Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis
Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis
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DOI：
10.1074/jbc.m414361200
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发表时间：
2005-07-22
影响因子：
4.8
通讯作者：
Häussinger, D
中科院分区：
文献类型：
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作者：
Reinehr, R;Becker, S;Häussinger, D
CD 95配体（CD 95 L）作为大鼠肝细胞中CD 95活化和凋亡诱导的上游事件，触发活性氧（ROS）的快速形成。这种ROS反应是敏感的抑制diphenyleneiodonium，夹竹桃，和新蝶呤，提示参与NADPH氧化酶。与此一致，肝细胞不仅表达吞噬细胞gp 91（phox）（Nox 2）的mRNA，而且表达同源物Nox 1和4以及Duox 1和2以及调节亚基p47（phox）的mRNA。在大鼠肝细胞中还在蛋白水平上鉴定了gp 91（phox）（Nox 2）和p47（phox）。CD 95 L在1分钟内诱导神经酰胺形成和p47（phox）的丝氨酸磷酸化，其对鞘磷脂酶和蛋白激酶C ζ（PKC ζ）的抑制剂敏感。这些抑制剂和p47（phox）蛋白敲低抑制了早期CD 95 L诱导的ROS反应，表明神经酰胺和PKC zeta是CD 95 L诱导的Nox/Duox激活的上游事件。CD 95 L还诱导Src家族激酶Yes的快速活化，随后是c-Src、Fyn和c-Jun-N-末端激酶（JNK）的活化。只有Yes和JNK激活对N-乙酰半胱氨酸、NADPH氧化酶抑制剂、PKC zeta或鞘磷脂酶敏感，表明CD 95 L诱导的ROS反应位于Yes和JNK的上游，而不是Fyn和c-Src激活的上游。激活的Yes与表皮生长因子受体（EGFR）迅速相关，EGFR在Tyr（845）和Tyr（1173）处磷酸化，但在Tyr（1045）处未磷酸化。活化的EGFR然后触发AG 1478敏感的CD 95-酪氨酸磷酸化，这是EGFR/CD 95复合物的膜靶向信号，随后招募Fas相关死亡结构域和半胱天冬酶8，以及诱导凋亡。鞘磷脂酶、PKC zeta、NADPH氧化酶、Yes或EGFR-酪氨酸激酶活性抑制剂以及p47（phox）、Yes或EGFR蛋白质敲低后，所有这些事件均显著减弱。这些数据表明，CD 95 L诱导的细胞凋亡涉及NADPH氧化酶亚型的鞘磷脂酶和PKC ζ依赖性激活，这是Yes/EGFR/CD 95相互作用作为CD 95激活的上游事件所必需的。
CD95 ligand (CD95L) triggers a rapid formation of reactive oxygen species (ROS) as an upstream event of CD95 activation and apoptosis induction in rat hepatocytes. This ROS response was sensitive to inhibition by diphenyleneiodonium, apocynin, and neopterin, suggestive of an involvement of NADPH oxidases. In line with this, hepatocytes expressed mRNAs not only of the phagocyte gp91(phox) (Nox 2), but also of the homologs Nox 1 and 4 and Duox 1 and 2, as well as the regulatory subunit p47(phox). gp91(phox) (Nox 2) and p47(phox) were also identified at the protein level in rat hepatocytes. CD95L induced within 1 min ceramide formation and serine phosphorylation of p47(phox), which was sensitive to inhibitors of sphingomyelinase and protein kinase C zeta (PKC zeta). These inhibitors and p47(phox) protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKC zeta are upstream events of the CD95L-induced Nox/Duox activation. CD95L also induced rapid activation of the Src family kinase Yes, being followed by activation of c-Src, Fyn, and c-Jun-N-terminal kinases (JNK). Only Yes and JNK activation were sensitive to N-acetylcysteine, inhibitors of NADPH oxidase, PKC zeta, or sphingomyelinase, indicating that the CD95L-induced ROS response is upstream of Yes and JNK but not of Fyn and c-Src activation. Activated Yes rapidly associated with the epidermal growth factor receptor ( EGFR), which became phosphorylated at Tyr(845) and Tyr(1173) but not at Tyr(1045). Activated EGFR then triggered an AG1478-sensitive CD95-tyrosine phosphorylation, which was a signal for membrane targeting of the EGFR/CD95 complex, subsequent recruitment of Fas-associated death domain and caspase 8, and apoptosis induction. All of these events were significantly blunted by inhibitors of sphingomyelinase, PKC zeta, NADPH oxidases, Yes, or EGFR-tyrosine kinase activity and after protein knockdown of either p47(phox), Yes, or EGFR. The data suggest that CD95L-induced apoptosis involves a sphingomyelinase- and PKC zeta-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation.