Dixit Madhulika, Bess Elke, Fisslthaler Beate, Härtel Frauke V, Noll Thomas, Busse Rudi, Fleming Ingrid
Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
Cardiovasc Res. 2008 Jan;77(1):160-8. doi: 10.1093/cvr/cvm017. Epub 2007 Sep 19.
Phosphorylation of forkhead box O (FoxO) transcription factors induces their nuclear exclusion and proteosomal degradation. Here, we investigated the effect of fluid shear stress on FoxO1a in primary cultures of human endothelial cells and the kinases that regulate its phosphorylation.
Shear stress (12 dynes/cm2) elicited the phosphorylation, nuclear exclusion, and degradation of FoxO1a. Inhibition of Akt signalling using either a dominant negative (DN) mutant of Akt or downregulation of Gab1 largely failed to affect the shear stress-induced changes in FoxO1a, while a DN-AMP-activated protein kinase (AMPK) abrogated its shear stress-induced phosphorylation and degradation. Similar effects were observed using the AMPK inhibitor compound C. Moreover, in an in vitro assay, the AMPK directly phosphorylated FoxO1a. As FoxO1a regulates the expression of angiopoietin-2 (Ang-2), we determined the role of shear stress and the AMPK in this phenomenon. Not only did the DN-AMPK increase the expression of Ang-2 in cells maintained under static conditions, it also abrogated the shear stress-induced decrease in FoxO1a and Ang-2 protein levels. Functionally, Ang-2 sensitizes endothelial cells to the effects of tumour necrosis factor (TNF)-alpha, and DN-AMPK increased basal endothelial cell E-selectin expression and permeability as well as the increase induced by TNF-alpha.
These data indicate that the AMPK activated by fluid shear stress is a novel regulator of FoxO1a phosphorylation and protein levels. Moreover, as the AMPK-dependent phosphorylation and degradation of FoxO1a attenuates Ang-2 expression and protects against the pro-inflammatory actions of TNF-alpha, this kinase may be a useful target to prevent the progression of vascular diseases.
叉头框O(FoxO)转录因子的磷酸化会导致其核输出及蛋白酶体降解。在此,我们研究了流体剪切应力对人内皮细胞原代培养物中FoxO1a的影响以及调节其磷酸化的激酶。
剪切应力(12达因/平方厘米)引发了FoxO1a的磷酸化、核输出及降解。使用Akt的显性负性(DN)突变体或下调Gab1抑制Akt信号传导,在很大程度上未能影响剪切应力诱导的FoxO1a变化,而DN-AMP激活蛋白激酶(AMPK)消除了其剪切应力诱导的磷酸化和降解。使用AMPK抑制剂化合物C也观察到了类似的效果。此外,在体外实验中,AMPK直接使FoxO1a磷酸化。由于FoxO1a调节血管生成素-2(Ang-2)的表达,我们确定了剪切应力和AMPK在此现象中的作用。DN-AMPK不仅增加了在静态条件下培养的细胞中Ang-2的表达,还消除了剪切应力诱导的FoxO1a和Ang-2蛋白水平的降低。在功能上,Ang-2使内皮细胞对肿瘤坏死因子(TNF)-α的作用敏感,而DN-AMPK增加了基础内皮细胞E-选择素的表达和通透性以及TNF-α诱导的增加。
这些数据表明,流体剪切应力激活的AMPK是FoxO1a磷酸化和蛋白水平的新型调节因子。此外,由于AMPK依赖的FoxO1a磷酸化和降解减弱了Ang-2的表达并抵御了TNF-α的促炎作用,这种激酶可能是预防血管疾病进展的有用靶点。