Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells.
The inflammatory effects of glycogen synthase kinase-3 (GSK-3) have been identified; however, the potential mechanism is still controversial. In this study, we investigated the effects of GSK-3-mediated interleukin-10 (IL-10) inhibition on lipopolysaccharide (LPS)-induced inflammation. Treatment with GSK-3 inhibitor significantly blocked LPS-induced nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression in BV2 murine microglial cells and primary rat microglia-enriched cultures. Using an antibody array and enzyme-linked immunosorbent assay, we found that GSK-3-inhibitor treatment blocked LPS-induced upregulation of regulated on activation normal T-cell expressed and secreted (RANTES) and increased IL-10 expression. The time kinetics and dose-response relations were confirmed. Reverse transcription-polymerase chain reaction showed changes on the messenger RNA level as well. Inhibiting GSK-3 using short-interference RNA, and transfecting cells with dominant-negative GSK-3beta, blocked LPS-elicited NO and RANTES expression but increased IL-10 expression. In contrast, GSK-3beta overexpression upregulated NO and RANTES but downregulated IL-10 in LPS-stimulated cells. Treating cells with anti-IL-10 neutralizing antibodies to prevent GSK-3 from downregulating NO and RANTES showed that the anti-inflammatory effects are, at least in part, IL-10-dependent. The involvement of Akt, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-kappaB that positively regulated IL-10 was demonstrated. Furthermore, inhibiting GSK-3 increased the nuclear translocation of transcription factors, that all important for IL-10 expression, including CCAAT/enhancer-binding protein beat (C/EBPbeta), C/EBPdelta, cAMP response binding element protein and NF-kappaB. Taken together, these findings reveal that LPS induces iNOS/NO biosynthesis and RANTES production through a mechanism involving GSK-3-mediated IL-10 downregulation.
Huang
Wei-Ching
WC
Institute of Clinical Medicine, National Cheng Kung University Medical College, Tainan, Taiwan.
Lin
Yee-Shin
YS
Wang
Chi-Yun
CY
Tsai
Cheng-Chieh
CC
Tseng
Hsiang-Chi
HC
Chen
Chia-Ling
CL
Lu
Pei-Jung
PJ
Chen
Po-See
PS
Qian
Li
L
Hong
Jau-Shyong
JS
Lin
Chiou-Feng
CF
D000818:Animals; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D002460:Cell Line; D018946:Chemokine CCL5 / Q000276:immunology / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D007249:Inflammation / Q000139:chemically induced / Q000276:immunology* / Q000378:metabolism; D016753:Interleukin-10 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000201:enzymology / Q000276:immunology*; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000276:immunology / Q000378:metabolism; D034741:RNA, Small Interfering / Q000276:immunology / Q000378:metabolism; D051381:Rats; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology; D048051:p38 Mitogen-Activated Protein Kinases / Q000276:immunology / Q000378:metabolism
D057134:Antibodies, Neutralizing; D018946:Chemokine CCL5; D008070:Lipopolysaccharides; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D016753:Interleukin-10; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051057:Proto-Oncogene Proteins c-akt; D048051:p38 Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3