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Induction of short heterodimer partner 1 precedes downregulation of Ntcp in bile duct-ligated mice

Gernot Zollner1, Peter Fickert1, Dagmar Silbert1, Andrea Fuchsbichler2, Conny Stumptner2, Kurt Zatloukal2, Helmut Denk2, and Michael Trauner1

1 Division of Gastroenterology and Hepatology, Department of Internal Medicine and 2 Department of Pathology, Karl-Franzens University, Graz, A-8036 Austria


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cholestasis is associated with retention of bile acids and reduced expression of the Na+/taurocholate cotransporter (Ntcp), the major hepatocellular bile acid uptake system. This study aimed to determine whether downregulation of Ntcp in obstructive cholestasis 1) is a consequence of bile acid retention and 2) is mediated by induction of the transcriptional repressor short heterodimer partner 1 (SHP-1). To study the time course for the changes in serum bile acid levels as well as SHP-1 and Ntcp steady-state mRNA levels, mice were subjected to common bile duct ligation (CBDL) for 3, 6, 12, 24, 72, and 168 h and compared with sham-operated controls. Serum bile acid levels were determined by radioimmunoassay. SHP-1 and Ntcp steady-state mRNA expression were assessed by Northern blotting. In addition, Ntcp protein expression was studied by Western blotting and immunofluorescence microscopy. Increased SHP-1 mRNA expression paralleled elevations of serum bile acid levels and was followed by downregulation of Ntcp mRNA and protein expression in CBDL mice. Maximal SHP-1 mRNA expression reached a plateau phase after 6-h CBDL (12-fold; P < 0.001) and preceded the nadir of Ntcp mRNA levels (12%, P < 0.001) by 6 h. In conclusion, bile acid-induced expression of SHP-1 may, at least in part, mediate downregulation of Ntcp in CBDL mice. These findings support the concept that downregulation of Ntcp in cholestasis limits intracytoplasmatic accumulation of potentially toxic bile acids.

cholestasis; bile acids; proinflammatory cytokines; transport; orphan nuclear receptors; transcription factors


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHOLESTASIS IS ASSOCIATED WITH reduced expression of hepatocellular transport systems, which contributes to impaired hepatic uptake and biliary excretion of bile acids and other biliary constituents (e.g., bilirubin) (24, 44). However, it is unclear whether these changes in transporter expression are primary events resulting in elevated bile acid levels or consequences of bile acid retention.

The Na+/taurocholate cotransporter (Ntcp; official gene code Slc10a1) is the major hepatocellular uptake system for bile acids (23) and is profoundly downregulated at a transcriptional level in animal models of cholestasis such as common bile duct-ligated (CBDL) or endotoxin-treated rats (14, 42). Hepatic steady-state mRNA levels of Ntcp are inversely related to serum bile acid levels in CBDL rats (15) and humans with various cholestatic disorders (47), suggesting that accumulating bile acids may suppress Ntcp gene expression (15).

Bile acids stimulate expression of short heterodimer partner 1 (SHP-1; official nuclear receptor name NR0B2), a transcriptional repressor inhibiting expression of several genes (17, 30, 38-40). As such, downregulation of cholesterol-7alpha -hydroxylase (cytochrome P-450 7a1, Cyp7a1), the rate limiting enzyme of bile acid synthesis, by bile acid-induced SHP-1 provides the molecular mechanism for feedback inhibition of bile acid synthesis through bile acids (7, 8, 17, 30). Furthermore, bile acid-induced SHP-1 also inhibits retinoid transactivation of Ntcp promoter activity in vitro (10). Mice with targeted disruption of the farnesoid X receptor (FXR, NR1H4), now known as the nuclear bile acid receptor (31, 35, 45), lack bile acid-mediated induction of SHP-1 and concurrent reduction of Ntcp in response to cholic acid (CA) feeding (40). These findings further suggest that SHP-1 may a play a pivotal role in the regulation of Ntcp expression in vivo. However, the role of endogenous bile acids (accumulating during cholestasis) and SHP-1 in mediating reduced Ntcp expression during cholestasis is unknown.

