Knockout of p47 phox uncovers a critical role of p40 phox in reactive oxygen species production in microvascular endothelial cells.
OBJECTIVE
p40(phox) is an important regulatory subunit of NADPH oxidase, but its role in endothelial reactive oxygen species (ROS) production remains unknown.


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.
Fan
Lampson M
LM
Faculty of Health and Medical Sciences, AY Building, University of Surrey, Guildford, Surrey GU2 7XH, UK.
Teng
Lei
L
Li
Jian-Mei
JM
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D008833:Microcirculation; D019255:NADPH Oxidases / Q000235:genetics / Q000502:physiology*; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012097:Repressor Proteins / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
C486329:Hbp1 protein, mouse; D006609:High Mobility Group Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; D000804:Angiotensin II; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D013755:Tetradecanoylphorbol Acetate