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Peroxisome Proliferator-activated Receptor Alpha (PPARα) Agonists Induce Constitutive Androstane Receptor (CAR) and Cytochrome P450 2B in Rat Primary Hepatocytes
Drug Metab. Pharmacokinet. 25 (1): 108–111 (2010).
Note Peroxisome Proliferator-activated Receptor Alpha (PPARa) Agonists Induce Constitutive Androstane Receptor (CAR) and Cytochrome P450 2B in Rat Primary Hepatocytes Kosuke SAITO, Kaoru KOBAYASHI*, Yuki MIZUNO, Yukina FUKUCHI, Tomomi FURIHATA 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: The constitutive androstane receptor (CAR; NR1I3) is a key transcriptional factor that regulates genes encoding drug-metabolizing enzymes and drug transporters. However, studies on regulation of CAR target genes via up- or down-regulation of CAR are limited. In this study, we examined the effects of PPARa agonists (ciprofibrate, bezafibrate, fenofibrate and WY14643) on the expression of CAR and its target gene CYP2B1/2 in rat primary hepatocytes. Results from real-time PCR analysis showed that CAR and CYP2B1/2 mRNAs exhibit increases in response to all PPARa agonists studied (5 to 10-folds of control). Pretreatment of cells with cycloheximide, an inhibitor of protein synthesis, completely suppressed increase in CYP2B1/2 mRNA in response to ciprofibrate, suggesting that protein synthesis is required in this process. In addition, the induction of CAR by ciprofibrate on the protein level was observed with nuclear extracts as well as total cell lysates. These results indicate that CYP2B1/2 mRNAs are induced by PPARa agonists and that this effect is accompanied by increase in the expression of CAR gene at both mRNA and nuclear protein levels. Activated PPARa may increase functional CAR protein, which can induce the expression of CAR target genes such as CYP2B. Keywords: constitutive androstane receptor (CAR); cytochrome P450 2B (CYP2B): peroxisome proliferator-activated receptor a (PPARa); nuclear receptor; rat primary hepatocytes
receptor a (RXRa) and binds to response elements in target gene promoters resulting in transcriptional activation of genes such as mouse, rat and human CYP2B.5) While CAR is known to activate many genes at the transcriptional level, studies on regulation of CAR target genes via up- or down-regulation of CAR are limited. Pascussi et al.6,7) reported that dexamethasone increases CAR mRNA at the transcriptional level in human primary hepatocytes and identified a glucocorticoid response element in the far upstream region of human CAR gene promoter. Recently, Patel et al.8) demonstrated that CAR is a gene regulated by the arylhydrocarbone receptor (AhR) in mice. Treatment with b-naphthoflavone, an AhR agonist, increased CAR mRNA in wild-type mice but not in AhR-null mice. Wieneke et al.9) reported that CAR is
Introduction Constitutive androstane receptor (CAR, NR1I3), a member of nuclear receptor superfamily, regulates transcription of many genes encoding cytochrome P450s (CYPs), UDP-glucronosyltransferases, sulfotransferases and drug transporters.1) CAR activity is modulated by many synthetic drugs and natural products, which can be classified as an agonist, an indirect activator or an inverse agonist.2) Most of CAR is normally retained in the cytoplasm by forming a complex with Hsp90 (heat shock protein 90) and the co-chaperone CCRP (cytoplasmic CAR retention protein).3,4) In response to indirect activators such as phenobarbital, CAR translocates into the nucleus where it forms a heterodimer with retinoid X
Received; September 11, 2009, Accepted; November 15, 2009 *To whom correspondence should be addressed: Kaoru KOBAYASHI, PhD, Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan. Tel/fax. ＋81-43-226-2895, E-mail: kaoruk＠ p.chiba-u.ac.jp This work supported in part by the Research Foundation for Pharmaceutical Sciences, a Global COE Program (Global Center for Education and Research in Immune System Regulation and Treatment) and Special Funds for Education and Research (Development of SPECT Probes for Pharmaceutical Innovation) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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induced by WY14643, a peroxisome proliferator-activated receptor a (PPARa) agonist, in rat hepatocytes, and identified a PPARa-binding motif in the rat CAR gene promoter. These studies show that the up-regulation of CAR in response to agonists of glucocorticoid receptor, AhR or PPARa enhances the induction of CYP2B genes by CAR activators. However, it is unclear whether the up-regulation of CAR affects the expression levels of CYP2B genes in the absence of CAR activators. This study, we examines whether PPARa agonists (ciprofibrate, bezafibrate, fenofibrate and WY14643) could increase the expression levels of CAR and CYP2B1/2 mRNAs in rat primary hepatocytes. CYP2B1/2 mRNAs were found to be induced by PPARa agonists and that this effect is accompanied by an increased expression of CAR gene at mRNA and nuclear protein levels.
