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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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.)

Cholestatic liver diseases are characterized by the accumulation of toxic bile acids, for example, glycochenodeoxycholic acid (GCDCA), causing damage to hepatocytes and cholangiocytes. Ursodeoxycholic acid (UDCA) is used as a treatment for patients with chronic cholestatic liver diseases. In primary biliary cirrhosis, doses of 13 to 15 mg/kg daily of UDCA decrease serum liver enzymes, improve liver histological test results, and delay the time to liver transplantation or death for up to 4 years.1, 2 However, the mechanisms of the beneficial effect of UDCA in these conditions remain unclear. From in vitro and in vivo studies, it is postulated that UDCA protects cholangiocytes against membrane damage induced by hydrophobic bile acids.3, 4 Furthermore, UDCA stimulates biliary secretion of bile acids and other toxic compounds.5, 6 In addition, anti-apoptotic effects of UDCA have been described, such as the inhibition of the mitochondrial membrane permeability transition in hepatocytes,7, 8 leading to prevention of mitochondrial release of cytochrome c.9 A recent study suggested that TUDCA does inhibit apoptosis by preventing the binding of Bax to mitochondria.10

Hepatocytes are exposed to many proapoptotic compounds. Therefore, antiapoptotic signaling pathways are important to limit programmed cell death. The antiapoptotic action of UDCA in part may be the result of activation of these anti-apoptotic pathways. A major survival pathway in hepatocytes is the activation of the transcription factor nuclear factor (NF)-κB. Activation of NF-κB-regulated survival genes causes inhibition of apoptosis.11, 12 Although we have shown that NF-κB is not activated by bile acids,13 its role in the protection of UDCA against bile acid-induced apoptosis is not clear. Furthermore, other cell survival pathways, like the activation of mitogen-activated protein kinases (MAPK), could be involved. These kinases are comprised of at least three different pathways: extracellular signal-regulated protein kinase (ERK), p38, and c-Jun amino-terminal kinase.14 Although the inhibition of ERK enhances UDCA-induced apoptosis,15 little is known about the role of MAPKs in the antiapoptotic actions of taurine-conjugated UDCA (TUDCA). Another survival pathway is the phosphatidylinositol-3 kinase (PI3K) pathway. This kinase cascade results in activation of a number of cellular intermediates, of which A serine threonine protein kinase (Akt) is one of the most important survival factors.16 The mechanisms by which PI3K promotes cell survival are diverse, and its role in the protection of UDCA against GCDCA-induced apoptosis has not been explored yet. In rats, TUDCA predominates compared with GUDCA.17, 18 Furthermore, TUDCA may be of benefit for patients with primary biliary cirrhosis.19 Therefore, we investigated the anti-apoptotic actions of TUDCA in primary rat hepatocytes.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animals.

Specified pathogen-free male Wistar rats (220–250 g) were purchased from Harlan (Zeist, The Netherlands). They were housed under standard laboratory conditions with free access to standard laboratory chow and water. Each experiment was performed following the guidelines of the local committee for care and use of laboratory animals.

Hepatocyte Isolation.

Hepatocytes were isolated and cultured as described previously.11, 20 Isolated hepatocytes were cultured in William's medium E (Life Technologies Ltd., Breda, The Netherlands) supplemented with 50 μg/mL gentamicin (BioWhittaker, Verviers, Belgium) without the addition of hormones or growth factors. During the attachment period (4 hours) 50 nmol/L dexamethasone (Sigma, St Louis, MO) and 5 % fetal calf serum (Life Technologies Ltd.) were added to the medium.

Experimental Design.

Experiments were started 24 hours after isolation. Hepatocytes were exposed to 50 μmol/L GCDCA (Calbiochem, La Jolla, CA), 50 μmol/L TUDCA (Calbiochem), 50 μmol/L glyco-ursodeoxycholic acid (GUDCA; Calbiochem), or 50 μmol/L TCDCA (Calbiochem) for 4 hours or the indicated period. In some experiments, hepatocytes were exposed to 20 ng/mL recombinant mouse tumor necrosis factor α (R&D Systems, Abingdon, United Kingdom) or a mixture of cytokines as described previously.11 Signal transduction or apoptosis pathways specifically were inhibited with the following compounds: 50 μmol/L of caspase-8 inhibitor Ac-IETD-CHO or the caspase-3 inhibitor Ac-DEVD-CHO (Biomol, Plymouth Meeting, PA), 10 μmol/L of the p38 inhibitor SB 203580 (Biomol), 10 μmol/L of the ERK1/2 inhibitor U0 126 (Promega, Madison, WI), 50 μmol/L PI3K inhibitor LY 294002 (Sigma-Aldrich), and 200 ng/mL of the transcriptional inhibitor actinomycin-D (Roche Diagnostics, Almere, The Netherlands). All inhibitors were added 30 minutes before bile acids or cytokines. Hepatocytes received adenovirus (multiplicity of infection of 10) 15 hours before exposure of bile acids. Each experimental condition was performed in triplicate wells. Each experiment was performed at least three times, using hepatocytes from different isolations.

