1. Volume 122, Issue 5, Pages 1411-1427 (May 2002)
Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N- terminal kinase–dependent manner 
Dirk Graf, Anna Kordelia Kurz, Richard Fischer, Roland Reinehr, Dieter Häussinger 
Gastroenterology 
Volume 122, Issue 5, Pages (May 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
TLCS-induced PARP cleavage is prevented by TUDC. Rat hepatocytes were kept in primary culture for 24 hours and subsequently exposed to TLCS, GCDC, and TUDC. Western blot analysis was performed by using an antibody against PARP (see Materials and Methods). Representative results, from a series of 3–4 independent experiments, are shown. (A) Time course of TLCS-induced PARP cleavage. Rat hepatocytes were incubated with TLCS (100 μmol/L) for the indicated time periods. (B) Concentration dependence of TLCS-induced PARP cleavage. Rat hepatocytes were treated with the indicated concentrations of TLCS for 6 hours. (C) Concentration dependence of the protective TUDC effect. TUDC was added together with TLCS (100 μmol/L) or GCDC (100 μmol/L) at the concentrations indicated for 6 hours. (D) Time dependence of the protective TUDC effect. Hepatocytes were exposed to TLCS (100 μmol/L) for 6 hours and TUDC (100 μmol/L) was further added 0.5, 1, 2, or 3 hours after TLCS addition. TLCS + TUDC refers to simultaneous addition of both bile acids. (E) Both 24- and 48-hour cultured rat hepatocytes were incubated with TLCS and/or TUDC. TLCS and TUDC were coincubated in equimolar concentrations (100 μmol/L) for 6 hours.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
TLCS-induced loss of symmetry of cell membrane phospholipids and DNA fragmentation is prevented by TUDC. For determination of the loss of symmetry of cell membrane phospholipids as an apoptotic index, FITC-conjugated Annexin-V was used. PI was used as a marker for necrotic cells (A-C, upper lane). For comparison, phase-contrast images are also given (A-C, lower lane). The 24-hour cultured rat hepatocytes were (A) incubated for 24 hours with TLCS (100 μmol/L), (B) or were left untreated and stained with Annexin-V–FITC and propidium iodide. Most TLCS-treated cells were fluoresceine-positive and only few cells showed signs of necrosis measured by nuclear PI staining (red signal in the upper lane). (C) Coincubation of TLCS (100 μmol/L) with TUDC (100 μmol/L) for 24 hours resulted in a significant reduction of TLCS-induced apoptosis as measured by Annexin-V staining. DNA single-strand breaks were determined with the TUNEL reaction. Apoptotic cell damage was indicated by positive nick end-labeled nuclear chromatin. Before the labeling reaction, cells were incubated with 0.5 μg/mL TRITC-conjugated phalloidin for detection of the cell shape (red staining). The 24-hour cultured rat hepatocytes were (D) incubated for another 24 hours with TLCS (100 μmol/L), (E) or were left untreated. (F) TUDC significantly reduced TLCS-induced DNA fragmentation. The percentage of apoptotic cells was determined with TUNEL reaction. *Significant reduction of TLCS-induced apoptosis by TUDC. Data are given as means ± SEM (n = 3).
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Modulation of TLCS-induced caspase 3, 8, and 9 activation by TUDC. The 24-hour cultured rat hepatocytes were exposed to TLCS (100 μmol/L) for 6 hours and the relative activities of caspases 3, 8, and 9, as well as the active subunits by means of Western blot were measured. Relative activity was related to corresponding controls from untreated cells, which was set to 1. Data are given as means ± SEM for 3–4 different experiments; *P < 0.05 vs. TLCS addition alone. (A) TUDC blocked the TLCS-induced activation of caspases 3, 8, and 9. TUDC (100 μmol/L) was coincubated with TLCS (100 μmol/L) for 6 hours. (B) Inhibition of either caspase 8 or 9 blocks TLCS-induced caspase 3 activation. The caspase 8 inhibitor Z-IETD-FMK (20 μmol/L) and the caspase 9 inhibitor Z-LEHD-FMK (20 μmol/L) were incubated 30 minutes before treatment with TLCS (100 μmol/L) for 6 hours. (C) Inhibitors of caspase 8 or 9 block TLCS-induced PARP cleavage. TLCS (100 μmol/L) was added for 6 hours in the absence or presence of Z-IETD-FMK (20 μmol/L) or Z-LEHD-FMK (20 μmol/L). Western blot analysis was performed by using an antibody against PARP. Representative results from a series of 3–4 independent experiments are shown. (D) Inhibition of TLCS-induced caspase 9 activation by the caspase 8 inhibitor Z-IETD-FMK (20 μmol/L).
