Modulation of calcium signaling by changes in STIM expression

通过 STIM 表达的变化调节钙信号传导

• 批准号: 8366449
• 负责人: Jonathan A Soboloff
• 金额: $ 3.51万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8366449
• 关键词: 2,4-Dinitrophenol; Affect; Agonist; Allergic; Anaphylaxis; Apoptosis; Asthma; Biological Assay; Calcium Signaling; Cell membrane; Cell model; Cells; Chronic; Coupled; Dependence; Early identification; Electrophoretic Mobility Shift Assay; Endocrine; Endoplasmic Reticulum; Event; Exposure to; Family; Family member; Fibroblasts; Fluorescence; Fura-2; Gene Expression; Genetic Transcription; Goals; Growth; Hour; Human; IgE; Immune response; Insulin; Insulin Receptor; Interleukin-13; Investigation; Jurkat Cells; Knock-out; Knockout Mice; Luciferases; Measurable; Measures; Mediating; Membrane Fusion; Muscle Contraction; Outcome; Pathway interactions; Phospholipase C; Physiological; Platelet-Derived Growth Factor; Production; Property; Proteins; Regulation; Response Elements; Rheumatoid Arthritis; Role; STIM1 gene; Signal Transduction; Signal Transduction Pathway; Stem Cell Factor; System; T-Lymphocyte; Time; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins; Work; Zinc Fingers; allergic response; autocrine; base; cell type; chromatin immunoprecipitation; cytokine; density; mast cell; member; novel; paracrine; promoter; receptor; receptor-mediated signaling; research study; response; sensor

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.

描述（由申请人提供）：胞质 Ca2+ 浓度的增加是多种信号转导途径的常见组成部分，调节多种反应，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+ 信号通常在几秒到几分钟的时间范围内发生，因此人们对 Ca2+ 瞬变如何调节几小时到几天内发生的事件知之甚少。我们实验室最近的研究发现早期生长反应 1 (EGR1) 是 STIM1 表达的调节因子，STIM1 是钙池操纵的 Ca2+ 进入的必需组成部分，而 STIM1 是 Ca2+ 进入非兴奋性细胞的主要方式。这一观察使我们得出这样的假设：Ca2+信号是通过协调EGR依赖性控制STIM表达来调节的。第一个目标是结合使用荧光素酶、电泳迁移率变化分析 (EMSA) 和染色质免疫沉淀 (ChIP) 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 STIM1 和 STIM2 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，在野生型和 EGR1 敲除小鼠的成纤维细胞和肥大细胞中检查受体介导的 EGR1 和 EGR4 表达变化改变 STIM1 和 STIM2 表达的能力。 Ca2+ 本身对受体介导的 EGR1 和 EGR4 表达的贡献将使用 STIM1 和 STIM2 敲除的成纤维细胞进行检查。最后，第三个目标是检查 EGR 介导的 Ca2+ 信号传导的生理控制。胰岛素与 SCF 诱导的 STIM 表达变化对肥大细胞中 Ca2+ 信号的影响将通过用抗 DNP IgE 引发后暴露于不同浓度的 DNP-BSA 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肥大细胞中 STIM1 和 STIM2 介导的通道激活变化，其中内源电流处于可测量水平。最后，将重点关注野生型、EGR1-null 和 STIM1-null 肥大细胞中细胞因子的产生和释放，确定 EGR 介导的胞质 Ca2+ 浓度变化对肥大细胞活化的影响。 综合考虑，这些研究将为新理解细胞中的 Ca2+ 信号如何通过自分泌、旁分泌和内分泌因子之间的串扰在较长时间内进行调节提供框架。