Bile acids inhibit interleukin-6 signaling via gp130 receptor-dependent and -independent pathways in rat liver

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Interleukin-6 (IL-6) is a major regulator of the acute phase reaction in the liver and is thought to mediate protective effects in response to hepatotoxins. In this study, the influence of bile acids on IL-6 signal transduction was analyzed. It was shown that hydrophobic bile acids such as glycochenodeoxycholate (GCDC) inhibited IL-6–induced tyrosine phosphorylation of signal transducer and activator of transcription (STAT) 3 in hepatocytes and in perfused rat liver. This inhibition was accompanied by GCDC-mediated downregulation of glycoprotein (gp) 130 expression, whereas gp130 and suppressor of cytokine signaling 3 messenger RNA and gp80 protein levels remained unaffected. The GCDC-induced downregulation of gp130 protein expression was insensitive to inhibition of proteasomal or lysosomal protein degradation but turned out to be sensitive to inhibition of caspase-3 or caspase-8 activity. Accordingly, treatment of cell extracts with active recombinant caspase-3 led to a decay of immunoreactive gp130. Moreover, activation of caspases by CD95 ligand or hyperosmotic stress also resulted in a downregulation of gp130 levels. This indicates that caspase activation antagonizes IL-6 signaling by decay of gp130 levels. However, caspase inhibition did not prevent GCDC-dependent inhibition of IL-6–induced STAT3 activation, which turned out to be at least partially sensitive to suppression of p38MAPK activation. In conclusion, hydrophobic bile acids compromise IL-6 signaling through both a caspase-mediated downregulation of gp130 and a p38MAPK-dependent inhibition of STAT3 phosphorylation. This may contribute to bile acid–induced hepatotoxicity in cholestasis through counteracting the known hepatoprotective effects of IL-6. (HEPATOLOGY 2006;44:1206–1217.)

Interleukin-6 (IL-6) is a pleiotropic cytokine with pro- and anti-inflammatory properties, and may exert beneficial effects on the liver.1 The IL-6 receptor complex consists of the ligand-binding subunit glycoprotein (gp) 80 and the signal-transducing subunit gp130, which is also involved in signal tranduction of other members of the IL-6–type cytokine family such as IL-11, oncostatin M, leukemia inhibitory factor, cardiotrophin-1, ciliary neurotrophic factor, the novel neurotrophin-1/B cell–stimulating factor 3, and the more recently discovered IL-12–type cytokine IL-27.1–3 Binding of IL-6 to gp80 receptors leads to the recruitment of the signal-transducing subunit gp130. This is the prerequisite for the activation of tyrosine kinases of the Janus kinase (Jak) family, constitutively associated to the cytoplasmic tail of gp130. Activation of Jak leads to phosphorylation of tyrosine residues within the cytoplasmic tail of gp130 and to phosphotyrosine/SH2 domain interaction-dependent recruitment of STAT factors. STATs in turn become phosphorylated on tyrosine705, dimerize, and translocate to the nucleus, where they bind to specific responsive elements within the 5′-regulatory region of their target genes, which are involved in control of the cell cycle, differentiation, apoptosis, proliferation, regeneration, and acute phase reaction (for reviews, see Barton,1 Heinrich et al.,2 and Pflanz et al.3).

IL-6 was shown to protect against liver injury by ethanol or CCl4 and has beneficial effects in ischemia/reperfusion models.4–6 Serum IL-6 levels are elevated in cholestatic liver diseases, and protective effects of this cytokine have been discussed previously.7–13 In cholestatic syndromes, bile acids accumulate and trigger hepatotoxicity (e.g., by inducing hepatocyte apoptosis). Apoptosis induction by hydrophobic bile acids occurs in vivo and in vitro and involves a ligand-independent CD95 activation with subsequent activation of caspase-8 and caspase-3.14–21 Some effects of hydrophobic bile acids are counteracted by tauroursodesoxycholate, which induces choleresis and can protect hepatocytes against bile acid–induced apoptosis.14, 22–24 Depending on their physical properties, bile acids affect a variety of signal transduction pathways involving mitogen-activated protein kinases, Src family kinases, protein kinase B, phosphatidylinositol-3 kinase, or protein kinase C isoforms and interfere with Ca2+ and cyclic adenosine monophosphate signaling.14, 16–18, 21, 22–27

This study showed that hydrophobic, proapoptotic bile acids inhibit IL-6 signaling at different levels. On the one hand, they induce downregulation of gp130 in a caspase-dependent manner; on the other hand, they inhibit IL-6–induced STAT3 phosphorylation via p38MAPK. This is of pathophysiological interest in view of the proposed hepatoprotective action of IL-6 in cholestatic liver disease and may contribute to bile acid–induced hepatotoxicity in cholestasis.

