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.
