De-orphanization of Cytochrome P450 2R1
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m307028200
ISSN1083-351X
AutoresJeffrey B. Cheng, Daniel Motola, David J. Mangelsdorf, David W. Russell,
Tópico(s)Diet and metabolism studies
ResumoThe conversion of vitamin D into an active ligand for the vitamin D receptor requires 25-hydroxylation in the liver and 1α-hydroxylation in the kidney. Mitochondrial and microsomal vitamin D 25-hydroxylase enzymes catalyze the first reaction. The mitochondrial activity is associated with sterol 27-hydroxylase, a cytochrome P450 (CYP27A1); however, the identity of the microsomal enzyme has remained elusive. A cDNA library prepared from hepatic mRNA of sterol 27-hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionarily conserved microsomal cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. Expression of CYP2R1 in cells led to the transcriptional activation of the vitamin D receptor when either vitamin D2 or D3 was added to the medium. Thin layer chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was 25-hydroxyvitamin D3. Co-expression of CYP2R1 with vitamin D 1α-hydroxylase (CYP27B1) elicited additive activation of vitamin D3, whereas co-expression with vitamin D 24-hydroxylase (CYP24A1) caused inactivation. CYP2R1 mRNA is abundant in the liver and testis, and present at lower levels in other tissues. The data suggest that CYP2R1 is a strong candidate for the microsomal vitamin D 25-hydroxylase. The conversion of vitamin D into an active ligand for the vitamin D receptor requires 25-hydroxylation in the liver and 1α-hydroxylation in the kidney. Mitochondrial and microsomal vitamin D 25-hydroxylase enzymes catalyze the first reaction. The mitochondrial activity is associated with sterol 27-hydroxylase, a cytochrome P450 (CYP27A1); however, the identity of the microsomal enzyme has remained elusive. A cDNA library prepared from hepatic mRNA of sterol 27-hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionarily conserved microsomal cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity. Expression of CYP2R1 in cells led to the transcriptional activation of the vitamin D receptor when either vitamin D2 or D3 was added to the medium. Thin layer chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was 25-hydroxyvitamin D3. Co-expression of CYP2R1 with vitamin D 1α-hydroxylase (CYP27B1) elicited additive activation of vitamin D3, whereas co-expression with vitamin D 24-hydroxylase (CYP24A1) caused inactivation. CYP2R1 mRNA is abundant in the liver and testis, and present at lower levels in other tissues. The data suggest that CYP2R1 is a strong candidate for the microsomal vitamin D 25-hydroxylase. Vitamin D regulates calcium and phosphate metabolism by activating the vitamin D receptor, a transcription factor and member of the nuclear receptor family. In the 1920s, McCollum, Mellanby, and Pappenheimer (see Ref. 1Simoni R.D. Hill R.L. Vaughan M. J. Biol. Chem. 2002; 277: 9623Abstract Full Text Full Text PDF Google Scholar) showed that deficiency of vitamin D caused rickets in experimental animals. Subsequently, the structure of vitamin D and its origins from plant and animal steroids were determined, and the roles of the vitamin in the mobilization of minerals from the diet and in bone were defined. The liver was shown to be required for the activation of vitamin D by 25-hydroxylation (2Ponchon G. DeLuca H.F. J. Clin. Invest. 1969; 48: 1273-1279Crossref PubMed Scopus (203) Google Scholar, 3Ponchon G. Kennan A.L. DeLuca H.F. J. Clin. Invest. 