Involvement of the Xenobiotic Response Element (XRE) in Ah Receptor-mediated Induction of Human UDP-glucuronosyltransferase 1A1 *

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an important physiological role by contributing to the metabolism of endogenous substances such as bilirubin in addition to xenobiotics and drugs. The UGT1A1 gene has been shown to be inducible by nuclear receptors steroid xenobiotic receptor (SXR) and the constitutive active receptor, CAR. In this report, we show that in human hepatoma HepG2 cells the UGT1A1 gene is also inducible with aryl hydrocarbon receptor (Ah receptor) ligands such as 2,3,7,8-tetrachlodibenzo- p -dioxin (TCDD), (cid:1) -naphthoflavone, and benzo[a]pyrene metabolites. Induction was monitored by increases in protein and catalytic activity as well as UGT1A1 mRNA. To examine the molecular interactions that control UGT1A1 expression, the gene was characterized and induction by Ah receptor ligands was regionalized to bases (cid:2) 3338 to (cid:2) 3287. Nucleotide sequence analysis of this UGT1A1 enhancer region revealed a xenobiotic response element (XRE) at (cid:2) 3381/ (cid:2) 3299. The dependence of the XRE on UGT1A1 luciferase activity was demonstrated by a loss of Ah receptor ligand inducibility when the m m m of Ah g of receptor or anti-Arnt (a Chris- topher Bradfield) was included in the binding reaction. Protein-DNA complex was then separated on a 6% nondenaturing oligonucleotide.

Glucuronidation has evolved in vertebrates to catalyze the transfer of glucuronic acid from uridine 5Ј-diphosphoglucuronic acid to soluble non-lipid dependent substances generated as byproducts of dietary and cellular metabolism (1). Some of the endogenous agents that are targets for glucuronidation are bilirubin and many of the steroids as well as several phenolic neurotransmitters. In addition, hundreds of drugs and xenobiotics are subject to glucuronidation (2,3). The vast numbers of endogenous and exogenous substances that are susceptible to glucuronidation in humans are catalyzed by the family of UDPglucuronosyltransferases (UGTs). 1 A comparison of the de-duced amino acid sequence of the UGTs in mammalian species has helped in classifying these proteins as members of the UGT1 or UGT2 gene family (4). In humans, 16 cDNAs have been identified and shown through expression experiments in tissue culture to encode proteins that display functional glucuronidation activity (3). It is generally felt that evolutionary constraints associated with the UGT1 family of proteins leads to more efficient glucuronidation of drugs and xenobiotics, whereas the UGT2 family of proteins displays far greater catalytic diversity toward endogenous agents such as steroids.
Regulation of the UGTs in human tissues is tightly controlled. Analysis of RNA expression patterns has demonstrated that no two tissues display the same pattern of UGT gene expression, indicating that regulatory control occurs in a tissue-specific manner (5). In addition, environmental influences on gene control clearly indicate that the UGTs are capable of undergoing differential regulation resulting in enhanced glucuronidation capacity. The treatment of Caco-2 cells with the antioxidant tert-butylhydroquinone leads to induction of UGT1A6, UGT1A9, and UGT2B7 (6,7). Transcriptional regulation of UGT1A6 and UGT1A9 occurs after exposure to Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (6,8). Human UGT1A1 has recently been shown to be under control by agents that induce gene expression in concordance with the constitutive active receptor (CAR) (9) and the steroid xenobiotic receptor (SXR) (33). The treatment of HepG2 and Caco-2 cells with the flavonoid chrysin leads to the induction of UGT1A1 (10 -12). Interestingly, flavonoids have also been shown to induce CYP1A1 (13) in a CYP1A1-luciferase reporter HepG2 cell line (14), implicating a potential role for the induction of UGT1A1 through a similar mechanism. One potential mechanism that may link the expression of UGT1A1 and CYP1A1 by flavonoids is the ability of these agents to activate the Ah receptor. Although the mechanisms surrounding expression of CYP1A1 after activation of the Ah receptor are well documented (15)(16)(17), there is little information linking expression of the human UGT1A1 gene through an Ah receptor-dependent mechanism. Experiments were undertaken in this study to examine the actions of several Ah receptor ligands to modulate expression of the UGT1A1 gene.  Taq polymerase and the  reporter plasmids PGL3-basic vector and PGL3-promoter vector were  from Promega (Madison, WI). Custom oligonucleotides used in PCR cloning, DNA sequencing, and electrophoretic mobility shift assay were purchased from Genbase (San Diego, CA). The ␤-galactosidase expression vector PCMV␤ Gal was purchased from Clontech (Palo Alto, CA). Thin-layer chromatography plates for enzyme analysis were from Whatman (Clifton, NJ).

