Drug Metab. Pharmacokinet. 22 (5): 391–398 (2007).

Note Hepatocyte Nuclear Factor 1 Alpha and 4 Alpha are Factors Involved in Interindividual Variability in the Expression of UGT1A6 and UGT1A9 but not UGT1A1, UGT1A3 andUGT1A4mRNAinHumanLivers†

Sasitorn AUEVIRIYAVIT,TomomiFURIHATA*,KaoriMORIMOTO, Kaoru KOBAYASHI and Kan CHIBA Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: UDP- (UGTs) catalyze phase-II biotransformation reaction of a var- iety of substances. Among the UGT1A isoforms, UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9 are predominantly expressed in the liver. Interindividual variability in expression of these isoforms would cause interindividual diŠerences in drug response, toxicity and cancer susceptibility. In the present study, we investigated the interindividual variability in UGT1A mRNA expression and whether hepatocyte nuclear factor 1a (HNF1a)andHNF4a were factors responsible for their variability in human livers. The amounts of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, HNF1a and HNF4a mRNA in 18 human livers were measured by quantitative real-time polymerase chain reaction. The largest and smallest interindividual diŠerences in expression levels were observed in UGT1A1 (8.6- fold) and UGT1A4 (2.5-fold) mRNA, respectively. The amounts of HNF1a and HNF4a mRNA were strongly correlated with the amount of UGT1A9 mRNA and moderately correlated with that of UGT1A6 mRNA, whereas no signiˆcant correlation was found with the amounts of UGT1A1, UGT1A3 and UGT1A4 mRNA. Our results suggest that HNF1a and HNF4a are the factors involved in the interin- dividual variability of UGT1A6 and UGT1A9 mRNA expression. Further studies of other transcription factors are needed to clarify the factor(s) determining the interindividual variations in UGT1A1, UGT1A3 and UGT1A4 mRNA expression.

Key words: UDP-; hepatocyte nuclear factor; regulation; interindividual variability; human liver

of amino acid sequences, UGTs have been categorized Introduction into two families, UGT1 and UGT2.2) UGT1A UDP-glucuronosyltransferases (UGTs) catalyze the isoforms, encoded from a single gene with alternative glucuronidation of many endogenous compounds, promoters located on 2q37, have been drugs and carcinogens, which is one of the important characterized in humans.2) Through a process of RNA detoxiˆcation pathways.1) On the basis of the homology splicing, nine UGT1A isoforms are generated with an individual ˆrst exon joined to identical exons 2 to 5.3) † This work was supported by grants-in-aid from the Ministry of Among the UGT1A isoforms, UGT1A1, UGT1A3, Health, Labor and Welfare of Japan (Health and Labor Sciences UGT1A4, UGT1A6 and UGT1A9 have been shown to Research Grants, Research on , Tissue Engineering; be predominantly expressed in the liver,4) where they Health and Labor Sciences Research Grants, Risk Analysis Research on Food and Pharmaceuticals), and was partially support- contribute to more than half of total drug glucuronida- 5) ed by grants (18890044 and 17790112) from the Ministry of Educa- tion. tion, Sciences, Sports and Culture of Japan. It is likely that interindividual variability of hepatic

Received; June 11, 2007, Accepted; July 23, 2007 *To whom correspondence should be addressed: Tomomi FURIHATA,Ph.D.,Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan. Tel. & Fax. ＋81-43-226-2894, E-mail: tomomif＠ p.chiba-u.ac.jp

391 392 Sasitorn AUEVIRIYAVIT, et al.

UGT1A expression plays an important role in drug (Kashiwa, Japan). The liver samples were part of tissues e‹cacy, xenobiotic toxicity and cancer susceptibility.6) surrounding tumor areas that had been surgically resect- To date, there have been only limited reports showing ed from donors with hepatocarcinoma metastasis from interindividual variability in the hepatic expression of colorectal or gastric cancer. The liver tissues were snap- UGT1A mRNA. Congiu et al.7) reported that interin- frozenandstoredinliquidnitrogenuntiluseforRNA dividual variations in the amounts of UGT1A and isolation. This study was approved by the Ethics Com- UGT2B mRNA ranged from 5- to 15-fold in human mittee of Chiba University (Chiba, Japan). livers and that the variations in UGT1A4, UGT2B4 and RNAisolationandcDNAsynthesis: Total RNA UGT2B7 mRNA levels were related to the degree of was isolated from liver tissues by using an SV Total Iso- liver in‰ammation. Two other studies showed remarka- lation System (Promega, Madison, WI) according to the ble interindividual variations in UGT1A1 and UGT1A6 manufacturer's instructions. The isolated total RNA mRNA expression levels in human livers.8,9) However, was then treated with RNase-free DNase I (Takara, Shi- the molecular mechanism(s) involved in the interindivid- ga, Japan) to remove contaminating genomic DNA, and ual diŠerence in UGT1A mRNA expression has not the integrity of each RNA sample was evaluated by de- been fully elucidated. Although it has been reported termination of the ratio of 28S rRNA to 18S rRNA by that genetic polymorphisms in the 5?-‰anking region formaldehyde-agarose gel electrophoresis. The cDNA and environmental factors, such as drugs, tobacco and was generated with a random hexamer by using Ready- alcohol consumption, are associated with the interin- to-GoTM RT-PCR Beads (GE Healthcare, Little Chal- dividual diŠerences in UGT1A1 and UGT1A6 mRNA font, UK). expression levels,8,9) these factors cannot entirely explain Quantiˆcation of hepatic UGT1A, HNF1a and their interindividual variations. HNF4a mRNA expression levels: The expression lev- Besides genetic and environmental factors, the in- els of UGT1A1, UGT1A3, UGT1A4, UGT1A6, terindividual variation in UGT1A mRNA expression UGT1A9, HNF1a and HNF4a mRNA in human liver may result from diŠerent expression levels of transcrip- tissues were measured by quantitative real-time PCR tion factors controlling their expression between in- carried out on an ABI PRISM 7000 (Applied dividuals. Recently, the proximal promoters of several Biosystems, Foster city, NJ) and using TaqMan Gene human UGT have been studied and the transcrip- Expression Assays speciˆc for each gene (FAM/MGB tion factors that regulate these promoters have been Probe, Applied Biosystems). The PCR ampliˆcation identiˆed.6,10–14) The results of these studies have shown was performed as described previously.16) The expres- that HNF1a and HNF4a, which are the major liver-en- sion levels of UGT1A, HNF1a and HNF4a mRNA were riched transcription factors,15) canbindandactivate normalized by the average values of four housekeeping UGT1A promoters. Potential binding sites for HNF1a genes (cyclophilin, b-glucuronidase, acidic ribosomal were found in UGT1A1, UGT1A3, UGT1A4, UGT1A9 protein and glyceraldehyde-3-phosphate dehydro- and rat Ugt1a6 gene promoters and their functions were genase) based on previous reports.17,18) The mRNA ex- characterized by luciferase assays and electrophoretic pression levels of genes are expressed in arbitrary units, mobility shift assays.6,11–14) In addition to HNF1a, with the lowest mRNA expression level of each gene HNF4a hasbeenshowntoactivateandbindtothe being assigned to 1. The amount of mRNA was meas- UGT1A9 gene promoter.13,14) Basedontheresultsof ured in duplicate, and each value presented is the mean these in vitro studies, it is reasonable to hypothesize that from three independent measurements. HNF1a and HNF4a are factors determining the hepatic Statistical analysis: Correlations between the expression of UGT1A mRNA in vivo. However, the sig- amounts of UGT1A mRNA and those of HNF1a and niˆcance of HNF1a and HNF4a in the expression of HNF4a mRNA were determined by univariated linear UGT1A mRNA in human livers has not been clariˆed. regression analysis. A p value º0.05 was considered In the present study, we investigated the interindivid- statistically signiˆcant. ual variability in UGT1A mRNA expression and Results whether HNF1a and HNF4a were factors responsible for their variability in human livers. The expression lev- Interindividual variability of UGT1A mRNA expres- els of UGT1A1, UGT1A3, UGT1A4, UGT1A6 and sion levels in human livers: The extents of interin- UGT1A9 mRNA in 18 human liver samples were quan- dividual variability of UGT1A mRNA expression were tiˆed and their correlations with the amounts of HNF1a diŠerent among isoforms (Fig. 1). The highest to lowest and HNF4a mRNA were examined. extents of interindividual diŠerences in expression levels were observed in UGT1A1 (8.6-fold), UGT1A3 (6.5- Materials and Methods fold), UGT1A9 (5.1-fold), UGT1A6 (4.9-fold) and Human liver samples: Japanese liver samples (n＝ UGT1A4 (2.5-fold) mRNA in that order. 18) were obtained from National Cancer Hospital East Interindividual variability of HNF1a and HNF4a HNF1a &4a are Determinants of UGT1A6 & 1A9 mRNA Expression 393

Fig. 1. Interindividual variability in expression levels of UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9 mRNA in human livers. The mRNA expression levels of each gene in 18 human liver samples were measured by real-time PCR and were normalized to average ex- pression levels of housekeeping genes as described in Materials and Methods. The data are expressed as arbitrary units, with the lowest mRNA expression level of each gene being assigned to 1. Each bar represents the mean of three independent measurements (±S.D.).

