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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:1651.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

Knockout of p47^phox Uncovers a Critical Role of p40^phox in Reactive Oxygen Species Production in Microvascular Endothelial Cells

Lampson M. Fan; Lei Teng; Jian-Mei Li

From the Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK.

Correspondence to Jian-Mei Li, MD, PhD, Professor of Cardiovascular Biology, Faculty of Health and Medical Sciences, AY Building, University of Surrey, Guildford, Surrey GU2 7XH, UK. E-mail j.li{at}surrey.ac.uk

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 (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 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.

Conclusion— 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.

p40^phox is prephosphorylated in microvascular endothelial cells and is critical in keeping the basal reactive oxygen species (ROS) production at a low level with or without p47^phox. Agonist stimulation induces p40^phox dephosphorylation and rephosphorylation, which interacts with p47^phox and is involved in agonist-induced ROS production.

Key Words: NADPH oxidase • endothelial cells • gene regulation • reactive oxygen species