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Novel CAR-mediated Mechanism for Synergistic Activation of Two Distinct Elements within the Human Cytochrome P450 2B6 Gene in HepG2 Cells

2004; Elsevier BV; Volume: 280; Issue: 5 Linguagem: Inglês

10.1074/jbc.m411318200

1083-351X

Karen E. Swales, Satoru Kakizaki, Yukio Yamamoto, Kaoru Inoue, Kaoru Kobayashi, Masahiko Negishi,

Estrogen and related hormone effects

The constitutive active receptor (CAR) regulates the induction of the cytochrome P450 2B6 (CYP2B6) gene by phenobarbital-type inducers, such as 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) via the distal phenobarbital-responsive enhancer module (PBREM, at –1732/–1685 bp). Activation of the PBREM by TCPOBOP generated a 10-fold induction of CYP2B6 mRNA in HepG2 cells stably expressing mouse CAR (Ym17). Co-treatment with the protein phosphatase inhibitor okadaic acid (OA) synergistically increased this induction over 100-fold without directly activating CAR or the PBREM. Although OA synergy required the presence of PBREM, deletion assays delineated the OA-responsive activity to a proximal 24-bp (–256/–233) sequence (OARE) in the CYP2B6 promoter. CAR did not directly bind to the OARE in electrophoretic mobility shift assays. However, both DNA affinity and chromatin immunoprecipitation assays showed a significant increase in CAR association with the OARE after co-treatment with TCPOBOP and OA, indicating the indirect binding of CAR to the OARE. The two cis-acting elements, the distal PBREM and the proximal OARE, within the chromatin structure are both regulated by CAR in response to TCPOBOP and OA, respectively, to maximally induce the CYP2B6 promoter. This functional interaction between the two sites expands the current understanding of the mechanism of CAR-mediated inducible transcription. The constitutive active receptor (CAR) regulates the induction of the cytochrome P450 2B6 (CYP2B6) gene by phenobarbital-type inducers, such as 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) via the distal phenobarbital-responsive enhancer module (PBREM, at –1732/–1685 bp). Activation of the PBREM by TCPOBOP generated a 10-fold induction of CYP2B6 mRNA in HepG2 cells stably expressing mouse CAR (Ym17). Co-treatment with the protein phosphatase inhibitor okadaic acid (OA) synergistically increased this induction over 100-fold without directly activating CAR or the PBREM. Although OA synergy required the presence of PBREM, deletion assays delineated the OA-responsive activity to a proximal 24-bp (–256/–233) sequence (OARE) in the CYP2B6 promoter. CAR did not directly bind to the OARE in electrophoretic mobility shift assays. However, both DNA affinity and chromatin immunoprecipitation assays showed a significant increase in CAR association with the OARE after co-treatment with TCPOBOP and OA, indicating the indirect binding of CAR to the OARE. The two cis-acting elements, the distal PBREM and the proximal OARE, within the chromatin structure are both regulated by CAR in response to TCPOBOP and OA, respectively, to maximally induce the CYP2B6 promoter. This functional interaction between the two sites expands the current understanding of the mechanism of CAR-mediated inducible transcription. The nuclear receptor CAR 1The abbreviations used are: CAR, constitutive active-androstane receptor; bHLH, basic helix loop helix; CBP, cAMP response element-binding protein; ChIP, chromatin immunoprecipitation; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; CPZ, chlorpromazine; CYP, cytochrome P450; E2, 17β-estradiol; OA, okadaic acid; OARE, Okadaic acid responsive element; OABP, OARE-binding protein; PB, phenobarbital; PBREM, phenobarbital-responsive enhancer module; RAR, retinoic acid receptor; RXR, retinoid X receptor; SOD3, superoxide dismutase 3; SREBP, sterol regulatory element-binding protein; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; UGT1A1, UDP-glucuronosyltransferase 1A1; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; mCAR, mouse CAR; RT, reverse transcription. transcriptionally modifies the expression of genes involved in the metabolism and elimination of xenobiotics and endogenous compounds, such as cytochrome P450 (CYP), in response to xenochemical exposure. The receptor is essential for regulating the induction of a set of CYP genes, particularly the CYP2B subfamily, by phenobarbital (PB) and PB-like inducers (e.g. chlorpromazine (CPZ) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP)) (1Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (655) Google Scholar, 2Honkakoski P. Moore R. Washburn K.A. Negishi M. Mol. Pharmacol. 1998; 53: 597-601Crossref PubMed Scopus (161) Google Scholar, 3Sueyoshi 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, 4Ueda A. Hamadeh H.K. Webb H.K. Yamamoto Y. Sueyoshi T. Afshari C.A. Lehmann J.M. Negishi M. Mol. Pharmacol. 2002; 61: 1-6Crossref PubMed Scopus (400) Google Scholar, 5Wei P. Zhang J. Egan-Hafley M. Liang S. Moore D.D. Nature. 2000; 407: 920-923Crossref PubMed Scopus (593) Google Scholar). As a heterodimer with the retinoid X receptor (RXR), CAR binds to direct repeats of nuclear receptor half-sites, such as the NR1 within the distal 51-bp PB-responsive enhancer module (PBREM) of Cyp2b10, and activates transcription (1Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (655) Google Scholar, 6Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (488) Google Scholar, 7Sueyoshi T. Negishi M. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 123-143Crossref PubMed Scopus (338) Google Scholar). Experiments with transgenic mice bearing a mutated PBREM have confirmed that it is an essential enhancer element (8Ramsden R. Beck N.B. Sommer K.M. Omiecinski C.J. Gene (Amst.). 1999; 228: 169-179Crossref PubMed Scopus (35) Google Scholar). In addition, several PB-responsive DNA elements have been reported in the proximal promoters of CYP2B genes, but their role as enhancer elements in PB induction is controversial (reviewed in Ref. 9Honkakoski P. Negishi M. J. Biochem. Mol. Toxicol. 1998; 12: 3-9Crossref PubMed Scopus (66) Google Scholar). Thus, the exact mechanism how CAR regulates inducible transcription still remains elusive. Signal transduction has been repeatedly suggested to regulate PB induction. Various inhibitors of protein kinases or phosphatases often repress PB induction in hepatocytes (10Corcos L. Lagadic-Gossmann D. Pharmacol. Toxicol. 2001; 89: 113-122Crossref PubMed Scopus (33) Google Scholar). Translocation of CAR from the cytoplasm to the nucleus is an essential step in PB induction (6Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (488) Google Scholar) and has recently been shown to involve a phosphorylation/dephosphorylation cascade (11Yoshinari K. Kobayashi K. Moore R. Kawamoto T. Negishi M. FEBS Lett. 2003; 548: 17-20Crossref PubMed Scopus (143) Google Scholar). The protein phosphatase inhibitor okadaic acid (OA) effectively repressed the induction by PB of CYP2B genes in rodent primary hepatocytes (12Honkakoski P. Negishi M. Biochem. J. 1998; 330: 889-895Crossref PubMed Scopus (95) Google Scholar, 13Sidhu J.S. Omiecinski C.J. J. Pharmacol. Exp. Ther. 1997; 282: 1122-1129PubMed Google Scholar) by preventing CAR nuclear translocation (6Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (488) Google Scholar, 11Yoshinari K. Kobayashi K. Moore R. Kawamoto T. Negishi M. FEBS Lett. 2003; 548: 17-20Crossref PubMed Scopus (143) Google Scholar). However, distinct activation processes occur in the nucleus, for example the Ca2+/calmodulin-dependent kinase inhibitor KN-62 repressed the induction of CYP2B10 by PB in mouse primary hepatocytes despite CAR accumulation in the nucleus (14Marc N. Galisteo M. Lagadic-Gossmann D. Fautrel A. Joannard F. Guillouzo A. Corcos L. Eur. J. Biochem. 2000; 267: 963-970Crossref PubMed Scopus (39) Google Scholar, 15Yamamoto Y. Kawamoto T. Negishi M. Arch. Biochem. Biophys. 2003; 409: 207-211Crossref PubMed Scopus (66) Google Scholar). It is possible that CAR interacts with other factors in the nucleus that are the target of phosphorylation because direct phosphorylation of CAR has not, as yet, been reported. Indirect mechanisms of gene induction by nuclear receptors, including the retinoic acid receptor (RAR), RXR, and the glucocorticoid receptor, independent of nuclear receptor half-site activation, have been reported previously (16Froeschle A. Alric S. Kitzmann M. Carnac G. Aurade F. Rochette-Egly C. Bonnieu A. Oncogene. 