1Laboratory of Molecular Gastroenterology and Hepatology and 2Division of Clinical Pharmacology and Toxicology, Department of Internal Medicine, University Hospital, CH-8091 Zurich, Switzerland
Submitted 23 October 2003 ; accepted in final form 25 December 2003
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ABSTRACT |
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organic anion transport; bile acids and salts; cholestasis; transcription factors; cytokines; liver receptor homolog; farnesoid X receptor
The exact molecular mechanism of decreased NTCP/Ntcp expression in cholestasis is unresolved. The rat Ntcp gene has been reported to be downregulated by the transcriptional repressor small heterodimer partner (SHP) 1 due to its interference with retinoid activation of the retinoid X receptor/retinoic acid receptor dimer (RXR/RAR
) (7, 8). However, the role of SHP is debatable. Bile acid feeding decreases Ntcp expression to the same degree in SHP knockout (SHP-/-) mice as in SHP+/+ mice, indicating that bile acids can repress the Ntcp gene through SHP-independent mechanisms (43). Such mechanisms could include activation of the xenobiotic pregnane X receptor (PXR), activation of the c-Jun NH2-terminal kinase, or bile acid-mediated repression of the transcriptional activator hepatocyte nuclear factor-1
(HNF1
) (20, 43). Whereas several mechanisms that regulate the rat Ntcp gene have been identified (7, 8, 11, 22, 39), little is known about the regulation of the NTCP/Ntcp gene in human and mouse. It is unknown, for instance, whether HNF1
and RXR
/RAR
have the same activating effect on human and mouse NTCP/Ntcp as on rat Ntcp (7, 8, 22).
To compare the transcriptional regulation of the human and mouse NTCP/Ntcp promoter with that of rat Ntcp, we isolated the 5'-regulatory regions of the human and mouse genes and compared sequences with the rat gene by computer alignment. A highly conserved sequence was identified that contained several cis-acting elements previously shown to regulate the rat Ntcp gene. We report the first in vitro characterization of the human and mouse NTCP/Ntcp promoters compared with the rat and show that the role of liver-enriched transcription factors and nuclear receptors in governing the transcriptional regulation of the NTCP/Ntcp gene differs considerably between species.
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MATERIALS AND METHODS |
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Cell culture and reporter gene assay. Huh7 and chicken hepatoma (LMH) cells were cultured and transfected as described (20, 21). In case of ligand treatment, the following ligands were added 18 h after transfection: 1 µM arotinoid acid, 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid/1 µM 9-cis retinoic acid (9cRA), 100 µM chenodeoxycholic acid (CDCA)/1 µM 9cRA, 100 µM deoxycholic acid (DCA)/1 µM 9cRA, 100 µM cholic acid (CA)/1 µM 9cRA or DMSO, and/or ethanol as controls.
Electrophoretic mobility shift assays. Dimerized oligonucleotides (Microsynth, Balgach, Switzerland) with sequences corresponding to the NTCP/Ntcp gene or a perfect HNF3 binding site (Table 1) were labeled with [
-32P]adenosine triphosphate (3,000 Ci/mmol; Amersham Pharmacia Biotechnology, Dübendorf, Switzerland) using T4 polynucleotide kinase (Stratagene). For gel mobility shift assays, 2 µl of in vitro translated HNF1
protein (TnT Quick coupled transcription/translation system; Promega Catalys) or 5 µl Huh7 or HepG2 nuclear extracts were incubated as described previously (20, 21). HNF3
(sc-6554), HNF4
(H-171), and RXR
(D-20) antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany).
Statistical analysis. Reporter gene activities are expressed as the means ± 1 SE of four to eight individual transfection experiments. All data were reproduced at least once using two different preparations of plasmid DNA.
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RESULTS |
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To compare the transcriptional regulation of the conserved 5'-regulatory region between species, appropriate fragments were PCR amplified from genomic DNA and cloned into the luciferase reporter vector pGL3-Basic. Constructs Hu-Luc, Mo-Luc, and Ra-Luc showed sequence identity with the accession numbers AF184235 [GenBank] , AF190698 [GenBank] , and L76612 [GenBank] , respectively. In transfected Huh7 hepatoma cells, all constructs showed relevant luciferase activity compared with the promoterless control vector pGL3-Basic (Fig. 2B).
