Regulation of Smooth Muscle Differentiation by RhoA

描述（由申请人提供）：血管平滑肌细胞（SMC）分化是血管发生和血管生成过程中非常重要的过程，众所周知，SMC表型的改变在几种主要心血管疾病状态（包括动脉粥样硬化、高血压和再狭窄）的进展中起作用。然而，我们刚刚开始了解调控SMC分化的转录机制，我们对调控这一过程的信号传导机制知之甚少。我们以前表明，小GTdR，RhoA，是SMC分化标志物基因表达的重要调节因子，我们已经扩展并证实了这些研究，证明了强RhoA激动剂，鞘氨醇-1磷酸，刺激SMC特异性转录，RhoA效应子mDia 1和mDia 2在这一过程中发挥关键作用，RhoA/mDia信号的作用是由心肌素相关转录因子（MRTF）的核定位介导的。目前的建议的目标是进一步确定RhoA依赖的信号机制，调节SMC分化标志物基因的表达，并研究RhoA信号在SMC分化过程中的参与。我们的具体目标如下：1）研究RhoA信号通路对SMC分化的影响。我们将使用他莫昔芬诱导的SM MHC和Wnt 1 Cre小鼠在SMC和假定SMC中表达组成型活性和显性阴性形式的RhoA。我们将研究小鼠在发育过程中和成年血管损伤后SMC分化的变化。2)鉴定RGS-RhoGEFs对SMC中RhoA活性和SMC分化标志基因表达的调节作用。我们将使用一种成熟的逆转录病毒介导的siRNA方法来敲低大鼠主动脉SMC中的LARG、p115和PDZ RhoGEF，强调对RhoA活化、SMC分化标记基因表达、SMC迁移和MRTFs核定位的影响。3)目的：探讨mDia 2在平滑肌细胞中的作用机制。我们将测试我们的假设，磷酸化的mDia 2的Rho激酶增强mDia 2的活性，我们将检查mDia 2的核结构，CRM-1依赖的核输出方面的核中的作用，和MRTF的转录活性。这些目标的完成应导致更好地了解SMC分化的调节。公共卫生相关性：血管平滑肌细胞分化是血管发育过程中一个非常重要的过程，众所周知，这一过程中的改变在几种主要心血管疾病状态（包括动脉粥样硬化、高血压和再狭窄）的进展中发挥作用。我们的建议检查调节平滑肌分化的分子机制，并应有助于确定治疗这些疾病的治疗靶点。

DESCRIPTION (provided by applicant): Vascular smooth muscle cell (SMC) differentiation is a very important process during vasculogenesis and angiogenesis, and it is well recognized that alterations in SMC phenotype play a role in the progression of several prominent cardiovascular disease states including atherosclerosis, hypertension, and restenosis. However, we are just beginning to understand the transcription mechanisms that regulate SMC differentiation, and we know very little about the signaling mechanisms that regulate this process. We previously showed that the small GTPase, RhoA, was an important regulator of SMC differentiation marker gene expression, and we have extended and confirmed these studies by demonstrating that the strong RhoA agonist, sphingosine-1 phosphate, stimulates SMC-specific transcription, that the RhoA effectors mDia1 and mDia2 play a critical role in this process, and that the effects of RhoA/mDia signaling are mediated by nuclear localization of the myocardin-related transcription factors (MRTFs). The goals of the current proposal are to further define the RhoA-dependent signaling mechanisms that regulate SMC differentiation marker gene expression and to study the involvement of RhoA signaling during SMC differentiation in vivo. Our specific aims are as follows; 1) To determine the contribution of RhoA signaling to SMC differentiation of in vivo. We will express constitutively active and dominant negative forms of RhoA in SMC and presumptive SMC using tamoxifen-inducible SM MHC and Wnt1 Cre mice. We will examine mice for changes in SMC differentiation during development and in adults following vessel injury. 2) to identify the RGS- RhoGEFs that regulate RhoA activity and SMC differentiation marker gene expression in SMC. We will use a well-established retroviral-mediated siRNA approach to knock down LARG, p115, and PDZ RhoGEF in rat aortic SMC emphasizing effects on RhoA activation, SMC differentiation marker gene expression, SMC migration, and nuclear localization of the MRTFs. 3) To identify the molecular mechanisms that regulate mDia2 function in SMC. We will test our hypothesis that phosphorylation of mDia2 by Rho-kinase enhances mDia2 activity, and we will examine mDia2's role in the nucleus in regard to nuclear structure, CRM-1- dependent nuclear export, and the transcriptional activity of the MRTFs. Completion of these aims should lead to a better understanding of the regulation of SMC differentiation. PUBLIC HEALTH RELEVANCE: Vascular smooth muscle cell differentiation is a very important process during the development of blood vessels and it is well recognized that alterations in this process play a role in the progression of several prominent cardiovascular disease states including atherosclerosis, hypertension, and restenosis. Our proposal examines the molecular mechanisms that regulate smooth muscle differentiation and should help to identify therapeutic targets for the treatment of these diseases.