Tumor Necrosis Factor Induces Ceramide Oscillations and Negatively Controls Sphingolipid Synthases by Caspases in Apoptotic Kym-1 Cells

Abstract

The role, origin, and mode of action of the lipid messenger ceramide in programmed cell death and its linkage to receptor-associated apoptotic signal proteins is still unresolved. We show here in Kym-1 rhabdomyosarcoma cells that tumor necrosis factor (TNF)-induced apoptosis is preceded by a multiphasic increase in intracellular ceramide levels. Distinct enzymes were found to contribute to three waves of ceramide, neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase, with peak activities at 1-2, 40, and around 200 min, respectively, the latter coinciding with progression to irreversible damage. In parallel with ceramide generation, TNF-mediated inhibition of glucosylceramide and sphingomyelin (SM) synthase prevents the immediate metabolization of this lipid mediator. In the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) or benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethyl ketone (Z-DEVD-cmk), a broad spectrum and a caspase 3-selective inhibitor, respectively, glucosylceramide and SM synthase activity remains unaffected by TNF, and intracellular ceramide accumulation is not observed. Our results show that several lipid enzymes contribute to generation of ceramide in response to TNF and identify glucosylceramide and SM synthase as important regulators of the kinetics and magnitude of intracellular ceramide accumulation. As glucosylceramide and SM synthase activity is caspase-sensitive, our data suggest a novel functional link between caspase(s) and ceramide during apoptotic processes.

Tumor necrosis factor signaling

“…170 The role of nSMase-derived ceramide is largely undefined, especially as at least in some cells nSMase becomes only activated very transiently during only the first 3 min of TNF stimulation. 171 A proapoptotic function of nSMase has been described in one report using a dominant-negative form of FAN, 172 whereas ERK1/2 and phospholipase A2 activation are not linked to FAN. 173 Other proteins binding TNF-R1 upstream the DD include a regulatory component of the 26S proteasome, called TRAP2, 55.11 or p97, that binds to aa residues 234-308 of TNF-R1, and which might be involved in TNF-mediated regulation of proteasomal functions.…”

“…288,290 Several studies have implicated acidic SMase and ceramide release in TNF-induced cell death. 171,[302][303][304] As cells deficient for acidic SMase are still TNF sensitive, 305 TNFinduced ceramide production seems to have no general, obligatory role in TNF-induced cell death. Nevertheless, the demonstration of reduced TNFsensitivity of aSMaseÀ/À mouse embryonic fibroblasts 306 and several other studies point to aSMase and ceramide production as celltype specific modulatory factors of TNF-induced cell death.…”

Integration of cytokine biology and lipid metabolism in stroke

“…TNF-α and IL-1 inhibit PC and SM synthesis by affecting CCT and SM synthase respectively PC homeostasis is regulated by the opposing actions of hydrolysis by phospholipases (174) and synthesis through CCT (Figure 10). TNF-α can activate caspases (175) which in turn can down-regulate CCT (176) and SM synthase (177,178) through proteolytic cleavage during apoptosis. Other studies have shown that TNF-α can stimulate degradation of CCT through the ubiquitin-proteasome and calpain-mediated proteolytic pathways (42).…”

Caspase-mediated inhibition of sphingomyelin synthesis is involved in FasL-triggered cell death

“…13 Different stimuli, including TNFa, photodynamic therapy and UVB, have been shown to inhibit de novo SM synthesis. [14][15][16][17] Although homotrimerization of CERT (ceramide transfer protein) has been implicated in UVB-mediated inhibition of SM synthesis, the underlying mechanisms involved are largely unknown. 17 Two different mammalian genes encoding SMS have been cloned so far.…”