CATHEPSIN-B CONTRIBUTES TO BILE SALT-INDUCED APOPTOSIS OF RAT HEPATOCYTES

Background & Aims: Bile salt-induced apoptosis is mediated by a trypsi n-like nuclear protease. The aims of this study were to identify this protease and to elucidate its mechanistic role in bile salt-induced he patocyte apoptosis. Methods: Rats, isolated rat hepatocytes, and a rat hepatoma cell line stably transfected with a bile salt transporter (M cNtcp.24) were used for this study. Results: In the bile duct-ligated rat, a threefold increase in apoptosis and a fourfold increase in tryp sin-like nuclear protease activity were observed. The nuclear protease activity was purified from bile duct-ligated rats and identified as c athepsin B. Specific, structurally dissimilar cathepsin B inhibitors b locked 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 re duced cathepsin B activity and GCDC-induced apoptosis by >75%. Next, c athepsin B cellular localization during apoptosis was determined by im munoblot 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 o f cathepsin B from the cytoplasm to the nucleus during GCDC-induced ap optosis. Conclusions: The data suggest that translocation of cathepsin B from the cytoplasm to the nucleus is a mechanism contributing to bi le salt-induced apoptosis of hepatocytes.