Figure B9-2. LC/MS detection of ceramide species. Example chromatogram of ceramides and sphingoid bases extracted from MCF-7 adenocarcinoma cells. Unnatural, synthetic sphingolipid species containing C13- or C17-sphingoid bases (e.g., C17/C16-ceramide) are used as internal standards for HPLC separation. Most mammalian ceramides contain C18-sphingoid bases and are therefore distinguishable from the synthetic standards by their retention time as well as mass transition. Using ceramide standards to generate calibration curves, ceramides are quantified and normalized to total protein or total lipid phosphate.

reception of a stimulus, the production and activation of signaling intermediates, the activation of effectors, and a resulting change in cell behavior (Figure 9-3). In the case of programmed cell death, there is a multitude of potential stimuli, and although the “reception” phase of cell death signaling may be divergent in response to various stimuli, at some level many signaling pathways converge to produce a seemingly unified response – apoptosis. A number of proapoptotic signaling modalities are engaged to bring about apoptosis, including Ca2+ release, production of lipid mediators, and the activation of proteases.

3.1. Basic cell signaling often involves small molecules

Most cellular signaling uses small molecules to convey information between protein components of each pathway. For example, Ca2+ signaling can occur by

releasing Ca2+ from compartments such as the ER or extracellular milieu where Ca2+ concentrations are relatively high. After stimulation and activation of Ca2+ channels, elevations in cytosolic Ca2+ can cause the modulation of multiple downstream effectors. In a similar fashion, signaling sphingolipids such as ceramide can be released from “storage” in the form of SM at the plasma membrane to produce local changes in the concentration of ceramide. However, unlike soluble free Ca2+ , the hydrophobic nature of ceramide confines it to the membrane, where it can interact with membrane proteins (e.g., receptor signaling complexes) or soluble proteins that might be recruited from the cytosol (e.g., protein phosphatases).

3.2. Sphingolipids are cell-signaling molecules

The study of sphingolipids as signaling molecules originally emerged from the finding that sphingosine inhibits

Figure 9-3. A simple signaling paradigm. The most conceptually simple lipid signaling paradigms involve ligand-induced activation receptor complex, the recruitment of signaling proteins, and the activation of enzymes that produce signaling lipids. These lipids either recruit proteins to the membrane from the hydrophilic compartments or bind to proteins within the membranes themselves. Lipid-protein interactions result in changes in e ector protein function, leading to the downstream activation of signaling components and a resultant biological response. TNFR, tumor necrosis factor receptor; GPCR, G- protein coupled receptor.

the activation of PKC both in vitro and as a component of signaling induced by the pleiotropic proinflammatory cytokine tumor necrosis factor-α (TNF-α). Additional studies found that TNF-α could induce ceramide production via SM hydrolysis and that SM-derived ceramide could also modify cell behavior. Thus the field of sphingolipid signaling was born. Once the connections between TNF-α signaling and ceramide were established, it was not long before this lipid was being studied in the context of apoptosis. Although many questions remain today, three main lines of evidence support the hypothesis that ceramide is a key mediator of programmed cell death.

3.2.1. Ceramide induces apoptosis

One of the first connections made between ceramide and apoptosis was the discovery that short-chain, cell-permeable analogs of ceramide (e.g., N-acetyl- sphingosine, or C2-ceramide) were able to induce apoptosis in leukemia cell lines. The closely related molecule C2-dihydroceramide, however, was unable to induce apoptosis, suggesting that the effect was highly specific to the molecular configuration of ceramide.

Several studies followed to establish the importance of ceramide’s 4–5 trans double bond for its proapoptotic effects. Short-chain ceramides (C2–8) induce apoptosis that involves activation of members of the mitogenactivated protein kinase (MAPK) family (e.g., p38-MAPK and c-Jun N-terminal kinase), although the dependence on these signaling pathways is controversial. Shortchain analogs also modulate the activities of protein phosphatases such as PP1 and PP2A that can function to de-phosphorylate prosurvival proteins such as Akt. In addition to its direct effects, C6-ceramide, and to a lesser extent C2-ceramide, can be metabolized by the salvage pathway to form long-chain ceramides. Moreover, C6-ceramide can serve as a substrate for the ceramide transfer protein CERT and thus may modify levels of endogenous ceramide.

Currently, many researchers use C2- and C6-ceramide applied exogenously to induce cell death, but several other forms of ceramide can also reproduce similar effects. In some cell types, the addition of the bacterial sphingomyelinase from Bacillus cereus is sufficient to produce ceramide and cause cell death. Long-chain ceramides (e.g., C16–ceramide) dissolved in appropriate solvent mixtures such as dodecane/ethanol are also able to promote apoptosis. Interest in the proapoptotic abilities of ceramide have led to the development of ceramide analogs and ceramide-containing liposomes for clinical use in the treatment of a variety of diseases.

3.2.2. Ceramide accumulates during programmed cell death

After stimulation of cells in culture with a deathpromoting ligand such as TNF-α or genotoxic stress such as doxorubicin, one or more SMases are activated, leading to the hydrolysis of SM and accumulation of ceramide (Figure 9-4). These events typically happen within the first 30 minutes after stimulation, and the activities of the SMases often return to basal levels within 1 hour (Figure 9-5). However, prolonged stimulation results in a second wave of ceramide production that, although less well characterized, often depends on de novo ceramide synthesis (e.g., CerS activation) (Figure 9-6). Both SMaseand de novo–mediated ceramide production have been described for a variety of diverse cell-death stimuli (Figure 9-7). Details about these two means of ceramide production, as well as their significance in cell death, are provided later in the chapter.

3.2.3. Inhibition of ceramide production alters cell death signaling

In many experimental models of apoptosis, deathinduced ceramide production can be inhibited using

Figure 9-4. Ceramide generation via SMase activation. Activation of SMases leads to hydrolysis of SM to form ceramide and phosphocholine.

pharmacological, genetic, or more recent RNA inter- ference-based approaches. In many cases, such inhibition impedes the cell death process. For example, activation of CD95 on lymphocytes leads to SMase activation, ceramide production, receptor clustering, and apoptosis. Cells deficient in a particular SMase (see Sections 4.2 and 4.3) fail to achieve the same responses and are protected from cell death. In other systems, inhibition of de novo ceramide synthesis using pharmacological inhibitors of SPT or CerS prevents apoptosis. As we discuss next, cell death signaling can occur via multiple ceramide-mediated pathways that differ in terms of time, subcellular localization, and involvement of particular enzymes of sphingolipid metabolism.

4. CERAMIDE MEDIATES APOPTOTIC CELL DEATH:

ROLE OF PARTICULAR ENZYME SYSTEMS

To illustrate the salient features of regulated sphingolipid metabolism and the ceramide-mediated death signaling, we discuss selected studies that highlight key features regarding the nature of sphingolipids as signaling molecules in cell death.