Transcriptional Regulation of CYP2C9 Gene
2002; Elsevier BV; Volume: 277; Issue: 1 Linguagem: Inglês
10.1074/jbc.m107228200
ISSN1083-351X
AutoresSabine Gerbal‐Chaloin, Martine Daujat‐Chavanieu, Jean‐Marc Pascussi, Lydiane Pichard‐Garcia, Marie‐José Vilarem, Patrick Maurel,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoAlthough cytochrome P450 2C9 (CYP2C9) is a major CYP expressed in the adult human liver, its mechanism of regulation is poorly known. In previous work, we have shown that CYP2C9is inducible in primary human hepatocytes by xenobiotics including dexamethasone, rifampicin, and phenobarbital. The aim of this work was to investigate the molecular mechanism(s) controlling the inducible expression of CYP2C9. Deletional analysis ofCYP2C9 regulatory region (+21 to −2088) in the presence of various hormone nuclear receptors suggested the presence of two functional response elements, a glucocorticoid receptor-responsive element (−1648/−1684) and a constitutive androstane receptor-responsive element (CAR, −1783/−1856). Each of these were characterized by co-transfection experiments, directed mutagenesis, gel shift assays, and response to specific antagonists RU486 and androstanol. By these experiments we located a glucocorticoid-responsive element imperfect palindrome at −1662/−1676, and a DR4 motif at −1803/−1818 recognized and transactivated by human glucocorticoid receptor and by hCAR and pregnane X receptor, respectively. Identification of these functional elements provides rational mechanistic basis for CYP2C9induction by dexamethasone (submicromolar concentrations), and by phenobarbital and rifampicin, respectively. CYP2C9 appears therefore to be a primary glucocorticoid-responsive gene, which in addition, may be induced by xenobiotics through CAR/pregnane X receptor activation. Although cytochrome P450 2C9 (CYP2C9) is a major CYP expressed in the adult human liver, its mechanism of regulation is poorly known. In previous work, we have shown that CYP2C9is inducible in primary human hepatocytes by xenobiotics including dexamethasone, rifampicin, and phenobarbital. The aim of this work was to investigate the molecular mechanism(s) controlling the inducible expression of CYP2C9. Deletional analysis ofCYP2C9 regulatory region (+21 to −2088) in the presence of various hormone nuclear receptors suggested the presence of two functional response elements, a glucocorticoid receptor-responsive element (−1648/−1684) and a constitutive androstane receptor-responsive element (CAR, −1783/−1856). Each of these were characterized by co-transfection experiments, directed mutagenesis, gel shift assays, and response to specific antagonists RU486 and androstanol. By these experiments we located a glucocorticoid-responsive element imperfect palindrome at −1662/−1676, and a DR4 motif at −1803/−1818 recognized and transactivated by human glucocorticoid receptor and by hCAR and pregnane X receptor, respectively. Identification of these functional elements provides rational mechanistic basis for CYP2C9induction by dexamethasone (submicromolar concentrations), and by phenobarbital and rifampicin, respectively. CYP2C9 appears therefore to be a primary glucocorticoid-responsive gene, which in addition, may be induced by xenobiotics through CAR/pregnane X receptor activation. cytochrome P-450 pregnane X receptor glucocorticoid receptor glucocorticoid-responsive element constitutive androstane receptor retinoid X receptor xenobiotic responsive enhancer module Dulbecco's modified Eagle's medium pregnenolone 16α-carbonitrile 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene constitutive androstane