Abbreviations

IL-6, interleukin-6; GCDC, glycochenodeoxycholate; STAT, signal transducer and activator of transcription; gp, glycoprotein; Jak, Janus kinase; SOCS, suppressor of cytokine signaling; mRNA, messenger RNA; PCR, polymerase chain reaction; HPRT1, hypoxanthine phosphoribosyltransferase 1; TLCS, taurolithocholic acid-3 sulfate; TCDC, taurochenodeoxycholate; TC, taurocholate; TUDC, tauroursodeoxycholate; NTCP, natrium taurocholate cotransporting peptide; TNF-α, tumor necrosis factor α.

Materials and Methods

Materials.

Antibodies against gp80, gp130, caspase-3, STAT1, and STAT3 were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Antibodies against phosphospecific STAT3 (tyrosine705) or phosphospecific STAT1 (tyrosine701) were obtained from New England Biolabs (Beverly, MA). IL-6 was purchased from Biosource (Camarillo, CA). The caspase-3 inhibitor (Z-DEVD-FMK), caspase-8 inhibitor (Z-IETD-FMK), and pan-caspase inhibitor (Z-VED-FMK) were obtained from R+D (Wiesbaden, Germany). MG132 was purchased from Calbiochem-Novabiochem Gmbh (Bad Soden, Germany). SB220025 and bafilomycin A were obtained from Alexis Biochemicals (San Diego, CA). Recombinant active caspase-3 was obtained from Pharmingen (San Diego, CA).

Fetal bovine serum was obtained from Biochrom (Berlin, Germany). Tauroursodeoxycholate, taurocholate, taurochenodeoxycholate, taurolithocholate-3-sulfate, glycochenodeoxy-cholate, and William's E medium were obtained from Sigma-Aldrich Chemie GmbH (Munich, Germany). All other chemicals were obtained from Merck (Darmstadt, Germany).

Cell Culture and Isolation of Primary Rat Hepatocytes.

Transfected HepG2 cells stably expressing the rat sodium-taurocholate cotransporting peptide coupled to the enhanced green fluorescence protein were cultured as previously described28 in the presence of geniticin (400 mg/L). Isolated hepatocytes were prepared from livers of male Wistar rats, fed ad libitum with a standard diet, by way of a collagenase perfusion technique and were cultured as previously described.14

Cells (1.5 × 106) were plated on collagen-coated culture dishes and were cultured for 24 hours in 1.5 mL William's medium E (Sigma Chemical Co., Munich Germany) supplemented with 2 mmol/L glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 10−7 mol/L insulin, 10−7 mol/L dexamethasone, and 5% fetal calf serum. Two hours before starting the experiments, cells were washed again and incubated with 1.5 mL William's E medium supplemented with penicillin, streptomycin, and fetal calf serum in the same concentrations as mentioned above.

Liver Perfusions.

The studies were approved by the local ethics committee, and all procedures were performed in accordance with the Declaration of Helsinki and the Guiding Principles on the Care and Use of Animals. Livers of male Wistar rats (120-180 g), fed ad libitum with a standard diet, were perfused in situ in a non-recirculating system as previously described.23

Liver lobes from perfused liver were excised at the time points indicated and minced with an Ultra Turrax homogenizer (Janke & Kunkel, Staufer, Germany) at 0°C in lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4), 140 mmol/L NaCl, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 1% Triton X-100, 1 mmol/L EDTA, 1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mmol/L sodium vanadate, 20 mmol/L β-glycerophosphate, and protease inhibitor cocktail (Boehringer, Mannheim, Germany).

Western Blot Analysis.

SDS-PAGE and Western blot analysis were performed as previously described.14 Blots were incubated for 3 to 4 hours with the respective first antibodies (gp130, gp80, STAT3, STAT1, phosphospecific STAT3 [tyrosine705], and phosphospecific STAT1 [tyrosine701], caspase-3; dilution 1:2,000). Following washing with TBST and incubation with horseradish peroxidase–coupled anti-rabbit immunoglobulin G antibody (dilution 1:10,000) at room temperature for 2 hours, the blots were developed using enhanced chemiluminescent detection (Amersham Pharmacia Biotech, Uppsala, Sweden).

Real-Time Polymerase Chain Reaction.

The expression of gp130 and suppressor of cytokine signaling (SOCS) 3 messenger RNA (mRNA) was analyzed via real-time polymerase chain reaction (PCR). The oligonucleotides were synthesized from MWG Biotech (Ebersberg, Germany). Primers for gp130, SOCS3, and hypoxanthine phosphoribosyltransferase 1 (HPRT1) were designed as follows: rat gp130, forward 5′-CCGTCAGTGCAAGTGTTCTCA-3′, reverse 5′-CACTATCCACCAGCTGCAGGT-3 ′; rat SOCS3, forward 5′-CGAGAAGATCCCGCTGGTACT-3′, reverse 5′-GTAGCCACGTTGGAGGAGAGA-3′; rat HPRT1, forward 5′-TGCTCGAGATGTCATGAAGGA-3′, reverse 5′-CAGAGGGCCACAATGTGATG-3′.

