QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: jpet.107.124610v1 323/2/586    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fang, H.-L. Articles by Runge-Morris, M. Search for Related Content PubMed PubMed Citation Articles by Fang, H.-L. Articles by Runge-Morris, M.

METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Positive and Negative Regulation of Human Hepatic Hydroxysteroid Sulfotransferase (SULT2A1) Gene Transcription by Rifampicin: Roles of Hepatocyte Nuclear Factor 4 and Pregnane X Receptor

Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan (H.-L.F., Z.D., J.F., Z.D.-D., T.A.K., M.R.-M.); Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania (S.C.S., E.E.); and the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama (C.N.F., J.L.F.)

Received April 19, 2007; accepted August 7, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The effects of rifampicin treatment on SULT2A1 mRNA expression were evaluated in 23 preparations of primary cultured human hepatocytes. In contrast to the consistently occurring induction of CYP3A4, a prototypical pregnane X receptor (PXR) target gene, rifampicin treatment increased SULT2A1 mRNA levels in 12 of the hepatocyte preparations, but it produced little change or even suppression in the others. Transient transfection of HepG2 cells with a series of reporter constructs implicated two SULT2A1 5'-flanking regions as containing rifampicin-responsive information. Each of these regions contained a hepatocyte nuclear factor 4 (HNF4) binding site (at nucleotide [nt] –6160 and –54), as demonstrated by in vitro binding and site-directed mutagenesis. HNF4 bound to the HNF4-54 region of the endogenous SULT2A1 gene, as indicated by chromatin immunoprecipitation. Cotransfection of HepG2 cells with pregnane X receptor (PXR) dose-dependently suppressed reporter expression from SULT2A1 constructs containing the HNF4 sites, and rifampicin treatment augmented the suppression. Rifampicin treatment concentration-dependently suppressed SULT2A1 reporter expression at the same concentrations that progressively induced expression from a PXR-responsive CYP3A4 reporter, whereas higher rifampicin concentrations reversed the SULT2A1 suppression. The suppressive effect of rifampicin was diminished, whereas the activating effect was augmented, in HepG2 cells with RNA interference-mediated PXR knockdown. These results suggest that HNF4 plays a central role in the control of SULT2A1 transcription and that rifampicin-liganded PXR suppresses SULT2A1 expression by interfering with HNF4 activity. By contrast, the rifampicin-inducible SULT2A1 expression that occurs in many human hepatocyte preparations seems to be mediated through a PXR-independent mechanism.

In mice and rats, hepatic SULT2A transcription is transactivated by the PXR in concert with its heterodimeric partner, RXR (Xu and Kong, 2005). The PXR·RXR transcription factor transactivates rodent SULT2A transcription by binding to an IR0 nuclear receptor motif [inverted repeat of (A/G)G(G/T)TCA with 0 intervening bases] in the 5'-flanking region of the gene (Runge-Morris et al., 1999; Sonoda et al., 2002). The regulation of rodent hepatic SULT2A by PXR plays an essential role in bile acid metabolism. Cholestatic liver disease is characterized by the accumulation of toxic hydrophobic secondary bile acids such as lithocholic acid (Sonoda et al., 2002). The activation of PXR by lithocholic acid transactivates murine hepatic SULT2A expression and protects the liver from the damaging effects of lithocholic acid retention (Sonoda et al., 2002; Kitada et al., 2003). PXR also controls the transcription of hepatic transporters that are required for the recognition and transport of sulfonated conjugates in the liver (Sonoda et al., 2002; Xu and Kong, 2005). As a wide spectrum "xenosensor," PXR, along with other nuclear receptors, coordinates the transcriptional control of the xenobiotic-metabolizing and transporter networks in the liver that are essential for the detoxication of both endogenous and xenobiotic substrates (Mohan and Heyman, 2003; Klaassen and Slitt, 2005; Runge-Morris and Kocarek, 2005).

Our previous investigations in primary cultured rat hepatocytes suggested a dual role for PXR- and GR-mediated mechanisms in the transcriptional regulation of rat hepatic SULT2A expression (Runge-Morris et al., 1999). Pharmacological concentrations of dexamethasone were demonstrated to induce rat hepatic SULT2A expression via a PXR-mediated mechanism, whereas treatment with a GR-activating concentration of glucocorticoid transactivated SULT2A transcription indirectly, through intermediary steps involving GR-inducible C/EBP (Fang et al., 2005a).

In contrast to rodent systems, the role of PXR and other nuclear receptors in the transcriptional regulation of human SULT2A1 is just emerging. Activation of CAR, a mediator of phenobarbital-inducible gene expression, was shown to induce the expression of murine hepatic SULT2A, as well as of the Mrp4 bile acid transporter (Assem et al., 2004). In a microarray study, phenobarbital treatment of primary cultured human hepatocytes increased the expression of SULT2A1 mRNA, implicating a possible role for CAR in the regulation of human SULT2A1 (Assem et al., 2004).

Our recent studies indicate that important species differences pertain to the regulation of hepatic SULT2A by members of the nuclear receptor superfamily. Primary hepatocyte culture studies suggest that human, but not rat, hepatic SULT2A is transactivated by PPAR, acting through a peroxisome proliferator response element in the distal 5'-flanking region of the SULT2A1 gene (Fang et al., 2005b). Initial studies also suggested that PXR-activating concentrations of the potent glucocorticoid, dexamethasone, or of the prototypical human PXR ligand rifampicin are capable of inducing SULT2A1 gene expression (Duanmu et al., 2002; Fang et al., 2005b). The current study was undertaken to investigate the impact of PXR activation on human hepatic SULT2A1 expression and to identify the cis-acting sequences in the 5'-flanking region of the SULT2A1 gene that are essential for the modulation of human hepatic SULT2A1 transcription by PXR. Our investigation indicates that human hepatic SULT2A1 expression is subject to a balance of positively and negatively acting mechanisms. HNF4 is identified as a major positive regulator of human hepatic SULT2A1 transcription, and ligand-activated PXR is capable of suppressing HNF4 function. Data are also provided suggesting that the positive effects of rifampicin treatment on SULT2A1 transcription are mediated through a PXR-independent mechanism.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. Rifampicin was purchased from Sigma-Aldrich (St. Louis, MO). Other supplies and reagents were purchased from the sources described previously (Fang et al., 2005a,b) or that are described below.

Primary Cultured Human Hepatocytes. Plated primary cultures of human hepatocytes were obtained from the Liver Tissue Procurement and Distribution System of the University of Minnesota (Minneapolis, MN), in collaboration with Dr. Stephen Strom (University of Pittsburgh, Pittsburgh, PA). Following hepatocyte preparation and overnight culture, the hepatocytes, in T25 flasks, were express shipped to our facility. Upon receipt, medium was replaced with our standard human hepatocyte medium, consisting of Williams' medium E supplemented with 0.25 U/ml insulin, 0.1 µM triamcinolone acetonide, 50 µg/ml gentamicin, and 2.5 µg/ml amphotericin B, and the cells were maintained at 37°C under a humidified atmosphere of 95% air, 5% CO₂. The following day, medium was replaced with standard medium containing 200 µg/ml Matrigel (BD Biosciences, San Jose, CA). The following day, the medium was replaced with standard medium containing 0.1% DMSO or 10 or 50 µM rifampicin (in DMSO). The cultures were either harvested after 24-h incubation, or they were retreated at 24 h and harvested after a total of 48-h incubation.

TaqMan Real-Time Reverse Transcription-Polymerase Chain Reaction and Enzyme Activity Analyses. Total RNA was prepared from individual T25 flasks of primary cultured human hepatocytes, by using the ToTALLY RNA kit (Ambion, The Woodlands, TX). Samples of total RNA were reverse transcribed, using either Superscript II (Invitrogen, Carlsbad, CA) or the Omniscript RT kit (QIAGEN, Valencia, CA), according to the manufacturers' instructions. The levels of SULT2A1 and CYP3A4 were measured, using TaqMan Gene Expression Assays Hs00234219_m1 and Hs00430021_m1, respectively (Applied Biosystems, Foster City, CA). Each PCR reaction included cDNA template, a primer/probe (5-carboxyfluorescein fluor, minor groove binder quencher), a primer-limited primer/probe (VIC-minor groove binder) set for 18S rRNA and Universal PCR Master Mix (Applied Biosystems), and amplifications were performed using an ABI Prism 7500 Sequence Detection System (Applied Biosystems). Standard thermocycling parameters were 94°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Cycle threshold values were obtained using the SDS software package (Applied Biosystems). For each RNA sample, the target mRNA level was normalized to the 18S rRNA level. These normalized data were then expressed relative to the DMSO control group. The Wilcoxon sign-ranked test was used to compare median -fold changes against the hypothetical value of 1.0 (GraphPad Prism, version 5.0; GraphPad Software Inc., San Diego, CA). Correlation analysis was performed, using the nonparametric Spearman test (Prism).