mRNA expression levels and their correlation in human between UGT1A9 mRNA and HNF1a mRNA expres- livers: Among individuals, HNF1a and HNF4a sion levels (r＝0.79, pº0.0001) (Fig. 3). The correlation mRNA expression levels varied 8.0- and 18-fold, respec- between UGT1A6 mRNA and HNF1a mRNA expres- tively (Fig. 2A). The high correlation was observed be- sion levels was also statistically signiˆcant (r＝0.52, tween the expression levels of HNF1a and HNF4a pº0.05). In contrast, there was no signiˆcant correla- mRNA (r＝0.70, pº0.005) (Fig. 2B), being consistent tion between UGT1A1, UGT1A3, UGT1A4 mRNA ex- with the results of the previous reports showing that pression levels and HNF1a mRNA expression level. HNF1a and HNF4a canbindtoeachother'spromoter Correlations between UGT1A mRNA and HNF4a to positively regulate the expression of each other.19–21) mRNA expression levels: Similar to HNF1a,asig- CorrelationsbetweenUGT1AmRNAandHNF1a niˆcant correlation was found between UGT1A9 mRNA expression levels: When compared among mRNA and HNF4a mRNA expression levels (r＝0.66, UGT1A isoforms, the strongest correlation was found pº0.005) (Fig. 4). A signiˆcant correlation was also 394 Sasitorn AUEVIRIYAVIT, et al.

Fig. 2. Interindividual variability in expression levels of HNF1a and HNF4a mRNA and their correlation in human livers. A, The mRNA expression levels of HNF1a and HNF4a in 18 human liver samples were measured by real-time PCR and were normalized to average ex- pression levels of housekeeping genes as described in Materials and Methods. The data are expressed as arbitrary units, with the lowest mRNA expression level of each gene being assigned to 1. Each bar represents the mean of three independent measurements (±S.D.). B, The correlation between the amount of HNF1a mRNA and the amount of HNF4a mRNA was analyzed by linear regression analysis. The correlation coe‹cient (r) and signiˆcant value (p) is shown. found between UGT1A6 mRNA and HNF4a mRNA tions －290 to －278 and －235 to －223, respectively.14) expression levels (r＝0.55, pº0.05), whereas there was Mutation of these binding sites dramatically reduced the no signiˆcant correlation between UGT1A1, UGT1A3, ability of HNF1a and HNF4a to activate a UGT1A9 UGT1A4 mRNA expression levels and HNF4a mRNA 2kb reporter construct. In addition, Gardner-Stephen et expression level. al.14) reported a synergistic role of HNF1a and HNF4a in regulation of the UGT1A9 promoter. However, the Discussion identiˆed HNF1a and HNF4a-binding elements in the We hypothesized that HNF1a and HNF4a were fac- UGT1A9 promoter were not conserved in the UGT1A7, tors determining the expression of UGT1A mRNA in UGT1A8 and UGT1A10 promoters, which are not ex- the human liver. Our results showed that the amounts of pressed in the liver.14) Therefore, both HNF1a and HNF1a and HNF4a mRNA were highly correlated with HNF4a are thought to be important transcription fac- the amount of UGT1A9 mRNA (Figs. 3 and 4), suggest- tors contributing to unique hepatic expression of ing that both HNF1a and HNF4a are factors determin- UGT1A9 amongst the UGT1A7-10 gene cluster. Taken ing the hepatic UGT1A9 mRNA expression. These ˆnd- together, our results suggest that both HNF1a and ings are consistent with the results of previous in vitro HNF4a are the factors determining the constitutive ex- studies characterizing the UGT1A9 promoter.13,14) pression of the UGT1A9 gene and are factors involved UGT1A9 is the only isoform of the UGT1A7-10 gene in the interindividual variability in UGT1A9 mRNA ex- cluster known to be expressed in the liver.2) Recently, pression in human livers. the functional binding sites of both HNF1a and HNF4a In addition to the UGT1A9 gene, our results suggest have been found in the UGT1A9 gene promoter at posi- that HNF1a and HNF4a are also factors determining HNF1a &4a are Determinants of UGT1A6 & 1A9 mRNA Expression 395

