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 i s 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 i s 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.
Control of STIM Expression and Function by Interplay between EGR Family Members Store Operated Calcium entry, a major pathway of Ca2+ entry in non-excitable cells, plays a critical role in T cell survival and activation. Here we propose tht regulation of STIM1 and other Ca2+ homeostatic proteins by EGR family members is crucial for Ca2+ dependent immunological activity. Deregulated activity in T cells leads to multiple pathophysiological outcomes including Severe Combined Immunodeficiency (SCID) and autoimmune disorders such as rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis.