Cells were harvested at the indicated times after the addition of bile acids as described before.11, 13

HepG2 Cell Experiments.

The human hepatoma cell line HepG2 and a stable derivative expressing rNtcp were cultured as described before.21, 22 Cells were incubated with indicated amounts of GCDCA or 1 μg/mL anti-Fas antibody (clone no. 7C11; Immunotech, Marseille, France) for 4 hours followed by harvesting in hypotonic cell lysis buffer.11

Adenoviral Constructs.

Adenoviral constructs have been described previously.23

Caspase-3 and Caspase-8 Enzyme Activity Assay.

Hepatocytes were scraped and cell lysates were obtained by three cycles of freezing (−80°C) and thawing (37°C) followed by centrifugation for 5 minutes at 13,000g. Caspase-3 and caspase-8 enzyme activity were assayed as described before.11, 13

Nuclear Staining.

Morphological features of apoptotic nuclei were demonstrated with acridine orange. Cells were seeded on glass coverslides and treated as indicated. These coverslides were fixed in methanol for 5 minutes, air-dried and rinsed twice in phosphate-buffered saline before incubating in acridine orange (1:1000) for 15 minutes in the dark. Fluorescent nuclei were visualized using a Leica confocal laser-scanning microscope.

Immunocytochemistry and Uptake of Fluorescent Bile Acids.

Analysis of active caspase-9 was performed on hepatocytes cultured on coverslips and exposed to 50 μmol/L bile acids for 4 hours. Coverslips were washed in phosphate-buffered saline and were fixed in 4% paraformaldehyde for 10 minutes, followed by incubation in 1% Triton-X100 for 5 minutes. Antibody against active caspase-9 was used at a dilution of 1:50 for 30 to 60 minutes. Fluorexein isothiocyanate–conjugated goat-anti-rabbit immunoglobulin (Molecular Probes, Eugene, OR) was added at a dilution of 1:600 for 45 minutes.

Uptake of bile acids was demonstrated using the fluorescent bile acid cholyl lysyl fluorescein (CLF).24 Cells were incubated with 2 μmol/L CLF at 37°C for 15 minutes. All slides were evaluated on a Leica confocal laser-scanning microscope.

Western Blot Analysis.

Western blot analysis of cell lysates was performed using polyclonal rabbit antibodies against cleaved caspase-9 and phosphorylated p38 MAPK, and monoclonal antibody against phosphorylated ERK1/2 (p44/42) MAPK (Cell Signaling Technology, Beverly, MA) at a dilution of 1:1000. Hepatocytes exposed to 50 μmol/L deoxycholic acid served as positive control for phophorylated ERK1/2.25 In addition, activated neutrophil extracts (kindly provided by Dr. Gwenny Fuhler, Department of Haematology, University Hospital Groningen) were used to confirm detection of phosphorylated ERK1/2 MAPK. For the detection of caspase-9 and phospho-p38, SARPO was used as a secondary antibody at a dilution of 1:2000. Phospho-ERK1/2 was detected with RAMPO (1:2000). Each lane contained the lysate of 150,000 cells. Equal loading was demonstrated by Ponceau-S staining. After Western blot analysis of phosphorylated p38 and ERK1/2 MAPKs, blots were stripped using 0.1% sodium dodecyl sulfate at 65°C for 30 minutes and incubated with 1:1000 antibody against total p38 MAPK or total ERK1/2 MAPK (Santa Cruz Biotechnology, Santa Cruz, CA). Western blot analysis for inducible nitric oxide synthase was performed as described before.26

Statistical Analysis.

Results are presented as the mean of at least three independent experiments ± standard deviation. A Mann-Whitney test was used to determine the significance of differences between two experimental groups. A P value of less than .05 (P < .05) was considered to be statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Ntcp Is Required for GCDCA-Induced Mitochondria-Controlled Apoptosis.

To investigate the protective mechanisms of TUDCA against GCDCA-induced apoptosis, we first determined whether GCDCA needs to be taken up by cells to induce apoptosis. Previous studies have postulated death receptor activation by toxic bile acids.27–29 However, a strong distinction between the role of the bile acid uptake transporter Ntcp and death receptor-mediated apoptosis has not been made yet. Recently, we have shown GCDCA-induced apoptosis in a mitochondrial-controlled manner in primary rat hepatocytes, which was FADD (Fas-associated protein with death domain) independent.13 In the present study, we examined whether Ntcp is required for GCDCA-induced apoptosis. For this purpose, we exposed Ntcp-negative and Ntcp-positive HepG2 cells21 to different concentrations of GCDCA. Both cell lines do express Fas death receptor. Anti-Fas antibody served as positive control. As shown in Fig. 1A, only HepG2 cells expressing Ntcp on their cell membrane are sensitive to GCDCA-induced apoptosis, which increased with increasing amounts of GCDCA. In contrast, anti-Fas antibody induced apoptosis in both Ntcp-positive and Ntcp-negative cell lines to the same extent. These data indicate that GCDCA first needs to be taken up by Ntcp before the onset of apoptosis and that ligand-dependent death receptor activation is not involved.