Regulation of the NTCP/Ntcp gene by HNF1 and CEBP-
. The rat Ntcp gene contains a highly conserved HNF1
recognition site located within the transcription start site, previously shown to bind HNF1
(22, 39). A 4-bp insertion in the corresponding DNA sequence of the human and mouse NTCP/Ntcp gene results in a disrupted HNF1
binding site but creates a consensus motif for the CCAAT/enhancer binding protein-
(CEBP-
; Fig. 1). To assess the effect of HNF1
and CEBP-
on the different NTCP/Ntcp reporter constructs, expression vectors were cotransfected. Coexpressed HNF1
led to a 3.5-fold increase in rat promoter activity, whereas CEBP-
did not affect the rat Ntcp promoter (Fig. 3, A and C). Conversely, the human and mouse NTCP/Ntcp constructs were not activated by HNF1
(Fig. 3A), whereas CEBP-
increased luciferase activity by 40% (Fig. 3C).
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Absence of HNF1 binding to the human and mouse sequence was further supported by mobility shift assays. Only a rat-derived oligonucleotide (Table 1) was able to bind in vitro translated HNF1
protein (Fig. 3B). The specificity of the complex was confirmed by competition and supershift analyses. No binding occurred using the corresponding sequence of the human or mouse NTCP/Ntcp genes.
HNF3 represses NTCP/Ntcp promoter function. HNF3
represents the only transcription factor with conserved binding sites in the 5'-regulatory region of the human, mouse, and rat NTCP/Ntcp genes (Fig. 1). In cotransfection experiments, HNF3
decreased luciferase activity of all promoter constructs (Hu-Luc -66%, Mo-Luc -41%, Ra-Luc -64%), suggesting that HNF3
may directly repress the NTCP/Ntcp gene (Fig. 4A). To verify that HNF3
binds to the 5'-regulatory region, electrophoretic mobility shift assays were performed using labeled oligonucleotides that corresponded to the HNF3
-I binding motif of the human and rodent genes (Fig. 1) or to a perfect HNF3
binding site (perHNF3
in Table 1). In the presence of nuclear extracts from Huh7 cells, a specific DNA-protein complex was formed with both the human and rodent binding motif (Fig. 4B). This complex was competed off in the presence of excess unlabeled human and mouse or rat oligonucleotides, respectively. Using a labeled perHNF3
oligonucleotide, complex formation was again competed off by the human and rodent binding motifs, whereas a mutated sequence (mutHNF3) did not inhibit (Fig. 4C). Specificity of the DNA-protein complexes formed with both the NTCP/Ntcp-derived binding motifs as well as the perHNF3
binding motif was confirmed by supershift analyses (Fig. 4, B and C).
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The nuclear receptors RXR/RAR
and HNF4
selectively activate the rat Ntcp construct. The nuclear receptor heterodimer RXR
/RAR
is an important activator of the rat Ntcp gene (7, 22, 26). The RXR
/RAR
response element is part of the conserved region and extends from nt -56 to -37 in the rat sequence. Computer analysis of the conserved 5'-regulatory region failed to identify an RXR
/RAR
binding site in the human and mouse NTCP/Ntcp genes. In accordance with the computer prediction, only the rat but not the human or mouse NTCP/Ntcp constructs were induced by ligand activated RXR
/RAR
(Fig. 5A). Of note, a putative binding site for HNF4
is located within the RXR
/RAR
sequence. Coexpressed HNF4
increased the activity of the rat Ntcp promoter construct twofold, suggesting a possible functional role of the identified consensus motif. In contrast, HNF4
had no effect on the activity of the human or mouse NTCP/Ntcp constructs (Fig. 5B), despite the presence of a consensus motif at nt -43/-26 of the human sequence.