Materials
Cell Culture-The human cell lines used in this study are the hepatoma-derived HepG2 and the human CYP1A1-luciferase reporter gene TV101 cell line (14). Both cell lines were maintained at 37°C in 95% air and 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were trypsinized 24 h before chemical treatment, and 10 6 cells were seeded in P100 plates. Cells were treated for 24 -72 h with either TCDD (10 nM) or BNF (20 M). For transient transfection experiments, 10 5 cells were split into 12-well plates ϳ24 h before transfection followed by chemical treatment for 48 h. Chemicals were first dissolved in Me 2 SO, and Me 2 SO concentration in media never exceeded 0.1% (v/v). Fresh media and chemical treatment were changed every 24 h.
Enzyme Analysis-UDP-glucuronosyltransferase analysis was determined using 1-naphthol and 17␣-ethynylestradiol as substrates (18). HepG2 cells were treated with either 10 nM TCDD or 20 M BNF, and the cells were collected after 24 -72 h of treatment. Whole cell lysates were prepared. Each UGT assay was conducted in a total volume of 100 l containing 50 mM Tris-HCl, pH 7.6, 10 mM MgCl 2 , 500 M uridine 5Ј-diphosphoglucuronic acid, 0.04 Ci of [ 14 C]uridine 5Ј-diphosphoglucuronic acid (0.14 nmol), 8.5 mM saccharolactone, 100 M substrate, and 100 g of protein. Each reaction was incubated at 37°C for 90 min. TLC plates were visualized with a Molecular Dynamics Storm 820 Phosphor-Imager. Resident glucuronides were then removed and quantitated by liquid scintillation counting.
Western Blot Analysis-HepG2 cells were collected and washed in cold phosphate-buffered saline and resuspended in ϳ5 volumes of phosphate-buffered saline. The cells were sonicated on a 10-s pulse cycle for 2 min at 6 watts with a Sonic Dismembrator (Fisher). Each extract was centrifuged first for 5 min at 1000 ϫ g in a refrigerated Eppendorf centrifuge followed by centrifugation for 10 min at 9000 ϫ g. This supernatant was then centrifuged at 100,000 ϫ g in a Beckman TL-100 tabletop ultracentrifuge, and the microsomes were resuspended in phosphate-buffered saline. Western blots were carried out on Nupage Bis-Tris 10% polyacrylamide gels as outlined by the manufacturer (Invitrogen). Protein (10 g) was run at 200 V for 50 min and transferred at 30 V for 1 h to nitrocellulose membranes. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline for 1 h at room temperature followed by incubation with anti-human UGT1A1 (19) (1:1000) or antihuman CYP1A1 (20) (1:5000) in Tris-buffered saline overnight at 4°C. The membranes were washed and then treated with horseradish peroxidase-conjugated anti-mouse (for UGT1A1) or antirabbit (for CYP1A1) antibody for 1 h at room temperature. Detection of the proteins was conducted by chemiluminescence.
Northern Blot Analysis-HepG2 cells were treated for 24 -72 h with 10 nM TCDD or 20 M BNF, and total RNA was prepared using TRIZOL Reagent (Invitrogen). For Northern blots, 15 g of total RNA was separated through 1% formaldehyde agarose gels. RNA was subsequently blotted onto GeneScreen membrane (PerkinElmer Life Sciences) by capillary transfer. After transfer, the blot was stained with methylene blue to visualize RNA for loading efficiency. A 423-bp fragment recovered by digesting the UGT1A1 cDNA with AvaI/ExoRI was 32 P-labeled by random priming (Invitrogen) and purified using a nucleotide removal kit (Qiagen). After boiling, the probe was added to hybridization solution (Stratagene) and incubated with the filters at 68°C for 6 h followed by washing in 0.1ϫ SSC (1ϫ SSC ϭ 0.15 M NaCl and 0.015 M sodium citrate) and 0.1% SDS at 60°C for 30 min. Visualization was performed using a Storm 860 PhosphorImager (Molecular Dynamics).
UGT1A1 Promoter Cloning-Genomic clones containing the UGT1 locus were characterized by screening a human bacterial artificial chromosome (BAC) library. Enhancer DNA fragments as well as a Ϫ3712/Ϫ7 UGT1A1 promoter fragment containing the TATA box were amplified by PCR using primers corresponding to sites on the promoter sequence, as published in NCBI GenBank TM accession number AF297093 (21). The cap site in AF297093 is at base pair 175,027, as characterized previously (38). The restriction enzyme sites SacI and XhoI were incorporated at the 5Ј end of the sense and antisense primers, respectively. The PCR product for the Ϫ3712/Ϫ7 UGT1A1 promoter was generated with oligonucleotides 5Ј-tttaggagctcTCAGACAAAAG-GAA-3Ј and 5-tcctgctcgagGTTCGCCCTCTCCT-3Ј, digested with SacI/ XhoI (the sites are in lowercase and underlined) and subcloned into SacI/XhoI-digested PGL3-basic vector. This plasmid was identified as pLUGT1A1. Using the pL1A1Neo plasmid originally cloned in the laboratory (14), the neomycin gene was removed and cloned into the SalI site of pLUGT1A1, generating the pLUGT1A1N plasmid.
For construction of the mutant UGT1A1-XRE enhancer plasmid, E4 was used as template. The primers used for amplification of the insert were 5Ј-tccttgagctcTTTTTGACACTGGA-3Ј and 5Ј-aaattctcgagCTCAT-TCCTCCTCT-3Ј. The two internal primers that carried the mutations were 5Ј-CTTGGTAAGACCGCAATGAAC-3 and 5-GTTCATTGCG-GTCTTACCAAG-3Ј. The underlined region represents the area of the Ah receptor core binding region, and the bold and italicized bases are those that were changed form the normal XRE sequence to disrupt the Ah receptor binding region (see Fig. 4A). After digestion of the amplified sequence with SacI and XhoI, the insert was cloned into these same sites in the PGL3-promoter vector.
Transfection Assays-HepG2 cells were plated in 12-well tissue culture plates at 30 -40% confluence and transfected after 24 h using LipofectAMINE Plus reagent as described by the manufacture's protocol (Invitrogen). In general, transfection mixtures contained 500 ng of UGT1A1-reporter plasmid and 300 ng of ␤-galactosidase expression vector (PCMV␤) as an internal control to monitor for transfection efficiency. The day after transfection, the cells were treated with 20 M BNF, 10 nM TCDD, or Me 2 SO for 48 h. The cells were harvested, lysed, and analyzed for luciferase and ␤-galactosidase activity. Luciferase activities were assessed by the methods described previously (22)  Generation of G418-resistant UGT1A1-luciferase MH1A1L Cells-Using HepG2 cells that were seeded at ϳ10 6 cells/100-mm tissue culture dish, the pLUGT1A1N plasmid was transfected as outlined above. After 48 h of growth, the cells were trypsinized, 1 ⁄10 volume of the collected cells was plated into a 100-mm tissue culture dish, and the cells were exposed to media containing 0.8 mg/ml G418. After 2-3 weeks, individual colonies of selected cells were removed and re-cultured in 60-mm plates with continued G418 selection. A final round of clonal selection was made, and each clone was expanded and treated with 5 M TCDD for 24 h followed by analysis of induced luciferase activity. For these studies, the cell line selected is referred to as MH1A1L cells.