Materials and Methods Reagents: Bezafibrate, ciprofibrate, cycloheximide, fenofibrate and WY14643 were purchased from Sigma (St. Louis, MO). Anti-CAR IgG was purchased from Perseus Proteomics (PP-N4111-00, Tokyo, Japan), anti-bactin IgG from Sigma and anti-TFIIB IgG from Active Motif (Carlsbad, CA). All other reagents were of the highest grade commercially available. Primary hepatocytes: Hepatocytes were isolated from collagenase-perfused livers of male Sprague-Dawley rats (6-weeks old, Japan SLC, Inc., Shizuoka, Japan) as described previously.10) Hepatocytes (4×105 cells/ml) were plated on Collagen Type-1 coated wells in 6-well plates and maintained in William's E media supplemented with 10% fetal bovine serum, 10 nM dexamethasone, 100×ITS-X (Invitrogen), 4 mM glutamine and 50 mg/ml gentamycin for 4 h. After cell attachment to the culture plate, medium was replaced with serum free medium and maintained for 16 h. Cells were then cultured in the presence of the specified chemicals for 48 to 72 h. The present study was conducted in accordance with Guiding Principles for the Care and Use of Laboratory Animals, as adopted by the Committee on Animal Research of Chiba University. Analysis of mRNA expression: Hepatocytes were treated with ciprofibrate (30 mM), WY14643 (10 mM), bezafibrate (30 mM), fenofibrate (30 mM) or vehicle (0.1% DMSO) alone for 48 h. To measure mRNA levels of CAR and CYP2B1/2, cDNAs prepared from total RNAs of hepatocytes were subjected to quantitative realtime PCR with be ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). mRNA levels of CAR were determined using Gene Expression Assay (Rn00576085_m1) and normalized against those of 18S rRNA determined by Eukaryotic 18S rRNA endogenous control (Applied Biosystems). mRNA levels of CYP2B1/2 were determined using Platinum SYBR Green
qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA) and specific primers (5?-TGG TGG AGG AAC TGC GGA AAT C-3? and 5?-TGA TGC ACT GGA AGA GGA AGG T-3?), and normalized against those of GAPDH determined by using specific primers (5?-TGC ACC ACC AAC TGC TTA-3? and 5?-GGA TGC AGG GAT GAT GTT C-3?). Western blotting analysis: Total cell lysates and nuclear extracts were prepared from hepatocytes treated with ciprofibrate (30 mM) or vehicle (0.1% DMSO) alone for 72 h. Total cell lysates were prepared by lysis of hepatocytes with RIPA buffer (0.01 M phosphate buffer, pH 7.4, 138 mM NaCl, 2.7 mM KCl, 2% Nonident P-40, 2% deoxycholic acid, 0.4% SDS and 0.02% sodium azide) containing 1% protease inhibitor cocktail (Calbiochem, Darnstadt, Germany). Nuclear extracts were prepared by using CellLytic Nuclear Extraction Kit (Sigma). Protein quantity was estimated by using the Bio-Rad protein assay with bovine serum albumin as standard. Total cell lysates (40 mg protein) or nuclear extracts (30 mg protein) were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated for 1 h at room temperature with antiCAR IgG (1:1000 dilution for total cell extracts and 1:500 dilution for nuclear extracts), anti-b-actin IgG (1:1000 dilution) or anti-TFIIB IgG (1:500 dilution) in 3% bovine serum albumin in phosphate buffered saline containing 0.05% Tween 20 and were incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:5000 dilution, Sigma). Protein bands were visualized by the ECL Western Blotting Analysis System (GE healthcare, Milwaukee, WI). Statistical analysis: Data are presented as means± S.D. of results of at least three independent experiments. Comparison of multiple groups was made with KruskalWallis test followed by Post-hoc test of Fisher's PLSD. A p value of less than 0.05 was considered significant.
