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: 8246228
• 负责人: Jiliang Zhou
• 金额: $ 39.5万
• 依托单位: ALBANY MEDICAL COLLEGE
• 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 2012-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8246228
• 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

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.

描述（由申请人提供）：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) 表型调节的新机制。 揭示平滑肌表型转换的机制是更好地了解平滑肌相关血管疾病病理学的重要一步。 Hippo 信号通路从果蝇到哺乳动物在进化上都是保守的，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着关键作用。在哺乳动物中，细胞接触和其他未知机制激活Hippo途径核心成分Mst1/2激酶，从而磷酸化并激活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，而未磷酸化形式的 YAP 易位到细胞核中，与各种转录因子结合，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 通路成分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。 该提案中描述的实验将批判性地评估 Hippo-YAP 通路在平滑肌表型调节中发挥综合作用的假设。在目标 1 中，首先我们将通过 YAP shRNA 腺病毒转导来敲低大鼠颈动脉球囊损伤模型中的 YAP 表达，以确定 YAP 在血管病变形成中的作用。 然后，我们将通过生成平滑肌特异性 YAP 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，我们将定义 Hippo 通路成分在调节平滑肌表型调节中的作用。 拟通过 SMC 功能获得和丧失检测来研究 Hippo 通路核心成分 Mst1/2 和 Lats1/2 在 SMC 表型调节中的功能，并通过使用大鼠球囊损伤模型确定血管损伤期间 YAP 上调与激活的 Hippo 通路信号传导和 YAP 负调节的相对重要性。 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 基序的相互作用对于其功能来说是可有可无的，而与 TEAD 的相互作用对于 YAP 通过消除 SRF 与平滑肌基因启动子内 CArG 盒的结合来消除平滑肌基因表达至关重要。因此，我们将通过凝胶位移、co-IP、报告基因和 ChIP 测定来确定 TEAD 在 Hippo-YAP 介导的平滑肌表型调节中的作用及其潜在机制。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 Hippo 通路的成员可能是改善血管疾病的适当治疗靶点。 公共卫生相关性：心血管疾病是美国人的第一大死因。 我们的工作重点是表征转录因子/信号级联的功能，并了解平滑肌表型调节的机制，这将为治疗或预防心血管系统中平滑肌相关疾病提供基础。