Preparation of Nuclear Proteins-Nuclear extracts from HepG2 cells were isolated as described previously (22) Electrophoretic Mobility Shift Assay-A complementary pair of synthetic oligonucleotides, 5Ј-GCTAGGCACTTGGTAAGCACGCAATGAA-CAGTCA-3Ј and 5Ј-GCTATGACTGTTCATTGCGTGCTTACCAAGTG-CC-3Ј, encoding the consensus core sequence (underlined) of the UGT1A1 XRE element were synthesized. For analysis of Ah receptor activation, the human CYP1A1 DRE3 oligonucleotides (5Ј-GATCCGG-CTCTTGTCACGCAACTCCGAGCTCA-3Ј and 5Ј-GATCTGAGCTCGG-AGTTGCGTGAGAAGAGCCG-3Ј) were used as previously described (22). Double-stranded oligonucleotides were assembled by annealing equal concentrations of either the XRE or DRE and then labeled with [␣-32 P]CTP in the presence of Klenow and 25 M dATP, dGTP, and dTTP. Binding assays were carried out on ice containing 3 ϫ 10 4 cpm of labeled oligonucleotide, 10 g of nuclear extract, 2 g of poly(dI-dC), and 1 g of salmon sperm DNA in a final reaction volume of 30 l containing 25 mM HEPES, pH 7.5, 1.5 mM EDTA, 1 mM DTT, 10% glycerol (22). To examine the specificity of Ah receptor binding, 100 g of anti-Ah receptor or anti-Arnt antibody (a generous gift from Christopher Bradfield) was included in the binding reaction. Protein-DNA complex was then separated on a 6% nondenaturing polyacrylamide gel using 45 mM Tris-borate, 10 mM EDTA as a running buffer. Competition assays were performed by adding a 50-fold excess of unlabeled CYP1A1 DRE or UGT1A1 XRE oligonucleotide. The gels were then dried, and protein-DNA complexes were visualized by a PhosphorImager.
Induction of CYP1A1 in TV101 Cells-Human TV101 cells were derived from the human hepatoma cell line HepG2 but carry the human CYP1A1 promoter fused to the firefly luciferase gene (14). The Ϫ1600 bp of the CYP1A1 promoter contains 3 Ah receptor-specific XRE sites. Luciferase activity results from Ah receptor activation after treatment with Ah receptor ligands. The TV101 cells were grown under the same conditions as HepG2 cells but supplemented with 0.8 mg/ml G418. The TV101 cells were treated with TCDD, BNF, or Me 2 SO at different time points to evaluate their ability to activate CYP1A1 gene transcription. Luciferase activity was measured and normalized for protein concentration.

Induction of UGT1A1 by TCDD and BNF-
The small phenolic compound 1-naphthol was used as a substrate to examine UGT activity in HepG2 cells (Fig. 1A). Treatment of HepG2 cells with 10 nM TCDD for 72 h led to a time-dependent increase in 1-naphthol UGT activity that consistently was determined to be 3-fold over untreated cells. Similar treatment of cells with 20 M BNF resulted in a 4 -5-fold increase in 1-naphthol UGT activity. Simple phenols have been shown to be glucuronidated by most of the UGT1A proteins (3), with a preference for UGT1A1, UGT1A6, UGT1A8, and UGT1A9. Glucuronidation of 17␣-ethynylestradiol, a substrate that is preferentially glucuronidated by UGT1A1, was increased 2.5-5fold in TCDD-or BNF-treated cells (Fig. 1A). Quantitation of UGT1A1 RNA transcripts by Northern blot analysis demonstrated that both TCDD and BNF induced UGT1A1 (Fig. 1B) in a time-dependent fashion. Slightly greater increases in RNA were observed with BNF-treated cells, a pattern that was also reflected in catalytic activity. It was also observed that induction of UGT1A1 RNA and 17␣-ethynylestradiol glucuronidation by TCDD and BNF correlated with increased levels of UGT1A1 protein (Fig. 1C), with BNF generating slightly greater levels of induced UGT1A1 in microsomes. In HepG2 cells, TCDD and BNF are capable of inducing CYP1A1, as shown by induction of CYP1A1 (Fig. 1C) and activation of the human CYP1A1-luciferase gene in TV101 cells (Fig. 2). Induction of CYP1A1-luciferase in TV101 cells has been linked to activation of the Ah receptor (22,23). Although maximal CYP1A1-luciferase activity is achieved between 8 -24 h in TV101 cells with TCDD and BNF, maximal levels of UGT1A1 RNA and protein are evident at around 48 h (Fig. 1C), indicating that slightly different regulatory events may control the CYP1A1 and UGT1A1 genes. Combined, these results indicate that induction of UGT1A1 may occur through an Ah receptordependent mechanism.
