Multiple NF-kappaB enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene

J Biol Chem. 1998 Jun 12;273(24):15148-56. doi: 10.1074/jbc.273.24.15148.


The human inducible nitric oxide synthase (iNOS) gene is overexpressed in a number of human inflammatory diseases. Previously, we observed that the human iNOS gene is transcriptionally regulated by cytokines and demonstrated that the cytokine-responsive regions are upstream of -3.8 kilobase pairs (kb). Therefore, the purpose of this study was to further localize the functional enhancer elements and to assess the role of the transcription factor NF-kappaB in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-kappaB inhibitors significantly suppressed cytokine-stimulated iNOS mRNA expression and NO synthesis, indicating that NF-kappaB is involved in the induction of the human iNOS gene. Analysis of the first 4.7 kb of the 5'-flanking region demonstrated basal promoter activity and failed to show any cytokine-inducible activity. However, promoter constructs extending to -5.8 and -7.2 kb revealed 2-3-fold and 4-5-fold induction, respectively, in the presence of cytokines. DNA sequence analysis from -3.8 to -7.2 kb identified five putative NF-kappaB cis-regulatory transcription factor binding sites upstream of -4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-kappaB motif at -5.8 kb is required for cytokine-induced promoter activity, while the sites at -5.2, -5.5, and -6.1 kb elicit a cooperative effect. Electromobility shift assays using a site-specific oligonucleotide and nuclear extracts from cells stimulated with cytokine-mixture, tumor necrosis factor-alpha or interleukin-1beta, but not interferon-gamma, exhibited inducible DNA binding activity for NF-kappaB. These data indicate that NF-kappaB activation is required for cytokine induction of the human iNOS gene and identifies four NF-kappaB enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-alpha and interleukin-1beta.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Cell Line
  • Cytokines / pharmacology*
  • DNA Mutational Analysis
  • DNA-Binding Proteins / analysis
  • Enhancer Elements, Genetic / genetics*
  • Gene Expression Regulation, Enzymologic / drug effects*
  • Humans
  • Interleukin-1 / pharmacology
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed / genetics
  • NF-kappa B / genetics*
  • Nitric Oxide Synthase / genetics*
  • Nitric Oxide Synthase Type II
  • Nuclear Proteins / analysis
  • Proline / analogs & derivatives
  • Proline / pharmacology
  • Promoter Regions, Genetic / genetics
  • RNA, Messenger / metabolism
  • Sequence Analysis, DNA
  • Thiocarbamates / pharmacology
  • Tumor Necrosis Factor-alpha / pharmacology


  • Cytokines
  • DNA-Binding Proteins
  • Interleukin-1
  • NF-kappa B
  • Nuclear Proteins
  • RNA, Messenger
  • Thiocarbamates
  • Tumor Necrosis Factor-alpha
  • prolinedithiocarbamate
  • Proline
  • NOS2 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II

Associated data

  • GENBANK/AF049872