BACKGROUND & AIMS: Bile salt-induced apoptosis is mediated by a trypsin-like nuclear protease. The aims of this study were to identify this protease and to elucidate its mechanistic role in bile salt-induced hepatocyte apoptosis. METHODS: Rats, isolated rat hepatocytes, and a rat hepatoma cell line stably transfected with a bile salt transporter (McNtcp.24) were used for this study. RESULTS: In the bile duct-ligated rat, a threefold increase in apoptosis and a fourfold increase in trypsin-like nuclear protease activity were observed. The nuclear protease activity was purified from bile duct-ligated rats and identified as cathepsin B. Specific, structurally dissimilar cathepsin B inhibitors blocked glycochenodeoxycholate (GCDC)-induced apoptosis in cultured rat hepatocytes. Furthermore, stable transfection of McNtcp.24 cells with the complementary DNA for cathepsin B in the antisense orientation reduced cathepsin B activity and GCDC-induced apoptosis by >75%. Next, cathepsin B cellular localization during apoptosis was determined by immunoblot analysis of nuclear cell fractions, immunocytochemistry, and by determining the compartmentation of expressed cathepsin B fused to green fluorescent protein. All three approaches showed translocation of cathepsin B from the cytoplasm to the nucleus during GCDC-induced apoptosis. CONCLUSIONS: The data suggest that translocation of cathepsin B from the cytoplasm to the nucleus is a mechanism contributing to bile salt-induced apoptosis of hepatocytes.
BACKGROUND & AIMS:Bile salt-induced apoptosis is mediated by a trypsin-like nuclear protease. The aims of this study were to identify this protease and to elucidate its mechanistic role in bile salt-induced hepatocyte apoptosis. METHODS:Rats, isolated rat hepatocytes, and a rathepatoma cell line stably transfected with a bile salt transporter (McNtcp.24) were used for this study. RESULTS: In the bile duct-ligated rat, a threefold increase in apoptosis and a fourfold increase in trypsin-like nuclear protease activity were observed. The nuclear protease activity was purified from bile duct-ligated rats and identified as cathepsin B. Specific, structurally dissimilar cathepsin B inhibitors blocked glycochenodeoxycholate (GCDC)-induced apoptosis in cultured rat hepatocytes. Furthermore, stable transfection of McNtcp.24 cells with the complementary DNA for cathepsin B in the antisense orientation reduced cathepsin B activity and GCDC-induced apoptosis by >75%. Next, cathepsin B cellular localization during apoptosis was determined by immunoblot analysis of nuclear cell fractions, immunocytochemistry, and by determining the compartmentation of expressed cathepsin B fused to green fluorescent protein. All three approaches showed translocation of cathepsin B from the cytoplasm to the nucleus during GCDC-induced apoptosis. CONCLUSIONS: The data suggest that translocation of cathepsin B from the cytoplasm to the nucleus is a mechanism contributing to bile salt-induced apoptosis of hepatocytes.
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Authors: W A Faubion; M E Guicciardi; H Miyoshi; S F Bronk; P J Roberts; P A Svingen; S H Kaufmann; G J Gores Journal: J Clin Invest Date: 1999-01 Impact factor: 14.808