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 i s 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 i s 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.
STIM-operated Ca2+ signals represent the primary means of Ca2+ entry in mast cells, with critical contributions to their activation. The current proposal is based on the concept that EGR-mediated changes in STIM expression modulate Ca2+ signals over extended time frames. This concept is uniquely relevant to mast cells;while brief induction of mast cell activity contributes positively to the immune response, chronic mast cell activation leads to multiple pathophysiological outcomes including allergic responses, anaphylactic shock, asthma, sclerderma and rheumatoid arthritis.
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|Hooper, Robert; Rothberg, Brad S; Soboloff, Jonathan (2016) Sterol hindrance of Orai activation. Sci Signal 9:fs4|
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