Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas

Cholestatic liver injury appears to result from the induction of hepatocyte apoptosis by toxic bile salts such as glycochenodeoxycholate (GCDC). Previ ous studies from this laboratory indicate that cathepsin B is a downstream effector protease during the hepatocyte apoptotic process. Because caspases can initiate apoptosis, the present studies were undertaken to determine t he role of caspases in cathepsin B activation. Immunoblotting of GCDC-treat ed McNtcp.24 hepatoma cells demonstrated cleavage of poly(ADP-ribose) polym erase and Iamin B-1 to fragments that indicate activation of effector caspa ses. Transfection with CrmA, an inhibitor of caspase 8, prevented GCDC-indu ced cathepsin B activation and apoptosis. Consistent with these results, an increase in caspase 8-like activity was observed in GCDC-treated cells. Ex amination of the mechanism of GCDC-induced caspase 8 activation revealed th at dominant-negative FADD inhibited apoptosis and that hepatocytes isolated from Fas-deficient lymphoproliferative mice were resistant to GCDC-induced apoptosis. After GCDC treatment, immunoprecipitation experiments demonstra ted Fas oligomerization, and confocal microscopy demonstrated Delta FADD-GF P (Fas-associated death domain-green fluorescent protein, aggregation in th e absence of detectable Fas Ligand mRNA. Collectively, these data suggest t hat GCDC-induced hepatocyte apoptosis involves ligand-independent oligomeri zation of Fas, recruitment of FADD, activation of caspase 8, and subsequent activation of effector proteases, including downstream caspases and cathep sin B.