Role of Hippo-YAP Pathway in Smooth Muscle Phenotypic Modulation

Hippo-YAP 通路在平滑肌表型调节中的作用

• 批准号: 8403797
• 负责人: Jiliang Zhou
• 金额: $ 33.75万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8403797
• 关键词: Ablation; Adenoviruses; Affect; American; Angioplasty; Apoptosis; Arterial Injury; Atherosclerosis; Attenuated; Binding; Biological Assay; Blood Vessels; Boxing; Cardiovascular Diseases; Cardiovascular system; Carotid Arteries; Cell Nucleus; Cell Proliferation; Cells; Contractile Proteins; Cytoplasm; Data; Disease; Dominant-Negative Mutation; Drosophila genus; Family member; Foundations; Gel; Gene Expression; Genes; Goals; Hypertension; Immunohistochemistry; In Vitro; Injury; Knockout Mice; Lesion; Ligands; Mammals; Measures; Mediating; Modeling; Mus; Muscle Development; Nuclear Import; Organ Size; Pathology; Pathway interactions; Phenotype; Phosphotransferases; Play; Prevention; Proteins; Rattus; Regulation; Relative (related person); Reporter; Reporter Genes; Research; Role; Serum Response Factor; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Stimulus; Subfamily lentivirinae; Therapeutic Agents; Tissues; Up-Regulation; Vascular Diseases; Vascular Smooth Muscle; Western Blotting; Work; cell dedifferentiation; cofactor; design; in vivo; injured; insight; knock-down; loss of function; member; migration; mortality; mutant; myocardin; novel; prevent; promoter; protein expression; protein function; research study; restenosis; small hairpin RNA; therapeutic target; transcription factor; tumorigenesis; upstream kinase; vascular smooth muscle cell migration

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.

拟议研究的总体目标是确定河马信号传导的新机制 途径调节血管平滑肌细胞（SMC）的表型调节。 解开 平滑肌表型转换涉及的机制是迈向更好的一步 了解平滑肌相关的血管疾病的病理。 河马信号通路是 从果蝇到哺乳动物的进化保守，在控制器官大小和 肿瘤发生通过调节细胞增殖和凋亡。 在哺乳动物中，细胞接触和其他未知 机制激活河马途径核心成分MST1/2激酶以磷酸化和激活 LATS1/2激酶，进而直接磷酸化转录调节剂YAP。磷酸化的YAP是 保留在细胞质中，而未磷酸化的YAP形式易位到核与它与 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在血管平滑肌和A 河马途径在表型调制中的新作用。 该提案中描述的实验将 批判性地评估了河马途径在平滑肌中起综合作用的假设 表型调制。在AIM 1中，首先，我们将在大鼠颈动脉球囊受伤中击倒YAP表达 通过用YAP SHRNA腺病毒转导的模型来确定YAP在血管病变中的作用 形成。 然后，我们将研究YAP在体内平滑肌发育中的功能作用 产生平滑肌肉特异性的YAP敲除鼠标。在AIM 2中，我们将定义河马途径的作用 调节平滑肌表型调节的组件。 提出了研究以研究 河马途径核心成分的功能，MST1/2和LATS1/2在SMC表型调制中通过增益和 在SMC中进行功能丧失测定，并确定YAP上调与激活的相对重要性 通过使用大鼠球囊损伤，血管损伤期间YAP的河马途径信号传导和负调节 模型。 在AIM 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基序的相互作用是可分配的 对于它的功能，与teads的相互作用对于消除平滑肌基因至关重要 通过消除平滑肌基因启动子中的carg盒的结合来表达。因此我们 将确定曲调在河马介导的平滑肌表型调制及其中的作用 凝胶移位，co-IP，记者和CHIP分析的基本机制。这些研究的完成将提供 对控制平滑肌分化和表型调制的机制的新见解以及 识别河马途径的成员，这可能是适当的治疗靶标，以改善血管 疾病。