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Institut National de la Santé et de la Recherche Médicale, Unité 128, Institut Fédératif de Recherche 24 (J.C.O., P.M., M.-J.V., J.M.P.), Centre National de la Recherche Scientifique, 34293 Montpellier Cedex 05, France; Laboratoire de Pharmacologie et Toxicologie (F.L., T.P.), Institut National de la Recherche Agronomique, 31931 Toulouse, Cedex 9, France; Service de Chirurgie (J.M.F.), Hôpital Saint Eloi, 34295 Montpellier Cedex 05, France; and Service de Chirurgie Digestive (A.S.-C.), Hôpital Haut Levèque, 33600 Pessac, France
Address all correspondence and requests for reprints to: Jean Marc Pascussi, Institut National de la Santé et de la Recherche Médicale, Unité 128, Institut Fédératif de Recherche 24, Centre National de la Recherche Scientifique, 1919 Route de Mend, 34293 Montpellier Cedex 05, France. E-mail: pascussi{at}montp.inserm.fr.
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ABSTRACT |
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-hydroxylase (CYP7) expression, the rate-limiting enzyme in bile acid production from cholesterol. PXR (pregnane X receptor, NR1I2) is a broad-specificity sensor that recognizes a wide variety of synthetic drugs as well as endogenous compounds such as bile acid precursors. Upon activation, PXR induces CYP3A and inhibits CYP7, suggesting that PXR can act on both bile acid synthesis and elimination. Indeed, CYP7 and CYP3A are involved in biochemical pathways leading to cholesterol conversion into primary bile acids, whereas CYP3A is also involved in the detoxification of toxic secondary bile acid derivatives. Here, we show that PXR is a target for SHP. Using pull-down assays, we show that SHP interacts with both murine and human PXR in a ligand-dependent manner. From transient transfection assays, SHP is shown to be a potent repressor of PXR transactivation. Furthermore, we report that chenodeoxycholic acid and cholic acid, two farnesoid X receptor ligands, induce up-regulation of SHP and provoke a repression of PXR-mediated CYP3A induction in human hepatocytes as well as in vivo in mice. These results reveal an elaborate regulatory cascade, tightly controlled by SHP, for both the maintenance of bile acid production and detoxification in the liver. |
INTRODUCTION |
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SHP (short heterodimer partner) is an orphan nuclear receptor that lacks a conventional DBD and is mostly expressed in the liver (2). In spite of the lack of a DBD or of a conventional nuclear localization signal, it appears to be located in the nucleus of mammalian transfected cells (3). A series of reports have shown that SHP is able to interact with and inhibit the transcriptional activity of several members of the nuclear receptor superfamily including the constitutive androstane receptor (CAR), thyroid hormone receptor (TR), retinoid X receptor (RXR), retinoid acid receptor (RAR), androgen receptor, estrogen receptor (ER), the liver receptor homolog-1 (LRH-1), liver X receptor and ß, and hepatocyte nuclear factor-4 (HNF4) (3, 4, 5, 6, 7, 8, 9, 10, 11). SHP has been shown to inhibit RAR:RXR heterodimer DNA binding and to inhibit the transcriptional activity of RAR, mCAR, and ER. Moreover, as SHP contains a strong transcriptional repression domain in its C terminus (3), it has been suspected to act as a direct transcriptional repressor by recruiting conventional corepressors such as nuclear receptor corepressor, as reported with DAX-1 (DSS-AHC critical region on X chromosome, gene 1) (12). However, further analysis failed to demonstrate a direct interaction between SHP and nuclear receptor corepressor (3). Alternatively, SHP has recently been proposed to represent a new category of nuclear receptor coregulator, interfering directly with AF-1 and AF-2 coactivator factors such as coactivator four-and-a-half-LIM-only protein FHL2, steroid receptor coactivator-1 (SRC-1), transcription intermediary factor 2, or receptor interacting protein with a molecular mass of 140 kDa, and two possibilities have been considered in this respect: 1) SHP and AF-1/2 coactivators may compete for a common site; or 2) binding of SHP to the receptor may induce conformational changes leading to the dissociation of AF-1/2 coactivators from the receptors (3, 5, 13). Taken together, these studies suggest that SHP inhibits the transcriptional activity of nuclear receptors by several mechanisms.
Recently, SHP has been shown to be involved in the control of bile acid biosynthesis, the first and rate-limiting step of which is catalyzed by cytochrome P450 7 (CYP7), a liver-specific enzyme (14). Two different laboratories (4, 9) have reported that upon activation by primary bile acids such as chenodeoxycholic acid (CDCA), farnesoid X receptor (FXR) induces the expression of SHP, which then binds to and inhibits LRH-1, an orphan receptor that regulates CYP7 expression (15).
CYP3A enzymes are known to be involved in the oxidative metabolism of many xenobiotics as well as of endogenous compounds such as steroids (16). More recently, these enzymes have been shown to play a role in bile acid catabolism and biosynthesis (17, 18). Fuster and Wikvall (18) demonstrated that microsomal 25-hydroxylation of 5ß-cholestane-3,7,12-triol, a known bile acid precursor, is catalyzed mainly by CYP3A4. In addition, Honda et al. (19) demonstrated that microsomal 25- and 26-hydroxylation of the cholesterol side chain are catalyzed by cyp3a11 in CYP27-/- mice, and they provided strong arguments in favor of the implication of human CYP3A4 in a similar function. Notably, using human liver microsomes, they reported that the rates of 5ß-cholestane-3,7,12-triol 25- and 26-hydroxylation and 5ß-cholestane-3,7,12, 25-tetrol 23R-,24R-, 24S- and 27-hydroxylation are strongly correlated with the rate of 6ß-hydroxylation of testosterone, a marker of CYP3A4. Finally, Cheng et al. (20) reported in 1977 that phenobarbital, a well-known CYP3A inducer, produces an increase in the rate of hydroxylation of 5ß-cholestane-3,7,12-triol and 5ß-cholestane-3,7,12,25-tetrol in rats. These metabolite products can be either excreted from the body in the bile or urine or converted to cholic acid (CA).
PXR (pregnane X receptor) was first reported to control CYP3A gene induction upon activation by xenobiotic inducers such as phenobarbital and rifampicin (RIF) (21). More recently, it has been shown that PXR is a functional receptor of the bile acid precursors (5ß-cholestan-3-7-12-triol; and 5ß-cholestan-3-7-12-25-tetrol) (22, 23), which represent CYP3A substrate and product, respectively. PXR is also activated by secondary bile acid derivatives such as lithocholic acid (24). Upon activation, PXR controls elimination of these compounds by inducing CYP3A4 (21) and Oatp2 (24, 25) expression. However, the concentration of lithocholic acid required to activate PXR appears to be higher than those that occurs in vivo.
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A Series of Recent Reports Suggest that CYP3A Expression Is Negatively Regulated by FXR Ligands and/or FXR |
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TOPABSTRACTINTRODUCTIONA Series of Recent...RESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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RESULTS |
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TOPABSTRACTINTRODUCTIONA Series of Recent...RESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 35S-methionine-labeled murine PXR.1 interacted with the GST-murine SHP fusion protein (mSHP) in the presence of pregnenolone 16-carbonitrile (PCN) in a dose-dependent manner (lanes 5 and 6), whereas a very faint interaction was observed in the absence of PCN (lane 4). No interaction was observed with the control GST protein (lane 3). As expected, a ligand-independent interaction was observed between GST-mSHP and mCAR (lane 2), but no interaction was observed with radiolabeled luciferase protein (lane 1). A similar interaction was observed with an opposite approach between the GST-human (h) PXR fusion protein and 35S-methionine-labeled mSHP in the presence of RIF (Fig. 1B, lanes 2 and 3). As expected, luciferase protein failed to interact with the GST-hPXR fusion protein (lane 5), whereas no interaction was observed with the control GST protein (lane 4). Overall, these in vitro assays demonstrate that SHP interacts with PXR in a ligand-enhanced manner and suggest that PXR could be a target for SHP-mediated repression.
