Modulation of calcium signaling by changes in STIM expression

描述（由申请方提供）：胞质Ca 2+浓度增加是多种信号转导途径的常见组分，调节从快速事件（如膜融合和肌肉收缩）到增殖、分化和凋亡控制的各种反应。由于Ca 2+信号通常发生在数秒至数分钟的时间范围内，因此Ca 2+瞬变如何调节数小时至数天内发生的事件尚不清楚。我们实验室最近的研究已经确定了早期生长反应1（EGR 1）作为STIM 1表达的调节因子，STIM 1是钙库操作的Ca 2+进入的必需组分，是非兴奋细胞中Ca 2+进入的主要手段。这一观察结果使我们假设，Ca 2+信号通过协调的EGFR依赖性控制STIM表达进行调制。第一个目的是使用荧光素酶、电泳迁移率改变试验（EMSA）和染色质免疫沉淀（ChIP）的组合来定义EGFR介导的STIM 1和STIM 2转录控制，以调查EGFR 1的特性可以应用于其他家族成员的程度。实验将在优化的细胞系统中进行，包括HEK 293细胞和Jurkat T细胞。第二个目的是分析受体介导的STIM 1和STIM 2表达控制。在此，将使用胰岛素、血小板衍生生长因子（PDGF;成纤维细胞）和干细胞因子（SCF;肥大细胞）作为激动剂，在源自野生型和EGFR 1敲除小鼠的成纤维细胞和肥大细胞中检查受体介导的EGFR 1和EGFR 4表达变化改变STIM 1和STIM 2表达的能力。将使用STIM 1和STIM 2敲除成纤维细胞检查Ca 2+本身对受体介导的EGR 1和EGR 4表达的贡献。最后，第三个目的是研究EGFR介导的Ca 2+信号生理控制。胰岛素与SCF诱导的STIM表达变化对肥大细胞中Ca 2+信号的影响将通过在用抗DNP IgE引发后暴露于不同浓度的DNP-BSA来确定。将在负载fura-2的细胞中确定对胞质Ca 2+浓度的总体影响，而Ca 2+电流的电生理学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STIM 1和STIM 2介导的通道激活变化。最后，将确定EGFR介导的胞质Ca 2+浓度变化对肥大细胞活化的影响，重点是野生型、EGFR 1缺失和STIM 1缺失肥大细胞中细胞因子的产生和释放。 结合考虑，这些调查将提供一个新的理解的框架，细胞中的Ca 2+信号是如何通过自分泌，旁分泌和内分泌因子之间的串扰在较长的时间内调制。

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.