Abstract 3202: GSK3β Negatively Regulates Cardiac Hypertrophy Through Activation of FoxO
Both glycogen synthase kinase-3 β (GSK3β), a serine/threonine kinase, and FoxO1, a member FoxO family transcription factor, negatively regulate cardiac hypertrophy, and their activity is negatively regulated by Akt. Despite their similarity, interaction between GSK3β and FoxO1 has not been investigated. Here we demonstrate the cooperative role of GSK3β and FoxO1 in inhibiting cardiac hypertrophy. Co-immunoprecipitation assays showed that GSK3β physically interacts with FoxO1. In vitro kinase assays showed that GSK3β phosphorylates FoxO1. Immunostaining and immunoblots indicated that overexpression of GSK3β induces the nuclear localization of FoxO1 in neonatal rat cardiomyocytes (CMs). On the other hand, reporter gene assays showed that the transcriptional activity of FoxO1 was inhibited by shRNA-mediated knock-down of GSK3β. These results suggest that GSK3β plays a critical role in mediating the nuclear localization/transcription of FoxO1 in CMs. Cardiac hypertrophy induced by 4 weeks of transverse aortic constriction (TAC) was significantly attenuated in GSK3β (S9A) knock-in mice (βKI), in which GSK3β is constitutively active. Staining of myocardial sections in wild type (WT) mice demonstrated that FoxO1 is localized primarily in the cytosol after TAC. However, FoxO1 was localized primarily in the nucleus in βKI mice after TAC. Both p27kip1 and atrogin-1, known targets of FoxO1 which negatively regulate hypertrophy, were significantly upregulated in βKI hearts after TAC. These results suggest that localization of FoxO1 is critically regulated by GSK3β in vivo and that active GSK3β induces nuclear localization of FoxO1. Overexpression of GSK3β or FoxO1 significantly reduced the size of CMs, and co-overexpression of GSK3β and FoxO1 enhanced the effects of each in vitro. However, co-expression of dominant negative (DN) FoxO1 significantly attenuated the effect of GSK3β upon the size of CMs (LacZ=100±4%, GSK3β=69±4%*, FoxO1=43±2%*#, GSK3β+FoxO1=31±2%*#, GSK3β+DN-FoxO1=87±5%#, *p<0.05 vs LacZ, #p<0.05 vs GSK3β, n=50). Collectively, these results show that GSK3β induces nuclear localization of FoxO1, possibly through phosphorylation, and FoxO1 plays an essential role in mediating the anti-hypertrophic action of GSK3β in CMs.