For measurement of SULT2A1 activity, human hepatocyte monolayers (3 T25s per treatment group) were washed with ice-cold phosphate-buffered saline, scraped (replicates pooled) into Matrisperse (Collaborative Research, Bedford, MA), mixed end-over-end at 4°C for 30 min to remove traces of Matrigel, and centrifuged. The cell pellets were homogenized by sonication in buffer (50 µl per flask of hepatocytes) consisting of 10 mM potassium phosphate, 0.15 M potassium chloride, 1 mM dithiothreitol, and 10% glycerol, pH 7.4. Homogenates were centrifuged at 20,000g at 4°C for 20 min, and supernatants were centrifuged at 105,000g at 4°C for 1 h. SULT2A1 enzyme activities were measured in the cytosolic fractions, as described previously (Fang et al., 2005b).

Preparation of SULT2A1 Reporter Plasmids. A luciferase reporter plasmid containing nt –492 to +48 (hereafter designated –492: 48) of the SULT2A1 5'-flanking region has been described previously (Fang et al., 2005b). Deletion constructs containing nt –225:48, –191: 48, –165:48, –141:48, and –107:48 of the SULT2A1 5'-flanking region were amplified by PCR, using –492:48 as template and the primers shown in Supplemental Table 1. These amplified fragments were ligated into the MluI and XhoI sites of pGL3-Basic (Promega, Madison, WI). Reporter constructs containing a series of upstream fragments (1000 nt each) of the SULT2A1 5'-flanking region, extending to 8.8 kb upstream from the transcription start site, in tandem with the –492:48 promoter region, have been described previously (Fang et al., 2005b). To prepare reporter constructs containing this series of upstream fragments in tandem with the –107:48 promoter region, the upstream fragments were removed from the original constructs with KpnI and MluI and ligated into the corresponding sites of the –107:48 construct. In the same manner, fragments corresponding to SULT2A1 nt –6248:–5812, –6832:–6225, –6044:–5813, and –6248:–6025 were prepared by PCR and ligated into the KpnI and MluI sites of –107:48.

Site-Directed Mutagenesis. Candidate transcription factor binding sites were identified using MatInspector (Quandt et al., 1995). Mutations were introduced into core regions of predicted transcription factor binding sites using the QuikChange II XL site-directed mutagenesis kit, according to the manufacturer's instructions (Stratagene, La Jolla, CA). The targeted sites and mutagenic primer pairs are shown in Supplemental Table 1. The sequences of all constructs were verified using the resources of the Applied Genomics Technology Center at Wayne State University (Detroit, MI).

Transient Transfection Analysis. Approximately 250,000 HepG2 cells were seeded into the wells of 12-well plates and cultured in 2 ml of Dulbecco's modified Eagle's medium supplemented with 10% charcoal-stripped fetal bovine serum (Invitrogen), at 37°C under a humidified atmosphere of 95% air, 5% CO₂. The following day, culture medium was replaced with 0.6 ml of Opti-MEM (Invitrogen) containing a premixed complex of 6.25 µl of Lipofectin Reagent (Invitrogen) and plasmid DNA consisting of selected combinations of the following: 800 ng of a SULT2A1 luciferase reporter plasmid; 40 ng (or as otherwise indicated in the figure legends) of pSG5 (Stratagene); 40 ng (or as otherwise indicated in the figure legends) of pSG5-PXR (provided by Dr. Steven Kliewer, University of Texas Southwestern, Dallas, TX); 50 ng of pcDNA3.1 (Invitrogen); 50 ng pCMV-HNF4, expressing human HNF4 (provided by Dr. Erin Schuetz, St. Jude Children's Research Hospital, Memphis, TN); 1.25 ng of pRL-SV40 (to normalize for differences in transfection efficiency; Promega); and sufficient pBluescript II KS⁺ (Stratagene) to adjust total amounts of DNA to 1 µg. A PXR-responsive luciferase reporter plasmid containing the proximal and distal enhancer regions of the CYP3A4 gene (XREM-CYP3A4-Luc; provided by Dr. Bryan Goodwin, GlaxoSmithKline, Research Triangle Park, NC) (Goodwin et al., 1999) was used as a positive control. After 5-h incubation, transfection medium was replaced with fresh, unsupplemented Opti-MEM (i.e., serum-free). The following day, cultures were incubated with Opti-MEM containing either 0.1% DMSO or rifampicin (at the concentrations indicated in the individual figure legends), and they were harvested 24 h later for the measurement of firefly and Renilla luciferase activities by using the Dual Luciferase Reporter Assay System (Promega) and a Dynex model MLX Luminometer. Data were statistically analyzed using one-way analysis of variance followed by either the Newman-Keuls multiple comparison test or Dunnett's test (Prism).

RNA Interference. A set of five transduction-ready lentiviral particles (from the MISSION library, the RNAi Consortium) expressing siRNAs targeting human PXR was purchased, together with a lentivirus expressing a nontargeting control siRNA (Sigma-Aldrich), and used to transduce HepG2 cells, according to the protocol provided by MISSION siRNA. The transduced cells were maintained in the presence of 1.5 µg/ml puromycin, to select for cells containing stably integrated sequences. After several passages, total RNA was prepared, and PXR mRNA levels were measured using a TaqMan Gene Expression Assay (assay ID Hs01114267_m1; Applied Biosystems). The HepG2 cells transduced with lentivirus expressing siRNA TRCN0000021623 showed the greatest reduction in PXR mRNA content relative to nontargeting siRNA-transduced cells, and they were used in transient transfection experiments as described above.

Electrophoretic Mobility Shift Assay. EMSA was performed using in vitro transcribed and translated transcription factors, which were prepared using the TNT T7-coupled reticulocyte lysate system (Promega). HNF4 and PXR were prepared using the expression plasmids described above. Mouse PPAR and human RXR were prepared using expression plasmids provided by Dr. Eric Johnson (Scripps Research Institute, La Jolla, CA) and Dr. Steven Kliewer (University of Texas Southwestern, Dallas, TX), respectively. EMSA probes and unlabeled competitors are shown in Supplemental Table 1. Each annealed oligonucleotide pair contained a 2-nt 5' overhang at each end, and the probes were 3' end-labeled by filling-in with Klenow DNA polymerase and [-³²P]dATP. The in vitro transcribed/translated proteins (5 µl) were incubated with 50,000 cpm of a labeled probe for 20 min at room temperature in buffer containing 12 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 12% glycerol, and 1 µg of poly(dI-dC), in a total volume of 15 µl. The DNA-protein complexes were separated on native 4% polyacrylamide gels in 0.5x TBE running buffer, at 30 mA, for 1.5 h. For competition analysis, incubations included various amounts of unlabeled double-stranded oligonucleotides. For supershift analysis, 1 µl of an antibody directed against HNF4 (H-171), PXR (H-160), or RXR (D-20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the binding reaction.

