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Calcium-dependent Regulation of Smooth Muscle Phenotype

平滑肌表型的钙依赖性调节

基本信息

批准号：
7911717
负责人：
Brian Robert Wamhoff
金额：
$ 25.23万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-15 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7911717
关键词：
Acetylation Actins Adult Agonist Alleles Atherosclerosis Binding Biological Assay Biological Process Blood Vessels CREB1 gene Calcineurin Calcineurin B Calcium Calcium Channel Calcium Signaling Calcium/calmodulin-dependent protein kinase Calmodulin Cell Differentiation process Cell Maturation Cell Nucleus Cell Proliferation Chromatin Chromatin Structure Coupled Cyclic AMP-Responsive DNA-Binding Protein Cyclosporine Data Development Differentiation Antigens EP300 gene ES Cell Line Elements FOS gene Family member Gene Expression Genes Genetic Growth Histones Hydrogen Immunohistochemistry In Vitro Injury Lead Lesion Maintenance Mediating Messenger RNA Methylation Modeling Molecular Molecular Profiling Mus Muscle Contraction Muscle Fibers Myosin Heavy Chains Nature Nifedipine Pathway interactions Phase Phenotype Phosphorylation Phosphotransferases Play Preparation Promoter Regions Protein Dephosphorylation Protein Isoforms Proteins Protons Rattus Reagent Receptor Gene Regulation Regulatory Element Research Personnel Reverse Transcriptase Polymerase Chain Reaction Rho-associated kinase Rodent Model Role Sarcoplasmic Reticulum Serum Response Factor Signal Pathway Signal Transduction Smooth Muscle Smooth Muscle Myocytes Smooth Muscle Myosins Solid Sphingosine-1-Phosphate Receptor System Technology Testing Time Transcriptional Regulation Vascular Diseases activating transcription factor calcineurin phosphatase channel blockers edg-1 Protein edg-3 Protein embryonic stem cell histone acetyltransferase histone modification laser capture microdissection loss of function myocardin novel nuclear factors of activated T-cells overexpression programs promoter receptor reconstitution response sphingosine 1-phosphate transcription factor voltage

项目摘要

DESCRIPTION (provided by applicant): The overall aim of this proposal is to determine the mechanisms by which calcium (Ca) signaling differentially regulates vascular smooth muscle cell (SMC) phenotype. SMC phenotypic modulation is characterized by alterations in SMC differentiation marker gene expression (SMGX) including SM cr-actin, smooth muscle myosin heavy chain (SMMHC) and SM22a. Transcriptional regulation of SM a-actin, SMMHC, and SM22a is regulated in part through the transcription factor SRF (serum response factor) binding to CArG c/s regulatory promoter elements. Multiple SRF-CArG-dependent signaling pathways have been described in regulating SMC phenotypic modulation during development, in mature contractile SMCs and in vascular disease (i.e. atherosclerosis. However, although Ca has connections to virtually every biological process in nature, including SMC contraction, it is still unclear what role Ca plays in regulating SMGX and SMC phenotypic modulation. We recently showed in adult SMCs that Ca influx via L-type voltage-gated Ca channels (VGCC) resulted in an increase in SMGX through mechanisms that are dependent on RhoA/Rho-kinase, myocardin (a SMC-selective SRF co-factor) and increased binding of SRF to CArG cis promoter regulatory elements required for SMGX. Exciting recent studies from our lab provide evidence showing that the contractile agonist sphingosine-1-phosphate increases SMGX in part via VGCCs/Rho-kinase/SRF and selective S1P receptor subtypes. However, sphingosine-1-phosphate, not VGCC activation alone, also mediates SMGX through calcineurin and enrichment of NFAT2, a Ca-activated transcription factor, within CArG promoter elements of intact chromatin. Taken together, the preceding studies clearly implicate a role for differential regulation of SMGX by sphingosine-1-phosphate- and depolarization-dependent Ca signaling. Thus, Aim 1 will determine molecular mechanisms by which Ca differentially regulates SMGX in adult SMCs. Our hypothesis is that sphingosine-1-phosphate and depolarization-induced Ca influx regulate SRF-dependent SMGX through RhoA/Rho-kinase/myocardin but differentially regulate the interaction of Ca-activated transcription factors mediated by calcineurin/NFAT signaling pathways and by inducing changes in chromatin structure that enhance binding of SRF to CArG elements. Aim 2 will determine the role of Ca-dependent signaling on differentiation, maturation and function of SMCs derived from embryonic stem cells. We will employ embryonic stem cells genetically null for select genes (defined in Aim 1) to determine the role of these factors in regulating SMC differentiation/maturation/function in the embryoid body model of SMC differentiation. Aim 3 will determine the role of Ca signaling pathways in SMC phenotypic modulation associated with vascular injury using SMC-selective Cre/lox technology. The overall hypothesis is that Ca-dependent molecular mechanisms regulate SMGX during SMC development and maintenance of the contractile phenotype, and that these control mechanisms are altered during phenotypic modulation associated with atherosclerosis.

