Control of STIM Expression and Function by Interplay Between EGR Family Members

通过 EGR 家族成员之间的相互作用控制 STIM 表达和功能

• 批准号: 8765623
• 负责人: Elsie Samakai
• 金额: $ 2.88万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-11-16 至 2015-11-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8765623
• 关键词: Affect; Antibodies; Antigen Presentation; Apoptosis; Autoimmune Diseases; Binding; Biological Assay; Biological Models; CD28 gene; CD3 Antigens; Ca(2+)-Transporting ATPase; Calcineurin; Calcium; Calmodulin; Cell Nucleus; Cell Survival; Cell membrane; Cell model; Cell physiology; Cells; Complex; Dependence; EMSA; Electrophoretic Mobility Shift Assay; Embryo; Ensure; Enzyme-Linked Immunosorbent Assay; Event; Family member; Fibroblasts; Fluorescence; Generations; Genetic Transcription; Growth; Health; Homeostasis; Hour; Inflammatory Bowel Diseases; Investigation; Knock-out; Luciferases; Measures; Mediating; Membrane; Metabolic Clearance Rate; Multiple Sclerosis; Mus; Outcome; Pathway interactions; Phosphoric Monoester Hydrolases; Physiological; Play; Process; Proteins; Receptor Activation; Regulation; Relative (related person); Response Elements; Rheumatoid Arthritis; Role; STIM1 gene; Series; Severe Combined Immunodeficiency; Signal Transduction; Signaling Molecule; Small Interfering RNA; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Time; Tissues; Transcriptional Regulation; Transfection; Zinc Fingers; base; cell type; chromatin immunoprecipitation; crosslink; cytokine; defined contribution; novel; nuclear factors of activated T-cells; promoter; receptor; receptor expression; receptor-mediated signaling; research study; response; transcription factor; vector

DESCRIPTION (provided by applicant): Use of Ca2+ as a signaling molecule is central to a wide variety of cellular responses including contraction, proliferation, membrane excitability and apoptosis. Store Operated Ca2+ Entry (SOCe) is a major pathway of Ca2+ entry in non-excitable cells. SOCe serves as a crucial mechanism, required to maintain Ca2+ stores which is essential for the generation of long term Ca2+ signals such as those required in T cell activation. Our focus is to try to understand how Ca2+ signals in these cells are modulated over extended time periods to ensure their survival and activation. Recent investigations in the lab have revealed Early Growth Response 1 (EGR1) as a direct regulator of STIM1 expression (a required component of SOCe). However, examination of EGR1 knockout tissues revealed that the requirement for EGR1 is celltype dependent. My preliminary investigations reveal a strong correlation between EGR4 expression and the ability to maintain basal STIM1 expression in EGR1 knockout cells. Irrespective of EGR4 expression, receptor-mediated STIM1 induction requires EGR1. This suggests that EGR1 and EGR4 may serve distinct roles in the control of STIM1 expression. We hypothesize that basal and inducible STIM1 expression is differentially regulated by interplay of EGR1 and EGR4 for the control of Ca2+ signaling. The first aim is to define the contributions of EGR1 and EGR4 to Ca2+ entry through transcriptional control of STIM1. Using a combination of ChIP and EMSA assays, we will assess the ability of EGR4 to bind to the STIM1 promoter. In addition, we will assess the effects of EGR1- and EGR4-dependent STIM1 transcription and subsequent effects on SOCe. Collectively, these studies will help us identify the relative contributions of EGR1 and EGR4 to modulation of Ca2+ signaling. The second aim is to assess the implications of EGR-mediated control of STIM1 to T cell activation. EGR-dependent receptor-mediated STIM1 increases will be measured in Jurkat T cells. Upon activation of cells with antiCD3/CD28 antibodies, the ability to increase STIM1 expression will be determined using Western analysis after knockdown of EGR1 and/or EGR4. To measure the effects of EGR-mediated changes in Ca2+ signaling on T cell function, we will look at NFAT induction and cytokine expression in cells lacking EGR1 and EGR4. Luciferase assays and ELISA will be used to measure NFAT induction and cytokine expression, respectively.

描述（由申请人提供）：使用Ca 2+作为信号分子对多种细胞反应（包括收缩、增殖、膜兴奋性和细胞凋亡）至关重要。钙池操纵的钙内流（SOCe）是非兴奋细胞内钙内流的主要途径。SOCe作为维持Ca 2+储存所需的关键机制，其对于产生长期Ca 2+信号（例如T细胞活化中所需的那些信号）是必不可少的。我们的重点是试图了解这些细胞中的Ca 2+信号如何在较长的时间内被调节，以确保它们的存活和激活。最近的实验室研究表明，早期生长反应1（EGR 1）是STIM 1表达（SOCe的必需成分）的直接调节因子。然而，对EGR 1敲除组织的检查显示，对EGR 1的需求是细胞类型依赖性的。我的初步研究揭示了在EGR 1敲除细胞中EGR 4表达和维持基础STIM 1表达的能力之间的强相关性。不管EGR 4的表达，受体介导的STIM 1诱导需要EGR 1。这表明，EGR 1和EGR 4可能在控制STIM 1表达中发挥不同的作用。我们假设，基础和诱导STIM 1的表达差异调节的相互作用的EGR 1和EGR 4的Ca 2+信号的控制。第一个目的是确定EGR 1和EGR 4通过转录控制STIM 1对Ca 2+进入的贡献。使用ChIP和EMSA测定的组合，我们将评估EGR 4结合STIM 1启动子的能力。此外，我们将评估EGR 1和EGR 4依赖性STIM 1转录的影响以及随后对SOCe的影响。总的来说，这些研究将帮助我们确定EGR 1和EGR 4对Ca 2+信号调节的相对贡献。第二个目的是评估EGFR介导的STIM 1控制对T细胞活化的影响。将在Jurkat T细胞中测量EGFR依赖性受体介导的STIM 1增加。在用抗CD 3/CD 28抗体活化细胞后，在敲低EGR 1和/或EGR 4后使用Western分析来确定增加STIM 1表达的能力。为了测量EGFR介导的Ca 2+信号传导变化对T细胞功能的影响，我们将研究缺乏EGFR 1和EGFR 4的细胞中的NFAT诱导和细胞因子表达。荧光素酶测定和ELISA将分别用于测量NFAT诱导和细胞因子表达。