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J. Biol. Chem., Vol. 280, Issue 29, 27179-27194, July 22, 2005

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Involvement of NADPH Oxidase Isoforms and Src Family Kinases in CD95-dependent Hepatocyte Apoptosis^*

Roland Reinehr, Stephan Becker, Andrea Eberle, Susanne Grether-Beck, and Dieter Häussinger¶

From the Clinic for Gastroenterology, Hepatology, and Infectiology, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany and Institut für Umweltmedizinische Forschung, D-40225 Düsseldorf, Germany

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 (PKC). These inhibitors and p47^phox protein knockdown inhibited the early CD95L-induced ROS response, suggesting that ceramide and PKC 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, 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 Tyr⁸⁴⁵ and Tyr¹¹⁷³ but not at Tyr¹⁰⁴⁵. 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, 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-dependent activation of NADPH oxidase isoforms, which is required for Yes/EGFR/CD95 interactions as upstream events of CD95 activation.

Received for publication, December 21, 2004 , and in revised form, May 23, 2005.

^* This study was supported by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 575 "Experimentelle Hepatologie" (Düsseldorf). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Universitätsklinikum Düsseldorf, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Moorenstrasse 5, D-40225 Düsseldorf. Tel.: 49-2118117569; Fax: 49-2118118838; E-mail: haeussin{at}uni-duesseldorf.de.

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