Ursodeoxycholic acid diminishes Fas-ligand-induced apoptosis in mouse hepatocytes

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Abstract
Ursodeoxycholic acid (UDCA) can protect hepatocytes from apoptosis induced by a variety of stimuli including anti-Fas antibody. However, in vivo the Fas receptor is activated by its natural ligand, Fas-L, whereas anti-Fas antibodies are not a physiologic stimulus. We therefore have assessed the anti-apoptotic effects of UDCA and other bile acids in a novel coculture model where apoptosis is induced in murine hepatocytes by membrane-bound Fas-L expressing fibroblasts. Primary hepatocytes were cultured overnight on collagen-coated coverslips with increasing concentrations of different bile acids and overlaid with either NIH 3T3 Fas-L+ or Fas-L− expressing fibroblasts. After 6 hours cells were fixed and apoptosis was evaluated by TUNEL assay and DAPI staining using digital imaging. Fas-L protein expression and Fas trimerization were evaluated by Western blot analysis. FITC-UDCA and Mitotracker Red were used to evaluate UDCA localization with mitochondria. UDCA (up to 100 μmol/L, P < .0001), TUDCA (up to 400 μmol/L, P < .0001), and TCDCA (up to 200 μmol/L, P < .0001), but not TCA (up to 500 μmol/L), significantly protected hepatocytes in Fas-L+ cocultures. UDCA had no significant effect on hepatocytes in Fas-L− cocultures. TUDCA, 50 μmol/L (P < .001) and TCDCA up to 200 μmol/L (P < .0001) also reduced the hepatocytes apoptotic rate in Fas-L− cocultures. Bile acids did not affect Fas-L expression in fibroblasts or Fas trimerization. FITC-UDCA colocalized with the mitochondrial probe. In conclusion, UDCA, TUDCA, and TCDCA but not TCA are capable of protecting hepatocytes from Fas-L-induced apoptosis. This protective effect is not associated with reductions in Fas trimerization, but may be related to a direct effect on the mitochondrial membrane.
Funding Information
  • USPHS (DK25636, DK48823)
  • NIDDK Liver Center Molecular Biology and Imaging Cores (P30-34989)