above, the strongest evidence points to a role for SK1, although other studies suggest proapoptotic roles for sphingosine kinase 2 (SK2). Ceramide levels are also highly regulated by metabolism into SM and GSL such that inhibition or loss of GlcCer synthase or SM synthase can result in increased cellular ceramide and, in some but not all cases, increased cell death.

Investigations into the details of sphingolipid metabolism, especially through the cloning and characterization of the enzymes involved (e.g., SPT, CerS1–6, aSMase), are beginning to bear fruit in terms of understanding the function of these enzymes and their products in the cell death process. Illustrative examples of this fact are seen in the case studies examining aSMase-mediated death using knockout animals (mentioned previously). As for de novo ceramide synthesis, a handful of studies have demonstrated an in vivo role for CerS-dependent ceramide generation in controlling apoptosis-mediated disease (see Clinical Cases 9-2 and 9-3).

Through the use of more specific pharmacological inhibitors, manipulations of specific enzymes via overexpression and knockdown, and mice with knockouts of the enzymes of de novo synthesis, the understanding of de novo ceramide-mediated signaling and apoptosis will be greatly enhanced.

5. CONCLUDING REMARKS AND FUTURE DIRECTIONS

The previous four sections have introduced the concept of bioactive lipids and the role of sphingolipids as bioactive lipid mediators in cell death and provided specific examples of studies implicating regulated ceramide

production in the cellular program activated by inducers of cell death. In response to various inducers of cell death, ceramide is generated through either the SMase or de novo pathway – or a combination of the two (i.e., the salvage pathway) – resulting in cell death. Inhibiting ceramide generation, either through pharmacological or genetic manipulation of enzymes of sphingolipid metabolism, often but not always rescues cells from death. Lastly, restoring ceramide formation re-engages the cell’s death program.

In addition to cell death, ceramide is emerging as a mediator of a variety of cellular processes (e.g., inflammation, cellular adhesion, senescence, and cell cycle arrest). It has become obvious that ceramide, although a key mediator of apoptosis, has signaling roles that are not restricted to cell death pathways. Differences in signaling pathways that connect ceramide to various biological effects likely occur in a cell typeand stimulus-specific manner. The current evidence points to ceramide signaling as a module that may be used in a variety of contexts, of which programmed cell death is only one. If this is true, then an understanding of the multiple ceramide-mediated pathways is of paramount importance to designing ceramide-based therapeutic interventions that have specificity for a particular target system (e.g., killing cancer cells vs. normal tissue).

For example, a comparison of aSMase-mediated signaling with de novo–mediated signaling raises the obvious question: “Are all ceramides created equal?” The available evidence indicates that ceramide signaling is sufficiently complex to necessitate a careful dissection of the individual pathways. For that reason, the future of research in the field of ceramide-mediated signaling

and apoptosis is concerned with several issues that can be framed into several key questions, outlined as follows.

5.1. Who? (Which enzyme?)

At the expense of anthropomorphizing the enzymes of sphingolipid metabolism, it is crucial to address the question of which enzyme – “who” – is responsible for particular signaling events in the context of cell death. As elaborated previously, numerous enzymes control the balance and flux of sphingolipids both at the basal state and during drastic changes in cell function such as apoptosis. aSMase and CerS have thus far been heavily implicated in death-induced ceramide increases, but the mechanisms of their involvement are ill-defined. As discussed below, the particular enzyme involved dramatically affects the location and composition of the ceramide produced (i.e., specific chain lengths), its ability to be metabolized by additional enzymes (e.g., CDases, SM synthases), and its interaction with putative effectors (e.g., protein phosphatases, cathepsin D). Identification of the specific genes and protein products regulating ceramide production in apoptosis is a key initial step to elucidating the mechanisms of ceramidemediated death.

5.2. What? (Which ceramide?)

With the development of more sensitive and specific techniques for analysis of the thousands of sphingolipid

species – the “sphingolipidome” (see Box 9-2) – it is becoming understood that “ceramide” does not represent a single molecule, but rather a large variety of distinct molecular species with variable acyl chain lengths, degrees of saturation, and other modifications (e.g. α- hydroxylation). An obvious question is “What functions or advantages does a particular ceramide repertoire impart?” Recent evidence suggests that acyl chain length can be controlled at the level of CerS. Mammals possess six CerS (CerS1–6) that have distinct preferences for the acyl-CoA used in the formation of ceramide. For example, CerS1 synthesizes ceramide from stearoyland oleolyl (C18- and C18:1-ceramides), whereas CerS2 synthesizes predominantly very long-chain ceramides (C22- through C24-ceramides). Moreover, several studies have suggested distinct roles for individual CerS and their ceramide products in regulated cell death.

