Functional Analysis of Diastrophic Dysplasia Sulfate Transporter
1998; Elsevier BV; Volume: 273; Issue: 20 Linguagem: Inglês
10.1074/jbc.273.20.12307
ISSN1083-351X
AutoresHideshi Satoh, Masakazu Susaki, Chisa Shukunami, Ken‐ichi Iyama, Takaharu Negoro, Yuji Hiraki,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoMutations in the diastrophic dysplasia sulfate transporter (DTDST) gene constitute a family of recessively inherited osteochondrodysplasias including achondrogenesis type 1B, atelosteogenesis type II, and diastrophic dysplasia. However, the functional properties of the gene product have yet to be elucidated. We cloned rat DTDST cDNA from rat UMR-106 osteoblastic cells. Northern blot analysis suggested that cartilage and intestine were the major expression sites for DTDST mRNA. Analysis of the genomic sequence revealed that the rat DTDST gene was composed of at least five exons. Two distinct transcripts were expressed in chondrocytes due to alternative utilization of the third exon, corresponding to an internal portion of the 5′-untranslated region of the cDNA. Injection of rat and human DTDST cRNA into Xenopus laevis oocytes induced Na+-independent sulfate transport. Transport activity of the expressed DTDST was markedly inhibited by extracellular chloride and bicarbonate. In contrast, canalicular Na+-independent sulfate transporter Sat-1 required the presence of extracellular chloride in the cRNA-injected oocytes. The activity profile of sulfate transport in growth plate chondrocytes was studied in the extracellular presence of various anions and found substantially identical to DTDST expressed in oocytes. Thus, sulfate transport of chondrocytes is dominantly dependent on the DTDST system. Finally, we demonstrate that undersulfation of proteoglycans by the chlorate treatment of chondrocytes significantly impaired growth response of the cells to fibroblast growth factor, suggesting a role for DTDST in endochondral bone formation. Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene constitute a family of recessively inherited osteochondrodysplasias including achondrogenesis type 1B, atelosteogenesis type II, and diastrophic dysplasia. However, the functional properties of the gene product have yet to be elucidated. We cloned rat DTDST cDNA from rat UMR-106 osteoblastic cells. Northern blot analysis suggested that cartilage and intestine were the major expression sites for DTDST mRNA. Analysis of the genomic sequence revealed that the rat DTDST gene was composed of at least five exons. Two distinct transcripts were expressed in chondrocytes due to alternative utilization of the third exon, corresponding to an internal portion of the 5′-untranslated region of the cDNA. Injection of rat and human DTDST cRNA into Xenopus laevis oocytes induced Na+-independent sulfate transport. Transport activity of the expressed DTDST was markedly inhibited by extracellular chloride and bicarbonate. In contrast, canalicular Na+-independent sulfate transporter Sat-1 required the presence of extracellular chloride in the cRNA-injected oocytes. The activity profile of sulfate transport in growth plate chondrocytes was studied in the extracellular presence of various anions and found substantially identical to DTDST expressed in oocytes. Thus, sulfate transport of chondrocytes is dominantly dependent on the DTDST system. Finally, we demonstrate that undersulfation of proteoglycans by the chlorate treatment of chondrocytes significantly impaired growth response of the cells to fibroblast growth factor, suggesting a role for DTDST in endochondral bone formation. In animals, most of the bone initially forms as cartilage (cartilaginous bone rudiments), which is later replaced by bone (endochondral ossification) (1Horton W.A. Growth Genet. Horm. 1990; 6: 1-3Google Scholar). In adults, cartilage is left in particular body portions such as rib, auricle, and joints and functions as a load-bearing tissue. Thus, cartilage is essential for growth and maintenance of animal skeletal systems. Biological functions of cartilage are mostly dependent on the properties of its extracellular matrix, whose