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Figure 1. Ntcp is required for glycochenodeoxycholic acid (GCDCA)-induced apoptosis. (A) Ntcp-positive and -negative HepG2 cells were treated with different amounts of GCDCA, as indicated in the figure, and 1 μg/μL anti-Fas antibody (positive control) for 4 hours. Only in Ntcp-positive HepG2 cells did caspase-3 activity increase with increasing amounts of GCDCA. *P < .05 for GCDCA 100 μM and anti-Fas antibody versus control. (B) Caspase-3 activity in primary rat hepatocytes exposed to 50 μmol/L GCDCA or 20 ng/mL tumor necrosis factor α in the presence of 200 ng/mL actinomycin-D (ActD) at 24 hours and 72 hours after isolation. (C) Uptake of bile acids demonstrated by fluorescence microscopy. Primary hepatocytes were exposed to 2 μmol/L of the fluorescent bile acid cholyl lysyl fluorescein (CLF) for 15 minutes. Accumulation was observed in the bile canaliculus between adjacent hepatocytes at 24 hours but not 72 hours after isolation. (Original magnification, ×400.)

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To exclude HepG2-specific artifacts, primary hepatocytes were exposed to GCDCA at 24 hours and 72 hours after isolation. Ntcp was expressed at high levels 24 hours after isolation (data not shown), but decreased in hepatocytes maintained in primary cultures for 72 hours, thereby confirming previous data.30 In addition, uptake of bile acids in these hepatocytes decreased in time, as shown with the fluorescent bile acid CLF (Fig. 1C). Accumulation of CLF was detected only in bile canaliculi of hepatocytes cultured for 24 hours, as previously reported.24 At this time point, GCDCA strongly induced caspase-3 activity. In contrast, GCDCA did not induce caspase-3 activity anymore in hepatocytes cultured for 72 hours (Fig. 1B). Induction of caspase-3 activity by tumor necrosis factor α in the presence of actinomycin-D remained unaffected. All together, these data provide evidence that Ntcp is required for GCDCA-induced apoptosis in primary hepatocytes.

TUDCA Inhibits But Does Not Delay GCDCA-Induced Caspase-3 Activity and Apoptotic Nuclear Morphological Features.

GCDCA-induced caspase-3 activity in hepatocytes peaks at approximately 4 hours.13 Therefore, at this point, we investigated the effect of TUDCA on GCDCA-induced caspase-3 activity in primary hepatocytes. TUDCA itself did not induce caspase-3 activation (Fig. 2A), confirming previous data.13 TUDCA, but not TCDCA and GUDCA, inhibited GCDCA-induced caspase-3 activity for 70%, as shown in Fig. 1A. A concentration-dependent curve displayed that the minimal concentration exerting the maximal protective effect is 50 μM of TUDCA (Fig. 2B). To demonstrate that TUDCA inhibits but does not delay GCDCA-induced caspase-3 activity, a time-course study was performed. Two to 15 hours after addition of GCDCA plus TUDCA, caspase-3 activity was inhibited significantly at all times (Fig. 2C). Nuclei staining was performed with acridine orange, confirming that GCDCA-induced activation of caspase-3 activity results in apoptosis. Nuclear fragmentation and condensation were observed 4 hours after the addition of GCDCA, which increased after 8 hours and persisted up to 15 hours. After 8 hours, 30% to 40% of hepatocytes displayed apoptotic nuclei, which were inhibited in the presence of TUDCA to 5% (supplemental material; Fig. 2D). Cytokine-exposed hepatocytes in which transcription was blocked with actinomycin-D served as positive control.

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Figure 2. Tauroursodeoxycholic acid (TUDCA), but not taurochenodeoxycholic acid (TCDCA) and glycoursodeoxycholic acid (GUDCA), inhibits glycochenodeoxycholic acid (GCDCA)-induced caspase-3 activity and nuclear fragmentation. (A) Primary rat hepatocytes were stimulated for 4 hours with 50 μmol/L GCDCA, 50 μmol/L TUDCA, 50 μmol/L TCDCA, 50 μmol/L GUDCA, or a combination thereof. Caspase-3 activity is presented as percentage of GCDCA alone. Data represent mean of at least three independent experiments with n = 3 per condition. *P < .05 for GCDCA plus TUDCA versus GCDCA alone. (B) Caspase-3 activity. Primary rat hepatocytes were stimulated for 4 hours with 50 μmol/L GCDCA and different amounts of TUDCA, as indicated in the figure. *P < .05 for GCDCA plus 50, 25, and 10 μM TUDCA versus GCDCA alone. (C) Time-course study. TUDCA (50 μmol/L) significantly inhibits (50 μmol/L) GCDCA-induced caspase-3 activity at all indicated time points. Representative data of three independent experiments are shown. (D) Nuclear morphological features in hepatocytes as determined by acridine orange staining. Cells were treated for 8 hours as indicated in the figure. Treatment with 50 μmol/L GCDCA induced nuclear condensation and fragmentation, which persisted for at least 15 hours and was blocked with 50 μmol/L TUDCA. Hepatocytes treated with cytokinemix (CM) plus 200 ng/mL actinomycin-D (ActD) for 15 hours served as positive control. See Materials and Methods for details. (Original magnification, ×1000.)

