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Tumor necrosis factor induces ceramide oscillations and negatively controls sphingolipid synthases by caspases in
S Bourteele1, A Hausser, H Döppler
1Institute of Cell Biology and Immunology, University of Stuttgart, D-70569 Stuttgart, Germany.
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.
Insights
Tumor necrosis factor (TNF) triggers apoptosis via multiphasic ceramide increases, regulated by specific lipid enzymes. Caspase activity is crucial for controlling ceramide levels during programmed cell death.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- The precise role of the lipid messenger ceramide in programmed cell death remains unclear.
- Its connection to receptor-associated apoptotic signaling proteins requires further elucidation.
Purpose of the Study:
- To investigate the origin, role, and mechanism of ceramide in tumor necrosis factor (TNF)-induced apoptosis.
- To identify the enzymes involved in ceramide generation and regulation during apoptosis.
Main Methods:
- Utilized Kym-1 rhabdomyosarcoma cells to study TNF-induced apoptosis.
- Measured intracellular ceramide levels and activities of neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase.
- Assessed the impact of caspase inhibitors (Z-VAD-fmk, Z-DEVD-cmk) on ceramide metabolism.
Main Results:
- TNF induced a multiphasic increase in intracellular ceramide levels.
- Neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase contributed to distinct ceramide waves.
- TNF inhibited glucosylceramide and sphingomyelin (SM) synthase, preventing ceramide metabolism.
- Caspase inhibitors blocked ceramide accumulation, indicating caspase-sensitive regulation.
Conclusions:
- Multiple lipid enzymes contribute to ceramide generation in response to TNF.
- Glucosylceramide and SM synthase are key regulators of ceramide accumulation kinetics and magnitude.
- A novel functional link between caspases and ceramide in apoptotic processes is suggested.