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. In these experiments, PCN and RIF were used as specific ligands of mPXR and hPXR, respectively. It clearly appears that cotransfection of increasing amounts of mSHP or hSHP expression plasmids resulted in a dose-dependent decrease in the ligand-induced transactivation of both reporter constructs by mPXR (Fig. 2A) or hPXR (Fig. 2B), with only approximately 20% of residual activity in the presence of the higher amount of SHP expression plasmids. This level of inhibition is close to that previously reported with mCAR and RXR, and stronger than that observed with RAR (2). Similar results were obtained in HepG2 cells (data not shown) so that SHP is able to interact with hPXR in two different cell lines. Consistent with the relatively weak ligand-independent interaction of SHP with PXR observed in vitro (Fig. 1
), SHP coexpression resulted in a moderate decreased of PXR transcriptional activity in the absence of ligand (20–40% of inhibition). This inhibition may result from different mechanisms including: 1) SHP vs. RXR competitive binding to PXR leading to impairment of PXR:RXR heterodimer binding to DNA as suggested for RAR:RXR (10); or 2) SHP-PXR interaction involving the repression domain and/or the LBD/AF-2 domain of PXR as reported for the ER (3).
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heterodimer does (Fig. 3, lane 4). We then verified that this complex is displaced by an excess of ER6 unlabeled oligonucleotide (lane 5). Interestingly, bacteria-expressed GST-SHP inhibited the binding of PXR:RXR heterodimer to the ER6 in a dose-dependent manner (lanes 8–9), whereas comparable amounts of GST did not (lanes 6–7). It is tempting to propose from these results that the inhibitory effect of SHP on PXR transcriptional activity results from the formation of a SHP:PXR heterodimer that is unable to bind the PXR-responsive element. However, as SHP is able to interact with RXR in the absence of ligand, this inhibitory effect could merely result from a depletion of RXR through the formation of SHP:RXR heterodimers.
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). The cotransfection of both mSHP or hSHP expression vectors resulted 1) in a moderate but dose-dependent reduction of pM-LBDhPXR basal transactivation of the reporter, and 2) a stronger decrease of the RIF-induced enhancement of luciferase expression. In addition, inhibitions of LBD-hPXR by hSHP appeared somewhat stronger that those observed with mPXR. These results show that the inhibitory effect of SHP on PXR might be mediated through the LBD/AF-2 domain of PXR.
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). More interestingly, whereas SHP inhibits PXR-mediated reporter gene expression, the cotransfection of increasing amounts of SRC-1 expression plasmid partially abrogates the inhibitory effect of SHP on the transcriptional activity of PXR and suggest that SHP may act as a negative AF-2 coregulator of PXR.
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) and in vivo in mice (Fig. 7). For this purpose, human hepatocytes were first pretreated for 72 h with increasing concentrations of CDCA (10–50 µM) and then treated for 16 h with 5 µM RIF. The level of CYP3A4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was examined at the same time by Northern blotting analysis. As expected, RIF provoked an induction of CYP3A4, whereas CDCA had no effect (Fig. 6, A and B). However, pretreatment of cells with CDCA resulted in a concentration-dependent inhibition of RIF-mediated induction of CYP3A4 mRNA, so that, in the presence of 50 µM CDCA, CYP3A4 mRNA expression was reduced by approximately 70% (n = 3, FT178, FT180, and FT215). As expected, CDCA treatment alone provoked an increase in SHP mRNA expression (5-fold induction) and a total inhibition of CYP7 mRNA expression as shown in Fig. 6, B and C. RIF-mediated CYP2B6 and CYP2C9 expressions were also decreased by CDCA pretreatment as shown in Fig. 6C, whereas PXR and CAR expression was not decrease by CDCA treatment. These results, obtained in a fully functional and highly differentiated cellular system, confirm that activation of SHP expression by a bile acid coincides with a strong reduction of PXR-mediated CYP expression. In addition, in CV1 cells cotransfected with the pM-LBDhPXR and a GAL4-responsive luciferase reporter gene, we observed no effect of CDCA nor CA on RIF-mediated luciferase expression (data not shown). These results rule out a possible antagonistic effect of these compounds on PXR.
