| Recent mechanistic studies indicate that pro-apoptotic signaling through ceramide is mediated by a unique biophysical capacity to self-associate, driving coalescence of microscopic cell surface lipid rafts into ceramide-rich platforms. These platforms serve as efficient sites for transmembrane signaling by promoting protein translocation, concentration and multimerization. Here, we report that ceramide-enriched platforms regulate apoptosis and clonogenic cell death in response to UV irradiation, demonstrating that environmental stresses, like cytokines of the TNF superfamily, signal via platforms in vitro. Stress, however, activates acid sphingomyelinase (ASMase)-mediated platform generation via a novel, caspase-independent mechanism distinct from the cytokine-activated pathway. Further, we identify the requirement for ceramide-enriched platforms in stress- and cytokine-induced apoptotic events in vivo, determined in mouse models of radiation-induced gastrointestinal (GI) syndrome and cytolytic T cell-induced acute graft-versus-host disease. Lethal radiation damage to GI stem cells within the crypts of Lieberkuhn and denudation of the intestinal mucosa are linked to an early wave of ASMase-mediated microvascular endothelial apoptosis. Antagonism of endothelial apoptosis, shown here by inactivation of proapoptotic Bax, enhances stem cell survival and organ regeneration, inhibiting the GI syndrome. Platforms are essential for this event, as pharmacologic antagonism of ceramide-enriched platform generation impacted apoptosis and improved survival. Similarly, we identified an essential role for target cell ASMase in CTL-induced apoptosis of the liver, intestine and skin during acute GvHD. Inactivation of ASMase decreased organ damage and enhanced survival in minor and major histocompatibility mismatched hematopoietic stem cell transplantation models. Deconstruction of GvHD into a 2-cell ex vivo model using hepatocytes as target cells identified a critical role for platforms in this apoptotic event. Abrogation of CTL-induced hepatocyte platform generation by inactivation of ASMase attenuated apoptosis, which was reversible by restoration of platform generation by exogenous ceramide. Extension of these studies to established models of T cell-induced cytotoxicity identified ceramide-enriched platforms regulate CD3-induced activation-induced cell death of splenocytes in vitro and target cell lysis in a in vitro -stimulated mixed lymphocyte reaction, demonstrating a central role for platforms in cell sensitivity to cytolytic T cell-induced apoptosis. Collectively, these studies identify ceramide-enriched platform generation as a critical event in ASMase-mediated pathologies including radiation damage to the GI mucosa and CTL-induced target cell apoptosis, suggesting that platforms represent previously-unrecognized targets for therapeutic intervention. |
