IRF-1 and NF-κB p50/cRel bind to distinct regions of the proximal murine IL-12 p35 promoter during costimulation with IFN-γ and LPS

LPS and IFN-gamma, which activate NF-kappa B 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 kappa B 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-kappa B cRel and p50 bind to the kappa B-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 kappa B-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, IFN-gamma/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-1 and LPS costimulation of macrophages. (C) 2005 Elsevier Ltd. All rights reserved.