major components are cartilage-specific collagens and sulfated proteoglycans (2Comper W.D. Hall B. Newman S. Cartilage: Molecular Aspects. CRC Press, Inc., Boca Raton, FL1991: 59-96Google Scholar). Recently, three human congenital chondrodysplasias, i.e. diastrophic dysplasia, atelosteogenesis type II, and achondrogenesis type 1B (ACG-1B), 1The abbreviations used are: ACG-1B, achondrogenesis type 1B; DIDS, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene; DTDST, diastrophic dysplasia sulfate transporter; FGF, fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; PBS, phosphate-buffered saline.1The abbreviations used are: ACG-1B, achondrogenesis type 1B; DIDS, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene; DTDST, diastrophic dysplasia sulfate transporter; FGF, fibroblast growth factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; PBS, phosphate-buffered saline. have been demonstrated to be caused by mutations in a single gene (3Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve Daly M.P. Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E.S. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (620) Google Scholar, 4Superti-Furga A. Hästbacka J. Wilcox W.R. Cohn D.H. van der Harten H.J. Rossi A. Blau N. Rimoin D.L. Steinmann B. Lander E.S. Gitzelmann R. Nat. Genet. 1996; 12: 100-102Crossref PubMed Scopus (186) Google Scholar, 5Hästbacka J. Superti-Furga A. Wilcox W.R. Rimoin D.L. Cohn D.H. Lander E.S. Am. J. Hum. Genet. 1996; 58: 255-262PubMed Google Scholar). The gene, the diastrophic dysplasia sulfate transporter (DTDST), is presumed to encode a Na+-independent sulfate transporter on the basis of its structural similarity with rat hepatocanalicular sulfate transporter (Sat-1) and human intestine-specific sulfate transporter (DRA;down-regulated in adenoma) (3Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve Daly M.P. Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E.S. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (620) Google Scholar,6Silberg D.G. Wang W. Moseley R.H. Traber P.G. J. Biol. Chem. 1995; 270: 11897-11902Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Rossi (7Rossi A. Bonaventure J. Delezoide A.-L. Cetta G. Superti-Furga A. J. Biol. Chem. 1996; 271: 18456-18464Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) recently demonstrated that chondrocytes isolated from a ACG-1B patient synthesized chondroitin sulfate proteoglycans that bore glycosaminoglycan chains that were of normal size but were undersulfated. The oocyte expression system has been proved to be a powerful tool for functional analysis of the DRA andSat-1 gene products (6Silberg D.G. Wang W. Moseley R.H. Traber P.G. J. Biol. Chem. 1995; 270: 11897-11902Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 8Bissig M. Hagenbuch B. Stieger B. Koller T. Meier P.J. J. Biol. Chem. 1994; 269: 3017-3021Abstract Full Text PDF PubMed Google Scholar). In the present study, we directly characterized the sulfate transport activity of rat DTDST by injection of its cRNA into Xenopus oocytes and compared it to that of normal rat costal chondrocytes. The activity profile of the gene product showed an ion dependence of transport distinct from that of Sat-1, suggesting that DTDST was a sulfate/chloride antiporter. Hästbacka and co-workers (3Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve Daly M.P. Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E.S. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (620) Google Scholar) demonstrated a ubiquitous expression of DTDST mRNA in the body. Histological observations of diastrophic dysplasia patients would suggest cartilage to be the most relevant tissue to examine. However, they did not include cartilage in their study. The present Northern blot analysis clearly indicated that the level of DTDST mRNA was particularly high in cartilage and intestine. Only a marginal level of the transcript was detected in other tissues. A unique ion dependence of sulfate transport in culture chondrocytes further supported the notion that cartilage is predominantly dependent on the DTDST system for its sulfate utilization. Chondrocytes