SGK1 Signaling Pathways in Vascular Remodeling

描述（申请人提供）：异常血管平滑肌细胞（SMC）增殖和存活是血管损伤后动脉粥样硬化和再狭窄的标志性血管病理。有丝分裂信号级联在损伤后被激活，刺激丝氨酸/苏氨酸蛋白激酶，快速调节控制细胞周期进程和细胞存活过程的关键基因和调节蛋白的磷酸化。在这方面，新出现的数据表明，血清和糖皮质激素诱导激酶1 （SGK1）的活性与肿瘤细胞增殖和存活过程的变化有关。尽管SGK1在上皮细胞生物学中的作用已经取得了很大进展；它在血管平滑肌细胞功能和病变形成过程中的作用；特别是，完全是未知的。我们已经证明，激活的SGK1过表达诱导A7r5大鼠主动脉SMC的增殖和存活表型。这与细胞周期进程的加速相关，因为G1到S转变的增加。此外，我们发现SGK1活性在损伤的颈动脉中增强。为了阐明这些作用的分子机制，我们进行了激酶底物蛋白微阵列筛选新的SGK1靶点。我们最初的研究确定了线粒体去乙酰化酶Sirt3作为假定的SGK1靶点。鉴于这些发现，我们假设SGK1通过依赖于SGK1介导的Sirt3磷酸化和线粒体功能调节的机制，刺激血管SMC生长和抑制血管SMC凋亡，从而促进体内新内膜病变的发展。为了验证这一假设，我们建立了一个独特的smc特异性SGK1敲除小鼠模型。我们将使用从该模型分离的SMC以及SMC稳定细胞系进行功能丧失和功能获得实验。此外，我们将利用这些资源来研究以下具体目标：1)验证血管SMC靶向敲除SGK1可减弱钢丝诱导的血管损伤中新生内膜形成的假设；2)验证SGK1可直接磷酸化Sirt3从而调节其蛋白质功能的假设；3)证明SGK1/Sirt3信号通路是血管SMC生长和存活的关键决定因素。

DESCRIPTION (provided by applicant): Aberrant vascular smooth muscle cells (SMC) proliferation and survival is a hallmark vascular pathology underlying atherosclerosis and restenosis following vascular injury. Mitogenic signaling cascades that become activated upon injury stimulate serine/threonine protein kinases that rapidly regulate the phosphorylation of key genes and regulatory proteins that control cell cycle progression and cell survival processes. In this regard, emerging data indicate that the activity of serum and glucocorticoid inducible kinase 1 (SGK1) is linked to changes in cellular proliferation and survival processes in tumor cells. Although much progress has been made regarding the role of SGK1 in epithelial cell biology; its role in vascular smooth muscle cell function and in the development of lesion formation; in particular, is completely unknown. We have shown that over-expression of activated SGK1 induces a proliferative and survival phenotype in A7r5 rat aortic SMC. This correlated with an acceleration of cell cycle progression owing to an increase in G1 to S transition. Further, we found that SGK1 activity is enhanced in injured carotid arteries. To elucidate the molecular mechanism underlying these effects, we conducted a kinase substrate protein microarray to screen for novel SGK1 targets. Our initial studies identified the mitochondrial deacetylase, Sirt3 as a putative SGK1 target. In light of these findings, we hypothesize that SGK1 promotes neointimal lesion development in vivo by stimulating vascular SMC growth and inhibiting vascular SMC apoptosis via a mechanism that relies upon SGK1-mediated phosphorylation of Sirt3 and modulation of mitochondrial function. To test this hypothesis, we established a unique SMC-specific SGK1 knockout mouse model. We will use SMC isolated from this model as well as SMC stable cell lines in loss- and gain-of-function experiments. In addition, we will utilize these resources as we examine the following specific aims: 1) to test the hypothesis that vascular SMC-targeted knockout of SGK1 attenuates the development of neointimal formation in response to wire-induced vascular injury, 2) to test the hypothesis that SGK1 can directly phosphorylate Sirt3 and thereby regulate its protein function, 3) to demonstrate that a SGK1/Sirt3 signaling pathway is a critical determinant of vascular SMC growth and survival.