Therefore, the aim of the present study was to test the hypotheses that 1) downregulation of Ntcp in obstructive cholestasis is a consequence of elevated bile acid levels and 2) is mediated by induction of the transcriptional repressor SHP-1. To address these questions, changes in serum bile acid levels and SHP-1 steady-state mRNA levels were studied in bile duct-ligated and sham-operated mice and compared with the time course of changes in Ntcp expression. The present study clearly demonstrates, that accumulation of bile acids and induction of SHP-1 precedes the downregulation of Ntcp by several hours, indicating that reduced Ntcp expression is a secondary event rather than the cause for elevated bile acid levels in obstructive cholestasis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Swiss albino mice (strain Him OF1 SPF), 25-30 g body wt, were obtained from the Institute of Laboratory Animal Research, University of Vienna, School of Medicine, Himberg, Austria. They were housed with a 12:12 h light-dark cycle and permitted ad libitum consumption of water and a standard mouse diet (Marek, Vienna, Austria). 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 (National Institutes of Health publication 86-23, revised 1985).

Materials. The following reagents were used: pCR II vector and One Shot competent cells (Invitrogen, Groningen, The Netherlands); Quantum Prep Plasmid Miniprep and Maxiprep kits (Bio-Rad Laboratories, Hercules, CA); deoxycytidine 5'-triphosphate ([32P]dCTP) and Random Prime II DNA Labeling Kit (Amersham, Little Chalfont, UK); avian myeloblastosis virus reverse transcriptase and restriction enzymes (Boehringer Mannheim, Mannheim, Germany); AmpliTaq DNA Polymerase (Perkin-Elmer, Branchburg, NJ). Cholate was obtained from Aldrich (Steinheim, Germany). Lipopolysaccharide (LPS) from Salmonella typhimurium, Escherichia coli O26:B6, and E. coli O55:B5 was purchased from Sigma (Steinheim, Germany). All other chemicals used were of the highest purity commercially available and purchased from Sigma.

CBDL. All surgical procedures were performed under sterile conditions. The common bile duct was ligated close to the liver hilum immediately below the bifurcation and dissected between the ligatures as described previously (43). In addition, cholecystectomy was performed after ligation of the cystic duct. Control animals underwent a sham operation with exposure, but without ligation of the common bile duct and removal, of the gallbladder. The livers were excised 3, 6, 12, 24, 72 h (3 days), and 168 h (7 days) after CBDL under general anesthesia, immediately snap-frozen, and stored in liquid nitrogen until RNA extraction and preparation of liver membranes.

Bile acid feeding and endotoxin treatment. To compare the effects of bile acids (retained during cholestasis) and proinflammatory cytokines [induced during biliary obstruction (4, 16)], mice were fed a diet supplemented with CA (1% wt/wt) or treated with intraperitoneal injections of LPS from S. typhimurium, E. coli O26:B6, and E. coli O55:B5 (at doses ranging from 0.5 to 15 mg/kg body wt). The CA and LPS doses were previously shown to be biologically active and to reduce Ntcp expression (13, 18). Control animals received a standard diet or were injected with vehicle (saline), respectively. Furthermore, CA feeding at this dose (1% wt/wt) results in enrichment of the bile acid pool with taurocholate by >95% (13). Livers were harvested after 7 days of CA-feeding or 16 h after LPS administration.

Serum bile acid measurements. Blood was collected at the time of death, and serum samples were stored at -70°C until analysis of total serum bile acid levels by a commercially available radioimmunoassay for conjugated bile acids (ICN Pharmaceuticals, New York, NY). Tests were performed in duplicate.

