Glucuronosyltransferase 1A7 Is Regulated by Thyroid Hormone ...

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Endocrinology 148(12):6124 – 6133 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2007-0443

Thyroxine-Metabolizing Rat Uridine DiphosphateGlucuronosyltransferase 1A7 Is Regulated by Thyroid Hormone Receptor Yoshikazu Emi, Shin-ichi Ikushiro, and Yoshihisa Kato Graduate School of Life Science (Y.E.), University of Hyogo, Harima Science Park City, Hyogo 678-1297, Japan; Biotechnology Research Center (S.I.), Faculty of Engineering, Toyama Prefectural University, Toyama 939-0398, Japan; and Faculty of Pharmaceutical Sciences of Kagawa Campus (Y.K.), Tokushima Bunri University, Kagawa 769-2193, Japan Exposure of rats to microsomal enzyme inducers perturbs thyroid hormone (TH) homeostasis through a variety of mechanisms. Glucuronidation is an important metabolic pathway for TH and is catalyzed by uridine diphosphate-dibenzo-glucuronosyltransferase (UGT) family proteins. Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats markedly increases the biliary clearance of glucuronidated T4 and results in reduced plasma T4 levels. Determination of the UGT1 isoforms responsible for glucuronidation of T4 has yet to be conclusively established. We here provide evidence for the involvement of TCDD-inducible UGT1A7 in the glucuronidation of T4 and TH-controlled UGT1A7 expression. Among a number of rat UGT1 isoenzymes examined in this study, UGT1A7 was the most active in catalyzing glucuronidation of T4. Expression of UGT1A7 was positively regulated by

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HYROID HORMONES (TH) play critical roles in the differentiation, growth, metabolism, and physiology of a wide variety of vertebrate tissues (1, 2). TH is released from the thyroid gland under the control of TSH. This control is strictly associated with feedback regulation by levels of circulating TH in serum. The thyroid gland predominantly produces the less active prohormone T4. Outer ring deiodination of T4 produces the more biologically active hormone T3 in peripheral tissues; alternatively, inner ring deiodination of T4 yields the inactive metabolite rT3 (3). In addition to deiodination, glucuronidation is an important metabolic pathway for TH (4). Glucuronidation involves transfer of the sugar moiety from uridine diphosphate (UDP)-glucuronic acid to substrates and is considered to play a crucial role in clearance of a wide variety of chemical compounds from the body. The resulting glucuronidated metabolites are more hydrophilic than the parent materials and therefore easily excretable into bile and urine. Over the last two decades there has been a growing interest in the glucuronidation of TH. Several chemical compounds may interfere with thyroid hoFirst Published Online September 20, 2007 Abbreviations: AhR, Aryl hydrocarbon receptors; MC, 3-methylcholanthrene; NR, nuclear receptors; PB, phenobarbital; PCB, polychlorinated biphenyls; PCN, pregnenolone-16␣-carbonitrile; RXR, retinoid X receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TH, thyroid hormone; TR, TH receptor; TRE, TH-responsive element; UDP, uridine diphosphate; UGT, UDP-glucuronosyltransferase. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

T4 through specific binding of TH receptor-retinoid X receptor heterodimers to a DR-5 sequence located between ⴚ109 and ⴚ93 in the UGT1A7 promoter. Overproduction of UGT1A7 protein decreased T4 responsiveness of a reporter gene containing the T4-responsive UGT1A7 promoter sequence. These results raise the possibility that UGT1A7 plays a key role in the glucuronidation of T4 leading to inactivation of T4, functioning via feedback regulation to control T4 levels in an autoregulatory manner, and that T4 regulates its own metabolism and subsequent clearance from cells. Our findings also predict that accumulation of TCDD-inducible UGT1A7 proteins in TH-target cells might disrupt the TH signaling by lowering the intracellular pool of T4. (Endocrinology 148: 6124 – 6133, 2007)

meostasis through a variety of mechanisms. Microsomal enzyme inducers, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (MC), polychlorinated biphenyls (PCB), phenobarbital (PB), and pregnenolone-16␣carbonitrile (PCN), have been shown to increase the clearance of TH by glucuronidation, resulting in decreased serum concentrations of TH (5– 8). UDP-glucuronosyltransferase (UGT) comprises a family of phase II drug-metabolizing enzymes and catalyzes glucuronidation of a wide variety of substrates, such as xenobiotics and endobiotics. UGT are classified into two subfamilies, UGT1 and UGT2 (9). Characterization of the UGT1 gene has revealed a striking genomic organization similar to that of the Ig gene cluster (10 –12). The UGT1 locus consists of multiple first exons that encode isoform-specific substratebinding domains and a single set of commonly used exons (exons II, III, IV, and V) that encode the same C-terminal UDP-glucuronic acid-binding domain for all UGT1 isoforms. Transcription from the unique first exon is independently regulated under the control of the corresponding promoter in the UGT1 gene complex. A long precursor transcript is transcribed at one of the unique exons and contains the entire downstream part of the gene complex. The first part of the variable exon at the 5⬘-end of transcript is selectively combined with the first common exon, which results in generation of diverse functional mRNA. Each UGT1 isoform arises from the complex gene through the alternative combination of a unique first exon and the common exons (see Fig. 2A). T4 and rT3 are believed to be glucuronidated to a large