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The Protective Action of TUDCA Depends on the Inhibition of Caspase-9 Activation.

Because we previously noted that GCDCA induces apoptosis in a mitochondria-controlled manner,13 the effects of TUDCA on caspase-9 and caspase-8 were investigated. As shown in Fig. 3, TUDCA also prevented GCDCA-induced activation of caspase-9. Both Western blot (Fig. 3A) and immunocytochemistry (Fig. 3B) demonstrated this effect. Although TUDCA inhibited GCDCA-induced caspase-8 activity for 50% (Fig. 4B), we previously showed that caspase-8 inhibition does not inhibit GCDCA-induced caspase-3 activity.13 In contrast, the caspase-3 inhibitor Ac-DEVD-CHO and the caspase-9 inhibitor Ac-LEHD-CHO inhibited GCDCA-induced activation of both caspase-3 (Fig. 4A) and caspase-8 (Fig. 4B). These data demonstrate that inhibition of caspase-9 is more important in the TUDCA-mediated protection against GCDCA-induced apoptosis than caspase-8 inhibition. In contrast to TUDCA, TCDCA and GUDCA did not inhibit GCDCA-induced caspase-9 activity (Fig. 3) and caspase-8 activity (Fig. 4B).

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Figure 3. Tauroursodeoxycholic acid (TUDCA), but not taurochenodeoxycholic acid (TCDCA) or glycoursodeoxycholic acid (GUDCA), prevents glycochenodeoxycholic acid (GCDCA)-induced activation of caspase-9. Primary rat hepatocytes were exposed for 4 hours to 50 μmol/L of GCDCA, 50 μmol/L of TUDCA, or both plus TCDCA or GUDCA. (A) Western blot analysis on cell lysates for active caspase-9. (B) Immunocytochemistry for active caspase-9 (original magnification, ×400). AFU, arbitrary fluorescent units.

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Figure 4. Caspase-3 and caspase-8 activation in glycochenodeoxycholic acid (GCDCA)-exposed primary rat hepatocytes in the presence of bile acids and caspase inhibitors. Representative data of three independent experiments are shown with n = 3 per condition. (A) Caspase-3 activity. Cells were treated for 4 hours with 50 μmol/L of GCDCA plus 50 μmol/L caspase-8 inhibitor, 50 μmol/L caspase-9 inhibitor, or 50 μmol/L caspase-3 inhibitor. *P < .05 for GCDCA plus caspase-9 inhibitor and GCDCA plus caspase-3 inhibitor versus GCDCA. (B) Caspase-8 activity. Cells were treated for 4 hours with 50 μmol/L of GCDCA plus 50 μmol/L tauroursodeoxycholic acid (TUDCA), 50 μmol/L taurochenodeoxycholic acid (TCDCA), or 50 μmol/L glycoursodeoxycholic acid (GUDCA), 50 μmol/L caspase-8 inhibitor, 50 μmol/L caspase-9 inhibitor, or 50 μmol/L caspase-3 inhibitor. *P < .05 for GCDCA plus TUDCA versus GCDCA. AFU, arbitrary fluorescent units.

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TUDCA Does Not Compete with GCDCA for Uptake at the Cell Membrane.

Next, we investigated whether TUDCA needs to be present at the same time with GCDCA, before GCDCA or after the addition of GCDCA, to exert its protective effect. For this purpose, we preincubated hepatocytes with TUDCA for 9, 6, and 3 hours and washed these cells and exposed them to GCDCA in fresh medium for 4 hours. Six and 3 hours of preincubation with TUDCA significantly inhibited GCDCA-induced caspase-3 activity for 50% (Fig. 5A). Preincubation with TUDCA for 3 hours also inhibited GCDCA-induced caspase-9 activation (supplemental material; Fig. 5B). Furthermore, exposure to TUDCA for up to 90 minutes after the addition of GCDCA still exerted protection against GCDCA-induced apoptosis (Fig. 5C). At this point, GCDCA-induced apoptosis had not reached its maximum yet (Fig. 5D), confirming previous results.13 These data indicate that the protective effect of TUDCA is not the result of competition with GCDCA for uptake at the cell membrane. In addition, these data imply that the antiapoptotic actions of TUDCA are induced very rapidly, because simultaneous addition or addition of TUDCA after GCDCA is still able to prevent apoptosis in these cells. This could mean that signaling cascades are activated by TUDCA.