were isolated from growth plate cartilage of ribs of young Wistar rats (weighing 90–110 g), as described (9Shimomura Y. Yoneda T. Suzuki F. Calcif. Tissue Res. 1975; 19: 179-187Crossref PubMed Scopus (170) Google Scholar). After removal of peripheral muscular tissues and perichondrium from whole ribs, a portion of the growth plate cartilage was isolated and minced. Cells were isolated by sequential treatment of cartilage with EDTA, trypsin, and collagenase. The isolated cells were plated at a density of 4 × 104 cells/well in 24-multiwell plates, 2 × 104 cells/well in 48-multiwell plates, or 1 × 104 cells/well in 96-multiwell plates in DMEM/F-12 medium containing 5% FBS. Cells were grown to confluency at 37 °C under 5% CO2 in air. The culture medium was renewed every other day. Rat UMR-106 osteosarcoma cells were maintained in DMEM containing 10% FBS as described (10Gutierrez G.E. Mundy G.R. Katz M.S. Endocrinology. 1984; 115: 2342-2346Crossref PubMed Scopus (15) Google Scholar). The oligonucleotide primers used in this study are summarized in Table I. Human placenta cDNA (Quick-Clone cDNA) was purchased fromCLONTECH (Palo Alto, CA) and was used to amplify human DTDST cDNA by polymerase chain reaction (PCR) with ExTaq DNA polymerase (Perkin-Elmer) and specific primers to the 5′- and 3′-ends of the entire coding sequence in human DTDST cDNA: primer 1 and primer 2. Further 5′-regions of human DTDST cDNA were amplified by rapid amplification of cDNA ends (RACE) using the Marathon cDNA amplification kit (CLONTECH) with the gene-specific antisense primer (hDTD-GSP1) and the nested gene-specific antisense primer (hDTD-NGSP1).Table ISummary of PCR primers used in this studyPrimersNucleotide sequencesSpecific primers for amplification of human DTDST coding region Primer 15′-GCTGAACCATCTATCTCC-3′ Primer 25′-CTAGACATTCTTCTATCTAC-3′The gene-specific and the nested gene-specific primers for 5′-RACE of human DTDST hDTD-GSP15′-TACTTGATTCCCTTTGAAGTTCCAG-3′ hDTD-NGSP15′-CTTCAGCTGAGTCTCTGGGTGAAAC-3′Specific primers for rat Sat-1 coding region Primer 35′-CAAAGAGCCTGGTGTGACAG-3′ Primer 45′-CTTGGCTCTTGAGGAAGCGG-3′Primers for amplification of rat DTDST cDNA fragment Primer 55′-TTGCCAACCAAAGAACTC-3′ Primer 65′-TGTGATCACACCAAGGAC-3′Primers for amplification of the entire coding region of rat DTDST cDNA Primer 75′-ATGTCTTCAGAAAGTAAAGA-3′ Primer 85′-ATTAGCAGAAGGACCAACAG-3′ Primer 95′-TTGCCAACCAAAGAACTC-3′ Primer 105′-TCACTACTAAGACTCAGACC-3′The gene-specific and the nested gene-specific primers for 5′-RACE of rat DTDST rDTD-GSP15′-GCAGTGGAAGAAGGAGGGTATG-3′ rDTD-GSP25′-CCGGCACCAATTCCAACTGAGC-3′ rDTD-NGSP15′-GCATACATTTCCTGATTGGCTTTC-3′ rDTD-NGSP25′-AGCTCATTGTCGTTGTGGCAGC-3′Specific primers for amplification of 5′-untranslated region of rat DTDST gene Primer 115′-AGACATCATCTTCTGGCTTC-3′ Primer 125′-TGGTTTCTCATGGAGTTCCA-3′Primers for amplification of further 5′-region of rat DTDST gene rDTD-GSP35′-ATCACTGTTACTCAGAGGGTGCTTC-3′ rDTD-NGSP35′-GACAGACACACATCACTTATGGAGG-3′ Open table in a new tab Total RNAs isolated from rat UMR-106 cells and rat kidney were reverse transcribed into cDNA with oligo(dT) primer using the cDNA synthesis Superscript preamplification system (Life Technologies, Inc.). The cDNA from rat kidney was used to amplify an entire coding sequence rat Sat-1 cDNA by PCR with the specific primer set (primer 3 and primer 4). The resulting cDNA was cloned into pCRII vector. The cDNA from UMR-106 was used to amplify rat DTDST cDNA by PCR with the primers designed from the human DTDST sequence (primer 5 and primer 6). Then two sets of specific primers were used to amplify the entire coding region in rat DTDST; one was composed of the 5′-end of human DTDST coding region (primer 7) and the internal site in rat DTDST (primer 8). The other set was a combination of the internal site of human DTDST coding region (primer 9) and the 3′-end of human DTDST coding region (primer 10). Further 5′- and 3′-regions of rat DTDST cDNA were amplified by the RACE method using the Marathon cDNA amplification kit (CLONTECH) with the gene-specific primers (rDTD-GSP1 and rDTD-GSP2) and the nested gene specific primers (rDTD-NGSP1 and rDTD-NGSP2). Since initial PCR with GSP1 and GSP2 did not give rise to any distinct bands, the initial PCR products were subjected to a nested PCR reactions. The resulting 5′-RACE products and 3′-RACE products were cloned into pCRII vector, respectively. To construct plasmids containing 5′-coding region, 3′-coding region, and entire coding region of rat DTDST cDNAs, the overlapping cDNA clones obtained by RT-PCR and RACE were digested with the appropriate restriction enzymes and cloned into Bluescript (Stratagene, La Jolla, CA). Rat genomic DNA was purchased from CLONTECH and used to amplify DTDST genomic DNA sequences. The 5′-untranslated region of rat DTDST gene was amplified by PCR with sense primer 11 and antisense primer 12. An approximately 1.3-kb genomic DNA fragment was amplified. Further 5′-regions of rat DTDST genomic sequence were amplified using the GenomeWalker kit (CLONTECH) by two steps of PCR reactions with the gene-specific antisense primer (rDTD-GSP3) and the nested gene specific antisense primer (rDTD-NGSP3), corresponding to the sequence in exon I. The PCR products were cloned into pCRII vectors by TA cloning for sequence analysis. The DTDST cDNA and genomic sequences were determined by dye terminator cycle-sequencing reactions using specific primers and the purified PCR fragments or cloned plasmids as templates and an automated sequencer (Applied Biosystems model 373A, Perkin-Elmer). Sequences were analyzed using DNASIS software (Hitachi Software Engineering) and the computer programs BLAST (11Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69707) Google Scholar) and MACAW (12Schuler G.D. Altschul S.F. Lipman D.J. Proteins. 1991; 9: 180-190Crossref PubMed Scopus (892) Google Scholar). Total RNA was prepared from various rat tissues by a single step method according to Chomczynski and Sacchi (13Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63088) Google Scholar). Isolated total RNA (20 μg/lane) was separated by electrophoresis on a 1% agarose/formaldehyde gel and transferred onto Hybond-N nylon membrane (Amersham Pharmacia Biotech). The cloned rat DTDST and Sat-1 cDNA fragments were labeled by the BcaBEST labeling kit (TAKARA, Ohtu, Japan) and [α-32P]dCTP (3,000 Ci/mmol; Amersham Pharmacia Biotech). Human β-actin cDNA was similarly labeled and used as a control probe. The blots were hybridized overnight at 42 °C with a probe (2 × 106 cpm/ml) in 50% formamide, 5 × saline/sodium/phosphate/EDTA, 2 × Denhardt's solution, 2% SDS, and 100 μg/ml denatured salmon testis DNA (Sigma). The filters were washed twice at room temperature for 15 min in 2 × SSC, 0.05% SDS and then washed twice at 50 °C for 40 min in 0.1 × SSC, 0.1% SDS and exposed to Hyperfilm-MP x-ray films (Amersham Pharmacia Biotech) at −80 °C with an intensifying screen. Total RNAs (2.5 μg each) isolated from UMR-106 cells and rat growth plate chondrocytes were reverse transcribed into cDNA with random hexamer using the cDNA synthesis Superscript preamplification system. One-fiftieth or one-hundredth of the cDNAs were used to amplify two alternative spliced transcripts of the DTDST gene by PCR. PCR amplification was performed by using AmpliTaq DNA polymerase (Perkin-Elmer) and sense primer 11 and antisense primer 12 (Table I). Thermal cycling was carried out for 28 cycles (30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C). Aliquots of the PCR products were resolved on 5% polyacrylamide gel along with molecular size markers, and the amplified products were stained with ethidium bromide. Plasmids harboring cloned sulfate transported cDNAs were linearized with appropriate restriction enzymes that have a cleavage site immediately downstream of the cDNA insert. Capped cRNAs were synthesized by T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase using the mCAP RNA capping kit (Stratagene). Unincorporated nucleotides were removed with Quick Spin columns (Boehringer Mannheim, Germany), and cRNA was recovered by ethanol precipitation and resuspended in water for injection into oocytes. The handling of Xenopus laevis oocytes and the transport assay were carried out