TY - JOUR
T1 - 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
AU - Kollet, Jutta I.
AU - Petro, Thomas M.
PY - 2006/2
Y1 - 2006/2
N2 - LPS and IFN-γ, which activate NF-κB 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-γ and LPS because it contains κB and IRF elements (E) with significant homology to the human p35 promoter. IFN-γ or LPS stimulate nuclear localization of IRF-1 or cRel/p50, respectively, in the RAW 264.7 macrophage cell line. EMSAs reveal that IFN-γ/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-κB cRel and p50 bind to the κB-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 κB-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-γ and LPS. IFN-γ, IFNγ/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-γ and LPS costimulation of macrophages.
AB - LPS and IFN-γ, which activate NF-κB 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-γ and LPS because it contains κB and IRF elements (E) with significant homology to the human p35 promoter. IFN-γ or LPS stimulate nuclear localization of IRF-1 or cRel/p50, respectively, in the RAW 264.7 macrophage cell line. EMSAs reveal that IFN-γ/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-κB cRel and p50 bind to the κB-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 κB-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-γ and LPS. IFN-γ, IFNγ/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-γ and LPS costimulation of macrophages.
KW - IL-12
KW - IRF-1
KW - Macrophages
KW - NF-κB
KW - Transcription factors
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U2 - 10.1016/j.molimm.2005.04.004
DO - 10.1016/j.molimm.2005.04.004
M3 - Article
C2 - 15871905
AN - SCOPUS:28944440894
VL - 43
SP - 623
EP - 633
JO - Molecular Immunology
JF - Molecular Immunology
SN - 0161-5890
IS - 6
ER -