Identification of a novel tumor necrosis factor alpha-responsive region in the NCF2 promoter

J Leukoc Biol. 2005 Feb;77(2):267-78. doi: 10.1189/jlb.0604329. Epub 2004 Oct 28.


The phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is a multiprotein enzyme that catalyzes the production of microbicidal oxidants. Although oxidase assembly involves association of several membrane and cytosolic oxidase proteins, one of the cytosolic cofactors, p67phox, appears to play a more prominent role in final activation of the enzyme complex. Based on the importance of p67phox, we investigated transcriptional regulation of the p67phox gene [neutrophil cytosolic factor 2 (NCF2)] and demonstrated previously that activator protein-1 (AP-1) was essential for basal transcriptional activity. As p67phox can be up-regulated by tumor necrosis factor alpha (TNF-alpha), which activates AP-1, we hypothesized that TNF-alpha might regulate NCF2transcription via AP-1. In support of this hypothesis, we show here that NCF2 promoter-reporter constructs are up-regulated by TNF-alpha but only when AP-1 factors were coexpressed. Consistent with this observation, we also demonstrate that NCF2 mRNA and p67phox protein are up-regulated by TNF-alpha in various myeloid cell lines as well as in human monocytes. It was surprising that mutagenesis of the AP-1 site in NCF2 promoter constructs did not eliminate TNF-alpha induction, suggesting additional elements were involved in this response and that AP-1 might play a more indirect role. Indeed, we used NCF2 promoter-deletion constructs to map a novel TNF-alpha-responsive region (TRR) located between -56 and -16 bp upstream of the translational start site and demonstrated its importance in vivo using transcription factor decoy analysis. Furthermore, DNase footprinting verified specific binding of factor(s) to the TRR with AP-1 binding indirectly to this region. Thus, we have identified a novel NCF2 promoter/enhancer domain, which is essential for TNF-alpha-induced up-regulation of p67phox.

Publication types

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

MeSH terms

  • Evaluation Studies as Topic
  • HL-60 Cells
  • Humans
  • Monocytes / metabolism
  • Myeloid Cells / drug effects
  • Myeloid Cells / metabolism
  • Oligodeoxyribonucleotides / pharmacology
  • Oxygen / metabolism
  • Phosphoproteins / drug effects*
  • Phosphoproteins / genetics*
  • Phosphoproteins / metabolism
  • Promoter Regions, Genetic / drug effects
  • Promoter Regions, Genetic / physiology*
  • RNA, Messenger / drug effects
  • RNA, Messenger / metabolism
  • Transcription Factor AP-1 / analysis
  • Transcription Factor AP-1 / drug effects
  • Transcription Factor AP-1 / metabolism
  • Tumor Necrosis Factor-alpha / pharmacology*
  • Up-Regulation


  • Oligodeoxyribonucleotides
  • Phosphoproteins
  • RNA, Messenger
  • Transcription Factor AP-1
  • Tumor Necrosis Factor-alpha
  • neutrophil cytosol factor 67K
  • Oxygen