Abstract
Regulator of G‐protein Signaling (RGS10) is a member of a family of proteins that canonically bind and terminate signaling downstream of G proteins. RGS10 is highly expressed in brain and immune cells, with specific enrichment in microglia. Microglial RGS10 serves an anti‐inflammatory and neuroprotective role by suppressing expression of pro‐inflammatory genes, such as tumor necrosis factor‐alpha (TNF‐α), and cyclooxygenase (COX‐2) in response to TLR4 receptor activation. We recently demonstrated that this anti‐inflammatory activity of RGS10 occurs through an undefined G protein‐independent mechanism. Further, RGS10 expression is suppressed by activation of TLR4 receptor, resulting in amplification of inflammatory gene expression. The molecular mechanisms directly linking TLR4 activation to RGS10 suppression are also undefined. In order to exploit the anti‐inflammatory effects of RGS10 in the development of therapeutics for the treatment of neuroinflammation and accompanying neurodegeneration, it is essential to define both the mechanism for RGS10 suppression of inflammatory gene expression, and the mechanism for RGS10 silencing. The current study implicates calcium signaling as playing a critical role in the regulation of both processes. We have identified STIM2 as a novel RGS10 interacting protein in BV2 microglia cells and in RAW macrophages. STIM2 is an ER‐resident calcium sensor that mediates store‐operated Ca2+ entry (SOCE) through Orai channels in the plasma membrane in response to ER calcium depletion. STIM2 activation and its coupling to Orai‐mediated calcium signaling is strongly implicated in regulation of inflammatory signaling in microglia. Therefore, we hypothesized that RGS10 suppression of TLR4‐stimulated inflammatory gene expression reflects regulation of STIM2‐Orai activity. Our results demonstrate that pharmacological inhibition of Orai channels completely blocks the amplification of TLR4‐stimulated COX‐2 and TNFα expression in response to loss of RGS10 expression. Further, RGS10 knockout microglia exhibit markedly enhanced SOCE in response to ER calcium depletion. These results suggest that RGS10 suppresses COX‐2 and TNF‐α expression downstream of TLR4 activation of Orai‐mediated SOCE. Further, we demonstrate that thapsigargin, which triggers transient rise in cytoplasmic calcium and ER calcium depletion, strongly enhanced LPS‐induced RGS10 silencing, suggesting a potential mechanism for feedback regulation of calcium signaling. These findings provide critical insight into the mechanism that RGS10 utilizes to regulate inflammatory signaling. Defining these mechanisms in microglia is essential to develop novel therapeutics that target RGS10 in treatment of neuroinflammatory diseases.
Support or Funding Information
National Institutes of Health
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.