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. 2009 Sep 4;284(36):24006-16.
doi: 10.1074/jbc.M109.025528. Epub 2009 Jul 10.

AP-1 activated by toll-like receptors regulates expression of IL-23 p19

Weicheng Liu  1 , Xinshou Ouyang, Jianjun Yang, Jianguo Liu, Qingshan Li, Yongpeng Gu, Masayuki Fukata, Tony Lin, John Cijiang He, Maria Abreu, Jay C Unkeless, Lloyd Mayer, Huabao Xiong
Affiliations

AP-1 activated by toll-like receptors regulates expression of IL-23 p19

Weicheng Liu et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
IL-23 p19 expression is dependent upon TLR4 and MyD88. A, thioglycollate-elicited peritoneal macrophages from WT (C57BL/6), TLR4−/−, and MyD88−/− mice were activated with LPS (1 μg/ml) for 4 h. Total RNA was analyzed by qRT-PCR for p19 and p35. *, p < 0.01 versus WT LPS (+). B, the peritoneal macrophages described in A were activated with LPS for 16 h, and brefeldin A was added for 6 h. The whole cell lysates were collected for immunoblotting analysis using anti-p19 antibody. C, bone marrow-derived dendritic cells from WT, TLR4−/−, and MyD88−/− mice were activated with LPS (1 μg/ml) for 4 h. Total RNA was analyzed by qRT-PCR for p19 and p35. *, p < 0.01 versus WT LPS (+). D, the peritoneal macrophages described in A were activated with LPS plus IFN-γ for 24 h, and supernatants were assayed for IL-23 and IL-12 p70. The data are representative of three similar experiments.
FIGURE 2.
FIGURE 2.
TLR signaling regulates p19 promoter activation. A, schematic of the p19 promoter with TIS that were determined by RLM-RACE. The promoter coordinates (from −246 down to +106) are relative to the most dominant TIS1, located at +1. B, RAW264.7 cells were transfected with the p19 luciferase reporter plasmid. The cells were incubated for 18–24 h, activated for 12 h with various TLR ligands (LPS (1 μg/ml), PGN (25 μg/ml), poly(I-C) (50 μg/ml), or IFN-γ (10 ng/ml)), after which luciferase activity from cell extracts was quantified. Results were normalized for β-galactosidase activity from a control (con) β-galactosidase expression plasmid. *, p < 0.01 versus LPS (+). C, RAW264.7 cells were co-transfected with the p19 luciferase reporter plasmid and a constitutive form of TLR4 for 30 h. *, p < 0.01 versus control. Luciferase activity was analyzed as described in A. The results are representative of three similar experiments.
FIGURE 3.
FIGURE 3.
Deletion analysis of p19 promoter activity. The constructs with progressive deletions of 5′-flanking region of the murine p19 promoter were used to transfect RAW264.7 cells, and 18–24 h after transfection, the cells were activated with LPS (1 μg/ml) for 12 h. Luciferase activity was quantified and normalized for β-galactosidase. *, p < 0.01 versus −413. The data are representative of three similar experiments.
FIGURE 4.
FIGURE 4.
NF-κB p65 binds to the p19 promoter region. A, LPS induces NF-κB DNA complex formation. RAW264.7 cells were activated with IFN-γ (10 ng/ml), LPS (1 μg/ml), or LPS plus IFN-γ for 4 h, and nuclear protein was extracted for EMSA. 5 μg of nuclear protein were added to the 32P-labeled probe (−228 to −198 of the p19 promoter). Extract and probe were incubated with 0.5 μg of poly(dI-dC) at room temperature for 30 min. B, p65 antibody attenuates DNA-protein complex formation. RAW264.7 cells were activated with LPS (1 μg/ml) for 4 h, and nuclear protein was extracted for EMSA. 5 μg of nuclear protein were incubated with 2 μg of p65, p50, or c-Rel antibody and the 32P-labeled probe used in A. C, chromatin co-immunoprecipitation assays. RAW264.7 cells were activated with LPS for 4 h. Formaldehyde (1% final concentration) was added for 30 min at room temperature. Cells were harvested, and the chromatin was sheared by sonication and purified by immunoprecipitation (IP) with an anti-p65 antibody. After reversal of cross-linking, DNA was amplified by PCR with primers spanning the p19 promoter. D, TLR ligands induce an NF-κB DNA complex. RAW264.7 cells were activated with LPS, PGN, and poly(I-C) for 4 h, and nuclear protein was extracted for EMSA. 5 μg of nuclear protein were added to the 32P-labeled probe used in A. con, control.
