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

Jutta I. Kollet, Thomas M. Petro

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)623-633
Number of pages11
JournalMolecular Immunology
Volume43
Issue number6
DOIs
StatePublished - Feb 1 2006

Keywords

  • IL-12
  • IRF-1
  • Macrophages
  • NF-κB
  • Transcription factors

ASJC Scopus subject areas

  • Immunology
  • Molecular Biology

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