Novel targets of rapamycin and Akt in vascular smooth muscle cell differentiation

描述（由申请人提供）：药物洗脱支架通过在很大程度上防止再狭窄，彻底改变了冠状动脉病变的血运重建。重要的是，新出现的长期安全性数据表明，雷帕霉素洗脱支架可能由于内皮细胞的不完全愈合而增加晚期血栓形成的风险。因此，理想的支架药物应选择性地抑制血管平滑肌细胞（VSMC）的增殖和分化，而不抑制再内皮化。在我们对VSMC表型调节的分子机制的研究中，我们发现mTOR抑制剂雷帕霉素通过诱导一个新的基因表达程序来促进VSMC分化，包括收缩蛋白。我们发现雷帕霉素对mTOR效应物S6K1的抑制，以及由此产生的Akt2的激活对于这种作用是必要的。令人惊讶的是，Akt1抑制VSMC分化。我们还令人兴奋地发现，雷帕霉素激活了VSMC转录因子GATA-6，该因子对于雷帕霉素诱导的分化是必需的。这种转录因子是VSMC特有的，在内皮细胞中没有发现。我们发现雷帕霉素还能诱导促进VSMC分化的主要调控转录辅激活因子心肌素的表达。众所周知，mTOR通路调节蛋白质合成。值得注意的是，我们发现vsmc特异性转录调控是该途径的一种新功能。我们假设雷帕霉素通过Akt2调控转录因子是该药物抑制VSMC增殖和促进分化的关键机制。我们的目的是了解雷帕霉素调节VSMC前分化转录因子的机制。(1)为了解决这一假设，我们的目标是确定雷帕霉素激活Akt2如何调节GATA-6。我们假设雷帕霉素诱导GATA-6磷酸化，导致其核易位和激活。我们将使用来自Akt1或Akt2敲除小鼠的siRNA、VSMC和GATA-6磷酸化位点突变体来确定哪种激酶磷酸化GATA-6，以及磷酸化如何影响GATA-6的活性。我们假设一种磷酸化模拟突变体GATA-6可能具有组成性活性，因此可能是一种潜在的vsmc特异性前分化治疗药物。(2)我们的目标是利用siRNA方法确定心肌素在雷帕霉素诱导的表达中的作用。我们将使用DNA结合、染色质免疫沉淀和启动子报告方法来确定雷帕霉素是否以及如何促进心肌蛋白的表达和/或活性。(3)我们将使用野生型、Akt1或Akt2敲除小鼠的损伤模型，确定Akt2缺失是否会加剧，Akt1缺失是否会减少内膜增生，以及雷帕霉素的治疗反应是否需要Akt2。我们建议，了解雷帕霉素的这些下游靶点及其调控的分子机制将为改进支架治疗的发展提供关键靶点。

DESCRIPTION (provided by applicant): Drug-eluting stents have revolutionized revascularization of coronary artery lesions by largely preventing restenosis. Importantly, emerging long term safety data suggests that rapamycin-eluting stents pose an elevated risk of late thrombosis, likely due to incomplete healing of the endothelium. An ideal stent drug would, therefore, selectively inhibit proliferation and promote differentiation of vascular smooth muscle cells (VSMC), without inhibiting re-endothelialization. In our studies of the molecular mechanisms underlying VSMC phenotypic modulation, a process necessary for angiogenesis, atherosclerosis, and restenosis, we have discovered that the mTOR inhibitor rapamycin promotes VSMC differentiation by inducing a new program of gene expression, including contractile proteins. We have found that rapamycin inhibition of the mTOR effector S6K1, and the resulting activation of Akt2 is necessary for this effect. Surprisingly, Akt1 inhibited VSMC differentiation. We have also made the exciting discovery that rapamycin activates a VSMC transcription factor, GATA-6, and that this factor is necessary for rapamycin-induced differentiation. This transcription factor is specific to VSMC and not found in endothelial cells. We find that rapamycin also induces expression of the master regulatory transcriptional coactivator myocardin that promotes VSMC differentiation. The mTOR pathway is well known to regulate protein synthesis. Notably, we identify regulation of VSMC-specific transcription as a novel function for this pathway. We hypothesize that rapamycin regulation of transcription factors via Akt2 is a critical mechanism by which this drug inhibits proliferation and promotes differentiation in VSMC. We aim to understand the mechanisms by which rapamycin regulates prodifferentiation transcription factors in VSMC. (1) In addressing this hypothesis, we aim to determine how rapamycin activation of Akt2 regulates GATA-6. We hypothesize that rapamycin induces phosphorylation of GATA-6 that leads to its nuclear translocation and activation. We will use siRNA, VSMC from Akt1 or Akt2 knockout mice, and GATA-6 phosphorylation site mutants to determine which kinase phosphorylates GATA-6, and how phosphorylation influences GATA-6 activity. We hypothesize that a phospho-mimetic mutant GATA-6 may be constitutively active, and therefore a potential VSMC-specific prodifferentiation therapeutic. (2) We aim to use siRNA methods to determine the role of myocardin in rapamycin-induced expression. We will determine whether and how rapamycin promotes myocardin expression and/or activity using DNA binding, chromatin immunoprecipitation, and promoter reporter methods. (3) We will determine whether Akt2 deletion exacerbates, and Akt1 deletion diminishes intimal hyperplasia, and whether the therapeutic response to rapamycin requires Akt2, using an injury model in wild type, Akt1 or Akt2 knockout mice. We propose that understanding these downstream targets of rapamycin and the molecular mechanisms by which they are regulated will provide key targets for development of improved stent therapeutics. PUBLIC HEALTH RELEVANCE Cardiovascular disease is a major cause of morbidity and mortality in the western world. While stents coated with the drug rapamycin have greatly reduced the risk of restenosis (re-blockage of the vessel) after coronary artery angioplasty, recent findings have revealed that they confer a small but significant risk of heart attack or death. This project aims to understand the molecular mechanisms underlying the beneficial anti-restenotic response of vascular smooth muscle cells to rapamycin, as this knowledge may allow us to tailor future therapeutics to inhibit these cells specifically, avoiding detrimental side effects on other cell types that can cause the dangerous complications.