Apoptosis induced by hydrophobic bile acids is thought to contribute to liver injury during cholestasis. Caspase-6 is an executioner caspase that also appears to have regulatory functions in hematopoetic cell lines. We aimed to elucidate the role of caspase-6 in bile acid-induced apoptosis. The major human hydrophobic bile acid, glycochenodeoxycholic acid (GCDCA, 75 mol/liter), rapidly induced caspase-6 cleavage in HepG2-Ntcp human hepatoma cells. GCDCA-induced, but not tumor necrosis factor ␣-or etoposide-induced activation of effector caspases-3 and -7 was significantly reduced by 50% in caspase-6-deficient HepG2-Ntcp cells as well as in primary rat hepatocytes pretreated with a caspase-6 inhibitor. Inhibition of caspase-9 reduced GCDCA-induced activation of caspase-6, whereas inhibition of caspase-6 reduced activation of caspase-8 placing caspase-6 between caspase-9 and caspase-8. GCDCA also induced apoptosis in Fas-deficient Hep3B-Ntcp and HuH7-Ntcp hepatoma cells. In addition, GCDCA-induced apoptosis was reduced by 50% in FADD-deficient HepG2-Ntcp cells, whereas apoptosis induced by tumor necrosis factor ␣ was reduced by 90%. Collectively, these observations suggest that GCDCA can induce hepatocyte apoptosis in the absence of death receptor signaling, presumably by a compensatory mitochondrial pathway. In conclusion, caspase-6 appears to play an important regulatory role in the promotion of bile acid-induced apoptosis as part of a feedback loop.Cholestasis is a feature of many chronic human liver diseases like primary biliary cirrhosis (1). Hepatocyte damage by toxic bile acids is assumed to represent a key event for progression of cholestatic liver diseases (2). Toxic bile acids induce hepatocellular apoptosis, thereby providing a cellular mechanism for bile acid-mediated liver injury and fibrogenesis (3-6). The glycine and taurine conjugates of chenodeoxycholic acid (GCDCA, TCDCA) 2 are the predominant dihydroxy bile acids in cholestatic patients and have been held responsible for cholestasisassociated liver injury (7). GCDCA is thought to induce hepatocyte apoptosis by Fas death receptor-dependent signaling that is independent of Fas ligand (8). In a recently proposed model, activation of NADPH oxidase isoforms by GCDCA is followed by generation of reactive oxygen species, which results in epidermal growth factor receptor-dependent tyrosine phosphorylation of Fas (5, 9). GCDCA-induced oligomerization of Fas then causes activation of the initiator caspase-8, which requires the adaptor protein FADD. In hepatocytes, caspase-8 then would cause apoptosis via the mitochondrial pathway (10).In cell-free systems, however, toxic bile acids like GCDCA can directly cause mitochondrial damage leading to cytochrome c leakage, which is followed by the activation of caspase-9 via the apoptosome (11). GCDCA can dose-dependently induce the mitochondrial permeability transition in isolated human mitochondria (12). In addition, a marked increase in reactive oxygen species production was observed within minutes in the m...
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