Identification of a novel tumor necrosis factor α‐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 phox , appears to play a more prominent role in final activation of the enzyme complex. Based on the importance of p67 phox , we investigated transcriptional regulation of the p67 phox gene [neutrophil cytosolic factor 2 ( NCF2 )] and demonstrated previously that activator protein‐1 (AP‐1) was essential for basal transcriptional activity. As p67 phox can be up‐regulated by tumor necrosis factor α (TNF‐α), which activates AP‐1, we hypothesized that TNF‐α might regulate NCF2 transcription via AP‐1. In support of this hypothesis, we show here that NCF2 promoter‐reporter constructs are up‐regulated by TNF‐α but only when AP‐1 factors were coexpressed. Consistent with this observation, we also demonstrate that NCF2 mRNA and p67 phox protein are up‐regulated by TNF‐α 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‐α 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‐α‐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‐α‐induced up‐regulation of p67 phox .