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Emi et al. • Regulation of Rat UGT1A7 by TH

extent by UGT1A1 and UGT1A6 in rats (6, 13) and by UGT1A1 and UGT1A9 in humans (14). T3 has been shown to be glucuronidated predominantly by UGT2B2 in rats, in a study using the UGT2B2-deficient rat strain WAG (15). However, several lines of evidence from recent studies suggest that determination of the UGT1 isoforms responsible for glucuronidation of TH remains to be conclusively established. For example, increases in T4 glucuronidation in MCtreated rat hepatocytes could not be fully explained by induction of UGT1A1 and UGT1A6 proteins by MC (16). More light could be shed on this issue by overexpressing each UGT1 isoform in COS cells. On the other hand, UGT are subject to regulation by aryl hydrocarbon receptors (AhR) (17) and nuclear receptors (NR) such as retinoid X-receptor (RXR), constitutive androstane receptor, peroxisome proliferator-activated receptor, and farnesoid X-receptor (18 –21). Transcription from the unique first exon is controlled by a corresponding promoter in a manner that is drug responsive, tissue specific, and age dependent. These receptor-mediated gene regulatory actions may partly account for the isoform-specific expression patterns. UGT can also metabolize ligands for NR, such as retinoids, bile acids, fatty acids, and steroid hormones (21–25). UGT may therefore play an important role in NR-controlled cellular functions such as hormonal homeostasis, energy metabolism, and xenobiotic detoxification. Understanding NRdependent UGT gene regulation has broader significance in elucidating physiological functions of individual UGT isoforms. Effects of TH are mediated by TH receptors (TR) that belong to the NR superfamily (2, 26). Interestingly, TH can modulate glucuronidation by rat liver microsomes (27). Treatment of adult rats with T3 resulted in up-regulation of UGT1A6 and down-regulation of UGT1A1 (28, 29). However, little is known about molecular and cellular mechanisms of TH-mediated transcriptional regulation of the UGT1 gene complex. In this report, UGT1 isoforms responsible for the glucuronidation of T4 were identified using transiently expressed rat UGT1 proteins in COS cells. An unexpected finding was that UGT1A7 was the most active in glucuronidation of T4, whereas UGT1A1 and UGT1A6 had much lower activities than UGT1A7. We show that expression of UGT1A7 is upregulated by T4 through specific binding of TR/RXR heterodimer to a DR-5 motif within the UGT1A7 promoter. We also focused on the physiological significance of rat UGT1A7 in the cellular response to T4. Materials and Methods Construction of plasmids To express rat UGT1 family proteins in cultured mammalian cells, cDNA species [apart from the previously isolated UGT1A1 (30) and UGT1A6 (31)] were amplified by RT-PCR. Total RNA was prepared from rat liver using a QuickPrep Total RNA extraction kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions. RT was carried out using SuperScript II (GIBCO BRL, Gaithersburg, MD), and PCR was carried out using Pyrobest DNA polymerase (TaKaRa, Kyoto, Japan). The 5⬘-portion, which contains an isoform-specific sequence and a part of the common sequence encoded in exons 2 and 3 (12), was amplified by PCR using an isoform-specific primer with the rCOM-R1 common sequence primer. The primers used for amplification were as follows (the BamHI recognition sites are un-

Endocrinology, December 2007, 148(12):6124 – 6133

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derlined): 1A2-F, 5⬘-GTGTGTGGATCCGCCACTGCTGTGT-3⬘; 1A3-F, 5⬘-AGCACATTAGGATCCTGCGGACTTC-3⬘; 1A5-F, 5⬘-GTCCACTGGGATCCCATGGGACTCC-3⬘; 1A7-F, 5⬘-CACGGATCCCATGGCTCCTGCAGAC-3⬘; and rCOM-R1, 5⬘-TCCGGTGTAGCGCCACAGGAC-3⬘. The resultant PCR product was treated with BamHI and ligated into a pUC119 vector. The nucleotide sequences of the amplified cDNA fragments were confirmed by dideoxy sequencing with a Prism 3100 sequencer (Applied Biosystems, Foster City, CA). The 3⬘-portion containing the rest of the common region was excised from a UGT1A6 cDNA previously cloned into pBR322 (31) by digestion with BamHI plus DraI. The resultant 1.0-kb fragment was inserted into a pUCDSR␣ vector (32) that had been digested with BamHI and HincII to generate pCOM/ SR␣. DNA fragments of the 5⬘-portion were excised from the pUC119 subclones by digestion with BamHI and then ligated separately into pCOM/SR␣ that had been cleaved with BamHI to generate expression plasmids for UGT1 isoforms. Rat RXR␣ (L06482), TR␣ (M18028), and TR␤ (J03819) cDNA were amplified by RT-PCR as described above. The primers used for amplification were as follows (the EcoRI recognition sites are underlined): RXR-F, 5⬘-CCGAATTCACATGGACACCAAACATTTCCTG-3⬘; RXR-R, 5⬘-CCGAATTCCTAGGTGGTTTGATGTGGGGCCTC-3⬘; TR1-F, 5⬘-CCGAATTCGAATGGAACAGAAGCCAAGCAAG-3⬘; TR1-R, 5⬘-CCGAATTCTTAGACTTCCTGATCCTCAAAGAC-3⬘; TR2-F, 5⬘-CCGAATTCCTATGACTCCTAACAGTATGACA-3⬘; and TR2-R, 5⬘-CCGAATTCTCAGTCCTCAAAGACTTCCAAGAA-3⬘. The amplified PCR products were treated with EcoRI and then ligated into pCMV-HA (Clontech, Palo Alto, CA) to generate HA-RXR␣, HATR␣, and HA-TR␤. For construction of reporter gene, a genomic DNA clone COS6 –1 (12) was digested with HindIII plus BamHI, and the resultant 5.9-kb fragment containing the UGT1A7 promoter was recloned into pUC119 to generate the subclone pHB7 (6 –1). Site-directed mutagenesis was carried out using the QuikChange kit (Stratagene, La Jolla, CA) to introduce a HindIII recognition sequence in front of the translation initiation site. The primers used for mutagenesis were as follows (the HindIII recognition sites are underlined): 1A7/Hd-F, 5⬘-GTCTTGCCTTGGTCTAAGCTTCTCCCATGGCTC-3⬘, and 1A7/Hd-R, 5⬘-GAGCCATGGGAGAAGCTTAGACCAAGGCAAGAC-3⬘. A 4.5-kb fragment was then excised by digestion with HindIII and ligated into a pGL3-Basic vector (Promega, Madison, WI) to generate the reporter plasmid pA7-Luc/⫺4476. For preparation of progressive 5⬘deletions of the UGT1A7 promoter, pA7-Luc/⫺4476 was processed by exonuclease III and mung bean nuclease digestion as described previously (17).