1998; 16: 3369-3378Crossref PubMed Scopus (32) Google Scholar, 17Stoecklin E. Wissler M. Moriggl R. Groner B. Mol. Cell. Biol. 1997; 17: 6708-6716Crossref PubMed Scopus (150) Google Scholar) but no such mechanism has been reported for CAR at present. Dissecting the CAR-mediated response has been challenging because in transformed cell lines CAR spontaneously accumulates in the nucleus and is constitutively active (6Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (488) Google Scholar). In the present investigation, we used a stable line (Ym17) of HepG2 cells that express mouse CAR-V5-His primarily in the nucleus that can be effectively activated by CAR ligands. These cells provided an opportunity to examine directly the function of the receptor in the nucleus, independent of translocation, and are an excellent system for investigating the nuclear effects of inhibitors (e.g. OA) on CAR-mediated transcription. Here we present experimental considerations to reveal the role of a proximal region of the CYP2B6 gene in synergistically up-regulating CAR-mediated transcription in response to TCPOBOP and OA. These investigations shed light on a unique role for a proximal sequence in maximizing PB-type induction and identify a novel role for CAR in the formation of a proximal protein-DNA complex. Materials—Okadaic acid was purchased from Calbiochem; penicillin, streptomycin, 17β-estradiol (E2), and chlorpromazine were from Sigma; 6-(4-chlorophenyl)imidazo[2,1-b](1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) was from Biomol (Plymouth Meeting, PA); and hygromycin B was from Invitrogen. 1, 4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) was by using the method of Kende et al. (18Kende A.S. Ebetino F.H. Drendel W.B. Sundaralingam M. Glover E. Poland A. Mol. Pharmacol. 1985; 28: 445-453PubMed Google Scholar). The plasmids pGL3-basic, pBind, and pSG5-luc were obtained from Promega (Madison, WI). The plasmid pcDNA3.1/Hygro© and the V5-antibody were obtained from Invitrogen. The –1.8-kbp 5′-flanking DNA of the CYP2B6 gene and other recombinant plasmids were prepared previously (2Honkakoski P. Moore R. Washburn K.A. Negishi M. Mol. Pharmacol. 1998; 53: 597-601Crossref PubMed Scopus (161) Google Scholar, 3Sueyoshi 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, 6Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (488) Google Scholar, 19Kobayashi K. Sueyoshi T. Inoue K. Moore R. Negishi M. Mol. Pharmacol. 2003; 64: 1069-1075Crossref PubMed Scopus (156) Google Scholar, 20Sugatani J. Kojima H. Ueda A. Kakizaki S. Yoshinari K. Gong Q.H. Owens I.S. Negishi M. Sueyoshi T. Hepatology. 2001; 33: 1232-1238Crossref PubMed Scopus (335) Google Scholar). Normal mouse IgG was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cloning and Plasmid—The 1.8-kbp 5′-flanking DNA of the CYP2B6 gene was cloned between the KpnI and XhoI sites of basic firefly luciferase reporter plasmid pGL3-Basic (–1.8k-pGL3). To construct deletions, either proper fragments were generated from the 1.8-kbp DNA using convenient restriction sites or from the –1.8k-pGL3 plasmid using the Stratagene QuikChange® site-directed mutagenesis kit (Cedar Creek, TX) and specific primers spanning the excised bases (and containing the insert sequence for UGT256233). Full-length mouse CAR cDNA was cloned into pBIND vector (pBIND-mCAR). All constructs were verified by their sequences. Cell Culture—Cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics (100 unit/ml of penicillin and 100 μg/ml of streptomycin). Ym17 cells, a stable cell line that expressed mCAR, and Yh18 cells, a stable cell line that expressed hCAR, were established from HepG2 cells that were transfected with a mouse or human pcDNA3.1-CAR-V5-His expression vector, respectively, and were selected for neomycin resistance. During cell treatment, OA was added to the culture medium 30 min before treatment with a given chemical inducer. RT-PCR—Total RNA isolation and subsequent synthesis of first strand