Upon LPS stimulation, the kinetics of cytokine production in macrophages is characterized by rapid induction of proinflammatory cytokines and followed by the production of anti-inflammatory cytokines, e.g., IL-10, hence, dampening the exaggeration of proinflammatory cytokine production and associated pathology. This kinetic cytokine production pattern is illustrated in the sepsis syndrome in vivo (46
). IL-10, as a potent anti-inflammatory cytokine, inhibits the expression of a number of inducible genes in macrophages, including several proinflammatory cytokines (47
). Despite its biological importance, relatively little is known about the IL-10 gene regulation. In the present study, we report that protooncogene c-Maf is an essential transcription factor for IL-10 gene expression in macrophages. There are four sets of experiments that point to this conclusion. First, c-Maf-null macrophages exhibit impaired IL-10 production upon LPS stimulation. Second, c-Maf up-regulates both the exogenous IL-10 promoter activity, as well as the endogenous IL-10 gene transcription stimulated by LPS. Third, a c-Maf binding site is localized in the IL-10 promoter to −196/−184, which resembles a TRE-type MARE, demonstrated by both in vitro (EMSA) and in vivo (ChIP assay) experiments. Fourth, functional studies reveal that this site is operational in the response to c-Maf induction by mutagenesis and chimeric promoter approaches. All of these results indicate that c-Maf is a nonredundant transcription factor and regulates IL-10 gene expression through physically binding to the IL-10 promoter. Transcriptional regulation is dependent on the interaction among transcription factors and cis
-elements. In addition, the cis
-elements are wrapped by histones and not always accessible by the transcription factors or transcription factor complexes. Therefore, modification of histones determines their state—loose or condense—and then determines the accessibility of these cis
-elements and then determines the gene transcription state (histone codon theory). The observation that c-Maf is expressed constitutively in resting macrophages initially prompted us to postulate that c-Maf may not bind the MARE-like site in IL-10 promoter until later time points after LPS stimulation, which is known to induce chromatin changes (48
). The ChIP analyses surprisingly revealed constitutive c-Maf binding to the MARE sequence in both the resting state and LPS-activated human macrophages (). There are a few possible explanations: firstly, c-Maf is constantly present and active, and LPS stimulation induces additional factors that, together with c-Maf, contribute to transcriptional activation of IL-10 in macrophages. Secondly, c-Maf may undergo posttranslational modifications. It is reported that c-Maf activity is regulated negatively by the protein kinase A-, protein kinase C-, and MAPK/ERK-signaling cascades; however, mutation of the two most likely phosphorylation sites didn’t change its transactivation ability (49
). Thirdly, c-Maf can interact with transcriptional coactivators CREB-binding protein/p300, as shown in the case of α
A-crystallin promoter. The coactivation of c-Maf by CREB-binding protein/p300 requires histone acetyltransferase activity for crystalline gene expression (50
), and their recruitment could modulate the chromatin structure, exposing the cis
-elements for access by LPS-induced transcription factors and then turn on the IL-10 gene.
Gene regulation is a complex process that involves the coordinated integration of distinct signal transduction pathways. Although c-Maf is essential for IL-10 transcription, c-Maf itself is not enough, and its ability to do so still requires LPS stimulation. This situation is analogous to many other factors that have been shown to be involved in the transcription of IL-10, e.g., CREB-1, activating transcription factor-1 (51
), C/EBP (53
), SV40 promoter 1 (6
), SV40 promoter (55
), and STAT3 (27
). All these factors require an accompanying LPS stimulation, suggesting that multiple signal transduction pathways are involved in the activation of IL-10 expression (56
). Furthermore, LPS-induced IL-10 gene transcription requires de novo protein synthesis because cycloheximide treatment before LPS stimulation can completely block IL-10 gene induction (6
). Thus, we think that c-Maf works in concert with LPS-induced, yet-to-be-identified factors to activate the IL-10 gene. In support of this hypothesis, blocking AP-1 or CREB by AP-1-specific DNM (A-Fos) or CREB-DNM (57
) strongly interfered with the capacity of c-Maf to activate IL-10 transcription (data not shown).
There is increasing evidence indicating that macrophages are a diverse and dynamic population of cells that can be activated through a number of distinct pathways and participate in a wide range of critical functions (41
). In this study, we demonstrated that the IL-10-enhancing effect of IL-4 in LPS-activated human monocytes () is similar to that shown in mouse macrophages (45
). Then we investigated the modulation of c-Maf by IL-4 in human monocytes because it was reported that IL-4 could up-regulate c-Maf through Stat6 in Th cells (42
). Our data demonstrates that IL-4 can regulate c-Maf expression and its binding to IL-10 promoter in human macrophages. Moreover, the characteristics of IL-4-enhanced IL-10 production () not only correlate well with its ability to stimulate c-Maf expression () in human monocytes but also show direct dependency on c-Maf (). Considering that c-Maf is an essential transcription activator for IL-10 gene transcription, our data suggests that IL-4, which is derived mainly from activated Th2 cells, may exert its macrophage-regulating activity through stimulation of c-Maf. This idea is supported by the c-Maf overexpression experiment in human monocyte-derived macrophages, in which c-Maf overexpression enhanced LPS-induced IL-10 expression (), and by the study in c-Maf-null macrophages where rIL-4 failed to enhance LPS-induced IL-10 production ().
Inflammation is a complex response, and it is an attempt to restore homeostasis (58
). Macrophages are one of the main players in inflammatory immune responses. In this study, we demonstrated that c-Maf is an essential transcription factor for IL-10 production in inflammatory macrophages. This observation, together with our previous report that c-Maf is an inhibitor of IL-12 production, suggests that it may be an important factor in the homeostatic regulation. Our findings are one step further to understanding the intriguing and complex mechanisms of macrophage-gene regulation.