receptor-responsive element dithiothreitol electrophoretic mobility shift assay tyrosine aminotransferase Cytochrome P-450 (CYP)1is the generic name of a superfamily of heme-thiolate proteins that play a critical role in the oxidative metabolism of xenobiotics, including drugs, environmental pollutants and contaminants, and biological signaling molecules such as steroid hormones and biliary salts. CYP2C9 is a member of the CYP2C subfamily, which in man includes at least three other members, i.e. CYP2C8, CYP2C18, and CYP2C19 (1Goldstein J.A. de Morais S. Pharmacogenetics. 1994; 4: 285-299Crossref PubMed Scopus (519) Google Scholar). Accumulating evidence indicates that CYPC9 ranks second, after CYP3A4, among the most expressed drug-metabolizing enzymes in human liver (2Miners J.O. Birkett D.J. Br. J. Clin. Pharmacol. 1998; 45: 525-538Crossref PubMed Scopus (761) Google Scholar). CYP2C9 is involved in the metabolism of numerous substrates including phenytoin, tolbutamide, torsemide, S-warfarin, and numerous nonsteroidal anti-inflammatory drugs (reviewed in Refs. 1Goldstein J.A. de Morais S. Pharmacogenetics. 1994; 4: 285-299Crossref PubMed Scopus (519) Google Scholar and 2Miners J.O. Birkett D.J. Br. J. Clin. Pharmacol. 1998; 45: 525-538Crossref PubMed Scopus (761) Google Scholar). In contrast to the large amount of data on the biochemistry, enzymology, pharmacology, and genetic polymorphism of CYP2C9, little is known on the inducibility of this gene in response to xenobiotics in humans. We recently demonstrated that CYP2C9 is inducible at the mRNA and protein levels in human hepatocytes in primary cultures in response to xenobiotics shown previously to beCYP3A4 and CYP2B6 inducers such as dexamethasone, rifampicin, and phenobarbital (3Gerbal-Chaloin S. Pascussi J.M. Pichard-Garcia L. Daujat M. Waechter F. Fabre J.M. Carrere N. Maurel P. Drug Metab. Dispos. 2001; 29: 242-251PubMed Google Scholar). The concentration and time dependence of CYP2C9 mRNA expression in response to these three inducers, compared with those of CYP3A4 and CYP2B6 mRNAs, were consistent with the possible implication of at least three receptors in the inducible expression of CYP2C9: the glucocorticoid receptor (GR), the pregnane X receptor (PXR, also named steroid and xenobiotic receptor and pregnane-activated receptor), and the constitutive androstane receptor (CAR), respectively (3Gerbal-Chaloin S. Pascussi J.M. Pichard-Garcia L. Daujat M. Waechter F. Fabre J.M. Carrere N. Maurel P. Drug Metab. Dispos. 2001; 29: 242-251PubMed Google Scholar, 4Kliewer S.A. Moore J.T. Wade L. Staudinger J.L. Watson M.A. Jones S.A. McKee D.D. Oliver B.B. Willson T.M. Zetterstrom R.H. Perlmann T. Lehmann J.M. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1344) Google Scholar, 5Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Recent reports on PXR and CAR, two new members of the steroid receptor superfamily, have considerably clarified our understanding of the inducible regulation of CYP genes from families 2 and 3, in response to xenobiotics in rodents and in man (4Kliewer S.A. Moore J.T. Wade L. Staudinger J.L. Watson M.A. Jones S.A. McKee D.D. Oliver B.B. Willson T.M. Zetterstrom R.H. Perlmann T. Lehmann J.M. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1344) Google Scholar, 5Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, 6Bertilsson G. Heidrich J. Svensson K. Asman M. Jendeberg L. Sydow B.M. Ohlsson R. Postlind H. Blomquist P. Berkenstam A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12208-12213Crossref PubMed Scopus (796) Google Scholar, 7Blumberg B. Sabbagh W.J. Juguilon H. Bolado J.J. van, M. C. Ong E.S. Evans R.M. Genes Dev. 1998; 12: 3195-3205Crossref PubMed Scopus (820) Google Scholar, 8Lehmann J.M. McKee D.D. Watson M.A. Willson T.M. Moore J.T. Kliewer S.A. J. Clin. Invest. 