Total RNA from hepatocytes was prepared with the RNeasy Mini Kit/ QIA Shredder (Qiagen, Hilden, Germany). The amount and quality of RNA was checked spectrometrically (absorption 280-320 nm) and via capillary electrophoresis (GeneQuant II, Amersham Pharmacia Biotech).

Reverse transcription and PCR were performed in a two-step reaction. Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen) with 1 μg total RNA as a template. This step includes a DNAse I digestion. The PCR was performed with the SYBR Green Universal PCR MasterMix (Qiagen). The reaction was performed with the 7500 Real-time PCR System (Applied Biosystems, Foster City, CA) in 96-well optical reaction plates (Applied Biosystems) capped with optical adhesive covers (Applied Biosystems).

Specificity of RT-PCR was controlled by no template and no reverse-transcriptase controls. Semiquantitative PCR results were obtained using the ΔΔCT method.29 Threshold values were normalized to HPRT1 and set in reference to unstimulated control cells.

In Vitro gp130 Cleavage.

The in vitro assay for cleavage of gp130 by active caspase-3 was performed as previously described for the epidermal growth factor receptor, with minor modifications.30 The reaction was performed in a medium containing 20 mmol/L HEPES (pH 7.4), 100 mmol/L KCl, 3 mmol/L NaCl, 1 mmol/L dithiothreitol, and 0.2 μmol/L CaCl2 and was started by addition of active caspase 3. Incubation was performed for 2 hours at 37°C. The reaction was stopped by adding 30 μL of SDS sample buffer and denaturation at 95°C for 5 minutes.

Statistical Analysis.

Results from different experiments are expressed as the mean ± SEM (n = number of independent experiments). Results were compared using the Student t test. A P value of less than .05 was considered statistically significant.

Results

Hydrophobic Bile Acids Inhibit IL-6–Induced STAT3-705Y Phosphorylation in Hepatocytes and NTCP-Transfected HepG2 Cells.

Stimulation of 24-hour cultured rat hepatocytes with IL-6 (10 ng/mL) for 20 minutes induces tyrosine phosphorylation of STAT3 at tyrosine705 (Fig. 1A). Preincubation with glycochenodeoxycholate (GCDC) inhibited STAT3-705Y phosphorylation time and concentration dependently, although STAT3 protein expression was not significantly affected (Figs. 1A-B). Significant inhibition of IL-6–induced STAT3-705Y phosphorylation was detectable after a preincubation period of at least 1 hour, and GCDC concentrations of 25 μmol/L were already sufficient to impair STAT3 activation (Fig. 1D). However, extension of the preincubation period with GCDC to 6 hours led to a decrease of STAT3 protein expression (Fig. 1C). Therefore, all further experiments were performed after a 3-hour preincubation with bile acids.

Figure 1.

Effects of GCDC on IL-6–induced STAT3 phosphorylation (Tyr705) in 24-hour cultured rat hepatocytes. (A) Rat hepatocytes were preincubated without (control) or with GCDC (100 μmol/L) for the indicated time points. When indicated, IL-6 (10 ng/mL) was added for another 20 minutes after the GCDC preincubation period. Total protein extracts were prepared thereafter. Sixty micrograms of protein were subjected to immunoblot analysis for STAT3-705Y phosphorylation or determination of total STAT3 protein levels using antibodies specific for STAT3 or for STAT3 phosphorylated at tyrosine705. Relative STAT3 phosphorylation (B) and total STAT3 protein levels after 3 hours (B) or 6 hours (C) of preincubation with GCDC (100 μmol/L) were determined via densitometry and related to the respective levels in unstimulated control cells, which were set to 1. Data are expressed as the mean ± SEM (n = 4). *P < .05. (D) Rat hepatocytes were preincubated with different concentrations of GCDC for 3 hours or were left untreated (control). Hepatocytes were then stimulated for 20 minutes with IL-6 (10 ng/mL) and total protein levels were analyzed for STAT3-705Y phosphorylation or total STAT3 as outlined above. GCDC, glycochenodeoxycholate; IL-6, interleukin-6; STAT3, signal transducer and activator of transcription 3; Tyr705, tyrosine705.

Apart from GCDC, other hydrophobic, proapoptotic bile acids such as taurolithocholyl-3-sulfate (TLCS) and taurochenodesoxycholate (TCDC) inhibited IL-6–induced STAT3-705Y phosphorylation, whereas the nonapoptotic bile acids taurocholate (TC) and tauroursodesoxycholate (TUDC) were ineffective (Fig. 2A-B).

Figure 2.