Chromatin Immunoprecipitation. ChIP analysis was performed using the ChIP assay kit, according to the manufacturer's instructions (Upstate Biotechnology, Chicago, IL). HepG2 cells were grown in 100-mm dishes to 70 to 80% confluence in DMEM containing 10% charcoal-stripped fetal bovine serum. Some of the cultures were then transfected with 0.8 µg of pSG5-PXR, alone or in combination with 0.8 µg of pCMV-HNF4, using Lipofectin Reagent (the total amount of DNA per transfection was adjusted to 5 µgby the addition of pBluescript II KS⁺). The following day, the cultures were incubated with either Opti-MEM alone or containing either 0.1% DMSO or 50 µM rifampicin for 24 h. After treatment, medium was changed to fixing medium containing 1% formaldehyde, and the cultures were incubated for 10 min. Chromatin samples (average length 200–1000 nt) were prepared and processed according to the manufacturer's instructions. Before immunoprecipitation, chromatin samples were precleared, first by using protein G beads only, and second by using normal rabbit IgG with protein G beads. The precleared chromatin samples were then incubated with anti-HNF4 polyclonal antibody (H-171; Santa Cruz Biotechnology, Inc.) or rabbit IgG as negative control. PCR was performed using 2 µl of the DNA extracted from each immunoprecipitation reaction and using the primer pairs shown in Supplemental Table 1. PCR amplifications were performed for 33 to 36 cycles, and products were visualized on ethidium bromide-stained 2.5% agarose gels.

	Results

Top Abstract Materials and Methods Results Discussion References

 
In a previous report, we observed an 2-fold increase in SULT2A1 mRNA content when primary cultured human hepatocytes were treated with rifampicin, a prototypical ligand of the human PXR (Fang et al., 2005b). The reproducibility and concentration dependence of this observation were initially evaluated in three different preparations of human hepatocytes that were treated with 10 or 50 µM rifampicin (Fig. 1A). The effects of rifampicin treatment on SULT2A1 mRNA expression were compared with those on CYP3A4 expression, a well validated PXR target gene. Treatment with 50 µM rifampicin increased SULT2A1 mRNA content in each of the hepatocyte preparations, from 1.8 to 4.1-fold relative to DMSO-treated controls, while increasing CYP3A4 mRNA levels from 8.8 to 62-fold. The concentration-induction relationships for these two genes were somewhat different, in that the magnitude of SULT2A1 mRNA induction was always greater at 50 µM rifampicin than it was at 10 µM. By comparison, in two of the three hepatocyte preparations, CYP3A4 mRNA induction was maximal at 10 µM rifampicin.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Effects of rifampicin treatment on SULT2A1 and CYP3A4 mRNA levels in primary cultured human hepatocytes. A, concentration-dependent effects of rifampicin treatment on CYP3A4 and SULT2A1 mRNA levels in three preparations of human hepatocytes. Human hepatocyte cultures were incubated for 24 or 48 h with medium containing 0.1% DMSO (0 µM rifampicin; RIF), 10 µM RIF, or 50 µM RIF. After treatment, hepatocytes were harvested for the preparation of total RNA, and levels of CYP3A4 and SULT2A1 mRNAs were measured using TaqMan Gene Expression Assays, as described under Materials and Methods. For each hepatocyte preparation and gene, mRNA levels are presented as -fold changes relative to the amount measured in DMSO-treated cultures (each bar represents the mean ± S.D. of triplicate measurements performed on RNA isolated from one flask of hepatocytes). B, correspondence of rifampicin-mediated effects on CYP3A4 and SULT2A1 mRNA contents in 23 preparations of human hepatocytes. Human hepatocyte cultures were treated for 24 or 48 h with 0.1% DMSO or 50 µM rifampicin, and CYP3A4 and SULT2A1 mRNA levels were measured using TaqMan Gene Expression Assays. Each bar represents the mean ± S.D. of triplicate measurements performed on RNA isolated from one flask of hepatocytes. The rifampicin-mediated fold changes in CYP3A4 mRNA levels were used to sort the hepatocyte preparations from lowest to highest response (top). The rifampicin-mediated fold changes in SULT2A1 mRNA content, and the relative amounts of SULT2A1 mRNA measured in untreated cultures from the hepatocyte preparations (basal) are shown in the middle and bottom panels, respectively. The mean values and coefficients of variations (cv) that are shown summarize the data from all 23 hepatocyte preparations. For the SULT2A1 (RIF/DMSO) data, the p value is shown for the Wilcoxon signed rank test. The scatter plots show the correlations, with Spearman correlation coefficients and p values, between rifampicin-inducible CYP3A4 and SULT2A1 mRNA levels (top plot) and between rifampicin-inducible and basal SULT2A1 mRNA levels (bottom plot).

The effects of rifampicin treatment on SULT2A1 enzymatic activity were also determined in one of the hepatocyte preparations. Activities of 1.14 and 2.28 pmol of dehydroepiandrosterone sulfate formed per minute per milligram of protein were measured in cytosols prepared from cultures treated with DMSO or 50 µM rifampicin, respectively. This rifampicin-mediated -fold increase in SULT2A1 enzymatic activity (2.0-fold) corresponded closely to the mRNA change that was measured in this hepatocyte preparation (1.7-fold).

To investigate possible mechanisms underlying the complex regulation of SULT2A1 expression by rifampicin, we performed a series of transient transfection experiments using reporter constructs containing various amounts of the 5'-flanking region of the SULT2A1 gene. HepG2 cells were used for these studies, because this human liver cell model was previously found to be useful for investigating SULT2A1 gene regulation by peroxisome proliferators (Fang et al., 2005b). In preliminary experiments, we evaluated rifampicin treatment effects on luciferase reporter expression from the series of constructs that was used in our earlier study (Fang et al., 2005b), which contained progressively upstream 1000-nt regions of the SULT2A1 5' gene (to 8.8 kb from the transcription start site) linked to the first 492 nt of the SULT2A1 promoter, and we found that rifampicin treatment increased reporter expression from several of these constructs (data not shown). We therefore considered the possibility that information essential for mediating rifampicin-inducible SULT2A1 transcription might be contained within the first 492 nt of the SULT2A1 5'-flanking region that constituted the common SULT2A1 "core promoter" for the aforementioned reporter series (Fang et al., 2005b). This series of transfection experiments was performed using a reporter construct containing 492 nt of 5'-flanking sequence (SULT2A1 reporters are hereafter referred to by the 5' and 3' positions of the fragment, e.g., –492:48) and several deletion constructs (–225:48, –191:48, –165:48, –141:48, and –107: 48), and it was done in HepG2 cells that either contained only the endogenous amount of PXR or that were supplemented by cotransfection of a human PXR expression vector (Fig. 2A). In control HepG2 cells (i.e., containing endogenous PXR and treated with DMSO), reporter expression was decreased significantly (by 15%) in construct –225:48 compared with –492:48, suggesting that one or more cis-acting elements contained in the –492 to –225 region contributed to basal promoter activity. For example, several computer-predicted binding sites for liver-enriched transcription factors, HNF1, HNF4, HNF3B, and C/EBP are contained within this region. A significant decrease in promoter activity was also observed in construct –191:48 compared with –225:48 (49% decrease), and then in construct –141:48 compared with –165:48 (61% decrease). No further decrease in promoter activity was observed when the 5'-flanking region was shortened to 107 nt. A computer-predicted C/EBP binding site was present within the –165 to –141 region (CEBP-146), and mutation of this site within the context of construct –225:48 reduced promoter activity to the level seen for construct –107:48 (Fig. 2B), indicating a functional role for this C/EBP site, which is consistent with a recent report (Song et al., 2006).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Transient transfection analysis of the proximal SULT2A1 promoter for regions controlling basal activity and PXR and rifampicin responsiveness in HepG2 cells. A, HepG2 cells were transiently transfected with each of the indicated reporter plasmids together with either pSG5 (–PXR) or pSG5-PXR (+PXR), as described under Materials and Methods. Transfected cells were treated for 24 h with 0.1% DMSO or 50 µM rifampicin (Rif), and they were harvested for measurement of luciferase activities. For comparison, effects on expression from a PXR-responsive luciferase reporter construct (XREM-CYP3A4-Luc) are shown. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements (three wells per treatment group). *, **, and *** represent significantly different from the previous deletion construct (e.g., –225:48 versus –492:48, –191:48 versus –225:48, etc.) treated in the same manner. #, ##, and ### represent significantly different from the corresponding DMSO-treated group. The numbers adjacent to the # symbols indicate the Rif-induced -fold changes. +, ++, and +++, significantly different from the corresponding pSG5-transfected group. Approximate positions of several computer-predicted liver-enriched transcription factor binding sites, including a C/EBP site at nt –146 and an HNF4 site at nt –54 relative to the transcription start site, are indicated. B, HepG2 cells were transfected with SULT2A1 luciferase reporter plasmid –107:48 or –224:48, or with –224:48(mutCEBP-146), in which the C/EBP-binding site at nt –146 was mutated. ***, Significantly different from the –107:48 construct treated in the same manner. For statistical symbols, one, two and three copies indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

