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Abstract

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

Cholestatic liver injury is associated not only with accumulation of bile acids but also with activation of proinflammatory cytokines. Common bile duct ligation (CBDL) induces sustained downregulation of the Na+/taurocholate cotransporter (Ntcp) in rodent liver. Although repression of Ntcp during endotoxemia is cytokine mediated, it is unclear whether inflammatory cytokines contribute to this downregulation in obstructive cholestasis. Cytokine inactivation in CBDL rats and mice was either performed directly with tumor necrosis factor alpha (etanercept) or interleukin 1 beta inactivation (anakinra/AMG 719) or indirectly Kupffer cell depletion via intraperitoneal administration of liposome-encapsulated dichloromethylene bisphosphonate. Protein and messenger RNA (mRNA) expression of Ntcp and short heterodimer partner (SHP) were analyzed via Western and Northern blotting. Key regulators of Ntcp (hepatocyte nuclear factor 1 alpha [HNF-1α], HNF-4α, retinoid X receptor alpha [RXRα]:retinoic acid receptor alpha [RARα]) were studied via electrophoretic mobility shift analysis and nuclear Western blot analysis. Both methods of cytokine inactivation failed to maintain Ntcp protein or mRNA expression within 3 days after CBDL in either rats or mice (20%-40% of sham controls), while SHP mRNA expression increased three- to five-fold. Decreased nuclear HNF-1α and HNF-4α protein levels (45% and 60% of sham controls, respectively) and HNF-1α binding activity (32% of sham controls) were not restored during cytokine inactivation after CBDL, indicating cytokine-independent mechanisms of Ntcp regulation. RXRα:RARα binding remained unchanged in all experimental conditions. In conclusion, during obstructive cholestasis accumulating bile acids per se, without major contribution of cytokines, leads to downregulation of Ntcp via repression of HNF-1α and HNF-4α. (HEPATOLOGY 2005;41:470–477.)

The Na+/taurocholate cotransporter (Ntcp; SLC10A1 in humans, Slc10a1 in rodents) represents the main hepatocellular sodium-dependent uptake system for conjugated bile acids from sinusoidal blood in human and rodent liver.1-3 Downregulation of Ntcp under cholestatic conditions has been identified as a common mechanism of protecting the liver from continued uptake of potentially toxic bile acids.4 Accordingly, Ntcp expression is decreased in various human cholestatic liver diseases5-7 and rodent models of cholestasis.4, 8-12

Downregulation of Ntcp in cholestasis primarily occurs at the transcriptional level.4, 10, 13 Hepatocyte nuclear factor 1 alpha (HNF-1α) and the retinoid X receptor alpha (RXRα):retinoic acid receptor alpha (RARα) heterodimer complex have been identified as the major transactivators in the proximal region of rat Ntcp.10, 14 Decreased binding activity at these two regulatory elements in vivo occurs either via induction of inflammatory cytokines or retention of bile acids and leads to downregulation of Ntcp gene expression.10, 15, 16 HNF-1α activity is decreased by tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β)16 or by accumulating bile acids which in turn inhibit HNF-4α–mediated HNF-1α transactivation.17 Accumulating bile acids bind to farnesoid X receptor, a bile acid receptor, thereby inducing the expression of short heterodimer partner (SHP),18, 19 which inhibits HNF-4α–mediated transactivation of the HNF-1α promoter.17 As another pathway, SHP interferes with RXRα:RARα binding to the Ntcp promoter.15 Alternatively, inflammatory mediators such as IL-1β mediate suppression of RXRα:RARα transactivation of the Ntcp promoter by a c-jun-N-terminal kinase–dependent inhibition of RXRα phosphorylation.20

Obstructive cholestasis is a complex pathophysiological situation associated not only with accumulation of bile acids but also with activation of proinflammatory cytokines,21 both of which may mediate Ntcp repression. In addition, hydrophobic bile acids per se have been shown to induce proinflammatory cytokines in macrophages.22 However, the potential role of cytokines in mediating Ntcp repression in obstructive cholestasis is currently unknown.

Therefore, the contribution of proinflammatory cytokines to Ntcp repression was analyzed after common bile duct ligation (CBDL). Using a pharmacological knockout approach, we characterized the effects of TNF-α and IL-1β in the downregulation of nuclear receptors (SHP, RXRα:RARα), hepatocyte-enriched transcription factors (HNF-1α, HNF-4α), and Ntcp expression in both rats and mice. In addition, Kupffer cell depletion experiments employing liposomal bisphosphonate targeting have been performed to determine a potential role of other inflammatory mediators as effectors of a bile acid–independent regulation.

Materials and Methods

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

Animals.

Male Swiss albino mice (20-25 g, strain Him OF1 SPF; Institute of Laboratory Animal Research, Himberg, Austria) and male Sprague-Dawley rats (200-250 g; Janvier, Le Genest–St. Isle, France) were used. All animals were kept on a 12-hour light/dark cycle and had free access to water and standard mouse or rat diet. The experimental protocols were approved by the local Animal Care and Use Committee according to criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences, as published by the National Institutes of Health (NIH publication 86-23, revised 1985).

