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

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