as reported by Bissig (8Bissig M. Hagenbuch B. Stieger B. Koller T. Meier P.J. J. Biol. Chem. 1994; 269: 3017-3021Abstract Full Text PDF PubMed Google Scholar). [35S]Sulfate (carrier-free) was purchased from NEN Life Science Products. Oocytes were washed twice in ion-free washing solution (200 mm sucrose, 10 mm HEPES/Tris, pH 7.5). The oocytes were then incubated in 100 μl of uptake solution per oocyte (200 mm sucrose or 100 mm various salts, 1 mm[35S]sulfate (40 μCi/ml), 10 mm HEPES/Tris, pH 7.5) for 1 h at room temperature. Sulfate uptake was stopped by washing the oocytes three times with ice-cold washing solution containing 5 mm K2SO4. Each oocyte was then dissolved in 0.2 ml of 10% SDS, and the oocyte-associated radioactivity was determined in a liquid scintillation spectrometer after the addition of scintillation fluid (ACS II, Amersham Pharmacia Biotech). Sulfate uptake was measured as described by Hästbacka (3Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve Daly M.P. Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E.S. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (620) Google Scholar) with slight modifications. Confluent chondrocytes cultured in 24-multiwell plates were washed three times in ion-free washing solution (300 mm sucrose, 10 mm HEPES/Tris, pH 7.5). The cells were then incubated in 500 μl of uptake solution (300 mm sucrose or 149 mm various salts), 1 mm [35S]sulfate (40 μCi/ml), 10 mm HEPES/Tris, pH 7.5) for 5 min at 37 °C. Uptake was stopped by washing the cells four times with ice-cold washing solution containing 5 mm K2SO4. The cells were then dissolved in 0.3 ml of 10% SDS, and the cell-associated radioactivity was determined. For measurement of sulfate efflux from the cells, confluent rat growth plate chondrocytes were prelabeled with [35S]sulfate by incubation in uptake solution (300 mm sucrose, 1 mm [35S]sulfate (40 μCi/ml), 10 mm HEPES/Tris, pH 7.5) for 5 min at 37 °C. Efflux of sulfate was initiated by replacing the uptake solution with the efflux solution (300 mm sucrose or 149 mmvarious salts, 1 mm cold sulfate, 10 mmHEPES/Tris, pH 7.5). Radioactivity retained in the cells was determined. For determination of thymidine incorporation, rat growth plate chondrocytes were grown to confluency in DMEM/F-12 medium or sulfate-free DMEM/F-12 medium containing 5% FBS with or without sodium chlorate (Sigma) in 96-multiwell plates. Sulfate-free DMEM/F-12 medium, in which all of the sulfate salts in the standard formula were substituted by chloride salts, was specially prepared and obtained from Nikken Biomedical Lab (Osaka, Japan). Antibiotics, which are the significant sources of sulfate, were also omitted. Humphrieset al. (14Humphries D.E. Sugumaran G. Silbert J.E. Methods Enzymol. 1989; 179: 428-429Crossref PubMed Scopus (23) Google Scholar) noted that the antibiotic- and sulfate-free medium thus prepared may still contain 0.01 mm sulfate as a possible minor contaminant. Sulfate concentration in serum was also reported to be 0.3–2.5 mm (15Mulder G.J. Mulder G.J. Sulfation of Drugs and Related Compounds. CRC Press, Inc., Boca Raton, FL1981: 31-52Google Scholar). In this study, serum sulfate concentration and a sulfate contaminant in the sulfate-free DMEM/F-12 were assumed to be 2.5 and 0.01 mm, respectively. Cells were preincubated in the same medium containing 0.3% FBS for 24 h. The medium was replaced by the same medium containing 0.3% FBS with human recombinant FGF-2 (R & D Systems, Minneapolis, MN) in the presence or absence of 10 μg/ml heparin (Wako Pure Chemical, Osaka, Japan). Cells were incubated for another 26 h and labeled with 2 μCi/ml [3H]thymidine (Amersham Pharmacia Biotech) for the last 4 h. Radioactivity incorporated into DNA was determined as described previously (16Hiraki Y. Inoue H. Shigeno C. Sanma Y. Bentz H. Rosen D.M. Asada A. Suzuki F. J. Bone Miner. Res. 1991; 6: 1373-1385Crossref PubMed Scopus (143) Google Scholar). For determination of proteoglycan synthesis, chondrocytes were grown to confluency in 48-multiwell plates. The medium was replaced with 0.3 ml of DMEM/F-12 medium or sulfate-free DMEM/F-12 medium containing 