CBDL and Pharmacological Cytokine Inactivation.

All surgical procedures were performed under sterile conditions. Mice and rats (n = 3 to 4) were bile duct ligated as previously described.4, 23, 24 Briefly, the abdomen was opened through a midline incision. The bile duct was identified and isolated and double ligated in his proximal part. In addition, cholecystectomy was performed in mice after ligation of the cystic duct. Controls underwent a sham operation with exposure, but without ligation of the common bile duct and removal of the gallbladder. During the operation rats and mice received either 8 mg/kg body weight etanercept, a soluble p75 TNF receptor fusion protein,25 or 100 mg/kg body weight of a long acting formulation of anakinra (AMG 719, kindly provided by Amgen Inc., Thousand Oaks, CA), a specific IL-1 receptor antagonist.26 Etanercept was injected intraperitoneally once again 48 hours after surgery, and AMG719 was administered daily. The livers were harvested 1 (mice) or 3 (rats) days after CBDL under general anesthesia, immediately snap frozen, and stored in liquid nitrogen until RNA extraction or were freshly used for isolation of liver nuclei and isolation of nuclear proteins.

Kupffer Cell Depletion Via Administration of Liposome-Encapsulated Dichloromethylene Bisphosphonate.

Kupffer cells were depleted in Swiss albino mice (n = 7) via intraperitoneal injection of 4 mL/kg BW liposome-encapsulated dichloromethylene bisphosphonate (Cl2MBP) 48 hours before CBDL, and mice were sacrificed 24 hours after CBDL.27-29 CL2MBP was a gift from Roche Diagnostics (Mannheim, Germany); Cl2MBP liposomes were prepared as described previously.27 Controls received intraperitoneal treatment with vehicle (i.e., liposomes in phosphate-buffered saline). The duration of 24 hours for CBDL was chosen because depletion of Kupffer cells as well as systemic macrophages for a longer period would be expected to lead to pronounced bacterial translocation from the gut or to loss of regenerative mechanisms induced by cytokines.30 Kupffer cell elimination was confirmed by staining paraffin sections with an antibody recognizing the F4/80 antigen expressed by macrophages (Serotec, Oxford, UK; dilution 1:50). In brief, 3-μm paraffin sections were deparaffinated, rehydrated, and digested with 0.1% protease (type XXIV, Sigma) for 10 minutes. Endogenous peroxidase was blocked with 1% H2O2 in methanol for 10 minutes. Specific binding of the F4/80 antibody was detected using a biotinylated anti-rat immunoglobulin G (Dako, Glostrup, Denmark) and the ABC System (Dako) with β-amino-9-ethyl-carbazole as substrate.

RNA Preparation and mRNA Analysis.

RNA was isolated from mouse and rat livers by standard phenol chloroform extraction procedure. Total RNA (10-20 μg) was analyzed by Northern blotting with specific and constitutively expressed probes of mouse or rat Ntcp, mouse SHP and mouse or rat glyceraldehyde-3-phosphate dehydrogenase as previously described.23, 31 RNA was quantified spectrophotometrically at 260 nm and the quality of total RNA was controlled by denaturing formaldehyde agarose gel electrophoresis. Real-time polymerase chain reaction was performed on a Gene-Amp 5700 Sequence Detection System and with GeneAmp 5700 SDS software (Applied Biosystems, Vienna, Austria) to semiquantify mRNA levels of IL-1β and TNF-α after Kupffer cell depletion as described previously.32 The TaqMan oligonucleotides corresponding to published sequences were as follows:

  • IL-1β: forward primer, 536-560; reverse primer, 687-667; probe, 638-665, corresponding to the published sequence, GenBank accession number M15131;

  • TNF-α: forward primer, 408-432; reverse primer, 582-560; probe, 443-468, corresponding to the published sequence, GenBank accession number M13049.

  • mRNA data were normalized to those of 28S ribosomal RNA.32

Electrophoretic Mobility Shift Analysis.

To determine binding activity of liver-enriched transcription factors and nuclear hormone receptors, electrophoretic mobility shift assays were performed as previously described.31 Five to 10 micrograms of nuclear extracts (protein) were incubated on ice for 30 minutes with a 2 × 104 cpm [32P] end-labeled oligonucleotide probe representing the HNF-1α or DR-1 (direct repeat-1; binding site for RXRα:RARα) element of the proximal rat Ntcp promoter.10, 14 For competition assays, 100-fold molar excess of unlabeled oligonucleotides were coincubated with the labeled probe. For supershift experiments nuclear extracts were preincubated for 30 minutes on ice with 1 μg of a polyclonal antibody against HNF-4α (Santa Cruz Biotechnology, Santa Cruz, CA) before addition of the labeled oligonucleotide probe. Separation of protein–DNA complexes from unbound labeled probes was performed via electrophoresis using a nondenaturing 6% polyacrylamide gel and quantified via phosphorimaging (Biorad, Munich, Germany).

