Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis

Abstract

Our previous results have indicated that the major cellular pool of sphingomyelin present on the outer leaflet of the plasma membrane is not involved in the ceramide pathway of apoptosis. Thus, in this study we aimed at defining which intracellular pools of sphingomyelin and ceramide are involved in cell death. The bacterial sphingomyelinase (SMase) gene fused with green fluorescent protein was subcloned into mammalian vectors containing sequences that target the fusion proteins to cytoplasm, plasma membrane, mitochondria, Golgi apparatus, endoplasmic reticulum, or nucleus. Transfection of MCF7 breast cancer cells showed for all constructs an increase in SMase activity ranging from 2- to 60-fold, concomitant with an increase in total cellular ceramide levels (10-100%) as compared with vector-transfected cells. Next, the effect of overexpression of the SMase on cell death was examined. Results demonstrate that only when bacterial SMase was targeted to mitochondria did cells undergo apoptosis; its targeting to the other intracellular compartments was ineffective. Further, the results show that apoptosis induced by mitochondrial targeting of bacterial SMase requires SMase catalytic activity, is prevented by the overexpression of Bcl-2, and is mediated by inducing cytochrome c release. These results demonstrate that ceramide induces cell death specifically when generated in mitochondria. The results highlight the significance of compartment-specific lipid-mediated cell regulation, and they offer a novel general approach for these studies.

Ceramide-mediated clustering is required for CD95-DISC formation

“…The present data indicate a central role of ceramide for the formation of signaling platforms that serve to cluster CD95. However, our data do not exclude other functions of ceramide in different locations in the cell, in particular in mitochondria, endoplasmic reticulum or lysosomes (Hirschberg et al, 1993;Shimeno et al, 1995;Birbes et al, 2001). In fact, we recently demonstrated that ionizing radiation induces a late-phase ceramide elevation exclusively within the mitochondria of HeLa cells by activation of a mitochondrial form of ceramide synthase (Kolesnick, unpublished data).…”

Ceramides and Sphingomyelins with High Proportions of Very Long-chain Polyunsaturated Fatty acids in Mammalian Germ Cells

“…[41][42][43]. Although Cer can be generated from SM in practically all cellular compartments via the activation of sphingomyelinases (SMases), it is the mitochondrial Cer that is associated with apoptosis, the Cer generated in other cellular compartments having no effect on this death mechanism (44). Our observation that mitochondria from adult rat seminiferous tubules have SM and Cer with virtually no VLCPUFA, although indirectly, adds support to the idea that VLCPUFA-containing species of these testicular sphingolipids probably are not involved as mediators in proapoptotic mechanisms.…”

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo

“…Indeed, the accumulation of ceramide in mitochondria has been reported to mediate tumor necrosis factor (TNF), Fas or radiation-induced cell death (Garcia-Ruiz et al, 1997;Matsko et al, 2001). In addition, the mitochondrial generation of ceramide upon forced expression of neutral sphingomyelinase in mitochondria leads to apoptotic cell death in MCF7 breast cancer cells (Birbes et al, 2001). Although we observed the correlation between enhanced ceramide levels by DNR following acid CDase and mitochondrial structural alterations, it remains to be established whether ceramide directly targeted mitochondria, as shown with GD3 (Garcia-Ruiz et al, 2002), thus recruiting these organelles to apoptosis signaling or was this mediated by some other mechanism?…”