CYTOKINE-STIMULATED HUMAN INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) GENE EXPRESSION IS MEDIATED BY ACTIVITY OF TRANSCRIPTION FACTORS NF-??B AND STAT1??

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153 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. Therefore, the purpose of this study was to define the transcription factors responsible for mediating the synergistic cytokine-activation of the human iNOS gene. Previously, we cloned the human iNOS promoter; sequence analysis revealed a number of putative DNA binding elements for NF-κB and STAT1α in the 5′flanking region. Human iNOS gene expression was characterized by treating both human liver (AKN-1) and lung (A549) epithelial cell lines with individual cytokines (TNFα, IL-1β, or IFNγ) or a combination (CM=TNFα+IL-1β+IFNγ). After cytokine-stimulation. Northern blot analysis was performed for iNOS mRNA. iNOS promoter activity was determined by liposome-mediated transfection experiments using human iNOS-luciferase reporter gene constructs. NF-κB and STAT1α DNA-binding activities were examined by electromobility shift assays (EMSA) using nuclear extracts: (Table)TableLow levels of iNOS mRNA were seen in both cell types following single cytokine-stimulation, while CM resulted in a marked increase in iNOS mRNA expression. Similarly, there was no increase in basal iNOS promoter activity in the presence of single cytokines; however, a 5-fold induction was noted in the presence of CM. Nuclear run-on assay confirmed that the increase in promoter activity reflected an increase in the rate of iNOS gene transcription. Finally. EMSA demonstrated increased DNA-binding activity for NF-κB in response to TNFα, IL-1β or CM, but not IFNγ alone. In contrast, IFNγ alone or CM resulted in an increase in STAT1α DNA-binding activity. These results indicate that TNFα and IL-1β induce iNOS transcription by activating NF-κB, while IFNγ signals through STAT1α. Despite the fact that single cytokines activate specific transcription factors, both NF-κB and STAT1α must be coactivated for significant iNOS production in human cells. These data identify two transcription factors required for iNOS gene expression and provide a molecular basis for cytokine-mediated signal transduction that may have implications for regulating other genes associated with infection or transplant rejection.