1969; 48: 2032-2037Crossref PubMed Scopus (242) Google Scholar). Although 25-hydroxyvitamin D was more active than vitamin D in many bioassays (4Blunt J.W. DeLuca H.F. Schnoes H.K. Biochemistry. 1968; 7: 3317-3322Crossref PubMed Scopus (405) Google Scholar), the most potent hormone was 1α,25-dihydroxyvitamin D (5Haussler M.R. Myrtle J.F. Norman A.W. J. Biol. Chem. 1968; 243: 4055-4064Abstract Full Text PDF PubMed Google Scholar, 6Holick M.F. Schnoes H.K. DeLuca H.F. Suda T. Cousins R.J. Biochemistry. 1971; 10: 2799-2804Crossref PubMed Scopus (372) Google Scholar), which was synthesized from 25-hydroxyvitamin D in the kidney (7Fraser D.R. Kodicek E. Nature. 1970; 228: 764-766Crossref PubMed Scopus (924) Google Scholar). The molecular mechanism of vitamin D action was manifest with the identification (8Haussler M.R. Norman A.W. Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 155-162Crossref PubMed Scopus (175) Google Scholar) and cDNA cloning (9McDonnell D.P. Mangelsdorf D.J. Pike J.W. Haussler M.R. O'Malley B.W. Science. 1987; 235: 1214-1217Crossref PubMed Scopus (420) Google Scholar) of the vitamin D receptor. Hydroxylation reactions catalyzed by cytochrome P450s (CYP) 1The abbreviations used are: CYP, cytochrome P450; Adx, adrenodoxin; h, human; m, mouse; GAL4, galactose 4; VDR, vitamin D receptor; VDRE, vitamin D response element; TK, thymidine kinase; HEK, human embryonic kidney; C T, threshold value; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus.1The abbreviations used are: CYP, cytochrome P450; Adx, adrenodoxin; h, human; m, mouse; GAL4, galactose 4; VDR, vitamin D receptor; VDRE, vitamin D response element; TK, thymidine kinase; HEK, human embryonic kidney; C T, threshold value; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus. activate and inactivate vitamin D as a ligand for the receptor. 25-Hydroxylation is performed in the liver by two different enzymes, one located in the mitochondria (10Bjorkhem I. Holmberg I. J. Biol. Chem. 1978; 253: 842-849Abstract Full Text PDF PubMed Google Scholar), identified as the CYP27A1 sterol 27-hydroxylase (11Guo Y.-D. Strugnell S.A. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (141) Google Scholar, 12Dahlback H. Wikvall K. Biochem. J. 1988; 252: 207-213Crossref PubMed Scopus (53) Google Scholar, 13Masumoto O. Ohyama Y. Okuda K. J. Biol. Chem. 1988; 263: 256-260Abstract Full Text PDF Google Scholar, 14Su P. Rennert H. Shayiq R.M. Yamamoto R. Zheng Y. Addya S. Strauss III, J.F. Avadhani N.G. DNA Cell Biol. 1990; 9: 657-665Crossref PubMed Scopus (107) Google Scholar, 15Usui E. Noshiro M. Okuda K. FEBS Lett. 1990; 262: 135-138Crossref PubMed Scopus (110) Google Scholar), and a second in microsomes (16Bhattacharyya M.H. DeLuca H.F. Arch. Biochem. Biophys. 1974; 160: 58-62Crossref PubMed Scopus (127) Google Scholar, 17Madhok T.C. DeLuca H.F. Biochem. J. 1979; 184: 491-499Crossref PubMed Scopus (85) Google Scholar), which has not been identified in most species. A second mitochondrial P450 (CYP27B1), for which an encoding cDNA was isolated by expression cloning (18Takeyama K. Kitanaka S. Sato T. Kobori M. Yanagisawa J. Kato S. Science. 1997; 277: 1827-1830Crossref PubMed Scopus (454) Google Scholar), catalyzes 1α-hydroxylation of 25-hydroxyvitamin D in the kidney, and a third mitochondrial P450, vitamin D 24-hydroxylase (CYP24A1), inactivates the vitamin in this tissue (19Ohyama Y. Noshiro M. Okuda K. FEBS Lett. 1991; 278: 195-198Crossref PubMed Scopus (218) Google Scholar, 20Chen K.S. Prahl J.M. DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4543-4547Crossref PubMed Scopus (143) Google Scholar, 21Itoh S. Yoshimura T. Iemura O. Yamada E. Tsujikawa K. Kohama Y. Mimura T. Biochim. Biophys. Acta. 1995; 1264: 