Receptor-regulated Ca2+ Entry Pathways in Smooth Muscle

描述(申请人提供)：血管平滑肌细胞(VSMC)的增殖和迁移对血管发育、血管生成和修复是必不可少的；它们的功能障碍导致血管疾病，如动脉粥样硬化、高血压微血管重塑和平滑肌肉瘤。在这些疾病中，VSMC从静止的分化和收缩表型转变为合成的增殖和迁移表型，这种情况可以在培养中概括。有明确的证据表明，这种VSMC表型改变在动脉粥样硬化和其他血管闭塞疾病中具有重要的临床意义，但这种改变的分子机制仍不完全清楚。VSMC的表型调控伴随着离子通道表达的改变：合成的VSMC下调L型钙通道的表达，上调典型的瞬时受体电位通道的表达。我们的初步研究表明，与静止的新鲜分离的细胞相比，合成培养的大鼠主动脉VSMC中TRPC6和新发现的钙感受器STIM1以及钙通道Orai1和Orai3的表达增加。我们假设STIM1是Orai1、Orai3和TRPC6通道功能所需的VSMC内钙信号的主调节因子，STIM1、Orai和TRPC上调导致的钙内流增加有助于疾病中VSMC的增殖和迁移。为了支持这一假设，我们发现利用沉默RNA(SiRNA)敲除STIM1抑制了培养的VSMC的增殖，我们发现激动剂特异性地激活了不同的钙通道，并发现STIM1在调节这些通道方面具有显著的多功能性。事实上，在合成的VSMC中，STIM1的敲除取消了：i)PDGF激活的Orai1通道；ii)异多聚体Orai1/3贡献的凝血酶激活的通道；iii)二酰甘油(DAG)激活的TRPC6通道。在AIM1中，我们将利用全细胞膜片钳对凝血酶激活的钙内流通路进行生物物理表征，并确定STIM1寡聚、细胞定位以及与Orai1和Orai3的相互作用在激活这一钙内流通路中的作用。在AIM2中，我们将确定肌浆网或质膜相关的STIM1是否参与DAG激活TRPC6，通过FRET显微镜检查STIM1寡聚的作用以及STIM1和TRPC6的相互作用，并使用免疫共沉淀检测它们之间的相互作用。在Aim3中，我们将在血管损伤的大鼠模型中检验Orai1、Orai3和TRPC6上调也在体内发生的假设，并利用编码siRNA的腺病毒确定体内沉默这些蛋白对新生内膜形成的影响。这一提议的结果将使人们更好地了解VSMC的生理学，并揭示旨在控制血管疾病(如动脉粥样硬化和高血压)中VSMC增殖和迁移的药物治疗的新靶点。

DESCRIPTION (provided by applicant): Vascular smooth muscle cell (VSMC) proliferation and migration are essential for vascular development, angiogenesis and repair; their dysfunction contributes to vascular diseases such as atherosclerosis, hypertensive microvessel remodeling and Leiomyosarcomas. In these diseases VSMC switch from a quiescent differentiated and contractile phenotype to a synthetic proliferative and migratory phenotype, a condition that can be recapitulated in culture. There is clear evidence that this VSMC phenotypic modulation is of paramount clinical importance in atherosclerosis and other vascular occlusive diseases, yet the molecular mechanisms of this modulation remain incompletely understood. VSMC phenotypic modulation is accompanied by a change in ion channel expression: synthetic VSMC downregulate the expression of L-type Ca2+ channels and upregulate that of canonical transient receptor potential (TRPC) channels. Our preliminary studies have demonstrated an increase in TRPC6 and the newly discovered calcium sensor STIM1, and calcium channels Orai1 and Orai3 expression in synthetic cultured rat aortic VSMC, as compared to quiescent freshly isolated cells. We hypothesize that STIM1 is a master regulator of Ca2+ signaling in VSMC required for Orai1, Orai3 and TRPC6 channel function and that increased Ca2+ entry as a result of STIM1, Orai and TRPC upregulation contribute to VSMC proliferation and migration in disease. In support of this hypothesis we found that STIM1 knockdown using silencing RNA (siRNA) inhibited VSMC proliferation in culture and we revealed agonist-specific activation of distinct Ca2+ channels and remarkable STIM1 versatility in regulating these channels. Indeed, knockdown of STIM1 in synthetic VSMC abrogated the function of: i) PDGF-activated Orai1 channels; ii) thrombin-activated channels contributed by heteromultimeric Orai1/3 and iii) Diacylglycerol (DAG)-activated TRPC6 channels. In Aim1 we will biophysically characterize thrombin-activated Ca2+ entry pathways using whole cell patch clamp and determine the role of STIM1 oligomerization, cellular localization and interaction with Orai1 and Orai3 in the activation of this Ca2+ entry pathway. In Aim2 we will determine whether sarcoplasmic reticulum- or plasma membrane- associated STIM1 is involved in TRPC6 activation by DAG, examine the role of STIM1 oligomerization and the interaction of STIM1 and TRPC6 by FRET microscopy and the interaction of their native counterparts using co-immunoprecipitations. In Aim3 we will test the hypothesis that Orai1, Orai3 and TRPC6 upregulation also occurs in vivo in a rat model of vascular injury and determine the effect on neointimal formation of in vivo silencing of these proteins using adenovirus encoding siRNA. The results from this proposal will generate a better understanding of VSMC physiology and unveil novel targets for drug therapy aimed at controlling VSMC proliferation and migration that occur during vascular diseases such as atherosclerosis and hypertension.