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: 8529840
• 负责人: Elsie Samakai
• 金额: $ 2.79万
• 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 2016-11-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8529840
• 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; 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; public health relevance; 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)：使用钙离子作为信号分子是多种细胞反应的中心，包括收缩、增殖、膜兴奋性和细胞凋亡。存储操作钙内流(SOCE)是非兴奋性细胞内钙内流的主要途径。SOCE是维持钙储存的关键机制，而钙储存是产生长期钙信号(如T细胞激活所需的信号)所必需的。我们的重点是试图了解这些细胞中的钙信号是如何在延长的时间段内调节的，以确保它们的存活和激活。实验室最近的研究表明，早期生长反应1(Egr1)是STIM1表达的直接调节因子(SOCE的一个必需成分)。然而，对Egr1基因敲除组织的检查显示，对Egr1的需求取决于细胞类型。我的初步研究显示，在Egr1基因敲除细胞中，EGR4的表达与维持基础STIM1表达的能力之间存在很强的相关性。无论EGR4的表达如何，受体介导的STIM1诱导都需要Egr1。提示Egr1和EGR4在STIM1表达调控中可能起着不同的作用。我们假设基础的和可诱导的STIM1的表达是通过Egr1和EGR4的相互作用来控制钙信号的差异调节的。第一个目的是通过转录调控STIM1来确定EGR1和EGR4在钙离子进入中的作用。结合使用芯片和EMSA检测，我们将评估EGR4与STIM1启动子结合的能力。此外，我们将评估依赖Egr1和EGR4的STIM1转录的影响以及随后对SOCE的影响。总之，这些研究将帮助我们确定Egr1和EGR4在钙信号调节中的相对贡献。第二个目的是评估EGR介导的STIM1调控对T细胞激活的影响。将在Jurkat T细胞中检测到EGR依赖的受体介导的STIM1增加。一旦用抗CD3/CD28抗体激活细胞，在Egr1和/或EGR4被敲除后，将使用Western分析来确定增加STIM1表达的能力。为了测量EGR介导的钙信号变化对T细胞功能的影响，我们将观察缺乏EGR1和EGR4的细胞中NFAT的诱导和细胞因子的表达。荧光素酶测定法和ELISA法将分别用于检测NFAT的诱导和细胞因子的表达。