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To investigate whether HNF4 activates rat Ntcp through direct binding to the putative binding site or through an indirect mechanism such as induction of HNF1
(45), two additional rat promoter deletional constructs (-12-Ra-Luc and +28-Ra-Luc) and a construct containing a mutated HNF1
binding site (mutH1-Ra-Luc) were generated (Fig. 6A). As shown in Fig. 6B, coexpressed HNF4
again increased the activity of the initial rat Ntcp promoter construct (Ra-Luc). In contrast, deletion of the HNF4
binding site in constructs -12-Ra-Luc and +28-Ra-Luc abolished this activation, whereas the full-length construct with a mutated HNF1
site was still activated (Fig. 6B). These data suggested that HNF4
transactivates through direct binding to the rat Ntcp promoter. To verify binding of HNF4
, electrophoretic mobility shift assays were performed using labeled oligonucleotides that corresponded to the HNF4
-RXR
/RAR
binding motif of the rat Ntcp gene (Fig. 1, Table 1). In the presence of nuclear extracts from HepG2 cells, two specific DNA-protein complexes were formed with the rat binding motif (Fig. 6C). These complexes were competed off in the presence of excess unlabeled rat HNF4
oligonucleotide but not by unlabeled human or mouse oligonucleotides, respectively. Specificity of the DNA-protein complexes formed with the rat HNF4
-RXR
/RAR
binding motif was confirmed by supershift analyses (Fig. 6C). Addition of antibody against HNF4
resulted in a supershift (top arrow in Fig. 6C) and attenuated the formation of the HNF4
DNA-protein complex (bottom arrow in Fig. 6C). Addition of antibody against RXR
abolished formation of the RXR
/RAR
DNA-protein complex (middle arrow in Fig. 6C). These data confirmed binding of both factors to the HNF4
-RXR
/RAR
response element in the rat Ntcp promoter.
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Neither bile acids nor the transcriptional repressor SHP inhibit baseline NTCP/Ntcp promoter function. The rat Ntcp gene is thought to be repressed by bile acids via induction of the small heterodimer partner SHP through bile acid-activated farnesoid X receptor (FXR). To test this hypothesis, we used the LMH cell line, previously shown to possess conserved FXR signaling pathways (17, 21). CDCA treatment of LMH cells transfected with the different NTCP/Ntcp promoter constructs resulted in only a slight decrease in luciferase activity of the mouse and rat promoters. In contrast, the bile acid DCA decreased the activity of the rat but not the mouse promoter, indicating that only the rat promoter is in any way responsive to bile acids (Fig. 7A). In contrast, a promoter construct of the human HNF1 gene, previously shown to contain a classic "bile acid response element" (20), was markedly suppressed as expected (Fig. 7A), confirming that the bile acid-controlled gene regulatory cascade was functional in LMH cells. It is of note that the bile acid CA, which has not been described as a functional FXR ligand (29), had no effect on any promoter construct analyzed (Fig. 7A).
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To further elucidate the role of SHP, Huh7 cells were cotransfected with the NTCP/Ntcp constructs and an expression plasmid coding for SHP. As a positive control, we again employed the HNF1 construct, which is transcriptionally repressed by SHP (20). As shown in Fig. 7B, baseline promoter function of all three NTCP/Ntcp constructs was not affected by SHP. Because one mechanism by which SHP represses transcription is through decreased transactivation of the target gene by the liver receptor homolog 1 [LRH; also called fetal transcription factor (FTF) in humans] (2, 13, 28), we studied whether cotransfection of LRH or FTF activates the NTCP/Ntcp constructs. Coexpression of LRH or FTF had no detectable effect on NTCP/Ntcp promoter activity (data not shown). Taken together, these results indicate that neither CDCA nor SHP repress the NTCP/Ntcp gene promoter via a classic bile acid response element, as shown for certain bile acid-synthesizing enzyme genes as well as for the human HNF1
gene (6, 13, 20, 28, 47). Decreased expression of Ntcp in rat models of cholestasis is probably not attributable to a direct repressive effect of bile acids but rather to indirect effects such as cytokine-mediated inhibition of RXR
/RAR
or bile acidmediated repression of HNF1
and HNF4
(5, 7, 20).