RNA extraction. Isolation of total RNA was performed according to a procedure described by Krieg et al. (22). RNA was quantified spectrophotometrically at 260 nm, and the quality of total RNA was controlled by denaturing formaldehyde agarose gel electrophoresis.

Northern blot analysis. Total RNA (20 µg) was electrophoresed on a 1.2% agarose-formaldehyde gel, transferred to Hybond N membranes (Amersham) by overnight capillary transfer blotting and cross-linked by ultraviolet light (Stratalinker 1800; Stratagene, La Jolla, CA). Membrane prehybridization and hybridization were performed at 45°C for 1 h and overnight, respectively, following a standard protocol (2). Probes were labeled with [32P]dCTP by a random primed method according to the manufacturer's instructions (Random Prime II DNA Labeling Kit; Amersham). Membranes were washed under medium-stringency conditions (two washing steps with 2× SSC/1% SDS followed by two washing steps with 0.2× SSC-1% SDS at room temperature, 10 min each). mRNA levels were detected by exposure to Kodak BioMax films (Kodak, Rochester, NY) and quantified using video-densitometry software (RFLP-Scan or ZeroD-Scan; Scanalytics, Billerica, MA). Membranes were stripped and reprobed for GAPDH to determine equal loading. The size of mRNA was estimated by a 0.24- to 9.5-kb RNA ladder (GIBCO-BRL, Gaithersburg, MD). Specific probes were generated for SHP-1 (NR0B2), Ntcp (Slc10a1), and GAPDH using RT-PCR with following primer pairs: Ntcp (Gene Bank Acc. No. U95131) (5) upstream 290-307, downstream 905-886; SHP-1 (GenBank accession no. NM-011850) (38) upstream 290-309, downstream 782-763; GAPDH (GenBank accession no. M32599) (37) upstream 428-445, downstream 551-535. The PCR products were cloned into pCR II vectors (Invitrogen) and their specificity was checked by sequencing with an Abi Prism automatic sequencer (Perkin-Elmer).

Preparation of liver membranes. Crude liver membranes were isolated, and protein concentrations were determined according to Bradford, as described previously (13). Protein yields were similar in controls and CBDL (data not shown).

Western blot analysis. Similar amounts of protein (150 µg) were loaded onto 10% SDS-polyacrylamide gels, without boiling, and subjected to electrophoresis (25). Equal protein loading was confirmed by Coomassie staining of gels. After electrotransfer onto nitrocellulose membranes (Bio-Rad, Richmond, CA), the blots were incubated with a polyclonal antibody against Ntcp (kindly provided by Drs. Peter Meier and Bruno Stieger, Zurich, Switzerland), and immune complexes were detected using horseradish-conjugated goat anti-rabbit IgG F(ab')2 fragments as described (13).

Immunofluorescence microscopy. Immunofluorescence staining for Ntcp was performed as described previously (13). In brief, cryosections of liver tissue were fixed in a 4% paraformaldehyde solution in PBS for 20 min, rinsed three times in 50 mM NH4Cl in PBS, and treated with 5% Triton X-100 in 50 mM NH4Cl in PBS for 5 min, followed by incubation with a polyclonal antibody against Ntcp (dilution, 1:25). A tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit antibody (Dako, Glostrup, Denmark) was used as secondary antibody. Negative controls were performed by omitting the primary antibody. Fluorescent staining was visualized using a MRC 600 laser scanning confocal device (Bio-Rad) attached to a Axiophot (Zeiss, Oberkochen, Germany). The fluorescent images were collected using the confocal photomultiplier tube as full frame (768 × 512 pixels). Only samples prepared in parallel in all steps were compared.