Western Blot Analysis.

Preparation of either liver microsomes or nuclear protein and analysis of rat Ntcp protein mass and protein mass of various transcription factors were performed as described previously.12, 32 Protein concentrations were determined according to Bradford33 and 75 μg of microsomal protein or 10 to 25 μg of nuclear protein extract was separated by SDS-PAGE as previously described.12, 32 After electrotransfer onto nitrocellulose membranes, the blots were incubated with anti-rat Ntcp fusion protein antiserum,34 monoclonal anti-Na+/K+-ATPase α-1 antibody (Upstate, Lake Placid, NY) or polyclonal antibodies against transcription factors HNF-1α, HNF-4α, RXRα, and RARα as well as histone (Santa Cruz Biotechnology). Immune complexes were detected according to the ECL kit (Amersham, Little Chalfont, England).

Statistical Analysis.

Three to four animals were studied in each group. Data are reported as the arithmetic mean ± SD. Differences between experimental groups were analyzed via ANOVA with Bonferroni posttesting using the SYSTAT statistic program (SYSTAT, Evanston, IL). A P value of less than .05 was considered significant.

Results

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

Effects of Cytokine Inactivation on Ntcp Expression in CBDL Rats.

To determine whether reduced Ntcp expression, which has been observed after bile duct ligation in rats,4 was prevented by inactivation of TNF-α or IL-1β, we quantified Ntcp steady-state mRNA levels via Northern blot analysis (Fig. 1) and protein expression via Western blot analysis (Fig. 2). As previously described,4 rat Ntcp mRNA and protein expression declined to 37% ± 10% and 20% ± 11% of controls, respectively (P < .05; n = 4) (Figs. 1, 2). Inactivation of TNF-α or IL-1β by pretreatment with etanercept or AMG719 had no significant effect on steady state mRNA levels of Ntcp, which remained below 40% of controls (Fig. 1). Similarly, inactivation of either cytokine did not significantly restore Ntcp protein expression (Fig. 2). In contrast, Mrp2 mRNA expression was preserved after cytokine inactivation as a positive control (data not shown). These data indicate that Ntcp downregulation after CBDL occurs via cytokine-independent pathways.

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Figure 1. Cytokine inactivation does not prevent Ntcp mRNA downregulation after CBDL in rats. Total RNA was isolated from CBDL-treated rats with and without anticytokine treatment 3 days after surgery and analyzed via Northern blotting using specific probes for Ntcp and glyceraldehyde-3-phosphate dehydrogenase as described in Materials and Methods. (A) Representative autoradiograph. (B) Densitometric analysis. Data represent the mean ± SD of 4 animals per group expressed as a percentage of sham-operated controls. CBDL leads to a repression of Ntcp mRNA that is not prevented by inactivation of TNF-α or IL-1β. *CBDL rats versus sham-operated controls (P < .05). Abbreviations: mRNA, messenger RNA; CO, controls; CBDL, common bile duct ligation; EN, etanercept; KIN, anakinra/AMG719; Ntcp, Na+/taurocholate cotransporter.

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Figure 2. Effects of cytokine inactivation on Ntcp protein expression in rats after CBDL. Microsomes were prepared via differential centrifugation from rat liver 3 days after CBDL with and without anticytokine treatment. Protein mass of Ntcp and Na+, K+-ATPase was determined via Western blotting as described in Materials and Methods. (A) Representative immunoblot. Molecular weight markers are given in kilodaltons. (B) Densitometric analysis. Data represent the mean ± SD of 4 animals per group expressed as a percentage of sham-operated controls. Inactivation of TNF-α or IL-1β fails to restore Ntcp protein mass 3 days after CBDL. Changes in Ntcp expression appear to be specific, because protein mass of the basolateral Na+, K+-ATPase remains unchanged in both treatment groups. *CBDL rats versus sham-operated controls (P < .05). Abbreviations: CO, controls; CBDL, common bile duct ligation; EN, etanercept; KIN, anakinra/AMG719; Ntcp, Na+/taurocholate cotransporter; NK, Na+, K+-ATPase.

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Binding Activity and Nuclear Protein of Transcription Factors After Cytokine Inactivation in CBDL Rats.

Electrophoretic mobility shift assay and nuclear Western blotting were performed to further elucidate the consequences of biological inactivation of TNF-α or IL-1β as potential regulators of transcription factors involved in Ntcp downregulation. Oligonucleotides representing HNF-1α and RXRα:RARα (DR-1) responsive elements from the proximal rat Ntcp promoter sequence10, 14 were used for gel shift analysis (Fig. 3A-B). Analysis of nuclear extracts isolated 72 hours after CBDL showed decreased binding activity of HNF-1α to 32% ± 14% (P < .05; n = 4), whereas binding to the DR-1 element was unaltered. Supershift assays excluded significant HNF-4α binding to the Ntcp DR-1 element (data not shown). To characterize the nature of decreased HNF-1 binding to its respective element, HNF-1α and HNF-4α nuclear protein was quantified via Western blotting using nuclear extracts isolated at the same time point (Fig. 3C). CBDL leads to a significant decrease in nuclear protein expression of both hepatocyte-enriched transcription factors to 45% ± 28% and 58% ± 5%, respectively (P < .05 each; n = 4). In parallel to HNF-1 binding activity in electrophoretic mobility shift assay, nuclear protein abundance of both transcription factors remained suppressed despite either anticytokine treatment (Fig. 3C). Therefore, HNF-1α and HNF-4α appear to be decreased after CBDL, primarily via cytokine-independent but bile acid–dependent pathways.

