Regulation of Smooth Muscle Cell Differentiation by RhoA

描述(申请人提供)：血管平滑肌细胞(SMC)分化是血管生成和血管生成过程中的一个非常重要的过程，人们认识到SMC表型的变化在几种主要心血管疾病的进展中发挥作用，包括动脉粥样硬化、高血压和再狭窄。本研究的总体目标是确定环境信号调节SMC分化标记基因表达的机制。血清反应因子(SRF)是目前发现的唯一能调控SMC分化标志物基因的转录因子。由于RhoA调节SRF，而且RhoA活性受许多环境信号(基质、收缩激动剂、生长因子、机械拉伸)的影响，我们假设Rho信号对调节SMC特异性转录并最终调节SMC表型很重要。我们的具体目标如下：目的1-确定有助于调节SMC特异性基因表达的Rho依赖的信号通路。我们将测量SMC中Rho的激活，并使用显性负向方法来确定Rho和Rho效应器在SMC特异性启动子/报告结构的调控中的作用程度。目的2-确定Rho影响SMC中SRF依赖转录的机制。我们将研究Rho对SRF磷酸化和DNA结合的调节，以及对SRF与其他转录因子相互作用的影响。目的3-确定RhoA信号在体内对SMC分化的影响。由于SMC的表型受许多不同的环境信号调节，而这些环境信号在培养模型中不能准确复制，因此在体内研究SMC的分化是很重要的。我们将使用SM肌球蛋白重链启动子在小鼠的SMC中特异性地表达优势的、阴性的和结构性的活性形式的Rho。检测SMC分化标志物基因表达，观察血管和器官形态，测定增殖指数，将评估对SMC分化的影响。这些目标的完成将有助于更好地理解调控SMC分化的信号和转录机制，并可能成为心血管治疗干预的新靶点。

DESCRIPTION (provided by applicant): Vascular smooth muscle cell (SMC) differentiation is a very important process during vasculogenesis and angiogenesis, and it is recognized that alterations in SMC phenotype play a role in the progression of several prominent cardiovascular diseases including atherosclerosis, hypertension, and restenosis. The overall goal of the proposed studies is to identify the mechanisms by which environmental signals regulate SMC differentiation marker gene expression. Serum response factor (SRF) is the only transcription factor that has been shown to regulate all of the SMC differentiation marker genes. Since RhoA regulates SRF, and because RhoA activity is affected by many environmental cues (matrix, contractile agonists, growth factors, mechanical stretch) we hypothesize that Rho signaling is important for regulating SMC-specific transcription, and ultimately, SMC phenotype. Our specific aims are as follows: Aim 1 - to identify the Rho-dependent signaling pathways that contribute to the regulation of SMC-specific gene expression. We will measure Rho activation in SMC, and we will use dominant negative approaches to determine the extent to which Rho and Rho effectors contribute to the regulation of SMC-specific promoter/reporter constructs. Aim 2 - to identify the mechanisms by which Rho affects SRF-dependent transcription in SMC. We will study the effects of Rho on the regulation of SRF phosphorylation and DNA binding, and on SRF's interaction with other transcription factors. Aim 3 - to determine the effects of RhoA signaling on SMC differentiation in vivo. Because SMC phenotype is regulated by many diverse environmental cues that cannot be accurately reproduced in culture models, it is important to study SMC differentiation in vivo. We will express dominant negative and constitutively active forms of Rho specifically in SMC in mice using the SM myosin heavy chain promoter. Measuring SMC differentiation marker gene expression, examining vessel and organ morphology, and determining proliferation indices will assess effects on SMC differentiation. Completion of these aims should lead to a better understanding of the signaling and transcription mechanisms that regulate SMC differentiation and perhaps to novel targets for cardiovascular therapeutic intervention.