Human inducible nitric oxide synthase (hiNOS) is responsible for nitric oxide (NO) synthesis in response to inflammatory mediators. Regulation of (hiNOS) occurs predominantly at the transcriptional level. The proximal 8296-bp region of the hiNOS promoter contains many potential cytokine-response elements including those for AP-1 and NF-kB. AP-1 and NF-kB are ubiquitous transcription factors and pleiotropic regulators of many genes encoding proteins involved in the modulation of inflammatory and host defense processes in eukaryotic cells. The involvement of AP-1 and NF-kB 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-kB sites, were transiently expressed in A549 cells (a human lung epithelial adenocarcinoma cell line) and promoter activity was determined after treatment with CM (a cytokine mixture containing IL-1beta, IFN-gamma, and TNF-alpha). Mutation of the AP-1 heptad -5301 bp upstream of the transcription start site decreased gene activation by 90%. Disruption of AP-1 (at -5115), and NF-kB (at -115 and -8283) sites resulted in reduction of promoter activity by 45%, 67%, and 52%, respectively. Responsiveness to CM was reduced by 85% in constructs mutated in both NF-kB sites. Gel retardation analyses revealed that CM increased AP-1- and NF-kB- binding. With NF-kB sequences, protein/DNA complexes were observed only after treatment with CM. Induction was highest when first tested at 1 h and decreased with longer stimulation. In contrast, constitutive AP-1-binding was found in untreated cells and cytokine stimulation increased binding in a biphasic manner; with maximal binding at 3 and 24 h. Supershift analysis identified Jun D and Fra-2 as components of AP-1 complexes. Interestingly, each kB site bound different complements of NF-kB/Rel family members (proximal site, Rel A/p50; distal site, Rel A/Rel A). At the protein level, Jun D and p50 were constitutively expressed and Fra 2 was induced in CM-treated cells. In agreement with the current model of NF-kB regulation, Rel A was maximally, while IkB-alpha was minimally, expressed in nuclei after 1 h of CM treatment, corresponding with the peak in NF-kB-binding activity. Altogether, these findings suggest that AP-1 and NF-kB are important cis-elements in the transcriptional induction of hiNOS gene by cytokines in A549 cells.
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