Transfection of plasmids into HepG2 cells and reporter assays HepG2 cells were cultured in DMEM supplemented with 10% fetal calf serum (GIBCO BRL). Cells were transfected using FuGENE6 reagent (Roche, Indianapolis, IN) according to the supplier’s protocol. Typically, in reporter assays, the following DNA mixtures were treated with 6 ␮l FuGENE6 reagent and 94 ␮l serum-free medium: 1.0 ␮g reporter plasmids for expression of firefly luciferase, 0.8 ␮g pCMV-HA plasmids for expression of TR and RXR, and 0.2 ␮g of pmiwZ for expression of ␤-galactosidase. Half portions of the DNA-reagent mixtures were added to two 3.5-cm culture dishes. Cells were incubated with the DNA-lipid complex for 16 h, washed with PBS, incubated for another 24 h in fresh medium with T4 or 0.1% ethanol, and then assayed for ␤-galactosidase and luciferase activities. Normalization of transfection efficiency was performed by measuring ␤-galactosidase activities in different cell extracts by a spectrophotometric assay using o-nitrophenyl-␤-d-galactopyranoside as substrates. The induction rate was calculated from the ratio of luciferase activity of induced cells to that of uninduced cells.

Preparation of microsomal fractions and nuclear extracts COS cells were cultured in DMEM supplemented with 10% fetal calf serum. For preparation of microsomes and nuclear extracts, 6 ␮g plasmid DNA was treated with 18 ␮l FuGENE6 reagent and added to a 10-cm culture dish. Preparation of microsomes and immunoblot analysis were performed as previously described (33). Expression of UGT1 family proteins was confirmed by immunoblot analysis of the microsomes

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using anti-UGT1 IgG (34). Glucuronidation activity toward T4 was determined by quantifying the amount of [125I]T4-glucuronide as previously described (35). Glucuronidation activities toward bilirubin and 4-nitrophenol were determined as previously described (34). Nuclear extracts were prepared from COS cell cultures by a miniextraction method as described (36). Nuclear extracts were mixed with 1 ␮g poly(dI-dC) in 14 ␮l buffer containing 30 mm HEPES-KOH (pH 7.9) 100 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, and 15% glycerol. The reaction mixture was preincubated for 30 min at 4 C followed by another incubation with 1 ␮l labeled probe DNA (100 fmol) for 30 min at 4 C. Labeled DNA had been amplified by PCR using Cy5-conjugated primers. The sequences of primers used for PCR were as follows: A7/DR5-F, 5⬘-(Cy5)CAAGAAAAGGCTCACAAGCCC-3⬘, and A7/DR5-R, 5⬘-(Cy5)GGGAGAACAGTAATAATTGAGTAAT-3⬘. Competition experiments were carried out under the same conditions except with addition of unlabeled oligonucleotides. In antibody supershift experiments, nuclear extracts were preincubated at 4 C with anti-HA monoclonal antibodies (Covance, Princeton, NJ) for 60 min. DNAprotein complexes were separated on a 5% polyacrylamide gel as previously described (17) and visualized with an FLA3000 image analyzer (Fuji, Tokyo, Japan).

Preparation of total RNA and analysis of mRNA expression Rat hepatoma cell line H4-II-E was grown in DMEM supplemented with 10% fetal calf serum. For RNA analyses, 5 ⫻ 106 cells were treated with T4 or T3 at the indicated concentrations. In all experiments, controls were incubated with 0.1% ethanol. Total RNA was prepared from H4II-E cells by use of a QuickPrep total RNA extraction kit (Amersham) according to the manufacturer’s instructions. To compare the level of expression of each UGT1 isoform in TH-treated and untreated cells, semiquantitative RT-PCR was performed by use of isoform-specific primers with the rCOM-R1 common primer as described (12). The radioactivity of the band corresponding to each UGT1 isoform was quantified using an FLA3000 image analyzer, and the induction rate was defined to be the ratio of the radioactivity of an isoform in the TH-treated cells to the radioactivity of the respective isoform in the untreated cells. Each ratio was normalized to the amount of radioactivity incorporated into glyceraldehyde-3-phoshate dehydrogenase.

Results UGT1A7 is the most active UGT1 isoform involved in glucuronidation of T4

Six rat UGT1 isoforms (UGT1A1, UGT1A2, UGT1A3, UGT1A5, UGT1A6, and UGT1A7) were separately expressed in COS cells. UGT1A4 and UGT1A9 are not functional because of the premature termination codon present in the respective isoform-specific first exon (12). A cDNA clone encoding UGT1A8 was not available in this study because of its very low expression level in rat liver. Microsomal fractions were tested for their ability to catalyze glucuronidation of T4. As shown in Fig. 1, control cells transfected with an empty vector were unable to glucuronidate T4, indicating that this COS cell system is suitable for an evaluation of glucuronidation of T4 mediated by recombinant UGT1 isoforms. UGT1A7 glucuronidated T4 at the highest rate (154.5 ⫾ 7.0 pmol/ min䡠mg protein) among the rat UGT1 isoforms examined. UGT1A2 displayed a moderate rate of T4-glucuronide formation at 39.8 ⫾ 1.6 pmol/min䡠mg protein. In contrast, UGT1A1 and UGT1A6 catalyzed the glucuronidation of T4 at considerably lower rates (3.4 ⫾ 0.1 and 3.1 ⫾ 0.3 pmol/ min䡠mg protein, respectively). However, UGT1A1 and UGT1A6 exhibited significant activity toward bilirubin (1.06 nmol/min䡠mg protein) and 4-nitrophenol (32.5 nmol/ min䡠mg protein), respectively. Rat liver microsomes catalyzed the glucuronidation of bilirubin and 4-nitrophenol at

FIG. 1. Glucuronidation of T4 by UGT1A7. Microsomal fractions were prepared from COS cells that had been transfected with pUCDSR␣ (vector), p1A7/SR␣ (UGT1A7), p1A6/SR␣ (UGT1A6), p1A5/SR␣ (UGT1A5), p1A3/SR␣ (UGT1A3), p1A2/SR␣ (UGT1A2), or p1A1/SR␣ (UGT1A1). Transiently expressed UGT1 proteins in COS cell microsomes (10 ␮g) were analyzed by immunoblot analysis using antiUGT1 IgG, and representative immunostaining is shown on the upper panel. The positions of standard molecular weight markers are indicated on the left side. Glucuronidation activity was measured using 0.3 mg of microsomal fractions in the presence of 90 ␮M T4. Aliquots of the reaction were subjected to silica gel thin layer chromatography followed by autoradiography. Activities were determined by measuring the percent incorporation of radioactivity into the glucuronidated products, and expressed as picomoles substrate converted per minute per milligram protein for three determinations performed with the same microsomal preparation at different times. Quantitative data for UGT1 protein bands were determined with an LAS1000 image analyzer. The results are summarized in the lower panel, and data are mean values ⫾ SD. Values for glucuronidation activities are normalized to the estimated relative protein expression levels, expressed as a ratio to that of UGT1A1, and presented below the graph. N.D., Not detectable (⬍0.1 pmol/min䡠mg protein).

rates of 1.44 and 42.7 nmol/min䡠mg protein, respectively. These results rule out the possibility of production of inactive UGT1A1 and UGT1A6 enzymes in cells. UGT1A3 and UGT1A5 showed similar low activities for glucuronidation at rates of 1.8 ⫾ 0.3 and 6.5 ⫾ 0.4 pmol/min䡠mg protein, respectively. Expression of each UGT1 protein in microsomes was confirmed by immunoblotting with anti-UGT1 IgG (Fig. 1). This antibody was raised against the C terminus of rat UGT1 proteins and recognizes all UGT1 isoforms of rat origin to the same extent (34). Quantitative data for the expressed UGT1 protein bands on immunoblots were determined with an LAS1000 image analyzer, and expression levels relative to UGT1A1 were estimated. It was demonstrated that similar amounts of recombinant UGT1 proteins were produced in cells, except for those expressing UGT1A1. The level of UGT1A1 protein expression was less than 25% of that of other UGT1 isoforms. The activities for T4-glucuronide formation were normalized to the estimated relevant protein expression levels and expressed as a ratio of the expression of UGT1A1. It can be seen that the estimated specific activity for T4 glucuronidation of UGT1A7 still remains 10.2-fold that of UGT1A1.


T4 up-regulates UGT1A7 expression

Several groups have demonstrated that expression of UGT1 isoforms in MC-treated rat hepatoma H4-II-E cells is similar to that in the livers of MC-treated rats (37, 38). Hence, this cell line seems to be a good model to study UGT1A7 expression. H4-II-E cells were treated with 1 ␮m T4 or 0.1% ethanol for 18 h. The level of mRNA expression of each UGT1 isoform was determined by semiquantitative RT-PCR analysis (Fig. 2A). The radioactivity of the band corresponding to each isoform was measured as described in Materials and Methods (12, 17). The intensity of the amplified products increased in proportion to the increasing number of PCR cycles from 18 to 22 (data not shown). To evaluate the changes in mRNA expression within a given UGT1 isoform, the amount of RNA and the number of PCR cycles were fixed at 0.1 ␮g and 20, respectively. Under these conditions, all assays were performed within the range of the linear relationship between band intensity and the number of PCR cycles. Comparison of the PCR products revealed that the level of UGT1A7 mRNA in the T4-treated cells was approximately 2.2 times higher than that in untreated cells. UGT1A1 and UGT1A5 showed about 2-fold and 1.4-fold increments in the T4-treated cells, respectively. On the other hand, UGT1A6 did not significantly change during treatment. The levels of


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other UGT1 isoforms, such as UGT1A2, in H4-II-E cells were very low and did not change when T4 was present in the culture medium. Dose-dependent effects of T4 on the amounts of UGT1A7 transcripts were then analyzed (Fig. 2B). Treatment with 1 ␮m T4 resulted in a 2.5-fold induction of UGT1A7 mRNA, whereas treatment with higher concentrations somewhat reduced the level of UGT1A7 expression compared with that seen with 1 ␮m T4. This inhibitory effect on the expression of UGT1A7 may be due to cytotoxicity caused by the presence of elevated levels of hormones and solvent in the culture medium. MC provoked a 3.3-fold increase in accumulation of UGT1A7 transcript at 1 ␮m compared with untreated cells. It is noteworthy that the UGT1A7 promoter was activated by T4 as well as by MC. We also found that T3 was much less effective than T4, even in the presence of 1 ␮m T3 (data not shown). A concentration of 1 ␮m was used for both T4 and T3, which is close to the physiological concentration of serum T4 (100 nm) but 500 times the physiological value for T3 (2 nm). These findings encouraged us to determine whether the T4-dependent regulation of the T4-metabolizing UGT1A7 expression is mediated by TR. The 4.5-kb DNA fragment containing the UGT1A7 promoter was fused to the luciferase gene to generate a reporter

FIG. 2. Transcriptional activation of UGT1A7 gene by T4. A, RT-PCR analysis of mRNA expression. Total RNA was prepared from H4-II-E cells, which had been treated for 18 h with 0.1% ethanol (untreated) or 1 ␮M T4, and subjected to RT-PCR. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed as a loading control. The structure of the rat UGT1 locus is illustrated above. The open boxes and the closed boxes represent isoform-specific first exons and common exons, respectively. B, Dose-dependent expression of UGT1A7 mRNA by T4. H4-II-E cells were treated with the indicated concentrations of T4 or 1 ␮M MC for 18 h, and expression of UGT1A7 was analyzed by RT-PCR. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed for normalization purposes. Bar graphs and numbers represent the induction rates of UGT1A7 transcripts. C, Activation of the UGT1A7-luciferase fusion gene by T4. The indicated reporter plasmids (0.5 ␮g) were transfected into HepG2 cells in the presence of a 0.4-␮g mixture of expression plasmids for nuclear receptors and pmiwZ (0.1 ␮g). The 0.4-␮g DNA mixture consisted of the indicated combinations of NR (0.2 ␮g each), and the empty vector pCMV-HA was supplied to adjust the total amount of plasmid DNA to 0.4 ␮g. After incubation with transfection reagents for 16 h, cells were treated for 24 h with 0.1% ethanol (white bars) or 1 ␮M T4 (black bars). Luciferase activity was measured in the cell extracts, normalized to ␤-galactosidase activity, and expressed as the ratio to control luciferase activity on transfection with pGL3-Basic set at 1. All values are averages from three different transfections of three individual cell cultures and expressed as means ⫾ SD.