cDNA were performed using TRIzol reagent (Invitrogen) and SuperScript™ preamplification system (Invitrogen), respectively. The resulting cDNA was subjected to semi-quantitative real time PCR using an ABI Prism 7700 (PE Applied Biosystems, Foster City, CA). Primer and probe sets used for PCR analysis were as follows: CYP2B6 5′- and 3′-primers, respectively, 5′-AAGCGGATTTGTCTTGGTGAA-3′, 5′-TGGAGGATGGTGGTGAAGAAG-3′, and probe 6FAM-CATCGCCCGTGCGGAATTGTTC-TAMRA; UDP-glucuronosyltransferase 1A1 (UGT1A1), 5′-GGCCCATCATGCCCAATAT-3′, 5′-TTCAAATTCCTGGGATAGTGGATT-3′, and 6FAM-TTTTTGTTGGTGGAATCAACTGCCTTCACTAMRA; CYP3A7, 5′-CCCAATTCTTGAAGCATTAAATATCAC-3′, 5′-GGCCATGAGCTCCAGATCA-3′, and 6FAM-CAGATAAAAGAAGGTCG-MGB; superoxide dismutase (SOD3), 5′-GCGGAGCCCAACTCTGACT-3′, 5′-GCATGACCTCCTGCCAGATC-3′, and 6FAM-CCGAGACATGTACGCCAAGGTCACG-TAMRA. The quantity of mRNA was normalized by β-actin mRNA and measured by using a Pre-Developed TaqMan Assay for human β-actin (PE Applied Biosystems, Foster City, CA). Transfection Assays—For transient transfection assays, Ym17 or Yh18 cells in 24-well plates were co-transfected with a given pGL3 luciferase reporter plasmid and pRL-SV40 plasmid (0.1 μg/well each) by the calcium phosphate co-precipitation method using CellPhect Transfection Kit (Amersham Biosciences). Sixteen hours after transfection, the cells were treated with chemical inducers. Luciferase activity was measured in cell lysates using the dual-luciferase reporter assay system (Promega, Madison, WI). The receptor-dependent enhancer activity was determined based on firefly luciferase activity normalized against Renilla luciferase activity, except for the Ym17S15 cells when firefly luciferase activity was normalized against protein concentration (μg/ml). Protein concentration was measured at 595 nm using Bio-Rad protein assay reagent (Bio-Rad). Nuclear Extraction—Ym17 cells were harvested from four confluent 75-cm2 tissue culture flasks for each treatment group, and nuclear extracts were prepared using the procedure described previously (19Kobayashi K. Sueyoshi T. Inoue K. Moore R. Negishi M. Mol. Pharmacol. 2003; 64: 1069-1075Crossref PubMed Scopus (156) Google Scholar). The extracts were dialyzed as described previously (21Honkakoski P. Moore R. Gynther J. Negishi M. J. Biol. Chem. 1996; 271: 9746-9753Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). DNase I Protection Assays and Electrophoretic Mobility Shift Assays—DNase I protection assays were carried out as described previously (22Yokomori N. Kobayashi R. Moore R. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1995; 15: 5355-5362Crossref PubMed Scopus (81) Google Scholar). The DNA fragments were separated on standard sequencing gels, which were dried and autoradiographed. Electrophoretic mobility shift assay binding was performed as described previously (1Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (655) Google Scholar) for 25 min with 5 μg of protein to 32P-labeled double-stranded oligonucleotides corresponding to –252/–237 bp of the CYP2B6 5′-flanking region (5′-catgACCCACACATTCACTT-3′) or to the NR1 motif from the Cyp2b10 PBREM (5′-gatcTCTGTACTTTCCTGACCTTG-3′). DNA Affinity Chromatography—For affinity purification of CAR, Dynabeads® M-280 streptavidin (Dynal ASA, Oslo, Norway) conjugated with multiple copies of the wild-type or mutant (5′-catgACCCAAAAAAACACTT-3′)–252/–237-bp oligonucleotide were prepared as described previously (1Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (655) Google Scholar). The bound proteins were eluted with 50 μl of 20 mm Hepes, pH 7.6, containing 0.5 m NaCl, 20% glycerol, 0.2 mm EDTA, and 1 mm Na2MO4. Chromatin Immunoprecipitation—6 × 106 Ym17 cells per treatment group were incubated with TCPOBOP (250 nm) and/or OA (10 nm) for 24 h. Chromatin immunoprecipitation was performed by using the chromatin immunoprecipitation (ChIP) assay kit (Upstate Biotechnologies, Charlottesville, VA), according to the manufacturer's instructions, and adapted as follows. Proteins and DNA were cross-linked with 1% formaldehyde in the media for 20 min at 37 °C. The cells were collected in 450 μl of SDS lysis buffer containing