on July 16, 2009
Published online before print July 16, 2009, doi: 10.1161/ATVBAHA.109.191502
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Submitted on January 21, 2009
Accepted on June 23, 2009
Knockout of p47phox Uncovers a Critical Role of p40phox in Reactive Oxygen Species Production in Microvascular Endothelial Cells
Lampson M. Fan ;
From the Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK.
* To whom correspondence should be addressed. E-mail: j.li{at}surrey.ac.uk.
Objective—p40phox 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 p47phox knockout mice, we found that knockout of p47phox increased the level of p40phox expression, whereas depletion of p40phox in wild-type cells increased p47phox expression. In both cases, the basal ROS production (without agonist stimulation) was well preserved. Double knockout of p40phox and p47phox dramatically reduced (
65%) ROS production and cells started to die. The transcriptional regulation of p40phox and p47phox expressions involves HBP1. p40phox was prephosphorylated in resting cells. PMA stimulation induced p40phox swift dephosphorylation (within 1 minute) in parallel with the start of p47phox phosphorylation. p40phox was then rephosphorylated, and this was accompanied with an increase in ROS production. Depletion of p40phox resulted in 67% loss in agonist-induced ROS production despite the presence of p47phox. These were further supported by experiments on mouse aortas stimulated with angiotensin II.
Conclusion—p40phox is prephosphorylated in resting endothelial cells and can compensate p47phox in keeping basal ROS production. Dephosphorylation of p40phox is a prerequisite for agonist-induced p47phox phosphorylation, and p40phox through its dynamic dephosphorylation and rephosphorylation is involved in the regulation of agonist-induced ROS production.