FIGURE 5.
FIGURE 5.
NF-κB p65 is required for p19 promoter activation. A, mutation of the NF-κB site abolishes p19 promoter activity. RAW264.7 cells were transfected with either WT p19 promoter or a p19 promoter with a mutated NF-κB site for 18–24 h, the cells were activated with LPS (1 μg/ml) for 12 h, and luciferase activity was quantified and normalized for β-galactosidase. *, p < 0.01 versus p19 WT LPS (+). B, a dominant negative form of IκBα significantly reduced p19 promoter activation. RAW264.7 cells were co-transfected with the p19 promoter and DN-IκBα for 18–24 h, the cells were activated with LPS for 12 h, and extracts were quantified for luciferase activity and normalized for β-galactosidase. *, p < 0.01 versus LPS only. C, p19 promoter activity was significantly reduced in p65−/− mouse embryo fibroblasts. Wild type and p65−/− fibroblasts were transfected with the p19 promoter luciferase plasmid for 10–24 h and activated with LPS for 12 h, and extracts were quantified for luciferase and normalized for β-galactosidase. *, p < 0.01 versus p19 WT LPS (+).
FIGURE 6.
FIGURE 6.
MAPKs are involved in the activation of the p19 promoter. A, an ERK inhibitor blocks LPS-induced p19 mRNA and p19 promoter activation. RAW264.7 cells were pretreated with either the ERK inhibitor UO126 or the inactive analogue UO124 (10 μm) for 1 h, the cells were activated with LPS for 4 h, and RNA was analyzed by qRT-PCR for p19 mRNA. For p19 promoter analysis, RAW264.7 cells were transfected with the p19 promoter-luciferase reporter construct for 18–24 h, treated with either UO124 or UO126 for 1 h, and activated with LPS for 12 h. Luciferase activity was quantified and normalized for β-galactosidase. The data are representative of two similar experiments. B, JNK inhibitor reduces LPS-induced p19 mRNA and p19 promoter activation. RAW264.7 cells were pretreated with either JNK inhibitor or control for 1 h, the cells were activated with LPS for 4 h, and p19 mRNA was quantified by qRT-PCR. For p19 promoter analysis, RAW264.7 cells were transfected with the p19 promoter for 18–24 h, incubated with either JNK control or JNK inhibitor for 1 h, and activated with LPS for 12 h, and luciferase activity in extracts was quantified and normalized for β-galactosidase. The data are representative of two similar experiments. C, p38 inhibitor inhibits LPS-induced p19 mRNA and p19 promoter activation. RAW264.7 cells were pretreated with SB203580 for 1 h, the cells were activated with LPS for 4 h, and RNA was quantified by qRT-PCR for p19 mRNA. For p19 promoter analysis, RAW264.7 cells were transfected with the p19 promoter for 18–24 h, incubated with either JNK control or JNK inhibitor for 1 h, and activated with LPS for 12 h, and luciferase activity in extracts was quantified and normalized for β-galactosidase. The data are representative of two similar experiments. D, thioglycollate-elicited peritoneal macrophages from C57BL/6 mice were pretreated with UO126, JNK inhibitor, or SB20350 for 1 h and activated with LPS plus IFN-γ for 24 h, and supernatants were assayed for IL-23. E, MAPK inhibitors block MAPK activation. RAW264.7 cells were pretreated with either ERK or JNK inhibitors for 1 h and activated with LPS for 10 min, and cell lysates were analyzed by immunoblotting (IB) for phosphorylated and total ERK and JNK. F, dominant negative ERK2 significantly reduces p19 promoter activation. RAW264.7 cells were co-transfected with the p19 promoter and DN ERK2 for 18–24 h and activated with LPS for 12 h, extracts were quantified for luciferase, and results were normalized for β-galactosidase. *, p < 0.01 versus LPS only. G, knockdown of ERK expression by siRNA significantly inhibits p19 promoter activation. Left, RAW264.7 cells were co-transfected with the p19 promoter and ERK siRNA or control siRNA for 18–24 h and activated with LPS for 12 h, extracts were quantified for luciferase, and results were normalized for β-galactosidase. *, p < 0.01 versus LPS only. The data are representative of two similar experiments. Right, 293T cells were transfected with control siRNA or ERK siRNA for 48 h. The whole cell lysates were immunoblotted with anti-ERK antibody.
