Calcium-dependent Regulation of Smooth Muscle Phenotype

平滑肌表型的钙依赖性调节

• 批准号: 7663254
• 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
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7663254
• 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

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）：本提案的总体目的是确定钙（Ca）信号差异调节血管平滑肌细胞（SMC）表型的机制。SMC表型调节的特征在于SMC分化标志物基因表达（SMGX）的改变，包括SM cr-肌动蛋白、平滑肌肌球蛋白重链（SMMHC）和SM 22 a。SM α-肌动蛋白、SMMHC和SM 22 a的转录调节部分地通过转录因子SRF（血清应答因子）结合CArG c/s调节启动子元件来调节。已经描述了多种SRF-CArG依赖性信号传导途径在发育期间、在成熟收缩性SMC中和在血管疾病（即动脉粥样硬化）中调节SMC表型调节。然而，尽管钙与自然界中几乎所有的生物学过程（包括SMC收缩）都有联系，但目前仍不清楚钙在调节SMGX和SMC表型调节中起什么作用。我们最近在成人SMC中表明，通过L型电压门控Ca通道（VGCC）的Ca内流通过依赖于RhoA/Rho激酶、心肌蛋白（SMC选择性SRF辅因子）的机制导致SMGX增加，并增加SRF与SMGX所需的CArG顺式启动子调节元件的结合。我们实验室最近令人兴奋的研究提供的证据表明，收缩激动剂鞘氨醇-1-磷酸盐部分通过VGCC/Rho激酶/SRF和选择性S1 P受体亚型增加SMGX。然而，鞘氨醇-1-磷酸，而不是单独的VGCC激活，也介导SMGX通过钙调磷酸酶和富集NFAT 2，钙激活的转录因子，在CArG启动子元件的完整染色质。综上所述，先前的研究清楚地暗示了通过鞘氨醇-1-磷酸和去极化依赖性Ca信号传导对SMGX进行差异调节的作用。因此，目标1将确定钙差异调节SMGX在成人SMC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调节SRF依赖的SMGX通过RhoA/Rho激酶/心肌蛋白，但差异调节钙激活的转录因子介导的钙调神经磷酸酶/NFAT信号通路的相互作用，并诱导染色质结构的变化，增强结合SRF CArG元素。目的2探讨钙依赖性信号在胚胎干细胞来源的平滑肌细胞分化、成熟和功能中的作用。我们将采用选择基因（目标1中定义）遗传无效的胚胎干细胞，以确定这些因子在SMC分化的胚状体模型中调节SMC分化/成熟/功能的作用。目的3：利用SMC选择性Cre/lox技术，探讨钙信号通路在血管损伤相关SMC表型调节中的作用。总的假设是，钙依赖性分子机制调节SMGX在SMC的发展和维持收缩表型，这些控制机制改变与动脉粥样硬化相关的表型调制。