Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

Background: Deciphering the mechanisms that modulate the inflammatory response induced by microglial activation not only improves our insight into neuroinflammation but also provides avenues for designing novel therapies that could halt inflammation-induced neuronal degeneration. Decreasing glycogen synthase kinase-3b (GSK-3 beta) activity has therapeutic benefits in inflammatory diseases. However, the exact molecular mechanisms underlying GSK-3 beta inactivation-mediated suppression of the inflammatory response induced by microglial activation have not been completely clarified. Tumor necrosis factor-alpha (TNF-alpha) plays a central role in injury caused by neuroinflammation. We investigated the regulatory effect of GSK-3 beta on TNF-alpha production by microglia to discern the molecular mechanisms of this modulation. Methods: Lipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Release of TNF-alpha was measured by ELISA. Signaling molecules were analyzed by western blotting, and activation of NF-kappa B and AP-1 was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Protein interaction was examined by coimmunoprecipitation. Results: Inhibition of GSK-3 beta by selective GSK-3 beta inhibitors or by RNA interference attenuated LPS-induced TNF-alpha production in cultured microglia. Exploration of the mechanisms by which GSK-3 beta positively regulates inflammatory response showed that LPS-induced I kappa B-alpha degradation, NF-kappa Bp65 nuclear translocation, and p65 DNA binding activity were not affected by inhibition of GSK-3 beta activity. However, GSK-3 beta inactivation inhibited transactivation activity of p65 by deacetylating p65 at lysine 310. Furthermore, we also demonstrated a functional interaction between mixed lineage kinase 3 (MLK3) and GSK-3 beta during LPS-induced TNF-alpha production in microglia. The phosphorylated levels of MLK3, MKK4, and JNK were increased upon LPS treatment. Decreasing GSK-3 beta activity blocked MLK3 signaling cascades through disruption of MLK3 dimerization-induced autophosphorylation, ultimately leading to a decrease in TNF-alpha secretion. Conclusion: These results suggest that inactivation of GSK-3 beta might represent a potential strategy to downregulate microglia-mediated inflammatory processes.