Apoptosis and Sphingomyelin Hydrolysis

Apoptosis, a form of cell death that is coordinated by a set of defined biochemical pathways, exists throughout the animal kingdom. Its critically important roles in development, homeostasis, and disease have made this a topic of intensive research and sometimes even more intensive controversy. One of these areas of controversy concerns the role of ceramide, produced by the hydrolysis of sphingomyelin. In this issue, Tepper et al. (2000) add to this debate with an interesting new theory. When we consider the central mechanisms of apoptosis, we have two general questions to ask: first, what determines whether a cell will live or die? And if a cell is to die, what determines the precise form of this cell death? The second question is often as important as the first, as we’ll see. Evidence from early experiments showed that ceramide is produced under a variety of conditions leading to apoptosis (Kolesnick and Kronke, 1998). The knowledge that synthetic ceramide was able to induce apoptosis, led to the unfortunately too simple conclusion that the production and action of ceramide is an obligatory step in the apoptotic process. Therefore, several studies (based predominantly on correlations) put forth the idea that sphingomyelin hydrolysis and signaling via ceramide are essential in the decision of whether a cell dies. This conclusion fell from favor as evidence accumulated against ceramide production as a major determinant of the life/death decision. Now, a new study by Tepper and colleagues in this issue (Tepper et al., 2000) has perhaps defined a new role for sphingomyelin hydrolysis in apoptosis, determining not whether but how a cell dies. To understand this role in the context of the apoptotic process (as it is currently understood), we will first review the process itself and examine the different roles that have been proposed for sphingomyelin hydrolysis in the different pathways to apoptotic cell death. When cells die via apoptosis, they undergo a number of morphological and biochemical changes that are stereotypical for this type of death. These changes are orchestrated by a set of cysteine proteinases that become active during apoptosis, the caspases (cysteine proteinases with specificity for aspartic acid residues; reviewed in Wolf and Green, 1999). The final throes of cell death (and the associated changes) have been named execution and the caspases responsible for coordinating these changes are the executioner caspases. Caspase-3 is the major executioner caspase, together with caspases-6 and -7. The executioner caspases, once activated, cleave key substrates in the cell that produce the apoptotic phenotype. For example, caspase-3 cleaves iCAD (inhibitor of caspase-activated DNase), releasing CAD, which in turn cuts the chromatin between nucleosomes to produce the DNA ladder seen in many cell types as they undergo apoptosis. Another protein, acinus, is activated by caspase cleavage to cause chromatin condensation. Similarly, caspase-3 cleaves and activates p21-activated kinase-2 and gelsolin, both of which appear to participate in contortions of the plasma membrane, referred to as blebbing. Other effects of the substrates of activated caspases are inferred from the effects of caspase inhibitors or by the use of cells lacking one or more caspases. These include loss of mitochondrial membrane potential, cell shrinkage, loss of cell adhesion, and loss of plasma membrane integrity, among others. One of the most important caspase-mediated changes in the cell is the loss of plasma membrane lipid asymmetry. That is, the lipids of the planar membrane are normally nonrandomly distributed, some are predominantly localized to one or the other side. For example, sphingomyelin is mostly found on the outer leaflet, while phosphatidylserine and phosphatidylethanolamine are mostly on the inner leaflet of the plasma membrane. This is due to the action of phospholipid translocases that maintain the orientations of some of the lipids (Bevers et al., 1999). During apoptosis, the plasma membrane scrambles, and this involves both loss of the translocase activity and the action of an uncharacterized scramblase. Although this is often (but not always, as I’ll discuss) caspase dependent, we do not know the molecular mechanism of this event. The loss of lipid asymmetry can be readily monitored by the use of fluorescence-conjugated annexin V, which specifically binds to phosphatidylserine (PS) 1 as it externalizes on apoptotic cells (Martin et al., 1995). The appearance of PS on the outer leaflet of the plasma membrane is especially relevant, because of all the changes we have so far mentioned, it is the one with the most defined function. PS is recognized and bound by a receptor (PSR) on macrophages and other phagocytic cells, and this results in the rapid clearance of the dying cell from the body (Fadok et al., 2000). One could even argue that this is the only change that a cell needs to go through to be functionally