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Previous studies have revealed that LPS can activate transcription of the IL-10 gene promoter through an SV40 promoter factor 1 (Sp1) binding site in mouse macrophage RAW264.7. In this study, we determined that, in addition to Sp1, C/EBPbeta and delta were also involved in LPS-induced gene expression of IL-10. By transient transfection with 5'-deletion mutants of the IL-10 promoter, we found that there were two LPS-responsive elements in the promoter of the mouse IL-10 gene. Analysis of these two regions by gel shift assay suggested that Sp1 and C/EBPbeta and delta were bound to these two regions, respectively. By site-directed mutagenesis, we found that disruption at both the Sp1 and C/EBP binding sites almost completely blocked the LPS response. By gel shift assay and Western blotting, we found that the DNA binding complex and protein expression of C/EBPbeta and delta were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBPbeta or C/EBPdelta, respectively, activated the promoter of the IL-10 gene, and they were enhanced by LPS. Coimmunoprecipitation experiments in intact cells indicated that LPS stimulated interaction between Sp1 and C/EBPbeta and delta. These results suggested that the interaction between Sp1 and C/EBPbeta and delta induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBPbeta and delta proteins and the enhancement of transactivation activity of C/EBPbeta and delta by LPS treatment, at least in part, explain the activation of IL-10 gene expression.

the journal of immunology/the journal of immunology

Functional Cooperation of Simian Virus 40 Promoter Factor 1 and CCAAT/Enhancer-Binding Protein β and δ in Lipopolysaccharide-Induced Gene Activation of IL-10 in Mouse Macrophages