26-28Crossref PubMed Scopus (27) Google Scholar). Together, these enzymes regulate the systemic and local levels of vitamin D through complex feedback mechanisms mediated in part by the vitamin D receptor (22Jones G. Strugnell S.A. DeLuca H.F. Physiol. Rev. 1998; 78: 1193-1231Crossref PubMed Scopus (1022) Google Scholar). The existence and physiological importance of the two hepatic vitamin D 25-hydroxylase enzymes are demonstrated in humans (23Cali J.J. Hsieh C. Francke U. Russell D.W. J. Biol. Chem. 1991; 266: 7779-7783Abstract Full Text PDF PubMed Google Scholar, 24Bjorkhem I. Boberg K.M. Leitersdorf E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2001: 2961-2988Google Scholar) and mice (25Rosen H. Reshef A. Maeda N. Lippoldt A. Shpizen S. Triger L. Eggertsen G. Bjorkhem I. Leitersdorf E. J. Biol. Chem. 1998; 273: 14805-14812Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 26Repa J.J. Lund E.G. Horton J.D. Leitersdorf E. Russell D.W. Dietschy J.M. Turley S.D. J. Biol. Chem. 2000; 275 (639): 685-692PubMed Google Scholar) by mutations in the mitochondrial CYP27A1 vitamin D 25-hydroxylase gene. Loss of this enzyme has profound effects on cholesterol metabolism, as CYP27A1 catalyzes an essential reaction in bile acid synthesis. In contrast, vitamin D metabolism is normal in individuals and mice with no functional CYP27A1. These findings indicate that the microsomal vitamin D 25-hydroxylase can compensate for loss of the mitochondrial activity. In the current study, a cDNA library made from hepatic mRNA of mice deficient in the gene encoding the mitochondrial CYP27A1 enzyme was screened using a vitamin D receptor-based, ligand activation assay (18Takeyama K. Kitanaka S. Sato T. Kobori M. Yanagisawa J. Kato S. Science. 1997; 277: 1827-1830Crossref PubMed Scopus (454) Google Scholar). A single cDNA specifying a microsomal P450 enzyme termed CYP2R1 with previously unknown substrate specificity was identified. The biochemical properties and tissue distribution of CYP2R1 are consistent with this enzyme being the microsomal vitamin D 25-hydroxylase. Expression Plasmids—A mouse adrenodoxin cDNA (mAdx, nucleotides 41–772 of GenBank™/EBI Data Bank accession no. L29123) was amplified by the polymerase chain reaction (PCR) from random hexamer-primed mouse hepatic cDNAs using the following oligonucleotide primers: Forward (5′-GCCTATGTCGACTCAGCACTGCGCAGGACTCC-3′) and Reverse (5′-GCGGGATCCGACAGCACAGCTACTCACAC-3′). The amplified DNA was digested with the enzymes SalI and BamHI and ligated into pCMV6 (GenBank™/EBI Data Bank accession no. AF239250). The adrenodoxin expression vector encoded the expected electron transport enzyme activity in transfected cells, as judged by the ability of the protein to stimulate the vitamin D 25-hydroxylase activity of sterol 27-hydroxylase. A mouse vitamin D 24-hydroxylase cDNA (mCYP24A1, nucleotides 281–2200 of GenBank™/EBI Data Bank accession no. D89669) was amplified by PCR from random hexamer-primed kidney cDNA. The RNA for this cDNA synthesis reaction was isolated from a mouse injected intraperitoneally with 36 pmol of 1α,25-dihydroxyvitamin D/g of body weight for 5 h prior to tissue harvest. Oligonucleotide primers for the amplification reaction were: Forward (5′-GCCTATGTCGACACTTCAGAACCCAACAGCAC-3′) and Reverse (5′-ATTATGCGGCCGCAGTGACATCAGGCTCTTGAG-3′). The amplified DNA was digested with the restriction enzymes SalI and NotI and ligated into the pCMV6 vector. Human cytochrome P450 subfamily 3A, polypeptide 4 (hCYP3A4, nucleotides 98–1617 of GenBank™/EBI Data Bank accession no. NM_017460), and cytochrome P450 2R1 (hCYP2R1) cDNAs were amplified by PCR from liver QUICK-clone cDNA (Clontech). Oligonucleotide primers for the hCYP3A4 cDNA were: Forward (5′-GCCTATGTCGACAGTGATGGCTCTCATCCCAG-3′) and Reverse (5′-ATTATGCGGCCGCTTCAGGCTCCACTTACGGTG-3′). The amplified DNA was digested with the restriction enzymes SalI and NotI and then ligated into the pCMV6-SPORT vector (Invitrogen). The CYP3A4 expression vector encoded testosterone 6β-hydroxylase enzyme activity in human embryonic kidney (HEK) 293 cells, as judged by thin layer chromatography. Oligonucleotide primers for the hCYP2R1 cDNA were: Forward (5′-GCCTATGTCGACTGTGGAGTTCGCACCTCCAG-3′) and Reverse (5′-ATTATGCGGCCGCAACCAAGTTCAGGGATAAGG-3′). The amplified DNA was digested with the enzymes SalI and NotI and then ligated into the pCMV6 vector. A DNA fragment encompassing the 5′-flanking region of the mouse osteopontin gene (nucleotides 1–862 of GenBank™/EBI Data Bank accession no. D14816) was amplified via PCR from mixed strain C57Bl/6J;129S6/SvEv mouse genomic DNA. Oligonucleotide primers were: Forward (5′-ATTATGCGGCCGCTTCAGGCTCCACTTACGGTG-3′) and Reverse (5′-CCGCTCGAGCTTGGCTGGTTTCCTCCGAGAATG-3′). The amplified DNA product was digested with the restriction enzymes HindIII and XhoI and then ligated into the pTK-LUC reporter plasmid (27Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (916) Google Scholar). A mouse 25-hydroxyvitamin D3 1α-hydroxylase cDNA (mCYP27B1, GenBank™/EBI Data Bank accession no. AB006034) was a kind gift of Professor Shigeaki Kato (University of Tokyo, Tokyo, Japan). The mouse sterol 27-hydroxylase cDNA (mCYP27A1) used in these studies was generated previously (28Lund E.G. Kerr T.A. Sakai J. Li W.-P. Russell D.W. J. Biol. Chem. 1998; 273: 34316-34348Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Expression Cloning—A cDNA library was made from 3 μg of poly(A)+ hepatic RNA isolated from mice deficient in sterol 27-hydroxylase (25Rosen H. Reshef A. Maeda N. Lippoldt A. Shpizen S. Triger L. Eggertsen G. Bjorkhem I. Leitersdorf E. J. Biol. Chem. 1998; 273: 14805-14812Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) using the SUPERSCRIPT plasmid system for cDNA synthesis (Invitrogen). cDNA synthesis was initiated by NotI oligo(dT) primer-adapters; the cDNA products were ligated to SalI adapters, digested with the restriction enzyme NotI, size-fractionated by gel filtration chromatography, and then ligated into NotI- and SalI-digested pCMV-SPORT 6 vector. Nucleic acids in the ligation mixture were precipitated with ethanol and resuspended in 5 μl of deionized, distilled H2O. Escherichia coli Electromax DH10B cells (Invitrogen) were transformed with 1 μl of the resuspended DNAs. The transformation mixture was diluted into 500 ml of Luria-Bertani medium supplemented with ampicillin (50 μg/ml). An aliquot (1.25 ml) of the diluted culture (containing ∼100 transformants) was added to each well of a 96-well block (Qiagen) and incubated with shaking for 20–24hat37 °C. Plasmid DNA was isolated from the bacterial cultures using the QIAprep96 Turbo Miniprep kit (Qiagen). HEK 293 cells (American Type Culture Collection CRL no. 1573) were transfected with pools of ∼100 cDNAs using the FuGENE™ 6 reagent (Roche Applied Science). The cells were maintained in mono-layer culture at 37 °C in an atmosphere of 8.8% CO2, 91.2% air in low glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate. On day 0 of an expression cloning experiment, 25,000 cells/well were plated in 96-well plates (Corning Costar, no. 3595) in a volume of 100 μl of high glucose DMEM supplemented with 10% (v/v) dextran-charcoal-stripped fetal calf serum. On day 1, cells were transfected with a mixture containing 0.45-μl FuGENE™ 6 and 150 ng of plasmid DNA comprising 20 ng of pCMX-β-galactosidase (29Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1216) Google Scholar), 20 ng of pTK-MH100X4-LUC (27Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (916) Google Scholar), 20 ng of pCMX/GAL4-VDR (see below), 10 ng of pVA1 (30Schneider R.J. Shenk T. Annu. Rev. Biochem. 1987; 56: 317-332Crossref PubMed Scopus (136) Google Scholar), 5 ng of pCMV6/mAdx, and 75 ng of cDNA pool. Cells were returned to the incubator for 8–10 h, and then vehicle (ethanol) or 1α-hydroxyvitamin D3 (Sigma) substrate was added to a final concentration of 10 nm. After an additional 16–20 h of incubation, cells were lysed by the addition of 100 μl/well Lysis Buffer (2% (v/v) CHAPS, 0.6% (w/v) phosphatidylcholine, 2.5 mm Tris phosphate, pH 7.8, 0.6% (w/v) bovine serum albumin, 15% (v/v) glycerol, 4 mm EGTA, 8 mm MgCl2, 1 mm dl-dithiothreitol, and 1% (w/v) Pefabloc SC (Centerchem)). The 96-well plates were shaken for 5 min at 23 °C, and then 20 μl of cell lysate from each well were transferred to a fresh 96-well assay plate (Corning Costar no. 3922). Thereafter, 100 μl of Luciferase Assay Buffer (4 mm ATP (Roche Applied Science), 20 mm MgCl2, and 0.1 m KPO4) was added to each well, and luciferase activity was measured in a Dynex MLX microtiter plate luminometer running the Revelation MLX version 4.25 software after the addition of 100 μl of Luciferin Solution (1 mm d-luciferin (Molecular Probes) in 0.1 m KPO4). β-Galactosidase enzyme activity was determined in cell lysates using a Dynex Opsys MR machine running the Revelation Quicklink software; an aliquot (40 μl) of cell lysate was transferred to each well of a fresh 96-well plate (Corning Costar no. 3598), followed by the addition of 125-μl of β-Galactosidase Assay Buffer (60 mm Na2HPO4, 40 mm NaH2PO4, 8 mm KCl, 8 mm MgCl2, 0.4% (w/v) 2-nitrophenyl-β-d-galactopyranoside (Roche Applied Science), and 3 mm β-mercaptoethanol), and an incubation of 10 min at 37 °C prior to measuring the absorbance at A 405. Relative luciferase units were calculated by dividing the measured luciferase activities by the absorbance values per min determined in the β-galactosidase assay. All values are expressed as means ± S.E. of measurement derived from triplicate wells. Vitamin D Receptor Activation Assay and Ligands—The GAL4-VDR/GAL4-luciferase receptor-reporter system was composed of the pTK-MH100X4-LUC plasmid (27Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (916) Google Scholar), and pCMX/GAL4-VDR, which specifies a fusion protein consisting of amino acids 1–147 of the Saccharomyces cerevisiae galactose 4 (GAL4) transcription factor (encoded by nucleotides 2901–3353 of GenBank™/EBI Data Bank accession no. X85976) and amino acids 90–427 of the human vitamin D receptor (hVDR, encoded by nucleotides 383–1397 of GenBank™/EBI Data Bank accession no. 44507882). The VDR/vitamin D response element (VDRE)-luciferase system used here consisted of the SPPX3-TK-LUC plasmid (31Choi M. Yamamoto K.R. Itoh T. Makishima M. Mangelsdorf D.J. Moras D. DeLuca H.F. Yamada S. Chem. Biol. 2003; 10: 261-270Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and a plasmid expressing the full-length mouse vitamin D receptor (pCMX/mVDR, nucleotides 109–1377 of GenBank™/EBI Data Bank accession no. D31969) (32Makishima M. Lu T.T. Xie W. Whitfield G.K. Domoto H. Evans R.M. Haussler M.R. Mangelsdorf D.J. Science. 2002; 296: 1313-1316Crossref PubMed Scopus (958) Google Scholar). The VDR/osteopontin-luciferase system was composed of the osteopontin-LUC plasmid (0.862-kb DNA fragment encompassing the mouse osteopontin gene promoter ligated into the pTK-LUC vector (Ref. 27Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (916) Google Scholar)), and the pCMX/mVDR DNA described above. Transfection experiments with the GAL4-VDR/GAL4-luciferase receptor-reporter systems were carried out as described under "Expression Cloning." For the VDR/VDRE-luciferase and VDR/osteopontin-luciferase systems, the plasmid DNA mixtures (150 ng of DNA total) contained 5 ng of pCMX/mVDR, 20 ng of pSPPX3-TK-LUC or pOsteopontin-LUC, 20 ng of pCMX-β-galactosidase, 10 ng of pVA1, and 95 ng of CYP expression vector or pCMV6 plasmid DNA. Secosteroids used in the experiments reported here (vitamin D2, vitamin D3, 1α-hydroxyvitamin D3, 25-hydroxyvitamin D3, and 1α,25-dihydroxyvitamin D3) were obtained from Sigma and added to the culture medium in ethanol (1 μl/ml of medium). Stock solutions of these compounds ranged in concentration from 10-2 to 5 × 10-7m and were stored at -20 °C. Thin Layer Chromatography—HEK 293 cells were plated at a density of 4 × 105 cells/60-mm dish in 2.5 ml of high glucose DMEM supplemented with 10% (v/v) dextran-charcoal-stripped fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate. On day 2, cells were transfected with FuGENE™ 6 reagent and a mixture of plasmid DNAs (3.5 μg total) containing 0.5 μg of pVA1, and either 3 μg of pCMV6, 3 μg of pCMV6-h2R1, 1.5 μg of pCMV6, and 1.5 μg of pCMV6-mAdx, or 1.5 μg of pCMV6-mCYP27A1 and 1.5 μg of pCMV6-mAdx. After 18 h, the transfection medium was removed from the dish and replaced with 2 ml of fresh medium supplemented as above and containing 4.6 × 10-7m [4-14C]vitamin D3 (Amersham Biosciences). For cells transfected with the pCMV6-mCYP27A1 plasmid, the medium was brought to a final concentration of 10-6m vitamin D3 by the addition of unlabeled secosteroid. Cells and medium were harvested 96 h later, and vitamin D metabolites were extracted with 8 ml of chloroform:methanol (2:1, v/v), and then dried under a stream of N2. The extracted metabolites were resuspended in 50 μl of acetone and resolved by thin layer chromatography on pre-scored LD5DF silica gel 150-Å plates (Whatman). Solvent systems of cyclohexane:ethyl acetate (3:2, v/v), chloroform:ethyl acetate (3:1, v/v), and 2,2,4-trimethylpentane:ethyl acetate:acetic acid (50:50:17, v/v/v) were used. Radiolabeled vitamin D metabolites were detected by phosphorimage analysis on a BAS1000 machine (Fuji Medical Systems, Tokyo, Japan). Aliquots (5 μl) of 10-2m stock solutions of unlabeled standards (vitamin D3, 25-hydroxyvitamin D3, 1α-hydroxyvitamin D3, and 1α,25-dihydroxyvitamin D3) dissolved in ethanol were chromatographed in adjacent lanes on the plates and visualized by iodine staining (33Touchstone J.C. Practice of Thin Layer Chromotography. 3rd ed. John Wiley & Sons, New York1992Google Scholar). Radioimmunoassays—The γB 25 hydroxyvitamin D radioimmunoassay kit (Alpco Diagnostics) was used for the quantitative determination of 25-hydroxyvitamin D in medium from HEK 293 cells cultured in the 96-well plate format described above. Cells were transfected via the FuGENE™ 6 reagent with 150 ng of total plasmid DNA comprising 5 ng of pCMX/mVDR, 20 ng of SPPX3-TK-LUC, 20 ng of pCMX-β-galactosidase, 10 ng of pVA1, and either 95 ng of pCMV6, pCMV6-h2R1, or pCMV6 SPORT-m2R1, or 45 ng of pCMV6-mCYP27A1 and 45 ng of pCMV6-mAdx. After 10 h, ethanol vehicle, or vitamin D3 at final concentrations of 0.5 or 1 μm, was added to the medium. Radioimmunoassays were performed 44 h later on 50-μl aliquots of pooled medium from triplicate wells following directions provided by the manufacturer. The correlation coefficient of accuracy for the radioimmunoassay kit was r = 0.95. Sensitivity was >3 nmol/liter. Luciferase and β-galactosidase assays also were performed on cell lysates to ensure that transfected plasmids were expressed. Real Time PCR—Total RNA was prepared from mouse tissues using RNA STAT-60 (Tel-Test, Friendswood, TX). Aliquots (100 μg) of total RNA were treated with deoxyribonuclease I (DNA-free; Ambion). cDNA synthesis was initiated from 2 μg of deoxyribonuclease I-treated RNA using random hexamer primers and Taqman reverse transcription reagents (Applied Biosystems (ABI)). For each real time PCR reaction, which was carried out in triplicate, cDNA synthesized from 20 ng of deoxyribonuclease I-treated RNA was mixed with 2× SYBR Green PCR Master Mix (ABI). Samples were analyzed on an ABI Prism 7900HT sequence detection system. Oligonucleotide primer sequences used in these experiments were: CYP2R1 mRNA, 5′-CAGAAAGACGCTGAAAGTGCAA-3′ and 5′-CAGTGTATTTGTGTTTACTTGGCTTTATAA-3′; CYP24A1 mRNA, 5′-CTCCCTATGGATGCAGTATGTATAGTG-3′ and 5′-TTTAAAAACGTTGTCAGTAGGTCATAACT-3′; CYP27A1 mRNA, 5′-GGAGGGCAAGTACCCAATAAGA-3′ and 5′-TGCGATGAAGATCCCATAGGT-3′; CYP27B1 mRNA, 5′-TCAGCAGGCATCGCAGAAC-3′ and 5′-GCATTGGATCCTGAGGAATGA-3′; cyclophilin mRNA, 5′-TGGAGAGCACCAAGACAGACA-3′ and 5′-TGCCGGAGTCGACAATGAT-3′. An expression cloning assay was established in cultured cells and optimized to isolate cDNAs encoding enzymes capable of activating vitamin D3 by 25-hydroxylation. The vitamin D-responsive reporter system consisted of a plasmid encoding a chimeric protein composed of the DNA binding domain of the yeast GAL4 transcription factor fused to the ligand binding domain of the human vitamin D receptor, and a reporter plasmid in which luciferase expression was directed by a heterologous promoter consisting of four GAL4 DNA response elements linked to basal expression elements from the herpes simplex 1 TK gene; this receptor-reporter system is referred to as GAL4-VDR/GAL4-luciferase. The introduction of these plasmids into HEK 293 cells followed by the addition of saturating levels of 1α,25-dihydroxyvitamin D3 to the culture medium led to a >700-fold increase over background of luciferase enzyme activity versus that observed in the absence of ligand (data not shown). To determine the maximum number of cDNAs that could be assayed at one time in the expression screen, dilution experiments were performed with the CYP27A1 expression plasmid, the receptor-reporter system, and the precursor substrate, 1α-hydroxyvitamin D3. A luciferase enzyme activity that was 3-fold over background was observed when the sterol 27-hydroxylase plasmid was diluted 190-fold with a control plasmid lacking a cDNA insert. These results indicated that a cDNA pool size of ∼100–200 could be reliably screened in the expression cloning assay when 1α-hydroxyvitamin D3 was used as the precursor. They also confirmed that the sterol 27-hydroxylase enzyme is capable of 25-hydroxylating both vitamin D3 and 1α-hydroxyvitamin D3 (11Guo Y.-D. Strugnell S.A. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (141) Google Scholar, 34Saarem K. Pedersen J.L. Biochim. Biophys. Acta. 1985; 840: 117-126Crossref PubMed Scopus (22) Google Scholar, 35Holmberg I. Berlin T. Ewerth S. Bjorkhem I. Scand. J. Clin. Lab. Invest. 1986; 46: 785-790Crossref PubMed Scopus (53) Google Scholar). To avoid isolating cDNAs encoding the sterol 27-hydroxylase, the cDNA library used in these studies was constructed using poly(A)+ mRNA isolated from livers of mice lacking sterol 27-hydroxylase (25Rosen H. Reshef A. Maeda N. Lippoldt A. Shpizen S. Triger L. Eggertsen G. Bjorkhem I. Leitersdorf E. J. Biol. Chem. 1998; 273: 14805-14812Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The resulting library was estimated to contain 7.9 × 106 independent transformants with an average cDNA insert size of 1.5 kb. Aliquots of the library containing ∼100 cDNAs were transfected together with the GAL4-VDR/GAL4-luciferase receptor-reporter plasmids into HEK 293 cells. The data generated from one such experiment is shown in Fig. 1. One cDNA pool (A11) produced a luciferase enzyme activity that was ∼2-fold over that generated in mock transfected cells. Subsequent cycles of cDNA purification and screening were performed on the A11 pool to identify a single cDNA that encoded the vitamin D-activating activity. A total of ∼110,000 cDNAs in 1056 pools were screened. Seven positive cDNA pools were identified, revealing four different encoded proteins. cDNAs for the retinoid X receptor α, hypoxia induction factor 1α, and the SEC14-related protein (GenBank™/EBI Data Bank accession no. NM_144520) produced a luciferase signal over background. Stimulation by the retinoid X receptor α protein may reflect a shortage of this vitamin D receptor heterodimerization partner in the HEK 293 cells. Similarly, enhancement by the hypoxia induction factor 1α is most likely the result of synergism between the receptor and this transcription factor. The mechanism by which the SEC14-related protein stimulated expression remains unknown, but the effect was modest (≤2-fold). The cDNA in the A11 pool that stimulated vitamin D receptor activity encoded cytochrome P450 2R1 (CYP2R1) based on sequence comparisons among gene family members (drnelson.utmem.edu/CytochromeP450.html). The amino acid sequence of the CYP2R1 protein was deduced from the isolated mouse cDNA (GenBank™/EBI Data Bank accession no. AY323818), and the amino acid sequence of the human enzyme (GenBank™/EBI Data Bank accession no. AY323817) was generated from a cDNA isolated from liver mRNA using PCR (Fig. 2A). The two enzymes share 89% sequence identity at the amino acid level (Fig. 2A), and the coding regions of the two cDNAs share 89% identity at the nucleic acid level. The human CYP2R1 gene is located on chromosome 11, band p15.2, and contains five exons distributed over ∼15.5 kb (Fig. 2B). The mouse Cyp2r1 gene occupies a syntenic location on chromosome 7, band 7E3. The mouse CYP2R1 mRNAs are ∼1.6 and 1.1 kb in length, with the larger form being more abundant than the smaller form (data not shown). The high degree of sequence conservation between mouse and human CYP2R1 protein sequences, and the ability of this enzyme to activate 1α-hydroxyvitamin D3, suggested that it might be a vitamin D 25-hydroxylase. This hypothesis was tested further in a series of transfection experiments with different cytochrome P450 cDNAs, vitamin D receptor constructs, and reporter genes. Vitamin D3 rather than 1α-hydroxyvitamin D3 was utilized as a precursor because 25-hydroxyvitamin D3 also activates the vitamin D receptor, albeit with decreased potency compared with the 1α,25-dihydroxy compound (18Takeyama K. Kitanaka S. Sato T. Kobori M. Yanagisawa J. Kato S. Science. 1997; 277: 1827-1830Crossref PubMed Scopus (454) Google Scholar). Transfection of the mouse or human CYP2R1 cDNA increased luciferase expression ∼7–10-fold in the GAL4-VDR/GAL4-luciferase reporter system (Fig. 3A). A similar stimulation was observed when a plasmid encoding the mouse sterol 27-hydroxylase (mCYP27A1) was introduced into the HEK 293 cells. As negative controls, neither the expression vector alone (pCMV6), nor a vector expr
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