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5. Fig. 4
Effects of TLCS on the activities of (A) MAP-kinases and (B) PI 3-kinase, and (C) the effect of different bile acids on PARP cleavage and JNK activation in primary rat hepatocytes. Hepatocytes were kept in primary culture for 24 hours and subsequently exposed to TLCS (A and B, 100 μmol/L) for the indicated time periods or (C) to different bile acids (TLCS, GCDC, TCDC, TC, TUDC) at a concentration of 100 μmol/L each for 6 hours. Thereafter, the supernatants of the lysed cultures were subjected to SDS-PAGE and analyzed by immunoblotting with a phosphorylation-specific antibody against p38MAPK or an antibody against ERK-1/2 and PARP. The antigen-antibody complexes were visualized by using enhanced chemiluminescence detection. JNK-1, p38MAPK, and PI 3-kinase activation were measured by immune complex assays. GST-Jun was used as substrate for immunoprecipitated JNK-1, myelin basic protein was used as substrate for the immunoprecipitated p38MAPK, and phosphatidylinositol was used as substrate for PI 3-kinase assay (see Materials and Methods). Representative results from a series of 3–4 independent experiments are shown. TLCS induces sustained activation of (A) Erk-2, p38MAPK, and JNK-1 and (B) a transient activation of PI 3-kinase. (C) Only bile acids that activate JNK-1 (TLCS and GCDC) induce PARP cleavage.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Effects of TLCS and TUDC on phosphorylation of PKB at serine 473. Rat hepatocytes were cultured for 24 hours and exposed to TLCS or TUDC for the time periods indicated. The supernatants of the lysed cultures were subjected to SDS-PAGE and analyzed by immunoblotting with a phosphorylation-specific antibody against serine 473 of PKB. Representative results from a series of 3–4 independent experiments are shown. (A) Concentration dependence of PKB phosphorylation. Rat hepatocytes were treated with the indicated concentrations of TLCS or TUDC for 10 minutes. (B) Time course of PKB phosphorylation. Rat hepatocytes were exposed to TLCS (100 μmol/L) or TUDC (100 μmol/L) for the indicated time periods. To exclude differences in protein amount the same supernatants were analyzed by immunoblotting with an antibody against total PKBα. (C) Time course of PKB phosphorylation by TUDC and TLCS. Relative density was related to corresponding controls from untreated cells, which was set to 1. ●, TUDC; ▴, TLCS. (D) Sensitivity of TUDC- and TLCS-induced PKB phosphorylation to PI 3-kinase inhibition. Cells were preincubated with LY (50 μmol/L) for 30 minutes or were left untreated and subsequently exposed to TUDC or TLCS (100 μmol/L each) for 10 minutes.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Effects of inhibitors of signal transduction on TLCS-induced apoptosis and JNK-1 activation and the protective effect of TUDC. The 24-hour cultured rat hepatocytes were treated with TUDC (100 μmol/L), TLCS (100 μmol/L), or were coincubated with these bile acids in equimolar concentrations for 6 hours. Representative results from a series of 3–4 independent experiments are shown. (A) Western blot analysis was performed by using an antibody against PARP. For determination of the loss of symmetry of cell membrane phospholipids as an apoptotic index, FITC-conjugated Annexin-V was used (see Figure 2 legend). Data are given as means ± SEM for 3–4 different experiments. If present, cells were preincubated with PD098059, an inhibitor of MEK1 (10 μmol/L) or SB203580, a p38MAPK inhibitor (10 μmol/L), for 30 minutes. (B) Western blot analysis was performed by using an antibody against PARP. When present, LY294002, a PI 3-kinase inhibitor (50 μmol/L), was preincubated for 30 minutes. (C) Activity of caspase 3 was related to corresponding controls from untreated cells, which was set to 1. Data are given as means ± SEM for 3–4 different experiments. Insensitivity of the protective TUDC effect on TLCS induced caspase 3 activation toward PI 3-kinase inhibition by LY (50 μmol/L).
