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. 2005 Mar 15;174(6):3484-92.
doi: 10.4049/jimmunol.174.6.3484.

The protooncogene c-Maf is an essential transcription factor for IL-10 gene expression in macrophages

Shanjin Cao  1 , Jianguo Liu, Lihua Song, Xiaojing Ma
Affiliations

The protooncogene c-Maf is an essential transcription factor for IL-10 gene expression in macrophages

Shanjin Cao et al. J Immunol. .

Abstract

IL-10 is an important immunoregulatory factor. However, our understanding of IL-10 gene regulation remains very limited. In this study, following up on our previous novel finding that the protooncogene c-Maf of the basic leucine zipper family of transcription factors is expressed in monocytes and macrophages, we investigate the role of c-Maf in the transcriptional regulation of IL-10 and the underlying molecular mechanism in macrophages. c-Maf-null macrophages exhibit strongly impaired IL-10 protein production and mRNA expression upon LPS stimulation. Ectopic expression of c-Maf stimulates not only exogenously transfected IL-10 promoter-driven luciferase activity in a dose-dependent manner but also enhances endogenous IL-10 gene expression stimulated by LPS. Both in vitro and in vivo experiments identify a c-Maf response element localized to nucleotides -196/-184 relative to the transcription initiation site in the IL-10 promoter. This site represents an atypical 12-O-tetradecanoate-13-acetate-responsive element for musculoaponeurotic fibrosarcoma recognition and functions as an enhancer element in a heterologous and orientation-independent manner. Furthermore, c-Maf is expressed constitutively in resting monocytes/macrophages. IL-4 can up-regulate c-Maf expression, its binding to IL-10 promoter, and dose dependently enhance IL-10 production induced by LPS; moreover, IL-4 failed to enhance LPS-induced IL-10 production in c-Maf-null macrophages. Taken together, these data demonstrate that c-Maf is an indispensable yet constitutive transcription factor for IL-10 gene expression in LPS-activated macrophages, and IL-4 modulates IL-10 production in inflammatory macrophages likely via its ability to induce c-Maf expression. Thus, this study uncovers a novel and important function of c-Maf in macrophages and elucidates its transcriptional mechanism in the regulation of IL-10 gene expression.