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DISCUSSION |
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To examine the regulation of the NTCP/Ntcp gene, we first studied HNF1, a known regulator of basolateral bile acid transporter genes (35). The rat Ntcp gene contains a highly conserved HNF1
response element, previously shown to bind HNF1
(22). In this study, we showed that coexpression of HNF1
increases rat Ntcp promoter activity, confirming its role as a transcriptional activator (Fig. 3). However, neither the human nor the mouse NTCP/Ntcp promoter constructs were activated by HNF1
. Mobility shift assays indicated that the 4-bp insertion in the human and mouse NTCP/Ntcp promoter region disrupts the HNF1
binding site present in the rat, explaining the lack of activation. In place of the HNF1
site, a consensus motif for the liver-enriched transcription factor CEBP-
is present in the human and mouse NTCP/Ntcp gene. Both human and mouse constructs are activated by CEBP-
in cotransfection assays, whereas the rat Ntcp construct shows no induction (Fig. 3C). Members of the CEBP family have been implicated in regulating the differentiation of certain mammalian cells, including adipocytes and hepatocytes (1, 30, 34). CEBP-
could, therefore, contribute to the liver-specific expression of the NTCP/Ntcp gene in human and mouse.
Notwithstanding these data, the HNF1 knockout mice reported by Shih et al. (35), were shown to have almost absent Ntcp expression, suggesting that HNF1
binds at a different site in the mouse Ntcp gene. Generally, HNF1
can activate gene expression either through binding to a site near the transcription start site, as shown for the SLCO1B1 (protein name OATP1B1, formerly called OATP-C/OATP2), ASBT (SLC10A2), and NPT1 genes (3, 18, 19, 35), or through binding to sites that function as enhancers and are often located within intronic sequences, as shown for the aldolase B, HNF4, and NPT1 genes (3, 15, 27). Using a computer approach, we screened the proximal 1,000 bp of the promoter and all intronic sequences of the human and mouse NCTP/Ntcp genes for potential HNF1
binding sites. Although no HNF1
binding site was found within the promoter regions, we localized highly conserved motifs within intronic sequences (introns 1, 2, and 4 in human NTCP, intron 2 in mouse Ntcp). The exact role of these intronic HNF1
binding sites remains to be investigated.
Of note, the HNF1-/- strain generated by Pontoglio et al. (32) in our hands had almost normal Ntcp expression in Western blot analysis (data not shown). In contrast, expression of the apical sodium-dependent bile acid transporter (Asbt, Slc10a2) was strongly decreased (data not shown). In addition, we found that luciferase constructs of the rat Ntcp promoter containing the 5'-UTR but no HNF1
binding site were still active (Fig. 6B), suggesting that HNF1
is not critical for baseline promoter activity. In view of these discrepancies, the exact role of HNF1
in regulating the NTCP/Ntcp gene remains to be elucidated.
HNF3 was the only factor with conserved binding sites in all species (Fig. 1). Coexpressed HNF3
repressed the NTCP/Ntcp promoter constructs (Fig. 4A), and binding of HNF3
to the HNF3
-I consensus motif (Fig. 1) was verified by mobility shift assays (Fig. 4, B and C). HNF3
is an essential transcription factor during embryonic development and is thought to be a genetic initiator of the hepatic differentiation program (37, 46). However, the function of HNF3
in adult liver is not fully understood. Ntcp mRNA levels are substantially reduced in transgenic mouse hepatocytes overexpressing HNF3
, and serum bile acid levels are increased 50-fold (33, 38). In humans, increased expression of HNF3
in hepatocellular carcinomas is associated with decreased NTCP expression in Northern blot and immunofluorescence analyses (23, 41). These results suggest that HNF3
may repress NTCP/Ntcp gene expression in vivo.