Statistical analysis. In each group, five animals were studied for each given time point. Data are reported as arithmetic means ± SE. Differences among experimental groups were analyzed by unpaired t-test and ANOVA with Bonferroni posttesting using the SigmaStat statistic program (Jandel Scientific, San Rafael, CA). A P value <0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Bile acid levels in CBDL mice. To establish a time course for the accumulation of serum bile acids compared with SHP-1 and Ntcp mRNA expression in biliary obstruction, mice were subjected to CBDL for 3, 6, 12, 24, 72 h (3 days), and 168 h (7 days) and compared with naive mice (0 h) and sham-operated controls. Compared with naive mice (1.3 ± 0.3 µmol/l), serum bile acid levels already were 270-fold increased in 3-h CBDL mice (343 ± 101 µmol/l) with a first peak around 6-h CBDL (370-fold; 464 ± 36 µmol/l), followed by a second and even higher peak around 24-h CBDL (690-fold; 1011 ± 83 µmol/l) and decreasing levels, thereafter (570-fold at 72 h; 180-fold at 168 h) (Fig. 1B). Sham-operated controls showed no significant differences compared with naive mice (Fig. 1B).


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Fig. 1.   Bile acid-induced expression of short heterodimer partner 1(SHP-1) mRNA precedes downregulation of Na+/taurocholate cotransporter (Ntcp) mRNA in common bile duct-ligated (CBDL) mice. A: representative Northern blots. Total RNA was prepared from mouse livers after CBDL or sham operation at given time points. Northern blot analysis was performed using specific probes for mouse SHP-1, Ntcp, and GAPDH as described in MATERIALS AND METHODS. For each time point, blots from 3 representative animals are shown; all samples were run on identical gels. To correct for potential differences in mRNA loading, blots were stripped and reprobed for GAPDH. B: time course for changes in serum bile acid levels, SHP-1, and Ntcp steady-state mRNA levels. Elevations of serum bile acids were accompanied by increased SHP-1 mRNA expression. Note that bile acid-induced SHP-1 mRNA levels peaked 6 h before the nadir of Ntcp mRNA. open circle , Sham-operated animals; , CBDL mice. Densitometry data (means ± SE) are expressed as relative abundance compared with naive mice (0 h). *P < 0.01, **P < 0.001 compared with sham-operated controls (n = 5 for each time-point per group).

Induction of SHP-1 expression parallels elevation of serum bile acid levels in CBDL mice. Because SHP-1 is induced by bile acids and represents a well-defined suppressor of Ntcp expression in vitro (10), SHP-1 steady-state mRNA expression was determined in CBDL mice at the time points indicated above (Fig. 1, A and B). SHP-1 mRNA levels paralleled the increase of bile acid levels. Maximal expression was observed 6 h after CBDL (coinciding with the first peak of serum bile acid levels) followed by a plateau despite ongoing bile acid accumulation culminating in a second peak at 24 h after CBDL. The decline of SHP-1 mRNA levels after 72 h paralleled the reduction of serum bile acid levels beyond this time point. Sham-operated controls showed no significant differences compared with naive mice (Fig. 1B).

Downregulation of Ntcp expression follows bile acid-induced SHP-1 expression in CBDL mice. Downregulation of Ntcp transcription has been previously shown in several rodent models of cholestasis (14, 42). However, it has remained an open question whether this is a cause or consequence of cholestasis (44). Therefore, the time course of Ntcp steady-state mRNA expression was compared with serum bile acid levels (as an indicator of bile acid retention) and SHP-1 mRNA expression in CBDL mice. Downregulation of Ntcp mRNA expression followed the elevation of serum bile acid levels and induction of SHP-1 mRNA expression with a lag period of several hours (Fig. 1, A and B). Of note, Ntcp mRNA levels remained almost unchanged after 3-h CBDL, a time point where serum bile acid levels and SHP-1 mRNA expression were already increased 270- and 6-fold, respectively. The nadir of Ntcp mRNA levels was reached at 12 h, representing a lag period of 6 h after the plateau of SHP-1 mRNA expression was reached (Fig. 1, A and B). Sham-operated controls showed no significant changes in Ntcp mRNA levels compared with naive mice (Fig. 1B).

