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Regular Article Broad but Distinct Role of Pregnane X Receptor on the Expression of Individual Cytochrome P450s in Human Hepatocytes

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Regular Article Broad but Distinct Role of Pregnane X Receptor on the Expression of Individual Cytochrome P450s in Human Hepatocytes Drug Metab. Pharmacokinet. 22 (4): 276–286 (2007). Regular Article Broad but Distinct Role of Pregnane X Receptor on the Expression of Individual Cytochrome P450s in Human Hepatocytes Koki KOJIMA1,2, Kiyoshi NAGATA1,3,TsutomuMATSUBARA1 and Yasushi YAMAZOE1,* 1Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: In the present study, we have utilized a target selective human pregnane X receptor-siRNA (hPXR-siRNA)-adenovirus expression system to examine the contribution of hPXR on the gene regula- tion of drug-metabolizing P450s in human hepatocytes. Introduction of the hPXR-siRNA adenoviral vec- tor reduced the level of PXR mRNA. After infection with Ad hPXR-siRNA, the basal and ligand-activat- ed CYP2A6, CYP2C8, CYP3A4 and CYP3A5 mRNA levels were decreased signiˆcantly in dose-depen- dent manners, whereas CYP2B6, CYP2C9 and CYP2C19 mRNA levels were moderately in‰uenced af- ter infection with Ad hPXR-siRNA. These data suggest the distinct PXR in‰uences on the regulation of these genes. The expression of CYP1A2 and CYP2D6 mRNA were not aŠected by the introduction of hPXR-siRNA, suggesting that PXR plays no functional role in the expression of either of these genes. This is the ˆrst report to compare simultaneously the relative contribution of hPXR on the expression of nine forms of P450 in primary cultured human hepatocytes. Mutual sharing among nuclear receptors of their binding cis-elements becomes clear now. Thus, the present method using the combination of adenovirus-mediated hPXR-siRNA expression and human hepatocytes may oŠer clear information on the relative role of nuclear receptors such as hPXR on the expression of drug metabolizing genes. Key words: PXR; CYP3A4; P450; siRNA; human hepatocytes including drugs (e.g., macrolide antibiotics, antimycot- Introduction ics, glucocorticoids), herbs (e.g., St. John's wort) and It is now widely recognized that a number of nuclear pesticides (e.g., DDT, pyributicarb).1,3–5) Ligand-activa- receptors are involved in P450 gene regulation. Among tion of PXR andWor CAR stimulates expression of the these nuclear receptors pregnane X receptor (PXR; target gene through binding to elements located in the NR1I2) and constitutive androstane receptor (CAR; regulatory regions following dimerization with retinoid NR1I3) are major determinants in the expression of X receptor (RXRa;NR2B1).6–8) genes associated with various aspects of drug meta- The induction of CYP3A4 and CYP2B6 is mainly bolism, including oxidative metabolism, conjugation, mediated through activation of PXR and CAR, respec- and transport.1) PXR is expressed mainly in the liver and tively.9–14) However,ithasbeenreportedthatPXRand intestine, where it plays important roles in detoxiˆcation CAR are also involved in basic transactivation of both and elimination of xenobiotics from the body.2) PXR is CYP2B6 and CYP3A4 genes.15–17) Hepatic levels of activated by a structurally diverse array of xenobiotics, other P450, such as CYP2A6, CYP2C8, CYP2C9 and Received; March 15, 2007, Accepted; May 22, 2007 *To whom correspondence should be addressed: Yasushi YAMAZOE, Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki-aoba 6-3, Aoba-ku, Sendai, Miyagi 980-8578, Japan. Tel. ＋81-22-795-6827, Fax. ＋81-22-795-6826, E-mail: yamazoe＠mail.tains.tohoku.ac.jp 2Current address: Exploratory Toxicology & DMPK Research Laboratories, Tanabe Seiyaku Co., Ltd., 2-50 Kawagishi 2-chome, Toda, Saitama 335-8505, Japan. 3Current address: Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aobaku, Sendai, Miyagi 981-8558, Japan. Abbreviations used are: PXR, pregnane X receptor; CAR, constitutive androstane receptor; HNF4a, hepatocyte nuclear factor-4a;RXRa,reti- noid X receptor-a; FXR, farnesoid X receptor; GRa, glucocorticoid receptor-a; SHP, small heterodimer partner; P450, cytochrome P450; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; Ad hPXR-siRNA, hPXR- siRNA-expression adenovirus; PB, phenobarbital; CLO, clotrimazole; PCR, polymerase chain reaction; RT-PCR, reverse transcription- polymerase chain reaction; TCID50,50z titer culture infectious dose; MOI, multiplicity of infection. 276 Contribution of PXR to P450 Gene Expression 277 CYP2C19 are also increased after treatment with rifam- sion andWor induction of nine drug-metabolizing P450s picin, a typical PXR activator.11,13,14) It is also reported to examine diŠerences in the contribution of PXR in the that CYP2C8, CYP2C9 and CYP2C19 are transactivat- expression of these genes. ed through CAR.18,19) At present, the exact contribution Materials and Methods of individual nuclear receptors on individual P450 gene expression in human liver remain unclear. Materials: William's medium E, insulin, transfer- For studies of gene transcriptional activation, report- rin, selenium, dexamethasone, phenobarbital and er gene assay systems are mostly used to investigate clotrimazole were obtained from Sigma Chemical (St. promoter and enhancer elements to provide information Louis, MO). Matrigel was purchased from BD Bioscien- on the gene regulation. The results, however, do not al- ce-Discovery Labware (Bedford, MA). Penicillin-Strep- ways re‰ect the induction proˆle that was observed in tomycin was purchased from Gibco BRL (Gaithersburg, vivo, possibly because of established cell lines, arti- MD). KHEM5310 was purchased from KAC (Shiga, ˆcially fused genes andWor over-expressed transfac- Japan). tors.20,21) In most studies of hepatic P450 gene regula- Cell cultures: Cryopreserved human hepatocytes tion, the HepG2 cell line, which is derived from human (lot. 582; Caucasian, female, aged 60 years) were pur- hepatocellular carcinoma, has been used.17–19,22–25) Ex- chased from XenoTech LLC (Lenexa, KS), and thawed pression of PXR is detectable but CAR is below the as recommended by the vendor prior to use for incuba- limit of detection in HepG2 cells.5) In addition, CAR tions. Viability of the cryopreserved hepatocytes was at translocates spontaneously to the nucleus in many cell least 85z at the start of the incubation. The hepatocytes lines, even after the over-expression. The nuclear trans- pellets were resuspended in KHEM5310 medium location of CAR is tightly regulated in the liver and pri- (KAC). Hepatocytes were plated at 2.5×105 cellsWwell mary hepatocytes.26) Recently, we have reported that the on 24-well plates, coated with collagen, type I (Iwaki, induction of both human CYP1A1 and CYP1A2 are Tokyo, Japan). Cells were allowed to attach for 4 h in a controlled simultaneously through common regulatory humidiˆed atmosphere of 5z CO2 at 379C. Culture dis- elements found inside the CYP1A1 gene.25) This ˆnding hes were gently mixed by swirling, and medium contain- suggests a possibility that gene expression might be ing unattached cells was then aspirated. Fresh ice-cold aŠected by neighboring genes. In addition, inconsistent serum-free William's medium E containing 0.1 mMdex- results are obtained from reporter gene assays using es- amethasone, 6.25 mgWmL insulin, 6.25 mgWmL transfer- tablished cell lines and from those in the liver. rin, 6.25 ngWmL selenium and 50 mgWmL Matrigel was To overcome these di‹culties, human primary added to each dish. hepatocytes are preferred for the analysis of the func- For virus experiments, the day after seeding, cells tion of PXR in the gene regulation of P450s. When were infected with 0.05 mL of the hPXR-siRNA hepatocytes are cultured under conditions that restore adenovirus expression vector (Ad hPXR-siRNA) stock 6 near-normal hepatocellular morphology and the expres- solution (1×10 TCID50 Wwell) for 1 h, and then medium sion of liver-speciˆc genes, P450 enzymes can be detect- was added to each well and the cells were cultured for an ed in hepatocytes in a manner re‰ecting that in vivo in additional 72 hrs. Multiplicity of infection (MOI) was terms of the magnitude and speciˆcity of P450 induc- calculated by dividing the TCID50 by the number of tion.27,28) cells. For the induction study including virus, medium Recently, target selective knock-down technology, us- containing 1 mM phenobarbital, 10 mM clotrimazole, or ing small interfering RNA (siRNA), has been devel- 0.1z (vWv) DMSO (vehicle alone) was added to the oped.29) This siRNA technology is applicable to speciˆ- wells 48 hrs after virus infection and cultured for an ad- cally suppress the expression of PXR and may contrib- ditional 24 hrs. 0.1z DMSO had little eŠect on expres- ute to the elucidation of the involvement of PXR in sion of target mRNAs. transcriptional activation of P450 genes involved in Construction of the Ad hPXR-siRNA and the infec- drug metabolism and disposition. However, the e‹cien- tion conditions have been reported previously.5) Ad cy of siRNA is largely dependent on the delivery system hPXR-siRNA strongly suppressed the expression of transports the siRNA into target cells. Adenoviral vec- hPXR protein to a similar degree as that of the mRNA. tors represent one of the most sophisticated expression Ad LacZ, which expresses b-galactosidase, was pro- systems available today.30) vided by Dr. Izumi Saito (Tokyo University). We The present study aims to investigate and understand demonstrated that this adenovirus and an adenovirus how PXR contributes to the regulation of endogenous expressing a non-targeting siRNA,
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