Modulation of calcium signaling by changes in STIM expression

描述（由申请人提供）：细胞质Ca2+浓度的增加是多种信号转导途径的共同组成部分，调节各种各样的反应，从快速事件如膜融合和肌肉收缩到控制增殖、分化和凋亡。由于Ca2+信号通常发生在几秒到几分钟的时间框架内，Ca2+瞬态如何调节发生在数小时到数天内的事件尚不清楚。我们实验室最近的研究已经确定了早期生长反应1 （EGR1）作为STIM1表达的调节剂，STIM1是储存操作Ca2+进入的必要成分，是Ca2+进入不可兴奋细胞的主要手段。这一观察结果使我们提出了Ca2+信号是通过协调egr依赖的STIM表达控制来调节的假设。第一个目标是使用荧光素酶、电泳迁移位移测定（EMSA）和染色质免疫沉淀（ChIP）的组合来定义egr介导的STIM1和STIM2转录控制，以研究EGR1的特性在多大程度上可以应用于其他家族成员。实验将在优化的细胞系统中进行，包括HEK293细胞和Jurkat T细胞。第二个目的是分析受体介导的对STIM1和STIM2表达的控制。本研究将使用胰岛素、血小板衍生生长因子（PDGF）和干细胞因子（SCF）作为激动剂，在野生型和EGR1敲除小鼠的成纤维细胞和肥大细胞中检测受体介导的EGR1和EGR4表达改变STIM1和STIM2表达的能力。Ca2+本身对受体介导的EGR1和EGR4表达的贡献将通过STIM1和STIM2敲除成纤维细胞进行检测。最后，第三个目的是检查egr介导的Ca2+信号的生理控制。胰岛素和scf诱导的STIM表达变化对肥大细胞Ca2+信号的影响将通过暴露于不同浓度的DNP-BSA后，用抗dnp IgE启动来确定。对胞质Ca2+浓度的总体影响将在fura-2负载细胞中确定，而Ca2+电流的电生理分析将用于测量内源性电流处于可测量水平的肥大细胞中STIM1-和stim2介导的通道激活变化。最后，egr介导的胞质Ca2+浓度变化对肥大细胞活化的影响将被确定，重点关注野生型、EGR1-null和STIM1-null肥大细胞的细胞因子产生和释放。综合考虑，这些研究将提供一个新的框架，了解细胞中的Ca2+信号是如何通过自分泌、旁分泌和内分泌因素之间的串扰在长时间内调节的。

DESCRIPTION (provided by applicant): Increases in cytosolic Ca2+ concentration are a common component of multiple signal transduction pathways regulating a wide variety of responses ranging from rapid events such as membrane fusion and muscle contraction to control of proliferation, differentiation and apoptosis. Since Ca2+ signals typically occur in a time frame of seconds to minutes, how Ca2+ transients can regulate events that occur over hours to days is poorly understood. Recent investigations from our lab have led to the identification of Early Growth Response 1 (EGR1) as a regulator of the expression of STIM1, a required component of store-operated Ca2+ entry, the primary means of Ca2+ entry in non-excitable cells. This observation has led us to the hypothesis that Ca2+ signals are modulated via coordinated EGR-dependent control of STIM expression. The first aim is to define EGR-mediated control of STIM1 and STIM2 transcription using a combination of luciferase, Electrophoretic Mobility Shift Assays (EMSA) and chromatin immunoprecipitation (ChIP) to investigate the extent to which the properties of EGR1 can be applied to other family members. Experiments will be performed in optimized cell systems including HEK293 cells and Jurkat T cells. The second aim is to analyze receptor-mediated control of STIM1 and STIM2 expression. Here, the ability of receptor-mediated changes in EGR1 and EGR4 expression to change STIM1 and STIM2 expression will be examined in both fibroblasts and mast cells derived from wild type and EGR1 knockout mice, using insulin, Platelet-Derived Growth Factor (PDGF; fibroblasts) and Stem Cell Factor (SCF; mast cells) as agonists. The contribution of Ca2+ itself to receptor-mediated EGR1 and EGR4 expression will be examined using STIM1 and STIM2 knockout fibroblasts. Finally, the third aim is to examine EGR-mediated physiological control of Ca2+ signaling. The impact of insulin vs. SCF-induced changes in STIM expression on Ca2+ signals in mast cells will be determined by exposure to varying concentration of DNP-BSA after priming with anti-DNP IgE. Overall impact on cytosolic Ca2+ concentration will be determined in fura-2 loaded cells, while electrophysiological analysis of Ca2+ currents will be used to measure both STIM1- and STIM2-mediated changes in channel activation in mast cells where endogenous currents are at measurable levels. Finally, the impact of EGR-mediated changes in cytosolic Ca2+ concentration on mast cell activation will be determined focusing on cytokine production and release in wild type, EGR1-null and STIM1-null mast cells. Considered in combination, these investigations will provide the framework for a new understanding of how Ca2+ signals in cells are modulated over extended time periods via crosstalk between autocrine, paracrine and endocrine factors.