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Published online before print October 28, 2004

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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0604329

Received for publication June 8, 2004.
Revised October 5, 2004.
Accepted for publication October 12, 2004.

Article

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

Department of Veterinary Molecular Biology, Montana State University, Bozeman

^@ To whom correspondence should be addressed. E-mail: mquinn{at}montana.edu.

	Abstract

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 cytosol factor 2 (NCF2)] and demonstrated previously that activated protein-1 (AP-1) was essential for basal transcriptional activity. As p67^phox can be up-regulated by tumor necrosis factor (TNF-), which activates AP-1, we hypothesized that TNF- 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- but only when AP-1 factors are coexpressed. Consistent with this observation, we also demonstrate that NCF2 mRNA and p67^phox protein are up-regulated by TNF- in 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 TNF- 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--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^phox.

Key Words: neutrophil • promoter • transcriptional regulation • NADPH oxidase • monocyte

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