protease inhibitors aprotinin (1 μg/ml), phenylmethanesulfonyl fluoride (1 μm), and pepstatin A (1 μg/ml) and were sonicated at power 10 for five 10-s pulses using the Misonix Microson™ XL 2000 ultrasonic cell disrupter. The sonication was optimized as described in the ChIP assay kit instructions and examined by agarose gel electrophoresis to determine generation of DNA fragments between 200 and 1000 bp in length, which avoided nonspecific immunoprecipitation of the OARE via cross-linking of CAR to the PBREM. The diluted lysates were precleared by incubation with 160 μl of protein A-agarose salmon sperm slurry, 60 μl of normal mouse IgG, and 0.05% bovine serum albumin for 2 h at 4 °C. The agarose was collected by centrifugation at 100 × g for 2 min at 4 °C. 1 ml of the cleared fraction was subjected to overnight immunoprecipitation with either 5 μg of V5 antibody, 5 μg of normal mouse IgG, or no antibody at 4 °C. 1 ml was retained as an input sample for each treatment group. The immunoprecipitated complexes were recovered, washed, and eluted; the cross-links were reversed and proteins digested as described in the manufacturer's protocol. A phenol chloroform/ethanol precipitation method purified the DNA with a final resuspension in 45 μl of water. The DNA was amplified by real time PCR as described under "RT-PCR" using a specific primer and probe set designed to span the OARE between –287 and –193-bp upstream of the CYP2B6 transcription start site, with the probe located within the OARE sequence. This amplicon was specific for the OARE and did not contain other CAR-binding sites within the CYP2B6 gene, such as the PBREM. The OARE 5′- and 3′-primers were 5′-CAAAAATAGACATACATATACCCACAAACC-3′ and 5′-ATTGGCAGGGATACAGTGGTAGAG-3′, respectively, and the probe was 6FAM-CACTTGCTCACCTGGACT-MGB. The precipitated DNA levels were normalized to the input levels using the following equation (2[caret]Ctinput – Ctprecipitate). Synergistic Induction of the CYP2B6 Gene—In Ym17 cells, the endogenous CYP2B6 gene was induced by TCPOBOP 8–10-fold (Fig. 1a). Unexpectedly, 24 h of co-treatment with OA and TCPOBOP synergistically up-regulated the induction more than 100-fold. OA alone induced CYP2B6 mRNA only 3-fold. This synergistic up-regulation became greater in a time-dependent fashion as the induction by TCPOBOP increased (Fig. 1b) but was not observed in normal HepG2 cells, indicating mCAR was essential for synergistic induction (Fig. 1a). The mRNAs of CAR-regulated UGT1A1, CYP3A7, and SOD3 genes (4Ueda A. Hamadeh H.K. Webb H.K. Yamamoto Y. Sueyoshi T. Afshari C.A. Lehmann J.M. Negishi M. Mol. Pharmacol. 2002; 61: 1-6Crossref PubMed Scopus (400) Google Scholar, 20Sugatani J. Kojima H. Ueda A. Kakizaki S. Yoshinari K. Gong Q.H. Owens I.S. Negishi M. Sueyoshi T. Hepatology. 2001; 33: 1232-1238Crossref PubMed Scopus (335) Google Scholar) were increased by TCPOBOP in Ym17 cells, yet no synergistic up-regulation was observed following co-treatment with OA (Fig. 2a). Thus, OA-dependent synergistic induction occurred specifically in the CYP2B6 gene. OA-dependent synergistic up-regulation, however, was not TCPOBOP-specific and may be a general phenomenon occurring in the CYP2B6 gene in response to PB-type inducers because induction by CPZ and E2 was also synergistically up-regulated from 2- and 4-fold to 27- and 57-fold, respectively, in the presence of OA (Fig. 2b).Fig. 2a, no synergistic induction of the UGT1A1, CYP3A7, and SOD3 genes. Ym17 cells were treated with TCPOBOP (250 nm) and/or OA (10 nm) for 24 h. b, synergistic induction by co-treatment with OA and CPZ or E2. Ym17 cells were treated with CPZ (10 μm) or E2 (10 μm) with or without OA (10 nm) for 24 h. Total cellular RNAs were prepared from the treated cells and subjected to quantitative real time RT-PCR of UGT1A1, CYP3A7, SOD3, or CYP2B6 mRNA. The levels of target gene mRNAs were normalized by the β-actin mRNA levels, and fold induction was calculated relative to the levels in Me2SO (DMSO)-treated cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Role of OA in Synergistic Induction—To determine whether OA directly activated mCAR, a Gal4-based mammalian