Ceramide Synthase-dependent Ceramide Generation and Programmed Cell Death

Abstract

The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.

Many Ceramides

“…Kolesnick and co-workers (56) recently implicated CerS5 and CerS6 in mediating cytotoxic responses to ionizing radiation, whereas CerS2, probably acting in mitochondria, offered partial protection. Very recent work from our laboratories has also implicated CerS5 and CerS6 in the generation of long chain ceramides by the salvage pathway, which were necessary for regulating membrane permeability in the programmed cell death execution phase in MCF-7 breast cancer cells in response to UV radiation (86). Thus, taken together, studies on CerSs not only implicate each of these enzymes in the formation of specific ceramides and perhaps in specific compartments but also clearly suggest ceramide species-specific functions.…”

Sphingolipid Metabolism and Neutral Sphingomyelinases

“…In general, these processes have been associated with the action of CerS enzymes, which have different chain-length specificities. Based on their chain-length specificities, the involvement of individual CerS enzymes in these pathologies has been either inferred or tested experimentally [20–23]. …”

Sphingolipids in Obesity, Type 2 Diabetes, and Metabolic Disease

“…While myriocin, which inhibits SPT, or fumonisin B1, which inhibits all isoforms of CerS, can function as powerful tools to screen for sphingolipid-dependence, they reveal very little about involvement of specific sphingolipid metabolic pathways (Wang et al 1991; Miyake et al 1995). In contrast, new approaches based on DNA transfection, RNAi, and knockout animals allow precise insight into the roles of specific CerS isoforms in cellular functions and disease, with the caveat that dysregulation of individual isoforms can perturb global CerS expression patterns (Mullen et al 2011). These approaches may also be applied to other enzymes of SL synthesis that are present in more than one isoform (e.g.…”

“…In particular, many studies consider sensitivity to myriocin or fumonisin B1 to indicate a dependence on ceramide itself, excluding potential roles for subsequent metabolites. In fact, although such results are suggestive, they can be misleading; inhibition of de novo sphingolipid synthesis can reduce not only levels of ceramide but of its downstream metabolites (Saito et al 2005; Mullen et al 2011). Thus, conclusions based on inhibitor studies actually indicate a dependence on either ceramide or its downstream metabolites.…”