Results Effects of PPARa agonists on CAR and CYP2B1/2 mRNA expression: As shown in Figure 1, CAR and CYP2B1/2 mRNAs exhibited increase in response to all PPARa agonists studied (5 to 10-folds of control), although increase in CAR by fenofibrate and that of CYP2B1/2 by bezafibrate were not statistically significant. Increase in CYP2B1/2 mRNA by ciprofibrate was slightly lower than that by phenobarbital (1 mM), a typical CAR activator. Effects of cycloheximide on ciprofibrate-mediated CYP2B1/2 mRNA induction: To determine the mechanism for CYP2B1/2 mRNA increase by PPARa agonists, we evaluated the effects of cycloheximide, an inhibitor of protein synthesis, on this process. As shown in Figure 2, pretreatment of cells with cycloheximide completely suppressed increase in CYP2B1/2 mRNA in
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Fig. 3. Induction of nuclear CAR protein by ciprofibrate Rat primary hepatocytes were treated with ciprofibrate (Cipro; 30 mM) or vehicle (DMSO) for 72 h. Total cell lysates (A) and nuclear extracts (B) were prepared as described in Materials and Methods and subjected to Western blotting analysis. b-Actin and TFIIB were used as internal controls of total cell lysates and nuclear extracts, respectively.
Fig. 1. Induction of CAR (A) and CYP2B (B) by PPARa agonists Rat primary hepatocytes were treated with ciprofibrate (Cipro; 30 mM), bezafibrate (Beza; 30 mM), fenofibrate (Feno; 30 mM), WY14643 (WY; 10 mM) or vehicle (DMSO) for 48 h. The expression levels of CAR mRNA and CYP2B1/2 mRNA were determined as described in Materials and Methods. Data are expressed as fold induction over the vehicle control. Each value is the mean±S.D. of three independent experiments. *, pº0.05 and ***pº0.001 compared with vehicle controls.
Fig. 2. Effects of cycloheximide on induction of CYP2B by ciprofibrate Rat primary hepatocytes were treated with ciprofibrate (Cipro; 30 mM) or vehicle (DMSO) for 48 h in the presence or absence of cycloheximide (20 mM). The expression levels of CYP2B1/2 mRNA were determined as described in Materials and Methods. Relative mRNA expression levels in the absence of ciprofibrate were designated as 1. Each value is the mean±S.D. of four independent experiments ***pº0.001 compared with vehicle controls.
response to ciprofibrate, suggesting that protein synthesis is required in this process. Induction of nuclear CAR protein by ciprofibrate: Since PPARa is a transcription factor that regulates CAR gene expression in rats,9) we thought that increase in CAR by PPARa agonists might consequently increase CYP2B1/2 mRNA in rat primary hepatocytes. CAR is constitutively active, whereas most of CAR is normally retained in the cytoplasm.11) Thus, we analyzed the expression of CAR protein in total cell lysates and nuclear extracts prepared from cells treated with or without ciprofibrate. As shown in Figure 3, the induction of CAR by ciprofibrate on the protein level was observed with nuclear extracts as well as total cell lysates.
Discussion The results of the present study show that PPARa agonists induced CAR and its target gene CYP2B1/2 in rat primary hepatocytes (Fig. 1). Experiment with cycloheximide suggested that protein synthesis is required in the induction of CYP2B1/2 by PPARa agonists (Fig. 2). In addition, PPARa agonist induced CAR protein in nucleus (Fig. 3). Therefore, it was thought that the induction of CYP2B1/2 by PPARa agonists was mediated by increase in functional CAR protein but not a transcriptional activation of CYP2B1/2 genes by activated PPARa. In this study, PPARa agonists induced CYP2B1/2 mRNA in rat primary hepatocytes (Fig. 1). This is inconsistent with a previous report that PPARa agonists do not induce hepatic Cyp2b10 in mice.12) However, it has been reported that ligand recognition of CAR shows speciesspecific differences and a typical CAR ligand in mice, 1,4bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), is not the same as in human and rats.13) In mice, PPARa agonists have been shown to induce the nuclear translocation of CAR in liver cell in vivo, but possibly act as mouse CAR antagonist by interfering with coactivator recruitment to the CAR ligand binding domain. Since
PPARa Agonists Induce CAR and CYP2B
CYP2B1/2 mRNAs were increased by PPARa agonists in rat primary hepatocytes (Fig. 1), it is not considered that PPARa agonists act as rat CAR antagonists. Although further study is needed to clarify why the effects of PPARa agonists on the expression of rat CYP2B1/2 genes differ from that of mouse Cyp2b10 gene, it seems that PPARa agonists induce functional CAR protein and CYP2B1/2 mRNA at least in rat primary hepatocytes. This study is the first to demonstrate that up-regulation of CAR affects the expression levels of CYP2B genes in the absence of CAR activators, although it has been reported that the up-regulation of CAR enhances the induction of CYP2B genes by CAR activators such as phenobarbital.6,8,9) This finding suggests that not only activation but also up-regulation of CAR possibly alter drug metabolism and disposition because CAR has been implicated in regulating the expression of numerous drugmetabolizing enzymes and drug transporters. In an earlier study,9) treatment with PPARa agonist revealed no or slight induction of CYP2B1 mRNA, which is inconsistent with our results. Although the reason for this discrepancy is unclear, it is possible that induction of CYP2B genes is dependent on the period of treatment with PPARa agonist. This is because protein synthesis appears needed for the induction of CYP2B1/2 mRNA by PPARa agonists. Otherwise, strain-differences may be involved in the difference of induction. CAR is a key transcription factor that regulates CYP2B genes, whereas it has been suggested that several potential regulatory factors (e.g., CAAT/enhancer binding protein and Sp1) contribute to basal transcription of the CYP2B1/2 genes.14–16) There is thus the possibility that other proteins were increased by PPARa agonists and consequently up-regulate the expression of CYP2B1/2 genes. In conclusion, our results show that activated PPARa may increase functional CAR protein, which alters drug metabolism and disposition. Although further studies are needed to determine the exact molecular mechanism, the present information is helpful for understanding the regulation of CAR and its targeted genes encoding various drug-metabolizing enzymes and drug transporters.
References 1)
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Maglich, J. M., Stoltz, C. M., Goodwin, B., Hawkins-Brown, D., Moore, J. T. and Kliewer, S. A.: Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. Mol. Pharmacol., 62: 638–646 (2002). Chang, T. K. and Waxman, D. J.: Synthetic drugs and natural products as modulators of constitutive androstane receptor (CAR) and pregnane X receptor (PXR). Drug Metab. Rev., 38: 51–73 (2006). Yoshinari, K., Kobayashi, K., Moore, R., Kawamoto, T. and Negishi, M.: Identification of the nuclear receptor CAR:HSP90
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complex in mouse liver and recruitment of protein phosphatase 2A in response to phenobarbital. FEBS Lett., 548: 17–20 (2003). Kobayashi, K., Sueyoshi, T., Inoue, K., Moore, R. and Negishi, M.: Cytoplasmic accumulation of the nuclear receptor CAR by a tetratricopeptide repeat protein in HepG2 cells. Mol. Pharmacol., 64: 1069–1075 (2003). Sueyoshi, T. and Negishi, M.: Phenobarbital response elements of cytochrome P450 genes and nuclear receptors. Annu. Rev. Pharmacol. Toxicol., 41:123–143 (2001). Pascussi, J. M., Gerbal-Chaloin, S., Fabre, J. M., Maurel, P. and Vilarem, M. J.: Dexamethasone enhances constitutive androstane receptor expression in human hepatocytes: consequences on cytochrome P450 gene regulation. Mol. Pharmacol., 58: 1441–1450 (2000). Pascussi, J. M., Busson-Le Coniat, M., Maurel, P. and Vilarem, M. J.: Transcriptional analysis of the orphan nuclear receptor constitutive androstane receptor (NR1I3) gene promoter: identification of a distal glucocorticoid response element. Mol. Endocrinol., 17: 42–55 (2003). Patel, R. D., Hollingshead, B. D., Omiecinski, C. J. and Perdew, G. H.: Aryl-hydrocarbon receptor activation regulates constitutive androstane receptor levels in murine and human liver. Hepatology, 46: 209–218 (2007). Wieneke, N., Hirsch-Ernst, K. I., Kuna, M., Kersten, S. and Puschel, G. P.: PPARalpha-dependent induction of the energy homeostasis-regulating nuclear receptor NR1i3 (CAR) in rat hepatocytes: potential role in starvation adaptation. FEBS Lett., 581: 5617–5626 (2007). Seglen, P. O.: Preparation of isolated rat liver cells. Methods Cell Biol., 13: 29–83 (1976). Kawamoto, T., Sueyoshi, T., Zelko, I., Moore, R., Washburn, K. and Negishi, M.: Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol. Cell Biol., 19: 6318–6322 (1999). Guo, D., Sarkar, J., Suino-Powell, K., Xu, Y., Matsumoto, K., Jia, Y., Yu, S., Khare, S., Haldar, K., Rao, M. S., Foreman, J. E., Monga, S. P., Peters, J. M., Xu, H. E. and Reddy, J. K.: Induction of nuclear translocation of constitutive androstane receptor by peroxisome proliferator-activated receptor alpha synthetic ligands in mouse liver. J. Biol. Chem., 282: 36766–36776 (2007). Kobayashi, K., Yamanaka, Y., Iwazaki, N., Nakajo, I., Hosokawa, M., Negishi, M. and Chiba, K.: Identification of HMG-CoA reductase inhibitors as activators for human, mouse and rat constitutive androstane receptor. Drug Metab. Dispos., 33: 924–929 (2005). Park, Y. and Kemper, B.: The CYP2B1 proximal promoter contains a functional C/EBP regulatory element. DNA Cell Biol., 15: 693–701 (1996). Luc, P. V., Adesnik, M., Ganguly, S. and Shaw, P. M.: Transcriptional regulation of the CYP2B1 and CYP2B2 genes by C/EBPrelated proteins. Biochem. Pharmacol., 51: 345–356 (1996). Muangmoonchai, R., Smirlis, D., Wong, S. C., Edwards, M., Phillips, I. R. and Shephard, E. A.: Xenobiotic induction of cytochrome P450 2B1 (CYP2B1) is mediated by the orphan nuclear receptor constitutive androstane receptor (CAR) and requires steroid co-activator 1 (SRC-1) and the transcription factor Sp1. Biochem. J., 355: 71–78 (2001).
Note Peroxisome Proliferator-activated Receptor Alpha (PPARa) Agonists Induce Constitutive Androstane Receptor (CAR) and Cytochrome P450 2B in Rat Primary Hepatocytes Kosuke SAITO, Kaoru KOBAYASHI*, Yuki MIZUNO, Yukina FUKUCHI, Tomomi FURIHATA 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: The constitutive androstane receptor (CAR; NR1I3) is a key transcriptional factor that regulates genes encoding drug-metabolizing enzymes and drug transporters. However, studies on regulation of CAR target genes via up- or down-regulation of CAR are limited. In this study, we examined the effects of PPARa agonists (ciprofibrate, bezafibrate, fenofibrate and WY14643) on the expression of CAR and its target gene CYP2B1/2 in rat primary hepatocytes. Results from real-time PCR analysis showed that CAR and CYP2B1/2 mRNAs exhibit increases in response to all PPARa agonists studied (5 to 10-folds of control). Pretreatment of cells with cycloheximide, an inhibitor of protein synthesis, completely suppressed increase in CYP2B1/2 mRNA in response to ciprofibrate, suggesting that protein synthesis is required in this process. In addition, the induction of CAR by ciprofibrate on the protein level was observed with nuclear extracts as well as total cell lysates. These results indicate that CYP2B1/2 mRNAs are induced by PPARa agonists and that this effect is accompanied by increase in the expression of CAR gene at both mRNA and nuclear protein levels. Activated PPARa may increase functional CAR protein, which can induce the expression of CAR target genes such as CYP2B. Keywords: constitutive androstane receptor (CAR); cytochrome P450 2B (CYP2B): peroxisome proliferator-activated receptor a (PPARa); nuclear receptor; rat primary hepatocytes
receptor a (RXRa) and binds to response elements in target gene promoters resulting in transcriptional activation of genes such as mouse, rat and human CYP2B.5) While CAR is known to activate many genes at the transcriptional level, studies on regulation of CAR target genes via up- or down-regulation of CAR are limited. Pascussi et al.6,7) reported that dexamethasone increases CAR mRNA at the transcriptional level in human primary hepatocytes and identified a glucocorticoid response element in the far upstream region of human CAR gene promoter. Recently, Patel et al.8) demonstrated that CAR is a gene regulated by the arylhydrocarbone receptor (AhR) in mice. Treatment with b-naphthoflavone, an AhR agonist, increased CAR mRNA in wild-type mice but not in AhR-null mice. Wieneke et al.9) reported that CAR is
Introduction Constitutive androstane receptor (CAR, NR1I3), a member of nuclear receptor superfamily, regulates transcription of many genes encoding cytochrome P450s (CYPs), UDP-glucronosyltransferases, sulfotransferases and drug transporters.1) CAR activity is modulated by many synthetic drugs and natural products, which can be classified as an agonist, an indirect activator or an inverse agonist.2) Most of CAR is normally retained in the cytoplasm by forming a complex with Hsp90 (heat shock protein 90) and the co-chaperone CCRP (cytoplasmic CAR retention protein).3,4) In response to indirect activators such as phenobarbital, CAR translocates into the nucleus where it forms a heterodimer with retinoid X
Received; September 11, 2009, Accepted; November 15, 2009 *To whom correspondence should be addressed: Kaoru KOBAYASHI, PhD, Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan. Tel/fax. ＋81-43-226-2895, E-mail: kaoruk＠ p.chiba-u.ac.jp This work supported in part by the Research Foundation for Pharmaceutical Sciences, a Global COE Program (Global Center for Education and Research in Immune System Regulation and Treatment) and Special Funds for Education and Research (Development of SPECT Probes for Pharmaceutical Innovation) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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induced by WY14643, a peroxisome proliferator-activated receptor a (PPARa) agonist, in rat hepatocytes, and identified a PPARa-binding motif in the rat CAR gene promoter. These studies show that the up-regulation of CAR in response to agonists of glucocorticoid receptor, AhR or PPARa enhances the induction of CYP2B genes by CAR activators. However, it is unclear whether the up-regulation of CAR affects the expression levels of CYP2B genes in the absence of CAR activators. This study, we examines whether PPARa agonists (ciprofibrate, bezafibrate, fenofibrate and WY14643) could increase the expression levels of CAR and CYP2B1/2 mRNAs in rat primary hepatocytes. CYP2B1/2 mRNAs were found to be induced by PPARa agonists and that this effect is accompanied by an increased expression of CAR gene at mRNA and nuclear protein levels.
Materials and Methods Reagents: Bezafibrate, ciprofibrate, cycloheximide, fenofibrate and WY14643 were purchased from Sigma (St. Louis, MO). Anti-CAR IgG was purchased from Perseus Proteomics (PP-N4111-00, Tokyo, Japan), anti-bactin IgG from Sigma and anti-TFIIB IgG from Active Motif (Carlsbad, CA). All other reagents were of the highest grade commercially available. Primary hepatocytes: Hepatocytes were isolated from collagenase-perfused livers of male Sprague-Dawley rats (6-weeks old, Japan SLC, Inc., Shizuoka, Japan) as described previously.10) Hepatocytes (4×105 cells/ml) were plated on Collagen Type-1 coated wells in 6-well plates and maintained in William's E media supplemented with 10% fetal bovine serum, 10 nM dexamethasone, 100×ITS-X (Invitrogen), 4 mM glutamine and 50 mg/ml gentamycin for 4 h. After cell attachment to the culture plate, medium was replaced with serum free medium and maintained for 16 h. Cells were then cultured in the presence of the specified chemicals for 48 to 72 h. The present study was conducted in accordance with Guiding Principles for the Care