Abstract 19995: p22phox-Based Nox4 Regulates Cellular Survival by Modulating FOXO3A Activity and Sirt1 Expression
Fasting triggers a survival response coordinated by the transcription factor FOXO3A and histone deacetylase SIRT1 by controlling the expression of antioxidant enzymes. We hypothesized that the NADPH oxidase Nox4 regulates these responses via FOXO3A-SIRT1 activation. We first decreased Nox4 or FOXO3A levels in human cardiac fibroblasts (HCFB) and measured apoptotic response induced by serum deprivation. Transfection with siNox4 caused a □300% increase in cellular apoptosis in response to serum deprivation. This was associated with a corresponding 50% increase in cellular H2O2 production (n=7, p<0.05). We hypothesized that oxidative stress caused by depletion of Nox4 or FOXO3A is due to downregulation of the antioxidant enzymes. Quantitative PCR analysis showed that deletion of Nox4 leads to downregulation of antioxidant enzymes catalase, peroxiredoxin III (Prx III) and the anti-apoptotic bcl2 while it triggered the upregulation of the pro-apoptotic factor Bim and atrogin1 in HCFB. Furthermore, the apoptotic response was rescued by treating siNox4-transfected cells with PEG-catalase (n=5, p<0.05). Similarly, the gene expression of catalase, PrxIII and Bcl2, are significantly downregulated after 24 hours fasting in murine cardiomyocytes isolated from mice in which we specifically deleted p22phox (functionally deleted Nox4) in cardiac myocytes (n=3, p<0.05). In contrast, deletion of Nox2 (another Nox homologue which also requires p22phox subunit for activation), did not affect cardiac expression of antioxidant enzymes after fasting (n=6). Because these proteins are regulated by FOXO3A, we postulated that Nox4 is upstream of FOXO3A and regulates its activation via SIRT1-dependent deacetylation. We next demonstrated that transfection with siNox4 of cardiac fibroblasts lead to ∼50% downregulation of SIRT1 RNA and protein expression and lead to enhanced hyperacetylation of nuclear FOXO3A (n=4, p<0.05). Furthermore co-transfection with SIRT1 is able to rescue the apoptotic response triggered by serum deprivation suggesting that SIRT1 is required for survival response downstream of Nox4. We conclude that p22phox-based Nox4 is essential for cardiac adaptation to prolonged fasting by regulating FOXO3A activation and expression of SIRT1.
- © 2010 by American Heart Association, Inc.