Abstract 15061: Mst1-Mediated Phophorylation of Foxo1 and C/EBP-β Stimulates Cell Protective Mechanisms In Cardiomyocytes
Forkhead box O (FoxO) family transcription factors regulate a wide variety of functions in the heart, including hypertrophy, cell death, autophagy and metabolism. However, the molecular mechanism by which FoxOs regulate both cell death and survival pathways is not well understood. Mst1 (mammalian sterile 20-like kinase 1), a pro-apoptotic kinase, phosphorylates FoxO1 at Ser209/216/231/232, thereby inducing nuclear translocation of FoxO1 in cardiomyocytes (CMs). Increased expression of FoxO1 significantly inhibited (0.55 fold, p<0.05), whereas knockdown of FoxO1 exacerbated (2 fold, p<0.05), Mst1-induced apoptosis and Mst1 activity, suggesting that FoxO1 acts as a negative feedback regulator of Mst1. FoxO1 upregulated antioxidant genes, such as catalase and PRDX2, but inhibited pro-apoptotic genes, such as FasL and NOXA, in the presence of Mst1. Chromatin immunoprecipitation assays showed that Mst1 inhibited FoxO1 binding to the FasL promoter, but promoted binding to the catalase promoter. Reporter gene assays showed that C/EBP-β binding elements, but not the FoxO binding elements, in the catalase promoter are critical for Mst1-mediated upregulation of the catalase gene. FoxO1 interacted with C/EBP-β, and their interaction was enhanced in the presence of Mst1. Mass spectrometry revealed that Mst1 phosphorylates C/EBP-β at Thr299, in the leucine zipper domain. Knockdown of C/EBP-β reversed the beneficial effects of FoxO1 expression on Mst1-induced apoptosis. The role of Mst1-mediated phosphorylation of FoxO1 and C/EBP-β in response to ischemia/reperfusion (I/R) was evaluated. The left ventricular (LV) infarct after I/R was significantly larger in cardiac-specific FoxO1 knockout (c-FoxO1-/-) mice than in NTg mice (Infarction area/area at risk: 59±2, 41±1%, p<0.05, n=6). Injection of adenovirus harboring 3xFlag-C/EBP-β Thr299Asp, a phospho-mimetic mutant, into the LV of c-FoxO1-/- significantly reduced the I/R-induced LV infarct size observed in c-FoxO1-/- (50±2%, p<0.05, n=6). In conclusion, phosphorylation of FoxO1 by Mst1 inhibits the binding of FoxO1 to DNA but enhances its binding to C/EBP-β, phosphorylation of C/EBP-β by Mst1, and C/EBP-β-mediated transcription, which in turn stimulates protective mechanisms in the heart.
- © 2012 by American Heart Association, Inc.