Fig. 3. Correlations between expression levels of UGT1A mRNA (UGT1A1, 1A3, 1A4, 1A6 and 1A9) and HNF1a mRNA in human livers. The correlations between the amounts of UGT1A mRNA and the amount of HNF1a mRNA were analyzed by linear regression analysis. The correla- tion coe‹cient (r) and signiˆcant value (p) for each correlation is shown. the hepatic expression of UGT1A6 mRNA, although that while all hepatic UGT1A genes possess an HNF1a the strength of the correlation was not high (Figs. 3 and -binding site in their proximal promoters,6,11,12,14) their 4). To date, there has been no study on the role of signiˆcance in the expression of UGT1A genes in the HNF1a and HNF4a in hepatic expression of the human liver varies. Another example of this type of discrepancy UGT1A6 gene. However, it has been shown that has been reported in the UGT2B15 gene. Although it HNF1a can stimulate activity of the P2 promoter of the was reported that the putative HNF1a-binding site was rat Ugt1a6 gene.12) This HNF1a-binding site has also found in the UGT2B15 promoter and that HNF1a was been conserved in the promoter of the human UGT1A6 found to occupy the UGT2B15 promoter in hepatocytes gene at positions －169 to －157 (GTTAAATAT- by using chromatin immunoprecipitation combined TAAT).6) As for HNF4a, examination of the DNA se- with promoter microarrays,6,21) the amount of HNF1a quence of the human UGT1A6 promoter by using com- mRNA did not correlate with the expression level of puter analysis revealed a possible HNF4a-binding site at UGT2B15 mRNA.23) These results suggest that even if positions －750 to －738 (TGTTCTTTGTACT) there are functional HNF1a-binding sites, the contribu- (http://www.gene-regulation.com).22) Although further tion of these sites to gene expression in the liver is de- studies on the function of HNF1a and HNF4a in the hu- pendent on the individual UGT genes. These variations man UGT1A6 gene promoter are needed, our results may be caused by larger regulatory networks of tran- suggest that constitutive expression of the UGT1A6 scription factors occurring in vivo. In a chromosomal gene in the human liver could be under the control of setting, HNF1a can cooperate with other transcription both HNF1a and HNF4a asinthecaseoftheUGT1A9 factors to form a regulatory network that is speciˆc in gene. Taken together, the results suggest that HNF1a each gene and cell type in which it is active.24) For exam- and HNF4a are likely to be factors determining the in- ple, it has been shown that HNF1a acted synergistically terindividual diŠerence in hepatic UGT1A6 mRNA ex- with octamer transcription factor-1 to enhance the pression. UGT2B7 promoter activity in HepG2 cells.25) In con- In contrast to the UGT1A6 and UGT1A9 genes, the trast, pre B cell homeobox-2 decreased the binding and amounts of UGT1A1, UGT1A3 and UGT1A4 mRNA transcriptional capacity of HNF1a to the UGT2B17 were not signiˆcantly correlated with those of HNF1a promoter.26) Although little is known about regulatory and HNF4a mRNA (Figs. 3 and 4). It is noteworthy networks for transcription control in the UGT1A genes, 396 Sasitorn AUEVIRIYAVIT, et al.

Fig. 4. Correlations between expression levels of UGT1A mRNA (UGT1A1, 1A3, 1A4, 1A6 and 1A9) and HNF4a mRNA in human livers. The correlations between the amounts of UGT1A mRNA and the amount of HNF4a mRNA were analyzed by linear regression analysis. The correla- tion coe‹cient (r) and signiˆcant value (p) for each correlation is shown. the results of these studies imply that the relationship tivity of the UGT1A1 gene is reduced by the length of between HNF1a and HNF4a expression and UGT1A1, the TATA box (UGT1A1*28,(TA)7TA) and polymor- UGT1A3 and UGT1A4 expression might be complicat- phism in phenobarbital-responsive enhancer module ed by interactions with other transcription factors. (UGT1A1*60, T3263G).8,29) Although we did not ana- Among the hepatic UGT1A isoforms, we observed lyze these variants in the present study, it is possible that the greatest interindividual diŠerence in UGT1A1 the variation in UGT1A1 mRNA level in the liver sam- mRNA expression (Fig. 1). It has been reported that ex- ples studied can be partly explained by genetic polymor- pression of the UGT1A1 gene can be modulated by hor- phisms. mones, drugs and other xenobiotics through interaction Finally, it should be noted that liver tissues used in the with the aryl hydrocarbon receptor and members of the present study were macroscopically normal part of tis- nuclear receptor superfamily, including constitutive an- sues surrounding tumor areas, which were removed dur- drostane receptor, pregnane X receptor and peroxisome ing the surgery of metastatic tumor in the liver. Thus, proliferator-activated receptor alpha.10,27,28) Therefore, the results of the present study should be interpreted in addition to the expression levels of transcription fac- with the limitation that they were derived from the liver tors, the variability of UGT1A1 mRNA level might be tissue samples obtained from the normal part of the explained by drugs, including phenytoin, phenobarbital liver bearing tumor(s) but not from the normal liver. and dexamethasone, and/or tobacco consumption, In conclusion, our results suggest that both HNF1a which can activate these receptors to bind to the and HNF4a are the factors involved in the interindivid- promoter region of the UGT1A1 gene.8) Although data ual variability of UGT1A6 and UGT1A9 mRNA expres- on environmental factors are not available for all the sion levels at least in human livers used in this study. liver samples, we could not ˆnd any signiˆcant relation Although our results did not rule out the possibility of of drugs used to the variability of UGT1A1 mRNA level involvement of HNF1a and HNF4a in the regulation of in the samples of which the environmental data are hepatic UGT1A1, UGT1A3 and UGT1A4 expression, available. 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