描述（由申请人提供）：本提案的总体目标是确定钙（Ca）信号传导差异调节血管平滑肌细胞（SMC）表型的机制。SMC表型调节的特征是SMC分化标志基因（SMGX）表达的改变，包括SM cr-actin、平滑肌肌球蛋白重链（SMMHC）和SM22a。sma -actin、SMMHC和SM22a的转录调控部分是通过转录因子SRF（血清反应因子）结合CArG c/s调控启动子元件来调控的。多种srf - carg依赖的信号通路在发育过程中、成熟的收缩性SMC和血管疾病（如动脉粥样硬化）中调节SMC的表型调节。然而，尽管Ca几乎与自然界的每一个生物过程都有联系，包括SMC收缩，但Ca在调节SMGX和SMC表型调节中所起的作用仍不清楚。我们最近在成人SMCs中发现，通过l型电压门控Ca通道（VGCC）的Ca内流导致SMGX的增加，其机制依赖于RhoA/ rho激酶、心肌素（smc选择性SRF辅助因子）和SRF与SMGX所需的CArG顺式启动子调控元件的结合增加。我们实验室最近令人兴奋的研究提供了证据，表明收缩激动剂鞘氨醇-1-磷酸部分通过VGCCs/ rho激酶/SRF和选择性S1P受体亚型增加SMGX。然而，除了VGCC激活外，鞘氨醇-1-磷酸也通过钙调磷酸酶和NFAT2（一种钙激活的转录因子）在完整染色质的CArG启动子元件中的富集介导SMGX。综上所述，上述研究清楚地表明鞘氨醇-1-磷酸和去极化依赖的Ca信号在SMGX的差异调节中起作用。因此，Aim 1将确定Ca在成人SMCs中差异调节SMGX的分子机制。我们的假设是鞘氨醇-1-磷酸和去极化诱导的钙内流通过RhoA/ rho激酶/心肌素调节SRF依赖性SMGX，但通过钙调磷酸酶/NFAT信号通路介导的钙活化转录因子的相互作用以及诱导染色质结构的变化，增强SRF与CArG元件的结合，对钙活化转录因子的相互作用进行差异调节。目的2将确定钙依赖信号在胚胎干细胞衍生的SMCs的分化、成熟和功能中的作用。我们将使用基因选择基因为零的胚胎干细胞（在Aim 1中定义）来确定这些因素在SMC分化的胚胎体模型中调节SMC分化/成熟/功能中的作用。目的3将利用SMC选择性Cre/lox技术确定Ca信号通路在与血管损伤相关的SMC表型调节中的作用。总的假设是，钙依赖的分子机制在SMC发育和收缩表型维持过程中调节SMGX，并且这些控制机制在与动脉粥样硬化相关的表型调节过程中发生改变。