Tauroursodeoxycholic acid exerts anti-apoptotic effects by a MKP-1- and PKA-dependent mechanism in rat liver

Introduction:

Ursodeoxycholic acid, which in vivo is converted to its taurine conjugate tauroursodeoxycholic acid (TUDC), is a mainstay for the treatment of cholestatic liver disease. TUDC is also effective in protecting hepatocytes for apoptosis induced by hydrophobic bile salts. The aim of the study was to elucidate the underlying signaling events.

Methods:

Rat livers were perfused with GCDC (20µmol/L) and TUDC (20µmol/L) for up to 120 min while cell culture experiments were performed in primary rat hepatocytes, which were stimulated with GCDC (50µmol/L) and TUDC (100µmol/L) for up to 360 min. Activation of kinases and association of proteins were determined by immunoprecipitation and Western blot technique using specific antibodies with subsequent densitometric analysis. Apoptosis was determined by caspase-3 cleavage and DNA fragmentation. Expression of MKP-1 was analyzed by mRNA induction. cAMP was measured by a commercial ELISA assay kit.

Results:

The hydrophobic bile acid glycochenodeoxycholic acid (GCDC) induces a rapid oxidative stress response, followed by EGFR activation in vivo and in vitro. Activated EGFR then associates in a JNK-dependent way with CD95 and catalyzes CD95 tyrosine phosphorylation, which triggers CD95 membrane trafficking, DISC formation, and induction of hepatocyte apoptosis. These latter effects were abolished by TUDC, which blunted GCDC-induced activation of JNK within 60 min. mRNA analyses show that the dual specificity MAP kinase phosphatase MKP-1, a transcriptionally regulated immediate early gene-encoded enzyme, is upregulated in response to TUDC after stimulation with GCDC for up to 60 min. Like cAMP, TUDC induced a H89-sensitive serine/threonine phosphorylation of the CD95 in presence of GCDC, which favors internalization of the CD95. TUDC in combination with 3-Isobutyl-1-methylxanthin (IBMX) also increased cAMP production in primary hepatocytes within 30 min compared to the IBMX-stimulated control cells.

Discussion/Conclusion:

Inhibition of bile salt-induced apoptosis by TUDC involves both PKA-dependent serine/threonine phosphorylation of the CD95 and inhibition of JNK due to MKP-1 induction. This prevents the association of EGFR and CD95 and subsequent CD95 tyrosine phosphorylation.