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

基本信息

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
项目类别：
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

项目摘要

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河马信号通路调节血管平滑肌细胞（SMC）表型调节的新机制。阐明平滑肌表型转换所涉及的机制是更好地理解平滑肌相关血管疾病病理学的重要一步。河马信号通路从果蝇到哺乳动物的进化保守，并通过调节细胞增殖和凋亡来控制器官的大小和肿瘤发生在控制器官的大小和肿瘤发生中。在哺乳动物中，细胞接触和其他未知机制激活了河马途径核心成分MST1/2激酶，以磷酸化和激活LATS1/2激酶，从而直接磷酸化转录调节剂YAP。磷酸化的YAP保留在细胞质中，而未磷酸化的YAP形式将其与各种转录因子结合，以调节控制细胞增殖和细胞凋亡所需的基因表达。我们的初步数据表明，血管平滑肌中河马-YAP途径成分的表达以及河马途径在表型调制中的新作用。本提案中描述的实验将严格评估以下假设：河马-YAP途径在平滑肌表型调制中起着综合作用。在AIM 1中，首先，我们将通过用YAP shRNA腺病毒转导大鼠颈动脉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，我们将通过产生平滑肌特异性YAP敲除小鼠来研究YAP在体内平滑肌发育中的功能作用。在AIM 2中，我们将定义河马途径成分在调节平滑肌表型调节中的作用。提出了研究以研究河马途径核心成分，MST1/2和LATS1/2在SMC表型调节中通过SMC中的功能丧失测定法在SMC表型调节中的功能，并确定YAP上调节的相对重要性与激活的HIPPO途径信号传导的相对重要性，并通过使用Rat Balloon Balloon模型在血管造成的YAP受伤期间对YAP受伤的负调节。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与含有含有转录因子的PY基序的相互作用对于其功能是可分配的，而与teads的相互作用对于通过废除SRF结合在平滑肌基因促进子中的Carg Box来消除平滑肌基因表达至关重要。因此，我们将通过凝胶移位，co-IP，报告基因和芯片测定法确定曲头在河马介导的平滑肌表型调制及其基本机制中的作用。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，并确定河马途径的成员可能是适当的治疗靶标，以改善血管疾病。公共卫生相关性：心血管疾病是美国死亡率的第一名。我们的工作着重于表征转录因子/信号级联的功能，并了解平滑肌表型调制的基础机制，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。