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, cyp3a11 expression was strongly increased (
14-fold) by PCN. More interestingly, CA pretreatment significantly decreased PCN-mediated cyp3a11 expression (50% inhibition compared with control mice treated with PCN), whereas it moderately induced cyp3a11 expression (2-fold). As expected, SHP expression was strongly increased by CA pretreatment. Thus, although there is a 14-fold induction of cyp3a11 gene expression by PCN in mice fed a normal diet, this value falls to approximately 3- to 4-fold induction in mice fed a 1% CA-supplemented diet. |
DISCUSSION |
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(36), Oatp2 (25), and CYP3A (21). Thus, it has been proposed that PXR serves as a physiological sensor not only for xenobiotics but also for bile acid precursors (22, 23). Our results suggest that SHP is a negative regulator of PXR transcriptional activity. This conclusion derives from in vitro, cell culture, and in vivo experiments. GST pull-down assays demonstrated a direct interaction between SHP and both murine and hPXR which is enhanced by the presence of PXR ligands such as PCN and RIF, respectively. This interaction is specific as no binding was observed with our internal negative controls (GST alone or luciferase). Transient transfections in different cell lines and with different reporter constructs (i.e. native CYP3A4 promoter or repetitions of the minimal PXR responsive element), demonstrated that expression of SHP produced a dose-dependent inhibition of both murine and hPXR transcriptional activity and that this inhibition was reversed by the expression of SRC-1. This reversibility argues in favor of a functional and specific effect of SHP on PXR, and improves the understanding of the mechanism by which inhibition proceeds. Two complementary mechanisms can thus be proposed: 1) SHP interacts directly with PXR and weakens its binding to DNA as proposed previously for RAR; and 2) SHP blocks the AF-2 activation domain as proposed previously for ER (6).
We speculated that an overexpression of SHP would lead to a decrease in PXR activity and eventually to a decrease in CYP3A inducibility. SHP expression is under the control of FXR, which is activated by primary bile acids such as CA and CDCA (26). We have developed primary cultures of human hepatocytes in which many of the regulatory proteins (including aryl hydrocarbon receptor, CAR, PXR, glucocorticoid receptor, FXR, SHP) are expressed, whereas xenobiotic metabolism and hormonal regulation are fully maintained. As a consequence, the CYPs and others drug metabolizing enzymes are expressed and inducible in these cultures to an extent that is close to the in vivo situation (37, 38). The pretreatment of human hepatocytes with these compounds resulted in a clear overexpression of SHP. This overexpression was confirmed by the concomitant down-regulation of CYP7, the rate-limiting enzyme in bile acid biosynthesis. Indeed, CYP7 is known to be subject to primary bile acid feedback regulation via SHP (4). In agreement with our hypothesis, PXR activation of CYP3A4, CYP2B6 and CYP2C9 genes was inhibited by FXR activators in a dose-dependent manner. These effects cannot result from a toxicity due to CDCA treatment because both GAPDH and PXR mRNA expression were unaffected, and SHP mRNA level was up-regulated. Moreover, no detectable toxicity on cell culture has been noticed macroscopically (by examination under a microscope) or in terms of total RNA recovery. We finally used mice in an attempt to validate in vivo the observations made in vitro and in cellular models. Indeed, mice fed a CA-enriched diet exhibited a drastic decrease in PCN-induced cyp3a11 expression, whereas SHP was up-regulated.