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Figures

FIGURE 1
FIGURE 1
Impaired IL-10 production by c-Maf−/− macrophages upon LPS stimulation. A, Fetal liver-derived macrophages from c-Maf−/− and wild-type littermate embryos of gestation day 14.5 were plated into 24-well plates at a density of 0.5 × 106 cells/ml/well. Cells were stimulated with 1 μg/ml LPS. At different time points as indicated, IL-10 mRNA level was analyzed by RPA. One representative of five embryos for each group (n = 5) in two independent experiments with similar results is shown. B and C, Human monocyte-derived macrophages were transduced with adenovirus-expressing c-Maf or EGFP 24 h after the transduction cells were treated with LPS (1 μg/ml) for4hor primed with rhIFN-γ (10 ng/ml) for 16 h, followed by LPS for 4 h. Total RNA was isolated for analysis by RT-PCR (B) or by RPA (C) to detect IL-10 mRNA expression. hIL, human IL; hGAPDH, human GAPDH.
FIGURE 2
FIGURE 2
Ectopic c-Maf expression activates IL-10 gene transcription. A, RAW264.7 cells were transfected by electroporation using 6.5 μg of pIL-10 (−1044/+30)-luc reporter construct together with indicated amounts of expression vector c-Maf (long form) or its control vector pCEFL. The total amount of the effector plasmids (pCEFL/c-Maf) was maintained at 1.8 μg with pCEFL. The result shown here is the summary of two independent experiments (mean ± SD). B, The long and short forms of c-Maf were cotransfected with the IL-10 promoter as described in A. Their respective luciferase activities were expressed as fold induction compared with the activity of pCEFL plus IL-10 promoter, which was set as 1. C, RAW264.7 cells were cotransfected with 6.5 μg of pIL-10 (−1044/ +30)-luc with control vectors or pIL-10 (−1044/+30)-luc plus 1.8 μgof c-Maf expression vector (reporter:effector = 1:1/4 in molarity) or pIL-10 (−1044/+30)-luc and c-Maf expression vector plus v-Maf-DNM (reporter:effector:Mxi-v-Maf = 1:1/4:1 in molarity). Luciferase activity was expressed as fold induction (relative luciferase activity) by arbitrarily setting the activity of pIL-10 (−1044/+30)-luc as 1. Statistical analysis indicated that c-Maf significantly up-regulated IL-10 promoter-driven luciferase activity (p < 0.001, lane 1 vs 2), and Mxi-v-Maf completely blocked the exogenously transfected c-Maf on the induction of pIL-10(−1044/+30)-luc (p < 0.001, lane 2 vs 3; p > 0.05, lane 1 vs 3). D, RAW264.7 cells were cotransfected with 6.5 μg of pIL-10 (−1044/+30)-luc with control vector or with Mxi-v-Maf. Transfected cells were either left alone (medium) or treated with 1 μg/ml LPS for 20 h. Statistical analysis indicated that Mxi-v-Maf significantly blocked the endogenous c-Maf-induced basal (p < 0.05, lane 1 vs 2) and the LPS-induced luciferase activity of pIL-10(−1044/+30)-luc (p < 0.05, lane 3 vs 4). One representative of three independent experiments is shown.
FIGURE 3
FIGURE 3
Identification of c-Maf-responsive regions in the IL-10 promoter. The full-length (−1044/+30) and a series of 5′ deletion mutants of the human IL-10 promoter-luciferase constructs were cotransfected with c-Maf expression vector into RAW264.7 cells at a molar ratio of 1:1/4 (reporter:effector). The relative luciferase activities were plotted against that of the −1044/+30 promoter, which is set as 100%. A representative of two experiments with similar results are shown.
FIGURE 4
FIGURE 4
Binding of c-Maf to IL-10 promoter in vitro. A and B, Recombinant human c-Maf-long protein, generated by the PTNT system, was used in EMSA with 32P-labeled −203/−170 sequence from the IL-10 promoter (A) or the MARE consensus (B). Competition for c-Maf binding was done by adding cold −203/−170 or MARE or the NF-κB consensus sequences in 50× molar excess. The terms “self” and “non-self” refer reciprocally to −203/−170 and MARE, respectively. Lane 6 in A and B is mock transcription-translation controls. C, A series of consecutive mutant probes of every 4- to 5-bp mutations (M1–M6) or a 13-bp mutation (M7) of the predicted c-MARE were generated in the context of the −203/−170 sequence (mutated sequences are in boldface and underlined). D, The mutant oligos described in C were used as cold competitors (50× molar excess) in competitive EMSA with the wild-type −203/−170 probe and recombinant human c-Maf protein. E, Supershift EMSA was conducted with the −203/−170 probe and recombinant human c-Maf protein. Anti-c-Maf Abs (Bethyl Laboratories or Santa Cruz Biotechnology (SC)), control rabbit IgG, and anti-c-fos Ab were used. F and G, Supershift EMSA was conducted using the −203/−170 probe and the nuclear extract isolated from RAW264.7 cells, which had been transfected with the c-Maf expression vector for 48 h, followed by4hof LPS stimulation (1 μg/ml; F) or nuclear extract isolated from HEK 293 cells transduced with c-Maf-expressing adenovirus (G). The same Abs were used as in E. The supershifted bands are indicated by an asterisk (lane 3 in E; lane 2 in F).
