DEXAMETHASONE SUPPRESSES INOS GENE-EXPRESSION BY UP-REGULATING I-KAPPA-B-ALPHA AND INHIBITING NF-KAPPA-B

Cytokine-stimulated inducible nitric oxide synthase (iNOS) gene expres sion is dependent on nuclear factor-kappa B (NF-kappa B) activation an d is suppressed by glucocorticoids (GC). In this study we examined the molecular mechanisms of GC inhibition of iNOS expression in rat hepat ocytes. Combinations of tumor necrosis factor-alpha, interleukin-1 bet a, and interferon-gamma (cytokine mixture CM) induced high levels of i NOS mRNA and NO synthesis. The synthetic GC dexamethasone markedly rep ressed iNOS mRNA and protein expression, and nuclear run-on assays sho wed that this inhibition was occurring at the level of transcription. In addition, transfection studies showed that CM-stimulated activity o f a 1.6-kb murine iNOS promoter fragment linked upstream of luciferase was suppressed by dexamethasone. Electromobility shift assays demonst rated that CM induced the appearance of an NF-kappa B complex composed of p50 and p65 subunits; the addition of dexamethasone markedly decre ased this band shift. I-kappa B alpha expression was decreased by CM a nd upregulated in the presence of dexamethasone. Subsequently, nuclear p65 levels were decreased by dexamethasone compared with CM-treated c ells. Thus GC repress NF-kappa B DNA-binding activity in rat hepatocyt es in part through the upregulation of its inhibitor I-kappa B alpha. These data indicate that one mechanism by which GC block iNOS expressi on is through the inhibition of NF-kappa B activation resulting in dec reased iNOS transcription.