Ntcp protein levels and localization in CBDL mice. To determine whether the observed changes of Ntcp mRNA levels affect Ntcp protein expression, Western immunoblotting was performed in crude liver membranes obtained from livers from sham-operated and CBDL mice (Fig. 2, A and B). Ntcp protein levels were still unchanged after 6- and 12-h CBDL (time points when SHP-1 mRNA expression was already induced 6- and 12-fold, respectively), but decreased significantly to 67 ± 5% after 24 h and reached the minimum with 44 ± 22% after 168 h (7 days) CBDL (Fig. 2B). Sham-operated controls showed no significant changes in Ntcp protein levels compared with naive mice (Fig. 2A). Immunofluorescence staining showed a regular basolateral staining pattern of Ntcp at 3-, 6-, and 12-h CBDL, but a disrupted and reduced staining pattern beyond 24-h CBDL (Fig. 3, A-F). Reduced Ntcp protein expression after the elevation of serum bile acid levels further indicates that downregulation of Ntcp expression in CBDL mice is consequence rather than cause of elevated serum bile acid levels.


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Fig. 2.   Representative Western blots of Ntcp protein expression in sham-operated and CBDL mice. Crude liver membranes were isolated from mouse livers after CBDL or sham operation at given time points, and Western blot analysis was performed. Two representative animals are shown for each time point; samples from CBDL and sham-operated animals were run on identical gels. Specificity of the changes for Ntcp was confirmed by unchanged beta -actin protein levels. A: in sham-operated animals, no significant changes in Ntcp protein expression were detected at all investigated time points. B: Ntcp protein levels remained unchanged until 12-h CBDL, but decreased significantly after 24 h, reaching the minimum after 168-h (7 days) CBDL. Densitometry data are expressed as the degree of change (n = 5 for each time-point per group). *P < 0.05 compared with naive mice (0 h).



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Fig. 3.   Immunofluorescence localization of Ntcp in CBDL mice. To detect potential changes in Ntcp tissue distribution, cryosections of liver tissue from controls and CBDL mice were incubated with a Ntcp antibody as described in MATERIALS AND METHODS. Normal basolateral fluorescence staining for Ntcp in control animals (A) is shown. Ntcp staining shows regular basolateral localization and preserved signal intensities after 6-h (B) and 12-h CBDL (C). Ntcp signals showed a reduced and disrupted staining at 24 h (D), 72 h (3 days) (E), and 168 h (7 days) (F) after CBDL. Bar = 20 µm.

CA feeding, but not LPS administration, induces SHP-1 mRNA, whereas both reduce Ntcp mRNA expression. In addition to retention of bile acids, CBDL also results in activation of proinflammatory cytokines (4, 16), which represent established inhibitors of Ntcp expression (9, 42). To distinguish between the impact of cytokines and bile acids on SHP-1 expression in CBDL, the effects of LPS administration (a potent stimulator of cytokine production in vivo) were compared with CA feeding [known to induce SHP-1 concomitantly with reducing Ntcp (40)]. In line with previous findings (40), CA feeding over 1 wk resulted in elevated serum bile acid levels (71.5 ± 20.9 µmol/l vs. 3.9 ± 0.8 µmol/l in pair-fed mice; P < 0.05), induced SHP-1 mRNA by 4.2-fold (P < 0.01), and reduced Ntcp mRNA and protein expression to 73 ± 15% (P < 0.05) and 52 ± 8% (P < 0.05), respectively (Figs. 4A and 5A), suggesting that induction of SHP-1 by bile acids may be linked to changes in Ntcp expression. The moderate changes in SHP-1 and Ntcp mRNA levels are consistent with the mild elevations of serum bile acid levels (when compared with the more profound changes after CBDL). To induce proinflammatory cytokines in vivo, mice were injected with various doses and types of LPS. In line with previous findings (18, 42), Ntcp steady-state mRNA levels were reduced to 28 ± 10% of controls (P < 0.01) 16 h after injection of LPS from S. typhimurium (15 mg/kg body wt) (Fig. 4B). At this time point, Ntcp protein levels were not yet significantly reduced (Fig. 5B), consistent with only minimally elevated serum bile acid levels (2.1 ± 1 µmol/l vs. 1.2 ± 0.4 µmol/l in saline injected mice; P < 0.05). In contrast to the effects of CA feeding and despite the profound LPS effects on Ntcp mRNA expression, SHP-1 mRNA levels were not induced by LPS (Fig. 4B), suggesting that SHP-1 may not mediate the downregulation of Ntcp by LPS-induced proinflammatory cytokines. LPS from E. coli O26:B6 and E. coli O55:B5 had similar effects on SHP-1 and Ntcp expression (data not shown).