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gene (pA7-Luc/⫺4473). This reporter gene was cotransfected into HepG2 cells with expression plasmids, HA-RXR␣ combined with either HA-TR␣ or HA-TR␤, to test its ability to respond to T4-mediated transactivation by these receptor proteins. HepG2 cells are a human hepatocarcinoma cell line that displays a wide variety of responsiveness to many chemical compounds including hormones. Furthermore, HepG2 cells are easily transfected by our standard liposome-mediated method in contrast to H4-II-E cells. As shown in Fig. 2C, only background levels of expression of luciferase activity were observed when pGL3-Basic was transfected into HepG2 cells. 1A6/P1-Luc is derived by cloning the MCresponsive 1.4-kb HindIII fragment of the UGT1A6 promoter from the COS6-1 genomic clone (17) into pGL3-Basic. 1A6/ P1-Luc showed only basal levels of expression and failed to respond to T4. In contrast, cells transfected with pA7-Luc/ ⫺4473 expressed approximately 10-fold higher luciferase enzyme than cells transfected with pGL3-Basic. Cotransfection of pA7-Luc/⫺4473 into cells in the presence of rat RXR␣ and TR␣ followed by subsequent incubation with 1 ␮m T4 for 24 h caused a further 2-fold increase in UGT1A7 promoter activity. Activation of the UGT1A7 reporter by TR␤ also occurred in an RXR␣-dependent manner when 1 ␮m T4 was added to the culture medium. These results demonstrate that the 4.5-kb promoter region of UGT1A7 contains possible THresponsive elements (TRE).

FIG. 3. Identification of a functional TRE in the rat UGT1A7 promoter. Reporter plasmids (0.5 ␮g) with various deletions or substitutions were transiently transfected into HepG2 cells in the presence of HA-TR␤ (0.2 ␮g) and HARXR␣ (0.2 ␮g) together with pmiwZ (0.1 ␮g). After incubation with transfection reagents for 16 h, cells were treated for 24 h with 0.1% ethanol (white bars) or 1 ␮M T4 (black bars) and harvested. Luciferase activity was measured in the cell extracts, normalized to ␤-galactosidase activity, and expressed as the ratio to control luciferase activity on transfection with pGL3-Basic set at 1. All luciferase assays were done in triplicate, and the results are expressed as means ⫾ SD. A, Expression of reporter genes containing successive 5⬘-deletions of the UGT1A7 promoter. The numbers shown at the left side of the panel indicate the deletion end point from the transcription initiation site of the UGT1A7 gene. B, Expression of reporter genes containing fine deletions and base substitutions. Three additional reporter plasmids (⌬⫺61/⫺46, ⌬⫺110/⫺73, and mDR5) were used for the luciferase assay. C, Nucleotide sequence of a functional TRE. Shown is the sequence of the 60-nucleotide region between ⫺120 and ⫺61 of the UGT1A7 promoter that is necessary for the enhanced expression of UGT1A7 by T4. A DR-5 motif is shown between ⫺109 and ⫺93, and the core hexamer sequence is indicated in the closed box.


Identification of a cis-acting element required for the transcriptional enhancement of UGT1A7 gene by T4

To identify potential TRE in the UGT1A7 promoter, reporter genes with successive deletions of the 4.5-kb promoter region in the 5⬘ to 3⬘ direction were constructed. The nucleotide sequence of the proximal 0.9-kb region of the UGT1A7 promoter and the transcription start site have previously been reported (39). These plasmids were separately mixed with HA-RXR␣ and HA-TR␤ and transfected into HepG2 cells. Transient expression of luciferase enzyme driven by the deleted sequences was determined in the presence or absence of 1 ␮m T4 (Fig. 3A). A series of unidirectional deleted constructs containing the UGT1A7 promoter region starting at ⫺4473 to ⫺118 upstream from the transcription start site resulted in roughly equivalent fold induction of luciferase activities by T4. In contrast, a further deletion to ⫺38 (pA7Luc/⫺38) failed to respond to T4 but retained the basal level expression of luciferase. When the deletion end point was ⫺12 (pA7-Luc/⫺12), luciferase expression was markedly reduced to background levels. These results established that the distal 81 nucleotides (between ⫺118 and ⫺38) are required for enhanced expression by T4 and that the proximal 26 nucleotides (between ⫺37 and ⫺12) are required for basal expression of UGT1A7 in cultured cells. To more precisely localize the regulatory sequences, fur-



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ther dissection of this 81-bp region was investigated. A 65nucleotide deletion between ⫺110 and ⫺46 completely abolished the response to T4 (data not shown). As seen in Fig. 3B, deletion of the proximal 16 nucleotides spanning from ⫺61 to ⫺46 did not reduce T4-responsive luciferase expression; by contrast, deletion of the distal 38-nucleotide region spanning from ⫺110 to ⫺73 markedly reduced the level of T4-inducible luciferase expression. Inspection of this 38-nucleotide region revealed a plausible DR-5 NR-binding motif (AGGACAACAAAAGAGCA) between ⫺109 and ⫺93 (Fig. 3C). Mutation of the DR-5 motif (ACCATAACAAAACACTA) in the context of pA7-Luc/⫺955 abolished T4-inducible luciferase expression (Fig. 3B), indicating that this motif acts as a TRE. TR␣/RXR␣ heterodimers bind the DR-5 motif within the UGT1A7 promoter