FIGURE 7.
FIGURE 7.
AP-1 is important for p19 promoter activation. A, c-Jun binds to the AP-1 site in the p19 promoter. RAW264.7 cells were activated with LPS (1 μg/ml) for 6 h, and nuclear protein was extracted for EMSA. 5 μg of nuclear protein were incubated with 2 μg of anti-c-Jun and anti-c-Fos antibodies (Ab) and added to the 32P-labeled probe (−210 to −182 of the p19 promoter). B, chromatin co-immunoprecipitation (IP) assays were performed as in Fig. 4C, using c-Jun and ATF2 antibodies, and the immunoprecipitated DNA after cross-linker reversal was amplified by PCR with primers spanning the p19 promoter. C, mutation of the AP-1 site abolishes p19 promoter activation. RAW264.7 cells were transfected with either WT p19 promoter or the promoter with a mutated AP-1 site for 18–24 h and activated with LPS for 12 h, and luciferase activity in extracts was quantified and normalized for β-galactosidase. The data are representative of three similar experiments. D, dominant negative c-Jun significantly reduces the p19 promoter activity. RAW264.7 cells were co-transfected with the p19 promoter and DN c-Jun for 18–24 h and activated with LPS for 12 h, and extracts were assayed for luciferase activity, which was normalized for β-galactosidase. E, c-Jun and ATF2 synergistically induce p19 promoter activation. RAW264.7 cells were co-transfected with the p19 promoter, c-Jun, and ATF2 for 36 h. The extracts were assayed for luciferase activity, which was normalized for β-galactosidase. The data are representative of three similar experiments. F, c-Jun physically interacts with ATF2. 293T cells were transfected with c-Jun and ATF2 for 48 h. 500 μg of cell lysates were immunoprecipitated with c-Jun antibody or normal IgG and blotted (IB) with ATF2 antibody.
FIGURE 8.
FIGURE 8.
IL-10 negatively regulates IL-23 p19 expression. A, enhanced IL-23 p19 expression in IL-10−/− macrophages. Thioglycollate-elicited peritoneal macrophages from WT and IL-10−/− mice were activated with LPS (1 μg/ml) for various time intervals, and total RNA was analyzed by qRT-PCR for IL-23 p19. B, IL-10 inhibits p19 mRNA expression. Thioglycollate-elicited peritoneal macrophages from IL-10−/− mice were activated with LPS (1 μg/ml) in the presence of IL-10 (10 ng/ml) for 4 h, and total RNA was extracted and analyzed by qRT-PCR for IL-23 p19. *, p < 0.01 versus LPS only. C, IL-10 inhibits IL-23 synthesis. Both WT and IL-10−/− peritoneal macrophages were activated with LPS for 16 h, and brefeldin A was added for 6 h. The whole cell lysates were collected for immunoblotting analysis using anti-p19 antibody.
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