Calcium-dependent Regulation of Smooth Muscle Phenotype

描述(由申请人提供)：本提案的总体目标是确定钙(Ca)信号对血管平滑肌细胞(SMC)表型的不同调控机制。SMC表型调控的特征是SMC分化标志基因表达(SMGx)的改变，包括SMC cr-肌动蛋白、平滑肌肌球蛋白重链(SMMHC)和SM22a。SMa-肌动蛋白、SMMHC和SM22a的转录调控部分是通过转录因子SRF(血清反应因子)与Cargc/S调控启动子元件结合来调节的。多个依赖于SRF-Carg的信号通路被描述为在发育过程中调节SMC的表型调节，在成熟的收缩SMC中以及在血管疾病(即动脉粥样硬化)中。然而，尽管钙与自然界中几乎所有的生物过程都有联系，包括SMC收缩，但钙在调节SMGx和SMC表型调节中扮演什么角色仍然不清楚。我们最近在成年SMC中发现，通过L型电压门控钙通道的钙内流导致SMGx增加，其机制依赖于RhoA/Rho-Kinase、myocardin(一种SMC选择性的SRF辅助因子)以及增加SRF与SmGx所需的Carg顺式启动子调控元件的结合。我们实验室最近令人兴奋的研究提供的证据表明，收缩激动剂1-磷酸鞘氨醇通过VGCCs/Rho-Kinase/SRF和选择性的S1P受体亚型增加SMGx。然而，鞘氨醇-1-磷酸，而不仅仅是VGCC的激活，也通过钙调神经磷酸酶和钙激活的转录因子NFAT2在完整染色质的Carg启动子元件中丰富来介导SMGx。综上所述，前面的研究清楚地暗示了鞘氨醇-1-磷酸和去极化依赖的钙信号对SMGx的不同调控作用。因此，目标1将确定钙对成年SMC中SMGx的差异调节的分子机制。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-Kinase/myocardin调节依赖于SRF的SMGx，但通过钙调神经磷酸酶/NFAT信号通路和诱导染色质结构的变化来不同地调节钙激活的转录因子之间的相互作用，从而增强SRF与Carg元件的结合。目的2确定钙依赖信号在胚胎干细胞来源的SMC分化、成熟和功能中的作用。我们将使用基因缺失的胚胎干细胞(在目标1中定义)来确定这些因素在SMC分化的拟胚体模型中调节SMC分化/成熟/功能的作用。目的3将利用SMC选择性Cre/LOX技术确定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，在SMC发育和维持收缩表型期间，钙依赖的分子机制调节SMGx，并且这些控制机制在与动脉粥样硬化相关的表型改变过程中改变。

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.