Knockout of p47phox Uncovers a Critical Role of p40phox 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. 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 (approximate to 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 approximate to 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-47(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. (Arterioscler Thromb Vasc Biol.2009;29:1651-1656.)