Identification of a novel tumor necrosis factor alpha-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, 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.
Gauss
Katherine A
KA
Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA.
Bunger
Peggy L
PL
Larson
Trina C
TC
Young
Catherine J
CJ
Nelson-Overton
Laura K
LK
Siemsen
Daniel W
DW
Quinn
Mark T
MT
D005069:Evaluation Studies as Topic; D018922:HL-60 Cells; D006801:Humans; D009000:Monocytes / Q000378:metabolism; D022423:Myeloid Cells / Q000187:drug effects / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D010750:Phosphoproteins / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000032:analysis / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation
D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; C079789:neutrophil cytosol factor 67K; D010100:Oxygen