The conversion of cholesterol to bile acids occurs exclusively in the liver. This process involves the translocation of cholesterol and intermediates through various compartments of the cell, where they encounter a wide variety of enzymes (CYP27A, CYP7, CYP8B, CYP3A, etc.) necessary for their ultimate conversion to the primary C24 bile acids, CDCA and CA. Many nuclear receptors (liver X receptor, FXR, PXR) control this process through a complex network in which substrates and end-products modulate the up- and down-regulation of specific enzymes. According to our results, PXR function is inhibited in the presence of CDCA or CA through the up-regulation of SHP. This is reminiscent of the SHP-mediated repression of LRH-1 induced CYP7 expression. CYP7, and to a lower extent CYP3A, promote cholesterol conversion into bile acids. The reason why primary bile acids repress PXR activity and, by consequence, the expression of those genes (including CYP3A) involved in the clearance of bile acids is still unclear. As CA and CDCA are nontoxic bile acid derivatives, in contrast to some bile acids precursors or secondary bile acids products, one explanation is that this process could prevent the over conversion of cholesterol into such toxic compounds through a regulatory feedback loop similar to that observed with CYP7 (Fig. 8).
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compared with wild-type mice (19, 22), whereas feeding of these mice diets containing CA or CDCA lead to up-regulation of SHP and down-regulation of Cyp3a11 or CYP7 (22). In conclusion, we have shown that SHP interacts with and inhibits the transcriptional activity of PXR. As PXR controls the inducible expression of CYPs and other genes involved not only in the metabolism and elimination of xenobiotics but also in the biosynthesis and catabolism of bile acids, the current results reveal a functional interaction between bile acid homeostasis and the xenobiotic-mediated CYP induction in which SHP appears to play a major role.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONA Series of Recent...RESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-carbonitrile (PCN), RIF, and culture medium additives were from Sigma (Saint Louis, MO). 
-(32P)deoxy (d)-ATP and -(32P)d-CTP were from Amersham International (Amersham, Buckinghamshire, UK).
Plasmids
The following plasmids have been described previously: pCR3-mCAR (39), pSG5-
ATG-hPXR, p(CYP3A4 XREM[-7800/+7200] -1100/+43)-pGL3-LUC (32), pSG5-mPXR.1, pSG5-hPXR and p(CYP3A1 DR3)3-tkCAT (21), CDM8-mSHP (2). pSG5-SRC-1 and GST-SRC-1 (amino acids 580–750) are from V. Cavailles, (Institut National de la Santé et de la Recherche Médicale, Montpellier, France). The pCMV-hSHP plasmid was given by Jun Takeda (40). The 17mx5-ßGlob-LUC containing five GAL4 binding sites upstream of the luciferase reporter gene is from P. Chambon (Institut de Génétique Moléculaire et de Biologie Cellulaire, Strasbourg, France). The pM-LBDhPXR expression vector was generated by inserting a PCR fragment corresponding to the +107/STOP amino acids of the hPXR ligand binding domain (LBD) in the pM vector (CLONTECH). GST-mSHP fusion construct was generated by cloning the SmaI/XhoI fragment of the CDM8-mSHP into the EcoIRC1/XhoI sites of the pGEX-4T (Amersham). GST-hPXR fusion construct was generated by inserting the PCR-generated hPXR (amino acids 1–431) using oligonucleotides 5'-CCTCAGCTACCTGTGATGCCG and 5'-GGGTGTGGGGAATTCACCACCATGGAGGTGAGACCCAAAGAAAGC primers into pGEX-4T digested by EcoIRC1.
Cell Culture and Transfections
CV1 cells (Monkey kidney) or HepG2 cells (human hepatocarcinoma) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 10% fetal calf serum, 100 mg/liter penicillin, and 100 mg/liter streptomycin (Life Technologies, Inc., Gaithersburg, MD). Transfection of plasmid DNA was performed in single batches with Fugene-6 (Roche Applied Science, Indianapolis, IN) as instructed by the manufacturer. Transfection was performed using 80,000 cells, and cell extracts were prepared and analyzed for luciferase or CAT and ß-galactosidase activities as described (32).
