CYTOKINE-MEDIATED TRANSCRIPTIONAL INDUCTION OF THE HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASE GENE REQUIRES BOTH ACTIVATOR PROTEIN-1 AND NUCLEARFACTOR KAPPA-B-BINDING SITES
J. Markskonczalik et al., CYTOKINE-MEDIATED TRANSCRIPTIONAL INDUCTION OF THE HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASE GENE REQUIRES BOTH ACTIVATOR PROTEIN-1 AND NUCLEARFACTOR KAPPA-B-BINDING SITES, The Journal of biological chemistry, 273(35), 1998, pp. 22201-22208
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.