Characterization of the UGT1A1 Promoter and Ah Receptor Binding Site-To examine the mechanism of UGT1A1 induction, an 11-kilobase region of the UGT1A1 promoter was cloned from a human BAC containing the entire UGT1A1 locus. UGT1A1 promoter and enhancer regions, cloned by PCR, were subcloned into the pGL3 basic or pGL3 promoter vectors, respectively. Portions of the regulatory region including the promoter constituted a fragment from Ϫ3712 to Ϫ7, whereas the individual enhancer sequences contained bases from Ϫ10998/ Ϫ8134 (Enhancer 1, E1), Ϫ8533/Ϫ4738 (Enhancer 2, E2), and Ϫ3712/Ϫ2081 (Enhancer 3, E3). Each plasmid was transfected transiently into HepG2 cells, and expression of luciferase activity was determined after treatment of cells for 48 h with TCDD or BNF (Fig. 3). Our selection of 48 h for the treatment time was selected because we had observed adequate accumulation of both RNA and protein in TCDD/BNF-treated HepG2 cells. The UGT1A1 Ϫ3712/Ϫ7 luciferase promoter fragment was induced after treatment with TCDD and BNF. An enhancer sequence from Ϫ3712 to Ϫ2081 (E3) relative to the transcriptional start site was also responsive. Enhancer sequences E2 and E1, which covered a region from Ϫ10998 to Ϫ4738, were refractory to both TCDD and BNF.
Induction of the Ϫ3712/Ϫ7 promoter-luciferase construct with TCDD indicates that the transcriptional activation may occur through an Ah receptor-dependent mechanism. Compounds that have been shown to be ligands for the Ah receptor are classically polycyclic aromatic hydrocarbons. To examine this possibility further, we developed MH1A1L cells carrying the UGT1A1-luciferase plasmid and demonstrated that classical polycyclic aromatic hydrocarbons composed of hydroxylated benzo[a]pyrene were capable of inducing UGT1A1-driven luciferase. We examined 1-, 2-, 3-, 4-, 6-, 8-, 9-, and 10-hydroxylated isomers of benzo[a]pyrene in addition to cis-and trans-4,5dihydrodiol benzo[a]pyrene (Fig. 4). Along with TCDD induction, we observed a 2-5-fold induction of luciferase activity with the 3-and 9-hydroxybenzo[a]pyrene and the trans-4,5dihydrodiol serving as the most efficient inducers. The use of cell lines deficient in Ah receptor function show that polycyclic aromatic hydrocarbons induce gene expression in an Ah receptordependent fashion (24). It has also been demonstrated through the use of reporter gene assays that are controlled by the Ah receptor enhancer sequence that polycyclic aromatic hydrocarbons induce transcription through activation of the Ah receptor (14,25,26). Combined, the results of TCDD, BNF, and B[a]P induction of the UGT1A1 promoter constructs strongly indicates that these agents elicit transcriptional activation through and Ah receptor-dependent pathway.
To localize the region on the UGT1A1 gene that controls induction, further mutational analysis on the E4 clone demonstrated that a sharp drop in induction was observed between bases Ϫ3338 and Ϫ3287 (Fig. 5A). Sequence analysis in this region revealed the presence of a single copy of the Ah receptor XRE motif (CACGCA) starting at position Ϫ3309 (Fig. 5B). Using DNA fragments spanning Ϫ3525 to Ϫ3144, site-directed mutagenesis was carried out on the conserved UGT1A1 XRE sequence, altering CACGCA to ACCGCA. Transient transfection of this plasmid demonstrated that the mutated UGT1A1-XRE resulted in a loss of inducibility (Fig. 5C) by TCDD and BNF.
Regulation of UGT1A1 by the XRE core sequence indicates that the CACGCA motif may be a binding site for the Ah receptor. Binding of Ah receptor complex to the XRE response element in the UGT1A1 promoter region was examined by gel  3. Activation of the UGT1A1 promoter in response to TCDD and BNF. The UGT1A1 promoter and ϳ11 kilobases of flanking DNA was cloned and characterized from a human BAC clone. Three fragments of DNA, from Ϫ10998 to Ϫ8134 (E1), Ϫ8533 to Ϫ4738 (E2), and Ϫ3712 to Ϫ2081 (E3) were subcloned into the pGL3-promoter luciferase plasmid. A heterologous SV40 promoter drove transcription. A fourth fragment spanning Ϫ3712 to Ϫ7 (promoter) was subcloned into the pGL3-basic vector. The UGT1A1 promoter drove transcription from this plasmid. Each of these plasmids were transfected into HepG2 cells followed by a 48-h treatment with TCDD or BNF. Transient transfection experiments were carried out using luciferase reporter plasmids cotransfected with ␤-galactosidase to normalize transfection efficiency. Luciferase activity was measured in the cytosolic fraction and normalized by ␤-galactosidase activity. The fold induction was calculated from those values of the treated cells compared with Me 2 SO-treated transfected cells. Each assay was conducted in triplicate. DMSO, dimethyl sulfoxide. mobility shift analysis (Fig. 6). When nuclear extract prepared from TCDD-treated HepG2 cells was incubated with a 32 Plabeled UGT1A1-XRE probe, an induced DNA-protein complex was detected. Competition for the labeled XRE was evident when excess unlabeled UGT1A1-XRE as well as CYP1A1-DRE was included in the reaction. A similar series of experiments were conducted using the CYP1A1 DRE as probe. Binding of a TCDD-inducible nuclear protein to the CYP1A1 DRE could be blocked when the binding reactions were conducted in the presence of unlabeled UGT1A1-XRE and CYP1A1-DRE.