DNA Binding Assays
A P32-labeled oligonucleotide containing the human CYP3A4 PXRE (ER6, 5'-ATATGAACTCAAAGGAGGTCAGTG) motif was incubated for 20 min at room temperature with 1.5 µl of in vitro synthesized ATGhPXR, mRXR (coupled transcription-translation (TNT) reticulocyte lysate system, Promega, Madison, WI), with or without purified GST or GST-mSHP, or 250-fold molar excess of competitor unlabeled ER6 oligonucleotide as previously reported (41). GST and GST-mSHP was purified from bacteria-expressed fusion protein and dialysis against 1x EMSA buffer [10 mM Tris (pH 8.0), 100 mM KCl]. mSHP and GST were stored at 1 mg/ml in 1x EMSA buffer complemented with 10% glycerol. DNA-protein complexes were resolved on a 4% polyacrylamide gel (30:1 acrylamide:bis-acrylamide) in 0.5x TBE (1x TBE = 89 mM Tris, 89 mM boric acid, and 2 mM EDTA). Gels were dried and subjected to autoradiography.
In Vitro Interaction
35S-Methionine-labeled proteins were prepared by in vitro translation using the TNT-coupled transcriptional translation system according to the manufacturer’s instructions (Promega). GST fusion proteins were expressed in the Escherichia coli BL21 strain and purified using glutathione-sepharose-4B bead affinity chromatography as suggested by the vendor (Pharmacia, Uppsala, Sweden). The beads were subsequently washed and resuspended in 20 mM Tris (pH 8.0), 100 mM NaCl, 0.1% Nonidet P-40 buffer (NETN). GST proteins bound to glutathione-sepharose were incubated with 5 µl of 35S-methionine-labeled proteins in the presence of NETN buffer and 50 µM of indicated compound or 1% DMSO. After overnight incubation at 4 C with gentle agitation, agarose beads were extensively washed with NETN buffer and bound proteins were eluted in sample buffer and analyzed by SDS-PAGE. Gels were then stained with coomassie blue, incubated in an autoradiography enhancer (Dupont NEN, Boston, MA), dried and subjected to autoradiography at -70 C.
Primary Culture of Human Hepatocytes
Hepatocytes were prepared from lobectomy segments resected from adult patients for medically required purposes unrelated to our research program. The use of these human hepatic specimens for scientific purposes has been approved by the French National Ethics Committee. Hepatocytes were prepared and cultured according to the previously published procedure (42). The cells were plated into 60-mm plastic dishes precoated with collagen at 4 x 106 cells per plate in a total volume of 3 ml of a hormonally and chemically defined medium elaborated from a mixture of Williams’ E and Ham F12 (1:1 in volume). Forty-eight hours after plating, cells were cultured in the presence or absence of CDCA (10–50 µM) for 72 h. Cells were then treated with DMSO or 5 µM RIF for 16 h.
Animals and Treatments
Male mice (C57/BL6 from Charles River Laboratories, L’Arbresle, France, BL3EV05919) were housed in a pathogen-free animal facility under a standard 12-h light, 12-h dark cycle. After 1 wk of acclimatization, mice were fed ad libitum for 3 d with standard rodent chow (diet-1820 (4.5% lipids) Harlan, Ganat, France) or supplemented in house with 1% CA. PCN was injected for two successive daily administrations of 40 mg/kg body wet, in corn oil. The mice were killed 24 h following the last PCN administration.
Total RNA Purification and Northern Blot
Total RNA was extracted from frozen mice liver tissues or human hepatocytes using Trizol reagent (GIBCO BRL, Cergy-Pontoise, France) according to the manufacturer’s instructions and its purity was confirmed by spectrophotometry. For Northern blot experiments, 30 µg of total RNA were analyzed using (32P)-dCTP-labeled CYP3A4 and rat GAPDH cDNA probes as previously described (32). Probe for SHP was obtained after SmaI/XhoI digestion and purification of the CDM8-mSHP plasmid. Probe for CYP7 (716 nucleotides) was generated by RT-PCR using the following primers 5'-TCCAGCGACTTTCTGGAGTT and 5'-AAAGGGACTGTGTGGTGAGG (NM_000780). After PCR, the cDNA was cloned into pCR2-TOPO (Invitrogen, Cergy-Pontoise, France) and verified by sequencing. The signals were analyzed by quantifying the radioactivity with a PhosphoImager apparatus and ImageQuant software (both from Molecular Dynamics, Sunnyvale, CA).