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Figure 3. DNA binding studies and nuclear protein levels in CBDL-treated rats with and without cytokine inactivation. Hepatic nuclear extracts were prepared from CBDL-treated rats with and without anticytokine treatment and sham-operated controls 3 days after either treatment. For electrophoretic mobility shift assays, nuclear extracts were incubated with radiolabeled oligonucleotides representing HNF-1– and RXRα:RARα (rNtcp DR-1)–binding sites, electrophoresed, and autoradiographed as described in Materials and Methods. For nuclear Western analysis, hepatic nuclear protein was separated by SDS-PAGE and probed to HNF-1α and HNF-4α antibodies. (A) Representative autoradiographs of electrophoretic mobility shift assays. (B) Densitometric analysis. (C) Representative immunoblot with molecular weight markers given in kilodaltons and densitometric analysis. Data represent the mean ± SD of 4 animals per group expressed as a percentage of sham-operated controls. CBDL leads to a repression of HNF-1 binding activity that remains unchanged despite inactivation of TNF-α or IL-1β. In contrast, binding to a rNtcp DR-1 element is unaltered in both treatment groups. Cytokine inactivation fails to restore HNF-1α and HNF-4α nuclear protein levels after CBDL. *CBDL rats versus sham-operated controls (P < .05). Abbreviations: CO, controls; CBDL, common bile duct ligation; EN, etanercept; KIN, anakinra/AMG719; SC, specific competition; NSC, nonspecific competition; HNF, hepatocyte nuclear factor.

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CBDL-Mediated SHP Induction and Ntcp Repression in Mice Is Not Mediated by Cytokines.

Previous studies have shown concomitant induction of SHP associated with reduction of Ntcp levels in CBDL-treated mice,23 but the relative contribution of proinflammatory cytokines to this SHP-dependent pathway regulating Ntcp repression is unknown. To determine the effects of cytokine inactivation on Ntcp regulation in CBDL, mRNA levels of SHP and Ntcp were studied in CBDL mice 24 hours after surgery (Fig. 4). After CBDL, Ntcp steady state mRNA levels were reduced to 14% ± 10% of sham-operated controls (P < .05). Concomitant with reduction of Ntcp levels, SHP mRNA was induced 1 day (or 24 hours) after CBDL (272% ± 92%). As observed in CBDL-treated rats, inactivation of TNF-α or IL-1β did not significantly alter Ntcp mRNA expression, and similar results were obtained even after combined blockade of both cytokines (Fig. 4). In contrast, SHP mRNA expression was further increased by anticytokine treatment compared with both sham-operated controls and CBDL mice (506% ± 34% of controls after etanercept, 316% ± 101% after AMG 719, and 507% ± 74% after combined blockade vs. 282% ± 58% in CBDL mice, respectively; P < .05 each; n = 3). These data exclude a causative role of TNF-α and IL-1β in the SHP-dependent downregulation of Ntcp in CBDL mice.

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Figure 4. Effects of cytokine inactivation on Ntcp and SHP mRNA expression in mice after common bile duct ligation (CBDL). Total RNA was isolated from CBDL mice with and without anticytokine treatment 1 day after surgery and analyzed by Northern blotting using specific probes for Ntcp and SHP as described in Materials and Methods. (A) Representative autoradiograph. (B) Densitometric analysis. Data represent the mean ± SD of 3 animals per group expressed as a percentage of sham-operated controls. CBDL leads to a repression of Ntcp mRNA that is paralleled by an induction of SHP mRNA. Inactivation of TNF-α, IL-1β, or both fails to restore Ntcp mRNA expression and even further promotes SHP induction. *CBDL mice versus sham-operated controls (P < .05). Abbreviations: CO, controls; CBDL, common bile duct ligation; EN, etanercept; KIN, anakinra/AMG719; Ntcp, Na+/taurocholate cotransporter; SHP, short heterodimer partner; mRNA, messenger RNA.

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Kupffer Cell Depletion Does Not Prevent Ntcp Repression in CBDL Mice.

