Endosomal acidification and activation of NADPH oxidase isoforms are upstream events in hyperosmolarity-induced hepatocyte apoptosis

Hyperosmotic exposure of rat hepatocytes induced a rapid oxidativestress(ROS) response as an upstream signal for proapoptotic CD95 activation. This study shows that hyperosmotic ROS formation involves a rapid ceramide- and protein kinase C zeta (PKC zeta)-dependent serine phosphorylation of p47(phox) and subsequent activation of NADPH oxidase isoforms. Hyperosmotic p47(phox) phosphorylation and ROS formation were sensitive to inhibition of sphingomyelinases and were strongly blunted after knockdown of acidic sphingomyelinase (ASM) or of p47(phox) protein. Hyperosmolarity induced a rapid bafilomycin- and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid disodium salt (DIDS)-sensitive acidification of a vesicular compartment, which was accessible to endocytosed fluorescein isothiocyanate-dextran and colocalized with ASM, PKC zeta, and the NADPH oxidase isoform Nox 2 (gp91(phox)). Bafilomycin and DIDS prevented the hyperosmolarity-induced increase in ceramide formation, p47(phox) phosphorylation, and ROS formation. As shown recently (Reinehr, R., Becker, S., Hongen, A., and Haussinger, D. (2004) J. Biol. Chem. 279, 23977 - 23987), hyperosmolarity induced a Yes-dependent activation of JNK and the epidermal growth factor receptor (EGFR), followed by EGFR-CD95 association, EGFR-catalyzed CD95-tyrosine phosphorylation, and translocation of the EGFR-CD95 complex to the plasma membrane, where formation of the death-inducing signaling complex occurs. These proapoptotic responses were not only sensitive to inhibitors of sphingomyelinase, PKC zeta, or NADPH oxidases but also to ASM knock-down, bafilomycin, and DIDS, i.e. maneuvers largely preventing hyperosmolarity-induced endosomal acidification and/or ceramide formation. In hepatocytes from p47(phox) knock-out mice, hyperosmolarity failed to activate the CD95 system. The data suggest that hyperosmolarity induces endosomal acidification as an important upstream event for CD95 activation through stimulation of ASM-dependent ceramide formation and activation of NADPH oxidase isoforms.