Role of Hippo-YAP Pathway in Smooth Muscle Phenotypic Modulation

描述(申请人提供)：拟议研究的总体目标是确定Hippo信号通路调节血管平滑肌细胞(SMC)表型变化的新机制。揭示平滑肌表型转换的机制是更好地理解血管疾病的病理基础的重要一步。河马信号通路从果蝇到哺乳动物在进化上是保守的，并通过调节细胞增殖和凋亡在控制器官大小和肿瘤发生中发挥关键作用。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保留在细胞质中，而非磷酸化的YAP转位到细胞核中，在那里它与各种转录因子结合，调控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，以及Hippo-Yap通路在表型调节中的新作用。这项建议中描述的实验将对河马-YAP通路在平滑肌表型调节中发挥综合作用的假设进行批判性评估。在目标1中，首先我们将通过YAP shRNA腺病毒转导大鼠颈动脉球囊损伤模型来下调YAP的表达，以确定YAP在血管病变形成中的作用。然后，我们将通过建立一只平滑肌特异的YAP基因敲除小鼠来研究YAP在体内平滑肌发育中的功能作用。在目标2中，我们将确定河马通路组件在调节平滑肌表型调节中的作用。本研究拟通过大鼠血管球囊损伤模型，通过SMC功能缺失和获得性分析，探讨河马通路核心成分Mst1/2和Lats1/2在SMC表型调控中的作用，并确定YAP上调与激活的河马通路信号和YAP负调控在血管损伤中的相对重要性。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基序的相互作用是其功能所必需的，而与Teads的相互作用是YAP通过取消SRF与平滑肌基因启动子中Carg盒的结合而取消平滑肌基因表达所必需的。因此，我们将通过凝胶漂移、共IP、报告和芯片分析来确定Teads在河马-YAP介导的平滑肌表型调节中的作用及其可能的机制。这些研究的完成将为控制平滑肌分化和表型调节的机制提供新的见解，并确定河马通路的成员，这些成员可能是改善血管疾病的合适的治疗靶点。 公共卫生相关性：心血管疾病是美国人的头号死亡原因。我们的工作集中在研究转录因子/信号转导通路的功能，了解血管平滑肌表型调控的机制，这将为治疗或预防心血管系统中的平滑肌相关疾病提供基础。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is American's number 1 cause of mortality. Our work focuses on characterizing the function of transcription factors/signaling cascades and understanding the mechanisms underlying smooth muscle phenotypic modulation that will provide the foundation for treating or preventing smooth muscle-related diseases in cardiovascular system.