Ursodeoxycholic acid diminishes Fas-ligand-induced apoptosis in mouse hepatocytes.
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 micromol/L, P <.0001), TUDCA (up to 400 micromol/L, P <.0001), and TCDCA (up to 200 micromol/L, P <.0001), but not TCA (up to 500 micromol/L), significantly protected hepatocytes in Fas-L(+) cocultures. UDCA had no significant effect on hepatocytes in Fas-L(-) cocultures. TUDCA, 50 micromol/L (P <.001) and TCDCA up to 200 micromol/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.
Azzaroli
Francesco
F
Liver Center, Department of Internal Medicine and Immunology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8019, USA.
Mehal
Wajahat
W
Soroka
Carol J
CJ
Wang
Lin
L
Lee
John
J
Crispe
Ian Nicholas
IN
Crispe
Nicholas
N
Boyer
James L
JL
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003432:Cross-Linking Reagents; D003840:Deoxycholic Acid / Q000494:pharmacology; D053222:Fas Ligand Protein; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D022781:Hepatocytes / Q000166:cytology*; D020287:In Situ Nick-End Labeling; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000032:analysis / Q000494:pharmacology*; D051379:Mice; D013655:Taurochenodeoxycholic Acid / Q000494:pharmacology; D014580:Ursodeoxycholic Acid / Q000494:pharmacology*; D019014:fas Receptor / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D019014:fas Receptor; D003840:Deoxycholic Acid; D013655:Taurochenodeoxycholic Acid; C031655:ursodoxicoltaurine; D014580:Ursodeoxycholic Acid