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/EBPβ and δ 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/EBPβ and δ 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/EBPβ and δ were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBPβ or C/EBPδ, 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/EBPβ and δ. These results suggested that the interaction between Sp1 and C/EBPβ and δ induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBPβ and δ proteins and the enhancement of transactivation activity of C/EBPβ and δ by LPS treatment, at least in part, explain the activation of IL-10 gene expression.
All Science Journal Classification (ASJC) codes
- Immunology and Allergy