To investigate the relative contribution of proinflammatory cytokines/mediators (other than TNF-α and IL-1β) in mediating Ntcp repression during CBDL, Kupffer cells, the main source of hepatic cytokines,35 were depleted via intraperitoneal injection of liposome-encapsulated CL2MBP before CBDL.27 Staining liver sections with red pulp F4/80 antibody revealed successful Kupffer cell depletion (Fig. 5). Three of 7 CBDL mice treated with CL2MBP developed portal vein thrombosis, and 1 mouse died, whereas no portal vein thrombosis or death occurred in vehicle (liposome)-injected animals. Kupffer cell depletion, however, neither restored Ntcp expression nor prevented induction of SHP 3 days after CBDL (Fig. 6). To investigate whether sustained Ntcp repression despite efficient Kupffer cell depletion was due to proinflammatory cytokines excreted by other cells (e.g., bile duct epithelial cells, endothelial cells),36 we compared hepatic TNF-α and IL-1β mRNA expression—both known to be involved in Ntcp regulation9, 16, 37—in Kupffer cell–depleted and naive CBDL animals. CL2MBP liposome administration before CBDL attenuated upregulation of TNF-α mRNA (229% ± 123% in Kupffer cell–depleted CBDL vs. 552% ± 64% in naive CBDL mice) (Fig. 6) and completely prevented IL-1β induction (47% ± 22% in Kupffer cell–depleted CBDL vs. 242% ± 29% in naive CBDL mice) (Fig. 7). The occurrence of portal vein thrombosis had no influence on Ntcp expression nor on TNF-α or IL1β mRNA levels. These results suggest that during CBDL effects of bile acid–induced SHP on Ntcp repression may dominate those of cytokines that were effectively suppressed by Kupffer cell depletion.

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Figure 5. Effect of liposome-encapsulated CL2MBP on Kupffer cells in CBDL mice. Microscopy of 3-μm sections of paraffin-embedded liver tissue 24 hours after CBDL in mice. (A) Kupffer cells (red) were stained with an antibody raised against the F4/80 antigen of macrophages. The asterisk marks a characteristic bile infarct occurring after CBDL. (B) Administration of liposomal clodronate 48 hours before CBDL results in loss of Kupffer cell staining. Original magnification ×20.

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Figure 6. Effects of liposome-encapsulated CL2MBP on Ntcp and SHP mRNA expression in CBDL mice. Total RNA was isolated 24 hours after surgery from sham-operated controls, CBDL, CL2MBP liposome-pretreated CBDL, and from vehicle (liposome)-injected CBDL mice and was analyzed via Northern blotting using specific probes for Ntcp and SHP as described in Materials and Methods. (A) Representative autoradiograph. (B) Densitometric analysis. Data represent the mean ± SD of 3 animals per group expressed as a percentage of sham-operated controls. Depletion of Kupffer cells by CL2MBP liposomes fails to restore Ntcp expression and does not prevent SHP induction after CBDL. Vehicle (liposome) injection has virtually no effect on Ntcp and SHP expression levels. *CBDL mice versus sham-operated controls (P < .05). Abbreviations: CO, controls; CBDL, common bile duct ligation; CL2MBP, clodronate liposomes; Lip/LIP, vehicle (liposomes); Ntcp, Na+/taurocholate cotransporter; SHP, short heterodimer partner; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA.

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Figure 7. Cytokine mRNA expression in CBDL mice treated with liposome-encapsulated CL2MBP. mRNA levels of (A) TNF-α and (B) IL-1β were determined using real-time polymerase chain reaction 24 hours after CBDL in sham-operated controls, CBDL, CL2MBP liposome-pretreated CBDL, and from vehicle (liposome)-injected CBDL mice as described in Materials and Methods. Kupffer cell depletion attenuates TNF-α induction and completely abolishes induction of IL-1β in response to CBDL. Vehicle (liposome) injection has virtually no effect on the expression levels of either cytokine. *CBDL mice versus sham-operated controls (P < .05). TNFα, tumor necrosis factor alpha; IL1β, interleukin 1 beta; mRNA, messenger RNA; CO, controls; CBDL, common bile duct ligation; CL2MBP, clodronate liposomes; LIP, vehicle (liposomes).

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Discussion

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

CBDL represents a model of obstructive cholestasis that is associated with both elevation of serum bile acid levels and induction of proinflammatory cytokines.23, 38 Rodent Ntcp gene expression is controlled by the hepatocyte-enriched transcription factors HNF-1α and HNF-4α as well as nuclear receptors, including RXRα:RARα and the transcriptional repressor SHP.10, 14, 15 Farnesoid X receptor–mediated SHP induction by bile acids has been shown to decrease RXRα:RARα binding to the Ntcp promoter and inhibits HNF-1α transactivation by HNF-4α.15, 17, 23, 39 In lipopolysaccharide-induced cholestasis, reduction of nuclear levels of HNF-1α and RXRα:RARα has previously been linked to Ntcp repression.10, 16, 20, 37 Increased levels of proinflammatory cytokines with concomitantly reduced binding activities of RXRα:RARα have also been suggested to downregulate Mrp2 (Abcc2) expression in livers and duodenums of CBDL rats.40, 41 However, the contribution of proinflammatory cytokines (including TNF-α and IL-1β) to Ntcp regulation during obstructive cholestasis has not been studied to date.