Rifampicin treatment of HepG2 cells transfected with construct –492:48 produced a significant increase of 1.5-fold in reporter expression, relative to the respective DMSO-treated control (Fig. 2A). Significant rifampicin-mediated increases of 1.3- to 1.4-fold in promoter activity were also observed for constructs –225:48, –191:48, and –165:48, and increases of 1.3-fold were still maintained for constructs –141:48 and –107:48, suggesting that the essential information for achieving rifampicin-mediated inducibility may be contained within the first 107 nt of the SULT2A1 promoter, and that other elements within the 492 region may function largely to amplify SULT2A1 expression levels. The –191 to –165 region of SULT2A1 contains an IR2 (inverted repeat with two intervening bases) motif that has recently been implicated as a vitamin D-response element (Song et al., 2006). There was no significant difference in the basal or rifampicin-inducible promoter activities obtained with constructs –191:48 and –165:48, suggesting that the IR2 motif does not play a functional role in rifampicin-mediated SULT2A1 promoter activation (Fig. 2A).

To evaluate the role of PXR in rifampicin-regulated SULT2A1 transcription, HepG2 cells were transiently transfected with human PXR (Fig. 2A). As a positive control for these experiments, reporter expression from the PXR-responsive XREM-CYP3A4-Luc construct was evaluated. Reporter expression from XREM-CYP3A4-Luc was increased 4.9-fold by rifampicin treatment in HepG2 cells containing only endogenous PXR, whereas in PXR-transfected cells, both basal and rifampicin-inducible reporter expression were increased, and the rifampicin/control -fold response was 7.3-fold. By comparison, PXR transfection suppressed the promoter activities of the SULT2A1 constructs, relative to the respective activities that were measured in HepG2 cells containing only endogenous PXR. Moreover, unlike the positive effects of PXR supplementation on rifampicin-inducible expression from XREM-CYP3A4-Luc, PXR transfection decreased, or even abolished, the rifampicin-mediated increases in SULT2A1 promoter activity (Fig. 2A).

To evaluate the SULT2A1 5'-flanking region for additional upstream sequences that might contribute substantially to rifampicin-mediated SULT2A1 regulation, constructs containing the aforementioned 1000-nt regions were transiently transfected into HepG2 cells (Fig. 3A). For these experiments, the upstream fragments were re-engineered into reporter constructs containing the –107:48 fragment as core promoter, since the above-described studies suggested that –107:48 region was not only sufficient to confer rifampicin-mediated regulation but also demonstrated that the magnitude of expression from the –107:48 construct was relatively low, thereby facilitating our ability to detect upstream sequences with enhancer activity. Although most of the upstream regions provided little enhancement to SULT2A1 promoter activity, the presence of the –6832:–5812 region markedly increased basal promoter activities while maintaining 1.4-fold rifampicin-mediated induction. When the –6832:–5812 region was divided, all activity was contained within the downstream portion (–6248:–5812), and when this latter region was subdivided, the rifampicin-inducible activity was contained within the –6248:–6025 fragment (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Transient transfection analysis of the SULT2A1 5'-flanking sequence for regions controlling basal activity and rifampicin responsiveness in HepG2 cells. A and B, HepG2 cells were transiently transfected with each of the indicated reporter plasmids. Transfected cells were treated for 24 h with 0.1% DMSO or 50 µM Rif, and they were harvested for measurement of luciferase activities. For comparison, effects on expression from a PXR-responsive luciferase reporter construct (XREM-CYP3A4-Luc) are shown. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements (3 wells per treatment group). The approximate position of a computer-predicated HNF4 binding site (at nt –6160 relative to the transcription start site) is indicated (arrowhead).

View larger version (39K): [in this window] [in a new window]   Fig. 4. EMSA analysis of HNF4 or PXR·RXR binding to a candidate HNF4 binding site located 6160 nt upstream of the SULT2A1 transcription start site. A, The sequence of a computer-predicted HNF4-binding site at nt –6160 of the SULT2A1 5'-flanking region is indicated, together with the matched TRANSFAC matrices for HNF4 and the PXR/CAR half-site. Capitalized letters in the matrices indicate core nt. n, A, T, G, or C; r, A or G; and k, G or T. B, in vitro binding reactions contained 32P-labeled double-stranded oligonucleotide probe corresponding either to a consensus HNF4-RE or the predicted HNF4-binding site at nt –6160 (6160) and in vitro transcribed/translated HNF4 [or no protein (–) or protein from in vitro transcribed/translated pcDNA3.1 empty vector (V)]. For competition experiments, reactions also contained the indicated -fold excess amounts of unlabeled oligonucleotide corresponding to the consensus HNF4-RE (HNF4), 6160, or 6160 with mutated HNF4 core sequence (6160m). For supershift experiments, reactions were supplemented with an antibody directed against HNF4 (+). Positions of free probe, HNF4-shifted bands and supershifted bands are indicated. C, binding reactions contained 32P-labeled double-stranded oligonucleotide probe corresponding either to a consensus PXRE or the predicted HNF4-binding site at nt –6160 (6160). The indicated samples (+) also contained in vitro transcribed/translated human PXR, human RXR or PXR and RXR in combination. Selected samples also contained 50-fold excess of unlabeled consensus PXRE (P) or 6160, or an antibody directed against PXR (P) or RXR (R). Positions of free probe, PXR·RXR-shifted bands and supershifted bands are indicated.

Computational analysis of the –107:48 region also suggested the possible presence of an HNF4 binding site at –54 (Fig. 5A), although this site was only identified when the search stringency was reduced somewhat from optimal (i.e., from matrix similarity > optimized matrix threshold to matrix similarity > optimized matrix threshold –0.04). The analysis also identified this site as a candidate PPAR binding site (Fig. 5A). As described above, EMSA was used to evaluate the ability of in vitro transcribed/translated HNF4 to bind to the HNF4-54 site (Fig. 5B), and it indicated that HNF4 bound specifically to both the consensus HNF4-RE probe and the HNF4-54 probe, with both bands completely supershifted by HNF4 antibody. Competition analysis revealed no difference in the abilities of unlabeled consensus HNF4-RE and HNF4-54 site to reduce the intensity of the HNF4-shifted band, whereas HNF4-54 competitor containing a mutated HNF4 core sequence was markedly less effective. EMSA was also used to evaluate the abilities of PPAR·RXR and PXR·RXR to bind to the HNF4-54 site (Fig. 5C), and it indicated that although both heterodimers were able to bind to probes containing their respective consensus motifs, PPAR·RXR, but not PXR·RXR, was able to bind to HNF4-54. ChIP analysis confirmed the ability of HNF4 to interact with the endogenous SULT2A1 gene in the region containing the HNF4-54 site (Fig. 6, top left, PCR product –164 to +66), but not a region predicted to be devoid of any nuclear receptor motifs (Fig. 6, top right, PCR product –11,748 to –11,605). HNF4 association with the HNF4-54 region was detected under several conditions of treatment (i.e., DMSO or 50 µM rifampicin) and transfection (i.e., PXR and HNF4).

