Elsevier

Methods in Enzymology

Volume 428, 2007, Pages 145-160
Methods in Enzymology

Chapter Eight - Hyperosmotic Activation of the CD95 System

https://doi.org/10.1016/S0076-6879(07)28008-5Get rights and content

Abstract

Cell shrinkage, nuclear condensation, DNA fragmentation, and apoptotic body formation are hallmarks of programmed apoptotic cell death. Herein, apoptotic volume decrease (AVD) is an early and ubiquitous event. Conversely, in hepatocytes, hyperosmotic cell shrinkage leads to an activation of the CD95 death receptor system, which involves CD95 tyrosine phosphorylation, CD95 oligomerization, and subsequent trafficking of the CD95 to the plasma membrane, and sensitizes hepatocytes toward CD95 ligand (CD95L)‐induced apoptosis. Early signaling events leading to CD95 activation by hyperosmolarity have been identified. In hepatocytes, hyperosmotic exposure induces an almost instantaneous acidification of an acidic sphingomyelinase (ASM) containing endosomal compartment, which is followed by an increase in the intracellular ceramide concentration. Inhibition of anion channels or the vacuolar‐type H+‐ATPase abolishes not only endosomal acidification and subsequent ceramide generation, but also the otherwise observed hyperosmotically induced generation of reactive oxygen species (ROS) by NADPH oxidase isoforms. Hyperosmolarity‐induced ROS formation then leads to a Src‐family kinase Yes‐mediated activation of the epidermal growth factor receptor (EGFR) and to an activation of the c‐Jun‐N‐terminal kinase (JNK). JNK then provides a signal for CD95/EGFR association and subsequent CD95 tyrosine phosphorylation, which is mediated by the EGFR tyrosine kinase activity. CD95 tyrosine phosphorylation then allows for CD95 receptor oligomerization, translocation of the CD95/EGFR protein complex to the plasma membrane, and formation of the death inducing signaling complex (DISC). Mild hyperosmotic exposure, that is, 405 mosmol/liter, does not lead to a reduction of cell viability, even if DISC formation and subsequent caspase 8 and 3 activation occur, but sensitizes hepatocytes to CD95L‐induced apoptosis. However, activation of the CD95 system by a more severe hyperosmotic challenge (>505 mosmol/liter) is followed by execution of the apoptotic cell death. Other covalent modifications of CD95, such as CD95 tyrosine nitration or CD95 serine/threonine phosphorylation, were shown to inhibit the CD95 activation process.

Section snippets

INTRODUCTION

Apoptosis is characterized by cell shrinkage, nuclear condensation, DNA fragmentation, and apoptotic body formation. These features separate apoptosis from other forms of cell death, such as necrosis (Kerr et al., 1974). It is well known that shrunken hepatocytes are more susceptible to stress‐induced cell death, which may explain the clinical observation that transplantation of livers from hypernatremic donors shows high rates of primary graft dysfunction. Apoptotic volume decrease (AVD) is an

HYPEROSMOTIC ACTIVATION OF THE CD95 SYSTEM IN HEPATOCYTES

Whereas CD95 in normosmotically exposed rat hepatocytes is localized inside the cells and exhibits no detectable membrane localization, this death receptor is rapidly targeted to the plasma membrane in response to proapoptotic stimuli, such as hyperosmotic exposure, CD95L, or bile acids (Eberle et al., 2005, Eberle et al., 2007, Reinehr and Häussinger, 2004, Reinehr et al., 2002, Reinehr et al., 2003a, Reinehr et al., 2003b, Reinehr et al., 2004a, Reinehr et al., 2004b, Reinehr et al., 2004c).

CONCLUDING REMARKS

Hyperosmotic hepatocyte shrinkage sensitizes the cells toward CD95 ligand‐induced apoptosis by activating the CD95 system. Figure 8.3 summarizes our current view on the signaling events involved in hyperosmotic activation of the CD95 system in hepatocytes. The hyperosmotically induced cascade of signaling events starts with endosomal acidification triggering an ASM‐derived increase in the intracellular ceramide concentration, which leads via PKCζ and p47phox to NADPH oxidase activation and

ACKNOWLEDGMENTS

Our studies were supported by Deutsche Forschungsgemeinschaft (DFG) through Sonderforschungsbereich 575 “Experimentelle Hepatologie” (Düsseldorf). Excellent work by Stephan Becker, Andrea Eberle, Boris Görg, Elisabeth Winands, and Claudia Rupprecht is gratefully acknowledged.

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