Calcium Signaling Roles of STIM1 and STIM2 in Smooth Muscle

描述（由申请人提供）：钙信号在控制细胞事件中是至关重要的。在平滑肌细胞（SMC）中，短期Ca2+信号控制收缩反应，而长期Ca2+信号通过Ca2+介导的转录控制调节细胞生长和增殖。提出的研究集中在两个中心重要的Ca2+信号转导，STIM1和STIM2。STIM蛋白是精细调谐的内质网（ER） Ca2+传感器。STIM蛋白被触发自结合并转运到专门的ER-PM连接中，在该连接中，STIM蛋白控制高度Ca2+选择性的“存储操作”Orai通道。STIM蛋白也靶向“电压操作”的l型Ca2+通道（LTCC），并对这两个通道靶点施加相互控制，激活Orai但使LTCC失活，这可能对SMC生长表型变化很重要，其中LTCC丢失，STIM-Orai信号占主导地位。虽然STIM1已被深入研究，但一个主要的挑战是了解研究较少的STIM2蛋白的作用：其结构、功能和表达的差异如何导致强大的信号传导和表型变化。虽然整个动物敲除STIM1或STIM2都是致命的，但我们在小鼠中产生了条件smc靶向的STIM1， STIM2和STIM+STIM2缺失。SMC特异性STIM1-KO导致发育不良的动物过早死亡，SMC在体内和体外的生长转变能力存在重大缺陷。SMC条件STIM1/STIM2双敲除是围产期致命的，这表明STIM1和STIM2在SMC功能和生长转变中发挥着重要但不同的作用。使用这些系统，我们有两个独立但相互依存的特定目标。了解STIM1和STIM2激活的机制和区别，并定义它们的分子偶联以靶Ca2+通道。利用突变修饰、高分辨率Ca2+和FRET成像以及电生理学，我们的目标是定义STIM1的传感和靶通道耦合的分子基础，定义在研究较少的STIM2蛋白中机制上的重要区别。在新的结构见解和STIM1和STIM2之间的主要功能区别的基础上，我们的研究重点是定义两种STIM蛋白与Orai和LTCC靶通道相互作用和抑制的相互作用。2. 了解STIM1和STIM2在丝裂原诱导的SMC从静止表型向增殖表型转变中的不同作用。我们利用SMC靶向的STIM1和STIM2零背景小鼠与分子探针一起特异性修饰STIM1和STIM2介导的靶通道耦合，旨在确定STIM1和STIM2在介导SMC生长转变中的差异有效性。研究将确定STIM1和STIM2表达的变化及其激活和通道耦合的区别，如何导致Ca2+信号的不同谱、NFAT激活和生长转变的改变。我们的研究应用：(a)关于STIM蛋白结构和通道偶联的新知识，(b)创新的STIM1和STIM2基因缺失动物模型，(c)评估STIM1和STIM2靶通道偶联的创新探针-为STIM1在控制SMC生长转变中的关键作用提供新的认识。我们的目标还解决了关于STIM2机制、作用和作用的基本信息缺乏的问题。来自这些研究的信息对于理解血管SMC损伤反应如何发生以及如何控制它们至关重要。我们的模型预测，STIM1和STIM2的不同表达模式可能是SMC是否发生表型改变的决定性因素。因此，这项工作对于理解和预防主要血管疾病（包括动脉粥样硬化、高血压和动脉再狭窄）背后的SMC生长变化，以及哮喘和支持肿瘤生长的血管生成中肺部反应背后的SMC表型变化具有重要意义。

DESCRIPTION (provided by applicant): Calcium signals are vital in controlling cellular events. In smooth muscle cells (SMC), short term Ca2+ signals control contractile responses whereas longer term Ca2+ signals regulate cell growth and proliferation through Ca2+-mediated transcriptional control. The proposed studies focus on the two centrally important Ca2+ signal transducers, STIM1 and STIM2. STIM proteins are finely-tuned endoplasmic reticulum (ER) Ca2+ sensors. STIM proteins are triggered to self-associate and translocate into specialized ER-PM junctions within whom the STIM proteins gate the highly Ca2+ selective "store-operated" Orai channels. STIM proteins also target "voltage-operated" L-type Ca2+ channels (LTCC) and exert reciprocal control over these two channel targets, activating Orai but deactivating LTCC, likely important to SMC growth phenotype change in which LTCC is lost and STIM-Orai signaling predominates. While STIM1 has been intensively examined, a major challenge is to understand the role of the poorly-studied STIM2 protein: how differences in its structure, function and expression lead to powerful signaling and phenotypic changes. While whole animal knockout of either STIM1 or STIM2 are lethal, we generated conditional SMC-targeted deletions of STIM1, STIM2, and STIM+STIM2 in mice. The SMC-specific STIM1-KO results in poorly-developed animals dying early, with major defects in the ability of SMC to undergo growth transition, in vivo and in vitro. The SMC conditional STIM1/STIM2 double knockout is perinatally lethal indicating important yet distinct roles of STIM1 and STIM2 in SMC function and growth transition. Using these systems, we have two independent but inter-dependent specific aims 1. To understand the mechanisms and distinctions between STIM1 and STIM2 activation, and to define their molecular coupling to target Ca2+ channels. Using mutational modifications, high resolution Ca2+ and FRET imaging, and electrophysiology, we aim to define the molecular basis of sensing and target channel coupling for STIM1, defining mechanistically important distinctions in the poorly studied STIM2 protein. Building on new structural insights and major functional distinctions between STIM1 and STIM2, our studies focus on defining the interactions through which the two STIM proteins interact with and gate Orai and LTCC target channels. 2. To understand the distinct roles of STIM1 and STIM2 in mediating mitogen-induced growth transition of SMC from the quiescent to proliferative phenotype. Using our SMC-targeted STIM1 and STIM2 null-back- ground mice together with molecular probes to specifically modify STIM1- and STIM2-mediated target channel coupling, we aim to define the differential effectiveness of STIM1 and STIM2 in mediating SMC growth transition. Studies will determine how changes in STIM1 and STIM2 expression and distinctions in their activation and channel coupling, lead to distinct profiles of Ca2+ signals, NFAT activation, and altered growth transition. Our studies apply: (a) new knowledge on the structure and channel-coupling of STIM proteins, (b) innovative STIM1 and STIM2 gene-deletion animal models, (c) innovative probes to assess STIM1 and STIM2 target channel coupling - to provide new understanding of the crucial role of STIM1 in controlling SMC growth transition. Our goals also address a fundamental paucity of information on understanding the mechanism, action and role of STIM2. Information from these studies is crucial to understanding how vascular SMC injury responses occur and how they may be controlled. Our model predicts that distinct patterns of STIM1 and STIM2 expression can be a determining factor in whether SMC undergo phenotype change. Hence the work has fundamental importance in understanding and preventing the SMC growth changes that underlie major vascular diseases including atherosclerosis, hypertension, and arterial restenosis, and SMC phenotype change that underlies lung responses in asthma and angiogenesis that supports growth of tumors.