Sphingomyelin Hydrolysis to Ceramide during the Execution Phase of Apoptosis Results from Phospholipid Scrambling and Alters Cell-Surface Morphology

Abstract

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]–sphingosylphosphorylcholine (C6-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca2+ ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-β-cyclodextrin, which is indicative of a loss of tight SM–cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C6- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.

Recent insights into the mechanism of glucocorticosteroid-induced apoptosis

“…93 The induction of ceramide appears to be secondary to induction of both acidic and neutral sphingmyelinases. Although recent evidence has pointed to phospholipid scrambling in the plasma membrane as the mechanism of sphingomyelin activation during apoptosis, 94 studies in corticosteroid-treated thymocytes suggest that sphingomyelinase activation and ceramide generation proceed by a different mechanism. 93 In thymocytes, dexamethasone treatment rapidly induces diacylglycerol through a protein kinase C and G-proteindependent phosphatidylinositol-specific phospholipase C (PI-PLC).…”

Ceramide regulates cellular homeostasis via diverse stress signaling pathways

“…41 In fact, ceramide formation with concomitant sphingomyelin hydrolysis appears to be necessary for the changes in membrane morphology (eg blebbing, vasculization, and externalization of phosphatidylserine) that are the hallmark features of the execution phase of apoptosis. 42 In addition to its metabolic and structural roles, it is becoming increasingly clear that ceramide plays a critical role in transmembrane signaling, particularly in response to stress challenges. [5][6][7][8][9][10][11] Therefore, it is not surprising that there are multiple physiologic pathways that produce ceramide.…”

PS externalization: from corpse clearance to drug delivery

“…1 PS externalization can, in some cases, be divorced from membrane blebbing; however, macrophage engulfment of PS-positive cell corpses appears to be less efficient when blebbing is prevented (unpublished findings). An interesting twist to the story was added when Tepper et al 12 reported that blebbing during apoptosis is related to sphingomyelin (SM) hydrolysis to ceramide and the loss of phospholipid asymmetry in the plasma membrane. Hence, ceramide production during the execution phase of apoptosis was shown to be derived from SM initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling.…”