Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B

The human inducible nitric oxide synthase (hiNOS) gene is expressed in seve ral disease states and is also important in the normal immune response. Pre viously, we described a cytokine-responsive enhancer between -5.2 and -6.1 kb in the 5 ' -flanking hiNOS promoter DNA, which contains multiple nuclear factor KP (NF-kappaB) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway fo r regulation of hiNOS gene transcription, In A549 human lung epithelial cel ls, a combination of cytokines tumor necrosis factor-cu, interleukin-1 beta , and lFN-gamma (TNF-alpha, IL-1 beta, and IFN-gamma) function synergistica lly for induction of hiNOS transcription. Pharmacological inhibitors of Jak 2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene express ion. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-indu ced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DN A element at -5.8 kb in the hiNOS promoter identifies a bifunctional NF-kap paB/Stat 1 motif, In contrast, gel shift assays indicate that only Stat 1 b inds to the DNA element at -5.2 kb in the hiNOS promoter. Interestingly, St at 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH hu man fibroblasts, which are refractory to iNOS induction. Overexpression of NF-KB activates hiNOS promoter-reporter expression in Stat 1 mutant fibrobl asts, but not in the wild type, suggesting that Stat 1 inhibits NF-KB funct ion in these cells. These results indicate that both Stat 1 and NF-KB are i mportant in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.