Role of Hippo-YAP Pathway in Smooth Muscle Phenotypic Modulation

拟议研究的总体目标是确定河马信号传递的新机制 该通路调节血管平滑肌细胞(SMC)的表型变化。解开 参与平滑肌表型转换的机制是朝着更好的方向迈出的重要一步 了解与平滑肌相关的血管疾病的病理学。河马的信号通路是 在进化上从果蝇到哺乳动物都是保守的，在控制器官大小和 通过调节细胞增殖和凋亡来促进肿瘤的发生。在哺乳动物中，细胞接触和其他未知 机制激活河马通路核心成分Mst1/2激酶，使其磷酸化并激活 Lats1/2激酶，进而直接磷酸化转录调节因子YAP。磷酸化的YAP是 保留在细胞质中，而非磷酸化形式的YAP转位到细胞核中，在那里它与 多种转录因子，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通路组件在血管平滑肌中的表达 河马-YAP通路在表型调控中的新作用。本提案中描述的实验将 对河马-YAP通路在平滑肌中发挥整合作用的假说进行批判性评估 表型调节。在目标1中，首先我们将下调YAP在大鼠颈动脉球囊损伤中的表达 YAP shRNA腺病毒转导模型研究YAP在血管损伤中的作用 队形。然后我们将通过以下方式研究YAP在体内平滑肌发育中的功能作用 产生一种平滑肌肉特异性YAP基因敲除小鼠。在目标2中，我们将定义河马途径的作用 参与调节平滑肌表型调节的成分。建议进行研究，以调查 河马通路核心成分Mst1/2和Lats1/2在Gain-And对SMC表型调控中的作用 SMC的功能丧失分析，并确定YAP上调与激活的相对重要性 大鼠球囊损伤血管过程中河马通路信号转导及YAP的负性调节 模特。在目标3中，我们将确定YAP调节平滑肌表型的机制。 初步数据表明，YAP与含有转录因子的PY基序的相互作用是必不可少的 由于其功能，而与Teads的相互作用是YAP消除平滑肌基因的必要条件 在平滑肌基因启动子中通过取消SRF与Carg盒的结合来表达。因此我们 将确定Teads在河马-YAP介导的平滑肌表型调节中的作用及其 通过凝胶移位、共IP、报告和芯片分析揭示其可能的作用机制。这些研究的完成将提供 对血管平滑肌分化和表型调控机制的新认识 确定河马通路的成员可能是改善血管的适当治疗靶点 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.