Hydrogen peroxide stress stimulates phosphorylation of FoxO1 in rat aortic endothelial cells
Hydrogen peroxide stress stimulates phosphorylation of FoxO1 in rat aortic endothelial cells
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DOI：
10.1038/aps.2009.201
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发表时间：
2010-02-01
影响因子：
8.2
通讯作者：
Li, Hao
中科院分区：
文献类型：
--
作者：
Wang, Ye-yu;Chen, Si-min;Li, Hao
目的：探讨叉头转录因子（FoxO） O亚家族磷酸化是否参与大鼠主动脉内皮细胞（RAECs）氧化应激反应。方法：用H2O2处理RAECs， Western blot法检测其磷酸化状态。FoxO1的亚细胞定位采用核分离、胞浆分离、Western blot和免疫细胞化学检测。荧光素酶报告基因法测定FoxO1在H2O2胁迫下的转录活性。实时荧光定量PCR检测FoxO1靶基因的表达。结果：H2O2胁迫刺激raec中FoxO1 Thr24和Ser256位点的磷酸化，并呈浓度和时间依赖性。用PI-3K抑制剂预处理raec可抑制Akt的活化，阻止fox01的磷酸化。akt介导的磷酸化促进fox01的核排斥。由外源性fox01反激活的irs驱动的荧光素酶活性被过氧化氢胁迫适度抑制。FoxO因子靶基因Bim的表达在过氧化氢刺激下受到akt介导的磷酸化的负调控。结论：我们的数据表明，PI-3K/Akt信号通路介导的FoxO1磷酸化参与了血管内皮细胞对氧化应激的反应。
Aim: To examine whether the phosphorylation of the O subfamily of forkhead transcription factors (FoxO) is involved in response to oxidative stress in rat aortic endothelial cells (RAECs).Methods: RAECs were treated with H2O2 and phosphorylation status of proteins were evaluated by Western blot analysis. The subcellular localization of FoxO1 was determined by nuclear and cytosolic fractionation followed by Western blot analysis as well as immunocytochemistry. The transcriptional activity of FoxO1 in H2O2 stress was assessed by luciferase reporter assay. Expression of FoxO1 target gene was determined by real-time PCR analysis.Results: H2O2 stress stimulated phosphorylation of FoxO1 at Thr24 and Ser256 in a concentration and time dependent manner in RAECs. Pretreatment of RAECs with PI-3K inhibitors abolished the activation of Akt and prevented the phosphorylation of FoxO1. Akt-mediated phosphorylation promoted nuclear exclusion of FoxO1. An IRS-driven luciferase activity transactivated by exogenous FoxO1 was modestly suppressed by hydrogen peroxide stress. The expression of Bim, a target gene of FoxO factors, was negatively regulated by Akt-mediated phosphorylation in response to hydrogen peroxide stimulation.Conclusion: Our data demonstrate that phosphorylation of FoxO1 by PI-3K/Akt signaling is implicated in response to oxidative stress in vascular endothelial cells.