Regulation of Smooth Muscle Cell Differentiation by RhoA

描述（申请人提供）：血管平滑肌细胞（Vascular smooth muscle cell， SMC）分化是血管发生和血管生成过程中一个非常重要的过程，SMC表型的改变在包括动脉粥样硬化、高血压和再狭窄在内的几种主要心血管疾病的进展中起着重要作用。提出的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）是唯一被证明能调控所有SMC分化标记基因的转录因子。由于RhoA调节SRF，并且由于RhoA活性受到许多环境因素（基质、收缩激动剂、生长因子、机械拉伸）的影响，我们假设Rho信号对于调节SMC特异性转录和最终SMC表型很重要。我们的具体目标如下：目标1 -确定有助于调节smc特异性基因表达的rho依赖性信号通路。我们将测量SMC中的Rho激活，我们将使用显性负性方法来确定Rho和Rho效应子对SMC特异性启动子/报告子结构的调节程度。目的2 -确定Rho影响SMC中srf依赖性转录的机制。我们将研究Rho对SRF磷酸化和DNA结合的调控作用，以及SRF与其他转录因子的相互作用。目的3 -确定RhoA信号传导对体内SMC分化的影响。由于SMC表型受许多不同环境因素的调控，而这些环境因素无法在培养模型中精确再现，因此研究SMC在体内的分化非常重要。我们将使用SM肌球蛋白重链启动子在小鼠SMC中特异性表达显性阴性和构成活性形式的Rho。测量SMC分化标记基因表达，检查血管和器官形态，确定增殖指数将评估SMC分化的影响。这些目标的完成将有助于更好地理解调节SMC分化的信号和转录机制，并可能为心血管治疗干预提供新的靶点。

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.