Modulation of calcium signaling by changes in STIM expression

通过 STIM 表达的变化调节钙信号传导

基本信息

批准号：
8883571
负责人：
Jonathan A Soboloff
金额：
$ 29.07万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
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/8883571
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 Wilms Tumor Suppressor 1 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

项目摘要

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共同组成部分，这些途径调节了各种各样的反应，包括快速事件（例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钟到几分钟的时间范围内，因此CA2+瞬变如何调节在数小时至几天内发生的事件的了解很少。我们实验室的最新研究导致了早期生长反应1（EGR1）作为STIM1表达的调节剂，这是Stim1的表达，这是商店操作的Ca2+进入的所需组成部分，这是Ca2+进入不可驱动的细胞中Ca2+进入的主要均值。这一观察结果使我们提出了这样的假设：Ca2+信号是通过协调的EGR依赖性控制刺激表达来调节的。第一个目的是使用荧光素酶，电泳迁移率转移测定法（EMSA）和染色质免疫沉淀（CHIP）来定义EGR介导的STIM1和STIM2转录的控制，以研究EGR1的性质在多大程度上可以应用于其他家族成员。实验将在包括HEK293细胞和Jurkat T细胞在内的优化细胞系统中进行。第二个目的是分析受体介导的对STIM1和STIM2表达的控制。在此，将在使用胰岛素，血小板衍生的生长因子（PDGF；成纤维细胞）和干细胞因子（SCF; scf; mast; mast; mast; mast; mast; mast; mast; mast cells cy oggf; mast; mast cells rygr1和scf; mast; mast; mast; mast; mast cells cy介导的EGR1和EGR4表达变化改变stim1和stim2表达的变化的能力。 Ca2+本身对受体介导的EGR1和EGR4表达的贡献将使用STIM1和Stim2基因敲除成纤维细胞进行检查。最后，第三个目的是检查Ca2+信号传导的EGR介导的生理控制。胰岛素与SCF诱导的Stim表达变化对肥大细胞中Ca2+信号的影响将通过抗抗DNP IgE启动后暴露于不同浓度的DNP-BSA来确定。在Fura-2负载的细胞中将确定对胞质Ca2+浓度的总体影响，而Ca2+电流的电生理分析将用于测量内源性电流在可测量水平处的肥大细胞中的刺激和刺激介导的通道激活中的变化。最后，EGR介导的胞质Ca2+浓度变化对肥大细胞激活的影响将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。结合考虑，这些研究将为对细胞中的Ca2+信号的新理解提供框架，以通过自分泌，旁分泌和内分泌因子之间的串扰在延长的时间段内调节细胞中的Ca2+信号。