Effects of different bile acids on IL-6–induced STAT3 phosphorylation (Tyr705). Primary rat hepatocytes were prepared as described in Materials and Methods. Twenty-four hours after preparation, cells were incubated with TC, TUDC, GCDC, TLCS, and TCDC (100 μmol/L each) for 3 hours or were left untreated. Thereafter, cells were stimulated with IL-6 (10 ng/mL) for 20 minutes. (A) Total cell lysates were analyzed for STAT3-705Y phosphorylation and total STAT3 expression as described in Fig. 1. (B) Relative phosphorylation was measured via densitometry and was related to IL-6–stimulated control cells, which were set to 1. Data are expressed as the mean ± SEM (n = 4). *P < .05. (C) Rat hepatocytes were pretreated with 100 μmol/L GCDC for 3 hours before stimulation with IL-6, which was incubated for 20 minutes. STAT1-701Y phosphorylation and total STAT1 protein levels were determined via immunoblotting using specific antibodies as described in Materials and Methods. (D) HepG2 cells stably transfected with NTCP were pretreated with GCDC (200 μmol/L) for 6 hours and stimulated with IL-6 for 20 minutes. STAT3-705Y phosphorylation and total STAT3 expression was analyzed as described in Fig. 1. GCDC, glycochenodeoxycholate; IL-6, interleukin-6; TC, taurocholate; TLCS, taurolithocholic acid-3 sulfate; TCDC, taurochenodeoxycholate; TUDC, tauroursodeoxycholate; STAT, signal transducer and activator of transcription; Tyr705, tyrosine705.

In addition, the IL-6–induced activation of STAT1 phosphorylation at tyrosine701 was reduced by GCDC (Fig. 2C), indicating that inhibition of IL-6–induced STAT activation by GCDC may be a more common phenomenon.

In contrast to native HepG2 cells (i.e., without natrium taurocholate cotransporting peptide [NTCP] transfection), which were insensitive to the inhibitory effect of GCDC (data not shown), inhibition of IL-6–induced STAT3-705Y phosphorylation by GCDC was also found in HepG2 cells stably transfected with the natrium taurocholate cotransporting peptide (NTCP) (Fig. 2D). However, most likely because of differences in bile acid uptake, longer GCDC preincubation periods (6 hours) and higher GCDC concentrations (200 μmol/L) were required to achieve this effect in HepG2 cells (Fig. 2D) when compared with primary rat hepatocytes. Nevertheless, one may conclude from these data that the effect of GCDC on IL-6–induced Jak/STAT signaling requires the transporter-dependent uptake of GCDC into the cell.

Potential Mechanisms of GCDC-Induced Inhibition of IL-6–Induced STAT3 Phosphorylation.

Suppressors of cytokine signaling are known inhibitors of the Jak/STAT pathway, and their expression is predominantly regulated at the level of transcription (for review, see Krebs and Hilton31). As shown in Fig. 3A, IL-6 induces SOCS3 mRNA expression with maximal levels within 1 hour as assessed via quantitative real-time PCR. However, GCDC had no significant effect on SOCS3 mRNA levels in rat hepatocytes (Fig. 3A), indicating that SOCS3 does not account for the inhibitory effect of GCDC on IL-6–induced STAT3 phosphorylation.

Figure 3.

Mechanisms of bile acid–induced inhibition of STAT3 phosphorylation. (A) Primary rat hepatocytes were treated with IL-6 (10 ng/mL) or GCDC (100 μmol/L) for the time points indicated, and total mRNA was prepared. SOCS3 mRNA levels were analyzed via real-time PCR using primers specific for rat SOCS3 mRNA (see Materials and Methods). Rat HPRT1 was used as a housekeeping gene, and semiquantitative mRNA levels were calculated using the ΔΔCT method. Data are expressed in relation to the respective levels of the unstimulated control, which was set to 1. (B) Primary rat hepatocytes were incubated with GCDC (100 μmol/L) for 3 hours or were left untreated. Thereafter, cells were stimulated with IL-6 (10 ng/mL) for 20 minutes. As indicated, cells were preincubated with SB220225 (20 μmol/L) 30 minutes prior to bile acid stimulation. Total protein lysates were analyzed for STAT3-705Y phosphorylation, total STAT3, or gp130 protein expression via immunoblot analysis using specific antibodies. SB220025 restored IL-6–induced STAT3 phosphorylation (Tyr705) in the presence of GCDC, but was without effect on the GCDC-induced downregulation of gp130. SOCS, suppressor of cytokine signaling; mRNA, messenger RNA; GCDC, glycochenodeoxycholate; IL-6, interleukin-6; STAT3, signal transducer and activator of transcription 3; Tyr705, tyrosine705; gp130, glycoprotein 130. *P < .05.

