Upstream NF-kappaB 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-kappaB site, the region from -973 to -925 which contains a potential binding site for NF-kappaB (upstream NF-kappaB 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-kappaB 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-kappaB 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-kappaB sites showed LPS-induced NF-kappaB binding to both probes and its higher affinity to the upstream NF-kappaB site. Taken together, these suggest that the upstream NF-kappaB site having enhancer function, besides the downstream NF-kappaB site as a core promoter, is essential for maximal expression of the iNOS gene.
Kim
Y M
YM
Department of Biology, Chungnam National University, Taejon, South Korea.
Lee
B S
BS
Yi
K Y
KY
Paik
S G
SG
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D007371:Interferon-gamma / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D016296:Mutagenesis; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014162:Transfection
D008070:Lipopolysaccharides; D016328:NF-kappa B; D007371:Interferon-gamma; D004247:DNA; D019001:Nitric Oxide Synthase