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Figure 5. Tauroursodeoxycholic acid (TUDCA) does not compete with glycochenodeoxycholic acid (GCDCA) for uptake at the cell membrane. (A) Preincubation with TUDCA inhibits GCDCA-induced apoptosis. Primary rat hepatocytes were exposed to 50 μmol/L of TUDCA for 9 (−9 hr), 6 (−6 hr), and 3 (−3 hr) hours, after which cells were washed and exposed to 50 μmol/L of GCDCA for 4 hours. Cells also were incubated for 4 hours with GCDCA alone or with simultaneous addition of 50 μmol/L TUDCA (simultaneous). Caspase-3 activity is presented as percentage of GCDCA alone. *P < .05 for −6 hours, −3 hours and simultaneous incubation of TUDCA plus GCDCA versus GCDCA alone (control). (B) Western blot analysis for active caspase-9 on cell lysates of hepatocytes preincubated for 3 hours with 50 μmol/L TUDCA followed by addition of 50 μmol/L GCDCA in refreshed medium for 4 hours. (C) Caspase-3 activity. Primary rat hepatocytes were incubated with 50 μmol/L of GCDCA for 4 hours (only GCDCA). Simultaneously (0–4 hr) or half an hour (0.5–4 hr), 1 hour (1–4 hr), 2 hours (2–4 hr), or 3 hours (3–4 hr) after the addition of GCDCA, 50 μmol/L TUDCA was added. All cells were harvested 4 hours after the addition of GCDCA. Representative data of three independent experiments are shown with n = 3 per condition. *P < .05 for 0–4 hr of TUDCA, 0.5–4 hr of TUDCA, 1–4 hr of TUDCA versus only GCDCA. (D) Time-course study (50 μmol/L) of GCDCA-induced caspase-3 activity. Representative data of three independent experiments are shown with n = 3 per condition. AFU, arbitrary fluorescent units.

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NF-κB Is Not Involved in the Protection of TUDCA Against GCDCA-Induced Apoptosis.

Previous results demonstrated that activation of NF-κB resulted in the transcription of survival genes protecting hepatocytes against apoptosis.11, 12 However, TUDCA does not activate NF-κB and does not induce the expression of NF-κB-regulated antiapoptotic genes.13 Because NF-κB can be activated indirectly, we investigated the role of NF-κB activation in relation to the antiapoptotic mechanisms of TUDCA. Therefore, primary hepatocytes were infected with recombinant adenovirus expressing dominant-negative IκB preventing NF-κB activation. Functionality of this virus was demonstrated by electrophoretic mobility shift assay and by sensitizing hepatocytes to cytokine-induced apoptosis (data not shown). As presented in Fig. 6, inhibition of NF-κB does not significantly reduce the protective effect of TUDCA against GCDCA-induced caspase-3 activity.

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Figure 6. NF-κB is not involved in the protection of tauroursodeoxycholic acid (TUDCA) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis. Primary rat hepatocytes were exposed for 4 hours to 50 μmol/L of GCDCA, TUDCA, or both, 15 hours after receiving 10 plaque-forming units/cell of recombinant adenovirus inhibiting nuclear factor-κB activation (Ad5IκBAA). Ad5LacZ served as the control virus. Protection of TUDCA against GCDCA-induced caspase-3 activity did not change significantly. Caspase-3 activity is presented as percentage of GCDCA alone. Data represent mean of at least three independent experiments with n = 3 per condition.

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Antiapoptotic Action of TUDCA Depends on the Activation of p38 MAPK, ERK MAPK, and PI3K, Whereas Gene Transcription Is Not Involved.

To investigate whether PI3K and MAPK pathways are involved in the antiapoptotic effects of TUDCA, specific inhibitors of PI3K (LY 294002), p38 MAPK (SB 203580), and ERK1/2 MAPK (U0 126) were used. Dimethyl sulfoxide was used as a solvent for inhibitors but did not have an effect itself (data not shown). Experiments with inhibitors in control hepatocytes and in hepatocytes exposed to TUDCA or GCDCA were included as well. Hepatocytes exposed to inhibitors of MAPK pathways in the absence of bile acids demonstrated caspase-3 values around control level (Fig. 7A). Caspase-3 activity increased slightly when PI3K was blocked in control hepatocytes, and this effect was enhanced in combination with MAPK inhibitors. This pattern was similar for hepatocytes exposed to inhibitors in the presence of TUDCA (Fig. 7A).

