Upstream NF-kappa B site is required for the maximal expression of mouse inducible nitric oxide synthase gene in interferon-gamma plus lipopolysaccharide-induced RAW 264.7 macrophages

Transient transfection assays with various deletion mutants of the mouse inducible nitric oxide synthase (iNOS) promoter linked to a CAT reporter gene demonstrated that, besides the downstream NF-kappa B site, the region from -973 to -925 which contains a potential binding site for NF-kappa B (upstream NF-kappa B site) also mediated lipopolysaccharide (LPS)-inducibility in mouse macrophage cell line RAW 264.7. Site-specific mutation of three conserved nucleotides within the upstream NF-kappa B site abolished additional induction by LPS as well as maximal expression of iNOS by IFN-gamma plus LPS. In contrast, site-specific mutation of the downstream NF-kappa B site caused almost all reduction in expression of the reporter gene by LPS or LPS plus IFN-gamma. Electrophoretic mobility shift assays with the two NF-kappa B sites showed LPS-induced NF-kappa B binding to both probes and its higher affinity to the upstream NF-kappa B site. Taken together, these suggest that the upstream NF-kappa B Site having enhancer function, besides the downstream NF-kappa B site as a core promoter, is essential for maximal expression of the iNOS gene. (C) 1997 Academic Press.