title: IRF-1 and NF-kappaB p50/cRel bind to distinct regions of the proximal murine IL-12 p35 promoter during costimulation with IFN-gamma and LPS.
abstract: LPS and IFN-gamma, which activate NF-kappaB cRel/p50 and IFN regulatory factor-1 (IRF-1), respectively, costimulate expression of the IL-12 p35 subunit in macrophages. The murine p35 promoter proximal to exon 2 is active during costimulation with IFN-gamma and LPS because it contains kappaB and IRF elements (E) with significant homology to the human p35 promoter. IFN-gamma or LPS stimulate nuclear localization of IRF-1 or cRel/p50, respectively, in the RAW 264.7 macrophage cell line. EMSAs reveal that IFN-gamma/LPS stimulates within 2 h, in RAW 264.7 cells or peritoneal macrophages, nuclear localization of proteins that target nt -137/-93 of the p35 promoter. DNA affinity assays utilizing nuclear extracts from RAW 264.7 cells show that NF-kappaB cRel and p50 bind to the kappaB-E within nt -122 to -93 of the p35 exon 2 promoter while IRF-1 binds to the IRF-E within nt -157 to -113 but not the one within nt -122 to -93. In addition, p50/cRel attachment to the kappaB-E was not dependent upon IRF-1 association with the IRF-E, and vice versa. Chromosome immunoprecipitation assays confirm inducible recruitment of IRF-1 and cRel to the endogenous p35 exon 2 promoter in both RAW 264.7 and primary macrophages costimulated with IFN-gamma and LPS. IFN-gamma, IFNgamma/LPS, or overexpression of IRF-1 plus cRel activated the wild-type p35 promoter reporter but not the p35 promoter reporter mutated at nt -110/-101 or in the presence of IRF-1 siRNA. Thus, cRel with IRF-1 induce p35 expression through a small region of the p35 exon 2 promoter during IFN-gamma and LPS costimulation of macrophages.
authors.affiliation: Department of Oral Biology and the Nebraska Center for Virology, University of Nebraska Medical Center, Lincoln, NE 68583-0740, USA.
authors.forename: Jutta I
authors.initials: JI
authors.lastname: Kollet
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Petro
mesh_terms: D000818:Animals; D001665:Binding Sites; D005091:Exons; D050856:Interferon Regulatory Factor-1 / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D018664:Interleukin-12 / Q000235:genetics*; D053765:Interleukin-12 Subunit p35; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages; D051379:Mice; D052002:NF-kappa B p50 Subunit / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics*; D051996:Transcription Factor RelA / Q000378:metabolism*
chemical_list: C508858:IL12A protein, human; D050856:Interferon Regulatory Factor-1; D053765:Interleukin-12 Subunit p35; D008070:Lipopolysaccharides; D052002:NF-kappa B p50 Subunit; D021122:Protein Subunits; D051996:Transcription Factor RelA; D018664:Interleukin-12; D007371:Interferon-gamma