Several studies (25, 40) have suggested a repressive effect of bile salts on the NTCP/Ntcp gene in view of the consistent decrease in NTCP/Ntcp expression that is found in cholestasis. In the case of the rat Ntcp gene, the repressive effect of bile acids has been proposed to involve inhibition of retinoid activation of the nuclear receptor dimer RXR/RAR
by the transcriptional repressor SHP (8). The RXR
/RAR
binding element in the rat Ntcp gene is part of the conserved region (Fig. 1). In the human and mouse genes, the binding site is disrupted by nucleotide exchanges (Fig. 1). Accordingly, only the rat Ntcp reporter construct is activated by retinoid ligand treatment, whereas the human and mouse promoters show no response (Fig. 5A). This probably represents a major difference in the regulation of the rat compared with the human and mouse NTCP/Ntcp genes, because the repressive effects of bile acids, cholestasis, and cytokines on Ntcp expression have been largely attributed to decreased transactivation by RXR
/RAR
(7, 8, 26). Clearly, binding of RXR
/RAR
to a sequence that is not part of the conserved 5'-regulatory region cannot be excluded for the human and mouse genes. The nucleotide sequence spanning the RXR
/RAR
site in the rat promoter also represents an HNF4
binding site, as shown by cotransfection and mobility shift assays in this study (Fig. 6). One can only speculate as to whether, in vivo, both factors, RXR
/RAR
and HNF4
, are corequisite for rat Ntcp gene expression or whether they have distinct functions under different physiological conditions.
Although bile acids were previously reported to inhibit retinoid activation of RXR/RAR
in the rat through an SHP-mediated mechanism, a direct suppressive effect of SHP on the Ntcp promoter has not been shown in an in vitro system. We, therefore, tested the hypothesis that SHP directly represses NTCP/Ntcp promoter activity. As an assay system, we employed LMH cells that possess a conserved FXR signaling pathway and have been used extensively as a model for ligand-dependent activation of endogenously expressed nuclear receptors (17, 18, 21). Using the LMH cell culture system, we did not find relevant repression of NTCP/Ntcp promoter activity by treatment of cells with the FXR ligands CDCA and DCA (Fig. 7A). In addition to the lack of repression by bile acids, overexpression of the nuclear receptor SHP was also without effect on the NTCP/Ntcp constructs (Fig. 6B). In contrast, a promoter construct of the HNF1
gene, previously shown to be negatively regulated by bile acids through an SHP-mediated pathway (20), was repressed by both bile acids (CDCA, DCA) and SHP as expected. The lack of a repressive effect of cotransfected SHP on the NTCP/Ntcp constructs is in agreement with in vivo studies in SHP-/- mice. CA feeding represses Ntcp expression to the same degree in SHP-/- mice as in wild-type SHP+/+ mice, indicating that the repressive effect of bile acids on Ntcp expression is not directly mediated by SHP (43). It is likely that other mechanisms are responsible for the negative regulation of NTCP/Ntcp by bile acids. One such mechanism could be the induction of cytokines by bile acids (4), because several transcription factors such as ligand-activated RXR
/RAR
, HNF1
, and HNF4
, which are important for the expression of rat Ntcp and other liver-specific genes, are suppressed by cytokines (39, 42). Because the rat Ntcp promoter binds and is transactivated by HNF4
(Figs. 1, 5B, and 6), decreased expression of rat Ntcp in cholestasis could be attributable to the known repressive effect of bile acids and cytokines on nuclear HNF4
levels (20, 42, 47). Moreover, bile acids block the association of HNF4
with its transcriptional coactivators, thereby inhibiting the transactivation of target genes of HNF4
(5). The latter mechanism has been shown to be a major pathway by which bile acids repress the cholesterol 7
-hydroxylase gene (5).
In summary, this study shows that the liver-enriched transcription factors HNF1, CEBP-
, HNF3
, and HNF4
and the nuclear receptor dimer RXR
/RAR
have an important but species-specific function in the regulation of the NTCP/Ntcp gene. The transcriptional regulation of NTCP/Ntcp thus differs among human, mouse, and rat. Of note, the conserved 5'-regulatory region of the NTCP/Ntcp gene does not possess a direct bile acid response element, suggesting that bile acids regulate NTCP/Ntcp expression through indirect mechanisms.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by Grant 632-062773 from the Swiss National Science Foundation (to G. A. Kullak-Ublick) and by a research grant from the University of Zurich (to D. Jung).
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FOOTNOTES |
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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.
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REFERENCES |
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