one-hybrid assay was performed. TCPOBOP enhanced Gal4-reporter activity in HepG2 cells co-transfected with an mCAR-GAL4 DNA binding domain fusion plasmid, whereas additional co-treatment with OA caused no synergistic up-regulation of the reporter gene (Fig. 3a). Thus, OA does not directly regulate mCAR to synergistically induce the CYP2B6 gene. To determine whether OA activated CYP2B6 transcription via the PBREM, a luciferase reporter gene containing five copies of the Cyp2b10 NR1 motif upstream of a thymidine kinase promoter was transfected into Ym17 cells. TCPOBOP enhanced luciferase reporter activity 9-fold, whereas additional co-treatment with OA caused no synergistic up-regulation of the reporter gene (Fig. 3b). However, when the –1.8-kbp 5′-flanking sequence of the CYP2B6 gene that included the PBREM was cloned into a luciferase reporter gene (–1.8k-pGL3) and transfected into Ym17 cells, TCPOBOP activated the reporter gene 3-fold (Fig. 3c). Moreover, the activity was synergistically up-regulated 12-fold after co-treatment with OA (Fig. 3c). The –1.8-kbp DNA fragment retained its ability to be synergistically activated by OA in the presence of TCPOBOP. No synergistic up-regulation of the reporter gene was observed after co-treatment with TCPOBOP and the inactive analogues of OA, nor-okadaone, or okadaol (Fig. 3c). Delineation of OA-responsive Activity—To delineate the cis-acting region of the CYP2B6 gene directly responsible for the synergistic induction, a series of deletion assays were performed. Initially, a large internal deletion from –1685 to –307-bp within the –1.8k-pGL3 reporter gene was made to remove various elements, such as the glucocorticoid receptor element and activator protein-1 conserved in a region of the CYP2B6 gene corresponding to a –1.4/–1.2-kbp region of the rat CYP2B1/2 and mouse Cyp2b10 genes (reviewed in Ref. 9Honkakoski P. Negishi M. J. Biochem. Mol. Toxicol. 1998; 12: 3-9Crossref PubMed Scopus (66) Google Scholar). This joining of the PBREM to the proximal 307-bp (PBREM-307) did not significantly affect the synergy (Fig. 4). However, as expected, the proximal –307-bp sequence alone did not display either activation by TCPOBOP or a synergistic response to OA, indicating that synergy was PBREM- as well as CAR-dependent. Subsequently, deletions were constructed within the context of the –1.8-kbp DNA sequence to investigate the activity in the –306/–35-bp region. Whereas deletion of –125/–35-bp retained the synergistic response, the –306/–35-bp deletion lost significant synergistic activity. The proximal –306/–125-bp region was further dissected by constructing smaller deletions within the context of the –1.8-kbp DNA as follows: –306/–166, –256/–213, and –256/–233-bp (Fig. 4), all of which lost significant OA synergistic activity. Deletions within the –256/–233-bp region, such as the –252/–237-bp deletion in Fig. 4, all lost significant OA synergistic activity. Thus the OA-response element (OARE) was identified as a proximal 24-bp sequence between –256 and –233-bp of the –1.8-kbp DNA. The 51-bp PBREM was placed in front of the –256-bp proximal sequence of –1.8-kbp DNA (PBREM-256), by deleting the –1685/–257-bp fragment (Fig. 4), and was found to retain a significant level of synergistic response to OA, whereas deletion of a further proximal 9 bp (–1685/–247-bp, PBREM-247) lost synergistic activity, further confirming the role of the proximal –256-bp sequence in OA-responsive up-regulation of the –1.8-kbp DNA. Replacement of the OARE within the –1.8k-pGL3 construct with the –256/–233-bp sequence from the nonsynergistic gene UGT1A1 to generate a negative control showed a loss of synergy. This supported the –256/–233-bp region (OARE) as the OA-responsive site. Nuclear Protein Binding to the OARE—To investigate protein interactions with this region, DNase I protection of the –308/–121-bp fragment incubated with TCPOBOP- and OA-treated Ym17 nuclear extracts was performed. A large protected region was observed at –264/–195 bp that included the sequence identified as responsible for OA synergy (Fig. 5, left panel). However no differences in protein binding were evident either