Octamer motif is required for the NF-kappaB-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 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.
Kim
Y M
YM
Department of Biology, Chungnam National University, Taejon, Korea.
Ko
C B
CB
Park
Y P
YP
Kim
Y J
YJ
Paik
S G
SG
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004790:Enzyme Induction / Q000235:genetics; D005786:Gene Expression Regulation; D051863:Host Cell Factor C1; D008070:Lipopolysaccharides / Q000494:pharmacology; D046911:Macromolecular Substances; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D050811:Octamer Transcription Factor-1; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D000097782:NF-kappaB-Inducing Kinase
D004268:DNA-Binding Proteins; D051863:Host Cell Factor C1; D008070:Lipopolysaccharides; D046911:Macromolecular Substances; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D050811:Octamer Transcription Factor-1; D017346:Protein Serine-Threonine Kinases; D014157:Transcription Factors; D000097782:NF-kappaB-Inducing Kinase; C497693:Hcfc1 protein, mouse; C494166:Pou2f1 protein, mouse; C496317:Nos2 protein, mouse