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Arterioscler Thromb Vasc Biol. 2009 Oct;29(10):1651-6. doi: 10.1161/ATVBAHA.109.191502. Epub 2009 Jul 16.

Knockout of p47 phox uncovers a critical role of p40 phox in reactive oxygen species production in microvascular endothelial cells.

Author information

1
Faculty of Health and Medical Sciences, AY Building, University of Surrey, Guildford, Surrey GU2 7XH, UK.

Abstract

OBJECTIVE:

p40(phox) is an important regulatory subunit of NADPH oxidase, but its role in endothelial reactive oxygen species (ROS) production remains unknown.

METHODS AND RESULTS:

Using coronary microvascular endothelial cells isolated from wild-type and p47(phox) knockout mice, we found that knockout of p47(phox) increased the level of p40(phox) expression, whereas depletion of p40(phox) in wild-type cells increased p47(phox) expression. In both cases, the basal ROS production (without agonist stimulation) was well preserved. Double knockout of p40(phox) and p47(phox) dramatically reduced (approximately 65%) ROS production and cells started to die. The transcriptional regulation of p40(phox) and p47(phox) expressions involves HBP1. p40(phox) was prephosphorylated in resting cells. PMA stimulation induced p40(phox) swift dephosphorylation (within 1 minute) in parallel with the start of p47(phox) phosphorylation. p40(phox) was then rephosphorylated, and this was accompanied with an increase in ROS production. Depletion of p40(phox) resulted in approximately 67% loss in agonist-induced ROS production despite the presence of p47(phox). These were further supported by experiments on mouse aortas stimulated with angiotensin II.

CONCLUSIONS:

p40(phox) is prephosphorylated in resting endothelial cells and can compensate p47(phox) in keeping basal ROS production. Dephosphorylation of p40(phox) is a prerequisite for agonist-induced p47(phox) phosphorylation, and p40(phox) through its dynamic dephosphorylation and rephosphorylation is involved in the regulation of agonist-induced ROS production.

PMID:
19608974
PMCID:
PMC2888064
DOI:
10.1161/ATVBAHA.109.191502
[Indexed for MEDLINE]
Free PMC Article

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