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NADPH oxidase‐dependent reactive oxygen species are important to the early stage of CD95 engagement in hepatocytes
Author(s) -
Yin XiaoMing
Publication year - 2005
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.20902
Subject(s) - nadph oxidase , reactive oxygen species , chemistry , stage (stratigraphy) , oxygen , microbiology and biotechnology , biochemistry , biology , paleontology , organic chemistry
Abstract 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 Cζ (PKCζ). These inhibitors and p47 phox protein knockdown inhibited the early CD95L‐induced ROS response, suggesting that ceramide and PKCζ are upstream events of the CD95Linduced 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, PKCζ, 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, PKCζ, 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 PKCζ‐dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation. (H EPATOLOGY 2005;42:956–958.)