CYTOKINE-MEDIATED TRANSCRIPTIONAL INDUCTION OF THE HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASE GENE REQUIRES BOTH ACTIVATOR PROTEIN-1 AND NUCLEARFACTOR KAPPA-B-BINDING SITES

The involvement of AP-1 and NF-kappa B transcription factors in cytoki ne-mediated induction of human inducible nitric oxide synthase (hiNOS) promoter activity was examined. Luciferase reporter plasmids, contain ing mutations in AP-1 and NF-kappa B sites, in a hiNOS promoter extend ing from -8.3 kilobase pairs (kb) to +168, were transiently expressed in A549 cells, and promoter activity was determined after treatment wi th a cytokine mixture (CM) containing interleukin 1-beta, interferon-g amma, and tumor necrosis factor-alpha. Mutation of the AP-1 heptad loc ated -5301 base pairs upstream decreased gene activation by 90% in a - 8.3-kb promoter and a shorter -5.574-kb promoter. Disruption of AP-1 ( at -5115) or NF-kappa B (at -115 and -8283) sites reduced promoter act ivity by 45, 67, and 52%, respectively. Responsiveness to CM was decre ased by 85% in constructs mutated in both NF-kappa B sites. By gel ret ardation analyses, CM increased AP-1- and NF-kappa B binding. Supershi ft analysis identified Jun D and Fra-2 as components of AP-1 complexes . Each kappa B Site bound different complements of NF-kappa B/Rel fami ly members (downstream site, Rel A/p50; upstream site, Rel A/Rel A). R el A was maximally, whereas I kappa B-alpha was minimally, expressed i n nuclei after 1 h of CM treatment, corresponding with the peak in NF- kappa B inding activity. Thus, AP-1 and NF-kappa B are important cis-e lements for induction of hiNOS gene transcription.