Knockout of p47 phox uncovers a critical role of p40 phox in reactive oxygen species production in microvascular endothelial cells.
Knockout of p47 phox uncovers a critical role of p40 phox in reactive oxygen species production in microvascular endothelial cells.
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DOI：
10.1161/atvbaha.109.191502
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发表时间：
2009-10
期刊：
影响因子：
--
通讯作者：
Li JM
中科院分区：
文献类型：
--
作者：
Fan LM;Teng L;Li JM
p40phox是NADPH氧化酶的重要调节亚基，但其在内皮细胞活性氧（ROS）产生中的作用尚不清楚。使用从野生型和p47phox基因敲除小鼠中分离的冠状动脉微血管内皮细胞，我们发现敲除p47phox增加了p40phox的表达水平，而野生型细胞中p40phox的耗竭增加了p47phox的表达。在这两种情况下，基础ROS产生（无激动剂刺激）得到很好的保留。p40 phox和p47 phox的双重敲除显着减少（约65%）活性氧的产生，细胞开始死亡。p40 phox和p47 phox表达的转录调控涉及HBP1。p40phox在静息细胞中被预磷酸化。PMA刺激诱导p40phox快速去磷酸化（1分钟内）与p47phox磷酸化的开始平行。p40phox然后再磷酸化，并且这伴随着ROS产生的增加。尽管存在p47phox，但p40phox的耗尽导致激动剂诱导的ROS产生损失约67%。用血管紧张素II刺激的小鼠动脉瘤实验进一步支持了这一点。p40phox在静息内皮细胞中被预磷酸化，并且可以补偿p47phox以保持基础ROS产生。p40 phox的去磷酸化是激动剂诱导p47 phox磷酸化的先决条件，并且p40 phox通过其动态去磷酸化和再磷酸化参与调节激动剂诱导的ROS产生。
p40phox is an important regulatory subunit of NADPH oxidase, but its role in endothelial reactive oxygen species (ROS) production remains unknown. 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. 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.