To determine whether the TCDD-activated nuclear protein that associates with the UGT1A1 XRE is the Ah receptor, gel mobility shift analysis experiments were carried out in the presence of antibodies directed toward the Ah receptor and its dimerization partner Arnt. Binding of the TCDD-induced nuclear protein to the UGT1A1 XRE sequence was blocked by the IgG-purified rabbit anti-mouse AhR and anti-mouse Arnt antibodies. No inhibition was observed when the binding reactions were incubated with a mouse anti-human monoclonal UGT antibody (27), demonstrating that the inhibition of UGT1A1 XRE binding by the Ah receptor and Arnt antibodies was specific. As a control experiment to assure the specificity of the antibodies in blocking the functional Ah receptor, a similar experiment was carried out using CYP1A1 DRE as probe. The Ah receptor and Arnt antibodies inhibited binding of the TCDD-induced nuclear protein to the labeled CYP1A1 DRE. Combined, these experiments demonstrate that the induction of UGT1A1 by TCDD is controlled in part by binding of the activated Ah receptor-Arnt complex to the UGT1A1-XRE sequence.

DISCUSSION
The human UGT1A1 gene plays an important role in normal physiology by serving as the only source for the glucuronidation of bilirubin (28), the byproduct of heme degradation. The gene is expressed differentially in a tissue-specific fashion in humans (29 -31), indicating that multiple regulatory factors are involved in UGT1A1 expression. Several recent findings con-firm that UGT1A1 can also be regulated by environmental exposure. Exposure of HepG2 (10) and Caco-2 Cells (11) by specific bioflavonoids (10,32) induces UGT1A1. In primary human hepatocytes, treatment with phenobarbital, oltipraz, and 3-methylcholanthrene led to the induction of UGT1A1 mRNA and protein (19). The phenobarbital-type inducer TCPOBOP activates the human UGT1A1 gene through CAR at a nuclear receptor sequence (NR1) between bases Ϫ3483/ Ϫ3194. Work in our laboratory has recently identified a human SXR binding site in this same region (33). These results demonstrate that the UGT1A1 gene undergoes differential regulation because of tissue-specific expression and inducibility with drugs and xenobiotics. In addition to these responses, we have demonstrated that the UGT1A1 gene is also regulated by the human Ah receptor in response to TCDD, BNF, and B[a]P metabolites. HepG2 cells exposed to TCDD and BNF induces UGT1A1, as shown by Western blot analysis and indirectly by an increase in 17␣-ethynylestradiol UGT activity. The Ah receptor is functional in these cells as evident from the induction of CYP1A1 protein as well as regulation of a CYP1A1-luciferase promoter. We have mapped a regulatory sequence on the UGT1A1 gene that contains an XRE core sequence, which is positioned in close proximity to the NR1 (9) and SXR binding sites (Fig. 5B). An oligonucleotide encoding bases Ϫ3318/Ϫ3294 containing the Ah receptor binding sequence CACGCA associates with the activated nuclear Ah receptor in HepG2 cells. Mutation of this sequence eliminates binding of the Ah receptor, whereas the generation of enhancer constructs containing the same mutation leads to a loss of TCDD and BNF induction of transfected reporter gene activity. It would appear that this single responsive element plays an important role in regulation of UGT1A1 after exposure to TCDD and BNF.
