title: Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades.
abstract: 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-3β (GSK-3β) activity has therapeutic benefits in inflammatory diseases. However, the exact molecular mechanisms underlying GSK-3β inactivation-mediated suppression of the inflammatory response induced by microglial activation have not been completely clarified. Tumor necrosis factor-α (TNF-α) plays a central role in injury caused by neuroinflammation. We investigated the regulatory effect of GSK-3β on TNF-α 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-α was measured by ELISA. Signaling molecules were analyzed by western blotting, and activation of NF-κB 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-3β by selective GSK-3β inhibitors or by RNA interference attenuated LPS-induced TNF-α production in cultured microglia. Exploration of the mechanisms by which GSK-3β positively regulates inflammatory response showed that LPS-induced IκB-α degradation, NF-κBp65 nuclear translocation, and p65 DNA binding activity were not affected by inhibition of GSK-3β activity. However, GSK-3β 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-3β during LPS-induced TNF-α production in microglia. The phosphorylated levels of MLK3, MKK4, and JNK were increased upon LPS treatment. Decreasing GSK-3β activity blocked MLK3 signaling cascades through disruption of MLK3 dimerization-induced autophosphorylation, ultimately leading to a decrease in TNF-α secretion.
  
  
  CONCLUSIONS
  These results suggest that inactivation of GSK-3β might represent a potential strategy to downregulate microglia-mediated inflammatory processes.
authors.affiliation: Department of Medical Research, Neuro-Medical Scientific Center, Buddhist Tzu Chi General Hospital, Hualien, Taiwan. mjwang@tzuchi.com.tw
authors.forename: Mei-Jen
authors.initials: MJ
authors.lastname: Wang
authors.forename: Hsin-Yi
authors.initials: HY
authors.lastname: Huang
authors.forename: Wu-Fu
authors.initials: WF
authors.lastname: Chen
authors.forename: Hui-Fen
authors.initials: HF
authors.lastname: Chang
authors.forename: Jon-Son
authors.initials: JS
authors.lastname: Kuo
mesh_terms: D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D020672:I-kappa B Proteins / Q000378:metabolism; D007249:Inflammation / Q000139:chemically induced / Q000276:immunology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D051379:Mice; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
chemical_list: D004791:Enzyme Inhibitors; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D020672:I-kappa B Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D008070:Lipopolysaccharides; D020930:MAP Kinase Kinase Kinases; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C000606205:Nfkbia protein, mouse; C000606206:Nfkbia protein, rat; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat