The binding of PKCε and MEG2 to STAT3 regulates IL-6-mediated microglial hyperalgesia during inflammatory pain

Studies have suggested that microglial IL-6 modulates inflammatory pain; however, the exact mechanism of action remains unclear. We therefore hypothesized that PKC epsilon and MEG2 competitively bind to STAT3 and contribute to IL-6-mediated microglial hyperalgesia during inflammatory pain. Freund's complete adjuvant (FCA) and lipopolysaccharide (LPS) were used to induce hyperalgesia model mice and microglial inflammation. Mechanical allodynia was evaluated using von Frey tests in vivo. The interaction among PKC epsilon, MEG2, and STAT3 was determined using ELISA and immunoprecipitation assay in vitro. The PKC epsilon, MEG2, t-STAT3, pSTAT3(Tyr705), pSTAT3(Ser727), IL-6, GLUT3, and TREM2 were assessed by Western blot. IL-6 promoter activity and IL-6 concentration were examined using dual luciferase assays and ELISA. Overexpression of PKC epsilon and MEG2 promoted and attenuated inflammatory pain, accompanied by an increase and decrease in IL-6 expression, respectively. PKC epsilon displayed a stronger binding ability to STAT3 when competing with MEG2. STAT3(Ser727) phosphorylation increased STAT3 interaction with both PKC epsilon and MEG2. Moreover, LPS increased PKC epsilon, MEG2, pSTAT3(Tyr705), pSTAT3(Ser727), IL-6, and GLUT3 levels and decreased TREM2 during microglia inflammation. IL-6 promoter activity was enhanced or inhibited by PKC epsilon or MEG2 in the presence of STAT3 and LPS stimulation, respectively. In microglia, overexpression of PKC epsilon and/or MEG2 resulted in the elevation of tSTAT3, pSTAT3(Tyr705), pSTAT3(Ser727), IL-6, and TREM2, and the reduction of GLUT3. PKC epsilon is more potent than MEG2 when competitively binding to STAT3, displaying dual modulatory effects of IL-6 production, thus regulating the GLUT3 and TREM2 in microglia during inflammatory pain sensation.