Abstract:Background/Aims: To clarify the mechanism of hepatocyte apoptosis induced by tumor necrosis factor-α (TNF-α), caspase cascade and ceramide formation were investigated in the liver of D-galactosamine (GalN)-sensitized mice treated with TNF-α. 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-α (0.5 μg/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-α (GalN/TNF-α), but not GalN or TNF-α alone. Activation of caspases-9 and -3, and cytochrome c release were observed only in liver from mice treated with GalN/TNF-α. 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-α-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-α. 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-α-induced hepatocyte apoptosis, but caspases-8 and -2 play, if any, a minimal role. Ceramide may be implicated in this apoptotic process.