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). Previous 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 the role of caspases in cathepsin B activation. Immunoblotting of GCDC-treated McNtcp.24 hepatoma cells demonstrated cleavage of poly(ADP-ribose) polymerase and lamin B1 to fragments that indicate activation of effector caspases. Transfection with CrmA, an inhibitor of caspase 8, prevented GCDC-induced cathepsin B activation and apoptosis. Consistent with these results, an increase in caspase 8-like activity was observed in GCDC-treated cells. Examination of the mechanism of GCDC-induced caspase 8 activation revealed that 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 demonstrated Fas oligomerization, and confocal microscopy demonstrated DeltaFADD-GFP (Fas-associated death domain-green fluorescent protein, aggregation in the absence of detectable Fas ligand mRNA. Collectively, these data suggest that GCDC-induced hepatocyte apoptosis involves ligand-independent oligomerization of Fas, recruitment of FADD, activation of caspase 8, and subsequent activation of effector proteases, including downstream caspases and cathepsin B.
Faubion
W A
WA
Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota 55905, USA.
Guicciardi
M E
ME
Miyoshi
H
H
Bronk
S F
SF
Roberts
P J
PJ
Svingen
P A
PA
Kaufmann
S H
SH
Gores
G J
GJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001647:Bile Acids and Salts / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism*; D002401:Cathepsin B / Q000378:metabolism*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005999:Glycochenodeoxycholic Acid / Q000494:pharmacology; D007700:Kinetics; D034921:Lamin Type B; D034882:Lamins; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics; D015843:Serpins / Q000235:genetics; D014764:Viral Proteins; D019014:fas Receptor / Q000378:metabolism*
D001647:Bile Acids and Salts; D015853:Cysteine Proteinase Inhibitors; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D009842:Oligopeptides; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D015843:Serpins; D014764:Viral Proteins; D019014:fas Receptor; D005999:Glycochenodeoxycholic Acid; C074641:interleukin-1beta-converting enzyme inhibitor; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D002401:Cathepsin B