and Use of Laboratory Animals, as adopted by the Committee on Animal Research of Chiba University. Analysis of mRNA expression: Hepatocytes were treated with ciprofibrate (30 mM), WY14643 (10 mM), bezafibrate (30 mM), fenofibrate (30 mM) or vehicle (0.1% DMSO) alone for 48 h. To measure mRNA levels of CAR and CYP2B1/2, cDNAs prepared from total RNAs of hepatocytes were subjected to quantitative realtime PCR with be ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). mRNA levels of CAR were determined using Gene Expression Assay (Rn00576085_m1) and normalized against those of 18S rRNA determined by Eukaryotic 18S rRNA endogenous control (Applied Biosystems). mRNA levels of CYP2B1/2 were determined using Platinum SYBR Green
qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA) and specific primers (5?-TGG TGG AGG AAC TGC GGA AAT C-3? and 5?-TGA TGC ACT GGA AGA GGA AGG T-3?), and normalized against those of GAPDH determined by using specific primers (5?-TGC ACC ACC AAC TGC TTA-3? and 5?-GGA TGC AGG GAT GAT GTT C-3?). Western blotting analysis: Total cell lysates and nuclear extracts were prepared from hepatocytes treated with ciprofibrate (30 mM) or vehicle (0.1% DMSO) alone for 72 h. Total cell lysates were prepared by lysis of hepatocytes with RIPA buffer (0.01 M phosphate buffer, pH 7.4, 138 mM NaCl, 2.7 mM KCl, 2% Nonident P-40, 2% deoxycholic acid, 0.4% SDS and 0.02% sodium azide) containing 1% protease inhibitor cocktail (Calbiochem, Darnstadt, Germany). Nuclear extracts were prepared by using CellLytic Nuclear Extraction Kit (Sigma). Protein quantity was estimated by using the Bio-Rad protein assay with bovine serum albumin as standard. Total cell lysates (40 mg protein) or nuclear extracts (30 mg protein) were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated for 1 h at room temperature with antiCAR IgG (1:1000 dilution for total cell extracts and 1:500 dilution for nuclear extracts), anti-b-actin IgG (1:1000 dilution) or anti-TFIIB IgG (1:500 dilution) in 3% bovine serum albumin in phosphate buffered saline containing 0.05% Tween 20 and were incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:5000 dilution, Sigma). Protein bands were visualized by the ECL Western Blotting Analysis System (GE healthcare, Milwaukee, WI). Statistical analysis: Data are presented as means± S.D. of results of at least three independent experiments. Comparison of multiple groups was made with KruskalWallis test followed by Post-hoc test of Fisher's PLSD. A p value of less than 0.05 was considered significant.
Results Effects of PPARa agonists on CAR and CYP2B1/2 mRNA expression: As shown in Figure 1, CAR and CYP2B1/2 mRNAs exhibited increase in response to all PPARa agonists studied (5 to 10-folds of control), although increase in CAR by fenofibrate and that of CYP2B1/2 by bezafibrate were not statistically significant. Increase in CYP2B1/2 mRNA by ciprofibrate was slightly lower than that by phenobarbital (1 mM), a typical CAR activator. Effects of cycloheximide on ciprofibrate-mediated CYP2B1/2 mRNA induction: To determine the mechanism for CYP2B1/2 mRNA increase by PPARa agonists, we evaluated the effects of cycloheximide, an inhibitor of protein synthesis, on this process. As shown in Figure 2, pretreatment of cells with cycloheximide completely suppressed increase in CYP2B1/2 mRNA in
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Fig. 3. Induction of nuclear CAR protein by ciprofibrate Rat primary hepatocytes were treated with ciprofibrate (Cipro; 30 mM) or vehicle (DMSO) for 72 h. Total cell lysates (A) and nuclear extracts (B) were prepared as described in Materials and Methods and subjected to Western blotting analysis. b-Actin and TFIIB were used as internal controls of total cell lysates and nuclear extracts, respectively.
Fig. 1. Induction of CAR (A) and CYP2B (B) by PPARa agonists Rat primary hepatocytes were treated with ciprofibrate (Cipro; 30 mM), bezafibrate (Beza; 30 mM), fenofibrate (Feno; 30 mM), WY14643 (WY; 10 mM) or vehicle (DMSO) for 48 h. The expression levels of CAR mRNA and CYP2B1/2 mRNA were determined as described in Materials and Methods. Data are expressed as fold induction over the vehicle control. Each value is the mean±S.D. of three independent experiments. *, pº0.05 and ***pº0.001 compared with vehicle controls.