Quantitative PCR
Quantification of GAPDH, CYP2B6, CYP2C9, PXR, and SHP mRNA was performed using the Roche Light Cycler apparatus. cDNA were synthesized from 1 µg of total RNA using the Superscript II first-strand synthesis system for PCR (Invitrogen) at 42 C for 60 min, in the presence of random hexamers. One tenth was used for PCR amplification. The following program was used: denaturation step 95 C, 8 min; 45 cycles of PCR (denaturation 95 C, 15 sec; annealing 65 C, 7 sec; elongation 72 C, 19 sec). In all cases, the quality of the PCR-product was assessed by monitoring a fusion step. Sense and reverse primers were as follows, respectively: GAPDH: 5'-GGTCGGAGTCAACGGATTTGGTCG and 5'-CAAAGTTGTCATGGATGACC, CYP2B6: 5'-GGCCATACGGGAGGCCCTTG and 5'-AGGGCCCCTTGGATTTCCG, CYP2C9: 5'-TCCTATCATTGATTACTTCCCG and 5'-AACTGCAGTGTTTTCCAAGC, PXR: 5'-TCCGGAAAGATCTGTGCTCT and 5'-AGGGAGATCTGGTCCTCGAT, SHP: 5'-CCAATGATAGGGCGAAAGAA and 5'-GCTGTCTGGAGTCCTTCTGG, CAR: 5'-CCGTGTGGGGTTCCAGGTAG, and 5'-CAGCCAGCAGGCCTAGCAAC, CYP7: 5'-CACCTTGAGGACGGTTCCTA and 5'-CGATCCAAAGGGCATGTAGT.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AF, Activation function; CA, cholic acid; CAR, constitutive androstane receptor; CDCA, chenodeoxycholic acid; CYP7, cholesterol 7-hydroxylase; DBD, DNA-binding domain; DMSO, dimtheylsulfoxide; FXR, farnesoid X receptor; GAL4, positive regulator of galactose inducible genes in yeast; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; HNF4, hepatocyte nuclear factor-4; h, human; LRH-1, liver receptor homolog-1; m, mouse; mSHP, murine SHP fusion protein; PCN, pregnenolone 16-carbonitrile; PXR, pregnane X receptor; RAR, retinoic acid receptor; RIF, rifampicin; RXR, retinoid X receptor; SHP, small heterodimer partner; SRC-1, steroid receptor coactivator-1; TR, thyroid hormone receptor; UT, untreated; XREM, xenobiotic-responsive element module.
Received for publication November 19, 2002. Accepted for publication June 4, 2003.
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 D. D. Moore, S. Kato, W. Xie, D. J. Mangelsdorf, D. R. Schmidt, R. Xiao, and S. A. Kliewer International Union of Pharmacology. LXII. The NR1H and NR1I Receptors: Constitutive Androstane Receptor, Pregnene X Receptor, Farnesoid X Receptor {alpha}, Farnesoid X Receptor beta, Liver X Receptor {alpha}, Liver X Receptor beta, and Vitamin D Receptor Pharmacol. Rev., December 1, 2006; 58(4): 742 - 759. [Abstract] [Full Text] [PDF]
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 A. K. Iyer, Y.-H. Zhang, and E. R. B. McCabe Dosage-Sensitive Sex Reversal Adrenal Hypoplasia Congenita Critical Region on the X Chromosome, Gene 1 (DAX1) (NR0B1) and Small Heterodimer Partner (SHP) (NR0B2) Form Homodimers Individually, as Well as DAX1-SHP Heterodimers Mol. Endocrinol., October 1, 2006; 20(10): 2326 - 2342. [Abstract] [Full Text] [PDF]
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T. Li and J. Y. L. Chiang RIFAMPICIN INDUCTION OF CYP3A4 REQUIRES PREGNANE X RECEPTOR CROSS TALK WITH HEPATOCYTE NUCLEAR FACTOR 4{alpha} AND COACTIVATORS, AND SUPPRESSION OF SMALL HETERODIMER PARTNER GENE EXPRESSION Drug Metab. Dispos., May 1, 2006; 34(5): 756 - 764. [Abstract] [Full Text] [PDF]