0.3% FBS with or without sodium chlorate and incubated for 48 h. Then the cultures were labeled with 5 μCi/ml [35S]sulfate or 10 μCi/ml [3H]glucosamine for another 24 h. After incubation, the medium was collected, and the cell layer was rinsed with phosphate-buffered saline (PBS). Proteoglycans recovered in the medium and PBS rinse were combined. [35S]Sulfate and [3H]glucosamine incorporated into proteoglycans were determined after Pronase E digestion and precipitation by 1% cetylpyridinium chloride in the presence of chondroitin sulfate, as described (16Hiraki Y. Inoue H. Shigeno C. Sanma Y. Bentz H. Rosen D.M. Asada A. Suzuki F. J. Bone Miner. Res. 1991; 6: 1373-1385Crossref PubMed Scopus (143) Google Scholar). Developing bovine tails were collected from 5-month-old fetuses and fixed overnight at 4 °C in periodate/lysine/paraformaldehyde solution in 0.01 m PBS (pH 7.4). The caudal vertebrae were dissected out, dehydrated in a graded series of ethanol, and embedded in paraffin. Longitudinal serial sections were cut at 6 μm. Deparaffinized sections were treated with 1% H2O2 in methanol for 30 min to reduce endogenous peroxidase activity and washed in PBS. Sections were treated with 500 units/ml testicular hyaluronidase (type V, Sigma) in PBS for 20 min at 37 °C and rinsed in PBS. The slides were covered with 5% normal goat serum in PBS for 30 min and then with anti-FGF-2 monoclonal antibody (bFM-1) at a dilution of 1:50 (17Matsuzaki K. Yoshitake Y. Matuo Y. Sakaki H. Nishikawa K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9911-9915Crossref PubMed Scopus (166) Google Scholar) and incubated overnight at 4 °C. Preimmune mouse IgG was used as a negative control. Immunoreactions were performed using a Vectastain peroxidase rabbit ABC kit (Vector, Burlingame, CA). Sections were washed with PBS, and the antigenic sites were demonstrated by treating the sections with 0.05% 3,3′-diaminobenzidine tetrahydrochloride (Dojin Chemicals, Tokyo, Japan) in 0.05 m Tris-HCl buffer (pH 7.6) and 0.01% H2O2 for 7 min. Nuclei were stained with methyl green. Then the sections were dehydrated in ethanol, cleared in xylene, and mounted in Permount (Fisher). Physicochemical properties of extracellular matrix profoundly affect growth and differentiation of cells in animals. Bone and cartilage produce a large amount of extracellular matrix components, which include sulfated proteoglycans. Thus, these tissues are assumed to develop an efficient transport system for sulfate anion to meet their demand for sulfate during proteoglycan synthesis. To study the properties of the sulfate transporting system and its role in the growth regulation, we attempted to clone rat DTDST cDNA. Using RT-PCR with human DTDST-specific primers 5 and 6 (TableI), we amplified rat cDNA from total RNA isolated from rat UMR-106 osteoblastic cells. Then the entire coding region and the 5′- and 3′-regions of the cDNA were isolated by RT-PCR and RACE reactions. In the coding region, the cloned cDNA sequence had a higher similarity to human DTDST cDNA (73% identical) than that of rat hepatocanalicular sulfate transporter Sat-1 cDNA (43% identical) (3Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve Daly M.P. Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E.S. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (620) Google Scholar, 8Bissig M. Hagenbuch B. Stieger B. Koller T. Meier P.J. J. Biol. Chem. 1994; 269: 3017-3021Abstract Full Text PDF PubMed Google Scholar). We therefore identified the cloned cDNA as rat DTDST cDNA. For comparison, we also cloned human DTDST cDNA from human placenta cDNA by PCR using primers 1 and 2 and then 5′-RACE (Table I). The resultant RACE fragments contained the 5′-flanking sequence (−77 to −1) of the putative initiator ATG of human DTDST cDNA (3Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve Daly M.P. Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E.S. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (620) Google Scholar). Comparison of the cDNA sequence with the previously reported genomic sequence revealed the presence of 3′-splice acceptor and an exon/intron junction at −26/−25 (3Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve Daly M.P. Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E.S. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (620) Google Scholar). Rat Sat-1 cDNA was also amplified from total RNA isolated from rat kidney and sequenced (8Bissig M. Hagenbuch B. Stieger B. Koller T. Meier P.J. J. Biol. Chem. 1994; 269: 3017-3021Abstract Full Text PDF PubMed Google Scholar). Interestingly, two forms of rat DTDST cDNA that differ in their 5′-untranslated region were isolated by RACE reactions. One contained an additional 130-base pair insert that was absent from the other. To explore the cause of the differences between the two cDNA clones, we analyzed the corresponding genomic sequences by PCR reactions using GenomeWalker kit. Comparison of the genomic sequence with the cDNA sequence revealed that the 5′-untranslated region of rat DTDST sequence is encoded by four exons (exons I–IV), as shown in Fig.1 A. These exons were separated by intron I (8 kilobase pairs), intron II (512 base pairs), and intron III (1 kilobase pair). We also confirmed the presence of an approximately 1.8-kilobase pair intron after codon 233, the position of which is identical to that previously reported in human DTDST (3Hästbacka J. de la Chapelle A. Mahtani M.M. Clines G. Reeve Daly M.P. Daly M. Hamilton B.A. Kusumi K. Trivedi B. Weaver A. Coloma A. Lovett M. Buckler A. Kaitila I. Lander E.S. Cell. 1994; 78: 1073-1087Abstract Full Text PDF PubMed Scopus (620) Google Scholar). Furthermore, it was shown that two forms of cDNA were generated by alternative utilization of exon III (Fig. 1 B). As shown in Fig. 1 C, RT-PCR using sense primer 11 and antisense primer 12 revealed the presence of these two alternative transcripts in UMR-106 osteoblastic cells and growth plate chondrocytes. These transcripts were also detected in articular cartilage (data not shown). The nucleotide sequence and the deduced amino acid sequence of rat DTDST cDNA are shown in Fig. 2.Figure 2The nucleotide sequence of rat DTDST cDNA and the deduced amino acid sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2The nucleotide sequence of rat DTDST cDNA and the deduced amino acid sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the anion transport function of DTDST gene product unequivocally, we injected cRNAs of human and rat DTDST intoXenopus oocytes and compared them to cRNA prepared from rat Sat-1 cDNA. First, we examined endogenous sulfate transport inXenopus oocytes using water-injected oocytes and found that water-injected oocytes displayed only low [35S]sulfate uptake (data not shown). In contrast, human or rat DTDST cDNA-injected oocytes displayed significant uptake of [35S]sulfate in the outside sulfate pool in the presence of sodium gluconate outside (Fig. 3). The level of [35S]sulfate uptake in the presence of sodium gluconate was nearly the same as that in the sucrose-containing medium without sodium gluconate (data not shown), indicating a Na+-independent sulfate transport of DTDST. The presence of outside chloride anion, however, resulted in a significant inhibition of the DTDST-directed sulfate uptake. In contrast to DTDST, Sat-1 cRNA-injected oocytes displayed a chloride-dependent sulfate uptake as previously reported (8Bissig M. Hagenbuch B. Stieger B. Koller T. Meier P.J. J. Biol. Chem. 1994; 269: 3017-3021Abstract Full Text PDF PubMed Google Scholar), demonstrating that DTDST constitutes a unique transport system distinct from that of Sat-1 (Fig.3). We then studied the cis-inhibition pattern of DTDST-directed sulfate transport activity (Fig. 4). Human DTDST-directed sulfate uptake was sensitive to thiosulfate and oxalate. 4,4′-Diisothiocyano-2,2′-disulfonic acid stilbene (DIDS; 1 mm) completely blocked the sulfate transport activity of DTDST. In agreement with a previous report (8Bissig M. Hagenbuch B. Stieger B. Koller T. Meier P.J. J. Biol. Chem. 1994; 269: 3017-3021Abstract Full Text PDF PubMed Google Scholar), Sat-1-directed sulfate uptake displayed an identical cis-inhibition pattern (
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