5.3. Where? (Which compartment?)

Perhaps the most important consideration in terms of the study of bioactive lipids is the question of where the lipid is located within the cell. Although some lipid mediators are soluble once released from a lipid precursor (e.g., IP3), ceramide is an extremely hydrophobic lipid, and thus ceramide-mediated biology is likely to occur in close proximity to biological membranes. Given that sphingolipid metabolic enzymes have been detected in various subcellular regions, one can easily posit that ceramide is restricted to specific membranes

to serve specific functions (Figure 9-8). Thus SMasederived ceramide at the plasma membrane is unlikely to serve the same signaling function as ceramide derived from upregulation of the de novo pathway, which localizes almost exclusively to the ER.

Additionally, the ability of ceramide to promote cell death signaling is counterbalanced by other sphingolipid enzymes that have the capacity to “detoxify” the cell of ceramide as it is generated. The ceramidases, for example, not only relieve the burden of accumulat-

ing ceramide, but also act as a shunt to generate other bioactive sphingolipids, such as sphingosine or the prosurvival lipid, sphingosine-1-phosphate. Thus a deathinducing signal can be transformed to a survival signal by virtue of other sphingolipid metabolic enzymes. Ceramide increases may also be buffered by metabolism into sphingomyelin and glycosphingolipids; in fact, the conversion of ceramide into glycosphingolipids via glucosylceramide synthase has been shown to be a mechanism of drug resistance in cancer cells.

pro-survival pathways

SPL

5.4. When? (At what steps?)

The temporal relationship between the time of ceramide generation and the onset of cell death continues to be an issue of confusion and contention. Activation of aSMase is commonly seen as an acute phenomenon, separated from the first signs of cell death by several hours. On the other hand and depending on the stimulus, late ceramide accumulation occurs after a few hours of stimulation and may be coincident with some of the known downstream apoptotic mediators (e.g., mitochondrial permeabilization, caspase activation). If ceramide by itself is essential for cell death, why are there two pathways to make it? Is this a form of redundancy, or are there distinct functions for SMase-derived ceramide and de novo ceramide? To answer these questions, it will be imperative to use the growing knowledge of the particular enzymes – aSMase, SPT, and CerS1–6 – and their subcellular localization to experimentally address the contribution of each to apoptotic signaling.

5.5. How? (Through what mechanisms?)

To ask how is to question the mechanism by which an event occurs. In the context of ceramide-mediated cell death, there are basically two general mechanisms of interest. First, how is ceramide generated? The answer to this question most highly depends on answering the first question of which enzyme is responsible for the accumulation of ceramide. Identification of responsible enzymes allows the experimenter to use molecular tools to dissect the role of that particular enzyme in regulating apoptosis.

The second issue is that of how ceramide exerts its effects. There are abundant data to suggest that

ceramide functions both as a second messenger (e.g., activating protein phosphatases PP1/PP2A) and as a modulator of membrane structure (e.g., promoting microdomain formation and CD95 clustering). Unlike DAG, for which a specific protein interaction domain has been identified and characterized, the direct interaction of ceramide with candidate effector proteins through a particular protein motif has yet to be demonstrated. Many studies have implicated certain ceramideinteracting proteins in cell death (e.g., ceramide/PP2A interaction controlling Bcl-2 and Bax phosphorylation, or ceramide/cathepsin D interaction inducing activation of Bid), but robust connections between ceramide, these mediators, and the control of apoptosis remain to be delineated. The dearth of mechanistic explanations for ceramide signaling may appear surprising given the abundant data supporting roles for ceramide in mediating programmed cell death; however, the study of lipidprotein interactions remains one of the most difficult and vexing areas of biochemical research. Despite these shortcomings, investigations into the detailed mechanisms of ceramide signaling remain promising. The identification of the numerous genes governing sphingolipid metabolism as well as new tools in the detection and manipulation of sphingolipid levels are allowing unprecedented insight into the complexities of this field of research.

5.6. What purpose?

Although the “purpose” of any biological phenomenon is a matter of philosophy, one can ask what the contributions of ceramide signaling are to the evolutionarily conserved program of apoptosis. The road to apoptosis involves a vast multitude of molecular factors, of which