Oxidative Stress Stimulates Phosphorylation Of Transcription Factor FoxO1 In Vascular Endothelial Cells

Objectives:Endothelial dysfunction is considered a common risk factor for many cardiovascular diseases. Oxidative stress-induced endothelial cell injury is an important mechanism of endothelial dysfunction. Forkhead box O (FoxO) transcription factors, a subgroup of the Forkhead transcription factor family, serve as one of redox-sensitive transcriptional factors. FoxO are involved in the regulation of several cellular responses including cell cycle progression, apoptosis and oxidative stress resistant. We used hydrogen peroxide (H2O2) to model oxidative stress in cultured vascular endothelial cells. The aim of this study was to examine whether the phosphorylation of the FoxO1 transcription factor is involved in response to oxidative stress in vascular endothelial cells. Furthermore, the effects of H2O2 stress on the transcriptional activity of FoxO1 was assessed.Methods:Rat aortic endothelial cells (RAECs) were isolated with the pieces of artery. The morphology of RAECs were observed with inverted phase-contrast microscopy. The purity of RAECs was evaluated by immunocytochemistry with VIII factor related antigen antibody and uptake of Dil-Ac-LDL. We first assessed the expression profile of the FoxO isoforms FoxO1, FoxO3a, and FoxO4 in RAECs and human vascular endothelial cell line EA.hy926 using western blotting of cell extracts. Sub-confluenced vascular endothelial cells were serum-starved for 24h and then used for all experiments. To detect the influence of different levels of oxidative stress on the FoxO1 phosphorylation, vascular endothelial cells were incubated with 200μmol/L, 500μmol/L or 1000μmol/L H2O2 for 20min, then the phosphorylation activity of FoxO1 at Ser-256 and Thr-24 as well as Akt were examined by western blot analysis. Vascular endothelial cells were treated 500μmol/L H2O2 for 20min, 30min or 60min and the relationship between stimulate time periods and the phosphorylation status of proteins were evaluated. In order to clarify whether the phosphorylation of FoxO1 is related to PI3K/Akt signaling pathway, we used two specific PI3K inhibitors LY294002 or wortmannin to block the signaling pathway ahead, then evaluated the phosphorylation of FoxO1 and Akt upon oxidative stress by western blot. After incubation RAECs with 500μmol/L H2O2 for 20min, the subcellular localization of phosphorylated FoxO1 was determined by nuclear and cytosolic fractionation followed by western blot analysis as well as immunocytochemistry under inverted fluorescence microscope. To further assessed the effects of H2O2 stress on the transcriptional activity of FoxO1, cotransfection of RAECs was performed with a forkhead-responsive IRS (insulin-responsive sequence) luciferase reporter plasmid (3×IRS-luc) and either an expression vector encoding FoxO1 or a control plasmid using lipofectiamine 2000 transfection reagent. At 24h after transfection, cells were incubation with 200μmol/L H2O2 for 12h and lysed, then luciferase activity was measured using the dual luciferase repoter assay kit. Finally, we checked the effect of Akt-mediated phosphorylation of FoxO1 on the transcriptional level of its target gene Bim in hydrogen peroxide stress, RAECs were pretreated with or without 10μmol/L LY294002 for 1h and treated with or without 200μmol/L H2O2 for 4h, then total RNA was isolated and the relative mRNA level of Bim was determined by Real-time PCR. Results:The purity of RAECs was above 90% identified by immunocytochemistry and the uptake of Dil-Ac-LDL. Western blot showed that FoxO1 and FoxO3a are the most abundant FoxO isoforms expressed in RAECs and EA.hy926. Under serum-starved conditions, following treatment endothelial cells with various concentration H2O2 for 20 min, the phosphorylation of FoxO1 at Ser-256 and at Thr-24 were increased in a concentration dependent manner. After incubation of endothelial cells with 500μmol/L H2O2 for various time periods, phosphorylation of FoxO1 at Ser-256 and at Thr-24 were increased at 20min,30min and 60min. Total FoxO1 was not significantly affected upon H2O2 treatment. H2O2 aslo resulted in the phosphorylation of Akt in a concentration-and time-dependent increase similar as FoxO1. Pretreatment of RAECs with PI3K inhibitors either 10μmol/L LY294002 or 500nmmol/L wortmannin abolished the activation of Akt and prevented the phosphorylation of FoxO1. The addition of 500μmol/L H2O2 to RAECs for 20min resulted in significant increased phosphorylated FoxO1 in cytosolic fraction and enhanced translocation of phosphorylated FoxO1(Thr-24)from the nucleus to the cytoplasm, compared with control. An IRS-driven luciferase activity was transactivated by exogenous FoxO1, while hydrogen peroxide stress resulted in modestly suppression of IRS promoter activity. Real-time PCR modified that the level of Bim transcript was increased in 4 h of H2O2 treatment, and pretreatment of RAECs with PI3K inhibitor LY294002 increased the induction of Bim in response to H2O2 stimulation.Conclusion:Oxidative stress stimulates phosphorylation of FoxO1 at Ser-256 and at Thr-24 in a concentration and time dependent manner in vascular endothelial cells. The phosphorylation of these sites are mediated by PI3K/Akt signaling. Phosphorylation of FoxO1 triggers its translocation from the nucleus to the cytoplasm and contributes to the negative regulation of its transcriptional activity.