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Molecular Endocrinology 20 (1): 65-79
Copyright © 2006 by The Endocrine Society

The Human Na+-Taurocholate Cotransporting Polypeptide Gene Is Activated by Glucocorticoid Receptor and Peroxisome Proliferator-Activated Receptor-{gamma} Coactivator-1{alpha}, and Suppressed by Bile Acids via a Small Heterodimer Partner-Dependent Mechanism

Jyrki J. Eloranta, Diana Jung and Gerd A. Kullak-Ublick

Laboratory of Molecular Gastroenterology and Hepatology, Department of Internal Medicine, University Hospital Zurich, CH-8091 Zurich, Switzerland

Address all correspondence and requests for reprints to: Gerd A. Kullak-Ublick, Department of Internal Medicine, University Hospital Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland. E-mail: gerd.kullak{at}usz.ch.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Na+-taurocholate cotransporting polypeptide (NTCP) is the major bile acid uptake system in human hepatocytes. NTCP and the ileal transporter ASBT (apical sodium-dependent bile acid transporter) are two sodium-dependent transporters critical for the enterohepatic circulation of bile acids. The hASBT gene is known to be activated by the glucocorticoid receptor (GR). Here we show that GR also induces the endogenous hNTCP gene and transactivates the reporter-linked hNTCP promoter, in the presence of its ligand dexamethasone. Mutational analysis of the hNTCP promoter identified a functional GR response element, with which GR directly interacts within living cells. The GR/dexamethasone activation of endogenous hNTCP expression was suppressed by bile acids, in a manner dependent on the bile acid receptor farnesoid X receptor. Overexpression of the farnesoid X receptor-inducible transcriptional repressor small heterodimer partner also suppressed the GR/dexamethasone-activation of the hNTCP promoter. The peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} enhanced the GR/dexamethasone activation of the hNTCP promoter. In conclusion, the hNTCP promoter is activated by GR in a ligand-dependent manner, similarly to the hASBT promoter. Thus, glucocorticoids may coordinately regulate the major bile acid uptake systems in human liver and intestine. The GR/dexamethasone activation of the hNTCP promoter is counteracted by bile acids and small heterodimer partner, providing a negative feedback mechanism for bile acid uptake in human hepatocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HEPATIC CONVERSION OF cholesterol into bile acids is a major pathway of cholesterol elimination from the body and thus an essential determinant of cholesterol homeostasis. Bile acids themselves are essential for maintenance of bile flow and for biliary secretion of lipids (1) and are efficiently recycled between the liver and the intestine, with minimal losses into feces in healthy individuals (2). The enterohepatic circulation of bile acids is maintained by bile acid uptake and efflux proteins expressed on the membranes of hepatocytes and enterocytes in a polarized manner (3). Among the transporters critically important for the cycling of bile acids are the two members of the human SLC10 family of sodium-bile acid cotransporters, hNTCP (human Na+-taurocholate cotransporting polypeptide; gene symbol SLC10A1) and hASBT (human apical sodium-dependent bile acid transporter; gene symbol SLC10A2) (4). Whereas hASBT is chiefly responsible for absorption of bile acids into ileal enterocytes, hNTCP, in turn, accounts for more than 80% of hepatic uptake of conjugated bile salts across the basolateral membrane of hepatocytes. Loss-of-function mutations in hASBT can cause primary bile acid malabsorption, an intestinal disorder characterized by congenital diarrhea, and reduced plasma cholesterol levels (5). No mutations in hNTCP leading to clinically manifest defects in hepatic bile acid uptake have been characterized thus far. However, a recent study identified ethnicity-dependent single-nucleotide polymorphisms in the hNTCP gene associated with decreased transport function in vitro (6), implying that genetic heterogeneity in the hNTCP gene may indeed play a role in the etiology of hypercholanemia. Furthermore, certain human diseases, such as advanced stage primary biliary cirrhosis (7) and cholestatic alcoholic hepatitis (8), are associated with reduced hNTCP expression.

The efficiency of bile acid uptake into hepatocytes is likely to be largely determined by the expression level of the hNTCP gene. Whereas several promoter elements and DNA-binding factors have been suggested to regulate the rat Ntcp gene (9, 10, 11, 12, 13), transcriptional regulation of the hNTCP gene has remained less well characterized. Unlike the rat Ntcp promoter, the human gene does not appear to contain an identifiable TATA box (14). The only transcription factor binding sites so far identified as conserved between the proximal human, rat, and mouse NTCP/Ntcp promoters are the two binding elements for the forkhead/winged helix transcription factor hepatocyte nuclear factor-3ß (HNF-3ß). HNF-3ß mediates transcriptional repression of the NTCP/Ntcp promoter in all three species via direct binding to its response elements (12). In addition, the members of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors can transactivate the hNTCP promoter in transient transfection assays (12, 14).

The crucial role for bile acids as solubilizers of cholesterol in bile and emulsifiers of lipids in the intestine has long been recognized (1). Recently, bile acids have been identified as ligands for specific members of the nuclear receptor family of transcription factors (15, 16, 17, 18, 19). Consequently, changes in intracellular levels of bile acids have a profound feedback effect on the transcriptional regulatory circuits involved in bile acid metabolism (20). The transcriptional responses to elevated bile acid levels serve to decrease de novo synthesis and cellular uptake of bile acids and to increase hepatic bile acid efflux and detoxification systems. This response protects the hepatocytes or enterocytes from damaging detergent effects of excessive levels of intracellular bile acids. Expression of the NTCP/Ntcp (8, 21, 22, 23, 24) gene is reduced in response to increased intracellular bile acid concentrations. Whereas the molecular mechanism for the bile acid-mediated suppression of the hNTCP gene remains unelucidated, the nuclear receptor heterodimer retinoic acid receptor-{alpha}-retinoid X receptor {alpha} (RAR{alpha}-RXR{alpha}) has been identified as the target for down-regulation of the rat Ntcp promoter (25). Bile acids are agonistic ligands for the farnesoid X receptor (FXR), which positively regulates the gene encoding the transcriptional repressor small heterodimer partner (SHP) (26, 27). SHP, in turn, interferes with the transcriptional activity of the RAR{alpha}-RXR{alpha} heterodimer, leading to decreased expression of the rat Ntcp gene. The expression of the human and the mouse ASBT/Asbt genes is similarly suppressed by bile acids (28, 29, 30). The heterodimer RAR{alpha}-RXR{alpha} and the monomeric nuclear receptor liver receptor homolog-1 have been proposed as targets for negative transcriptional interference in the context of the human and the mouse ASBT/Asbt promoters, respectively.

