title: Role of caspases in CD95-induced biphasic activation of acid sphingomyelinase.
abstract: Acid sphingomyelinase (A-SMase) plays an important role in the initiation of CD95 signaling by forming ceramide-enriched membrane domains that enable clustering and activation of the death receptors. In TNF-R1 and TRAIL-R1/R2 signaling, A-SMase also contributes to the lysosomal apoptosis pathway triggered by receptor internalization. Here, we investigated the molecular mechanism of CD95-mediated A-SMase activation, demonstrating that A-SMase is located in internalized CD95-receptosomes and is activated by the CD95/CD95L complex in a biphasic manner.Since several caspases have been described to be involved in the activation of A-SMase, we evaluated expression levels of caspase-8, caspase-7 and caspase-3 in CD95-receptosomes. The occurrence of cleaved caspase-8 correlated with the first peak of A-SMase activity and translocation of the A-SMase to the cell surface which could be blocked by the caspase-8 inhibitor IETD.Inhibition of CD95-internalization selectively reduced the second phase of A-SMase activity, suggesting a fusion between internalized CD95-receptosomes and an intracellular vesicular pool of A-SMase. Further analysis demonstrated that caspase-7 activity correlates with the second phase of the A-SMase activity, whereas active caspase-3 is present at early and late internalization time points. Blocking caspases-7/ -3 by DEVD reduced the second phase of A-SMase activation in CD95-receptosomes suggesting the potential role of caspase-7 or -3 for late A-SMase activation.In summary, we describe a biphasic A-SMase activation in CD95-receptosomes indicating (I.) a caspase-8 dependent translocation of A-SMase to plasma membrane and (II.) a caspase-7 and/or -3 dependent fusion of internalized CD95-receptosomes with intracellular A-SMase-containing vesicles.
authors.affiliation: Institute of Immunology, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Stephan
authors.affiliation: Department of Hematology and Oncology, University Hospital Magdeburg, Magdeburg, Germany.
authors.forename: Bärbel
authors.initials: B
authors.lastname: Edelmann
authors.affiliation: Institute of Immunology, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Supandi
authors.initials: S
authors.lastname: Winoto-Morbach
authors.affiliation: Institute of Immunology, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Ottmar
authors.initials: O
authors.lastname: Janssen
authors.affiliation: Institute of Immunology, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Bertsch
authors.affiliation: Department of Biomedical and Clinical Sciences "Luigi Sacco" (DIBIC), Università degli Studi di Milano, Milano, Italy.
authors.forename: Cristiana
authors.initials: C
authors.lastname: Perrotta
authors.affiliation: Institute of Immunology, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schütze
authors.affiliation: Institute of Immunology, Christian-Albrechts-University of Kiel, Kiel, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Fritsch
mesh_terms: D017209:Apoptosis; D001402:B-Lymphocytes / Q000201:enzymology / Q000473:pathology*; D061945:Caspase Inhibitors / Q000494:pharmacology; D020169:Caspases / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D004789:Enzyme Activation; D053222:Fas Ligand Protein / Q000378:metabolism*; D006801:Humans; D015398:Signal Transduction / Q000187:drug effects; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D061945:Caspase Inhibitors; D053222:Fas Ligand Protein; D019014:fas Receptor; D013108:Sphingomyelin Phosphodiesterase; D020169:Caspases
keywords: CD95 ligand; CD95-receptosomes; acid sphingomyelinase; ceramide; internalization