Caspase activation during hepatocyte apoptosis induced by tumor necrosis factor-alpha in galactosamine-sensitized mice

Background/Aims: To clarify the mechanism of hepatocyte apoptosis induced b y tumor necrosis factor-alpha (TNF-alpha), caspase cascade and ceramide for mation 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 mug/mouse). Cytochrome c rele ase and processing of procaspases in the liver were analyzed by Western blo tting. Activities of caspases were measured using chromogenic peptides as s ubstrates. Ceramide content was determined using Escherichia coli diacylgly cerol kinase. Results: Apoptosis of hepatocytes was observed in mice treate d 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 obser ved only in liver from mice treated with GalN/TNF-alpha. In a cell-free sys tem, processing of procaspases-9 and -3, and cytochrome c release were obse rved in the postnuclear fraction of liver obtained from GalN/TNF-alpha -tre ated mice, but not in that from control mice. Processing of procaspase-3 wa s inhibited by a caspase-9 inhibitor, but not by inhibitor for caspase-8 or -2. In a reconstitution assay system, procaspase-9 processing occurred, wh en both cytosol and membrane fractions were obtained from the liver of mice treated with GalN/TNF-alpha. Ceramide accumulation was observed only in ap optotic liver and preceded cytochrome c release and caspase activation. Con clusion: Cytochrome c release and caspase-9 activation are required for the activation of executor caspase-3 in TNF-alpha -induced hepatocyte apoptosi s, but caspases-8 and -2 play, if any, a minimal role. Ceramide may be impl icated in this apoptotic process.