We have recently shown that the hASBT gene is transactivated by a member of the steroid receptor family, namely the glucocorticoid receptor (GR), in the presence of an agonistic GR ligand (31). Here we show that GR is also a transactivator of the hNTCP gene, another member of the same transporter gene superfamily. We have studied the coactivator requirements for GR-mediated transactivation of the hNTCP and hASBT genes and discovered that only the former gene is coactivated by PGC-1{alpha} (peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha}). We further show that bile acids, as well as the bile acid-induced transcriptional repressor SHP, can strongly antagonize GR-dependent induction of the hNTCP promoter. Thus, we describe the first potential mechanism mediating the bile acid-dependent negative feedback of the major hepatic bile acid uptake system in humans. Similarly to hNTCP, the GR-dependent induction of the hASBT gene can be suppressed by SHP, implying that GR may also be a target for bile acid-mediated gene suppression of hASBT.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GR Induces the Endogenous hNTCP Gene and the Reporter-Linked hNTCP Promoter in a Ligand-Dependent Manner
The two members of the SLC10 gene superfamily encode the major bile acid uptake systems in human liver (hNTCP, gene symbol SLC10A1) and in human intestine (hASBT, gene symbol SLC10A2). Because previous studies showed that GR can transactivate the hASBT gene (31), we first investigated whether the hNTCP gene is similarly regulated by GR. In untreated Huh7 cells, expression of the endogenous hNTCP mRNA is below the detection level, as measured by quantitative real-time PCR. Upon exogenous expression of GR and treatment of cells with the agonistic GR ligand dexamethasone, the hNTCP mRNA became readily detectable (Fig. 1Go). Neither expression of GR in the absence of a ligand, nor dexamethasone treatment of cells that do not exogenously express GR, increased hNTCP mRNA levels. This indicates that the level of GR endogenously present in Huh7 cells is insufficient to mediate induction by glucocorticoids.



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  		Fig. 1. GR Induces the Endogenous hNTCP Gene in a Ligand-Dependent Manner

Huh7 cells were transfected with either the pSG5-GR expression construct (GR) or the pSG5 vector. The cells were treated 12 h after transfections with dexamethasone (dexa) or the vehicle ethanol, after which total cellular RNAs were extracted 24 h later and used for reverse transcription, and levels of the endogenous hNTCP mRNA were determined by real-time PCR.

 
Having established that the endogenous hNTCP gene is inducible by GR in a ligand-dependent manner, we next examined whether the luciferase reporter-linked hNTCP promoter construct is also responsive to GR. Indeed, the proximal hNTCP promoter harboring the nucleotides (nt) –178 to +67 relative to the transcriptional start site was strongly transactivated by GR in Huh7 cells. Similarly to the endogenous hNTCP gene, GR-mediated transactivation of the hNTCP(–178/+67)luc was completely dependent on the treatment of the cells with dexamethasone (Fig. 2AGo). GR-mediated transactivation exhibited dose dependence, as transfection with increasing amounts of the GR expression plasmid in the presence of dexamethasone resulted in a corresponding increase in the activity of the hNTCP(–178/+67) promoter (Fig. 2BGo). Increasing the concentration of dexamethasone above 100 nM did not have any additional effect on GR-dependent transactivation of the hNTCP promoter (data not shown).



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  		Fig. 2. GR Induces the Luciferase-Linked Proximal hNTCP Promoter in a Ligand- and Dose-Dependent Manner

A, GR transactivates the luciferase-linked hNTCP promoter in a ligand-dependent manner. Huh7 cells were transfected with either the reporter construct hNTCP(–178/+67)luc or the pGL3basic vector, together with 100 ng of either the pSG5-GR expression construct (GR) or the empty pSG5 vector. The cells were treated with dexamethasone (dexa) or the vehicle ethanol 12 h after transfection and lysed for reporter assays 24 h later. B, GR transactivates the luciferase-linked hNTCP promoter in a dose-dependent manner. Huh7 cells were transfected with either the construct hNTCP(–178/+67)luc or the pGL3basic vector, together with the indicated amount of the pSG5-GR expression construct (GR). The pSG5 vector was used to normalize the amount of transfected effector plasmid to 300 ng. The cells were treated with dexamethasone (dexa) or the vehicle ethanol 12 h after transfection and lysed 24 h later.

 
We next studied whether the antagonistic GR ligand, the antiglucocorticoid RU486 (32), has an effect on GR-activated transcription of the hNTCP promoter. Simultaneous treatment of GR-transfected cells with 100 nM dexamethasone and 1 µM RU486 efficiently counteracted induction of both the endogenous hNTCP gene (Fig. 3AGo) and the luciferase-linked hNTCP(–178/+67) promoter (Fig. 3BGo), when compared with GR-transfected cells treated with dexamethasone alone.



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  		Fig. 3. Antiglucocorticoids Suppress the Induction of the hNTCP Promoter by GR

A, The antiglucocorticoid RU486 reverses GR/dexamethasone induction of the endogenous hNTCP gene. Huh7 cells were transfected with the pSG5-GR expression construct (GR) or the pSG5 vector (control). The cells were treated 12 h after transfections with dexamethasone, both dexamethasone and RU486, or the vehicle ethanol, for 24 h. The relative levels of the endogenous hNTCP mRNA were determined by real-time PCR. B, The antiglucocorticoid RU486 reverses GR/dexamethasone induction of the luciferase-linked hNTCP promoter. Huh7 cells were transfected with either the reporter construct hNTCP(–178/+67)luc or pGL3basic, together with 300 ng of either the pSG5-GR plasmid (GR) or of the pSG5 vector (control). The cells were treated with dexamethasone, both dexamethasone and RU486, or the vehicle ethanol, 12 h after transfection, and lysed 24 h later. dexa, Dexamethasone.

 
Functional Mapping of the GR-Responsive Region within the hNTCP Promoter
To localize the putative response element mediating the induction of hNTCP by GR and dexamethasone, we created progressive deletions of the proximal hNTCP promoter and subcloned these upstream of the luciferase reporter gene. When tested in transient transfections, the deletion variant hNTCP(–87/+67) was as responsive to exogenous GR expression and dexamethasone treatment as the hNTCP(–178/+67) variant (Fig. 4Go). A further deletion variant hNTCP(–36/+67) could still be induced by GR and dexamethasone, albeit to a lower degree than the hNTCP(–178/+67) construct. The induction by GR and dexamethasone was completely abolished upon deletion of 14 additional nucleotides [construct hNTCP(–22/+67)], indicating that critically important nucleotides are located between nt –36 and –22 of the hNTCP promoter. When the 5'-untranslated region was removed [construct hNTCP(–178/+1)], the inducibility was maintained, although to a lesser degree relative to the construct hNTCP(–178/+67).



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  		Fig. 4. Mapping of the Glucocorticoid-Inducible Element of the hNTCP Promoter

Progressive 5'-deletions of the hNTCP promoter (–178/+67, –87/+67, –36/+67, –22/+67) and a 3'-deletion removing the untranslated region (–178/+1) were created by PCR. Huh7 cells were transfected with the luciferase-linked hNTCP promoter variants, or the pGL3basic vector, together with 100 ng of the pSG5-GR expression construct where indicated. dexa, Dexamethasone.

 
In Silico Analysis of the GR-Responsive Region of the hNTCP Promoter
Having functionally localized the hNTCP promoter region mediating inducibility by GR and dexamethasone to the region between nt –36 and +1, we next identified potential GR-binding sites using NUBIscan (33) and MatInspector (www.genomatix.de) algorithms. A putative binding site for GR was found between nt –32 and –12, the sequence of which was compared with the glucocorticoid response element (GRE) in the rat tyrosine aminotransferase (Tat) promoter (34), commonly used as a consensus GRE. The rTat GRE represents an imperfect inverted repeat-3 configuration of two hexamers separated by three nucleotides. A clear similarity is evident between the hexameric arms of the putative hNTCP GRE and the rTat GRE, with an identity of 10 of 12 nucleotides; however, NUBIscan algorithm identified the sequence between the nt –32 and –12 as an imperfect direct repeat of two hexamers separated by nine nucleotides (DR-9), instead of three as in rTat (Fig. 5Go). Interestingly, a previous study of potential GR DNA-binding elements has identified DR-9 as a motif that can directly interact with the homodimeric GR (35).



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  		Fig. 5. The Sequence of the GRE of the hNTCP Promoter

The sequence of the putative GRE in the hNTCP promoter (top row), as identified by NUBIscan and MatInspector algorithms, and its comparison to a consensus GRE of the rat Tat promoter (bottom row). Also shown are the mutated variants of the hNTCP GRE (three middle rows) used in experiments presented in Figs. 6–8GoGoGo. The point mutations engineered into the hNTCP GRE are shown in bold letters. WT, Wild type.

 
The DR-9 Motif in the hNTCP Promoter Is a Functional GR Responsive Element
To functionally confirm that the identified DR-9 within the hNTCP promoter confers responsiveness to GR and dexamethasone, we created point mutants within the DR-9 element and subjected the mutated GRE variants to transient transfection assays in the context of the hNTCP(–36/+67) promoter construct. One conserved nucleotide predicted to be critical for interaction with GR within each hexamer (nt positions –28 and –13, relative to the transcription start site) of the DR-9 element was changed into a nonconserved nucleotide either individually or together (Fig. 5Go). The mutation in both hexamers completely abolishes the GR-dexamethasone induction of the hNTCP(–36/+67) promoter (Fig. 6Go). The single mutation at either nucleotide position –28 or –13 renders the hNTCP promoter considerably less inducible by GR and its ligand than the wild type, indicating that both hexameric arms of the DR-9-like element are required for the maximal induction.



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  		Fig. 6. The DR-9 motif in the hNTCP Promoter is a Functional GRE

The reporter-linked hNTCP promoter construct (–36/+67) harboring either the wild-type (wt), double-mutated (mut-28/mut-13) GRE, or single-mutated (mut-28 and mut-13) GRE, or the pGL3basic vector, were cotransfected into Huh7 cells together with 200 ng of the expression construct pSG5-GR where indicated. dexa, Dexamethasone.

 
To investigate whether the DR-9 element in the hNTCP promoter can confer glucocorticoid responsiveness to a heterologous core promoter, we subcloned the region corresponding to nt –38 to –7 in front of the thymidine kinase (tk) core promoter linked to the luciferase reporter gene. A mutant construct containing the same point mutations within the hNTCP GRE as employed above (Figs. 5Go and 6Go), and a positive control construct that contained the consensus GRE derived from the rTat promoter (Fig. 5Go) were also engineered. As expected, the hybrid promoter harboring the consensus GRE from the rTat promoter was strongly inducible by exogenously expressed GR in the presence of 100 nM dexamethasone (Fig. 7Go). The wild-type DR-9 element from the hNTCP promoter also conferred inducibility by GR and dexamethasone, albeit to a lesser degree than the consensus GRE. Consistent with the point mutations abolishing the responsiveness to GR and dexamethasone within the native hNTCP promoter, the variant of the DR-9 element containing point mutations in each hexameric arm also failed to respond in a heterologous promoter context. A single mutation at either nucleotide position –28 or –13 significantly reduced or abolished, respectively, the GR/dexamethasone induction of the hNTCP promoter also in the context of the heterologous tk promoter.



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  		Fig. 7. The hNTCP GRE Confers Glucocorticoid-Responsiveness to a Heterologous Promoter

The consensus GRE derived from the rat Tat promoter, the wild-type GRE of the hNTCP promoter, or the hNTCP GRE variant harboring either double (mut-28/mut-13) or single (mut-28 and mut-13) point mutations were subcloned upstream of the tk promoter and the luciferase reporter gene. These constructs, as well as the parental tk-luciferase vector, were cotransfected into Huh7 cells together with 100 ng of the pSG5-GR plasmid (GR) where indicated. dexa, Dexamethasone; cons, consensus; wt, wild type.

 
GR Directly Interacts with the DR-9 Element in the hNTCP Promoter
To study whether there is a direct interaction between the DR-9 element and GR in the context of living cells, we performed an antibody-facilitated DNA precipitation analysis. Huh7 cells were transfected with promoter constructs containing either the wild-type DR-9 element of the hNTCP promoter (hNTCP(–36/+67)wtGRE), or the variant DR-9 harboring point mutations within the GRE [hNTCP(–36/+67)mutGRE]. Cells cotransfected with the GR expression plasmid were also treated with 100 nM dexamethasone. Before lysing the cells, the proteins were cross-linked (36 h after transfection) to DNA in vivo using formaldehyde. After shearing the DNA into 400- to 500-bp fragments, we performed immunoprecipitations using an antibody specific for GR. Next, the cross-links were reversed, and the DNA fragments that were coimmunoprecipitated with GR were purified. These pools of DNA fragments were then used as templates for PCR with oligonucleotide primers specific for the transfected constructs and flanking the insert containing the hNTCP promoter variant. As shown in Fig. 8Go, efficient amplification was achieved only when the wild-type, but not the mutant DR-9 element, was present in the construct transfected into Huh7 cells. Furthermore, consistent with our functional studies in reporter assays, the interaction between GR and the wild-type DR-9 element was only evident in the presence of exogenously expressed GR and 100 nM dexamethasone. The results indicate that there is indeed a specific interaction between GR and the DR-9 element of the hNTCP promoter in living cells.



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  		Fig. 8. GR Directly Interacts with the DR-9 Element of the hNTCP Promoter

Huh7 cells were transfected with the hNTCP promoter construct (hNTCP(–36/+67)luc) containing either the wild-type or the mutated variant of GRE. Each promoter construct was cotransfected with the pSG5-GR expression plasmid (GR) or the pSG5 vector (control). Cells transfected with the pSG5-GR construct were treated with dexamethasone (dexa) 12 h after transfection, whereas the pSG5-transfected cells were treated with ethanol (control). The cells were treated 36 h after transfection with the cross-linker formaldehyde, and coimmunoprecipitations were performed using a GR-specific antibody (+Ab), or with no antibody added (no Ab). The input samples were taken before the addition of the GR antibody. The quantitation of the promoter fragments bound by GR, and thus coimmunoprecipitated with the GR-specific antibody, was achieved by PCR using vector-specific primers flanking the insert containing the hNTCP promoter variant. Samples from a representative experiment, taken after 35 PCR cycles and run on a 2% agarose gel, are shown. The size of the correct PCR product is 272 bp. wt, Wild type.

 
Bile Acids and SHP Suppress the Induction of the Endogenous hNTCP Gene by GR and Dexamethasone
In several rodent models of cholestasis both the mRNA and protein levels of Ntcp are down-regulated by elevated levels of bile acids (21, 22, 23, 24). In rats, the target for bile acid-mediated down-regulation has been identified as the RAR{alpha}-RXR{alpha} nuclear receptor heterodimer, which binds to its response element within the rat Ntcp promoter (25, 36). The binding site for RAR{alpha}-RXR{alpha} is not conserved in the hNTCP promoter. We therefore investigated whether transactivation by GR could be a target for the suppressive effect that bile acids exert on the hNTCP gene.

As shown in Fig. 9AGo the inducing effect on endogenous hNTCP mRNA levels that is achieved by exogenous expression of GR and treatment with 100 nM dexamethasone can be efficiently suppressed by simultaneous treatment with 50 µM of the bile acid chenodeoxycholic acid (CDCA). CDCA is an agonistic ligand for the bile acid receptor FXR (17, 18, 19); however, bile acids are also known to affect gene expression via FXR-independent pathways (37, 38). To assess whether the suppression of GR/dexamethasone-mediated induction of the hNTCP gene by CDCA involves an FXR-dependent mechanism, we treated cells with the synthetic FXR agonist GW4064 (39) in parallel with CDCA. Whereas GW4064 would be expected to enhance FXR-dependent activation of gene transcription, it should not affect FXR-independent pathways of bile acid signaling. As shown in Fig. 9AGo, treating cells with GW4064 suppressed the induction of hNTCP by GR and dexamethasone, indicating the involvement of FXR.



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  		Fig. 9. The Bile Acid CDCA, the Synthetic FXR Ligand GW4064, and the FXR-Inducible Transcriptional Repressor SHP Can Suppress Induction of the Endogenous hNTCP Gene by Glucocorticoids

A, FXR ligands CDCA and GW4064 suppress the induction of the endogenous hNTCP gene by GR/dexamethasone. Huh7 cells were transfected with 100 ng of the pSG5-GR expression construct (GR) where indicated. The cells were treated 12 h after transfection with dexamethasone, CDCA, GW4064, and/or the corresponding vehicles for 24 h. The relative levels of the hNTCP mRNA were determined by real-time PCR. B, The SHP mRNA and protein are induced by treatment of cells with CDCA and GW4064. The real-time PCR analysis of the SHP mRNA levels is shown above, and immunoblot using an antibody raised against either the human SHP protein, or the constitutively expressed Ku-70 antigen to verify equal loading, is shown below. C, Exogenous expression of SHP suppresses the GR/dexamethasone induction of the endogenous hNTCP gene. Huh7 cells were transfected with 100 ng of the pSG5-GR (GR) and the pCMX-SHP (SHP) expression plasmids where indicated. The relative hNTCP mRNA levels were determined by real-time PCR. dexa, Dexamethasone.

 
There is limited evidence that FXR can repress transcription through direct binding to negative response elements in the target genes (40, 41). More commonly, FXR negatively regulates transcription via an indirect mechanism involving another member of the nuclear receptor family, SHP (42). The SHP gene is a direct transcriptional target for activation by ligand-stimulated FXR. SHP does not itself bind to DNA, but interacts with, and negatively affects, the transcriptional activity of a range of DNA-binding transcription factors (20). SHP has also been suggested to interfere with the RAR{alpha}-RXR{alpha} heterodimer binding to the rat Ntcp promoter (25), thus providing a molecular mechanism for its down-regulation by bile acids.

Having shown that bile acids can suppress the induction of the hNTCP gene by GR and dexamethasone via an FXR-mediated mechanism, we next examined whether this also occurs through a pathway involving the FXR-induced repressor SHP. As shown by real-time and immunoblot analyses, treatment of Huh7 cells with both CDCA and GW4064 indeed induces the expression of both the SHP mRNA and protein (Fig. 9BGo). Whereas GW4064 induced SHP expression more strongly than CDCA, the latter was more efficient in suppressing the induction of the hNTCP mRNA expression by GR/dexamethasone. This implies that the bile acid CDCA may also suppress hNTCP expression through additional mechanisms that do not depend on SHP. However, in support of the role for SHP in the regulation of the hNTCP gene, when a SHP expression plasmid was cotransfected together with the GR expression construct, the dexamethasone-induced expression of the endogenous hNTCP mRNA was completely reversed (Fig. 9CGo).

SHP Suppresses Induction of Reporter-Linked hNTCP and hASBT Promoters by GR and Dexamethasone
After showing that the expression of the endogenous hNTCP gene is down-regulated by SHP, we next examined whether SHP can also suppress the GR/dexamethasone-dependent induction of the luciferase-linked hNTCP promoter region containing the newly identified GRE (hNTCP(–178/+67)). Cotransfection of SHP indeed counteracted the GR/dexamethasone induction of the reporter-linked hNTCP promoter (Fig. 10AGo), consistent with the results obtained for the endogenous hNTCP gene above.



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  		Fig. 10. Exogenous Expression of SHP Suppresses the Induction of the Reporter-Linked hNTCP and hASBT Promoters by GR/Dexamethasone

A, The transcriptional repressor SHP suppresses the induction of the luciferase-linked hNTCP promoter by GR/dexamethasone. Huh7 cells were transfected with the reporter construct hNTCP(–178/+67)luc or the pGL3basic vector, together with 100 ng of the pSG5-GR and pCMX-SHP expression constructs where indicated. B, The transcriptional repressor SHP suppresses the induction of the hASBT promoter by GR/dexamethasone. Huh7 cells were transfected with either the reporter construct hASBT(–1688/+525)luc or the pGL3basic vector, together with 100 ng of the pSG5-GR and pCMX-SHP expression constructs where indicated. dexa, Dexamethasone.

 
We have previously shown that the gene encoding the chief bile acid uptake system in human ileal enterocytes, hASBT, can be transactivated by GR in a ligand-dependent manner (31). Here we further tested whether the GR/dexamethasone stimulation of the luciferase-linked hASBT promoter can be suppressed by SHP, similarly to the hNTCP promoter. As shown in Fig. 10BGo, SHP overexpression was efficient in reversing the GR/dexamethasone induction of the hASBT(–1688/+525) construct, implying that GR and RAR{alpha}-RXR{alpha} (30) may be alternative or parallel targets for negative transcriptional interference by the bile acid-induced transcriptional repressor SHP.

PGC-1{alpha} Coactivates GR/Dexamethasone-Dependent Transcription of the hNTCP Gene
Several factors have been suggested to function as transcriptional coactivators for GR, in specific promoter contexts (43). We next tested whether the two factors PGC-1{alpha} and p300 coactivate GR in the context of the hNTCP and hASBT promoters. The exogenous expression of p300 had no effect on the basal or GR/dexamethasone-induced activity of either the hNTCP or hASBT promoter in transient transfections in Huh7 cells (data not shown). However, cotransfection of an expression plasmid for the coactivator PGC-1{alpha} further enhanced the GR/dexamethasone-induced activity of the hNTCP(–178/+67) promoter by a factor of two (Fig. 11Go), whereas baseline activity of this promoter construct was not affected by exogenous expression of PGC-1{alpha} alone (data not shown). Interestingly, PGC-1{alpha} was unable to coactivate the GR/dexamethasone- induced activity of the hASBT(–1688/+525) promoter (Fig. 11Go), implying that although both genes of the human SLC10 family are responsive to GR and glucocorticoids, the coactivators mediating transcriptional activation may be different for the two promoters.



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  		Fig. 11. PGC-1{alpha} Coactivates Transactivation of the hNTCP Promoter, but not the hASBT Promoter, by GR/Dexamethasone

Huh7 cells were transfected with either the reporter construct hNTCP(–178/+67)luc, hASBT(–1688/+525)luc, or the pGL3basic vector, together with 100 ng of the pSG5-GR (GR) and the pcDNA3.1-HA-PGC-1{alpha} expression construct where indicated. dexa, Dexamethasone.

 
It has been shown recently that the transcriptional repressor SHP can antagonize PGC-1{alpha}-mediated coactivation of GR, in the context of the phosphoenolpyruvate carboxykinase and mouse mammary tumor virus promoters (44). Given the inducibility of SHP expression by bile acids, and our finding in this study that hNTCP gene expression can be down-regulated by both bile acids and SHP, we next investigated whether SHP can negatively target coactivation of the hNTCP promoter by PGC-1{alpha}. Indeed, in addition to suppressing the GR/dexamethasone induction of the hNTCP(–178/+67) promoter, SHP also decreases the activity of the hNTCP promoter in the presence of exogenously expressed PGC-1{alpha} (Fig. 12Go).



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  		Fig. 12. SHP Suppresses the hNTCP Promoter Activity in the Presence of GR, Dexamethasone, and the Coactivator PGC-1{alpha}

Huh7 cells were transfected with the reporter construct hNTCP(–178/+67)luc or the pGL3basic vector, together with 100 ng of the pSG5-GR, pcDNA3.1-HA-PGC-1{alpha}, and the pCMX-SHP expression construct where indicated. dexa, Dexamethasone.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This study shows that the hNTCP gene, encoding the chief uptake system for conjugated bile acids from portal circulation into human hepatocytes, is a novel transcriptional target for induction by glucocorticoids via their affinity to the GR. Both the endogenous hNTCP gene in hepatoma cells (Fig. 1Go) and the reporter-linked hNTCP promoter construct in transiently transfected cells (Fig. 2Go) were inducible by GR and its agonistic ligand dexamethasone. Whereas the rat Ntcp gene has been previously shown to be subject to multihormonal regulation (45, 46, 47, 48), our study is the first to demonstrate the effects of a steroid hormone on hNTCP gene expression.

GR is not the only transcription factor that is activated by glucocorticoids. In addition, the master regulator of xenobiotic metabolism, the pregnane X receptor (PXR), responds to glucocorticoid ligands, such as dexamethasone (15). The dependence of hNTCP induction upon exogenous expression of GR (Figs. 1Go and 2Go) strongly implies that the effect of dexamethasone on the hNTCP promoter involves GR, but not PXR. Moreover, the low concentration (100 nM) of dexamethasone used here is not sufficient to activate PXR, which requires significantly higher micromolar concentrations of dexamethasone (49). Finally, the antiglucocorticoid RU486 is an antagonist for GR; however, it efficiently activates PXR (50). In our study, treatment of cells with RU486 led to potent suppression of the dexamethasone induction of the hNTCP promoter (Fig. 3Go), further confirming that GR, but not PXR, is responsible for the induction of hNTCP by glucocorticoids.

A DNA element arranged as a direct repeat of hexamers separated by nine nucleotides (DR-9) at nt –32/–12 of the hNTCP promoter mediates the glucocorticoid response (Figs. 4–7GoGoGoGo), and GR directly interacts with this motif, as shown in antibody-facilitated DNA precipitation assays (Fig. 8Go). Of note, promoter regions both up- and downstream of this element are required for maximal GR/dexamethasone induction of the hNTCP promoter (Fig. 4Go), suggesting that there are other factors binding to DNA sequences elsewhere within the promoter that assist or cooperate with GR in the glucocorticoid induction, e.g. through enhanced recruitment of coactivators, or via stabilization of GR DNA binding through protein-protein interactions. GR has previously been shown to be engaged in protein-protein interactions resulting in either synergistic or antagonistic actions on its target promoters (51, 52, 53).

The DR-9 element of the hNTCP promoter identified here as a functional GRE is not entirely conserved in mice and rats. Although the sequence of the hexameric repeats is identical in the regulatory regions of the two rodent Ntcp genes (mouse: nt –45/–27, rat: nt +26/+44; relative to the respective transcription start sites) when compared with the human counterpart, the spacing between the hexamers is seven instead of nine nucleotides in the context of the rodent promoters (12). We have tested the responsiveness of the luciferase-linked mouse Ntcp promoter containing the DR-7 element to GR and its ligand dexamethasone and observed that it also responds to glucocorticoids, albeit to a lower degree than the human counterpart (data not shown). These experiments were performed in human hepatoma cells, and it is conceivable that, to achieve full induction of the mouse promoters, a complete mouse-specific transcriptional machinery may be required. Consistent with a transactivating role for glucocorticoids, administration of corticosterones could rescue the levels of hepatic Ntcp mRNA expression reduced by adrenalectomy in rats (48). Furthermore, treatment of rat primary hepatocytes with dexamethasone resulted in moderate induction of the endogenous rNtcp gene.

We have previously identified the gene encoding the other member of the human SLC10 family of Na+/bile acid cotransporters, hASBT, as a direct target for agonist-dependent transactivation by GR (31). It is thus interesting to speculate that glucocorticoids could in a coordinated fashion regulate the expression of the two major bile acid uptake systems in the liver and intestine, thereby controlling the efficiency of the enterohepatic circulation of bile acids. We note that the configuration and relative location of the GREs differ greatly for the two glucocorticoid-regulated genes discussed here.

Induction of the hNTCP gene by GR in the presence of glucocorticoids can be enhanced by coexpression of the transcriptional coactivator PGC-1{alpha} (Fig. 11Go). Whereas PGC-1{alpha} was originally isolated through its interaction with the nuclear receptor, peroxisome proliferator-activated receptor-{gamma} (54), it has subsequently been shown capable of coactivating several other DNA-binding factors (55). Consistent with our results showing coactivation of GR by PGC-1{alpha} in the context of the hNTCP promoter, Knutti et al. (56) isolated PGC-1{alpha} in a genetic yeast screen on the basis of its interaction and ligand-dependent coactivation of GR. Recently, it has been reported that PGC-1{alpha} is also a potent coactivator of the nuclear bile acid receptor FXR (57, 58, 59). Our current finding that PGC-1{alpha} regulates a crucial bile acid transporter further supports a pivotal role for this transcriptional coactivator in the control of bile acid metabolism. Interestingly, activation of the other member of the human SLC10 gene family, hASBT, by GR was not enhanced by PGC-1{alpha}, indicating that PGC-1{alpha} can coactivate GR in a promoter-specific manner. Alternatively, the endogenous level of the PCG-1{alpha} protein in Huh7 cells is sufficient to achieve maximal coactivation of the hASBT promoter. Several other coactivators, such as the members of the p160 family and several ATP-dependent chromatin remodeling complexes, have been suggested to interact with GR (43), and future studies will reveal whether any of these regulate the hASBT promoter.

Elevated levels of intracellular bile acids, such as in cholestasis, initiate complex transcriptional programs in hepatocytes, aiming to protect cells from the inherent toxicity of bile acids (20). The uptake of bile acids into hepatocytes from sinusoidal blood is reduced in cholestasis, due to reduced expression of NTCP/Ntcp in both rodents (22, 23) and humans (8). In rats, bile acid-mediated suppression involves inhibition of the activity of the RAR{alpha}-RXR{alpha} heterodimer binding to the Ntcp promoter (25, 36). However, no transcriptional targets for the bile acid-mediated down-regulation of the hNTCP promoter have been previously suggested. In this study we show that transactivation of the hNTCP gene by GR/dexamethasone can be reversed by the bile acid CDCA (Fig. 9AGo) and propose that GR is a target for negative feedback regulation of hNTCP expression by bile acids in humans. Bile acids suppress transcription of their negative target genes either by inducing a FXR target gene encoding the transcriptional repressor SHP (26, 27) or by inducing FXR- and SHP-independent pathways (60, 61). We achieved efficient suppression of GR/dexamethasone-mediated induction of the hNTCP gene by cotreatment of cells with the specific FXR ligand GW4064 (Fig. 9AGo). Thus, although we cannot exclude that there may be parallel FXR-independent elements involved in the suppression of the hNTCP promoter by bile acids, our results demonstrate that the FXR-dependent pathway can suppress the induction of the hNTCP gene by glucocorticoids. Exogenously expressed SHP similarly suppresses the induction of both endogenous hNTCP mRNA expression (Fig. 9CGo), and of the reporter-linked hNTCP promoter (Fig. 10AGo). In addition, the glucocorticoid induction of the reporter-linked hASBT promoter was efficiently suppressed by overexpression of SHP (Fig. 10BGo), implying that GR is an alternative target for negative feedback regulation of the hASBT gene by bile acids, in addition to the RAR{alpha}-RXR{alpha} heterodimer suggested previously (30). Finally, SHP can decrease the GR/dexamethasone-dependent hNTCP promoter activity even in the presence of the coactivator PGC-1{alpha} (Fig. 12Go). Borgius et al. (44) have suggested that SHP-mediated suppression of PGC-1{alpha}-mediated coactivation of the phosphoenolpyruvate carboxykinase and mouse mammary tumor virus promoters may be due to competition between PGC-1{alpha} and SHP over the same interaction surface in GR. A similar mechanism may be operational on the hNTCP promoter. Alternatively, SHP may negatively interfere with the binding of GR to the hNTCP promoter, as recently suggested in the case of inhibition of the transcription factor HNF-3 by SHP (62). Additionally, SHP-mediated recruitment of transcriptional corepressors may contribute to suppression of the hNTCP promoter (63).

Could glucocorticoids or antiglucocorticoids be employed therapeutically to alter the expression level of hNTCP, and consequently the efficiency of hepatic extraction of bile acids? No data are available describing secondary effects of glucocorticoids or antiglucocorticoids on bile acid homeostasis in patients receiving such treatments. Our recent data (unpublished) indicate that in the case of the hASBT gene, endogenous glucocorticoids are sufficient to achieve high levels of GR-dependent gene expression in healthy human intestine. Thus, GR may be important for maintaining a sufficient level of hASBT gene expression in the normal intestine, rather than being an inducer of hASBT gene expression in states of elevated glucocorticoid levels. It is of interest to establish whether glucocorticoid levels in normal human liver are similarly sufficient to contribute to the basal level of hNTCP transcription, or whether the baseline activity of the hNTCP gene is maintained by other transcription factors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals
GW4064 was a gift from Dr. Daniel Berger (GlaxoSmithKline, Uxbridge, UK). Restriction enzymes were purchased from Roche Diagnostics (Rotkreuz, Switzerland), the pureTaq Ready-to-Go PCR beads were obtained from Amersham Biosciences (Otelfingen, Switzerland), and the LigaFast Rapid Ligation Kit was purchased from Promega Catalys (Wallisellen, Switzerland). FuGENE 6 (Roche Diagnostics) was used for all transfections at a ratio of 3 µl per µg of DNA. The oligonucleotides were synthesized by Microsynth (Balgach, Switzerland). Other chemicals were purchased from Sigma-Aldrich (Buchs, Switzerland), unless stated otherwise.

Plasmids
The pSG5-GR, pcDNA3.1-HA-PGC-1{alpha}, and pCMX-SHP constructs were kind gifts from Dr. K. Yamamoto (San Francisco, CA), Dr. A. Kralli (La Jolla, CA), and Dr. David Mangelsdorf (Dallas, TX), respectively. All hNTCP promoter deletions and their variants containing point mutations were created by standard PCR methods, using human genomic DNA (BD Biosciences CLONTECH, Basel, Switzerland) as a template and oligonucleotide primers with engineered restriction sites, listed in Table 1Go. The PCR-derived fragments were subcloned into the pGL3basic luciferase reporter vector (Promega Catalys) using appropriate restriction enzymes. To create the heterologous promoter constructs, single-stranded oligonucleotides corresponding to the GRE variants (Table 1Go) were annealed, and the double-stranded products with sticky ends compatible with HindIII and BamHI were subcloned into the tk-luc vector (64). All hNTCP promoter constructs were verified by DNA sequencing. The hASBT(–1688/+525)luc construct has been described previously (65).


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  		Table 1. Sequences of Oligonucleotides Used for Cloning, Mutagenesis, Real-Time PCR, and Antibody-Facilitated DNA Precipitation Analysis

 
Cell Culture
The human hepatoma cell line Huh7 (LGC Promochem, Molsheim Cedex, France) was cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, Basel, Switzerland). Cells were cultured at +37 C in a humidified atmosphere containing 5% CO2.

Transient Transfections and Reporter Assays
Cells were seeded in 48-well plates at a density of 1 x 105 cells per well and cotransfected with 400 ng of the luciferase constructs, together with the indicated amounts of the effector expression plasmids. To normalize the total amount of DNA transfected, pcDNA3.1 vector (Invitrogen) or pSG5 vector (Stratagene, La Jolla, CA) was added. To control for transfection efficiency, 100 ng of the pSV-ß-galactosidase reporter plasmid (Promega Catalys) was cotransfected. Where indicated, the medium was supplemented with 100 nM dexamethasone, 1 µM RU486, or the corresponding volumes of the vehicle ethanol 12 h after transfection. Cells were harvested 36 h after transfection in Passive Lysis Buffer (Promega Catalys), and luciferase activities were determined using the Luciferase assay system (Promega Catalys) in a Lumat LB 9507–2 luminometer (Berthold Technologies, Regensdorf, Switzerland). ß-Galactosidase activities were quantified by a chlorophenol red-ß-D-galactopyranoside-based colorimetric assay (66) in a microplate reader (Molecular Devices, Sunnyvale, CA). Reporter activities obtained for the empty pGL3basic corresponding to each test condition, as well as for the test construct containing the test promoter in the control conditions, are set to 1, and fold activities are shown relative to this. All transfection experiments were performed at least three times, each experiment containing triplicate wells for each set of conditions. Results from representative experiments are expressed as mean fold activities ± SDs.

RNA isolation, Reverse Transcription, and Real-Time PCR
Cells were seeded in six-well plates and, after reaching 80% confluency, transfected with 2 µg of the indicated expression plasmids or empty vectors. The medium was supplemented, 12 h after transfection, with 100 nM dexamethasone, 1 µM RU486, 50 µM CDCA, 200 nM FXR agonist GW4064, and the vehicles ethanol (for dexamethasone and RU486) or dimethylsulfoxide (for CDCA and GW4064) as indicated. Total RNAs were extracted 36 h after transfection using the Trizol reagent (Invitrogen). Of each RNA preparation, 2 µg was reverse transcribed by random priming (Reverse Transcription System, Promega Catalys). Of each resulting cDNA, 10 µl, from a final reaction volume of 100 µl, was used for real-time PCR, which was performed on an ABI PRISM 7700 sequence detection system (Applied Biosystems, Rotkreuz, Switzerland) using the SYBR Green PCR Master Mix (QIAGEN, Hombrechtikon, Switzerland) and the primers listed in Table 1Go. Constitutively expressed 18S rRNA was used as an internal standard for sample normalization. Relative levels of the hNTCP and SHP mRNAs were calculated using the {Delta}{Delta}CT (comparative threshold cycle) method. The cut-off limit was set to 40 cycles, and where no detectable mRNA levels were achieved by this, the value 40 was used in the {Delta}{Delta}CT formula. Each test was performed as a triplicate. The levels of the hNTCP and SHP mRNAs are expressed relative to the control sample, which was set to 1. All experiments were repeated three times, and representative results are shown.