To study the possibility that the DR-5 motif identified in the UGT1A7 promoter is a target of heterodimers of TR and RXR, gel mobility shift assays were carried out using nuclear extracts prepared from COS cells that had been transfected with expression plasmids for HA-tagged nuclear receptors. Expression of a roughly equivalent amount of TR␣ and RXR␣ proteins was confirmed from quantitative data for protein bands (48,000 and 58,000, respectively) determined by using an LAS1000 image analyzer (Fig. 4A). As shown in Fig. 4B, neither RXR␣ nor TR␣ alone was able to interact with this probe DNA (lanes 2 and 3). Coexpression of HA-RXR␣ and HA-TR␣ gave a prominent shifted band (lane 4), indicating the possible involvement of the TR␣/RXR␣ heterodimers in the complex formation. Furthermore, this DNA-protein complex was supershifted by anti-HA monoclonal antibodies (lane 9). Under these gel shift assay conditions, the DNAbinding complex appeared to contain heterodimers of HAtagged nuclear receptors. Competition experiments confirmed specific binding of the TR␣/RXR␣ heterodimers to the DR-5 motif. The binding was effectively competed with a 50-fold molar excess of unlabeled wild-type DR-5 oligonucleotides (lane 6) but not with a 100-fold molar excess of mutant DR-5 oligonucleotides (lane 8). This indicates that the mutated DR-5 motif failed to form a DNA-protein complex with a TR␣/RXR␣ heterodimer. Taken together, these results clearly demonstrate that the DR-5 site found in the UGT1A7 promoter is a target of the TR␣/RXR␣ heterodimers. It is noteworthy that the loss of capacity of the mutant DR-5 to bind to the TR␣/RXR␣ heterodimers shown in the gel shift assays (Fig. 4B) coincided with its failure to respond to the T4-triggered transactivation of the UGT1A7 gene observed in the reporter assays (Fig. 3B). Possible depression of intracellular thyroid hormone signaling by excess UGT1A7 proteins in the target cells

TH are excluded from cells as inactive glucuronidated metabolites (4), and in this study, rat UGT1A7 was found to be the most potent enzyme involved in glucuronidation of T4. UGT1A7 is also known to be highly inducible by various chemicals such as TCDD, MC (12), and oltipraz (39). Under such circumstances, overproduction of UGT1A7 proteins in the TH-targeted cells can facilitate the glucuronidation of T4. This may in turn reduce intracellular steady-state concen-

FIG. 4. Binding of transiently expressed TR␣ and RXR␣ to the DR-5 motif in the UGT1A7 promoter. A, Confirmation of the transiently expressed TR␣ and RXR␣ in nuclei of transfected cells. Nuclear extracts were prepared from COS cells that had been transfected with the indicated combinations of HA-TR␣ and HA-RXR␣. Five micrograms of each nuclear extract were subjected to immunoblot analysis using anti-HA monoclonal antibodies. The positions of standard molecular weight markers are indicated on the left side. B, TR␣/RXR␣ heterodimer binds to the DR-5 motif of the UGT1A7 promoter. The Cy5-labeled 84-bp DNA probe was incubated with crude nuclear extracts. The shifted bands are indicated by the black arrowhead. The specificity of their interaction was confirmed by competition with unlabeled DNA fragments. The indicated sequences of synthetic oligonucleotides were annealed and used as competitors in an indicated molar excess over the DNA probe. A supershift experiment was carried out using anti-HA monoclonal antibodies (0.1 ␮g), and the white arrowhead indicates a band shifted by antibodies. These results are representative of three independent experiments.

trations of the active ligands for TR, which then leads to disruption of normal cellular responses to TH. To explore this possibility, we examined whether overexpression of UGT1A7 could influence T4-mediated transactivation of the reporter gene in a dose-dependent manner. A reporter plasmid (pA7-Luc/⫺4473) was cotransfected into COS cells with constant amounts of expression vectors for nuclear receptors (HA-RXR␣ and HA-TR␤) and increasing amounts of an expression vector for UGT1A7 (p1A7/SR␣) (Fig. 5). Immunoblot analysis using anti-UGT1 IgG demonstrated that UGT1A7 protein content in transfected cells increased in proportion to increasing dose of p1A7/SR␣. When UGT1A7 was absent from the cells, treatment with 1 ␮m T4 increased pA7-Luc/⫺4473 activity 3.33-fold compared with untreated cells. In contrast, expression of UGT1A7 decreased basal expression levels and T4-mediated transactivation of the pA7-Luc/⫺4473 reporter gene. Cotransfection of 0.4 ␮g p1A7/SR␣ resulted in a 65% loss of T4-induced luciferase activity with respect to control cells not expressing UGT1A7, representing a 2.50-fold transactivation of the reporter gene. Taken together, these results suggest that overproduction of

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FIG. 5. Depression of intracellular thyroid hormone signaling by overexpression of UGT1A7 proteins. Luciferase reporter plasmid pA7-Luc/⫺955 (0.3 ␮g) was transiently transfected into COS cells in the presence of a DNA mixture of expression plasmids (0.6 ␮g) and pmiwZ (0.1 ␮g). This 0.6-␮g DNA mixture consisted of 0.1 ␮g HA-TR␤, 0.1 ␮g HA-RXR␣, and the indicated amounts of p1A7/SR␣. The empty vector pUCDSR␣ was supplied to adjust the total amount of DNA to 1.0 ␮g. Cells were treated for 24 h with 0.1% ethanol (white bars) or 1 ␮M T4 (black bars) and harvested at 40 h after transfection. Luciferase activity was measured in the cell extracts, normalized to ␤-galactosidase activity, and expressed as the ratio to uninduced luciferase activity on cotransfection with pUCDSR␣ (none) set at 100%. All values are expressed as means ⫾ SD of three independent experiments. Expression of UGT1A7 proteins was also confirmed by immunoblot analysis with anti-UGT1 IgG.

