title: Functional cooperation of simian virus 40 promoter factor 1 and CCAAT/enhancer-binding protein beta and delta in lipopolysaccharide-induced gene activation of IL-10 in mouse macrophages.
abstract: 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.
authors.affiliation: Graduate Institute of Biopharmaceutics, College of Life Science, National Chiayi University, Chiayi, Taiwan.
authors.forename: Yi-Wen
authors.initials: YW
authors.lastname: Liu
authors.forename: Hui-Ping
authors.initials: HP
authors.lastname: Tseng
authors.forename: Lei-Chin
authors.initials: LC
authors.lastname: Chen
authors.forename: Ben-Kuen
authors.initials: BK
authors.lastname: Chen
authors.forename: Wen-Chang
authors.initials: WC
authors.lastname: Chang
mesh_terms: D000818:Animals; D020029:Base Pairing; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D051743:CCAAT-Enhancer-Binding Protein-delta; D022762:CCAAT-Enhancer-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D004330:Drosophila; D005786:Gene Expression Regulation / Q000276:immunology*; D016753:Interleukin-10 / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000276:immunology*; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000276:immunology; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; C497253:Cebpd protein, mouse; D008070:Lipopolysaccharides; D016328:NF-kappa B; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D016753:Interleukin-10; D051743:CCAAT-Enhancer-Binding Protein-delta