A Novel NF-κB Enhancer Region Regulates Cytokine Induction Of The Human Inducible Nitric Oxide Synthase (iNOS) Gene Promoter

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123 Nitric oxide (NO) is a potent immunomodulator which has diverse roles during infection, inflammation, and solid organ transplant rejection. Previously, we have shown that a combination of cytokines stimulate human inducible NO synthase (iNOS) expression, however, almost nothing is known about the specific cytokine-activated nuclear factors that control iNOS gene transcription. To determine the molecular mechanisms and transcription factors regulating expression of the human iNOS gene, we cloned and sequenced 7.2 kilobases (kb) of the gene's 5′-flanking DNA. To characterize this region, we generated deletional constructs of the human iNOS promoter (1.3 to 7.2 kb) linked to the luciferase reporter gene. The deletional DNA promoter constructs were transfected into human liver or lung epithelial cells using liposomes and then stimulated with cytokine mix (CM) of TNFα (500 U/ml)+ IL-1β (100 U/ml) + IFNγ (250 U/ml). Cell extracts were assayed for luciferase activity as a measure of iNOS promoter strength (graph, mean± SEM, n=6 per group): No increase in luciferase activity was noted with the 1.3 or 4.7 kb iNOS promoter constructs in response to CM. However, in both cell types a 3 and 5-fold increase in promoter activity was seen with the 5.8 kb and 7.2 kb constructs, respectively. Nuclear runon assay showed a 4-fold increase in transcription rate in response to CM. These data indicate that the human iNOS gene is transcriptionally induced by cytokines and localizes the cytokine-responsive promoter elements between 4.7 and 7.2 kb upstream in the gene. Computer analysis of the DNA sequence reveals several potential binding sites for the NF-κB transcription factor. Addition of PDTC (a NF-κB inhibitor) abrogated the CM-induced promoter activity, and gel shift assays showed inducible NF-κB DNA binding activity (data not shown), further demonstrating a role for NF-κB in regulating transcription of the human iNOS gene. Site-directed mutagenesis of 4 novel NF-κB sites from -5.2 to -6.0 kb upstream in the gene abolished the CM-induced iNOS promoter activity, thus identifying a previously unrecognized NF-κB enhancer region. Characterization of the human iNOS gene promoter is likely to provide insight into the mechanisms of cytokine signalling and suggests new strategies for modulating iNOS expression during infection or solid organ transplant rejection. Figure