Phosphoproteome and Ang II-induced VSMC Gene Expression

描述（由申请方提供）：血管平滑肌细胞（VSMC）表型在健康和疾病状态下显示出显著的异质性。然而，VSMC表型调节的分子机制尚不清楚。在血管紧张素II刺激的VSMC模型系统中，我们发现磷酸化蛋白质组和转录组的全局变化。细胞骨架蛋白|β-肌动蛋白被磷酸化，导致磷酸化可能改变肌动蛋白动力学的假设，这是细胞运动，生长和基因表达变化所需的过程。基因表达谱分析表明，几个细胞表面受体，转录调节因子和蛋白质参与器官损伤的差异表达。这些基因的启动子区域的生物信息学分析显示，在遗传上保守的DMA c/s元件。我们推测这些基因组调控元件结合转录调节因子，其是Ang II诱导的信号转导的靶点并改变基因表达。本研究的短期目标是：（i）通过使用免疫纯化和质谱法（MS）鉴定Ang II处理的VSMC中的磷酸化蛋白靶标。我们将测试通过基因表达变化调节VSMC表型的磷酸化蛋白的网络，（ii）确定Ang II诱导的磷酸化的位点和β-肌动蛋白的磷酸化影响VSMC增殖、迁移和基因表达的机制，（iii）确定基因的Ang II反应性的分子基础。我们的长期目标是了解血管紧张素II（Ang II）对VSMC功能的调节以及VSMC表型调节的分子机制。人类主要疾病如动脉粥样硬化和血管成形术后再狭窄的发病机制涉及VSMC表型转换。

DESCRIPTION (provided by applicant): Vascular smooth muscle cell (VSMC) phenotypes display remarkable heterogeneity in health and disease states. Yet the molecular mechanisms underlying modulation of VSMC phenotypes are not known. In Ang ll-stimulated VSMC model system, we find global changes in both the phosphoproteome and transcriptome. The cytoskeletal protein |3-actin is phosphorylated, leading to the hypothesis that phosphorylation could alter actin-dynamics, a process required for cell motility, growth and gene expression changes. Gene expression profiling indicated that several cell surface receptors, transcription regulators and proteins involved in organ damage are differentially expressed. Bioinformatics analysis of the promoter regions of these genes showed phylogenetically conserved DMA c/s-elements. We speculate that these genome regulatory elements bind transcriptional regulators, which are targets of Ang ll-induced signal transduction and alter gene expression. The short-term goals of this study are: (i) Identify phosphorylated protein targets in Ang ll-treated VSMCs by using immuno-purification and mass spectrometry (MS). We will test the network of phosphorylated proteins that modulate VSMC phenotype through gene expression changes, (ii) Determine site(s) of Ang ll- induced phosphorylation and the mechanism by which phosphorylation of p-actin affects VSMC proliferation, migration and gene expression, (iii) Define the molecular basis for Ang ll-responsiveness of genes. Our long-term goal is to understand regulation of VSMC function by angiotensin II (Ang II) and the molecular mechanism of phenotypic modulation of VSMC. Pathogenesis of major human diseases such as atherosclerosis and post-angioplasty restenosis involves VSMC phenotype switching.