A Novel Interaction between Regulator of G‐protein Signaling (RGS10) and STIM2 Identifies New Mechanism for the Regulation of COX‐2 in Microglia

Abstract

Regulator of G‐protein Signaling 10 (RGS10) mediates strong anti‐inflammatory and neuroprotective effects. RGS10 is highly enriched in microglia where it suppresses the expression of many proinflammatory genes, including the key inflammatory enzyme cyclooxygenase 2 (COX‐2). The neuroprotective and anti‐inflammatory effects of RGS10 position it as a potential therapeutic target in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Our previous work demonstrated that microglial RGS10 strongly suppresses lipopolysaccharide (LPS)‐induced COX‐2 and tumor necrosis factor alpha (TNFα) expression, and this suppression is independent of its canonical G protein‐targeted mechanism. In this study, we further define the G protein‐independent anti‐inflammatory mechanism of RGS10 in microglia, focusing on the regulation of COX‐2. We hypothesized that novel binding partners link RGS10 to LPS‐stimulated inflammatory signaling pathways. Using an unbiased RGS10 co‐immunoprecipitation approach coupled with LC‐MS/MS, we identified the endoplasmic reticulum (ER)‐localized calcium sensor STIM2 as a major RGS10 interacting protein. STIM2 is an essential component of the store‐operated calcium entry (SOCE) machinery, which functions to detect Ca2+ depletion from the ER, and subsequently activate extracellular Ca2+ entry through plasma membrane Orai channels. Orai‐mediated Ca2+ entry is implicated in regulating inflammatory signaling in microglia through activation of the Ca2+‐dependent phosphatase, calcineurin, and its downstream pro‐inflammatory transcription factor target, NFAT. We have validated the RGS10‐STIM2 interaction in microglia and defined the specificity of interaction and its regulation by calcium and G protein signaling. To delineate the role of STIM2 and downstream calcineurin signaling in mediating the anti‐inflammatory effect of RGS10, we determined the effect of STIM2 knockdown and calcineurin inhibition on LPS‐stimulated COX‐2 expression in wildtype and RGS10 knockout microglial cells. Our findings demonstrate that LPS‐stimulated COX‐2 amplification in RGS10 knockout microglial cells is blocked by inhibition of either STIM2 expression or calcineurin activity. Furthermore, the increased inflammatory signaling observed in RGS10 knockout microglia correlates with enhanced SOCE, as measured by Ca2+ imaging using a fluorescent calcium indicator. Collectively, these results suggest that RGS10 strongly suppresses LPS‐stimulated inflammatory signaling in microglia by inhibiting STIM2‐induced calcium entry and the subsequent activation of calcineurin‐dependent inflammatory gene expression.Support or Funding InformationNational Institute of Neurological Disorders and Stroke (NINDS)[Grant NS101161]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.