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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-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 upstream sequences of the NF-kappaB 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-gamma and/or LPS-induced CAT activity. Similar results were obtained from site-directed mutants at either the NF-kappaB site or both the NF-kappaB 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-kappaB site or the octamer motif-containing oligonucleotide probe revealed that NF-kappaB 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-kappaB 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-kappaB site and the octamer motif identified two LPS-induced complexes. Competition assays with each NF-kappaB site or octamer motif competitor revealed that NF-kappaB and Oct-1 were present in these two complexes. These data suggest that, besides the NF-kappaB 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-kappaB is important for the regulation of this gene.
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