Regulation of Smooth Muscle 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表型的改变在动脉粥样硬化、高血压和再狭窄等几种主要心血管疾病的进展中起着重要作用。然而，我们刚刚开始了解调节SMC分化的转录机制，我们对调节这一过程的信号传导机制知之甚少。我们之前的研究表明，小GTPase RhoA是SMC分化标记基因表达的重要调节因子，我们通过证明强RhoA激动剂鞘氨醇-1磷酸刺激SMC特异性转录，RhoA效应物mDia1和mDia2在这一过程中发挥关键作用，以及RhoA/mDia信号传导的作用是由心肌素相关转录因子（mrtf）的核定位介导的，从而扩展和证实了这些研究。本研究的目标是进一步明确调节SMC分化标记基因表达的RhoA依赖的信号机制，并研究RhoA信号在SMC体内分化中的作用。我们的具体目标如下：1)确定RhoA信号传导对体内SMC分化的贡献。我们将使用他莫昔芬诱导的SM MHC和Wnt1 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。我们将检测小鼠在发育期间和成年血管损伤后SMC分化的变化。2)鉴定在SMC中调控RhoA活性和SMC分化标志基因表达的RGS- RhoGEFs。我们将使用一种成熟的逆转录病毒介导的siRNA方法来敲除大鼠主动脉SMC中的LARG、p115和PDZ RhoGEF，强调对RhoA激活、SMC分化标记基因表达、SMC迁移和mrtf的核定位的影响。3)明确mDia2在SMC中调控功能的分子机制。我们将验证rho激酶磷酸化mDia2增强mDia2活性的假设，并将研究mDia2在核结构、依赖于CRM-1的核输出和mrtf转录活性方面在细胞核中的作用。这些目标的完成应该导致更好地理解SMC分化的调控。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，这一过程的改变在几种主要心血管疾病的进展中起作用，包括动脉粥样硬化、高血压和再狭窄。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.