Regulation and Function of Myocardin in Vascular Pathobiology

描述(由申请人提供)：血清反应因子(SRF)和Myocardin(MYOCD)构成了一个分子触发开关，用于激活一组包含SRF结合Carg盒的血管平滑肌细胞(VSMC)细胞收缩和离子通道基因。我们首先证明了MYOCD(而不是SRF)水平与VSMC分化程度相关。由于VSMC的分化程序不是固定的，在许多疾病过程中MYOCD水平发生变化，因此我们有兴趣了解MYOCD表达的调节及其作为维持VSMC静止、收缩状态的动态平衡开关的功能。因此，我们在一系列论文中报道，MYOCD(A)足以通过依赖于SRF的收缩和离子通道基因表达的变化来赋予VSMC收缩能力；(B)完全抑制骨骼肌分化程序；(C)控制越来越多的microRNAs，包括microRNA143/145基因；以及(D)通过依赖p38MAPK的途径被转化生长因子β1正向诱导。初步数据进一步证明了转录抑制因子和新的microRNA的顺式元件，以及功能数据首次表明MYOCD表达在血管闭塞性疾病、脂质摄取和炎症标志物表达方面具有有利作用。总体而言，我们不断增长的工作为检验MYOCD是正常VSMC分化的内稳态开关这一假设提供了关键基础。本论文旨在通过一系列相互关联的特定目标来阐明MYOCD表达的转录和转录后调控，以及在损伤诱导的新生内膜扩张和动脉粥样硬化形成过程中MYOCD在血管重塑中的作用。在目标1中，细菌人工染色体转基因小鼠将阐明导致MYOCD激活或抑制的新的顺式作用元件的功能。在目标2中，新型MYOCD功能丧失和功能获得的小鼠将直接评估这种强大的辅助因子在实验性血管疾病过程中的作用，包括对VSMC炎症、增殖和转分化状态的影响。在目标3中，将进行将我们生成的人和小鼠CArGome数据(&gt；84,000 Carg盒)与VSMC的RNA-SEQ整合的基因组研究，其中MYOCD在没有SRF或存在SRF的情况下表达。这样的分析将揭示对MYOCD有反应的CArGome的一个子集，以及血管疾病中新的不依赖SRF的MYOCD靶基因。因此，计划中的研究将为MYOCD在正常出生后发育和血管疾病过程中的体内调控提供新的见解，并提供与MYOCD相关的新信息，MYOCD可能是与身体损伤和动脉粥样硬化疾病相关的血管重塑的抑制因子。这些研究的结果将在设计新的治疗策略方面具有巨大的应用价值，以对抗急性和慢性血管疾病，以及可能改变MYOCD表达/活性的其他疾病(例如哮喘、高血压、阿尔茨海默病)。

DESCRIPTION (provided by applicant): Serum response factor (SRF) and Myocardin (MYOCD) constitute a molecular trigger switch for the activation of a battery of vascular smooth muscle cell (VSMC) cyto-contractile and ion channel genes containing SRF- binding CArG boxes. We first showed that levels of MYOCD (but not SRF) correlate with the degree of VSMC differentiation. Since the VSMC differentiation program is not fixed and subject to adaptation in a number of disease processes where levels of MYOCD change, we have been interested in understanding the regulation of MYOCD expression and its function as a homeostatic switch for the preservation of a quiescent, contractile state in VSMC. Accordingly, we have reported in a series of papers that MYOCD (a) is sufficient for conferring VSMC contractile competence through SRF-dependent changes in contractile and ion channel gene expression; (b) completely represses the program of skeletal muscle differentiation; (c) controls a growing number of microRNAs, including the microRNA143/145 gene; and (d) is positively induced by TGF¿1 through a p38MAPK-dependent pathway. Preliminary data further demonstrate cis elements for a transcriptional repressor and new microRNA as well as functional data showing, for the first time, the favorable effects of MYOCD expression on vascular occlusive disease, lipid uptake, and inflammatory marker expression. Collectively, our growing body of work serves as a critical foundation to test the hypothesis that MYOCD is a homeostatic switch for normal VSMC differentiation. This thesis will be tested through a series of inter-related specific aims designe to elucidate transcriptional and post-transcriptional control of MYOCD expression and the role of MYOCD in vascular remodeling during injury-induced neointimal expansion and atherogenesis. In Aim 1, bacterial artificial chromosome transgenic mice will elucidate the function of novel cis-acting elements leading to activation or repression of MYOCD. In Aim 2, novel MYOCD loss- and gain-of-function mice will directly assess the role of this powerful cofactor in experimental vascular disease processes, including effects on VSMC inflammatory, proliferative, and transdifferentiative states. In Aim 3, genomic studies integrating human and mouse CArGome data we have generated (> 84,000 CArG boxes) with RNA-seq of VSMC where MYOCD is expressed in the absence or presence of SRF will be carried out. Such an analysis will reveal a subset of the CArGome that is responsive to MYOCD as well as new SRF-independent MYOCD target genes of import in vascular disease. Thus, the planned studies will yield new insight into the in vivo regulation of MYOCD expression during normal postnatal development and in vascular disease processes as well as novel information related to MYOCD as a likely inhibitor of vascular remodeling associated with physical injury and atherosclerotic disease. The results of these studies will have enormous applications for devising new therapeutic strategies to combat acute and chronic vascular diseases and perhaps other diseases where MYOCD expression/activity may be altered (e.g., asthma, hypertension, Alzheimer's disease).