The current study yielded several important new findings: (1) down-regulation of Ntcp within 3 days of obstructive cholestasis does not involve TNF-α and IL-1β; (2) Kupffer cell depletion experiments without recovery of Ntcp expression after CBDL also exclude a role of other inflammatory mediators as effectors of bile acid–independent gene regulation; (3) decreased Ntcp mRNA expression upon CBDL is paralleled by a cytokine-independent reduction of both HNF-1α and HNF-4α; and (4) maintained RXRα:RARα binding despite reduced Ntcp expression supports a role for HNF-1α as the major gene regulator.

Selective inactivation of TNF-α and IL-1β without effects on Ntcp expression in the present study clearly demonstrates that downregulation of Ntcp in obstructive cholestasis is independent of these cytokines. Moreover, the lack of Ntcp restoration in Kupffer cell–depleted CBDL animals with attenuated (i.e., TNF-α) or even absent (i.e., IL-1β) cytokine induction further indicates that the effects of bile acids (e.g., via induced SHP) may dominate those of cytokines in mediating Ntcp repression in biliary obstruction. However, in contrast to CBDL, there may be a significant contribution of proinflammatory cytokines to Ntcp repression in rodent models of inflammation-induced cholestasis. In these models, Kupffer cell depletion and anticytokine strategies are able to prevent downregulation of Ntcp,16, 42 because inflammatory cytokines are higher in endotoxin-induced cholestasis while bile acid levels are much lower than in CBDL.

Elevated bile acid levels in CBDL lead to induction of SHP, which may subsequently reduce HNF-4α and HNF-1α transactivation.17, 43 However, this mechanism remains to be determined for β-muricholic acid, the major bile acid accumulating in rodents following CBDL.32, 44 HNF-1α and the RXRα:RARα heterodimer complex have been identified as the major transactivators of rat Ntcp.10, 14 In contrast, regulation of the mouse Ntcp promoter by HNF-1α and RXRα:RARα was recently questioned by Jung et al.,45 who did not identify respective response elements in a minimal mouse Ntcp promoter. However, the presence of specific binding elements upstream of the studied minimal promoter has to be postulated from other studies: A principal role of HNF-1α is suggested by findings in HNF-1α knockout mice that have demonstrated an almost absent baseline Ntcp mRNA expression in these animals.46 Furthermore, in extended studies on the full-length mouse Ntcp promoter gene, transactivation occurs by a more distal HNF-1α element which is also present in rat Ntcp (A. Geier et al., unpublished data, June 2004). The present study supports a role of HNF-1α as the predominant of these two Ntcp regulators, because gene expression in rats is remarkably reduced after CBDL even in the presence of maintained RXRα:RARα binding. However, RXRα:RARα may play a supplemental role in Ntcp regulation, which is in line with decreased binding activity and nuclear levels during endotoxin-induced cholestasis and prolonged (i.e., 7 days) bile duct obstruction.10, 40 During the first 3 days of CBDL, SHP mRNA expression correlates inversely with Ntcp expression, while after 7 days SHP mRNA levels approximate normal values while Ntcp remains repressed.23 This apparent discrepancy could be attributed to rising levels of proinflammatory cytokines such as TNF-α and IL-1β over time (G. Zollner et al., unpublished data, June 2004), because these cytokines are known to inhibit RXRα:RARα activity, which is decreased primarily during the later stages of biliary obstruction.20, 40 Moreover, bile acids have been shown to suppress HNF-4α transcription through SHP-independent mechanisms,17 which could explain the reduction of HNF-1α and subsequent Ntcp expression despite low SHP levels. We conclude that cytokine-independent effects are the dominant cause for Ntcp downregulation after CBDL. Such effects may involve bile acid–dependent suppression of the HNF-4α/HNF-1α pathway via SHP-dependent and independent mechanisms17 as a central pathway of Ntcp gene regulation.

In summary, taken together, our findings support a new model of distinct transcriptional pathways involved in the regulation of Ntcp expression during cholestasis depending on the type of cholestatic liver injury. In endotoxin-induced cholestasis, proinflammatory cytokines have been demonstrated to repress both HNF-4α/HNF-1α and RXRα:RARα pathways, resulting in downregulation of Ntcp.10, 16, 23, 47 Bile acids per se accumulating during the early stage of obstructive cholestasis without major contribution of cytokines may lead to Ntcp downregulation via cytokine-independent repression of HNF-4α/HNF-1α transactivation.

