Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis

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.