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and 
in Lipopolysaccharide-Induced Gene Activation of IL-10 in Mouse Macrophages1
* Graduate Institute of Biopharmaceutics, College of Life Science, National Chiayi University, Chiayi, Taiwan; and
Department of Pharmacology, Medical College, National Cheng Kung University, Tainan, Taiwan
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/EBP
and 
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/EBP and 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/EBP and were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBP or C/EBP, 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/EBP and . These results suggested that the interaction between Sp1 and C/EBP and induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBP and proteins and the enhancement of transactivation activity of C/EBP and by LPS treatment, at least in part, explain the activation of IL-10 gene expression.
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