Article: article from journal or magazin.
Ceramide enables fas to cap and kill.
Journal of Biological Chemistry
Recent studies suggest that trimerization of Fas is insufficient for apoptosis induction and indicate that super-aggregation of trimerized Fas might be prerequisite. For many cell surface receptors, cross-linking by multivalent ligands or antibodies induces their lateral segregation within the plasma membrane and co-localization into "caps" on one pole of the cell. In this study, we show that capping of Fas is essential for optimal function and that capping is ceramide-dependent. In Jurkat T lymphocytes and in primary cultures of hepatocytes, ceramide elevation was detected as early as 15-30 s and peaked at 1 min after CH-11 and Jo2 anti-Fas antibody treatment, respectively. Capping was detected 30 s after Fas ligation, peaked at 2 min, and was maintained at a lower level for as long as 30 min in both cell types. Ceramide generation appeared essential for capping. Acid sphingomyelinase -/- hepatocytes were defective in Jo2-induced ceramide generation, capping, and apoptosis, and nanomolar concentrations of C(16)-ceramide restored these events. To further explore the role of ceramide in capping of Fas, we employed FLAG-tagged soluble Fas ligand (sFasL), which binds trimerized Fas but is unable to induce capping or apoptosis in Jurkat cells. Cross-linking of sFasL with M2 anti-FLAG antibody induced both events. Pretreatment of cells with natural C(16)-ceramide bypassed the necessity for forced antibody cross-linking and enabled sFasL to cap and kill. The presence of intact sphingolipid-enriched membrane domains may be essential for Fas capping since their disruption with cholesterol-depleting agents abrogated capping and prevented apoptosis. These data suggest that capping is a ceramide-dependent event required for optimal Fas signaling in some cells.
Animals, Antibodies/immunology, Antigens, CD95/immunology, Antigens, CD95/metabolism, Apoptosis/drug effects, Cells, Cultured, Ceramides/biosynthesis, Ceramides/pharmacology, Fas Ligand Protein, Hepatocytes/cytology, Hepatocytes/drug effects, Humans, Jurkat Cells, Kinetics, Membrane Glycoproteins/pharmacology, Membrane Microdomains/drug effects, Mice, Mice, Knockout, Receptor Aggregation, Sphingomyelin Phosphodiesterase/genetics, T-Lymphocytes/cytology, T-Lymphocytes/metabolism
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