Abstract
Interleukin (IL)-23, a new member of the IL-12 family, plays a central role in the Th17 immune response and in autoimmune diseases. It is clear that activated macrophages and dendritic cells produce IL-23, but the molecular mechanisms whereby inflammatory signals stimulate IL-23 expression are not fully understood. We demonstrate that induction of IL-23 p19 gene expression by LPS depends on the TLR4 and MyD88 pathways. All three MAPK pathways (ERK, JNK, and p38) that are activated by lipopolysaccharide (LPS) stimulation were shown to exert a positive effect on p19 expression. We cloned a 1.3-kb putative p19 promoter and defined its transcription initiation sites by the 5'-rapid amplification of cDNA ends method. By analyzing IL-23 p19 promoter mutants, we have identified a promoter region (-413 to +10) that contains several important elements, including NF-kappaB and AP-1. In addition to NF-kappaB, we have demonstrated that the proximal AP-1 site is important for p19 promoter activation. Mutation of the AP-1 site resulted in the loss of p19 promoter activation. Electrophoretic mobility shift assay (EMSA) analysis showed that c-Jun and c-Fos bind to the AP-1 site, which was confirmed by a chromatin immunoprecipitation assay. Furthermore, co-transfection of c-Jun and ATF2 synergistically induced p19 promoter activation, and c-Jun and ATF2 formed a protein complex, demonstrated by co-immunoprecipitation. Finally, LPS-stimulated peritoneal macrophages from IL-10-deficient mice expressed significantly higher IL-23 p19 than macrophages from wild type mice, and the addition of recombinant IL-10 strongly inhibited LPS-induced p19 expression. Thus, this study suggests that MyD88-dependent Toll-like receptor signaling induces IL-23 p19 gene expression through both MAPKs and NF-kappaB.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Activating Transcription Factor 2 / genetics
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Activating Transcription Factor 2 / immunology
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Activating Transcription Factor 2 / metabolism
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Animals
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Autoimmune Diseases
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Cell Line
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Dendritic Cells / immunology
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Dendritic Cells / metabolism
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Gene Expression Regulation / drug effects
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Gene Expression Regulation / physiology*
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Interleukin-10 / immunology
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Interleukin-10 / pharmacology
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Interleukin-23 Subunit p19 / biosynthesis*
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Interleukin-23 Subunit p19 / genetics
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Interleukin-23 Subunit p19 / immunology
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Lipopolysaccharides / pharmacology
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MAP Kinase Signaling System / drug effects
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MAP Kinase Signaling System / physiology
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Macrophages, Peritoneal / cytology
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Macrophages, Peritoneal / immunology
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Macrophages, Peritoneal / metabolism*
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Mice
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Mice, Knockout
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Mitogen-Activated Protein Kinase Kinases / genetics
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Mitogen-Activated Protein Kinase Kinases / immunology
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Mitogen-Activated Protein Kinase Kinases / metabolism
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Mutation
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Myeloid Differentiation Factor 88 / genetics
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Myeloid Differentiation Factor 88 / immunology
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Myeloid Differentiation Factor 88 / metabolism*
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NF-kappa B / genetics
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NF-kappa B / immunology
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NF-kappa B / metabolism
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Proto-Oncogene Proteins c-jun / genetics
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Proto-Oncogene Proteins c-jun / immunology
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Proto-Oncogene Proteins c-jun / metabolism
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Response Elements / genetics
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Response Elements / immunology
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Toll-Like Receptor 4 / genetics
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Toll-Like Receptor 4 / immunology
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Toll-Like Receptor 4 / metabolism*
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Transcription Factor AP-1 / genetics
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Transcription Factor AP-1 / immunology
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Transcription Factor AP-1 / metabolism*
Substances
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Activating Transcription Factor 2
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Atf2 protein, mouse
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Il23a protein, mouse
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Interleukin-23 Subunit p19
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Lipopolysaccharides
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Myd88 protein, mouse
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Myeloid Differentiation Factor 88
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NF-kappa B
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Proto-Oncogene Proteins c-jun
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Tlr4 protein, mouse
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Toll-Like Receptor 4
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Transcription Factor AP-1
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Interleukin-10
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Mitogen-Activated Protein Kinase Kinases