View larger version (44K): [in this window] [in a new window]   Fig. 5. EMSA analysis of HNF4, PPAR·RXR, or PXR·RXR binding to a candidate HNF4 binding site located 54 nt upstream of the SULT2A1 transcription start site. A, sequence of a computer-predicted HNF4-binding site at nt –54 of the SULT2A1 5'-flanking region is indicated, together with the matched TRANSFAC matrices for HNF4 and PPAR. Capitalized letters in the matrices indicate core nt. n, A, T, G, or C; r, A or G; k, G or T; and w, A or T. B, in vitro binding reactions contained 32P-labeled double-stranded oligonucleotide probe corresponding either to a consensus HNF4-RE or the predicted HNF4-binding site at nt –54 (54) and in vitro-transcribed/translated HNF4 [or no protein (–) or protein from in vitro transcribed/translated pcDNA3.1 empty vector (V)]. For competition experiments, reactions also contained the indicated amounts of unlabeled oligonucleotide corresponding to the consensus HNF4-RE (HNF4), 54 or 54 with mutated HNF4 core sequence (54m). For supershift experiments, reactions were supplemented with an antibody directed against HNF4 (+). Positions of free probe, HNF4-shifted bands and supershifted bands are indicated. C, binding reactions contained 32P-labeled double-stranded oligonucleotide probe corresponding either to a consensus HNF4-RE (C under HNF4), a consensus PPRE (C under PPAR·RXR), a consensus PXRE (C under PXR·RXR), the predicted HNF4-binding site at nt –54 (54) or 54 with a mutated core sequence (54m). The indicated samples also contained in vitro transcribed/translated HNF4, PPAR and RXR, or PXR and RXR. Positions of free probe and shifted bands are indicated.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Chromatin immunoprecipitation analysis of HNF4 association with the SULT2A1 5'-flanking region containing HNF4-54 in HepG2 cells. HepG2 cells were either not transfected or were transfected with pSG5-PXR alone or in combination with pCMV-HNF4. Each transfection group was then treated for 24 h with either 0.1% DMSO (–) or 50 µM rifampicin (+). Cultures were fixed and harvested for the preparation of sheared chromatin, and immunoprecipitations were performed using an antibody directed against HNF4, or with nonspecific IgG, as a negative control. Following immunoprecipitation, associated DNA was amplified with a primer pair targeting the SULT2A1 gene region –164 to +66 (containing HNF4-54) or –11,748 to –11,605 (containing no predicted HNF4 site). Ethidium bromide-stained agarose gels of the PCR products are shown. For comparison, amplifications derived from equivalent amounts of unprecipitated chromatin DNA are also shown (input).

To evaluate the functional significance of the HNF4-54 and HNF-6160 sites in the regulation of SULT2A1 transcription, transient transfection experiments were performed using various SULT2A1 reporter constructs containing either intact or mutated HNF4-54 or HNF4-6160 sites (Fig. 7). In the –107:48 construct, containing only the HNF4-54 site, mutation of that site reduced reporter expression by 80% (Fig. 7A). The simple linkage of the HNF4-6160 site to the –107:48 fragment produced a construct with 2.4 times greater promoter activity than the –107:48 construct, and mutation of HNF4-54 in this construct again markedly suppressed reporter expression (Fig. 7A). When the two HNF4 sites were evaluated within the context of a longer SULT2A1 reporter construct, specifically the (–6832:–5812)(–107:48) construct that displayed the greatest activity in our earlier experiment (Fig. 3A), mutation of HNF4-54 caused a significant reduction in promoter activity, whereas mutation of HNF4-6160 had no significant effect, and the double mutation of HNF4-54 and HNF4-6160 did not cause any greater reduction than occurred following mutation of HNF-54 alone (Fig. 7A). Also in this experiment, the two (–6832:–5812)(–107:48) constructs containing an intact HNF-54 site showed significant increases in reporter expression following rifampicin treatment, whereas those containing a mutated HNF-54 site were not significantly increased by rifampicin (Fig. 7A). When the HNF4-6160 site was mutated within the context of the smaller –6248:–6025 fragment, which retained substantial constitutive and rifampicin-inducible activity (Fig. 3B), a significant reduction of promoter activity occurred that was further reduced when the HNF4-54 site was also mutated (Fig. 7B). Overall, these results indicate that the HNF4-54 site is functionally important for the control of basal SULT2A1 transcription, and may also play a role in rifampicin-mediated regulation. Although the HNF4-6160 site is also functional, other information contained within the –6832:–5812 region contributes substantially to SULT2A1 transcription.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Impact of HNF4-54 and HNF4-6160 mutations on transcription from SULT2A1 reporter constructs. A, HepG2 cells were transiently transfected with –107:48-Luc, (HNF4-6160)(–107:48)-Luc or (–6832: –5812)(–107:48)-Luc. The HNF4 binding sites at –54 and –6160 are identified with arrowheads, and site-directed mutations of these sites are indicated with cross-outs. Transfected cells were treated for 24 h with 0.1% DMSO or 50 µM rifampicin, and cells were harvested for measurement of luciferase activities. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements (3 wells per treatment group). *, ***, Significantly different from the group treated with the same agent and transfected with the corresponding reporter containing intact HNF4 sites. +++, Significantly different from the corresponding DMSO-treated group. B, HepG2 cells were transiently transfected with either –107:48-Luc, (–6832:–5812)(–107:48)-Luc, (–6248:–5812) (–107:48)-Luc, or (–6248:–6025)(–107:48)-Luc. The HNF4 binding sites at –54 and –6160 are identified with arrowheads, and site-directed mutations of these sites are indicated with cross-outs. Transfected cells were treated for 24 h with 0.1% DMSO or 50 µM rifampicin, and cells were harvested for measurement of luciferase activities. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements (3 wells per treatment group). *, ***, Significantly different from the group transfected with (–6832:–5812)(–107:48)-Luc and treated with the same agent. ###, Significantly different from the group transfected with (–6248:–6025)(–107:48)-Luc and treated with the same agent. xxx, Significantly different from the group transfected with (–6248:–6025mutHNF4-6160)(–107:48)-Luc and treated with the same agent. ++, +++, Significantly different from the corresponding DMSO-treated group. For statistical symbols, one, two, and three copies indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

To gain further insight into the interacting roles of PXR and HNF4 in regulating SULT2A1 transcription, additional studies were performed in which HepG2 cells were transfected with the simple reporter construct containing only the HNF4-6160 element and the –107:48 core promoter [i.e., construct (HNF4-6160)(–107:48)], in combination with expression constructs for HNF4 and/or PXR, with or without rifampicin treatment (Fig. 8). In the experiment that is shown, rifampicin treatment increased reporter expression, although such a clear increase was not always evident when this simple construct was used. Transfection with HNF4 alone increased promoter activity, and rifampicin treatment had no further effect (Fig. 8A). PXR transfection significantly reduced promoter activity, and rifampicin treatment augmented this reduction (Fig. 8A). When cotransfected with HNF4, PXR significantly reduced promoter activity relative to that seen in cells transfected with HNF4 alone, and again, rifampicin treatment augmented the reduction (Fig. 8A).

View larger version (23K): [in this window] [in a new window]   Fig. 8. Effects of HNF4 and/or PXR cotransfection on basal and rifampicin-regulated reporter expression from SULT2A1 constructs containing intact or mutated HNF4-54 and HNF4-6160 sites. A, HepG2 cells were transiently transfected with (HNF4-6160)(–107:48)-Luc (reporter containing the HNF4-6160 site and the first 107 nt of the SULT2A1 promoter with intact HNF4-54 site) together with 50 ng of pcDNA3.1, (pcDNA, empty vector control for HNF4), 50 ng of pCMV-HNF4, 100 ng of pSG5 (empty vector control for PXR), 100 ng of pSG5-PXR, or 50 ng of pCMV-HNF4, and 100 ng of pSG5-PXR in combination. Transfected cells were treated for 24 h with 0.1% DMSO or 50 µM rifampicin and harvested for measurement of luciferase activities. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements (3 wells per treatment group). ***, Significantly different from the corresponding pcDNA-transfected, DMSO-treated group. ##, ###, Significantly different from the corresponding pSG5-transfected, DMSO-treated group. +++, Significantly different from the corresponding DMSO-treated group. xxx, Significantly different from corresponding HNF4-transfected group. B, HepG2 cells were transiently transfected with (HNF4-6160)(–107:48)-Luc together with amounts of pSG5-PXR ranging from 0 to 160 ng (the amount of expression plasmid in each transfection was kept constant at 160 ng by the addition of pSG5). Transfected cells were treated for 24 h with 0.1% DMSO or 50 µM rifampicin, and cells were harvested for measurement of luciferase activities. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements (3 wells per treatment group). *, **, and ***, Significantly different from the group receiving 0 ng of PXR and treated with DMSO. +, ++, +++, Significantly different from the corresponding DMSO-treated group. C, HepG2 cells were transiently transfected with the (–6832:–5812)(–107:48)-Luc reporter, containing intact or mutated HNF4-54 and HNF4-6160 sites as indicated, together with pSG5 or pSG5-PXR. Transfected cells were treated for 24 h with 0.1% DMSO or 50 µM rifampicin and harvested for measurement of luciferase activities. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements (three wells per treatment group). ***, significantly different from the group cotransfected with pSG5 and reporter containing intact HNF4 sites and treated with DMSO. #, ##, ###, Significantly different from the group transfected with the same reporter and pSG5 and treated with DMSO. ++, +++, Significantly different from the group transfected with the same reporter and PXR and treated with DMSO. For statistical symbols, one, two and three copies indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