with nuclear extracts from treated Ym17 cells or from HepG2 cells (Fig. 5, right panel). To search for a specific binding site, electrophoretic mobility shift assays were performed using probes spanning the OARE (–256/–233-bp) and smaller overlapping regions –256/–247, –252/–237, and –242/–233 bp to dissect any treatment effects on this complex. One protein band showed similar electrophoretic mobility shift profiles for both –256 probes and corresponded to a single signal observed with the –252/–237-bp probe (Fig. 6a, lanes 1 and 3). The protein binding to the –252/237-bp probe increased in the presence of OA (Fig. 6a, lane 5) and was further enhanced in the presence of both TCPOBOP and OA (lane 7). The binding was specific because it was competed by an excess of unlabeled –252/–237-bp probe (Fig. 6a, lanes 2, 4, 6, and 8). Competition increased concentration dependently between 25- and 100-fold excess. For practical reasons, hereafter we will call this putative binding complex OARE-binding protein or OABP.Fig. 6a, treatment and CAR-dependent binding of a nuclear protein to the central region of the OARE. Electrophoretic mobility shift assay using double-stranded 32P-end-labeled probe –252/–237 with 5 μg of nuclear extracts from Ym17 cells incubated with TCPOBOP (250 nm) with or without OA (10 nm) for 48 h. A specific protein denoted OABP and a nonspecific protein bound to the probe. Specificity of binding was confirmed by competition with 100-fold excess of unlabeled –252/–237 probe. b, no OABP binding from HepG2 nuclear extracts. Electrophoretic mobility shift assay was as described for a with 5 μg of nuclear extracts from HepG2 cells incubated with TCPOBOP (250 nm) with or without OA (10 nm) for 48 h. c, no direct CAR binding to the OARE. Recombinant mouse CAR-V5-His and human RXRα, generated by TNT® T7 Quick-coupled transcription/translation system (Promega), were used for electrophoretic mobility shift assay using double-stranded 32P-end-labeled probes –252/–237 or NR1 incubated with 4 μl of the receptors alone or in combination.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Involvement of CAR in OABP Binding—As the OA synergistic up-regulation of CYP2B6 appeared to be CAR-dependent, experiments were designed to investigate the role of CAR in the binding of the OABP protein to –252/–237 bp. First, electrophoretic mobility shift assays were performed by using nuclear extract from HepG2 cells. No specific protein binding was observed for the –252/–237-bp probe, indicating that CAR was necessary for OABP binding (Fig. 6b, lanes 1–3). However, no binding of recombinant mCAR-V5-His or hRXRα to the –252/–237-bp probe was observed, either alone or in combination, despite strong binding of the two receptors in combination with a probe consisting of the Cyp2b10 NR1 motif (Fig. 6c, lane 6), suggesting that CAR did not bind directly to the OARE. To investigate the possibility that CAR interacted indirectly with the OARE and whether the interaction was treatment-dependent, DNA affinity chromatography was performed using magnetic beads with multiple copies of the –252/237-bp wild-type oligonucleotides or mutant oligonucleotides with the central 6 bp mutated, attached by biotinylation, and incubated with Ym17 nuclear extract. 4% of the input nuclear extracts were electrophoresed alongside the eluted fractions and Western-blotted with the V5 antibody to detect the V5-tagged mCAR from Ym17 cells. The eluted fractions from the –252/–237-bp wild-type beads indicated a background CAR association with the –252/–237-bp region in the presence of Me2SO, TCPOBOP, or OA alone (Fig. 7a). However, after co-treatment with TCPOBOP and OA, CAR interaction with the –252/–237-bp region was greatly enhanced. An extremely weak association after a much longer exposure was observed with the mutant beads, which was not changed by treatment. Thus, co-treatment of TCPOBOP and OA significantly increased CAR association with the OARE. To test whether CAR interacted with the endogenous CYP2B6 OARE in Ym17 cells, ChIP assays were performed with V5 antibody. DNA from –287 to –193 bp, encompassing the OARE but not the PBREM,