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Figure 7. Tauroursodeoxycholic acid (TUDCA) protects against glycochenodeoxycholic acid (GCDCA)-induced apoptosis by activation of the p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated protein kinase (ERK) 1/2 MAPK, and the phosphatidylinositol-3 kinase (PI3K) pathway. (A) Caspase-3 activity in primary rat hepatocytes treated as indicated in the figure with 50 μmol/L of GCDCA, 50 μmol/L TUDCA, or both, with or without inhibitors of ERK MAPK (10 μmol/L of U0 126; U0), p38 MAPK (10 μmol/L of SB 203580; SB), PI3K (50 μmol/L of LY 294002; LY), or a combination thereof. Caspase-3 activity is presented as a percentage of GCDCA alone. Data represent the mean of at least three independent experiments with n = 3 per condition. ▾P < .05 for control + LY, control + SB + LY, and for control + UO + LY versus control. ▴P < .05 for TUDCA + SB + LY versus TUDCA and for TUDCA + UO + LY versus TUDCA. ×P < .05 for GCDCA + LY versus GCDCA and for GCDCA + SB + LY versus GCDCA and for GCDCA + UO + LY versus GCDCA. ▪P < .05 for GCDCA + TUDCA versus GCDCA. #P < .05 for GCDCA + TUDCA + inhibitors versus GCDCA + TUDCA. (B) Western blot analysis for phosphorylated p38 MAPK in control lysates (con) and cell lysates of 50 μmol/L TUDCA-exposed hepatocytes (20 or 60 minutes or both) with or without 10 μmol/L of p38 MAPK inhibitor (SB). The same blots for total p38 MAPK are presented. (C) Western blot analysis for phosphorylated ERK1/2 MAPK in cell lysates of 50 μmol/L TUDCA-exposed hepatocytes (30 minutes) with and without 10 μmol/L of ERK1/2 inhibitor U0 126 and control lysates (con). Positive control was 50 μmol/L of deoxycholic acid (DCA) for ERK1/2 phosphorylation in hepatocytes, whereas an activated neutrophil extract was included for Western blot detection of ERK1/2 (pos. con). Upper panel presents same blot for total ERK1/2 MAPK.

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GCDCA-induced apoptosis was slightly, but not significantly, enhanced with inhibitors of p38 or ERK MAPKs (Fig. 7A). In contrast, inhibition of PI3K pathway aggravated GCDCA-induced caspase-3 activity significantly (Fig. 7A). In the presence of both MAPK and PI3K inhibitors, exposure to GCDCA significantly increased caspase-3 values compared with GCDCA alone.

The protective effect of TUDCA against GCDCA-induced apoptosis was partially, but significantly, abolished by inhibition of p38 MAPK pathway (Fig. 7A). In support, TUDCA activated p38, which was blocked by SB 203580, as shown in Fig. 7B. Western blot demonstrated equal presence of total p38 MAPK in all lanes (Fig. 7B). Blocking of the PI3K survival cascade resulted in abrogation of TUDCA protection against apoptosis as well (Fig. 7A). The protective effect of TUDCA against GCDCA-induced apoptosis was completely abolished when both p38 MAPK and PI3K pathways were blocked (Fig. 7B).

Next, the ERK1/2 MAPK was investigated. As shown in Fig. 7A, specific inhibition of ERK1/2 MAPK using U0 126 significantly prevented the protective effect of TUDCA against GCDCA-induced apoptosis. In addition, Western blot analysis demonstrated that TUDCA activates ERK1/2, which can be blocked with U0 126 (Fig. 7C). The bile acid deoxycholic acid was included as positive control for hepatocytes, which was confirmed with a neutrophil extract displaying a high level of phopho-ERK1/2. U0 126 also inhibited deoxycholic acid-mediated ERK1/2 phosphorylation (data not shown). Total ERK1/2 MAPK was present equally in all lanes (Fig. 7C). Inhibition of both ERK1/2 MAPK and PI3K abolished the protective effect of TUDCA against GCDCA-induced apoptosis completely (Fig. 7A).

Finally, the role of transcription in the protection of TUDCA against bile acid-induced apoptosis was studied. Inhibition of transcription using actinomycin-D, at a dose sensitizing hepatocytes to cytokine-induced apoptosis (supplemental material; Fig. 2D) had no influence on the protective effect of TUDCA (Fig. 8). Blocking of transcription was confirmed with Western blot for cytokine-induced transcription of inducible nitric oxide synthase (Fig. 8, inset).11