Acknowledgements

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

The excellent technical assistance of Sabine Beutelspacher, Andrea Fuchsbichler, Aline Müller, Petra Schmitz, Dagmar Silbert, and Sonja Strauch is gratefully acknowledged.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Hagenbuch B, Meier PJ. Molecular cloning, chromosomal localization, and functional characterization of a human liver Na+/bile acid cotransporter. J Clin Invest 1994; 93: 13261331.
  • 2
    Hagenbuch B, Stieger B, Foguet M, Lübbert H, Meier PJ. Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system. Proc Natl Acad Sci U S A 1991; 88: 1062910633.
  • 3
    Cattori V, Eckhardt U, Hagenbuch B. Molecular cloning and functional characterization of two alternatively spliced Ntcp isoforms from mouse liver. Biochim Biophys Acta 1999; 1445: 154159.
  • 4
    Gartung C, Ananthanarayanan M, Rahman MA, Schuele St, Nundy S, Soroka C, et al. Down-regulation of expression and function of the hepatic sodium-dependent bile acid cotransporter in extrahepatic cholestasis in the rat. Gastroenterology 1996; 110: 199209.
  • 5
    Shneider BL, Fox VL, Schwarz KB, Watson CL, Ananthanarayanan M, Thevananther S, et al. Hepatic basolateral sodium-dependent-bile acid transporter expression in two unusual cases of hypercholanemia and in extrahepatic biliary atresia. HEPATOLOGY 1997; 25: 11761183.
  • 6
    Zollner G, Fickert P, Zenz R, Fuchsbichler A, Stumptner C, Kenner L, et al. Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases. HEPATOLOGY 2001; 33: 633646.
  • 7
    Zollner G, Fickert P, Fuchsbichler A, Silbert D, Wagner M, Arbeiter S, et al. Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter regulation by cholic and ursodeoxycholic acid in mouse liver, kidney and intestine. J Hepatol 2003; 39: 480488.
  • 8
    Moseley RH, Wang W, Takeda H, Lown K, Shick L, Ananthanarayanan M, et al. Effect of endotoxin on bile acid transport in rat liver: a potential model for sepsis-associated cholestasis. Am J Physiol 1996; 271: G137G146.
  • 9
    Green RM, Beier D, Gollan JL. Regulation of hepatocyte bile salt transporters by endotoxin and inflammatory cytokines in rodents. Gastroenterology 1996; 111: 193198.
  • 10
    Trauner M, Arrese M, Lee H, Boyer JL, Karpen SJ. Endotoxin down-regulates rat hepatic ntcp gene expression by decreased activity of critical transcription factors. J Clin Invest 1998; 101: 20922100.
  • 11
    Simon FR, Fortune J, Iwahashi M, Gartung C, Wolkoff A, Sutherland E. Ethinyl estradiol-induced cholestasis involves alterations in expression of liver sinusoidal membrane transporters. Am J Physiol 1996; 34: G1043G1052.
  • 12
    Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Pojer C, Zenz R, et al. Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver. Gastroenterology 2001; 121: 170183.
  • 13
    Kim PK, Chen J, Andrejko KM, Deutschman CS. Intraabdominal sepsis down-regulates transcription of sodium taurocholate cotransporter and multidrug resistance-associated protein in rats. Shock 2000; 14: 176181.
  • 14
    Karpen SJ, Sun A-Q, Kudish B, Hagenbuch B, Meier PJ, Ananthanarayanan M, et al. Multiple factors regulate the rat liver basolateral sodium-dependent bile acid co-transporter gene promotor. J Biol Chem 1996; 271: 15211-15221.
  • 15
    Denson LA, Sturm E, Echevarria W, Zimmerman TL, Makishima M, Mangelsdorf DJ, et al. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology 2001; 121: 140147.
  • 16
    Geier A, Dietrich CG, Voigt S, Kim SK, Gerloff T, Kullak-Ublick GA, et al. Effects of proinflammatory cytokines on rat organic anion transporters during toxic liver injury and cholestasis. HEPATOLOGY 2003; 38: 345354.
  • 17
    Jung D, Kullak-Ublick GA. Hepatocyte nuclear factor 1 alpha: a key mediator of the effect of bile acids on gene expression. HEPATOLOGY 2003; 37: 622631.
  • 18
    Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, et al. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell 2000; 6: 507515.
  • 19
    Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, et al. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 2000; 6: 517526.
  • 20
    Li D, Zimmerman TL, Thevananther S, Lee HY, Kurie JM, Karpen SJ. Interleukin-1 beta-mediated suppression of RXR:RAR transactivation of the Ntcp promoter is JNK-dependent. J Biol Chem 2002; 277: 3141631422.
  • 21
    Trauner M, Fickert P, Zollner G. Genetic disorders and molecular mechanisms in cholestatic liver disease—a clinical approach. Semin Gastrointest Dis 2001; 12: 6688.
  • 22
    Miyake JH, Wang SL, Davis RA. Bile acid induction of cytokine expression by macrophages correlates with repression of hepatic cholesterol 7alpha-hydroxylase. J Biol Chem 2000; 275: 2180521808.
  • 23