UGT1A7 proteins may potentiate elimination of glucuronidated T4 from cells, reduce intracellular concentration of T4, and thereby lead to a decrease in the cellular response to T4. Discussion

Rat UGT1A7 was originally identified as an MC-inducible isoform through our previous analysis of UGT1 genomic clones (12). UGT1A7 mRNA is expressed in many tissues including liver, intestine, kidney, and lung and has been reported to be inducible by administration of MC (12), oltipraz (40), and ␤-naphthoflavone (41). Ritter and his collaborators (40, 42– 44) have revealed that UGT1A7 catalyzes glucuronidation of a broad range of xenobiotics such as benzo(a)pyrene metabolites, chrysene, bisphenol A and acetaminophen. UGT1A7 has also been reported to glucuronidate endobiotics such as 17␤-estradiol and serotonin (44). Its expression is under the control of hepatocyte nuclear factor 1 in rat liver (45). However, few data are available regarding its substrate preference toward endogenous compounds and transcriptional regulation of the UGT1A7 gene. In this report, our findings particularly focus on the following features of UGT1A7: 1) its involvement in the glucuronidation of T4, 2) possible disruption of normal cellular responses to TH with overproduction of UGT1A7 protein, and 3) T4-regulated expression of the UGT1A7 isoform. Glucuronidation is one of the most important pathways for TH metabolism, facilitating the elimination of circulating TH into bile (4); this has generated considerable interest in


the function of UGTs in TH metabolism. UGT1A1 and UGT1A6 are believed to be important isoforms involved in the glucuronidation of T4 in rat liver (6, 13), however, we demonstrated that UGT1A7 and UGT1A2 were more potent in the glucuronidation of T4 than UGT1A1 and UGT1A6. Jeminitz et al. (16) predicted that isoforms additional to UGT1A1 and UGT1A6 are responsible for glucuronidation of T4 in studies using MC-treated primary rat hepatocyte cultures. Our study indicates that UGT1A7 could be one such enzyme. UGT1A2 may be an additional candidate for involvement in glucuronidation of T4 because culture-associated accumulation of UGT1A2 proteins occurs in primary cultures of rat hepatocytes (33). Vansell and Klaassen (46) also demonstrated that increased glucuronidation of T3 mediated by PCN treatment of rats is due to a mechanism other than induction of UGT2B2. More studies are required to analyze the ability of PCN to induce UGT isoforms. Relative contributions of individual rat UGT1 isoforms, such as UGT1A1, UGT1A2, UGT1A5, UGT1A6, and UGT1A7, to glucuronidation of T4 will depend on their relative protein expression levels in various tissues. Our previous study demonstrated that rat liver microsomes catalyzed the glucuronidation of T4 at 12.6 ⫾ 0.7 pmol/min䡠mg protein (47). In liver microsomes of untreated mature rats, UGT1A1 is a major UGT1 component; UGT1A6 together with UGT1A5 exist as additional minor components, whereas UGT1A2 and UGT1A7 proteins are virtually undetectable by standard immunoblot analysis (34). It is likely that hepatic UGT1A1 plays a critical functional role in the control of steady-state concentrations of circulating TH in untreated adult rats. On the other hand, UGT1A6 and UGT1A7 are known to be highly inducible in the liver after exposure of rats to polycyclic aromatic hydrocarbon-type inducers such as TCDD and MC (12). In MC-treated rats, UGT1A6 is increased not more than 3-fold, whereas UGT1A7 exhibits a dramatic increase from virtually undetectable levels. Our previous work showed that the level of UGT1A7 protein in the liver of MC-treated rats was between one fourth and one third the amount of UGT1A1 and UGT1A6 (34). The calculated specific activity for glucuronidation of T4 by UGT1A7 was 10.2-fold that of UGT1A1 and about 50 times higher than that of UGT1A6. These observations led us to estimate that the contribution of UGT1A7 to hepatic glucuronidation of T4 is at least three times and 10 times higher than that of UGT1A1 and UGT1A6 in MC-treated rats, respectively. Taken together, under these conditions of enzyme induction in rat liver, T4 is most likely to be glucuronidated by UGT1A7, and thereby the overall glucuronidation activity for T4 is presumably increased up to 3- to 4-fold with the additional contribution of newly induced UGT1A7. Accumulating evidence confirms that MC provokes a 4- to 6-fold induction of glucuronidation of T4 in rat liver (8, 16) and strongly supports the validity of our proposal. Determination of enzyme kinetic parameters of major hepatic UGT1 isoforms (UGT1A1, UGT1A5, UGT1A6, and UGT1A7) toward T4 would allow insight into the contribution of each UGT1 isoform to the metabolism of T4 in rat liver. In contrast to liver, rat intestinal epithelia exhibit constitutive expression of UGT1A7, UGT1A6, UGT1A2, and UGT1A1. Existence of an enterohepatic circulation is well


established, where enterobacteria hydrolyze glucuronidated conjugates to produce aglycons; some of the resultant aglycons, including TH, then return to the liver after absorption into mesenteric veins (48, 49). It is likely that intestinally expressed UGT1 isoforms are of functional importance for additional rounds of glucuronidation of absorbed aglycons, facilitating an extensive reexcretion of glucuronides into the intestinal tract. Further investigation is needed to explore this possibility. TH levels are regulated not only at the levels of synthesis and secretion by the thyroid gland under the influence of the hypothalamic-pituitary-thyroid axis but also at the levels of catabolism and elimination by peripheral tissues, especially the liver (50). Thus, the hepatic-endocrine axis is an important component in the homeostatic control of circulating TH levels; furthermore, induction of hepatic microsomal enzymes alters the metabolism not only of xenobiotics but also of various endobiotics, including TH. Many microsomal enzyme inducers have been shown to increase the clearance of TH by glucuronidation, resulting in decreased serum concentrations of TH (5– 8). Chronic exposure to environmental contaminants, industrial chemicals, and therapeutic drugs that induce UGT isoforms can lead to hypothyroidism by depleting the levels of TH. In particular, the increased glucuronidation of T3, rather than T4, enhances the secretion of TSH from the pituitary gland; the resulting elevation of TSH levels stimulates thyroid gland function and growth. Thus, chronic stimulation of thyroid follicular cells causes morphological changes of the thyroid, eventually resulting in thyroid hyperplasia (7, 8). Among these chemicals, only PB and PCN increase the glucuronidation and biliary excretion of T3 (6) but fail to induce UGT1A7 (12, 46). TCDD, MC, and PCB increase glucuronidation of T4 but have no effect on serum TSH, despite reducing serum T4. These three compounds strongly induce UGT1A7 (12, 46). Growth and development in the fetus and in childhood are dependent on normal levels of TH. Hypothyroidism during this critical phase of life causes many developmental deficiencies, such as abnormal development of the central nervous system. TCDD given to pregnant animals is easily transferred to the offspring via transplacental and lactational routes; hence, maternal exposure of rats to TCDD during gestation can cause many impairments in offspring such as decreased circulating T4, cognitive deficiencies, and reduction of body weight (51, 52). Recently, it is demonstrated that these impairments are mediated entirely via AhR (53). UGT1A6 is a predominant UGT1 component in fetal rat liver and may play an important role in control of steady-state concentrations of TH in the fetus. Rat UGT1A6 and UGT1A7 are inducible under the control of AhR and likely to be induced in the liver of fetus and pups upon exposure to TCDD. It is probable that eventual dramatic accumulation of UGT1A7 proteins causes pronounced depletion of circulating TH. In pregnant rats exposed to PCB, hydroxylated PCB metabolites are accumulated in fetal tissues, such as liver and brain. Moreover, mammals, including humans (54), perinatally exposed to PCB exhibit decreased circulating T4, abnormal thyroid function, and neurological impairment. More research is needed to clarify whether UGT1A7 is induced in