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Figure 8. Transcription-independent protection of tauroursodeoxycholic acid (TUDCA) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis. Caspase-3 activity in rat hepatocytes treated with 50 μmol/L GCDCA, 50 μmol/L TUDCA, or both with or without 200 ng/mL of transcription inhibitor actinomycin-D (ActD) for 4 hours. ActD did not significantly change the protective effect of TUDCA. Cytokine-exposed hepatocytes (CM) for 6 hours served as positive control for ActD. Inset: 200 ng/mL ActD blocks transcription. Western blot for cytokine (CM)-induced expression of inducible nitric oxide synthase (iNOS), 11 hours after exposure. See Materials and Methods for details. RLU, relative light units.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we investigated the protective mechanisms of TUDCA against GCDCA-induced apoptosis in primary rat hepatocytes. We previously showed that GCDCA induces apoptosis in a mitochondria-dependent manner, in which is not involved and caspase-8 activation is not initially required.13 In the present study, we investigated this further by blocking caspase-9. Indeed, peptide inhibitors of caspase-9 and caspase-3 blocked caspase-8 activation. Although these caspase inhibitors could have overlapping inhibitory effects, previous results with the human homolog of inhibitor of apoptosis protein 1 (HIAP1) demonstrated that overexpression of the human HIAP1 inhibited GCDCA-induced apoptosis. The human HIAP1 exclusively inhibits caspase-3 and caspase-9 activation, but not caspase-8.31 These data indicate that GCDCA induces apoptosis in a mitochondria-controlled manner.

In the present study, we demonstrated that TUDCA inhibits, but does not delay, GCDCA-induced caspase-9 and caspase-3 activity and the formation of apoptotic nuclei. Furthermore, we have shown that Ntcp is required for GCDCA-induced apoptosis in primary hepatocytes, indicating that ligand-dependent death receptor activation is not likely to occur. This report demonstrates that the antiapoptotic action of TUDCA against GCDCA-induced apoptosis is not the result of a direct competitive effect on the cell membrane. For example, TUDCA could compete with GCDCA for uptake by bile acid importer Ntcp, thus preventing uptake of proapoptotic GCDCA into hepatocytes. Preincubation for several hours with TUDCA followed by removal of TUDCA inhibits GCDCA-induced caspase-3 and caspase-9 activity, indicating the activation of survival pathways. Nine-hour preincubation with TUDCA does not protect cells against GCDCA, likely because of quenching of survival signals.

Overall, TUDCA seems to protect mitochondria from GCDCA-induced injury by preventing GCDCA-induced caspase-9 activation rather than by preventing death receptor-mediated apoptosis.

The inhibitory effect on GCDCA-induced apoptosis is exclusively exerted by TUDCA. Our results demonstrate that TCDCA does not inhibit GCDCA-induced caspase-3, caspase-9, and caspase-8 activity. This is in contrast to others showing inhibition of Fas-mediated caspase-8 activation by TCDCA-induced PI3-kinase.32 However, the regulation of cell survival and apoptosis in a hepatoma cell line and primary nontransformed hepatocytes differs, and this may explain the different results.

GUDCA could be clinically relevant because in humans, contrary to rats, GUDCA is much more abundant compared to TUDCA.17, 18 However, TUDCA has been reported to be beneficial in humans.19 Nevertheless, TUDCA abundance in rats could explain why GUDCA does not inhibit GCDCA-induced apoptosis in rat hepatocytes in vitro. Primary human hepatocytes should give more information about the protective effect of TCDCA and GCDCA against bile acid-induced apoptosis.

Our data demonstrate that the p38 and ERK MAPK and the PI3K pathway are involved in the protection of TUDCA against GCDCA-induced apoptosis. Because blocking of both PI3K and MAPK exaggerates caspase-3 activity compared with blocking a single pathway, these pathways are partly redundant. Moreover, inhibition of PI3K alone or in combination with MAPK inhibitors aggravates GCDCA-induced apoptosis and sensitizes hepatocytes slightly to TUDCA. Indeed, we have demonstrated that in addition to TUDCA (Fig. 7B), GCDCA activates p38 and ERK MAPKs (data not shown). These results are important for the interpretation of the protective effects of TUDCA. Blocking of protective kinases induces higher caspase-3 activity in GCDCA-exposed hepatocytes compared with GCDCA plus TUDCA-exposed hepatocytes, implying additional protective effects of TUDCA, for example, direct alterations of the mitochondrial membrane environment. Evidence for this was given recently by showing that TUDCA stabilizes the lipid and protein structure of mitochondrial outer membranes, thus inhibiting Bax binding to the outer membrane.10

Because simultaneous addition of TUDCA and GCDCA, or addition of TUDCA after GCDCA, blocks apoptosis in hepatocytes, TUDCA very rapidly exerts its protective effect. Indeed, no transcription or NF-κB activation is needed for protection, indicating that survival signaling involves posttranslational mechanisms exerted within 30 minutes, as shown by Western blot (Fig. 7B, C). TUDCA protection against GCDCA-induced apoptosis can be exerted only up to 1.5 hours after GCDCA addition. An explanation could be that in this time frame, GCDCA-induced apoptosis has not reached its maximum yet, whereas after 2 hours, apoptosis has proceeded beyond the point that TUDCA can be protective. Further evidence for rapid anti-apoptotic mechanisms of TUDCA is inferred from our previous results using cytokine-mediated protection against GCDCA. Cytokines do not protect against bile acid-induced apoptosis after the addition of GCDCA, because their protective mechanism depends on NF-κB-mediated transcription with a time frame of hours instead of minutes.13