    Zollner G, Fickert P, Silbert D, Fuchsbichler A, Stumptner C, Zatloukal K, et al. Induction of short heterodimer partner 1 precedes downregulation of Ntcp in bile duct-ligated mice. Am J Physiol Gastrointest Liver Physiol 2002; 282: G184G191.
  • 24
    Trauner M, Arrese M, Soroka CJ, Ananthanarayanan M, Koeppel TA, Schlosser SF, et al. The rat canalicular conjugate export pump (mrp2) is down-regulated in intrahepatic and obstructive cholestasis. Gastroenterology 1997; 113: 255264.
  • 25
    Feldmann M, Maini RN. Anti-TNF alpha therapy of rheumatoid arthritis: what have we learned? Annu Rev Immunol 2001; 19: 163196.
  • 26
    Cvetkovic RS, Keating G. Anakinra. BioDrugs 2002; 16: 303311.
  • 27
    Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 1994; 174: 8393.
  • 28
    Boulton RA, Alison MR, Golding M, Selden C, Hodgson HJ. Augmentation of the early phase of liver regeneration after 70% partial hepatectomy in rats following selective Kupffer cell depletion. J Hepatol 1998; 29: 271280.
  • 29
    Yamamoto T, Naito M, Moriyama H, Umezu H, Matsuo H, Kiwada H, et al. Repopulation of murine Kupffer cells after intravenous administration of liposome-encapsulated dichloromethylene diphosphonate. Am J Pathol 1996; 149: 12711286.
  • 30
    Ju C, Reilly TP, Bourdi M, Radonovich MF, Brady JN, George JW, et al. Protective role of Kupffer cells in acetaminophen-induced hepatic injury in mice. Chem Res Toxicol 2002; 15: 15041513.
  • 31
    Geier A, Kim SK, Gerloff T, Dietrich CG, Lammert F, Karpen SJ, et al. Hepatobiliary organic anion transporters are differentially regulated in acute toxic liver injury induced by carbon tetrachloride. J Hepatol 2002; 37: 198205.
  • 32
    Wagner M, Fickert P, Zollner G, Fuchsbichler A, Silbert D, Tsybrovskyy O, et al. Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice. Gastroenterology 2003; 125: 825838.
  • 33
    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248254.
  • 34
    Stieger B, Hagenbuch B, Landmann L, Höchli M, Schroeder A, Meier PJ. In situ localization of the hepatocytic Na+/taurocholate cotransporting polypeptide in rat liver. Gastroenterology 1994; 107: 17811787.
  • 35
    Ramadori G, Armbrust T. Cytokines in the liver. Eur J Gastroenterol Hepatol 2001; 13: 777784.
  • 36
    Loffreda S, Rai R, Yang SQ, Lin HZ, Diehl AM. Bile ducts and portal and central veins are major producers of tumor necrosis factor alpha in regenerating rat liver. Gastroenterology 1997; 112: 20892098.
  • 37
    Denson LA, Auld KL, Schiek DS, McClure MH, Mangelsdorf DJ, Karpen SJ. Interleukin-1beta suppresses retinoid transactivation of two hepatic transporter genes involved in bile formation. J Biol Chem 2000; 275: 88358843.
  • 38
    Plebani M, Panozzo MP, Basso D, De Paoli M, Biasin R, Infantolino D. Cytokines and the progression of liver damage in experimental bile duct ligation. Clin Exp Pharmacol Physiol 1999; 26: 358363.
  • 39
    Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, Gonzalez FJ. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 2000; 102: 731744.
  • 40
    Denson LA, Bohan A, Held MA, Boyer JL. Organ-specific alterations in RAR alpha:RXR alpha abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis. Gastroenterology 2002; 123: 599607.
  • 41
    Dietrich CG, Geier A, Salein N, Lammert F, Roeb E, Oude Elferink RPJ, et al. Consequences of bile duct obstruction on intestinal expression and function of multidrug-resistance associated protein 2. Gastroenterology 2004; 126: 10441053.
  • 42
    Sturm E, Havinga H, Baller JFW, Wolters H, van Roojien N, Kamps JAAM, et al. Kupffer cell depletion with liposomal clodronate prevents suppression of Ntcp expression in endotoxin-treated rats. J Hepatol 2005; 42: 102103.
  • 43
    Lee YK, Dell H, Dowhan DH, Hadzopoulou-Cladaras M, Moore DD. The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: two mechanisms for repression. Mol Cell Biol 2000; 20: 187195.
  • 44
    Setchell KD, Rodrigoues, CM, Clerici C, Solinas A, Morelli A, Gartung C, et al. Bile acid concentrations in human and rat liver tissue and in hepatocyte nuclei. Gastroenterology 1997; 112: 226235.
  • 45
    Jung D, Hagenbuch B, Fried M, Meier PJ, Kullak-Ublick GA. Role of liver-enriched transcription factors and nuclear receptors in regulating the human, mouse, and rat NTCP gene. Am J Physiol Gastrointest Liver Physiol 2004; 286: G752G761.
  • 46
    Shih DQ, Bussen M, Sehayek E, Ananthanarayanan M, Shneider BL, Suchy FJ, et al. Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism. Nat Genet 2001; 27: 375382.
  • 47
    Wang B, Cai SR, Gao C, Sladek FM, Ponder KP. Lipopolysaccharide results in a marked decrease in hepatocyte nuclear factor 4 alpha in rat liver. HEPATOLOGY 2001; 34: 979989.