Antibody-Facilitated DNA Precipitation Assays
Cells grown in 10-cm plates to 80% confluency were transfected with 2 µg of constructs containing either the wild-type GRE of the hNTCP promoter or the nonfunctional GRE containing two base changes (see Fig. 5Go). Both constructs were cotransfected with 2 µg of the pSG5-GR expression plasmid or of the empty vector pSG5. The medium of the cells exogenously expressing GR was supplemented with 100 nM dexamethasone 12 h after transfection, whereas the pSG5-transfected cells were treated with the vehicle ethanol. The samples were subjected 24 h later to antibody-facilitated DNA precipitation assays, performed as described (67, 68), with certain modifications. Briefly, the cells were cross-linked with 1% formaldehyde for 10 min at room temperature, shaking gently. Cross-linking was terminated by adding glycine to a final concentration of 0.125 M. After washing with ice-cold PBS including Complete protease inhibitors (Roche Diagnostics), the cells were scraped into 1 ml wash buffer and pelleted by centrifugation at 700 x g for 3 min at +4 C. Cell pellets were resuspended in 1.0 ml of the cell lysis buffer (67) and incubated on ice for 10 min. Nuclei were collected by a centrifugation at 2000 x g for 5 min at +4 C, resuspended in 1.0 ml of the nuclear lysis buffer (67) and incubated on ice for 10 min. Next, the DNAs were sheared into fragments of an average length of 400–500 bp using a Branson Sonifier 250 (Branson Ultrasonics, Danbury, CT). After this, the debris was removed by centrifugation for 10 min at 20,000 x g at +4 C. The supernatants were diluted 5-fold in dilution buffer [16.7 mM Tris-HCl, pH 8.0; 1.2 mM EDTA; 167 mM NaCl; 0.01% (wt/vol) sodium dodecyl sulfate (SDS); 1.1% (vol/vol) Triton X-100; Complete protease inhibitors]. The samples were precleared using 80 µl of Protein G Sepharose Fast Flow (Amersham Biosciences) beads coated with herring sperm DNA, rotating for 30 min at +4 C. After pelleting the beads with a 15-sec centrifugation at 10,000 x g, 0.5 ml of each supernatant was removed to be used as an input sample in the PCR (see Fig. 8Go). The remainder of the precleared supernatants was divided into two 2-ml aliquots, and 2 µg of the rabbit polyclonal anti-GR antibody (E-20 X; Santa Cruz Biotechnology, Santa Cruz, CA) was added to one aliquot. Samples were rotated overnight at +4 C. Herring sperm-coated Protein G Sepharose (40 µl) was added to all samples, and the immune complexes were collected by rotating for 1 h at +4 C. The sepharose-bound immunocomplexes were pelleted by a centrifugation at 3220 x g for 10 min at +4 C. The beads were then successively washed with 5 ml of low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and twice with TE buffer (68). Each wash was performed by rotating for 5 min at +4 C, and by collecting the beads by centrifugations at 3220 x g for 10 min at +4 C. After the washes the immune complexes were eluted by adding 0.25 ml elution buffer (68) per sample and shaking for 15 min at room temperature. The elution step was repeated and the eluates were combined. The reversal of cross-links and proteinase K treatment were performed as described (67). The final volume of each coimmunoprecipitated DNA sample was 50 µl, of which 1 µl was used as a template in PCR using pureTaq beads (Amersham Biosciences) and primers (400 nM of each) flanking the promoter region in the transfected constructs (GL2 and RV3 in Table 1Go). Samples of 5 µl were taken after 25, 30, and 35 cycles and analyzed on 2% agarose gels.

Preparation of Protein Extracts and Immunoblotting
Cells were seeded in six-well plates and, after reaching 80% confluency, transfected with 2 µg of the pSG5-GR expression construct or the empty pSG5 vector., the medium was supplemented 12 h after transfection with 100 nM dexamethasone, 50 µM CDCA, 200 nM FXR agonist GW4064, and the vehicles ethanol (for dexamethasone) or dimethylsulfoxide (for CDCA and GW4064) as indicated. To prepare whole-cell extracts, cells were washed with ice-cold PBS 36 h after transfection, lysed by a 5 min incubation in 250 µl of ice-cold lysis buffer [50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% (vol/vol) Igepal CA-630; 0.5% (wt/vol) Na-deoxycholate; 1 mM EDTA; 0.1% (wt/vol) SDS; 10% (vol/vol) glycerol], supplemented with Complete protease inhibitors (Roche Diagnostics). The debris were removed by centrifugation for 30 min at 14,000 rpm at +4 C. Protein concentrations were determined with the bicinchoninic acid protein assay (Pierce) and the samples were stored at –80 C until usage. whole-cell extracts (4 µg) were separated on 15% SDS polyacrylamide gels (Mini-PROTEAN 3 cell, Bio-Rad Laboratories, Reinach, Switzerland) and electroblotted (Mini Trans-Blot Cell, Bio-Rad Laboratories) onto nitrocellulose membranes (Hybond ECL, Amersham Biosciences). Membranes were blocked overnight in 5% (wt/vol) nonfat milk in PBS-T [0.1% (vol/vol) Tween-20 in PBS]. After this, the membranes were probed with an antibody against the human SHP (SH2G5-C; Alexis Corp., Lausen, Switzerland) at a concentration of 0.3 µg/ml in 5% (wt/vol) nonfat milk/PBS-T, for 1 h. After three washes with 5% (wt/vol) nonfat milk/PBS-T, the horseradish peroxidase-conjugated goat antirat antibody (Santa Cruz Biotechnology) was added at a concentration of 0.2 µg/ml in 5% (wt/vol) nonfat milk/PBS-T for 1 h. Blots were then washed three times with 5% (wt/vol) nonfat milk/PBS-T and twice with PBS, followed by detection with the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and exposure on Hyperfilm ECL (Amersham Biosciences). To verify equal loading of the protein samples, 9% SDS polyacrylamide gels were run in parallel, electroblotted, and probed for constitutively expressed Ku-70 antigen. The Ku-70 probing and detection were performed as above, except that the Ku-70 antibody (C-19; Santa Cruz Biotechnology) was used at a concentration of 50 ng/ml, and a horseradish peroxidase-conjugated rabbit antigoat antibody (DakoCytomation, Zug, Switzerland) was used as the secondary antibody at a concentration of 167 ng/ml.


   ACKNOWLEDGMENTS
 
We thank Dr. Keith Yamamoto, Dr. David Mangelsdorf, Dr. Anastasia Kralli, and Dr. Daniel Berger for generously donating the GR expression plasmid, the SHP expression plasmid, the PGC-1{alpha} expression plasmid, and the compound GW4064, respectively. The expert technical assistance of Ms. Claudia Seitz and Mr. Christian Hiller is gratefully acknowledged. We also thank Drs. Stephan Vavricka, Jean-François Landrier, Michael Saborowski, and Yvonne Meier for discussions and helpful comments on the manuscript. Professors Michael Fried, Peter Meier-Abt, and Bruno Stieger are acknowledged for their support.


   FOOTNOTES
 
This work was supported by Grant PP00B-108511/1 from the Swiss National Science Foundation.

First Published Online August 25, 2005

Abbreviations: ASBT, Apical sodium-dependent bile acid transporter; CDCA, chenodeoxycholic acid; DR-9, direct repeat of two hexamers separated by nine nucleotides; FXR, farnesoid X receptor; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HNF, hepatocyte nuclear factor; nt, nucleotide; NTCP, Na+-taurocholate cotransporting polypeptide; PBS-T, PBS-Tween 20; PGC, peroxisome proliferator-activated receptor-{gamma} coactivator; PXR, pregnane X receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; SHP, small heterodimer partner; SDS, sodium dodecyl sulfate.

Received for publication April 20, 2005. Accepted for publication August 17, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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