Sphingolipid metabolism in metazoan cells consists of a complex interconnected web of numerous enzymes, metabolites and modes of regulation. At the centre of sphingolipid metabolism reside CerSs (ceramide synthases), a group of enzymes that catalyse the formation of ceramides from sphingoid base and acyl-CoA substrates. From a metabolic perspective, these enzymes occupy a unique niche in that they simultaneously regulate de novo sphingolipid synthesis and the recycling of free sphingosine produced from the degradation of pre-formed sphingolipids (salvage pathway). Six mammalian CerSs (CerS1–CerS6) have been identified. Unique characteristics have been described for each of these enzymes, but perhaps the most notable is the ability of individual CerS isoforms to produce ceramides with characteristic acyl-chain distributions. Through this control of acyl-chain length and perhaps in a compartment-specific manner, CerSs appear to regulate multiple aspects of sphingolipid-mediated cell and organismal biology. In the present review, we discuss the function of CerSs as critical regulators of sphingolipid metabolism, highlight their unique characteristics and explore the emerging roles of CerSs in regulating programmed cell death, cancer and many other aspects of biology.

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.

Ceramide metabolism has come under recent scrutiny because of its role in cellular stress responses. CerS2 (ceramide synthase 2) is one of the six mammalian isoforms of ceramide synthase and is responsible for the synthesis of VLC (very-long-chain) ceramides, e.g. C24, C24:1. To study the role of CerS2 in ceramide metabolism and cellular homoeostasis, we down-regulated CerS2 using siRNA (small interfering RNA) and examined several aspects of sphingolipid metabolism and cell stress responses. CerS2 down-regulation had a broad effect on ceramide homoeostasis, not just on VLC ceramides. Surprisingly, CerS2 down-regulation resulted in significantly increased LC (long-chain) ceramides, e.g. C14, C16, and our results suggested that the increase was due to a ceramide synthase-independent mechanism. CerS2-down-regulation-induced LC ceramide accumulation resulted in growth arrest which was not accompanied by apoptotic cell death. Instead, cells remained viable, showing induction of autophagy and activation of PERK [PKR (double-stranded-RNA-dependent protein kinase)-like endoplasmic reticulum kinase] and IRE1 (inositol-requiring 1) pathways [the latter indicating activation of the UPR (unfolded protein response)].

The BCL-2 family members BAK and BAX are required for apoptosis and trigger mitochondrial outer membrane permeabilization (MOMP). Here we identify a MOMP-independent function of BAK as a required factor for long-chain ceramide production in response to pro-apoptotic stress. UV-C irradiation of wild-type (WT) cells increased long-chain ceramides; blocking ceramide generation prevented caspase activation and cell death, demonstrating that long-chain ceramides play a key role in UV-C-induced apoptosis. In contrast, UV-C irradiation did not increase long-chain ceramides in BAK and BAX double knock-out cells. Notably, this was not specific to the cell type (baby mouse kidney cells, hematopoietic) nor the apoptotic stimulus employed (UV-C, cisplatin, and growth factor withdrawal). Importantly, long-chain ceramide generation was dependent on the presence of BAK, but not BAX. However, ceramide generation was independent of the known downstream actions of BAK in apoptosis (MOMP or caspase activation), suggesting a novel role for BAK in apoptosis. Finally, enzymatic assays identified ceramide synthase as the mechanism by which BAK regulates ceramide metabolism. There was no change in CerS expression at the message or protein level, indicating regulation at the post-translational level. Moreover, CerS activity in BAK KO microsomes can be reactivated upon addition of BAKcontaining microsomes. The data presented indicate that ceramide-induced apoptosis is dependent upon BAK and identify a novel role for BAK during apoptosis. By establishing a unique role for BAK in long-chain ceramide metabolism, these studies further demonstrate that the seemingly redundant proteins BAK and BAX have distinct mechanisms of action during apoptosis induction.Programmed cell death (apoptosis) is a complex, multistep process essential for normal tissue homeostasis and development. A key event in apoptotic signaling is mitochondrial outer membrane permeabilization (MOMP), 3 whereby pro-apoptotic factors are released from the mitochondrial intermembrane space into the cytosol resulting in the activation of caspases and triggering the execution phase of apoptosis. Multiple cell death pathways converge on MOMP, and it is considered a necessary step for the commitment of cells to apoptosis. Anti-apoptotic BCL-2 proteins like BCL-2 and BCL-X L can block MOMP and cell death when overexpressed. On the other hand, the proapoptotic BCL-2 proteins BAK and BAX are necessary for MOMP in response to a wide variety of death-inducing signals. In fact, cells lacking both BAK and BAX are highly resistant to apoptotic stimuli (1-4).Sphingolipids have been extensively implicated in the apoptotic response. Ceramide, a central molecule of sphingolipid metabolism, has been shown to be involved in programmed cell death through several lines of evidence. First, cellular levels of ceramide are elevated by a variety of apoptotic stimuli. Second, blocking ceramide generation delays or abrogates cell death in response to many different signals (5-9). Moreover, cancer cells ...

We have previously shown that the death receptor ligand TRAIL induces an increase in intracellular C16-ceramide in sensitive SW480 but not in resistant SW620 cells. Resistance in SW620 cells was overcome by exogenous ceramide leading us to propose that defective ceramide signaling contributes to TRAIL resistance. In this study we found that the increase in C16-ceramide in SW480 cells was inhibited by fumonisin B1, an inhibitor of ceramide synthases (CerS). Protein analysis revealed that TRAIL resistant SW620 cells expressed lower levels of ceramide synthase 6 (CerS6, also known as longevity assurance homologue 6), which prompted us to investigate the effect of CerS6 modulation on TRAIL phenotype. RNAi against CerS6 resulted in a specific and significant decrease of the C16-ceramide species, which was sufficient to inhibit TRAIL-induced apoptosis. In cells with decreased levels of CerS6, caspase-3 was activated but failed to translocate into the nucleus. CerS6 localized primarily to the perinuclear region, suggesting this enzyme may play a role in regulation of nuclear permeability. Moderate elevation in CerS6 expression was sufficient to reverse TRAIL resistance in SW620 cells. These results suggest that modulation of CerS6 expression may constitute a new therapeutic strategy to alter apoptotic susceptibility.