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S. Aouabdi, G. Gibson, and N. Plant TRANSCRIPTIONAL REGULATION OF THE PXR GENE: IDENTIFICATION AND CHARACTERIZATION OF A FUNCTIONAL PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR {alpha} BINDING SITE WITHIN THE PROXIMAL PROMOTER OF PXR Drug Metab. Dispos., January 1, 2006; 34(1): 138 - 144. [Abstract] [Full Text] [PDF]
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J. Orans, D. G. Teotico, and M. R. Redinbo The Nuclear Xenobiotic Receptor Pregnane X Receptor: Recent Insights and New Challenges Mol. Endocrinol., December 1, 2005; 19(12): 2891 - 2900. [Abstract] [Full Text] [PDF]
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 Y. Li, M. Choi, K. Suino, A. Kovach, J. Daugherty, S. A. Kliewer, and H. E. Xu Structural and biochemical basis for selective repression of the orphan nuclear receptor liver receptor homolog 1 by small heterodimer partner PNAS, July 5, 2005; 102(27): 9505 - 9510. [Abstract] [Full Text] [PDF]
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M. G. Yeo, Y.-G. Yoo, H.-S. Choi, Y. K. Pak, and M.-O. Lee Negative Cross-Talk between Nur77 and Small Heterodimer Partner and Its Role in Apoptotic Cell Death of Hepatoma Cells Mol. Endocrinol., April 1, 2005; 19(4): 950 - 963. [Abstract] [Full Text] [PDF]
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 T. Li and J. Y. L. Chiang Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7{alpha}-hydroxylase gene transcription Am J Physiol Gastrointest Liver Physiol, January 1, 2005; 288(1): G74 - G84. [Abstract] [Full Text] [PDF]
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J.-Y. Kim, H.-J. Kim, K. T. Kim, Y.-Y. Park, H.-A Seong, K. C. Park, I.-K. Lee, H. Ha, M. Shong, S. C. Park, et al. Orphan Nuclear Receptor Small Heterodimer Partner Represses Hepatocyte Nuclear Factor 3/Foxa Transactivation via Inhibition of Its DNA Binding Mol. Endocrinol., December 1, 2004; 18(12): 2880 - 2894. [Abstract] [Full Text] [PDF]
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J.-Y. Kim, K. Chu, H.-J. Kim, H.-A Seong, K.-C. Park, S. Sanyal, J. Takeda, H. Ha, M. Shong, M.-J. Tsai, et al. Orphan Nuclear Receptor Small Heterodimer Partner, a Novel Corepressor for a Basic Helix-Loop-Helix Transcription Factor BETA2/NeuroD Mol. Endocrinol., April 1, 2004; 18(4): 776 - 790. [Abstract] [Full Text] [PDF]
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C. Stedman, G. Robertson, S. Coulter, and C. Liddle Feed-forward Regulation of Bile Acid Detoxification by CYP3A4: STUDIES IN HUMANIZED TRANSGENIC MICE J. Biol. Chem., March 19, 2004; 279(12): 11336 - 11343. [Abstract] [Full Text] [PDF]
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Y. Shimamoto, J. Ishida, K. Yamagata, T. Saito, H. Kato, T. Matsuoka, K. Hirota, H. Daitoku, M. Nangaku, K. Yamagata, et al. Inhibitory Effect of the Small Heterodimer Partner on Hepatocyte Nuclear Factor-4 Mediates Bile Acid-induced Repression of the Human Angiotensinogen Gene J. Biol. Chem., February 27, 2004; 279(9): 7770 - 7776. [Abstract] [Full Text] [PDF]
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C. Handschin and U. A. Meyer Induction of Drug Metabolism: The Role of Nuclear Receptors Pharmacol. Rev., December 1, 2003; 55(4): 649 - 673. [Abstract] [Full Text] [PDF]
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