and renin-angiotensin systems are proximal initiat- in heart development and a GATA4 mutation has been reported ing stimuli for cardiac hypertrophy. Binding of ligands to func- in patients with congenital heart disease.10,11) GATA4-knockout tional receptors activates intracellular signaling involved in mice have been reported to die at early embryonic stages 12,13) phosphorylation and calcium influx and alters nuclear gene ex- and GATA4 transgenic mice have been reported to show sig- pression in cardiomyocytes.2) Various studies have demonstrat- nificant cardiac growth.14) These data strongly indicate that the ed that transcriptional regulation in the nucleus is important for cardiomyocyte-specific transcription factor GATA4 regulates pathological cardiac hypertrophy and heart failure develop- gene transcription associated with cardiac hypertrophy. Various ment. Transcription factors such as myocyte enhancer factor 2 reports have demonstrated that GATA4 in cardiomyocytes (MEF2),3) serum response factor (SRF),4) GATA binding pro- plays important roles in the transcription of hypertrophic genes tein 4 (GATA4),5) AP-1,6) neuron-restrictive silencer factor such as α-myosin and β-myosin heavy chain (α and β-MHC), (NRSF),7) nuclear factor of activated T cells (NFAT),8) and my- myosin light chain 1/3 (MLC1/3), cardiac troponin C, cardiac ocardin 9) have been implicated as mediators of the fetal gene troponin I, atrial natriuretic factor (ANF), brain natriuretic pep- program that are associated with cardiac hypertrophy and heart tide (BNP), and endothelin 1 (ET-1).15-18) failure. Among these, we have investigated the function of In addition to its established roles in cardiac hypertrophy, GATA4 and p300, a histone acetyltransferase (HAT) and a GATA4 regulates cell survival and anti-apoptotic signaling. In GATA4 coactivator, in the progression of cardiac hypertrophy adult cardiac myocytes, Kakita, et al reported that the cal- and heart failure. In this review, we describe GATA4-mediated cineurin/NFAT/GATA4 pathway is required for ET-1-mediated nuclear signaling in cardiac hypertrophy and heart failure. protection against cardiac myocyte apoptosis.19) Cardiac-spe- cific deletion of GATA4 induces apoptosis by alteration of the Gata4 gene expression implicated in cellular apoptosis after pressure- overload.20) The overexpression of GATA4 has been shown to GATA transcription factors are zinc finger domains con- prevent cardiac myocyte apoptosis induced by anthracyclines From the 1 Division of Molecular Medicine, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, 2 Division of Translational Research, Kyoto Medi- cal Center, National Hospital Organization, Kyoto, and 3 Shizuoka General Hospital, Shizuoka, Japan. Address for correspondence: Tatsuya Morimoto, MD, Division of Molecular Medicine, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526 Shizuoka, Japan. E-mail: [email protected] Received for publication August 19, 2016. Revised and accepted August 30, 2016. Released in advance online on J-STAGE November 4, 2016. (This is “Advance Publication”.) All rights reserved by the International Heart Journal Association. 1 Int Heart J 2 KATANASAKA, ET AL November 2016 Table. GATA4 Post-Translational Modifications and Functions Modification Residue Function Mechanism Enzyme References Acetylation K312/319/ Transcription activation Increase in DNA binding and transcriptional p300 26) 321/323 activity Phosphorylation S105 Transcription activation Increase in DNA binding activity ERK1/2, p38 32, 33) S160 Transcription activation Increase in interaction with co-activators CDK4 35) Unknown Transcription inhibition, Increase in interaction with Crm1 GSK3-β 37) nuclear shuttling S261 Transcription activation Increase in acetylation by p300 ERK1/2, RSK2, PKA 39-41) S419/420 Transcription activation Increase in DNA binding activity PKC 42) Metylation K299 Transcription inhibition Decrease in interaction with p300 EZH2 43) SUMOylation K366 Transcription activation Facilitation of nuclear localization SUMO-1, PIAS1 44-46) such as daunorubicin and doxorubicin.21) Additionally, GATA4 has been demonstrated to regulate the expression of Bcl-2, an anti-apoptotic protein, in cardiac myocytes, and has been shown to be involved in erythropoietin-induced cardioprotec- tion against ischemia/reperfusion injury in the mouse heart.22,23) Regulation of GATA4 activity by post-translational modifica- tion and protein interaction GATA4 expression and post-translational modifications are altered in the heart during cardiac hypertrophy and heart failure. GATA4 post-transcriptional modifications are impor- tant for transcriptional activity, and GATA4 activity is report- edly regulated by modifications such as acetylation, phosphor- ylation, methylation, and SUMOylation (Table). Interactions between GATA4 and other proteins are also important to regu- late DNA binding activity and gene transcription. Acetylation: We have shown that acetylation of GATA4 by Figure. Scheme of GATA4 regulation in hypertrophic responses. p300, a histone acetyltransferase (HAT), is essential to induce hypertrophic gene transcription, cardiomyocyte hypertrophy, and heart failure development (Figure).22) Transcriptional co- regulated by histone deacetylases. Histone deacetylase 2 activator p300 was first identified as an E1A binding protein; it (HDAC2) and a small homeodomain factor, Hopx, mediate serves as a HAT, a scaffold protein or bridge for transcription deacetylation of GATA4.28) However, the details of regulation factors and other components of the basal transcription ma- of GATA4 deacetylation are still unclear. chinery to facilitate chromatin remodeling, and it activates Phosphorylation: GATA4 activation induced by hypertrophic gene transcription. Cardiac-specific p300 transgenic mice have stimulation has been demonstrated to be associated with shown acceleration of left ventricular remodeling after myo- GATA4 phosphorylation.29,30) The mitogen-activated protein cardial infarction, but the acceleration of remodeling in HAT kinase (MAPK) cascade is a key biochemical signal which activity-lacking p300 transgenic mice is attenuated,24,25) indi- mediates hypertrophic responses.31) Because GATA4 with the cating that the HAT activity of p300 is necessary for left ven- S105A point mutation, which cannot be phosphorylated by tricular remodeling after myocardial infarction. The zinc finger ERK, inhibited MEK1-induced hypertrophic responses in cul- domain of the c-terminus of GATA4 contains some residual tured cardiomyocytes, GATA4 may function downstream from lysine groups and plays important roles in the capacity to bind the ERK signaling pathway in hypertrophic responses.32) to DNA and to other factors. We analyzed the sites of p300-in- GATA4 is also activated through direct serine phosphorylation duced GATA4 acetylation, and found that 4 lysine resides by the p38 MAPK pathway, which is a main branch of the (K311, K318, K320, and K322) were acetylated by p300; ad- MAPK cascade and mediates hypertrophic growth in cultured ditionally, point mutations of these residues to alanine inhibited cardiomyocytes.33) It has been reported that the Rho/Rho ki- cardiomyocyte hypertrophy as a dominant-negative mutant.26) nase (ROCK) pathway is upstream of GATA4 phosphorylation We have identified cyclin-dependent kinase 9 (Cdk9), which is by ERK and p38 MAPK. In cardiomyocytes, the Rho/ROCK a component of positive transcription elongation factor b, as a pathway is involved in GATA4 phosphorylation and hyper- novel GATA4 binding partner. We reported that Cdk9 pro- trophic responses.34) In embryonic cardiomyocytes, CDK4 motes cardiomyocyte hypertrophy and p300/GATA4