Octamer motif is required for the NF-kappa B-mediated induction of the inducible nitric oxide synthase gene expression in RAW 264.7 macrophages

The promoter of the mouse inducible nitric oxide synthase (iNOS) has a puta tive 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 v arious deletions and site-directed mutants of the iNOS promoter linked to t he chloramphenicol acetyltransferase (CAT) reporter gene, transfected the c onstructs into RAW 264.7 macrophages, and stimulated the cells with interfe ron-gamma (IFN-gamma) 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 ups tream sequences of the NF-kappa B Site (-91 to +82), However, a site-direct ed 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-g amma and/or LPS-induced CAT activity. Similar results were obtained from si te-directed mutants at either the NF-kappa B site or both the NF-kappa B si te and octamer motif in these two constructs. In addition, we demonstrated that the conversion of the iNOS octamer motif into a consensus sequence inc reased CAT activity. Electrophoretic mobility shift assay (EMSA) performed with the NF-kappa B site or the octamer motif-containing oligonucleotide pr obe revealed that NF-kappa B binding was induced by LPS treatment, while th e Oct-1 binding was constitutive, Competition assays performed with octamer -related oligonucleotide competitors derived from the immunoglobulin-kappa B 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-kappa B site and the octamer moth identified two LPS-induced complex es. Competition assays with each NF-kappa B site or octamer motif competito r revealed that NF-kappa B and Oct-1 were present in these two complexes, T hese data suggest that, besides the NF-kappa B site, the octamer motif is e ssential for the maximal expression of the iNOS gene in murine macrophages, and the direct interaction of Oct-1 and NF-kappa B is important for the re gulation of this gene,