In additional experiments, the concentration-dependent effects of rifampicin treatment on reporter expression from the SULT2A1 (HNF4-6160)(–107:48) construct were evaluated in comparison to effects on the known PXR-responsive construct, XREM-CYP3A4-Luc (Fig. 9). For the CYP3A4 construct, rifampicin maximally increased reporter expression at a concentration of 3 µM, and expression was always augmented when the cells were supplemented with additional PXR. For the SULT2A1 construct, rifampicin treatment produced a progressively suppressive effect up to a concentration of 3 µM, and the suppression was always augmented by PXR transfection. However, treatment with higher concentrations of rifampicin caused progressive reversal of the SULT2A1 suppression. These data suggest that the major PXR-mediated effect of rifampicin on SULT2A1 transcription is suppression, with SULT2A1 induction only occurring at rifampicin concentrations higher than those sufficient for obtaining maximal PXR activation.

View larger version (34K): [in this window] [in a new window]   Fig. 9. Concentration-dependent effects of rifampicin treatment on luciferase expression from a SULT2A1 reporter construct in comparison with a known PXR-responsive reporter construct. HepG2 cells were transfected with either the empty vector, pSG5 (open bars), or with pSG5-PXR (closed bars) in combination with either the PXR-responsive luciferase reporter construct XREM-CYP3A4-Luc (top) or (HNF4-6160)(–107:48)-Luc (bottom). Transfected cells were treated for 24 h with either 0.1% DMSO or rifampicin at concentrations ranging from 0.1 to 50 µM and harvested for measurement of luciferase activities. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements (three wells per treatment group). **, ***, Significantly different from the group transfected with the same expression plasmid and treated with DMSO. +, +++, Significantly different from the corresponding group (i.e., same treatment) transfected with pSG5. For statistical symbols, one, two and three copies indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

View larger version (29K): [in this window] [in a new window]   Fig. 10. Effect of PXR knockdown on luciferase expression from SULT2A1 reporter constructs containing intact or mutated HNF4-54 and HNF4-6160 sites. A, HepG2 cells expressing either a nontargeting siRNA or a PXR-targeting siRNA (PXR knockdown) were transfected with either the PXR-responsive luciferase reporter construct XREM-CYP3A4-Luc (left) or (HNF4-6160)(–107:48)-Luc (right). Transfected cells were treated for 24 h with either 0.1% DMSO or rifampicin at concentrations ranging from 0.1 to 50 µM and harvested for measurement of luciferase activities. Each bar represents the mean ± S.D. of normalized (firefly/Renilla) luciferase measurements (three wells per treatment group). *, **, ***, Significantly different from the corresponding group (i.e., same cell line and reporter) treated with DMSO, p < 0.05, p < 0.01, and p < 0.001, respectively. B, HepG2 cells expressing nontargeting siRNA (siNT) or PXR-targeting siRNA (siPXR) were transfected with either XREM-CYP3A4-Luc or SULT2A1 (–6832:–5812)(–107:48)-Luc, treated for 24 h with either 0.1% DMSO or 50 µM rifampicin, and harvested for luciferase measurements as described above. Groups not sharing a letter are significantly different from each other, p < 0.05. C, PXR knockdown HepG2 cells were transfected with SULT2A1 (–6832:–5812)(–107:48)-Luc, containing either intact (left) or mutated (right) HNF4-54 and HNF4-6160 sites, treated for 24 h with either 0.1% DMSO or 50 µM rifampicin, and harvested for luciferase measurements as described above. Groups not sharing a letter are significantly different from each other, p < 0.05.

	Discussion

Top Abstract Materials and Methods Results Discussion References

PXR is activated by a wide spectrum of xenobiotic and endogenous ligands (Kliewer et al., 1998; Goodwin et al., 1999), and liganded PXR transactivates CYP3A4 at proximal and distal PXREs in the 5'-flanking region of the gene (Barwick et al., 1996; Goodwin et al., 1999). Because CYP3A4 is responsible for up to 60% of drug metabolism in humans (Li et al., 1995), PXR is recognized as a major node for the mediation of drug-drug interactions. CAR also partners with RXR to regulate transcription of xenobiotic-metabolizing enzyme genes, and the same regulatory motifs in target genes can often bind PXR or CAR (Xie et al., 2000; Wang et al., 2003). In contrast to PXR and CAR, HNF4 is a homodimer, and it is not known to be ligand-activated (Dhe-Paganon et al., 2002). HNF4 contains fatty acid as a structural component of its ligand-binding pocket (Dhe-Paganon et al., 2002), and mutations in the HNF4 gene have been linked to maturity onset diabetes of the young (Yamagata et al., 1996; Dhe-Paganon et al., 2002).

The integrated roles of nuclear receptors in gene regulation are complex and likely to contribute to interindividual differences in drug metabolism. The transactivation of CYP2C8 is mediated by separate cis-acting sites that confer PXR/CAR, GR, and HNF4 binding activity (Ferguson et al., 2005). Inducible CYP2C9 transcription is synergistically activated by proximal HNF4 binding and distal PXR/CAR binding to the CYP2C9 promoter (Chen et al., 2005). The cotransfection of HNF4 and PXR into cells produced a synergistic increase in CYP3A4 reporter activity, and mice with a conditional deletion of hepatic HNF4 lost both basal and PXR/CAR-inducible CYP3A expression (Tirona et al., 2003). The induction of CYP3A4 transcription by activated PXR was shown to require both the suppression of small heterodimer partner and the recruitment of HNF4 and coregulators to the CYP3A4 promoter (Li and Chiang, 2006).

By comparison with CYP3A4, CYP2C8, and CYP2C9, the genes encoding the bile acid-biosynthesizing cytochromes P450 CYP7A1 and CYP8B1 are negatively regulated by PXR (Bhalla et al., 2004; Li and Chiang, 2005). Current evidence indicates that these suppressive effects occur as a consequence of PXR interactions with HNF4. In one study (Bhalla et al., 2004), competition between HNF4 and ligand-activated PXR for recruitment of the PGC-1 coactivator was demonstrated. Through this "squelching" mechanism, PXR activation would be expected to suppress the expression of multiple HNF4-regulated genes, and indeed suppression of phosphoenolpyruvate carboxykinase was demonstrated (Bhalla et al., 2004). In another study, the cis-acting site controlling rifampicin-repressible CYP7A1 transcription was localized to a motif, termed BARE-I, in a proximal region of the CYP7A1 promoter (Li and Chiang, 2005). EMSA studies suggested that PXR·RXR binds to BARE-I, whereas HNF4 binds to a more distal BARE-II motif (Li and Chiang, 2005). Mammalian two-hybrid and coimmunoprecipitation analyses suggested that rifampicin treatment suppresses CYP7A1 transcription by causing PXR to disengage from BARE-I in favor of protein-protein interaction with BARE-II-bound HNF4 (Li and Chiang, 2005). These ligand-induced nuclear receptor interactions block HNF4 interaction with PGC-1, leading to suppression of CYP7A1 transcription (Li and Chiang, 2005).

We began the current investigation by examining the effects of rifampicin treatment on SULT2A1 expression in primary cultured human hepatocytes, as this system represents the only authentic cell culture model of the normal human hepatocyte that is currently available. Inextricable from this model is the same heterogeneity that exists among humans, which includes genetic polymorphisms and environmental influences that exert durable effects on hepatocellular phenotype. When the effects of rifampicin treatment were evaluated in several preparations of human hepatocytes, CYP3A4 expression was consistently enhanced multifold, consistent with the well described model of PXR·RXR activation. By comparison, the effects of rifampicin treatment on SULT2A1 expression were complicated, in that induction, suppression, or little change was seen, depending on the hepatocyte preparation. The rifampicin-mediated effects on CYP3A4 and SULT2A1 expression were not correlated, suggesting that the mechanisms underlying these effects are not identical. These findings indicate that SULT2A1 transcription is regulated by both positive- and negative-acting factors, both of which are sensitive to rifampicin treatment.