1998; 102: 1016-1023Crossref PubMed Scopus (1389) Google Scholar, 9Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 10Kim J. Kemper B. J. Biol. Chem. 1997; 272: 29423-29425Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). PXR forms a heterodimer with RXR, and this complex has been shown to activateCYP3A4 transcription through binding to an ER6 element present at position −160 in the proximal promoter (8Lehmann J.M. McKee D.D. Watson M.A. Willson T.M. Moore J.T. Kliewer S.A. J. Clin. Invest. 1998; 102: 1016-1023Crossref PubMed Scopus (1389) Google Scholar). More recently, Goodwin et al. (11Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google Scholar) reported the presence of a distal element called xenobiotic responsive enhancer module (XREM) harboring both a DR3 and an ER6 motif, located at −7 kb, and they demonstrated that this element cooperates with the proximal ER6 to activateCYP3A4 transcription. PXR is activated by numerous compounds known to induce CYP3A expression, such as rifampicin, phenobarbital, clotrimazole, and dexamethasone (12Moore L.B. Parks D.J. Jones S.A. Bledsoe R.K. Consler T.G. Stimmel J.B. Goodwin B. Liddle C. Blanchard S.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar). The apparentKd values of these compounds for the human PXR are in the micromolar (rifampicin, clotrimazole) or supramicromolar (phenobarbital, dexamethasone) range. In contrast to PXR, CAR is sequestered in the cytosol and translocates into the nucleus upon activation, notably in response to phenobarbital (13Honkakoski P. Negishi M. Biochem. J. 1998; : 889-895Crossref PubMed Scopus (95) Google Scholar), presumably through several steps of phosphorylation (14Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (488) Google Scholar). Like PXR, CAR forms a heterodimer with RXR. Several groups have identified a complex phenobarbital-responsive element module in CYP2B genes (15Park Y. Li H. Kemper B. J. Biol. Chem. 1996; 271: 23725-23728Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar,16Trottier E. Belzil A. Stoltz C. Anderson A. Gene (Amst.). 1995; 158: 263-268Crossref PubMed Scopus (163) Google Scholar), which was further characterized by Honkakoski et al.(9Honkakoski P. Negishi M. J. Biol. Chem. 1997; 272: 14943-14949Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The active element called NR1 is located at position −1663/−1683 in CYP2B6 and exhibits a DR4 motif (5Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Only a few molecules among CYP inducers were shown to bind directly to human CAR. These include androstenol (and related compounds at supramicromolar concentrations), and clotrimazole or 5β-pregnane-3,20-dione (in the micromolar range). However, both androstenol and clotrimazole appear to be deactivators instead of activators of hCAR, whereas 5β-pregnane-3,20-dione appears as a true activator. Phenobarbital, a compound that has been shown to activate CAR through indirect mechanism (13Honkakoski P. Negishi M. Biochem. J. 1998; : 889-895Crossref PubMed Scopus (95) Google Scholar), and other compounds known as CYP inducers (like rifampicin and dexamethasone) are not ligands of CAR (12Moore L.B. Parks D.J. Jones S.A. Bledsoe R.K. Consler T.G. Stimmel J.B. Goodwin B. Liddle C. Blanchard S.