Abstract 9477: Retinoblastoma Protein-associated Protein 48 and 46 Form Repressor Complex With p300/GATA4 And Inhibit Hypertrophic Responses in Cardiomyocytes

Abstract

Introduction: A zinc finger protein GATA4 associates with an intrinsic histone acetyltransferase p300 and regulates myocardial transcriptional activities in response to hypertrophic stimuli. Retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48, RbAp46) form a repressor complex with HDACs and are implicated in chromatin remodeling and transcriptional repression. We showed that RbAp48 and 46 are novel component of the p300/GATA4 complex. However, precise functional relationships among p300, GATA4 and RbAp48/46 and their roles in hypertrophic responses remain unknown. Hypothesis: We have assessed the hypothesis that RbAp48/46 regulate hypertrophic responses in cardiomyocytes by modulating functions of the p300/GATA4 complex. Methods and Results: Immunoprecipitation followed by Western blotting in HEK293T cells demonstrated that overexpression of RbAp48/46 inhibited p300-induced GATA4 acetylation and the association between p300 and GATA4. Conversely, knockdown of RbAp48/46 by siRNA reversed these changes. RbAp48/46 inhibited not only p300-induced DNA binding activity of GATA4 onto the atrial natriuretic factor (ANF) and endotheline-1 (ET-1) promoters, but also p300/GATA4-induced ANF and ET-1 promoters activities. In cardiomyocytes, overexpression of RbAp48/46 significantly inhibited phenylephrine (PE)-induced GATA4 acetylation, activation of ANF and ET-1 promoters, and myocardial cell hypertrophy. On the other hand, knockdown of both RbAp48 and 46 by shRNA reversed these changes. PE decreased the binding between GATA4 and RbAp48/46, and the recruitment of RbAp48/46 onto the GATA element of the ANF promoter. Although RbAp48/46 were co-localized with GATA4 in the nucleus of cardiomyocytes at the basal state, PE stimulus induced the export of RbAp48/46 to cytoplasm. LMB, a inhibitor of chromosomal region maintenance 1 (CRM1), inhibited nuclear export of RbAp48/46. Conclusion: These findings demonstrate that RbAp48/46 interact with GATA4, disasociate GATA4 from p300, and inhibit its acetylation as well as hypertrophic responses in cardiomyocytes. The nuclear export of RbAp48/46 by hypertrophic stimuli may trigger hypertrophy-responsive transcription by releasing these inhibitions.