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Fig. 4.   Cholic acid (CA) feeding, but not lipopolysaccharide (LPS) administration, induces SHP-1 mRNA, whereas both reduce Ntcp mRNA expression. To distinguish between the effects of bile acids and proinflammatory cytokines on SHP-1 and Ntcp mRNA expression, mice were fed a CA (1% wt/wt) -enriched diet (A) or injected intraperitoneally with LPS from Salmonella typhimurium (15 mg/kg body wt) (B). Total RNA was extracted from mouse livers, and Northern blot analysis was performed using specific probes for mouse SHP-1, Ntcp, and GAPDH, as described in MATERIALS AND METHODS. For each group, 3 representative blots are shown; all samples were run on identical gels. To correct for potential differences in mRNA loading, blots were stripped and reprobed for GAPDH. Both CA and LPS reduced Ntcp mRNA levels (A and B). SHP-1 mRNA expression was induced exclusively by CA (A) but remained unchanged after LPS (B). Densitometry data are expressed as the degree of change (n = 5 for each group). *P < 0.05, **P < 0.01 compared with pair-fed (for CA) or vehicle (saline) -injected controls (for LPS).



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Fig. 5.   CA feeding, but not LPS administration, reduces Ntcp protein expression in mice. To investigate the effects of proinflammatory cytokines compared with bile acid feeding on Ntcp protein expression, mice were fed a CA (1% wt/wt) -enriched diet (A) or injected intraperitoneally with LPS from S. typhimurium (15 mg/kg body wt) (B). Crude liver membranes were isolated from mouse livers, and Western blot analysis was performed using specific antibodies against Ntcp and beta -actin. For each group, 3 representative blots are shown; all samples were run on identical gels. Specificity of the changes for Ntcp was determined by blotting for beta -actin. In contrast to the reduction of Ntcp protein levels after CA feeding (A), Ntcp protein expression remained unchanged after a single LPS administration (B). Densitometry data are expressed as the degree of change (n = 5 for each group). *P < 0.05, compared with pair-fed (for CA) or vehicle (saline) -injected controls (for LPS).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first study demonstrating bile acid-mediated induction of a transcriptional repressor (SHP-1), which precedes downregulation of the major hepatic bile acid uptake system (Ntcp) in a mouse model of cholestasis (CBDL) in vivo.

Ntcp was the first cloned bile acid transport system (20) and is among the best characterized hepatocellular transport systems under physiological and cholestatic conditions (23). Previous studies have demonstrated profound reduction of Ntcp gene transcription in various animal models of cholestasis including CBDL and LPS-treated rats (14, 42), although the mediators and molecular mechanisms responsible for these changes have not been entirely clarified (44). Furthermore, it was not clear whether changes in transporter expression are primary (causing elevations of bile acid levels) or secondary (a consequence of bile acid retention). Using Ntcp as a model system, the present study demonstrates that reduction of Ntcp expression is secondary to bile acid retention, resulting in induction of the transcriptional repressor SHP-1.

The recent discovery of bile acids as ligands for orphan nuclear receptors allows a better understanding of bile acid effects on gene transcription (6, 36). SHP-1 is an atypical member of the orphan nuclear receptor family lacking a DNA-binding domain and inhibits gene transcription by interaction with other nuclear transcription factors (29, 38, 39). Bile acids stimulate SHP-1 transcription via activation of farnesoid X receptor (FXR) (17, 30, 40); in addition, activation of the c-Jun NH2-terminal kinase pathway resulting in formation of activating protein-1 (AP-1) may also contribute to the effects of bile acids on SHP-1 expression (19). Interaction of bile acid-induced SHP-1 with liver receptor homolog 1, a monomeric orphan receptor required for hepatic expression of Cyp7a1, plays a critical role in the negative feedback repression of Cyp7a1 catalyzing the rate limiting step in bile acid biosynthesis (17, 30).

What could be the molecular mechanisms for suppression of Ntcp gene expression in CBDL mice? Constitutive expression of Ntcp critically depends on transactivation by a heterodimer (formerly known as footprint B binding protein) composed of the orphan nuclear receptors retinoic X receptor-alpha (RXRalpha ; NR2B1) and retinoic acid receptor-alpha (RARalpha ; NR1B1) (9, 21). SHP-1 inhibits DNA binding of RXR:RAR heterodimers (38) and competes with coactivators for binding to ligand-activated RXR (29). Decreased activity of RXR:RAR has been demonstrated in animal models of cholestasis and is thought to play a critical role for reduced Ntcp transcription under these conditions (9, 42). Bile acid-induced SHP-1 also inhibits Ntcp promotor activity in vitro by inhibition of RXR:RAR transactivation (10). In line with previous findings (40), CA feeding to mice in the present study induced SHP-1 concurrently with reducing Ntcp expression. These changes are absent in FXR-/- mice (40), further suggesting a role for bile acid-induced SHP-1 in the regulation of Ntcp expression in vivo. Therefore, bile acid-mediated induction of SHP-1 in CBDL mice as demonstrated in the present study is likely to be responsible for reduced Ntcp gene expression in mechanical cholestasis (Fig. 6). This concept is further supported by the time course of events with induction of SHP-1 steady-state mRNA levels paralleling serum bile acid levels and preceding the reduction of Ntcp by several hours as demonstrated in this study. Future studies will have to explore the role of SHP-1 in the downregulation of other genes (e.g., the canalicular conjugate export pump), which also critically depends on transactivation by RXR:RAR (9), to explain the coordinated downregulation of several basolateral and canalicular transporter genes in cholestasis (23, 44).


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Fig. 6.   Proposed sequence of events leading to downregulation of Ntcp gene expression in common bile duct-ligated (CBDL) mice. Biliary obstruction results in (1) accumulation of bile acids with (2) activation of farnesoid X receptor (FXR)/bile acid receptor and (3) induction of SHP-1-expression. SHP-1 interacts with (4) retinoid X receptor/retinoic acid receptor (RXR:RAR) resulting in (5) reduced Ntcp transcription. In addition, (6) SHP-1 inhibits transcription of its own gene via interaction with liver receptor homolog 1 (LRH-1). Downregulation of Ntcp (7) aggravates serum bile acid retention, but limits hepatocellular bile acid accumulation.