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar). The aim of the present work was to investigate the molecular mechanism(s) of induction of CYP2C9 by dexamethasone, rifampicin, and phenobarbital. For this purpose, the 5′-flanking region of this gene was analyzed between +21 and −2088 by several tests, including transcriptional analysis of deletion fragments, co-transfection with nuclear receptor expression plasmids, directed mutagenesis, and gel shift assays. Our results suggest the existence of two functional responsive elements in the regulatory region ofCYP2C9: an imperfect palindromic glucocorticoid-responsive element (GRE) at −1662/−1676 and a CAR-responsive element (DR4) at −1803/−1818. DMEM culture medium was purchased from Invitrogen (Cergy Pontoise, France). Dexamethasone, mifepristone (RU486), androstenol (5α-androst-16-en-3α-ol), rifampicin, pregnenolone 16α-carbonitrile (PCN), cycloheximide, and dimethyl sulfoxide (Me2SO) were purchased from Sigma. 1,4-Bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP) was gift from P. Lesca (INRA, Toulouse, France). [γ-32P]ATP was purchased from Amersham Biosciences, Inc. (Amersham, England). A 2.1-kb XbaI/BglII fragment ofCYP2C9 5′-flanking region was cloned in pBluescript vector (Stratagene, La Jolla, CA) after amplification by PCR using human DNA as a template and oligonucleotides sense (p2C9−2088/XbaI) 5′-ATCTACACATTATCTAGAATTCTTTCT-3′ and reverse (p2C9+21/BglII) 5′-GAGATCTTCTCTTCTTGTTAAGACAACCA-3′.CYP2C9−1545/+21 deletion was then obtained byBspEI digestion, blunt-ended with Klenow enzyme, and cloned into pBlueScript-cut SmaI. CYP2C9−340/+21 deletion was obtained by an EcoRI/EcoRI deletion. These fragments were then cloned into pGL3-basic vector usingKpnI/SacI enzymes. CYP2C9−1856/+21 deletion was obtained by a StuI/SmaI deletion in pGL3-basic vector. CYP2C9−1783, −1684, and −1648/+21 were amplified by PCR using pBS-CYP2C9−2088/+21 as a template and oligonucleotides sense p2C9−1783/KpnI 5′-CGGGGTACCCTGTAATTATTAATG-3′, p2C9−1684/KpnI 5′-CGGGGTACCCAACTGA-ACTGAATG-3′ and p2C9−1648/KpnI 5′-CGGGGTACCTTTGAGATGCAGGGCTTATG-3′ and reverse p2C9+21/BglII. They were then cloned into pGL3-basic digested with BglII/KpnI. Oligonucleotides corresponding to 2C9-GRE (5′-ACCCAACTGAACTGAATGTTTTGCTTGAA-3′) and 2C9-DR4 (5′-AAACCAAACTCTTCTGACCTCTCA-3′) were cloned into pGL3 promoter digested with SmaI. The pTAT-GRE-TK-luc and pTAT-(GRE)2-TK-luc plasmids containing one or two copies of the consensus GRE upstream of a minimal herpes simplex virus thymidine kinase promoter and a luciferase reporter gene was kindly provided by Dr L. Poellinger (Karolinska Institute, Stockholm, Sweden). The wild hGR expression vector (pSG5-hGR) was kindly provided by Dr. J. C. Nicolas (INSERM, Montpellier, France). The pΔATG-hPXR expression plasmid was generated by PCR amplification of cDNA encoding amino acids 1–434 of hPXR (S. Kliewer, Glaxo-Wellcome, Research Triangle Park, NC) using oligonucleotides 5′-GGGTGTGGGGAATTCACCACCATGGAGGTGAGACCCAAAGAAAGC-3′ and 5′-GGGTGTGGGGGATCCTCAGCTACCTGTGATGCCG-3′ and insertion into pSG5 plasmid digested by EcoRI/BamHI (Stratagene, La Jolla, CA). The hCAR expression vectors were generated by PCR, using pCDM8-hCAR vector as a template (kindly provided by M. Negishi) and oligonucleotides sense hCAR/ATG 5′-CGGAATTCATGGCCAGTAGGGAAG-3′ and reverse hCAR/TGA 5′-AAAAAAGCGGCCGCCTCAGCTGCAGATCTCCTGG-3′ and cloned into plasmids pcDNA3 (Invitrogen, Groningen, The Netherlands) and pBSEN (17Pallisgaard N. Pedersen F.S. Birkelund S. Jorgensen P. Gene (Amst.). 1994; 138: 115-118Crossref PubMed Scopus (12) Google Scholar) digested byEcoRI/NotI. The mCAR expression vector (pCR3-mCAR) was kindly provided by M. Negishi. Primary cultures of human hepatocytes were prepared and cultured as described elsewhere (3Gerbal-Chaloin S. Pascussi J.M. Pichard-Garcia L. Daujat M. Waechter F. Fabre J.M. Carrere N. Maurel P. Drug Metab. Dispos. 