Abstract 12259: Enhanced Proteasomal Degradation of p300 by its Deacetylation Underlies Inhibition of Hypertrophic Response by SIRT1 in Cardiomyocytes
Background: We previously found that activation of the protein deacetylase SIRT1 suppresses cardiomyocyte hypertrophy, but its molecular mechanism has not been explored. In this study, we examined whether SIRT1 counteracts p300, a transcriptional activator possessing intrinsic lysine acetyltransferase activity.
Methods and Results: First, the relationship between SIRT1 and p300 level was examined in phenylephrine (PE)-induced myocyte hypertrophy. Treatment of neonatal rat cardiomyocytes (NRCM) with PE (50 μ M for 48 hrs) increased cell surface area, atrial natriuretic peptide (ANP) mRNA, and p300 protein level. Silencing of p300 by siRNA abolished PE-induced hypertrophy, and p300 dose-dependently increased ANP promoter activity in the presence of GATA4 transcription factor in HEK293 cells. Treatment of NRCM with a SIRT1 activator, resveratrol (RSV, 30 μ M), blocked PE-induced p300 upregulation and cell enlargement, which was sensitive to SIRT1 knockdown. Wild-type SIRT1, but not deacetylase inactive SIRT1 (H355Y), downregulated p300 protein level. Co-transfection of SIRT1 suppressed p300-induced increase in ANP promoter activity. The second series of experiments was carried out to determine how SIRT1 decreases p300 protein level. Neither PE nor RSV changed p300 mRNA level in NRCM. The reduction in p300 protein level by RSV or SIRT1 was suppressed by a proteasome inhibitor, MG132. Furthermore, SIRT1 promoted p300 deacetylation and poly-ubiquitination. Conversely, blocking SIRT1 activity by Ex527, caused p300 acetylation and increase in p300 protein level. Co-transfection of p300 with p300-CBP associated factor (PCAF), which acetylates p300, markedly elevated p300 level, and this p300 upregulation was inhibited by SIRT1. A mutant p300 in which lysine residues known as sites of deacetylation by SIRT1 were replaced with alanines was resistant to both downregulation and ubiquitination induced by SIRT1. Importantly, tamoxifen-inducible cardiomyocyte-specific SIRT1 knockout also caused upregulation of p300 protein in the heart.
Conclusion: The findings suggest that SIRT1 suppresses cardiomyocyte hypertrophy by promoting p300 degradation through removal of acetyl groups that competitively inhibit poly-ubiquitination.
- © 2011 by American Heart Association, Inc.