A specific p47phox-serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites
A specific p47phox-serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites
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DOI：
10.1172/jci27544
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发表时间：
2006-07-01
影响因子：
15.9
通讯作者：
El-Benna, Jamel
中科院分区：
文献类型：
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作者：
Dang, Pham My-Chan;Stensballe, Allan;El-Benna, Jamel
神经元NADPH氧化酶通过释放大量的超氧化物和其他ROS在宿主防御和炎症中起关键作用。促炎细胞因子如GM-CSF和TNF-α通过未知机制引发中性粒细胞产生ROS。在这里，我们使用串联质谱法进行肽测序，以表明GM-CSF和TNF-α诱导人中性粒细胞中NADPH氧化酶的胞质组分p47（phox）上Ser 345的磷酸化。由于Ser 345位于MAPK共有序列中，我们测试了MAPK抑制剂的作用。ERK 1/2通路抑制剂消除GM-CSF诱导的Ser 345磷酸化，而p38 MAPK抑制剂消除TNF-α诱导的Ser 345磷酸化。用突变的p47（phox）（S345 A）转染HL-60细胞抑制GM-CSF和TNF-α诱导的ROS产生的引发。这一事件也被抑制在中性粒细胞的细胞渗透肽含有TAT-p47（phox）-Ser 345序列。此外，ROS的产生，p47（phox）-Ser 345磷酸化，ERK 1/2和p38 MAPK磷酸化增加滑膜中性粒细胞类风湿关节炎（RA）患者，和TAT-Ser 345肽抑制这些引发的中性粒细胞ROS的产生。因此，本研究鉴定了Ser 345上的会聚MAPK通路，其参与GM-CSF和TNF-α诱导的中性粒细胞的引发并在RA中被激活。抑制这些通路的会聚点可能是一种新的抗炎策略。
Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-alpha prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem mass spectrometry to show that GM-CSF and TNF-alpha induce phosphorylation of Ser345 on p47(phox), a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM-CSF-induced phosphorylation of Ser345, while p38 MAPK inhibitor abrogated TNF-alpha-induced phosphorylation of Ser345. Transfection of HL-60 cells with a mutated p47(phox) (S345A) inhibited GM-CSF- and TNF-a-induced priming of ROS production. This event was also inhibited in neutrophils by a cell-permeable peptide containing a TAT-p47(phox)-Ser345 sequence. Furthermore, ROS generation, p47(phox)-Ser345 phosphorylation, and ERK1/2 and p38 MAPK phosphorylation were increased in synovial neutrophils from rheumatoid arthritis (RA) patients, and TAT-Ser345 peptide inhibited ROS production by these primed neutrophils. This study therefore identifies convergent MAPK pathways on Ser345 that are involved in GM-CSF- and TNF-a-induced priming of neutrophils and are activated in RA. Inhibition of the point of convergence of these pathways might serve as a novel andinflammatory strategy.