Bile acids were shown to activate mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinases and p38MAPK.14, 22–23 Mitogen-activated protein kinases are also known regulators of IL-6 signaling.2, 32–33 Preincubation of rat hepatocytes with SB220025 (20 μmol/L), an inhibitor of p38MAPK, partially prevented the GCDC-induced inhibition of STAT3 phosphorylation by IL-6 in rat hepatocytes (Fig. 3B), whereas PD089059 (10 μmol/L), an inhibitor of MEK1, had no effect (data not shown). These findings suggest that bile acid–induced activation of p38MAPK, but not of extracellular signal-regulated kinase–type mitogen-activated protein kinases,14, 22, 23, 25 may participate in the inhibition of IL-6–induced STAT3 phosphorylation. Interestingly, SB220025 had no effect on GCDC-induced gp130 downregulation (Fig. 3B).

Downregulation of gp130 Protein by Hydrophobic Bile Acids in Rat Hepatocytes and NTCP-Transfected HepG2 Cells.

Glycoprotein 130 is the signal-tranducing subunit of the IL-6 receptor complex, and it is also involved in the signaling of other IL-6–type cytokines.2 As shown in Fig. 4, GCDC downregulates gp130 expression at the protein level in rat hepatocytes. Simultaneously with the course of inhibition of STAT3 activation, downregulation of gp130 starts after 1 hour of GCDC exposure and leads to an significant decay within 3 hours (Fig. 4A). Downregulation of gp130 was also induced by TLCS and TCDC, which represent two other known caspase-activating bile acids14–20 (Fig. 4B). On the other hand, TC and TUDC, which do not induce hepatocyte apoptosis,14 failed to downregulate gp130 expression (Fig. 4B). GCDC-induced downregulation of gp130 was also observed in NTCP-transfected HepG2 cells (Fig. 4C). However, GCDC had no influence on expression of the ligand-binding subunit gp80 in rat hepatocytes (Fig. 4D) and in NTCP-transfected HepG2 cells (data not shown).

Figure 4.

Effects of bile acids on gp130 expression in different liver cells and perfused rat liver. (A) Primary rat hepatocytes were preincubated without (control) or with GCDC (100 μmol/L) for the indicated time points. When indicated, IL-6 (10 ng/mL) was added for another 20 minutes after the GCDC preincubation period. (B) Rat hepatocytes were incubated with TCDC, TUDC, TC, TLCS, and GCDC (100 μmol/L each) for 3 hours or were left untreated. (C) HepG2 cells stably transfected for NTCP were treated with GCDC (200 μmol/L) for 6 hours. After the respective time points, cells were lysed and total protein extracts were prepared. Sixty micrograms of protein were subjected to immunoblot analysis for total gp130 levels using an antibody specifically raised against gp130. GAPDH expression was determined for loading control using an antibody specific for GAPDH. (D) Primary rat hepatocytes were exposed to GCDC (100 μmol/L) for 3 hours, and expression of gp80 was measured via immunoblot analysis using a specific antibody as described in Materials and Methods. (E) Isolated rat livers were perfused with GCDC (50 μmol/L) and liver lobes were taken at the time points indicated (t = 0 minutes immediately before addition of GCDC to the perfusate starts). Sixty micrograms of protein were subjected to immunoblot analysis for determination of total gp130 and GAPDH protein levels. (F) Primary rat hepatocytes were treated with GCDC (100 μmol/L) as indicated, and total mRNA was prepared. Glycoprotein 130 mRNA levels were analyzed via real-time PCR using primers specific for rat gp130 mRNA (see Materials and Methods). Rat HPRT1 was used as a housekeeping gene, and semiquantitative mRNA levels were calculated using the ΔΔCT method. Data are expressed in relation to the respective levels of the unstimulated control, which was set to 1. GCDC, glycochenodeoxycholate; IL-6, interleukin-6; TCDC, taurochenodeoxycholate; TUDC, tauroursodeoxycholate; TC, taurocholate; TLCS, taurolithocholic acid-3 sulfate; gp, glycoprotein.

In support of the data obtained from cell culture experiments, perfusion of rat livers with perfusion medium substituted with GCDC at a final concentration of 50 μmol/L leads to an almost complete disappearance of gp130 within 2 hours, which was not observed when GCDC-free perfusion media were used (Fig. 4E).

Whereas GCDC strongly lowered gp130 expression in rat hepatocytes, no significant effect of GCDC was detectable in gp130 mRNA levels (Fig. 4F). These findings suggest that posttranscriptional mechanisms may account for the downregulation of gp130 by GCDC.

Downregulation of gp130 by GCDC Does Not Involve Lysosomal or Proteasomal Pathways.

