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J. Biol. Chem., Vol. 283, Issue 4, 2211-2222, January 25, 2008

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Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes^*

Roland Reinehr, Annika Sommerfeld, Verena Keitel, Susanne Grether-Beck, and Dieter Häussinger¹

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

Although in rat hepatocytes CD95 is predominantly located inside the cell with almost undetectable immunostaining at the plasma membrane, the addition of CD95-ligand (CD95L) induces hepatocyte apoptosis, which is preceded by a targeting and activation of intracellularly localized CD95 to the plasma membrane including formation of the death-inducing signaling complex. This process involves an NADPH oxidase-dependent generation of reactive oxygen species (ROS) through a ceramide- and protein kinase C-dependent pathway, which leads to an activating phosphorylation of p47^phox. The mechanisms underlying CD95L-induced ceramide formation were addressed in the present study. It was found that CD95L lowered within seconds the apparent vesicular pH from 6.0 to 5.7 in a fluorescein isothiocyanate-dextran-accessible endosomal compartment, which was previously shown to contain acidic sphingomyelinase, and decreased N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide fluorescence, suggestive for an increase of cytosolic [Cl^-]. Bafilomycin or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid disodium salt largely abolished the CD95L-induced endosomal acidification, ceramide formation, and downstream events, such as p47^phox phosphorylation, ROS formation, CD95 activation, and apoptosis. These responses were also abolished after knock-down of acidic sphingomyelinase in rat hepatocytes. Interestingly, caspase 8 inhibitors abolished these CD95L-induced signaling events, including the increase in cytosolic [Cl^-], endosomal acidification, ceramide formation, and ROS generation as well as CD95 targeting to the plasma membrane and CD95 activation. The data suggest that CD95L initiates a rapid caspase 8-dependent endosomal acidification, which triggers ceramide-dependent ROS formation as an upstream event of trafficking of intracellularly stored CD95 to the plasma membrane. It is concluded that a rapid caspase 8 activation in response to CD95L signals to intracellularly stored CD95, which becomes activated and targeted to the plasma membrane. This autoamplification of CD95-activation is required for apoptosis induction.

Received for publication, August 16, 2007 , and in revised form, October 24, 2007.

^* This study was supported by 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Films 1–4.

¹ To whom correspondence should be addressed: Universitätsklinikum Düsseldorf; Klinik für Gastroenterologie, Hepatologie und Infektiologie; Moorenstrasse 5; D-40225 Düsseldorf, Germany. Tel.: 49-2118117569; Fax: 49-2118118838; E-mail: haeussin{at}uni-duesseldorf.de.

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