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the livers of fetal rats that had suffered from maternal exposure to these chemicals. Regulation of cellular TH signaling is controlled by a number of coordinated mechanisms, including cell-specific expression of TR and metabolism of TH. Environmental chemicals and therapeutic drugs can alter TH-mediated gene regulation, but the precise molecular mechanisms executing these changes are still not clear (51). Chronic exposure of animals to many kinds of chemicals can stimulate expression of UGT1 proteins including UGT1A7. In practice, TCDD and MC induce the accumulation of UGT1A7 proteins (34); PCB can also induce UGT1A7 mRNA to the same extent as MC (46). This can facilitate elimination of T4 from the target cells and then may impart an increased risk of depression of cellular response to T4 by lowering the intracellular concentrations of TR ligands. Our finding presented in Fig. 5 is of significance in predicting one possible cellular event whereby overproduction of UGT1A7 proteins in the TH target cells might disrupt gene regulation governed by TRmediated signal transduction. Our data show convincingly that T4 rather than T3 is involved in the regulatory mechanism for the expression of UGT1A7 through specific binding of TR to the TRE in the UGT1A7 promoter. However, it is generally accepted that TR has a much higher affinity for T3 than T4 (Kd values are 0.2 nm for T3 and 2 nm for T4) (55). Binding of T3 to TR derepresses TRE-dependent genes and induces the expression of target genes. In addition to this genomic or TRE-dependent action of T3, nongenomic or TRE-independent pathways of T3 have been described. Nongenomic effects are characterized by onset within minutes, do not require nuclear TRs, and in some cases are more responsive to T4 (56). Thus, our finding raises an interesting possibility that some kind of nongenomic pathway could be partly involved in the T4regulated expression of the UGT1A7 gene. More research is required to explore this possibility. It is noteworthy that expression of T4-metabolizing UGT1A7 was positively regulated by T4 and that UGT1A7 is expressed in many tissues (12, 40). These findings imply the potential for T4-inducible metabolism of T4 by UGT1A7 in a wide variety of TH target cells. They also provide a mechanism whereby circulating T4 may play a critical role in maintaining proper levels of UGT1A7 and can act as a regulator of cellular TH metabolism leading to inactivation of T4. Generation of T3 by deiodination of T4, their glucuronidation to more water-soluble forms, and eventual elimination of TH are counterbalanced by metabolic pathways that regulate TH pools in the target tissues. UGT1A7 is assumed to play a key role in metabolism of T4 by functioning in a feedback loop in the target tissues, where physiological T4 levels are controlled in an autoregulatory manner. Thus, glucuronidation may function to protect TH-sensitive tissues from accumulation of deleterious TH concentrations. Further investigation is needed to determine the physiological significance of these significant findings. TR binds preferentially to most TRE as a heterodimer with the RXR. It is generally considered that the TR/RXR heterodimer binds to the DR-4 motif where two TRE half-sites (AGGTCA) are arranged in direct repeats separated by a four-nucleotide spacer (2, 26). In the present study, we found

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a DR-5 motif in the promoter region of the exon 1A7 and clearly demonstrated that this motif plays an important role in T4-mediated transactivation of the UGT1A7 gene. Our finding seems to deviate from the commonly held view of TRE. It has recently been shown that a more extended sequence is optimal for TR binding and TH responsiveness. Interestingly, the 8DR-5 motif (5⬘-TAAGGTCANNNTAAGGTCA-3⬘), which signifies a tandem arrangement of the extended octamer half-sites (TAAGGTCA) and five-nucleotide spacing with the core hexamer sequence (AGGTCA), exhibits robust responsiveness to TH (57). 8DR-5 corresponds to the DR-5 motif where two tandemly arranged hexamer half-sites are separated by a five-nucleotide spacer. An analogous sequence (5⬘-TCAGGACAACAAAAGAGCA-3⬘) was found in the promoter region of exon 1A7 and demonstrated to be of functional importance for responsiveness to TH. Inspection of TRE of many different target genes reveals that there is a relatively low degree of sequence conservation among these elements. This suggests the possibility that naturally occurring TRE may have diverged from an ancestral consensus element during evolution as a means to modulate the degree of TH responsiveness. Acknowledgments We gratefully acknowledge Dr. Masao Sakaguchi (University of Hyogo), Dr. Yuichiro Kida (University of Hyogo), and Dr. Takashi Osumi (University of Hyogo) for critical comments on the manuscript and helpful technical advice. We also thank Mr. Hiroshi Suzuki (School of Pharmaceutical Sciences, University of Shizuoka) for the invaluable technical assistance regarding determination of the glucuronidation activity toward T4. Received April 5, 2007. Accepted September 10, 2007. Address all correspondence and requests for reprints to: Yoshikazu Emi, Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1297, Japan. E-mail: [email protected]. This work was supported in part by the Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (18510061, Y.K.), Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (09780580, Y.E.), and Grants for Scientific Research from the Foundation of Himeji Institute of Technology (Y.E.). Disclosure Statement: The authors have nothing to declare.

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