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Effects of TLCS, TUDC, and geldanamycin on RIP, JNK-1 activation, and PARP cleavage. The 24-hour cultured rat hepatocytes were treated with TUDC (100 μmol/L), TLCS (100 μmol/L), or were coincubated with these bile acids in equimolar concentrations for 6 hours. If present, cells were preincubated with geldanamycin (1 μg/mL) for 12 hours before bile acid addition. Representative results from a series of 3–4 independent experiments are shown. Western blot analysis was performed by using an antibody against PARP and against RIP. JNK-1 activation was measured by immune complex assay. Down-regulation of RIP has no effect on TLCS-induced JNK activation and PARP cleavage and the protective TUDC effect.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 8
Modulation of TLCS-induced dephosphorylation of PKB and JNK-1 activation by TUDC, caspase inhibitors, and Gö6850. The 24-hour cultured rat hepatocytes were treated with TLCS (100 μmol/L), TUDC (100 μmol/L), or coincubated with both bile acids in equimolar concentrations for 6 hours. (A) TUDC abolishes the TLCS-induced JNK activation and PKB dephosphorylation. Analysis of phosphorylation of PKB at serine 473 and JNK-1 activity was performed as described in Figures 4 and 5 legends. (B) Inhibition of PI-3 kinase has no effect on TLCS-induced JNK activation and its abolition by TUDC. Cells were preincubated with LY (50 μmol/L for 30 minutes). (C) Caspase inhibition by Z-IETD-FMK (20 μmol/L for 30 minutes) or Z-LEHD-FMK (20 μmol/L for 30 minutes) does not prevent TLCS-induced JNK activation. (D) PKC inhibition by Gö6850 (10 μmol/L) has no effect on TLCS-induced JNK activation.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

10. Fig. 9
Time course of TLCS-induced JNK-1 activation, caspase 8 activation, and PARP cleavage. The 24-hour cultured rat hepatocytes were exposed to TLCS (100 μmol/L) for the time periods indicated and the relative activities of JNK-1 (●), caspase 8 (■), and PARP (▴) cleavage were measured. Relative activities were related to corresponding controls from untreated cells and set to 1. Data are given as means ± SEM for 3–4 different experiments. TLCS-induced JNK activation is persistent and precedes caspase 8 activation and PARP cleavage.
Gastroenterology  , DOI: ( /gast )
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11. Fig. 10
TLCS- and GCDC-induced CD95 surface trafficking and clustering and its prevention by TUDC. For experimental details see Materials and Methods. CD95 staining of (A) permeabilized and (B) unpermeabilized control cells. (C) CD95 ligand (100 ng/mL), (D) TLCS (100 μmol/L), or (E) GCDC (100 μmol/L) lead within 3 hours to CD95 surface trafficking/clustering in unpermeabilized cells. (F) TUDC (100 μmol/L), (G) the JNK inhibitor L-JNK-1 (5 μmol/L), or (H) the PKC inhibitor Gö6850 (10 μmol/L) prevent the TLCS (100 μmol/L)-induced CD95 surface trafficking/clustering in unpermeabilized cells. Images were obtained after 3 hours of exposure to the respective bile acids or CD95 ligand. TUDC was added together with TLCS. For statistical analysis see Table 1.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

12. Fig. 11
Effects of JNK and PKC inhibition on TLCS-induced PARP cleavage and Annexin-V staining. The 24-hour cultured rat hepatocytes were treated with TLCS (100 μmol/L) for 6 hours. When indicated, cells were preincubated for 30 minutes with L-JNK-1 (5 μmol/L), an inhibitor of JNK-1, or Gö6850, a PKC-inhibitor (10 μmol/L). Both inhibitors blunt the TLCS-induced apoptosis as assessed by (A) PARP cleavage and (B) Annexin-V staining. (A) Relative density was related to control data from untreated cells. Data are given as means ± SEM for 3–4 different experiments. For experimental details see Materials and Methods. *P < 0.05 vs. TLCS addition alone.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions