Drugs Mediate the Transcriptional Activation of the 5-Aminolevulinic Acid Synthase (ALAS1) Gene via the Chicken Xenobiotic-sensing Nuclear Receptor (CXR)
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m204699200
ISSN1083-351X
AutoresDavid Fraser, Michael Podvinec, Michel Kaufmann, Urs Meyer,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoHeme is an essential component in oxygen transport and metabolism in living systems. In non-erythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the first and rate-limiting enzyme in the heme biosynthesis pathway. ALAS1 expression and heme levels are increased in vivo by drugs and other chemical inducers of cytochrome P450 hemoproteins through mechanisms that are poorly understood. In the present studies, a chicken genomic cosmid library was employed to isolate a major portion of the ALAS1 gene. Two drug-responsive enhancer sequences, 176 and 167 base pairs in length, were identified in the 5′-flanking region of the gene in reporter gene assays in the hepatoma cell line LMH. The relative potency of inducers to activate these enhancers corresponds to induction of ALAS1 mRNA levels in LMH cells. Analysis of putative transcription factor binding sites within the enhancers revealed DR5 and DR4 type recognition sequences for nuclear receptors. Drug activation of the enhancer elements was reduced at least 60% after mutagenesis of individual nuclear receptor binding sites and was virtually eliminated following alteration of both recognition sites within the respective elements. Electrophoretic mobility shift assays and transactivation studies demonstrate direct interactions between the nuclear receptor binding sites and the recently described chicken xenobiotic-sensing receptor, (CXR) implicating drug activation mechanisms for ALAS1 similar to those found in inducible cytochrome(s) P450. This is the first report describing direct transcriptional activation of ALAS1 by drugs via drug-responsive enhancer sequences. Heme is an essential component in oxygen transport and metabolism in living systems. In non-erythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the first and rate-limiting enzyme in the heme biosynthesis pathway. ALAS1 expression and heme levels are increased in vivo by drugs and other chemical inducers of cytochrome P450 hemoproteins through mechanisms that are poorly understood. In the present studies, a chicken genomic cosmid library was employed to isolate a major portion of the ALAS1 gene. Two drug-responsive enhancer sequences, 176 and 167 base pairs in length, were identified in the 5′-flanking region of the gene in reporter gene assays in the hepatoma cell line LMH. The relative potency of inducers to activate these enhancers corresponds to induction of ALAS1 mRNA levels in LMH cells. Analysis of putative transcription factor binding sites within the enhancers revealed DR5 and DR4 type recognition sequences for nuclear receptors. Drug activation of the enhancer elements was reduced at least 60% after mutagenesis of individual nuclear receptor binding sites and was virtually eliminated following alteration of both recognition sites within the respective elements. Electrophoretic mobility shift assays and transactivation studies demonstrate direct interactions between the nuclear receptor binding sites and the recently described chicken xenobiotic-sensing receptor, (CXR) implicating drug activation mechanisms for ALAS1 similar to those found in inducible cytochrome(s) P450. This is the first report describing direct transcriptional activation of ALAS1 by drugs via drug-responsive enhancer sequences. 5-Aminolevulinate synthase (ALAS) 1The abbreviations used are: ALAS, 5-aminolevulinic acid synthase; ADRES, aminolevulinic acid drug-responsive enhancer sequence; PB, phenobarbital; DR, hexamer half-site direct repeat; LMH, leghorn male hepatoma; NF1, nuclear factor 1; CYP, cytochrome(s) P450; CXR, chicken xenobiotic receptor; PXR, pregnane X receptor; RXR, 9-cis-retinoic acid receptor; PIA, propylisopropylacetamide; PCN, 5-pregnen-3β-ol-20-one-16α-carbonitrile; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; LUC, luciferase; mifepristone, RU-486; clotrimazole, 1-[o-chlorotrityl]-imidazole; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; NR, nuclear receptor. is the first and rate-limiting enzyme in the heme biosynthesis pathway (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar). In eukaryotes, there exist two isoforms of ALAS that are encoded by distinct genes located on different chromosomes. The erythroid form ALAS2 is expressed in hematopoietic tissue and is essential for the generation of functional hemoglobin in erythrocytes, whereas ALAS1 is the drug-responsive, housekeeping form that is expressed ubiquitously, providing heme for CYPs and other hemoproteins. Defects in genes encoding enzymes in the heme biosynthesis pathway are associated with a family of serious disorders known as porphyrias, in which neuropsychiatric symptoms are precipitated by drugs and are associated with increased ALAS1 (2Sassa S. Int. J. Hematol. 2000; 71: 1-17PubMed Google Scholar). Because ALAS is the rate-limiting enzyme in the heme pathway, it has been the focus of numerous studies examining the mechanisms of coordinated heme and apocytochrome synthesis during drug induction of cytochromes P450 (1May B.K. Dogra S.C. Sadlon T.J. Bhasker C.R. Cox T.C. Bottomley S.S. Prog. Nucleic Acids Res. Mol. Biol. 1995; 51: 1-51Crossref PubMed Scopus (123) Google Scholar, 3Jover R. Hoffmann F. Scheffler-Koch V. Lindberg R.L. Eur. J. Biochem. 2000; 267: 7128-7137Crossref PubMed Scopus (57) Google Scholar, 4Lindberg R.L. Porcher C. Grandchamp B. Ledermann B. Burki K. Brandner S. Aguzzi A. Meyer U.A. Nat. Genet. 1996; 12: 195-199Crossref PubMed Scopus (136) Google Scholar, 5Cable E.E. Miller T.G. Isom H.C. Arch. Biochem. Biophys. 2000; 384: 280-295Crossref PubMed Scopus (32) Google Scholar). The mechanism of ALAS transcriptional regulation by xenochemicals has remained enigmatic. Under normal physiological conditions, free heme levels are low and tightly regulated, as toxicity can occur with increased cellular concentrations of unincorporated heme. Following administration of drugs such as phenobarbital (PB) or other prototypical CYP inducers, heme concentrations are elevated in the liver to accommodate the increased levels of heme-dependent enzymes (2Sassa S. Int. J. Hematol. 2000; 71: 1-17PubMed Google Scholar, 6Granick S. Sinclair P. Sassa S. Grieninger G. J. Biol. Chem. 1975; 250: 9215-9225Abstract Full Text PDF PubMed Google Scholar). This is achieved by induction of ALAS1 and assures an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoproteins. After accumulation of ALAS1 mRNA and protein, free heme represses hepatic ALAS1 by a number of negative feedback mechanisms that can inhibit the transport of ALAS1 into the mitochondria, increase heme degradation by inducing heme oxygenase, and decrease ALAS1 mRNA stability directly (5Cable E.E. Miller T.G. Isom H.C. Arch. Biochem. Biophys. 2000; 384: 280-295Crossref PubMed Scopus (32) Google Scholar). In this way, the cell can provide an adequate supply of heme when required while preventing the potentially dangerous accumulation of heme and heme precursors. Recent studies have demonstrated that the induction of CYPs by drugs is mediated by several orphan nuclear receptors (NRs), members of a superfamily of DNA-binding proteins that act as transcription factors. NRs regulate genes as homodimers, heterodimers, or monomers by binding to specific DNA response elements (7Gronemeyer H. Laudet V. Protein Profile. 1995; 2: 1173-1308PubMed Google Scholar, 8Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar, 9Steinmetz A.C. Renaud J.P. Moras D. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 329-359Crossref PubMed Scopus (179) Google Scholar). A number of NRs heterodimerize with retinoid X receptor (RXR), and these dimers then bind to cognate DNA recognition elements, which normally consist of two hexamer half-sites spaced by a variable number of nucleotides, and subsequently modify transcription rates of the targeted genes. In particular, NRs such as chicken xenobiotic receptor (CXR), pregnane X receptor (PXR), and constitutive androstane receptor have been shown to play crucial roles in the induction of members of the drug metabolizing cytochromes from the subfamilies 2H and 2C in chicken (10Handschin C. Podvinec M. Meyer U.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10769-10774Crossref PubMed Scopus (110) Google Scholar, 11Baader M. Gnerre C. Stegeman J.J. Meyer U.A. J. Biol. Chem. 2002; 277: 15647-15653Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) 3A and 2C in humans (12Kliewer 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, 13Lehmann 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 (1388) Google Scholar, 14Blumberg B. Sabbagh Jr., W. Juguilon H. Bolado Jr., J. van Meter C.M. Ong E.S. Evans R.M. Genes Dev. 1998; 12: 3195-3205Crossref PubMed Scopus (819) Google Scholar, 15Bertilsson G. Heidrich J. Svensson K. Asman M. Jendeberg L. Sydow-Backman 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, 16Gerbal-Chaloin S. Daujat M. Pascussi J.M. Pichard-Garcia L. Vilarem M.J. Maurel P. J. Biol. Chem. 2002; 277: 209-217Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), and 2B in rodents (17Paquet Y. Trottier E. Beaudet M.J. Anderson A. J. Biol. Chem. 2000; 275: 38427-38436Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 18Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (654) Google Scholar). In the present work, we describe the characterization of two drug-responsive elements isolated from the 5′-flanking region of the gene encoding ALAS1. These regions respond to a wide range of drugs and are referred to as aminolevulinic acid synthase drug-responsive enhancer sequence (ADRES) elements. Site-directed mutagenesis data demonstrate ADRES-mediated drug response to be conferred by DR4 and DR5 NR recognition sequences. Our data also suggest an important role for additional transcription factors including potential co-activators and/or co-repressors in conferring full drug response. Gel-shift assays and transactivations support the hypothesis that CXR is responsible for the transcriptional activation of the ALAS1 gene by drugs. The observed effects of drugs on ALAS1 mRNA transcription in LMH cells closely mirror the pattern of induction exhibited by the ADRES elements in response to diverse chemical inducers. These studies are the first to demonstrate the direct transcriptional activation of the ALAS1 gene by drugs via well defined drug-responsive enhancer units. Dexamethasone, 2-methyl-1,2-di-3-pyridyl propadone (metyrapone), 5-pregnene-3β-ol-20-one-16α-carbonitrile (PCN), and rifampicin were purchased from Sigma. Propylisopropylacetamide (PIA) was a gift from Dr. Peter Sinclair (Veterans Affairs Hospital, White River Junction, VT). Glutethimide was purchased from Aldrich. Mifepristone (RU-486) was obtained from Roussel-UCLAF. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) was generously provided by U. Schmidt (Institute of Toxicology, Bayer, Wuppertal, Germany). Phenobarbital sodium salt (5-ethyl-5-phenylbarbituric acid sodium salt) was purchased from Fluka. Tissue culture reagents, media, and sera were purchased from Invitrogen. All other reagents and supplies were obtained from standard sources. The pGL3LUC luciferase reporter containing an SV40 promoter was purchased from Promega. The reporter plasmid was modified by the addition of the fragment spanning the SacI to theXhoI restriction endonuclease sites of the multiple cloning site of the pBluescript SK vector (Stratagene) to the pGL3LUC vector, thus greatly enhancing the cloning versatility of the new pLucMCS reporter. The pBLCAT5 chloramphenicol acetyltransferase reporter vector was described previously (19Boshart M. Kluppel M. Schmidt A. Schutz G. Luckow B. Gene (Amst.). 1992; 110: 129-130Crossref PubMed Scopus (230) Google Scholar). Chicken CXR and RXR were cloned into the pSG5 expression vector (Stratagene) as previously reported (10Handschin C. Podvinec M. Meyer U.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10769-10774Crossref PubMed Scopus (110) Google Scholar). The pRSV β-galactosidase vector used for normalization of transfection experiments was kindly provided by Anastasia Kralli (Biozentrum, University of Basel, Basel, Switzerland). A specific probe for the ALAS1 gene was generated via PCR using chicken embryo liver genomic DNA as template and forward primer 5′-cgg gca gca ggt cga gga ga-3′ and reverse primer 5′-cag gaa cgg gca ttt tgt agc a-3′. The probe was32P-radiolabeled using the random primer labeling kit (Roche Molecular Biochemicals) according to instructions from the manufacturer. A genomic cosmid library generated from adult male Leghorn chicken liver was purchased from CLONTECH. The ALAS1 probe was used to identify an individual cosmid clone containing the ALAS1 gene, and at least 15 kb of 5′-flanking region was isolated and confirmed by sequencing. The cosmid containing the ALAS1 gene and flanking region was digested with EcoRI restriction endonuclease, and subfragments of the ∼35 kb of new sequence were cloned into the EcoRI site of the pLucMCS vector. Eight fragments ranging in size from 10 kb to 900 bp in length were cloned. In addition, a 3282-bp SmaI fragment encoding the ALAS1 promoter region and proximal 5′-flanking region was cloned into pLucMCS. The drug-responsive 8-kb EcoRI region was then further subdivided using standard subcloning procedures and restriction endonucleases to isolate the Sau3AI-SmaI 176-bp element and the PvuII-HaeIII 167-bp element. Single copies of the 176- and 167-bp wild type and mutated elements were cloned into pBLCAT5 by excising a 222-bp fragment containing the desired sequences with BamHI and BglII restriction endonucleases and ligating them intoBamHI-linearized pBLCAT5 vector. Multiple repeats of the 176-bp wild type and mutant elements were subcloned by inserting the 222-bp fragment four times in succession into theBamHI-linearized pBLCAT5 vector. Leghorn male hepatoma (LMH) cells were obtained from the American Type Culture Collection and cultivated in 10-cm dishes in Williams E medium supplemented with 10% fetal calf serum, 1% glutamine (2 mm), and 1% penicillin/streptomycin (50 IU/ml). Dishes coated with 0.1% gelatin were used for routine culture of LMH cells to facilitate proper seating of the cells onto the plastic plate surface. For transfections, cells were seeded onto 12-well Falcon 3043 dishes and expanded to 70–80% surface density. Cells were then maintained in serum-free Williams E media for 24 h and transfected using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to the protocol from the manufacturer. Cells were treated with drugs or vehicle for 16 h and harvested. For luciferase assays, lysis was performed with 200 μl/well Passive Lysis Buffer (Promega) and extracts were centrifuged for 1 min to pellet cellular debris. Luciferase assays were performed on supernatants using the luciferase assay kit (Promega) and a Microlite TLX1 luminometer (Dynatech). Relative β-galactosidase activities were determined as described (20Iniguez-Lluhi J.A. Lou D.Y. Yamamoto K.R. J. Biol. Chem. 1997; 272: 4149-4156Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). For CAT assays, cells were lysed with 600 μl/well CAT lysis buffer and extracts were centrifuged for 1 min to pellet cellular debris. Assays were performed using a CAT enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals) according to the protocol from the manufacturer. Mutations in the putative NR binding sites were introduced into the ADRES elements by PCR using standard overlap techniques. Briefly, subfragments were amplified with overlapping primers carrying the desired mutations and vector primers. These subfragments were then combined and used as template in a second PCR using vector primers to amplify the full-length mutated fragment, which was subsequently digested with appropriate enzymes and cloned into pLucMCS. The forward vector primer was the RV primer 3, and the reverse vector primer was the GL primer 2 within the pGL3 luciferase vector (Promega). All mutations are shown in bold. DR4-1 double mutation constructs were generated with 5′-gga gga act cga cac gat acc aac ata gca at-3′ forward and 5′-cta tgt tgg tat cgt gtc gag ttc ctc cct g-3′ reverse primers. DR5 double mutants were amplified with 5′-gaa ttcgcc aac tgc agc cag gct gtc c-3′ forward and 5′-cag cct ggc tgc agt tgg cga att ctc ctc-3′ reverse primers. DR4-2 double mutants were generated with 5′-ccc cacgca gcc cca ccg ctc ggc tga act cgt g-3′ forward and 5′-gtg ggg ctg cgt ggg gca gca gag aaa gtt cag g-3′ reverse primers. DR4-3 double mutants were amplified using a 5′-gaa ttc aca gcc atg gtg aag atc agc-3′ forward primer and a 5′-cca tgg ctg tga att cag tca cga g-3′ reverse primer. Avian NF1 consensus sequence was generated using 5′-gtt taa agc tgg cac tgt ccc aaa-3′ and 5′-ctt tgg cac agt gcc agc ttt aaa c-3′ forward and reverse primers (21Rupp R.A. Kruse U. Multhaup G. Gobel U. Beyreuther K. Sippel A.E. Nucleic Acids Res. 1990; 18: 2607-2616Crossref PubMed Scopus (150) Google Scholar). Following PCR overlap, the products were digested with BglII and eitherEcoRI or NotI restriction endonucleases and cloned into pLucMCS. All constructs were verified by sequencing. LMH cells were plated onto 12-well plates, expanded to 70–80% surface density, and incubated in serum-free media for 24 h. Cells were then exposed to either drug or vehicle, and RNA was isolated with TRIzol reagent (Invitrogen) according to the protocol from the manufacturer. One μg of total RNA was reverse transcribed with the Moloney murine leukemia virus reverse transcriptase kit (Roche Molecular Biochemicals). PCR was performed using the Taqman PCR core reagent kit (PerkinElmer Applied Biosystems) and transcript levels quantitated with an ABI Prism 7700 sequence detection system (PerkinElmer Applied Biosystems). Relative transcript levels were determined using the relative quantitation method measuring the ΔΔCt. The following primers and probes were used in these reactions: ALAS1 (probe, 5′-ttc cgc cat aac gac gtc aac cat ctt-3′; forward primer, 5′-gca ggg tgc caa aac aca t-3′; reverse primer, 5′-tcg atg gat cag act tct tca aca-3′) and glyceraldehyde-3-phosphate dehydrogenase (probe, 5′-tgg cgt gcc cat tga tca caa ttt-3′; forward primer, 5′-ggt cac gct cct gga aga tag t-3′; reverse primer, 5′-ggg cac tgt caa ggc tga ga-3′). Transcript levels were measured in separate tubes, and glyceraldehyde-3-phosphate dehydrogenase values were used for normalization of ALAS1 values. Chicken CXR and RXR proteins were expressed using the TNT T7 quick coupled translation system (Promega) according to the protocol from the manufacturer. Ends were filled in with the Klenow fragment of Escherichia coli DNA polymerase I in the presence of radiolabeled [α-32P]ATP and purified over a Biospin 6 chromatography column. A volume of labeled oligonucleotide corresponding to 100,000 cpm was used for each reaction in 10 mm Tris-HCl, pH 8.0, 40 mm KCl, 0.05% Nonidet P40, 6% glycerol (v/v), 1 mm DTT containing 0.2 μg of poly(dI-dC) and 2.5 μl in vitro synthesized proteins as described previously (10Handschin C. Podvinec M. Meyer U.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10769-10774Crossref PubMed Scopus (110) Google Scholar, 12Kliewer 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). To test for supershifts, 0.5 μl of monoclonal anti-mouse-RXR rabbit antibody (kindly provided by P. Chambon, Université Louis Pasteur, Illkirch, France) were added to the reaction mix. This antibody has been previously tested for interactions with chicken RXR in Western blots (data not shown). The reaction mix was incubated for 20 min at room temperature and electrophoresed on a 6% polyacrylamide gel in 0.5× Tris borate/EDTA buffer followed by autoradiography. Experiments to determine the ability of the nuclear receptors CXR, human PXR, and mouse PXR to mediate induction of ALAS-1 were done in COS-1 monkey kidney cells according to methods previously described (10Handschin C. Podvinec M. Meyer U.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10769-10774Crossref PubMed Scopus (110) Google Scholar). PXR clones were the generous gift of S. Kliewer (University of Texas Southwestern Medical Center, Dallas, TX). Briefly, cells were expanded for 3 days on 10-cm Falcon 3003 dishes in Dulbecco's modified Eagle's medium/F-12 medium (Invitrogen) without phenol red supplemented with 10% charcoal-stripped fetal bovine serum. Cells were then plated onto six-well dishes and expanded overnight to ∼30% density. Cells were then rinsed with PBS and maintained for transfection in OptiMEM (Invitrogen) without further additions. Transfection of 1 μg of reporter plus 800 ng of pSV β-galactosidase construct and 50 ng of CXR expression vector was performed using 3 ml/well LipofectAMINE, according to the protocol from the manufacturer. After a 24-h incubation, cells were rinsed with PBS and Dulbecco's modified Eagle's medium/F-12 containing 10% delipidated/charcoal-stripped fetal bovine serum containing either drugs or vehicle control was added. After a 16-h induction, cells were rinsed with PBS, lysed in 600 μl of CAT lysis buffer, and assayed for CAT enzyme using the CAT enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals). CAT levels were then normalized against β-galactosidase levels to compensate for variations in transfection efficiency. A cosmid clone containing an insert ∼35 kb in length, spanning the chicken ALAS1 gene and 15 kb of the 5′-flanking region, was isolated and its sequence analyzed. Three major subclones were generated from the region upstream of the transcriptional start site, including a 3282-bp SmaI fragment and 5056- and 7973-bpEcoRI segments (Fig.1 A). The SmaI clone extends from bp −167 to bp −3449, whereas the EcoRI subfragments span the regions from bp −2347 to bp −7402 and bp −7403 to bp −15376, respectively. These subfragments were cloned into the pLucMCS modified luciferase vector containing an SV40 promoter as described under "Experimental Procedures." Drug inducibility was measured in transiently transfected LMH cells treated with 600 μm PB and compared with control values. The results revealed the 7973-bp subfragment to be highly inducible with PB, displaying a 32-fold increase in transcriptional activation relative to control values. In comparison, the 5056- and 3282-bp subfragments exhibited virtually no transcriptional activation in response to drug treatment (Fig. 1 C). The 7973-bp subfragment (−15376/−7403) was chosen for further analysis and was divided into numerous subclones in the pLucMCS reporter vector, resulting in the isolation of 176-bp Sau3AI-SmaI and 167-bpPvuII-HaeIII elements (Fig. 1, A andB). These sequences routinely exhibit 25–60-fold induction over control values in reporter gene assays when exposed to PB in LMH cells (Fig. 1 C). All other portions of the 7973-bp fragment were also subcloned but displayed no drug response when tested in LMH cells (data not shown). Because the 176- and 167-bp fragments retain high drug response regardless of orientation or distance from the promoter (data not shown), they are referred to as ADRES enhancers. Recent discoveries have implicated NRs in drug-mediated enzyme induction (10Handschin C. Podvinec M. Meyer U.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10769-10774Crossref PubMed Scopus (110) Google Scholar, 13Lehmann 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 (1388) Google Scholar, 18Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (654) Google Scholar). For this reason, we scanned the responsive elements for potential nuclear receptor response sites using a computer algorithm based on a weighted nucleotide distribution matrix compiled from published functional hexamer half-sites (22Podvinec M. Kaufmann M.R. Handschin C. Meyer U.A. Mol. Endocrinol. 2002; 16: 1269-1279Crossref PubMed Scopus (152) Google Scholar). Two potential binding sites for orphan NRs were identified in each ADRES element, having two direct repeats with 4 nucleotide (DR4) and 5 nucleotide (DR5) separations between half-sites in the 176-bp sequence and two direct repeats with 4 nucleotide (DR4) separations between half-sites in the 167-bp sequence (Fig. 1 B). For clarity, the three DR4 binding sites are labeled according to their occurrence in the gene, with the furthest upstream from the transcription start site called DR4-1 and the closest to the start site DR4-3. The putative DR4-1 is defined by one perfect half-site (AGGTCA) and one imperfect half-site (AGTTGA) at −14186/−14181 and −14176/−14171 respectively, whereas the DR5 site is characterized by an imperfect upstream half-site (AGCTGA) and a perfect downstream half-site (AGGTCA) at −14251/−14246 and −14240/−14235. In the 167-bp sequence, DR4-2 consists of one imperfect upstream half-site (GGATGA) and one perfect downstream half-site (AGTTCA) at −13563/−13558 and −13553/−13548 and DR4-3 has two imperfect half-sites (GTGTCA and GGGGCA) at −13526/−13521 and −13516/−13511. It is interesting to note that the 176-bp ADRES also contains a putative binding site for nuclear factor 1 which overlaps the DR5, spanning bp −14255 to bp −14242, whereas the 167-bp ADRES does not. We next wanted to compare ADRES-mediated ALAS1 induction levels from reporter gene assays with stimulation of transcription in a physiological system. Therefore, ALAS1 mRNA levels were quantified in LMH cells cultured in serum-free medium and 16 h of exposure to a variety of chemical inducers and compared with the induction pattern observed with the same compounds in transient transfections of the ADRES (Fig. 2). The compounds examined include PB (600 μm) and the PB-like inducers PIA (250 μm), glutethimide (500 μm), and the potent mouse CYP 2B inducer TCPOBOP (10 μm). In addition, the common CYP3A inducers dexamethasone (50 μm), metyrapone (400 μm), and 10 μm mifepristone (RU-486) were employed for comparison. We were also interested in the effects of 10 μm PCN and rifampicin (100 μm) because of their species-specific effects on PXR activation and CYP3A induction. Messenger RNA was reverse transcribed, and levels of ALAS1 cDNA were quantified using the Taqman real-time PCR quantification system as described under "Experimental Procedures." PB was a strong inducer of ALAS1 in LMH cells, increasing RNA levels an average of 16-fold relative to basal transcript levels (Fig. 2). This value was chosen to represent 100% induction, against which all other values are compared. The general inducers PIA and glutethimide, as well as the 3A-specific inducer metyrapone, exhibited the strongest effects upon the ADRES elements, stimulating transcription in excess of levels obtained from PB treatment. In comparison, dexamethasone, PCN, RU-486, and rifampicin had minor or no effects on either mRNA levels or ADRES activation. Moreover, the mouse-specific compound TCPOBOP elicited no response in either mRNA transcription or stimulation of the ADRES in reporter assays. When comparing the induction profiles of the two ADRES elements to each other, very few differences are in evidence. The 167-bp element responds to PB with twice the activation when compared with the 176-bp element. Additionally, the 176-bp element has slightly more affinity for glutethimide than metyrapone, whereas the 167-bp element exhibits a stronger response to metyrapone than glutethimide. These experiments indicate a high degree of similarity in the relative activation of the ADRES elements in reporter gene assays to each other and to mRNA transcript levels from chemically induced LMH cells. Site-specific mutagenesis was used to examine the roles of specific nucleotides within the putative DR5 and DR4 recognition sequences in conferring drug response to the ADRES elements (Fig.3). Mutant constructs of the DR4 and DR5 core recognition sites destroying the putative NR binding sites were generated as described under "Experimental Procedures." Briefly, primers were used in conjunction with PCR to convert the 5′ and 3′ half-sites of the DR5 to EcoRI and PstI restriction endonuclease sites, respectively. Similarly, the DR4-3 half-sites were converted to EcoRI and NcoI restriction endonuclease sites. Data from a nucleotide distribution matrix for half-sites developed by M. Podvinec in this laboratory was applied to ascertain that the mutated half-sites least resemble functional half-sites. DR4-1 half-sites were obliterated by converting AGGTCA and AGTTGA half-sites to unconserved ACTCGA and ATACCA bases, respectively. Similarly, DR4-2 half-sites were both converted from GGATGA and AGTTCA nucleotides to CCCCAC bases. Primers were used to generate constructs mutated at each individual and both NR binding sites within both of the ADRES elements as shown in Fig. 3. The modified enhancers were examined for response to 600 μm PB in luciferase reporter gene assays, and the results are presented in Fig. 3. These findings indicate that both the DR5 and DR4 recognition sites in the 176-bp ADRES and b
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