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

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, p67, appears to play a more prominent role in final activation of the enzyme complex. Based on the importance of p67, we investigated transcriptional regulation of the p67 gene [neutrophil cytosol factor 2 (NCF2)] and demonstrated previously that activated protein-1 (AP-1) was essential for basal transcriptional activity. As p67 can be up-regulated by tumor necrosis factor (TNF), which activates AP-1, we hypothesized that TNFmight regulate NCF2 transcription via AP-1. In support of this hypothesis, we show here that NCF2 promoterreporter constructs are up-regulated by TNFbut only when AP-1 factors are coexpressed. Consistent with this observation, we also demonstrate that NCF2 mRNA and p67 protein are up-regulated by TNFin various myeloid cell lines as well as in human monocytes. It is surprising that mutagenesis of the AP-1 site in NCF2 promoter constructs did not eliminate TNFinduction, 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-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-induced up-regulation of p67. J. Leukoc. Biol. 77: 000–000; 2005.