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

描述（由申请人提供）：Ca2+作为信号分子的使用是多种细胞反应的核心，包括收缩，增殖，膜兴奋性和凋亡。储存操作Ca2+进入（SOCe）是Ca2+进入不可兴奋细胞的主要途径。SOCe是维持Ca2+储存所需的关键机制，这对于产生长期Ca2+信号（如T细胞激活所需的Ca2+信号）至关重要。我们的重点是试图了解这些细胞中的Ca2+信号如何在长时间内被调节，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 （EGR1）是STIM1表达的直接调节剂（SOCe的必需成分）。然而，对EGR1敲除组织的检查显示，对EGR1的需求是细胞类型依赖的。我的初步研究显示，EGR4表达与EGR1敲除细胞中维持基础STIM1表达的能力之间存在很强的相关性。无论EGR4表达如何，受体介导的STIM1诱导都需要EGR1。这表明EGR1和EGR4可能在控制STIM1表达中起着不同的作用。我们假设基础和诱导的STIM1表达通过EGR1和EGR4的相互作用来控制Ca2+信号传导而受到差异调节。第一个目的是确定EGR1和EGR4通过STIM1的转录控制对Ca2+进入的贡献。结合ChIP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EGR1和egr4依赖性STIM1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EGR1和EGR4对Ca2+信号调节的相对贡献。第二个目的是评估egr介导的STIM1控制对T细胞活化的影响。egr依赖性受体介导的STIM1增加将在Jurkat T细胞中测量。在使用antid3 /CD28抗体激活细胞后，在敲除EGR1和/或EGR4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Ca2+信号变化对T细胞功能的影响，我们将研究缺乏EGR1和EGR4的细胞中的NFAT诱导和细胞因子表达。荧光素酶法和ELISA法分别检测NFAT诱导和细胞因子表达。

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.