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Caspase activation during hepatocyte apoptosis induced by tumor necrosis factor-alpha in galactosamine-sensitized
1First Department of Internal Medicine, Gifu University School of Medicine, Gifu, Japan.
Background/Aims:
To clarify the mechanism of hepatocyte apoptosis induced by tumor necrosis factor-alpha (TNF-alpha), caspase cascade and ceramide formation were investigated in the liver of D-galactosamine (GalN)-sensitized mice treated with TNF-alpha.
Methods:
Seven-week-old male BALB/c mice were intraperitoneally injected with 20 mg GalN 30 min prior to the intravenous injection of recombinant mouse TNF-alpha (0.5 microg/mouse). Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting. Activities of caspases were measured using chromogenic peptides as substrates. Ceramide content was determined using Escherichia coli diacylglycerol kinase.
Results:
Apoptosis of hepatocytes was observed in mice treated with both GalN and TNF-alpha (GalN/TNF-alpha), but not GalN or TNF-alpha alone. Activation of caspases-9 and -3, and cytochrome c release were observed only in liver from mice treated with GalN/TNF-alpha. In a cell-free system, processing of procaspases-9 and -3, and cytochrome c release were observed in the postnuclear fraction of liver obtained from GalN/TNF-alpha-treated mice, but not in that from control mice. Processing of procaspase-3 was inhibited by a caspase-9 inhibitor, but not by inhibitor for caspase-8 or -2. In a reconstitution assay system, procaspase-9 processing occurred, when both cytosol and membrane fractions were obtained from the liver of mice treated with GalN/TNF-alpha. Ceramide accumulation was observed only in apoptotic liver and preceded cytochrome c release and caspase activation.
Conclusion:
Cytochrome c release and caspase-9 activation are required for the activation of executor caspase-3 in TNF-alpha-induced hepatocyte apoptosis, but caspases-8 and -2 play, if any, a minimal role. Ceramide may be implicated in this apoptotic process.
Insights
Tumor necrosis factor-alpha (TNF-alpha) induces hepatocyte apoptosis via cytochrome c release and caspase-9 activation. Ceramide accumulation precedes these events, suggesting its role in TNF-alpha-induced liver cell death.
Area of Science:
- Cell Biology
- Biochemistry
- Toxicology
Background:
- Hepatocyte apoptosis is a critical process in liver injury.
- Tumor necrosis factor-alpha (TNF-alpha) can induce apoptosis in hepatocytes, but the precise mechanism remains unclear.
- D-galactosamine (GalN) sensitizes mice to TNF-alpha-induced liver injury.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying TNF-alpha-induced hepatocyte apoptosis.
- To investigate the roles of caspase activation and ceramide formation in this process.
Main Methods:
- BALB/c mice were treated with GalN and TNF-alpha.
- Western blotting was used to analyze cytochrome c release and procaspase processing.
- Caspase activities were measured using chromogenic substrates.
- Ceramide levels were quantified using diacylglycerol kinase assay.
Main Results:
- Hepatocyte apoptosis was observed only in mice treated with both GalN and TNF-alpha.
- Cytochrome c release and activation of caspase-9 and caspase-3 were detected in the livers of GalN/TNF-alpha-treated mice.
- Ceramide accumulation preceded cytochrome c release and caspase activation.
- Caspase-3 activation was dependent on caspase-9, not caspase-8 or -2.
Conclusions:
- Cytochrome c release and caspase-9 activation are essential for TNF-alpha-induced hepatocyte apoptosis.
- Caspase-8 and caspase-2 play minimal roles in this pathway.
- Ceramide accumulation is implicated in the mechanism of TNF-alpha-induced hepatocyte apoptosis.