Based on these initial findings, we conducted transfection studies in HepG2 cells, with the goal of identifying the factors conferring the positive and/or negative effects of rifampicin on SULT2A1 expression. The results indicated that several elements located within the first 492 nt of the SULT2A1 promoter contribute to basal SULT2A1 transcription. In addition, multiple elements contained within upstream regions, particularly the –6832:–5812 region, enhance SULT2A1 transcription. Although some of the tested constructs displayed increased reporter expression in response to 50 µM rifampicin treatment, none of the responsive constructs contained a typical PXR binding site, and rifampicin-mediated induction, albeit somewhat variable among experiments, was retained in a reporter construct containing only the first 107 nt of the SULT2A1 promoter. This region contained a high-affinity and functional HNF4-binding site, as determined by EMSA and site-directed mutagenesis. Likewise, the –6832: –5812 region contained an HNF4-binding site, although the requirement of this site for maintaining a high level of transcription was less apparent.

Simple SULT2A1 reporter constructs, containing only the proximal 107 nt or the upstream HNF4-6160 site in cis with the 107 nt promoter, were used to evaluate the roles of PXR and HNF4 in rifampicin-mediated transcriptional regulation. Rifampicin treatment caused progressive suppression of reporter expression from the SULT2A1 reporters at the same concentrations that caused progressive activation of a known PXR-responsive reporter. Rifampicin-mediated suppression of SULT2A1 promoter activity was reversed only at rifampicin concentrations higher than necessary to achieve maximal CYP3A4 reporter induction. Thus, the SULT2A1 "induction" that was seen at 50 µM rifampicin was actually the net ability of the positive-acting effect of rifampicin to counteract its suppressive effect. In addition to the concentration-response analysis, the finding that PXR supplementation augmented rifampicin-mediated suppression of SULT2A1 reporter expression, while producing the expected enhancement of rifampicin-inducible CYP3A4 reporter expression, suggests that the primary PXR-mediated effect on SULT2A1 transcription is suppression, and that the positive effect on SULT2A1 transcription that occurs at higher concentrations of rifampicin is mediated through a PXR-independent mechanism. Most importantly, the suppressive effect of rifampicin was diminished in HepG2 cells with knocked down PXR expression, whereas the activating effect remained intact. The involvement of both PXR-mediated and PXR-independent mechanisms in SULT2A1 regulation provides a plausible explanation for the lack of correlation between rifampicin-mediated effects on CYP3A4 and SULT2A1 expression that was observed among human hepatocyte preparations.

As noted above, rifampicin-regulated SULT2A1 promoter constructs contained HNF4-, but not PXR-, binding sites. For the PXR-dependent suppressive effect, we hypothesize that, as for CYP7A1, rifampicin-activated PXR engenders protein-protein interactions that disrupt the transactivation of SULT2A1 transcription by HNF4, possibly through the impaired recruitment of one or more coactivators. Although PXR overexpression, without or with rifampicin treatment, was able to cause suppression of SULT2A1 promoter activity in constructs with intact or mutated HNF4 binding sites (Figs. 8 and 10), we suspect that the mutations reduced, but did not abolish the ability of HNF4 to bind (the mutants retained some ability to compete for probe binding in EMSAs) and that the residual transactivation could be suppressed by PXR overexpression.

Although the above-described PXR-mediated mechanism provides a plausible explanation for the finding that rifampicin treatment suppressed SULT2A1 expression in some human hepatocyte cultures, the mechanism that mediates the rifampicin-inducible SULT2A1 expression that occurs in many human hepatocyte preparations is not yet known. Rifampicin is not known to bind to any nuclear receptor other than PXR. Our data, demonstrating the retention of rifampicin-mediated SULT2A1 promoter activation in HepG2 cells lacking PXR expression but the loss of induction when HNF4-binding sites are mutated, suggest that the activating effect of rifampicin, like the suppressive effect, is transduced through HNF4.

Regulation of gene expression by nuclear receptors is gaining appreciation for its shades of complexity that are likely to translate into interindividual differences in xenobiotic and endogenous metabolism. For example, the impact on human health of natural PXR variants that lack DNA-binding and transactivation capacity is unknown (Koyano et al., 2004). It has also been proposed that the relative cellular contents of nuclear receptors, and not just the absolute amounts, are important determinants of CYP3A inducibility (Swales et al., 2003). Future investigations will focus on the interactive dynamics that occur between cis-acting regions, transcription factors, and coregulator complexes to orchestrate the net regulation of human SULT2A1 gene expression. Extensions of these studies will also shed light on the physiological implications of PXR interactions with liver-enriched transcription factors, such as HNF4, thereby strengthening the mechanistic bridge that links the chemical environment with the cellular physiology of the human hepatocyte.

	Footnotes

 
This work was conducted with support from National Institutes of Health Grants ES05823 (to M.R.-M.), HL50710 (to T.A.K.), and DK92310 (Liver Tissue Procurement and Distribution System, to S.C.S) and services provided by the Cell Culture Facility Core, Microarray and Bioinformatics Facility Core, and Imaging and Cytometry Facility Core of Environmental Health Sciences Center Grant ES06639.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.124610.

ABBREVIATIONS: PXR, pregnane X receptor; RXR, retinoid X receptor; GR, glucocorticoid receptor; C/EBP, CCAAT/enhancer-binding protein; CAR, constitutive androstane receptor; HNF4, hepatocyte nuclear factor 4; HNF4-RE, hepatocyte nuclear factor 4 response element; SULT, sulfotransferase; PPAR, peroxisome proliferator-activated receptor; DMSO, dimethyl sulfoxide; PCR, polymerase chain reaction; nt, nucleotide(s); kb, kilobase(s); siRNA, small interfering RNA; PXRE, pregnane X receptor-response element; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; BARE, bile acid response element; RIF (Rif), rifampicin.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Melissa Runge-Morris, Institute of Environmental Health Sciences, Wayne State University, 2727 Second Ave., Room 4000, Detroit, MI 48201. E-mail: m.runge-morris{at}wayne.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Assem M, Schuetz EG, Leggas M, Sun D, Yasuda K, Reid G, Zelcer N, Adachi M, Strom S, Evans RM, et al. (2004) Interactions between hepatic Mrp4 and sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice. J Biol Chem 279: 22250–22257.[Abstract/Free Full Text]

Barwick JL, Quattrochi LC, Mills AS, Potenza C, Tikey RH, and Guzelian PS (1996) Trans-species gene transfer for analysis of glucocorticoid-inducible transcriptional activation of transiently expressed human CYP3A4 and rabbit CYP3A6 in primary cultures of adult rat and rabbit hepatocytes. Mol Pharmacol 50: 10–16.[Abstract]

Bhalla S, Ozalp C, Fang S, Xiang L, and Kemper JK (2004) Ligand-activated pregnane X receptor interferes with HNF-4 signaling by targeting a common coactivator PGC-1. Functional implications in hepatic cholesterol and glucose metabolism. J Biol Chem 279: 45139–45147.[Abstract/Free Full Text]

Chen Y, Kissling G, Negishi M, and Goldstein JA (2005) The nuclear receptors constitutive androstane receptor and pregnane X receptor cross-talk with hepatic nuclear factor 4 to synergistically activate the human CYP2C9 promoter. J Pharmacol Exp Ther 314: 1125–1133.[Abstract/Free Full Text]

Dhe-Paganon S, Duda K, Iwamoto M, Chi Y-I, and Shoelson SE (2002) Crystal structure of the HNF4 ligand binding domain in complex with endogenous fatty acid ligand. J Biol Chem 277: 37973–37976.[Abstract/Free Full Text]

Duanmu Z, Locke D, Smigelski J, Wu W, Dahn MS, Falany CN, Kocarek TA, and Runge-Morris M (2002) Effects of dexamethasone on aryl (SULT1A1)- and hydroxysteroid (SULT2A1)-sulfotransferase gene expression in primary cultured human hepatocytes. Drug Metab Dispos 30: 997–1004.[Abstract/Free Full Text]

