SGK1 Signaling Pathways in Vascular Remodeling

描述(由申请人提供)：异常血管平滑肌细胞(SMC)的增殖和存活是导致动脉粥样硬化和血管损伤后再狭窄的标志性血管病理。丝裂原信号通路在损伤后被激活，刺激丝氨酸/苏氨酸蛋白激酶，迅速调节控制细胞周期进程和细胞生存过程的关键基因和调节蛋白的磷酸化。在这方面，新的数据表明，血清和糖皮质激素诱导蛋白1(SGK1)的活性与细胞增殖和肿瘤细胞生存过程的变化有关。尽管SGK1在上皮细胞生物学中的作用已经取得了很大进展，但它在血管平滑肌细胞功能和病变形成过程中的作用还完全不清楚。我们发现激活的SGK1过表达可诱导A7r5大鼠主动脉SMC的增殖和存活表型。这与细胞周期进程加快有关，这是由于G1向S转变的增加。此外，我们还发现在损伤的颈动脉中SGK1的活性增强。为了阐明这些效应背后的分子机制，我们进行了一种激酶底物蛋白微阵列来筛选新的SGK1靶点。我们的初步研究确定线粒体脱乙酰酶SIRT3可能是SGK1的靶标。根据这些发现，我们假设SGK1通过刺激血管SMC生长和抑制血管SMC凋亡而促进体内新生内膜病变的发展，其机制依赖于SGK1介导的SIRT3的磷酸化和线粒体功能的调节。为了验证这一假设，我们建立了一个独特的SMC特异性SGK1基因敲除小鼠模型。我们将使用从该模型中分离的SMC以及SMC稳定的细胞系进行功能丧失和功能获得的实验。此外，我们还将利用这些资源来检验以下具体目标：1)检验针对血管SMC的SGK1基因敲除可抑制线状血管损伤后新生内膜形成的假说；2)检验SGK1可直接磷酸化SIRT3从而调节其蛋白功能的假说；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。 公共卫生相关性：这项提案试图确定SGK1是动脉支架或血管成形术导致的血管闭塞的重要介体。此外，它还将提供对SGK1在血管平滑肌细胞中作用的分子机制的洞察。最终，这些研究可能确定SGK1作为一种新的治疗靶点，用于治疗因动脉粥样硬化、再狭窄或高血压而导致的闭塞性血管疾病。

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. PUBLIC HEALTH RELEVANCE: This proposal seeks to identify SGK1 as an important mediator of vascular occlusion that occurs as a consequence of arterial stenting or angioplasty. In addition, it will provide insight into the molecular mechanisms responsible for the actions of SGK1 in vascular smooth muscle cells. Ultimately, these studies may identify SGK1 as a novel therapeutic target for occlusive vascular diseases that occur as a consequence of atherosclerosis, restenosis or hypertension.