Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites

J Biol Chem. 1998 Aug 28;273(35):22201-8. doi: 10.1074/jbc.273.35.22201.


The involvement of AP-1 and NF-kappaB transcription factors in cytokine-mediated induction of human inducible nitric oxide synthase (hiNOS) promoter activity was examined. Luciferase reporter plasmids, containing mutations in AP-1 and NF-kappaB sites, in a hiNOS promoter extending from -8.3 kilobase pairs (kb) to +168, were transiently expressed in A549 cells, and promoter activity was determined after treatment with a cytokine mixture (CM) containing interleukin 1-beta, interferon-gamma, and tumor necrosis factor-alpha. Mutation of the AP-1 heptad located -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-kappaB (at -115 and -8283) sites reduced promoter activity by 45, 67, and 52%, respectively. Responsiveness to CM was decreased by 85% in constructs mutated in both NF-kappaB sites. By gel retardation analyses, CM increased AP-1- and NF-kappaB binding. Supershift analysis identified Jun D and Fra-2 as components of AP-1 complexes. Each kappaB site bound different complements of NF-kappaB/Rel family members (downstream site, Rel A/p50; upstream site, Rel A/Rel A). Rel A was maximally, whereas IkappaB-alpha was minimally, expressed in nuclei after 1 h of CM treatment, corresponding with the peak in NF-kappaB inding activity. Thus, AP-1 and NF-kappaB are important cis-elements for induction of hiNOS gene transcription.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Blotting, Western
  • Cytokines / physiology*
  • DNA Primers
  • Gene Expression Regulation, Enzymologic / physiology*
  • Humans
  • NF-kappa B / metabolism*
  • Nitric Oxide Synthase / genetics*
  • Nitric Oxide Synthase Type II
  • Promoter Regions, Genetic
  • RNA, Messenger / genetics
  • Transcription Factor AP-1 / metabolism*
  • Transcription, Genetic / physiology*
  • Transcriptional Activation
  • Tumor Cells, Cultured


  • Cytokines
  • DNA Primers
  • NF-kappa B
  • RNA, Messenger
  • Transcription Factor AP-1
  • NOS2 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II