Role of Hippo-YAP Pathway in Smooth Muscle Phenotypic Modulation

拟议研究的总体目标是确定Hippo信号转导的新机制。 信号通路调节血管平滑肌细胞（SMC）的表型调节。 Unraveling the 参与平滑肌表型转换的机制是朝着更好的 了解平滑肌相关血管疾病的病理学。 河马信号通路是 从果蝇到哺乳动物进化上保守，在控制器官大小和 通过调节细胞增殖和凋亡来调节肿瘤的发生。 在哺乳动物中，细胞接触和其他未知的 机制激活Hippo途径核心组件Mst 1/2激酶磷酸化并激活 Lats 1/2激酶，其又直接磷酸化转录调节因子雅普。磷酸化的雅普是 保留在细胞质中，而非磷酸化形式的雅普易位到细胞核中，在那里它与 各种转录因子，以调节控制细胞增殖和凋亡所需的基因表达。 我们的初步数据表明，Hippo-YAP通路组分在血管平滑肌中的表达， Hippo-YAP通路在表型调节中的新作用。 本提案中描述的实验将 批判性地评估Hippo-YAP通路在平滑肌中起整合作用的假设 表型调节在目标1中，首先我们将在大鼠颈动脉球囊损伤中敲低雅普表达 通过用雅普shRNA腺病毒转导的模型来确定雅普在血管损伤中的作用 阵 然后，我们将研究雅普在体内平滑肌发育中的功能作用， 产生平滑肌特异性雅普敲除小鼠。在目标2中，我们将定义Hippo通路的作用， 调节平滑肌表型调节的组分。 建议开展研究， Hippo通路核心组分Mst 1/2和Lats 1/2在SMC表型调节中的功能， SMC中的功能丧失测定，并确定雅普上调与活化的 大鼠血管球囊损伤中Hippo信号通路与雅普的负性调节 模型 在目标3中，我们将确定雅普调节平滑肌表型的机制。 初步数据表明，雅普与含有PY基序的转录因子的相互作用是不稳定的。 由于其功能，而与TEAD的相互作用对于雅普废除平滑肌基因至关重要 通过消除SRF与平滑肌基因启动子内的CArG盒的结合来表达。因此我们 将确定TEAD在Hippo-YAP介导的平滑肌表型调节中的作用， 通过凝胶迁移、co-IP、报告基因和ChIP测定的潜在机制。完成这些研究将提供 对控制平滑肌分化和表型调节的机制的新见解， 确定Hippo通路的成员，这些成员可能是改善血管紧张素转换酶活性的适当治疗靶点。 疾病

The overall goal of the proposed research is to determine the novel mechanisms by which the Hippo signaling pathway regulates the phenotypic modulation of vascular smooth muscle cells (SMCs). Unraveling the mechanisms involved in smooth muscle phenotypic switching is an important step towards better understanding the pathology of smooth muscle-related vascular diseases. The Hippo signaling pathway is evolutionarily conserved from Drosophila to mammals and plays a critical role in controlling organ size and tumorigenesis by regulating cell proliferation and apoptosis. In mammals, cell contact and other unknown mechanisms activate the Hippo pathway core component Mst1/2 kinases to phosphorylate and activate Lats1/2 kinases, which in turn directly phosphorylate the transcriptional regulator YAP. Phosphorylated YAP is retained in cytoplasm whereas unphosphorylated form of YAP translocates into the nucleus where it binds with various transcription factors, to regulate gene expression required for control of cell proliferation and apoptosis. Our preliminary data indicate expression of Hippo-YAP pathway components in vascular smooth muscle and a novel role of Hippo-YAP pathway in phenotypic modulation. Experiments described in this proposal will critically evaluate the hypothesis that the Hippo-YAP pathway plays an integrative role in smooth muscle phenotypic modulation. In Aim 1, first we will knock down YAP expression in a rat carotid artery balloon injury model through transduction with a YAP shRNA adenovirus to determine the role of YAP in vascular lesion formation. Then we will investigate the functional role of YAP in smooth muscle development in vivo by generating a smooth muscle-specific YAP knock-out mouse. In Aim 2, we will define the role of Hippo pathway components in regulating smooth muscle phenotypic modulation. Studies are proposed to investigate the function of Hippo pathway core components, Mst1/2 and Lats1/2 in SMC phenotypic modulation by gain- and loss-of-function assays in SMCs and determine the relative importance of YAP up-regulation versus activated Hippo pathway signaling and negative regulation of YAP during vascular injury by using rat balloon injury model. In Aim 3, we will determine the mechanism by which YAP modulates smooth muscle phenotype. Preliminary data demonstrate that YAP interaction with PY motif containing transcription factors is dispensable for its function while the interaction with TEADs is essential for YAP to abrogate smooth muscle gene expression through abolishing SRF binding to CArG box within smooth muscle gene promoters. Therefore we will determine the role of TEADs in Hippo-YAP mediated smooth muscle phenotypic modulation and its underlying mechanism by gel shift, co-IP, reporter and ChIP assays. Completion of these studies will provide new insights into the mechanisms controlling smooth muscle differentiation and phenotypic modulation and identify members of the Hippo pathway that may be appropriate therapeutic targets for ameliorating vascular diseases.