2001; 29: 242-251PubMed Google Scholar). The HepG2 and HuH7 cell lines were obtained from the NIH ATCC repository (Bethesda, MD) and maintained without antibiotics. Human hepatocytes, HepG2, and HuH7 were transfected in suspension with Fugene-6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions; 80,000 cells were transfected with 250 ng of reporter plasmids, 25 ng of pSV-β-galactosidase control vector (Promega, Madison, WI), and either 25 ng of pSG5 (Stratagene, La Jolla, CA) or pSG5-hGR or pCR3-mCAR, or 250 ng of pBSEN or pBSEN hCAR expression vector in Opti-MEM I medium (Invitrogen). HepG2 and HuH7 cells were seeded in 24-well plates in DNA:liposome mix in DMEM supplemented with 10% fetal calf serum medium (Invitrogen) for 24 h. Then the medium was replaced by DMEM, and cells were treated for 16 h with dexamethasone, RU486, TCPOBOP, androstenol, rifampicin, PCN, or Me2SO. Luciferase and β-galactosidase assays were performed according to the specifications of the manufacturer (Promega). Site-directed mutagenesis was performed using a QuikChange™ site-directed mutagenesis kit (Stratagene) according to the recommendation of the manufacturer, and oligonucleotides including, a mutated 2C9-GRE-m1 (mutated bases in 5′-half underlined): 5′-GGTGGACCCAACTGCCCTGAATGTTTTGCTTGAAATGAAACC-3′, a mutated 2C9-GRE-m2 (mutated bases in 3′-half underlined): 5′-GGTGGACCCAACTGAACTGAATGCCTTGCTTGAAATGA-AACC-3′, a mutated 2C9-DR4-m1 (mutated bases in 5′-half underlined): 5′-CTAAATGTTATAAAACCCTTGTCTTCTGACCTCTCAATCTAGTC-3′, and a mutated 2C9-DR4-m2 (mutated bases in 3′-half underlined) 2C9-DR4-m2: 5′-GTTTATAAACCAAACTCTTCTCTGGTCTCAATCTAGTCAACTGGGG-3′. EMSA was performed using 5′-32P-labeled oligonucleotides TAT-GRE: 5′-GACCCTAGAGGATCTGTACAGGATGTTCTAGAT-3′ (Santa Cruz) or 2C9-GRE: 5′-ACCCAACTGAACTGAATGTTTTGCTTGAA-3′. Fifty thousand or 100,000 cpm of TAT-GRE or 2C9-GRE oligonucleotides, respectively, were incubated for 15 min at 4 °C in 12 μl of 10% glycerol, 10 mm Tris-HCl, pH 7.2, 100 mm KCl, 1 mm DTT in the presence of 4 μg of total proteins from Sf9 cells transfected with a recombinant GR-expressing baculovirus and Nonidet P-40 (0.1%) to minimize the binding of accessory proteins to GR. Competitions were performed with unlabeled oligonucleotides, including TAT-GRE, TAT-GRE mutant (5′-GACCCTAGAGGATCTCAACAGGATCATCTAGAT-3′), and a mutated 2C9-GRE-m3 mutant (mutated bases underlined: 5′-ACCCAACCAAACTGAATCATTTGCTTGAA-3′). For supershift assays, extracts were pre-incubated with 1 μg of GR antibody (Santa Cruz). Samples were loaded on a 4% polyacrylamide gel and submitted to electrophoresis at 20 mA in 0.25× TBE. The gel was analyzed using a PhosphorImager apparatus and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). EMSA was performed using 5′-32P-labeled oligonucleotides 2B6-NR1 (5′-ACTGTACTTTCCTGACCCTGAAGA-3′) (5Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar) or 2C9-DR4 (5′-AAACCAAACTCTTCTGACCTCTCA-3′). Fifty thousand cpm were incubated for 20 min at 4 °C in 20 μl of 10 mm Hepes, pH 7.9, 10% glycerol, 1 mmMgCl2, 0.1 mm EDTA, 100 mm KCl, and 0.3 mm DTT in the presence of 15 μg of total proteins from COS cells transfected or not with pcDNA3-hCAR. Competitions were performed with unlabeled oligonucleotides including 2B6-NR1, 2C9-DR4, 2C9–5′-CAR-RE (5′-GCCTTTGACTTACCTAAGTACTAAATGTTATAAAACC-3′, position −1856/−1818), 2C9–3′-CAR-RE (5′-AACCAAACTCTTCTGACCTCTCAATCTAGTCAACTGGGG-3′, position −1822/−1783), 2C9-DR4-m1 (5′-AAACCCTTGTCTTCTGACCTCTCA-3′), 2C9-DR4-m2 (5′-AAACCAAACTCTTCTCTTGTCTCA-3′), and 2C9-DR4-m3 (in which both half-sites were mutated). For supershift assays, extracts were pre-incubated with 1 μg of RXR antibody (Santa Cruz) or hCAR antibody (kindly provided by M. Negishi). Samples were loaded on a 4% polyacrylamide gel and submitted to electrophoresis at 20 mA in 0.25× TBE. The gel was analyzed using a PhosphorImager apparatus and ImageQuant software. EMSA was performed using 5′-32P-labeled oligonucleotides 3A4-ER6 (5′-TAGAATATGAACTCAAAGGAGGTCAGTGAGT-3′) (8Lehmann J.M. McKee D.D. Watson M.A. Willson T.M. Moore J.T. Kliewer S.A. J. Clin. Invest. 1998; 102: 1016-1023Crossref PubMed Scopus (1389) Google Scholar) or 2C9-DR4 (described above). Fifty thousand cpm were incubated for 20 min at 20 °C in 20 μl of 5 mm Hepes, pH 7.8, 9% glycerol, 4 mm MgCl2, 0.05 mm EDTA, 1 mm DTT, 4 mm spermidine, 250 ng of dI-dC, and 1 μg of salmon sperm DNA in the presence of 2 μl of PXR and/or RXR proteins expressed using in vitro coupled transcription and translation (Promega). Competitions were performed using excess of unlabeled oligonucleotides, including 2C9-DR4, 3A4-ER6, and 3A4-DR3 (5′-GAATGAACTTGCTGACCCTCT-3′) (11Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google Scholar). Samples were loaded on a 4% polyacrylamide gel and submitted to electrophoresis at 20 mA in 0.25× TBE. The gel was analyzed using a PhosphorImager apparatus and ImageQuant software. Total RNA was isolated using Trizol reagent (Invitrogen), from 107 cultured hepatocytes according to the manufacturer's instructions. For quality control, 30 μg of total RNA were analyzed by Northern blot using a rat glyceraldehyde-3-phosphate dehydrogenase cDNA probe (J. M. Blanchard, IGMM, Montpellier France). Tyrosine aminotransferase (TAT) mRNA was quantified by Northern blot using a specific probe (kindly provided by Dr. T. Grange, Institut J. Monod, Paris, France), and CYP2C9 mRNA was quantified by RNase protection assay as described (3Gerbal-Chaloin S. Pascussi J.M. Pichard-Garcia L. Daujat M. Waechter F. Fabre J.M. Carrere N. Maurel P. Drug Metab. Dispos. 2001; 29: 242-251PubMed Google Scholar). In recent work, we observed that induction of CYP2C9 mRNA by dexamethasone paralleled that of TAT, a gene product known to be controlled by GR (18Schmid E. Schmid W. Jantzen M. Mayer D. Jastorff B. Schutz G. Eur. J. Biochem. 1987; 165: 499-506Crossref PubMed Scopus (106) Google Scholar) in terms of time and concentration dependence in primary human hepatocytes (3Gerbal-Chaloin S. Pascussi J.M. Pichard-Garcia L. Daujat M. Waechter F. Fabre J.M. Carrere N. Maurel P. Drug Metab. Dispos. 2001; 29: 242-251PubMed Google Scholar). This prompted us to look for a functional glucocorticoid-responsive element (GRE) in gene CYP2C9. For this purpose, the 5′-flanking region of CYP2C9 from −2088 to +21 was cloned upstream of a luciferase reporter gene driven by a SV40 promoter in pGL3 basic vector (p2088-luc), and several plasmids containing deletion constructs of this region were generated. The various plasmids harboring CYP2C9 deletions were transfected into HepG2 cells (in which GR expression is very low), with or without cotransfection of a plasmid expressing hGR (pSG5-hGR). Cells were then cultured for 16 h in the presence or absence of dexamethasone, and luciferase activity was measured. Plasmid pTAT-GRE-TK-luc harboring one GRE from TAT was used as control. As shown in Fig. 1, both p2088-luc and pTAT-GRE-TK-luc exhibited parallel response to increasing concentrations of dexamethasone (a plateau being reached at 10 nm) and similar extent of induction (15-fold). As these results suggested the presence of a GRE located on the −2088/+21 region of CYP2C9, a more detailed analysis of hGR-mediated transactivation of CYP2C9 deletion constructs was undertaken. The results are presented in Fig.2. No significant induction of the reporter gene was observed (2-fold induction maximum) without hGR transfection in any of the constructs examined, including the empty pGL3 basic vector. In contrast, when these plasmids were cotransfected with the hGR expression vector, a significant induction (10–20-fold) was observed in response to 100 nm dexamethasone for p2088-luc, p1856-luc, p1783-luc, and p1684-luc constructs. However, a major decrease in luciferase activity was observed with construct p1648-luc and shorter constructs including p1545-luc and p340-luc. These results suggest the existence of a GRE between −1684 and −1648.Figure 2Identification of a functional GRE in theCYP2C9 promoter.Left panel, schematic representation of CYP2C9promoter constructs (from −2088 to +21). Right panel, transactivation of CYP2C9 promoter constructs by hGR. Plasmids harboring the CYP2C9 promoter constructs were cotransfected in HepG2 cells with pSG5 (control vector,white bars) or pSG5-hGR (hGR-expressing vector,black bars), and with pSV-β-galactosidase for transfection control. hGR was activated by treatment of cells with 100 nm dexamethasone for 16 h. Cell extracts were assayed for luciferase activity, which was normalized to β-galactosidase activity. Induction is expressed as the ratio of normalized luciferase activity in the presence of DEX to this activity in the absence of DEX.Error bars represent the standard deviations of 10 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) An oligonucleotide corresponding to the region −1684 to −1654 was then cloned in three copies in the pGL3-promoter to generate plasmid p2C9-(GRE)3-luc. As shown in Fig.3A, this construct was strongly transactivated by hGR (150-fold) in the presence of 100 nm dexamethasone. To verify that this transactivation was hGR-dependent, cells were treated with 1 μmRU486 (a prototypical hGR antagonist), in the absence or presence of dexamethasone. Although RU486 per se produced a moderate induction of luciferase activity, this compound drastically inhibited the dexamethasone-mediated transactivation of the construct. The full-length 2C9 promoter responded only modestly to dexamethasone when transfected in hepatocytes under the same conditions (data not shown). In parallel experiments carried out with plasmid pTAT-(GRE)2-TK-luc, similar observations were made (Fig.3B). Plasmid p2C9-(GRE)3-luc was also transfected in primary human hepatocytes and was transactivated by 100 nm dexamethasone as shown in Fig. 3C. Here again, RU486 completely suppressed dexamethasone-mediated transactivation. Computer analysis of theCYP2C9−1684/−1654 region demonstrated the presence of two putative GRE half-sites separated by three nucleotides. To evaluate the role of these two sites, they were mutated sequentially by directed mutagenesis using p2088-luc as a template (Fig.4A). Plasmids p2088-GREwt-luc (wild type GRE sequence), p2088-GREm1-luc (mutations in the 5′-half-site of GRE), and p2088-GREm2-luc (mutations in the 3′-half-site of GRE) were then transfected in HepG2 cells in the presence of hGR expression vector and of 100 nmdexamethasone. As shown in Fig. 4B, the mutation of either half-site was sufficient to abolish transcriptional activation of the construct. The CYP2C9 region −1662/−1676 is hereafter referred to as 2C9-GRE. To determine whether hGR interacts directly with 2C9-GRE, a gel shift analysis was performed using baculovirus-expressed hGR. In a control experiment (Fig. 5A), we verified that the consensus TAT-GRE effectively binds hGR. A clear band revealing the complex was observed (lane 2), and this band was supershifted by anti-GR antibodies, as expected (lane 3). TAT-GRE oligonucleotide efficiently competed with itself (lanes 4 and 5), whereas the TAT-GRE mutant (see "Experimental Procedures") did not (lane 6). 