Amplification of CD95 activation by caspase 8-induced endosomal acidification in rat hepatocytes.
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 Czeta-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.
Clinic for Gastroenterology, Hepatology, and Infectiology, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany.
Roland
R
Reinehr
Annika
A
Sommerfeld
Verena
V
Keitel
Susanne
S
Grether-Beck
Dieter
D
Haüssinger
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053181:Caspase 8 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000201:enzymology; D002518:Ceramides / Q000096:biosynthesis; D011992:Endosomes / Q000201:enzymology*; D053222:Fas Ligand Protein / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D019255:NADPH Oxidases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D019014:fas Receptor / Q000819:agonists / Q000378:metabolism*
D002518:Ceramides; D053222:Fas Ligand Protein; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D019014:fas Receptor; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D051744:Protein Kinase C-epsilon; D013108:Sphingomyelin Phosphodiesterase; C505530:CASP8 protein, human; C505528:Casp8 protein, rat; D053181:Caspase 8