Glycoprotein 130 has a half-life of about 2.5 hours,34 and its degradation involves lysosomal proteolysis35, 36 and, under certain experimental conditions, proteasomal degradation.35 To assess the role of the lysosomal proteolysis for GCDC-induced gp130 downregulation, bafilomycin A (250 nmol/L), an inhibitor of lysosomal acidification, was used. A possible involvement of GCDC-induced proteasomal degradation of gp130 was investigated using MG132, a specific inhibitor of proteasomal degradation. As shown in Fig. 5A treatment of primary rat hepatocytes with bafilomycin A alone resulted in an increased expression of gp130 when compared with the untreated control; this probably reflects the inhibition of the basal gp130 turnover, which may normally involve lysosomal degradation. However, in the presence of bafilomycin, the GCDC-induced downregulation of gp130 in rat hepatocytes and NTCP-transfected HepG2 cells was largely preserved compared with respective controls (Fig. 5A). Similarly, treatment of primary rat hepatocytes with the inhibitor of proteasomal degradation MG132 did not affect the GCDC-induced downregulation of gp130. These findings suggest that the GCDC effect on gp130 protein levels is not explained by enhanced lysosomal or proteasomal degradation. Comparable results were obtained in NTCP-transfected HepG2 cells; however, in this experimental system, GCDC induced in the presence of MG132 the appearance of a band with lower molecular mass (≈115 kd), which was immunoreactive for the anti-gp130 antibody used for gp130 detection (Fig. 5B). This may suggest the appearance of an immunoreactive gp130 cleavage product in presence of GCDC, which accumulates upon inhibition of the proteasome. This is underlined in Fig. 5B.

Figure 5.

Effects of lysosomal or proteasomal inhibitors on GCDC-induced downregulation of gp130 expression. (A) Rat primary hepatocytes were stimulated with GCDC (100 μmol/L; incubation for 3 hours) or were untreated. When indicated, cells were preincubated with bafilomycin A (250 nmol/L) 30 minutes before bile acid stimulation. Furthermore, NTCP-transfected HepG2 cells were stimulated with GCDC (200 μmol/L) for 6 hours in the presence or absence of bafilomycin A (250 nmol/L), which was given 30 minutes before GCDC. Total protein extracts were prepared, and 60 μg of protein were analyzed via immunblotting for the expression of gp130 and GAPDH using specific antibodies. Relative gp130 expression was measured via densitometry and was related to unstimulated control cells, which were set to 1. Data are expressed as the mean ± SEM (n = 4). (B) Hepatocytes and NTCP-HepG2 cells were treated with GCDC as outlined above in presence or absence of MG132 (25 μmol/L), which was added to culture medium 30 minutes before bile acid stimulation. Total protein was analyzed for expression of gp130 and GAPDH via immunoblotting. Relative gp130 expression was measured in hepatocytes via densitometry and was related to unstimulated control cells, which were set to 1. Data are expressed as the mean ± SEM (n = 4). In NTCP-transfected HepG2 cells, GCDC induced in the presence of MG132 the appearance of a band with lower molecular mass (→), which is immunoreactive for the anti-gp130 antibody. This might be a truncated form of gp130. Because of this band, which we could not identify more specifically, we did not use densitometry for gp130 in NTCP-transfected HepG2 cells in the presence of MG132. GCDC, glycochenodeoxycholate; gp, glycoprotein; NTCP, natrium taurocholate cotransporting peptide.

GCDC-Induced gp130 Downregulation Is Caspase-Dependent.

Hydrophobic, proapoptotic bile acids such as GCDC, TLCS, and TCDC are well-known inducers of caspase activation in hepatocytes.14–21 In line with this, GCDC induced activation of caspase-3 within 1 hour in rat hepatocytes (Fig. 6A). Because this time period correlated well with the beginning of GCDC-induced gp130 downregulation, the possibility was addressed whether GCDC-induced downregulation of gp130 is mediated by a caspase-dependent pathway. In line with this hypothesis, the CD95 ligand, which activates caspases in rat hepatocytes,37 also strongly diminished the expression of gp130 in hepatocytes within 3 hours (Fig. 6B). Likewise, hyperosmotic hepatocyte exposure (405 mosmol/L), which also activates caspase-8 and caspase-3 but does not execute hepatocyte apoptosis,37 triggered gp130 downregulation in rat hepatocytes within 6 hours (Fig. 6C). GCDC-induced downregulation of gp130 was nearly completely blocked in rat hepatocytes after inhibition of caspase-3 by Z-DEVD-FMK (Fig. 6D) or of caspase-8 by Z-IETD-FMK (Fig. 6E). Likewise, the pan-caspase inhibitor Z-VAD-FMK prevented the GCDC-induced downregulation of gp130 in NTCP-transfected HepG2 cells (data not shown).

Figure 6.