2C9-GRE oligonucleotide was a modest competitor for this binding (lanes 7 and 8), whereas the mutant 2C9-GREm3 (mutations in both half-sites of GRE, see "Experimental Procedures") was not (lane 9). Similar experiments carried out with 2C9-GRE are shown in Fig. 5B. The efficient binding of hGR to 2C9-GRE is revealed by a clear band (lane 2); this band was supershifted by anti-hGR antibodies (lane 3). TAT-GRE in excess efficiently competed with this binding (lanes 4 and5), whereas mutated TAT-GRE did not (lane 6). These observations suggest that 2C9-GRE binds to hGR, although with a lower affinity than for TAT-GRE. hGR is expressed constitutively in our hepatocyte cultures (19Pascussi J.M. Drocourt L. Fabre J.M. Maurel P. Vilarem M.J. Mol. Pharmacol. 2000; 58: 361-372Crossref PubMed Scopus (330) Google Scholar, 20Pascussi J.M. Gerbal-Chaloin S. Fabre J.M. Maurel P. Vilarem M.J. Mol. Pharmacol. 2000; 58: 1441-1450Crossref PubMed Scopus (226) Google Scholar). We therefore anticipated that, if induction of CYP2C9 mRNA by dexamethasone is primarily mediated by hGR, protein synthesis should not be necessary for this process. To test this hypothesis, human hepatocytes were cultured in a dexamethasone-depleted medium for 48 h and then re-exposed to 100 nm dexamethasone for 24 h, either in the absence or presence of 10 μg/ml cycloheximide, a typical inhibitor of protein synthesis (control experiments indicated that protein synthesis is inhibited by more than 90% in these conditions). Analysis of CYP2C9 mRNA expression by RNase protection assay is reported in Fig.6. Clearly, cycloheximide affected neither CYP2C9 nor TAT mRNA induction by dexamethasone. In contrast, induction of CYP2C9 mRNA by rifampicin and phenobarbital was inhibited by 50% and by more than 75%, respectively, in cells cultured in the presence of cycloheximide. Similarly, induction of CYP3A4 mRNA by rifampicin was inhibited by more than 75% in cycloheximide-treated cells (not shown). In aggregate, our previous studies (3Gerbal-Chaloin S. Pascussi J.M. Pichard-Garcia L. Daujat M. Waechter F. Fabre J.M. Carrere N. Maurel P. Drug Metab. Dispos. 2001; 29: 242-251PubMed Google Scholar), the data reported above on CYP2C9-GRE characterization, and the absence of any effect of cycloheximide on mRNA induction demonstrate that CYP2C9 is a primary hGR-responsive gene, in cultured human hepatocytes. We have previously observed that induction of CYP2C9 mRNA in response to phenobarbital in primary human hepatocytes parallels that of CYP2B6 mRNA in terms of time and concentration dependence (3Gerbal-Chaloin S. Pascussi J.M. Pichard-Garcia L. Daujat M. Waechter F. Fabre J.M. Carrere N. Maurel P. Drug Metab. Dispos. 2001; 29: 242-251PubMed Google Scholar). It was therefore suspected that CAR could account for the phenobarbital-mediated induction of CYP2C9. We looked for possible CAR-responsive element(s) in theCYP2C9 regulatory region by deletion analysis of transcriptional activity. The plasmids harboring the variousCYP2C9 deletion constructs described above were transfected into HuH7 cells, with or without cotransfection of a plasmid expressing either the mouse or human CAR (mCAR or hCAR). Cells were then cultured for 36 h and the luciferase activity measured. Plasmid p2B6-(NR1)3-luc, harboring
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