FIGURE 5
FIGURE 5
Binding of c-Maf to IL-10 promoter in vivo. A, Partial sequence of the human IL-10 promoter that covers the c-Maf binding site (−196/−184, boldfaced and underlined). The region from −206 to −132 amplified by PCR in the ChIP assay is marked by the two directional arrows. B and C, Human monocyte-derived macrophages, either unstimulated or treated, with LPS (1 μg/ml) for different times as indicated and then subjected to the ChIP procedure with anti-c-Maf Ab (Santa Cruz Biotechnology) or its isotype control (normal rabbit IgG). Two pairs of primers were used to amplify the −203/−132 region (B) and a separate region 3 kb upstream (C). The PCR amplified a 75-bp product in B and a 211-bp product in C. D, Kinetics of c-Maf binding to the IL-10 promoter in LPS-activated human macrophages.
FIGURE 6
FIGURE 6
Enhancer activity of the MARE-like sequence. A, RAW264.7 cells were cotransfected with pIL-10(−206/+30)-luc, pIL-10(−206/+30)M4-luc, or pIL-10(−171/+30)-luc with the c-Maf expression vector (reporter:effector is 1:1/4 in molarity). The crosses in the M4 construct denote the 13-bp mutations. Statistic analysis indicates that, compared with pIL-10(−206/+30)-luc, pIL-10 (−206/+30)M4-luc significantly lost its response to c-Maf induction (p < 0.001) and decreased to the level comparable with the downstream deletion mutant reporter pIL-10(−171/+30)-luc (p > 0.05). Mean values ± SD of three experiments are shown. B, The sequences of −206/−170, −206/−171M4 (mutant), and MARE consensus were cloned into a minimal TK promoter in both orientations (forward and reverse indicated by directional arrows). RAW264.7 cells were cotransfected with these chimeric reporter constructs or the parental pTK-luc, with c-Maf expression vector (reporter:effector = 1:1/4 in molarity). Luciferase activity was expressed as relative stimulation to that of the minimal pTK-luc reporter, which is arbitrarily set as 1. Compared with the minimal pTK-luc reporter, c-Maf significantly induced the response of p(MARE)-TK-luc and p(−206/−170)-TK-luc (p < 0.01 and p < 0.05, respectively) in both forward and reverse orientations; however, p(−206/−170)M4-TK-luc lost its response to c-Maf induction in both orientations (p > 0.05 and p > 0.05, respectively). Mean values ± SD of three experiments are shown.
FIGURE 7
FIGURE 7
Modulation of LPS-induced IL-10, IL-12 p40, and c-Maf expression by IL-4. A and B, Primary human monocytes were treated with LPS (1 μg/ ml) alone or recombinant human IL (hIL)-4 (1 ng/ml at +2 h) alone or LPS plus IL-4 added at different times relative to LPS. Twelve hours later, cell culture supernatants were collected for IL-10 (A) or IL-12 p40 (B) production by ELISA. *, A value of p < 0.05 compared with LPS treatment alone. C and D, Human monocytes were treated with different doses of rhIL-4 and added 2 h after LPS stimulation. Cell culture supernatants were collected 12 h after LPS stimulation for IL-10 and IL-12 p40 secretion by ELISA. *, A value of p < 0.05 and formula image, p < 0.01, compared with LPS treatment alone. E, Human monocytes were treated with different concentrations of IL-4 as indicated for 2 h, and total RNA was isolated for RT-PCR analysis of c-Maf expression (both short and long forms). GAPDH was analyzed as the internal control. F, Human monocytes were treated with IL-4 (1 ng/ml) or LPS (1 μg/ml) or both (IL-4 was added together with LPS) for 8 h, and total RNA was isolated for RT-PCR to examine the long- and short-form c-Maf mRNA expression. GAPDH was analyzed as the internal control. G, Human monocytes were treated with recombinant murine IL-4 (1 ng/ml) or LPS (1 μg/ml) or both (IL-4 was added at +2h) for 8 h, and ChIP assay was conducted as described in Fig. 5B. H, Fetal liver-derived macrophages from c-Maf−/− and c-Maf+/− littermate embryos of gestation day 14.5 were plated into 48-well plates at a density of 0.2 × 106 cells/ml/well. Cells were stimulated with 1 μg/ml LPS, murine IL-4 (1 ng/ml), or LPS + IL-4 (added at +2 h). Supernatants were collected 24 h after LPS treatment for ELISA. Results shown are the summary of four heterozygous and three homozygous knockout embryos. ND, nondetectable.
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