Ceramide Enables Fas to Cap and Kill

Abstract

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.The current model of Fas (CD95 or APO-1) signaling suggests that engagement of Fas by its ligand or anti-Fas antibody leads to receptor trimerization and recruitment of the cytoplasmic adapter protein FADD 1 (MORT-1) and pro-caspase 8 (Flice/ MACH-1), (1, 2), thus forming a death-inducing signaling complex. Assembly of the death-inducing signaling complex results in release of active caspase 8, initiating the apoptotic process (1, 2). Peter and co-workers (2) provide evidence that after caspase 8 activation, Fas signals apoptosis via two different mechanisms. In Type I cells, caspase 8 directly activates a hierarchical cascade of effector caspases, involving among others caspases 3 and 7. Apoptosis in Type II cells occurs after minimal caspase 8 activation and proceeds through an amplification cascade involving mitochondrial dysfunction, the release of mitochondrial cytochrome c, and activation of Apaf-1 and caspase 9. Commitment to apoptosis ensues subsequently via effector caspase activation. Whereas this model is widely accepted, recent data are incompatible with portions of this paradigm. Siegel et al. (3) reported recently that Fas may already exist in a trimerized state before ligand interaction and that preassembly may be required for binding Fas ligand and t...

Plasma sphingolipids and depressive symptoms in coronary artery disease

“…found an association between ceramide C18:0 and depressive symptom severity in their study population (Demirkan et al., 2013). Importantly, both species have been implicated in apoptosis (Babenko, Hassouneh, Budvytiene, Liesiene, & Geilen, 2010; Cremesti et al., 2001; Grassme et al., 2001; Senkal et al., 2007; Thomas, Matsko, Lotze, & Amoscato, 1999). For ceramide C16:0, concentrations were found to be elevated in apoptotic cells within 2 hr of being exposed to ionizing radiation (Thomas et al., 1999).…”

“…For ceramide C16:0, concentrations were found to be elevated in apoptotic cells within 2 hr of being exposed to ionizing radiation (Thomas et al., 1999). Moreover, C16:0 has been shown to be required for the initiation of Fas‐induced apoptosis and may act as a second messenger after Fas ligation by inducing Ras activation and subsequent apoptosis (Cremesti et al., 2001; Grassme et al., 2001; Gulbins et al., 1995). C18:0 has been observed to induce apoptosis of keratinocytes in a concentration‐dependent manner (Babenko et al., 2010).…”

The Role of Sphingolipids and Ceramide in Pulmonary Inflammation in Cystic Fibrosis

“…Other receptors, for instance CD95 or DR5, or infections with P. aeruginosa trigger a translocation of the acid sphingomyelinase onto the extracellular leaflet [39, 40, 43]. Translocation of the enzyme onto the outer leaflet of the cell membrane is very likely mediated by the fusion of vesicles, in particular secretory lysosomes that contain the enzyme [76], with the plasma membrane.…”

TRAIL activates acid sphingomyelinase via a redox mechanism and releases ceramide to trigger apoptosis

“…Several studies indicated that rafts are required to cluster CD95 and to recruit FADD, caspase 8 and 3 to CD95 upon stimulation (Grassme et al, 2001bHueber et al, 2002;Scheel-Toellner et al, 2002;Delmas et al, 2003;Aouad et al, 2004;Eramo et al, 2004). Accordingly, disruption of rafts prevented CD95-induced apoptosis, at least in most cells (Grassme et al, 2001b;Cremesti et al, 2001;Hueber et al, 2002;Scheel-Toellner et al, 2002). Likewise, a recent study by Delmas et al (2004) reported that DR4 and DR5 translocate into rafts upon stimulation.…”

“…For instance, the extracellular domain of CD95 contains a motif, the PLAD domain that mediates trimerization of CD95 and seems to contribute to the specificity of the interaction between CD95 ligand and CD95 . However, trimerization of the CD95 receptor is not sufficient to induce apoptosis (Schneider et al, 1998) and several studies demonstrated that CD95 signaling is initiated by oligomerization and aggregation (clustering), respectively, of the receptor in distinct domains of the cell membrane (Cremesti et al, 2001;Grassme et al, 2001a, b).…”