Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites.
The involvement of AP-1 and NF-kappaB transcription factors in cytokine-mediated induction of human inducible nitric oxide synthase (hiNOS) promoter activity was examined. Luciferase reporter plasmids, containing mutations in AP-1 and NF-kappaB sites, in a hiNOS promoter extending from -8.3 kilobase pairs (kb) to +168, were transiently expressed in A549 cells, and promoter activity was determined after treatment with a cytokine mixture (CM) containing interleukin 1-beta, interferon-gamma, and tumor necrosis factor-alpha. Mutation of the AP-1 heptad located -5301 base pairs upstream decreased gene activation by 90% in a -8.3-kb promoter and a shorter -5.574-kb promoter. Disruption of AP-1 (at -5115) or NF-kappaB (at -115 and -8283) sites reduced promoter activity by 45, 67, and 52%, respectively. Responsiveness to CM was decreased by 85% in constructs mutated in both NF-kappaB sites. By gel retardation analyses, CM increased AP-1- and NF-kappaB binding. Supershift analysis identified Jun D and Fra-2 as components of AP-1 complexes. Each kappaB site bound different complements of NF-kappaB/Rel family members (downstream site, Rel A/p50; upstream site, Rel A/Rel A). Rel A was maximally, whereas IkappaB-alpha was minimally, expressed in nuclei after 1 h of CM treatment, corresponding with the peak in NF-kappaB inding activity. Thus, AP-1 and NF-kappaB are important cis-elements for induction of hiNOS gene transcription.
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590, USA. marksj@gwgate.nhlbi.nih.gov
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Chu
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D016207:Cytokines / Q000502:physiology*; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D016207:Cytokines; D017931:DNA Primers; D016328:NF-kappa B; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II