Calcium-dependent Regulation of Smooth Muscle Phenotype

平滑肌表型的钙依赖性调节

• 批准号: 7837497
• 负责人: Brian Robert Wamhoff
• 金额: $ 17.36万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7837497
• 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）：该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 分化标记基因表达 (SMGX) 的改变，包括 SM cr-肌动蛋白、平滑肌肌球蛋白重链 (SMMHC) 和 SM22a。 SM a-肌动蛋白、SMMHC 和 SM22a 的转录调节部分是通过转录因子 SRF（血清反应因子）与 CArG c/s 调节启动子元件结合来调节的。多种 SRF-CArG 依赖性信号通路已被描述用于调节发育过程中的 SMC 表型调节、成熟的收缩性 SMC 和血管疾病（即动脉粥样硬化）。然而，尽管 Ca 与自然界中几乎所有生物过程（包括 SMC 收缩）都有联系，但它目前尚不清楚 Ca 在调节 SMGX 和 SMC 表型调节中发挥什么作用我们最近在成人 SMC 中发现 Ca 通过 L 型电压门控 Ca 通道流入。 (VGCC) 通过依赖 RhoA/Rho 激酶、心肌素（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 顺式启动子调控元件的结合。我们实验室提供的证据表明，收缩激动剂 1-磷酸鞘氨醇部分通过 VGCC/Rho-激酶/SRF 和选择性 S1P 受体增加 SMGX然而，1-磷酸鞘氨醇（不仅仅是 VGCC 激活）还通过钙调神经磷酸酶和完整染色质 CArG 启动子元件内的 NFAT2（一种 Ca 激活的转录因子）介导 SMGX。综上所述，前面的研究清楚地表明了 1-磷酸鞘氨醇和去极化依赖性 Ca 信号传导对 SMGX 的差异调节作用。因此，目标 1 将确定 Ca 差异调节成体 SMC 中 SMGX 的分子机制。我们的假设是，1-磷酸鞘氨醇和去极化诱导的 Ca2+ 流入通过 RhoA/Rho-kinase/myocardin 调节 SRF 依赖性 SMGX，但差异性地调节由钙调磷酸酶/NFAT 信号通路介导的 Ca2+ 激活转录因子的相互作用并诱导变化染色质结构中增强 SRF 与 CArG 元件的结合。目标 2 将确定 Ca 依赖性信号传导对胚胎干细胞衍生的 SMC 的分化、成熟和功能的作用。我们将使用对选定基因（目标 1 中定义）基因无效的胚胎干细胞来确定这些因子在 SMC 分化的胚状体模型中调节 SMC 分化/成熟/功能中的作用。目标 3 将使用 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。总体假设是，Ca 依赖性分子机制在 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。