Effect of caspases on IL-6–induced STAT3 phosphorylation and overall gp130 expression. Primary rat hepatocytes were cultured for 24 hours as described in Materials and Methods. (A) Cells were stimulated with GCDC (100 μmol/L) for the indicated time points. Sixty micrograms of total protein was analyzed via immunoblotting using an antibody specific for p17 caspase-3. (B) Primary rat hepatocytes were treated with CD95 ligand (100 ng/mL) for 3 hours or (C) cells were cultured under normo-osmotic or hyperosmotic conditions (405 mosmol/L) for 6 hours. Sixty micrograms of total protein was analyzed via immunoblotting for gp130 and GAPDH expression using specific antibodies. (D, E) Primary rat hepatocytes were stimulated with GCDC (100 μmol/L) for 3 hours; thereafter, incubation was continued in the presence or absence of IL-6 (10 ng/mL) for 20 minutes. As indicated, (D) cells were pretreated for 30 minutes with the caspase-3 inhibitor Z-DEVD-FMK (50 μmol/L) or (E) the caspase-8 inhibitor Z-IETD-FMK (50 μmol/L). Total protein was determined for gp130 protein expression, STAT3-705Y phosphorylation, and total STAT3 expression as described in Figs. 1 and 4. (F) Rat hepatocytes were incubated with GCDC, TUDC, or the combination of both bile acids (each 100 μmol/L) for 3 hours. IL-6 (10 ng/mL) was added as indicated and the incubation was continued for additional 20 minutes. Total protein was analyzed via immunoblotting as outlined above. (G) Total cell lysates prepared from rat hepatocytes (100 μg protein) was incubated with active recombinant caspase-3 in vitro as described in Materials and Methods. After a period of 2 hours at 37°C, reaction was stopped and the remaining gp130 and gp80 protein level was determined via immunoblot analysis of 60 μg of protein using the specific antibodies. GCDC, glycochenodeoxycholate; gp, glycoprotein; IL-6, interleukin-6; STAT3, signal transducer and activator of transcription 3; Tyr705, tyrosine705; TUDC, tauroursodeoxycholate.

TUDC, an antiapoptotic bile acid, was recently shown to prevent caspase-3 and -8 activation in response to hydrophobic bile acids.14 As shown in Fig. 6F, TUDC largely prevented the GCDC-induced decrease of gp130 protein expression.

Although caspase inhibitors and TUDC largely prevented GCDC-induced gp130 disappearance, these compounds were without effect on the GCDC-induced inhibition of STAT3-Y705 phosphorylation by IL-6 (Fig. 6D-F).

Further support for the view that activated caspases trigger the GCDC-induced downregulation of gp130 in rat hepatocytes came from in vitro experiments, which demonstrated that addition of recombinant active caspase-3 triggered gp130 disappearance in crude rat hepatocyte lysates (Fig. 6G). Glycoprotein 80 levels were not downregulated by recombinant active caspase-3 (Fig. 6G).

Discussion

This study showed that bile acids can inhibit IL-6–induced STAT3 tyrosine705 phosphorylation in rat hepatocytes and NTCP-transfected HepG2 cells. This effect is time-dependent, concentration-dependent, and bile acid–dependent in such a way that toxic and proapoptotic bile acids, such as GCDC or TLCS, but not TC or TUDC inhibited IL-6–mediated STAT3 activation. Furthermore, hydrophobic bile acids downregulate gp130 protein expression in primary rat hepatocytes, NTCP-transfected HepG2 cells, and perfused rat liver without affecting gp130 mRNA levels, suggesting that enhanced degradation of gp130 protein by hydrophobic bile acids is involved. Normally, gp130 is degraded via a lysosomal pathway, but evidence for a proteasomal degradation has also been presented.35, 36 However, pharmacological inhibition of either lysosomal or proteasomal proteolysis did not prevent the GCDC-induced decrease of gp130 protein expression. Evidence is provided that downregulation of gp130 protein levels by proapoptotic bile acids is due to caspase activation. This is supported by several observations: (1) apart from hydrophobic bile acids, other caspase-activating stimuli such as addition of CD95 ligand or hyperosmotic exposure induced gp130 downregulation; (2) GDCD-induced gp130 downregulation was sensitive to caspase-3 or -8 inhibitors and to the antiapoptotic bile acid TUDC, which was shown to prevent caspase activation in response to hydrophobic bile acids14; (3) recombinant active caspase-3 was able to induce gp130 degradation in vitro. Previous studies have indicated that specific proteins are cleavage targets for caspases, such as the receptors for epidermal growth factor or tumor necrosis factor α (TNF-α).30, 38, 39 This also holds for gp130, which is an essential signaling component for a variety of cytokines, suggesting that one role of caspases may be an induction of cellular refractoriness toward multiple cytokines irrespective of apoptosis induction. It should be kept in mind that caspase activation occurs in response to mild hyperosmotic stress without induction of apoptosis.37 In line with a role of caspases in triggering gp130 degradation is also the recent finding that in a multiple myeloma cell line, inhibition of the proteasome resulted in a pan-caspase inhibitor-sensitive decay of gp130 and inhibition of STAT3 activation.40