Serum bile acid levels showed a biphasic pattern, with a first peak around 6 h followed by a second and an even higher peak 24 h after CBDL. Interestingly, serum bile acid levels were already significantly elevated at time points when Ntcp protein levels were still normal and Ntcp was regularly localized to the basolateral membrane. The most likely explanation for this finding could be regurgitation of bile acids through leaky canalicular and ductular tight junctions (1), because bile infarcts as a result of biliary leakage are observed as early as 3 h after CBDL (P. Fickert and M. Trauner, unpublished observation). Alternatively, posttranslational modifications of Ntcp impairing its function (34) and basolateral efflux of bile acids from cholestatic hepatocytes via induced multidrug resistance protein 3 (11, 41) could also contribute to increased serum bile acid levels, although this remains to be determined in future studies. Of interest, maximal SHP-1 mRNA expression already reached a plateau 6 h after CBDL, despite a further increase in serum bile acid levels. Because SHP-1 is known to repress its own gene transcription (30), one may speculate that the stagnation of SHP-1 expression represents a negative feedback mechanism limiting further induction of SHP-1. SHP-1-induced downregulation of Ntcp resulting in impaired bile acid uptake may, at least in part, contribute to the further increase of serum bile acid levels between 12 and 24 h after CBDL. Decreasing bile acid levels beyond 24-h CBDL, despite continued biliary obstruction, have also been described in CBDL rats (14). This phenomenon may be explained by partial recovery of the canalicular bile salt export pump (28) and renal elimination of bile acids facilitated by upregulation of the apical sodium-dependent bile acid transporter in cholangiocytes and reciprocal downregulation in proximal renal tubules (26).

In addition to retention of bile acids, induction of proinflammatory cytokines could also contribute to downregulation of Ntcp in biliary obstruction (4, 16). Cytokines could mediate at least part of the bile acid effects on gene expression, as suggested by absent bile acid-mediated feedback inhibition of Cyp7a1 in cytokine-resistant mice (32). Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha ) and interleukin-1beta are established inhibitors of Ntcp transcription (18, 33, 42). These effects may, at least in part, be mediated by inhibition of retinoid transactivation of Ntcp, because LPS and proinflammatory cytokines inhibit RXR:RAR binding activity (9, 42) and reduce RXRalpha transcription (3). In addition, proinflammatory cytokines could also be expected to inhibit Ntcp expression by induction of SHP-1 via a recently identified AP-1 response element in the SHP-1 promoter, because cytokines (similar to bile acids) activate the c-Jun NH2-terminal kinase pathway (19). However, our results demonstrate that LPS-induced cytokines reduce Ntcp expression without inducing SHP-1. These data further indicate that, in contrast to bile acids, proinflammatory cytokines may not contribute to the induction of SHP-1 mRNA in CBDL mice. Moreover, the administration of anti-TNF-alpha antiserum and suppression of cytokine synthesis by dexamethasone did not prevent downregulation of Ntcp in CBDL rats (16). In line with our findings, these data suggest that proinflammatory cytokines may not contribute to downregulation of Ntcp in biliary obstruction.

In summary, the present study demonstrates for the first time that bile acid-mediated induction of a transcriptional repressor (SHP-1) precedes downregulation of Ntcp as a model transport system during cholestasis. Our findings suggest that bile acid-induced SHP-1 expression mediates, at least in part, downregulation of Ntcp, which aggravates cholestasis with systemic bile acid retention but may protect hepatocytes from accumulation of potentially toxic bile acids.


    ACKNOWLEDGEMENTS

This work was supported by Grant 8522 from Jubilee Funds of the Austrian National Bank (to M. Trauner), the Joseph Skoda Prize from the Austrian Society of Internal Medicine (to M. Trauner) and Grant S7401-MOB from the Austrian Science Foundation (to K. Zatloukal).


    FOOTNOTES

First published September 21, 2001; 10.1152/ajpgi.00215.2001

This study was presented, in part, at the 52nd Annual Meeting of the American Association for the Study of Liver Diseases, Dallas, TX, November 9-13, 2001, and published in abstract form (Hepatology; 34:469A, 2001.

Address for reprint requests and other correspondence: M. Trauner, Division of Gastroenterology and Hepatology, Dept. of Internal Medicine, Karl-Franzens Univ., Auenbruggerplatz 15, A-8036 Graz, Austria (E-mail: michael.trauner{at}kfunigraz.ac.at).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 May 2001; accepted in final form 1 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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