Octamer Motif is Required for the NF-κB-mediated Induction of the Inducible Nitric Oxide Synthase Gene Expression in RAW 264.7 Macrophages

Yong Man Kim, Chang Bo Ko, Yuk Pheel Park, Yung Jin Kim, Sang Gi Paik

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

The promoter of the mouse inducible nitric oxide synthase (iNOS) has a putative octamer motif (ATGCAAAA) which exists 24 bp upstream from the TATA box and is mismatched at a single residue from the consensus octamer motif. To examine whether this site is involved in iNOS expression, we constructed various deletions and site-directed mutants of the iNOS promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene, transfected the constructs into RAW 264.7 macrophages, and stimulated the cells with interferon-γ (IFN-γ) and/or lipopolysaccharide (LPS). CAT activity was not induced by LPS in constructs containing only the octamer motif (-71 to +82), but was induced with constructs containing the octamer motif and the upstream sequences of the NF-κB site (-91 to +82). However, a site-directed mutation of the octamer motif in the context of the -91 to +82 promoter construct or an extended promoter construct (-1542 to +82) abolished IFN-γ and/or LPS-induced CAT activity. Similar results were obtained from site-directed mutants at either the NF-κB site or both the NF-κB site and octamer motif in these two constructs. In addition, we demonstrated that the conversion of the iNOS octamer motif into a consensus sequence increased CAT activity. Electrophoretic mobility shift assay (EMSA) performed with the NF-κB site or the octamer motif-containing oligonucleotide probe revealed that NF-κB binding was induced by LPS treatment, while the Oct-1 binding was constitutive. Competition assays performed with octamer-related oligonucleotide competitors derived from the immunoglobulin-κB or SV40 promoter confirmed the identity of the iNOS promoter sequence as being a Oct-1 binding site. EMSA carried out using a probe containing both the NF-κB site and the octamer motif identified two LPS-induced complexes. Competition assays with each NF-κB site or octamer motif competitor revealed that NF-κB and Oct-1 were present in these two complexes. These data suggest that, besides the NF-κB site, the octamer motif is essential for the maximal expression of the iNOS gene in murine macrophages, and the direct interaction of Oct-1 and NF-κB is important for the regulation of this gene.

Original languageEnglish (US)
Pages (from-to)99-109
Number of pages11
JournalMolecules and Cells
Volume9
Issue number1
StatePublished - Feb 28 1999
Externally publishedYes

Keywords

  • Inducible Nitric Oxide Synthase
  • NF-κB
  • Octamer Motif
  • RAW 264.7 Cell
  • Transcription Regulation

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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