The identification of the UGT1A1-XRE suggests that Ah receptor ligands may regulate UGT1A1 in a fashion comparable with CYP1A1. Along with results that we have presented FIG. 6. Ah receptor binding to UGT1A1-XRE. HepG2 cells were treated with Me 2 SO or 10 nM TCDD for 48 h. As outlined under "Experimental Procedures," nuclear extract was isolated from Me 2 SOtreated (DMSO-E) or TCDD-treated (TCDD-E) HepG2 cells, and 10 g of protein from each extract was incubated with labeled UGT1A1-XRE or CYP1A1-DRE probe (indicated at the bottom of the autoradiographs) and subjected to 6% non-denaturing acrylamide gel electrophoresis. Competition was performed in the presence of a 50-fold excess of unlabeled UGT1A1-XRE (XRE ϫ50) or CYP1A1-DRE (DRE ϫ50) To determine whether the induced nuclear protein represented the Ah receptor/ Arnt complex, binding reactions were also carried out in the presence of antibody generated toward the mouse Ah receptor (Anti-AhR) or mouse Arnt (Anti-Arnt). Control experiments were also conducted with an antibody generated toward the UDP-glucuronosyltransferases (Anti-UGT). The arrow indicates the TCDD inducible protein-DNA complex.
FIG. 5. Functional characterization of the UGT1A1-XRE sequence. A, an additional series of expression plasmids were generated from E1 (Fig. 3) to identify the TCDD-responsive region. A region of ϳ200 bases (E5) was identified that supports enhancer activity after treatment with TCDD (blue bars) and BNF (brown bars). B, nucleotide sequence of a 130 base pair region spanning from Ϫ3425 to Ϫ3295. Shown in bold are binding regions for SXR, CAR (NR1), and the Ah receptor (XRE). C, activity of an enhancer region that contains a mutation in the XRE sequence. The reporter plasmid containing either wild type or mutated UGT1A1-XRE (see "Experimental Procedures") was inserted into the PGL3-promoter vector and then used in transient transfections. The core binding sequence of CACGCA was changed to ACCGCA. This mutation resulted in a lack of TCDD-dependent induction of transcriptional activity.
for TCDD and BNF, other polycyclic aromatic hydrocarbons such as metabolites of B[a]P are capable of inducing UGT1A1. In addition, there is building evidence that some of the flavonoids modulate gene regulation in part through the Ah receptor. Chrysin is a potent inducer of UGT1A1 (10) and is able to induce the expression of CYP1A1, as demonstrated through induction of CYP1A1-luciferase in TV101 cells. 2 Studies in rats show that Ah receptor ligands such as 3-methylcholanthrene are capable of inducing intestinal Ugt1a1 (34), and it is well known that 3-methylcholanthene is a potent Ah receptor ligand. Omeprazole, a benzimidazole used in the treatment of peptic ulcer disease, activates the Ah receptor and induces CYP1A1 (23). Although not directly demonstrating induction of UGT1A1, omeprazole therapy has been shown to increase duodenal 3-hydroxybenzo[a]pyrene UGT activity greater than 5-fold (35). UGT1A1 is abundantly expressed in the small intestine (31). However, it is important to appreciate that dual regulation of UGT1A1 and CYP1A1 may not always occur. Apigenin, a flavonoid that is a potent inducer of human UGT1A1 (32), has very limited capacity to induce CYP1A1, as measured by induction of CYP1A1-luciferase in TV101 cells (13). Apigenin may regulate UGT1A1 in a manner that is independent of the Ah receptor.
As described by Sugatani et al. (9) and expanded by these studies and others (33), the UGT1A1 gene can be regulated by ligands that activate nuclear receptors CAR, SXR, and the Ah receptor. These cis-acting regulatory elements are positioned within a 125-base pair region on the UGT1A1 gene between bases Ϫ3424 and Ϫ3299. The location of these xenobiotic receptors in close proximity to each other may serve an important biological role in maintaining adequate expression levels UGT1A1. SXR and CAR are part of the orphan nuclear receptors that are structurally related to nuclear hormone receptors. It has been proposed that the xenobiotic nuclear receptors compose a family of regulatory proteins that are involved in steroid and xenobiotic sensing, leading to altered gene expression patterns essential for normal homeostasis (36,37). Originally postulated to regulate CYP3A genes, these nuclear receptors are now known to regulate a number of phase I and phase II xenobiotic enzymes. Although not part of the nuclear receptor family, the Ah receptor also serves to modulate phase I and phase II enzymes in response to environmental stimuli. Thus, regulation of UGT1A1 can be controlled by numerous endogenous agents that are ligands for SXR and CAR as well as xenobiotics that are ligands for SXR, CAR, and the Ah receptor.