Our PI3K data support other reports, although in these studies unconjugated UDCA was found apoptotic in itself.15 Activation of Akt protects against apoptosis via several mechanisms, including the phosphorylation of proapoptotic Bcl-2 family member Bad, which can no longer associate with and inhibit antiapoptotic Bcl-XL.33, 34 Akt activation also results in phosphorylation and inactivation of caspase-935 and may suppress proapoptotic Bax translocation to the mitochondria.36 It is reported that bile acids activate the ERK1/2 MAPK pathway via activation of the EGF receptor.15, 25, 37, 38 The exact mechanism is still unclear, but mitochondrial-derived reactive oxygen species may be involved, as recently suggested,37, 39 and TUDCA prevents the generation of ROS.10 The inhibition of Bax relocation to the mitochondria also could be mediated by ERK1/2 MAPK.40 Because TUDCA prevents Bax-induced membrane perturbation,10 TUDCA-activation of ERK1/2 MAPK and PI3K could be a mechanism to act on Bax. All together, our data fit very well with mitochondria-controlled bile acid-induced apoptosis in primary hepatocytes and provide more information about the link between TUDCA-activated survival pathways and mitochondria.

Interestingly, our data are in contrast to a recent study describing that the protection of TUDCA against taurolithocholic acid-3 sulfate–induced apoptosis is p38 MAPK, ERK1/2 MAPK, and PI3K independent and depends on inhibition of Fas trafficking and caspase-8 activation.41 The authors suggest that TUDCA does inhibit TLCS-induced apoptosis upstream of caspase-8 activation. However, they also suggest that TUDCA inhibits TLCS-triggered mitochondrial reactive oxygen species formation,41 which is needed for Fas trafficking.39 Therefore, TUDCA may inhibit TLCS-induced apoptosis in a mitochondria-controlled manner. Alternatively, GCDCA and TLCS may induce apoptosis via different mechanisms, which could explain the discrepancies between the protective mechanisms of TUDCA against these bile acids.

The activation of the p38 MAPK pathway by TUDCA to inhibit GCDCA-induced stress in rat hepatocytes is in line with the activation of p38 MAPKs during other forms of environmental stress.14 Several reports describe the involvement of these kinases in mRNA stabilization.42 Because we demonstrated that the protective effect of TUDCA is at a posttranscriptional level, this mechanism could be present in primary rat hepatocytes as well.

Although NF-κB is involved in protection against cytokine-induced stress in primary rat hepatocytes,11, 12 we did not find evidence that the NF-κB pathway is involved in the protective action of TUDCA against GCDCA-induced apoptosis. Bile acids do not activate NF-κB directly.13 Moreover, inhibition of the NF-κB pathway did not change the protective action of TUDCA.

In summary, we have shown that the antiapoptotic effect of TUDCA against GCDCA-induced apoptosis in primary rat hepatocytes is independent of caspase-8 inhibition, but is the result activation of p38, ERK MAPK, and PI3K survival pathways. Furthermore, we have demonstrated that TUDCA protects against GCDCA-induced mitochondrial injury in primary rat hepatocytes. Our data provide more information about the mechanism of action of UDCA in cholestatic liver diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors are grateful to Gwenny Fuhler (Hematology Department, University Hospital Groningen) for her generous gift of antibodies and her advice concerning Western blot analysis.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
suppmat_1563_fig2d.jpg18KNuclear morphology in hepatocytes as determined by acridine orange staining. Cells were treated for 8 hours as indicated in the figure. 50 µmol/L GCDCA induces nuclear condensation and fragmentation, which persists for at least 15 hours and is blocked with 50 µmol/L TUDCA. Hepatocytes treated with cytokinemix (CM) + 200 ng/mL actinomycin-D (ActD) for 15 hours served as positive controls. See Materials and Methods section for details. Orignial magnification is 1000x.
suppmat_1563_fig5b.tif49KWestern blot analysis for active caspase-9 on cell lysates of hepatocytes pre-incubated for 3 hours with 50 µmol/L TUDCA followed by addition of 50 µmol/L GCDCA in refreshed medium for 4 hours. (C) Caspase-3 activity. Primary rat hepatocytes were incubated with 50 µmol/L of GCDCA for 4 hours (only GCDCA). Simultaneously (0-4 hr) or half an hour (0.5-4 hr), 1 hour (1-4 hr), 2 hours (2-4 hr), or 3 hours (3-4 hr) after the addition of GCDCA, 50 µmol/L TUDCA was added. All cells were harvested 4 hours after the addition of GCDCA. Representative data of 3 independent experiments are shown with n = 3 per condition. *P < .05 for (0-4 hr) of TUDCA, (0.5-4 hr) of TUDCA, (1-4 hr) of TUDCA vs. only GCDCA. (D) Tim course study of (50 µmol/L) GCDCA-induced caspase-3 activity. Representative data of 3 independent experiments are shown with n = 3 per condition.

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