Caspases are cysteine proteases with specificity for an aspartic acid residue at position 1 of the substrate and the cleavage motif DXXD. The cytoplasmic tail of human and rat gp130 contains two overlapping DXXD motifs. Thus, it is conceivable that the decay of gp130 in response to apoptotic bile acids, CD95 ligand, or hyperosmolarity is due to caspase activation. Cleavage of the receptor at this site would lead to the removal of a 125–amino acid fragment from the C-terminus of gp130. Interestingly, cleavage of gp130 at these motifs would result in a truncated gp130 receptor lacking the three distal tyrosine motifs (Tyr814, Tyr905, Tyr915), which are important for STAT activation (for review, see Heinrich et al.2 and Pflanz et al.3)—whereas the remaining proximal tyrosine motifs are essential for the recruitment of the tyrosine phosphatase SHP2 and the SOCS3 to gp130. Thus, the resulting truncated receptor is expected to mediate still an activation of the MAPK cascade via SHP2 recruitment, whereas induction of STAT3-mediated signals in response to IL-6–type cytokines will no longer occur.

In addition to the downregulation of gp130 protein by hydrophobic bile acids, inhibition of IL-6–dependent STAT3 activation was observed. Although inhibition of STAT3 phosphorylation roughly paralleled the time course of gp130 disappearance, inhibition of gp130 disappearance by caspase inhibitors did not restore IL-6–mediated STAT3 activation in the presence of hydrophobic bile acids. This suggests that other mechanisms contribute to inhibition of the IL-6–mediated activation of STAT3. Downregulation of IL-6–induced STAT3 activation was at least partially restored by inhibition of p38MAPK, whereas gp130 protein levels remained unaffected. This indicates that a p38MAPK-dependent mechanism contributes to the bile acid–mediated inhibition of STAT3 activity, independent of the decay of gp130 levels.

The signaling events responsible for the p38 MAPK -sensitive downregulation of STAT3 activation by proapoptotic bile acids are not clear. Similar to bile acids, TNF-α, lipopolysaccharides, and IL-1β have been shown to suppress IL-6–mediated activation of STAT3 in a p38MAPK-dependent way.32, 33, 41, 42 Inhibition of STAT3 activation by TNF-α was shown to be accompanied by a p38 MAPK-dependent induction of SOCS3, a potent inhibitor of IL-6–mediated STAT3 activation. This suggests that TNF-α inhibits STAT3 activation at least to some extent through induction of SOCS3.32–33 However, we observed that hydrophobic bile acids did not induce significant SOCS3 mRNA, suggesting that SOCS3 induction may not explain the inhibition of STAT3 activation by bile acids. It remains unclear whether other recently proposed potential mechanisms for the inhibition of STAT3 activation, such as recruitment of SHP2 or postulated p38 MAPK-mediated posttranslational effects on gp13043 are involved.

It is well documented that STAT3 activation is a mediator of signals important for cellular proliferation and survival. STAT3 activates several genes, which are involved in the control of the cell cycle, such as cyclins D1, D2, and D3 and antiapoptosis such as c-myc, bcl-xl, or bcl.44, 45 Thus, induction of apoptosis by hydrophobic bile acids may not only involve the induction of signaling cascades toward CD95 activation,14–18 but also a downregulation of antiapoptotic signals through STAT3 inhibition.

IL-6 is thought to be essential for the homeostasis during inflammation because IL-6 deficient animals are more susceptible for hepatotoxic challenges such as endotoxin.5, 8, 11 However the role of IL-6 in liver regeneration is discussed controversialy. On the one hand IL-6 ist thought to be a positive inducer of hepatocyte proliferation. This assumption is supported by the observation that liver regeneration after partial hepatectomy is strongly impaired in IL-6 deficient animals.10 On the other hand it was shown that IL-6 induces protective signaling pathways after partial hepatectomy without significant influence on hepatocyte proliferation. IL-6 hyperstimulation even inhibited hepatocyte proliferation.46, 47 Compared with wild-type mice, IL-6−/− mice develop a more advanced stage of biliary fibrosis 12 weeks after bile duct ligation, which is associated with higher serum bilirubin levels and mortality.11 Moreover, mice with hepatocyte-specific gp130 gene knockout suffer from increased bacterial infections of the biliary tract and higher mortality during chronic cholestasis. These studies support the view that the IL-6/gp130/Jak/STAT cascade mediates hepatoprotective effects9, 12, 13 that are counteracted by TNF-α, ethanol, and, as suggested by our study, proapoptotic bile acids.

Cholestatic patients with cirrhosis have a decreased capacity for liver regeneration; in addition, obstructive jaundice is associated with enhanced susceptibility to toxins in human and animal models.48–50 Increased serum bile acid concentrations may at least in part be responsible for this through inhibition of IL-6–induced STAT3 signaling.

In summary, this study showed that apoptotic bile acids inhibit IL-6 signaling via gp130-dependent and -independent pathways. This may contribute to the hepatotoxic effects of bile acids during cholestatic syndromes by inhibiting the protective effects of IL-6 on liver regeneration or acute phase response.

Acknowledgements

The authors gratefully acknowledge Verena Fuchs for technical assistance.

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