Tauroursodeoxycholic acid protects rat hepatocytes from bile acid‐induced apoptosis via activation of survival pathways

Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine‐conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)‐induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco‐conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol‐3 kinase pathway (PI3K) and nuclear factor‐κB were inhibited using LY 294002 and adenoviral overexpression of dominant‐negative IκB, respectively. The role of p38 and extracellular signal‐regulated protein kinase mitogen‐activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin‐D. Apoptosis was determined by measuring caspase‐3, ‐9, and ‐8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase‐8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCA‐induced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF‐κB activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA‐induced mitochondria‐controlled apoptosis by activating survival pathways. Supplemental material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/supplmat/index.html). (HEPATOLOGY 2004;39:1563–1573.)

[1]  D. Brenner,et al.  NF-κB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-α– and Fas-mediated apoptosis , 2001 .

[2]  D. Häussinger,et al.  Bile salt-induced hepatocyte apoptosis involves epidermal growth factor receptor-dependent CD95 tyrosine phosphorylation. , 2003, Gastroenterology.

[3]  U. Leuschner,et al.  Molecular aspects of membrane stabilization by ursodeoxycholate [see comment]. , 1993, Gastroenterology.

[4]  P. Fisher,et al.  Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced apoptosis. , 2001, Molecular biology of the cell.

[5]  Jiahuai Han,et al.  Activation of p38α MAPK Enhances Collagenase-1 (Matrix Metalloproteinase (MMP)-1) and Stromelysin-1 (MMP-3) Expression by mRNA Stabilization* , 2002, The Journal of Biological Chemistry.

[6]  G. Johnson,et al.  Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases , 2002, Science.

[7]  D. Mignault,et al.  Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. , 2001, Journal of lipid research.

[8]  C. Steer,et al.  A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation. , 1998, The Journal of clinical investigation.

[9]  C. Trautwein,et al.  Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-kappaB-regulated inhibitor of apoptosis protein 2 (cIAP2) prevents apoptosis. , 2002, Journal of hepatology.

[10]  U. Beuers,et al.  Ursodeoxycholic acid in cholestatic liver disease: Mechanisms of action and therapeutic use revisited , 2002, Hepatology.

[11]  G. Gores,et al.  Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas. , 1999, Gastroenterology.

[12]  C. Trautwein,et al.  Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation. , 2003, Journal of hepatology.

[13]  J C Reed,et al.  IAPs block apoptotic events induced by caspase‐8 and cytochrome c by direct inhibition of distinct caspases , 1998, The EMBO journal.

[14]  C. Steer,et al.  Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation , 1999, Cell Death and Differentiation.

[15]  D. Brenner,et al.  NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis. , 2001, Gastroenterology.

[16]  G. Tytgat,et al.  Effects of Ursodeoxycholate and cholate feeding on liver disease in FVB mice with a disrupted mdr2 P-glycoprotein gene. , 1996, Gastroenterology.

[17]  D. Häussinger,et al.  Tauroursodesoxycholate-induced choleresis involves p38(MAPK) activation and translocation of the bile salt export pump in rats. , 2001, Gastroenterology.

[18]  G. Gores,et al.  Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism of UDCA cytoprotection. , 1995, The Journal of pharmacology and experimental therapeutics.

[19]  A. Gouw,et al.  Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats. , 1997, Gastroenterology.

[20]  J. Boyer,et al.  Biliary bile acids in primary biliary cirrhosis: Effect of ursodeoxycholic acid , 1999, Hepatology.

[21]  P. Hersey,et al.  Activation of ERK1/2 protects melanoma cells from TRAIL-induced apoptosis by inhibiting Smac/DIABLO release from mitochondria , 2003, Oncogene.

[22]  K. Faber,et al.  Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump , 2002, Hepatology.

[23]  S. R. Datta,et al.  Cellular survival: a play in three Akts. , 1999, Genes & development.

[24]  C. Lieber,et al.  Acetaldehyde selectively stimulates collagen production in cultured rat liver fat‐storing cells but not in hepatocytes , 1990, Hepatology.

[25]  Fuminori Tsuruta,et al.  The Phosphatidylinositol 3-Kinase (PI3K)-Akt Pathway Suppresses Bax Translocation to Mitochondria* , 2002, The Journal of Biological Chemistry.

[26]  U. Leuschner,et al.  Molecular aspects of membrane stabilization by ursodeoxycholate , 1993 .

[27]  P. Allen,et al.  Interaction of 14-3-3 with Signaling Proteins Is Mediated by the Recognition of Phosphoserine , 1996, Cell.

[28]  D. Häussinger,et al.  Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N-terminal kinase-dependent manner. , 2002, Gastroenterology.

[29]  G. Gores,et al.  Bile acids induce cyclooxygenase-2 expression via the epidermal growth factor receptor in a human cholangiocarcinoma cell line. , 2002, Gastroenterology.

[30]  P. Dent,et al.  Activation of the Raf‐1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytes , 2002, Hepatology.

[31]  S. Korsmeyer,et al.  BH3 Domain of BAD Is Required for Heterodimerization with BCL-XL and Pro-apoptotic Activity* , 1997, The Journal of Biological Chemistry.

[32]  A. Larghi,et al.  Differences in the metabolism and disposition of ursodeoxycholic acid and of its taurine‐conjugated species in patients with primary biliary cirrhosis , 1999, Hepatology.

[33]  John Calvin Reed,et al.  Regulation of cell death protease caspase-9 by phosphorylation. , 1998, Science.

[34]  C. Steer,et al.  Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria. , 2003, Biochemistry.

[35]  R. Millikan,et al.  The bile acid-activated phosphatidylinositol 3-kinase pathway inhibits Fas apoptosis upstream of bid in rodent hepatocytes. , 2001, Gastroenterology.

[36]  M. Müller,et al.  Different pathways of canalicular secretion of sulfated and non-sulfated fluorescent bile acids: a study in isolated hepatocyte couplets and TR- rats. , 1999, Journal of hepatology.

[37]  D. Keppler,et al.  Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C–dependent mechanisms in cholestatic rat liver , 2001, Hepatology.

[38]  P. Dent,et al.  Inhibition of the MAPK and PI3K pathways enhances UDCA‐induced apoptosis in primary rodent hepatocytes , 2002, Hepatology.

[39]  P. Meier,et al.  Cholestatic expression pattern of sinusoidal and canalicular organic anion transport systems in primary cultured rat hepatocytes , 2001, Hepatology.

[40]  D. Häussinger,et al.  Stable expression and functional characterization of a Na+-taurocholate cotransporting green fluorescent protein in human hepatoblastoma HepG2 cells , 2000, Cytotechnology.

[41]  G. Gores,et al.  Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. , 1999, The Journal of clinical investigation.

[42]  G. Gores,et al.  The Bile Acid Glycochenodeoxycholate Induces TRAIL-Receptor 2/DR5 Expression and Apoptosis* , 2001, The Journal of Biological Chemistry.

[43]  E. Dickson,et al.  Combined analysis of randomized controlled trials of ursodeoxycholic acid in primary biliary cirrhosis. , 1997, Gastroenterology.

[44]  D. Brenner,et al.  NFkappaB prevents apoptosis and liver dysfunction during liver regeneration. , 1998, The Journal of clinical investigation.