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...
Background: Bile acid induced apoptosis in hepatocytes can be antagonised by nuclear factor kB (NFkB) dependent survival pathways. Sulfasalazine modulates NFkB in different cell types. We aimed to determine the effects of sulfasalazine and its metabolites sulfapyridine and 5-aminosalicylic acid (5-ASA) on bile acid induced apoptosis in hepatocytes. Methods: Apoptosis was determined by caspase assays and immunoblotting, NFkB activation by electrophoretic mobility shift assay and reporter gene assays, generation of reactive oxygen species (ROS) fluorometrically, bile secretion gravimetrically, and bile acid uptake radiochemically and by gas chromatography in HepG2-Ntcp cells and isolated perfused rat livers. Results: Glycochenodeoxycholic acid (GCDCA 75 mmol/l) induced apoptosis was reduced by sulfasalazine dose dependently (1-1000 mmol/l) in HepG2-Ntcp cells whereas its metabolites 5-ASA and sulfapyridine had no effect. Sulfasalazine significantly reduced GCDCA induced activation of caspases 9 and 3. In addition, sulfasalazine activated NFkB and decreased GCDCA induced generation of ROS. Bile acid uptake was competitively inhibited by sulfasalazine. In perfused rat livers, GCDCA (25 mmol/l) induced liver injury and extensive hepatocyte apoptosis were significantly reduced by simultaneous administration of 100 mmol/l sulfasalazine: lactate dehydrogenase and glutamate-pyruvate transaminase activities were reduced by 82% and 87%, respectively, and apoptotic hepatocytes were observed only occasionally. GCDCA uptake was reduced by 45 (5)% when sulfasalazine was coadministered. However, when 50% of GCDCA (12.5 mmol/l) was administered alone, marked hepatocyte apoptosis and liver injury were again observed, questioning the impact of reduced GCDCA uptake for the antiapoptotic effect of sulfasalazine. Conclusion: Sulfasalazine is a potent inhibitor of GCDCA induced hepatocyte apoptosis in vitro and in the intact liver. C holestasis is a common feature of many human liver diseases. Elevated bile acid concentrations in hepatocytes, a hallmark of cholestasis, promote liver cell death resulting in liver injury and liver cirrhosis.1 Toxic bile acids induce hepatocellular apoptosis, thereby providing a cellular mechanism for bile acid mediated liver injury.2-4 The glycine and taurine conjugates of chenodeoxycholic acid (GCDCA, TCDCA) are the predominant dihydroxy bile acids in cholestatic patients and have been held responsible for cholestasis associated liver injury.5 Glycochenodeoxycholic acid (GCDCA) is thought to induce hepatocyte apoptosis by a Fas death receptor dependent process that is independent of Fas ligand 6 but induces oligomerisation of Fas by increasing cell surface trafficking of Fas.7 GCDCA induced generation of reactive oxygen species followed by epidermal growth factor receptor dependent tyrosine phosphorylation of Fas also appears to be required for GCDCA induced Fas signalling. 4 The transcription factor nuclear factor kB (NFkB) has been shown to reduce hepatocyte apoptosis induced by toxic bile acids, 8 t...
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