Falany CN, Comer KA, Dooley TP, and Glatt H (1995) Human dehydroepiandrosterone sulfotransferase purification, molecular cloning, and characterization. Ann N Y Acad Sci 774: 59–72.[Medline]

Fang H-L, Abdolalipour M, Duanmu Z, Smigelski JR, Weckle A, Kocarek TA, and Runge-Morris M (2005a) Regulation of glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription in primary cultured rat hepatocytes: role of CCAAT/enhancer-binding protein liver-enriched transcription factors. Drug Metab Dispos 33: 147–156.[Abstract/Free Full Text]

Fang H-L, Strom SC, Cai H, Falany CN, Kocarek TA, and Runge-Morris M (2005b) Regulation of human hepatic hydroxysteroid sulfotransferase gene expression by the peroxisome proliferator-activated receptor transcription factor. Mol Pharmacol 67: 1257–1267.[Abstract/Free Full Text]

Ferguson SS, Chen Y, LeCluyse EL, Negishi M, and Goldstein JA (2005) Human CYP2C8 is transcriptionally regulated by the nuclear receptors constitutive androstane receptor, pregnane X receptor, glucocorticoid receptor, and hepatic nuclear factor 4. Mol Pharmacol 68: 747–757.[Abstract/Free Full Text]

Goodwin BA, Hodgson E, and Liddle C (1999) The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module. Mol Pharmacol 56: 1329–1339.[Abstract/Free Full Text]

Kitada H, Miyata M, Nakamura T, Tozawa A, Honma W, Shimada M, Nagata K, Sinal CJ, Guo GL, Gonzalez FJ, et al. (2003) Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity. J Biol Chem 278: 17838–17844.[Abstract/Free Full Text]

Klaassen CD and Slitt AL (2005) Regulation of hepatic transporters by xenobiotic receptors. Curr Drug Metab 6: 309–328.[CrossRef][Medline]

Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, et al. (1998) An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92: 73–82.[CrossRef][Medline]

Koyano S, Kurose K, Saito Y, Ozawa S, Hasegawa R, Komamura K, Ueno K, Kamakura S, Kitakaze M, Nakajima T, et al. (2004) Functional characterization of four naturally occurring variants of human pregnane X receptor (PXR): one variant causes dramatic loss of both DNA binding activity and the transactivation of the CYP3A4 promoter/enhancer region. Drug Metab Dispos 32: 149–154.[Abstract/Free Full Text]

Li AP, Kaminski DL, and Rasmussen A (1995) Substrates of human hepatic cytochrome P450 3A4. Toxicology 104: 1–8.[CrossRef][Medline]

Li T and Chiang JY (2005) Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7-hydroxylase gene transcription. Am J Physiol Gastrointest Liver Physiol 288: G74–G84.[Abstract/Free Full Text]

Li T and Chiang JYL (2006) Rifampicin induction of CYP3A4 requires pregnane X receptor cross talk with hepatocyte nuclear factor 4 and coactivators, and suppression of small heterodimer partner gene expression. Drug Metab Dispos 34: 756–764.[Abstract/Free Full Text]

Li X and Anderson RJ (1999) Sulfation of iodothyronines by recombinant human liver steroid sulftotransferases. Biochem Biophys Res Commun 263: 632–639.[CrossRef][Medline]

Mohan R and Heyman RA (2003) Orphan nuclear receptor modulators. Curr Top Med Chem 3: 1637–1647.

Nishiyama T, Ogura K, Nakano H, Kaku T, Takahashi E, Ohkubo Y, Sekine K, Hiratsuka A, Kadota S, and Watabe T (2002) Sulfation of environmental estrogens by cytosolic human sulfotransferases. Drug Metab Pharmacokinet 17: 221–228.[CrossRef][Medline]

Quandt K, Frech K, Karas H, Wingender E, and Werner T (1995) MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 23: 4878–4884.[Abstract/Free Full Text]

Radominska A, Comer KA, Zimniak P, Falany JL, Iscan M, and Falany CN (1990) Human liver steroid sulphotransferase sulphates bile acids. Biochem J 272: 597–604.[Medline]

Runge-Morris M and Kocarek TA (2005) Regulation of sulfotransferases by xenobiotic receptors. Curr Drug Metab 6: 299–307.[CrossRef][Medline]

Runge-Morris M, Wu W, and Kocarek TA (1999) Regulation of rat hepatic hydroxysteroid sulfotransferase (SULT2-40/41) gene expression by glucocorticoids: evidence for a dual mechanism of transcriptional control. Mol Pharmacol 56: 1198–1206.[Abstract/Free Full Text]

Song CS, Echchgadda I, Seo Y-K, Oh T, Kim S, Kim S-A, Cho S, Shi L, and Chatterjee B (2006) An essential role of the CAAT/enhancer binding protein- in the vitamin D-induced expression of the human steroid/bile acid-sulfotransferase (SULT2A1). Mol Endocrinol 20: 795–808.[Abstract/Free Full Text]

Sonoda J, Xie W, Rosenfeld JM, Barwick JL, Guzelain PS, and Evans RM (2002) Regulation of a xenobiotic sulfonation cascade by nuclear pregnane X receptor (PXR). Proc Natl Acad Sci U S A 99: 13801–13806.[Abstract/Free Full Text]

Swales K, Plant N, Ayrton A, Hood S, and Gibson G (2003) Relative receptor expression is a determinant in xenobiotic-mediated CYP3A induction in rat and human cells. Xenobiotica 33: 703–716.[CrossRef][Medline]

Tirona RG, Lee W, Leake BF, Lan L-B, Cline CB, Lamba V, Parvitz F, Duncan SA, Inoue Y, Gonzalez FJ, et al. (2003) The orphan nuclear receptor HNF4 determines PXR- and CAR-mediated xenobiotic induction of CYP3A4. Nat Med 9: 220–224.[CrossRef][Medline]

Wang H, Faucette S, Sueyoshi T, Moore R, Ferguson S, Negishi M, and LeCluyse EL (2003) A novel distal enhancer module regulated by pregnane X receptor/constitutive androstane receptor is essential for the maximal induction of CYP2B6 gene expression. J Biol Chem 278: 14146–14152.[Abstract/Free Full Text]

Xie W, Barwick JL, Simon CM, Pierce AM, Safe S, Blumberg B, Guzelian PS, and Evans RM (2000) Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR. Genes Dev 14: 3014–3023.[Abstract/Free Full Text]

Xu C and Kong AN (2005) Induction of phase I, II and III metabolism/transport by xenobiotics. Arch Pharmacol Res 28: 249–268.[Medline]

Yamagata K, Furuta H, Oda N, Kaisaki PJ, Menzel S, Cox NJ, Fajans SS, Signorini S, Stoffel M, and Bell GI (1996) Mutations in the hepatocyte nuclear factor-4 gene in maturity onset diabetes of the young (MODY1). Nature 384: 458–460.[CrossRef][Medline]

This article has been cited by other articles:

				Z. Duniec-Dmuchowski, H.-L. Fang, S. C. Strom, E. Ellis, M. Runge-Morris, and T. A. Kocarek Human Pregnane X Receptor Activation and CYP3A4/CYP2B6 Induction by 2,3-Oxidosqualene:Lanosterol Cyclase Inhibition Drug Metab. Dispos., April 1, 2009; 37(4): 900 - 908. [Abstract] [Full Text] [PDF]

				Y. Alnouti Bile Acid Sulfation: A Pathway of Bile Acid Elimination and Detoxification Toxicol. Sci., April 1, 2009; 108(2): 225 - 246. [Abstract] [Full Text] [PDF]

				N. Anderson and J. Borlak Molecular Mechanisms and Therapeutic Targets in Steatosis and Steatohepatitis Pharmacol. Rev., September 1, 2008; 60(3): 311 - 357. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: jpet.107.124610v1 323/2/586    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fang, H.-L. Articles by Runge-Morris, M. Search for Related Content PubMed PubMed Citation Articles by Fang, H.-L. Articles by Runge-Morris, M.