Abstract 20541: Cytoglobin Regulates Proliferation and Differentiation by Homeostatic and Stress Induced Repression of p53 Transcriptional Activity
Cytoglobin (Cygb) is a stress-responsive hemoprotein that is abundantly expressed in the heart and it is transcriptionally regulated by the redox sensitive transcription factors AP1 and NFAT. Cygb is present in the cytosol and nucleus of C2C12 cells and cardiomyocytes. Overexpression of Cygb protects C2C12 myoblasts from oxidative stress and promotes proliferation, while knockdown promotes differentiation and apoptosis. Gene expression data (microarray, PCR array, qRT-PCR; n=6-9) show a reciprocal expression pattern between Cygb levels and various p53 target genes (p21, Gadd45A, Plk3). Therefore, utilizing in vitro assays and generation of cardiac-specific knockout of Cygb (Cygb CKO) and Cygb overexpressing (TgCygb) mouse models, we tested the hypothesis that Cygb modulates myocyte proliferation by inhibiting p53 transcriptional activity. Cygb inhibited p53 activity in transcription assays (p<0.05; n=6-9). ChIP and EMSA verified formation of a Cygb-p53 complex on the p21 and Puma promoters as well as Cygb's ability to inhibit p53 occupancy on the promoters under stress. Protein-protein interaction assays (Co-IP, GST-pull down) demonstrated a direct physical interaction between Cygb and the DNA-binding domain of p53. Cardiac function was normal in Cygb CKO mice; however, there was a 7 fold decrease in BrdU incorporation and an increase in expression of p53 target genes involved in differentiation (p21, Gadd45a, Plk3) (p<0.05; n=10-20). In contrast, TgCygb mice had a 20% increase in heart mass, 20% decrease in LV systolic function and activation of the fetal gene program (p<0.05; n=5). There was a 3 fold increase in BrdU incorporation and a 40-140 fold increase in genes involved in proliferation (Ki67, Cyclin D1 and D3) (p<0.05; n=10-20), suggesting that myocytes in these hearts may not have fully differentiated. Infusion of angiotensin (AngII) in Cygb CKO mice resulted in enhanced p53 occupancy on p53 target gene promoters while in AngII treated TgCygb mice there was marked blunting of p53 transcriptional activity due to a failure to augment p53 binding to its target promoters (p<0.05; n=5). Collectively, our data support the hypothesis that Cygb influences myocyte proliferation and differentiation via repression of p53 transcriptional activity.
- © 2010 by American Heart Association, Inc.