Cystatin A expression reduces bile salt-induced apoptosis in a rat hepatoma cell line.
We have previously demonstrated abrogation of bile salt-induced apoptosis by cathepsin B inhibitors. However, caspases have been strongly implicated in apoptosis, and the mechanistic interface between caspase and cathepsin B activation is unclear. Thus our aims were to determine the mechanistic relationship between caspases and cathepsin B in bile salt-induced apoptosis in a rat hepatoma cell line. Expression of cystatin A was used to inhibit cathepsin B, whereas Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) was used to inhibit caspases. Cystatin A expression prevented cathepsin B activation and apoptosis during treatment with glycochenodeoxycholate (GCDC), a toxic bile salt. Caspase N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) hydrolytic activity increased in both wild-type and cystatin A-transfected cells treated with GCDC, demonstrating caspase activation despite inhibition of cathepsin B. In contrast, Z-VAD-FMK blocked both DEVD-AMC hydrolytic activity and cathepsin B activity during GCDC treatment. Our data demonstrate that 1) bile salt-induced apoptosis can be inhibited by the cystatin A transgene and 2) caspase and cathepsin B activation are linked mechanistically with cathepsin B downstream of caspases.
Jones
B
B
Center for Basic Research in Digestive Diseases, Mayo Clinic, Rochester, Minnesota 55905, USA.
Roberts
P J
PJ
Faubion
W A
WA
Kominami
E
E
Gores
G J
GJ
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001647:Bile Acids and Salts / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D015891:Cystatins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D005999:Glycochenodeoxycholic Acid / Q000494:pharmacology*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000473:pathology*; D009842:Oligopeptides / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D001647:Bile Acids and Salts; D003374:Coumarins; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D011994:Recombinant Proteins; C102580:acetyl-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarin; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005999:Glycochenodeoxycholic Acid; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D002401:Cathepsin B