Fig. 2. Effects of cycloheximide on induction of CYP2B by ciprofibrate Rat primary hepatocytes were treated with ciprofibrate (Cipro; 30 mM) or vehicle (DMSO) for 48 h in the presence or absence of cycloheximide (20 mM). The expression levels of CYP2B1/2 mRNA were determined as described in Materials and Methods. Relative mRNA expression levels in the absence of ciprofibrate were designated as 1. Each value is the mean±S.D. of four independent experiments ***pº0.001 compared with vehicle controls.
response to ciprofibrate, suggesting that protein synthesis is required in this process. Induction of nuclear CAR protein by ciprofibrate: Since PPARa is a transcription factor that regulates CAR gene expression in rats,9) we thought that increase in CAR by PPARa agonists might consequently increase CYP2B1/2 mRNA in rat primary hepatocytes. CAR is constitutively active, whereas most of CAR is normally retained in the cytoplasm.11) Thus, we analyzed the expression of CAR protein in total cell lysates and nuclear extracts prepared from cells treated with or without ciprofibrate. As shown in Figure 3, the induction of CAR by ciprofibrate on the protein level was observed with nuclear extracts as well as total cell lysates.
Discussion The results of the present study show that PPARa agonists induced CAR and its target gene CYP2B1/2 in rat primary hepatocytes (Fig. 1). Experiment with cycloheximide suggested that protein synthesis is required in the induction of CYP2B1/2 by PPARa agonists (Fig. 2). In addition, PPARa agonist induced CAR protein in nucleus (Fig. 3). Therefore, it was thought that the induction of CYP2B1/2 by PPARa agonists was mediated by increase in functional CAR protein but not a transcriptional activation of CYP2B1/2 genes by activated PPARa. In this study, PPARa agonists induced CYP2B1/2 mRNA in rat primary hepatocytes (Fig. 1). This is inconsistent with a previous report that PPARa agonists do not induce hepatic Cyp2b10 in mice.12) However, it has been reported that ligand recognition of CAR shows speciesspecific differences and a typical CAR ligand in mice, 1,4bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), is not the same as in human and rats.13) In mice, PPARa agonists have been shown to induce the nuclear translocation of CAR in liver cell in vivo, but possibly act as mouse CAR antagonist by interfering with coactivator recruitment to the CAR ligand binding domain. Since
PPARa Agonists Induce CAR and CYP2B
CYP2B1/2 mRNAs were increased by PPARa agonists in rat primary hepatocytes (Fig. 1), it is not considered that PPARa agonists act as rat CAR antagonists. Although further study is needed to clarify why the effects of PPARa agonists on the expression of rat CYP2B1/2 genes differ from that of mouse Cyp2b10 gene, it seems that PPARa agonists induce functional CAR protein and CYP2B1/2 mRNA at least in rat primary hepatocytes. This study is the first to demonstrate that up-regulation of CAR affects the expression levels of CYP2B genes in the absence of CAR activators, although it has been reported that the up-regulation of CAR enhances the induction of CYP2B genes by CAR activators such as phenobarbital.6,8,9) This finding suggests that not only activation but also up-regulation of CAR possibly alter drug metabolism and disposition because CAR has been implicated in regulating the expression of numerous drugmetabolizing enzymes and drug transporters. In an earlier study,9) treatment with PPARa agonist revealed no or slight induction of CYP2B1 mRNA, which is inconsistent with our results. Although the reason for this discrepancy is unclear, it is possible that induction of CYP2B genes is dependent on the period of treatment with PPARa agonist. This is because protein synthesis appears needed for the induction of CYP2B1/2 mRNA by PPARa agonists. Otherwise, strain-differences may be involved in the difference of induction. CAR is a key transcription factor that regulates CYP2B genes, whereas it has been suggested that several potential regulatory factors (e.g., CAAT/enhancer binding protein and Sp1) contribute to basal transcription of the CYP2B1/2 genes.14–16) There is thus the possibility that other proteins were increased by PPARa agonists and consequently up-regulate the expression of CYP2B1/2 genes. In conclusion, our results show that activated PPARa may increase functional CAR protein, which alters drug metabolism and disposition. Although further studies are needed to determine the exact molecular mechanism, the present information is helpful for understanding the regulation of CAR and its targeted genes encoding various drug-metabolizing enzymes and drug transporters.
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