Functional Analysis of Diastrophic Dysplasia Sulfate
Transporter
ITS INVOLVEMENT IN GROWTH REGULATION OF CHONDROCYTES MEDIATED BY
SULFATED PROTEOGLYCANS*
Hideshi
Satoh,
Masakazu
Susaki,
Chisa
Shukunami
§,
Ken-ichi
Iyama¶,
Takaharu
Negoro, and
Yuji
Hiraki
From Discovery Research Laboratories 1, Sumitomo Pharmaceuticals
Research Center, Osaka 544, the ¶ Department of Surgical
Pathology, Kumamoto University School of Medicine, University Hospital,
Kumamoto 860, and the
Department of Biochemistry, Osaka
University Faculty of Dentistry, Osaka 565, Japan
 |
ABSTRACT |
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.
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INTRODUCTION |
In animals, most of the bone initially forms as cartilage
(cartilaginous bone rudiments), which is later replaced by bone (endochondral ossification) (1). 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 (2). Recently, three human congenital
chondrodysplasias, i.e. diastrophic dysplasia,
atelosteogenesis type II, and achondrogenesis type 1B
(ACG-1B),1 have been
demonstrated to be caused by mutations in a single gene (3-5). 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) (3,
6).
Rossi (7) 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 and
Sat-1 gene products (6, 8). 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 (3) 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.
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MATERIALS AND METHODS |
Cell Culture--
Chondrocytes were isolated from growth plate
cartilage of ribs of young Wistar rats (weighing 90-110 g), as
described (9). 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
(10).
Cloning of DTDST cDNA and Sat-1 cDNA--
The
oligonucleotide primers used in this study are summarized in Table I.
Human placenta cDNA (Quick-Clone cDNA) was purchased from
CLONTECH (Palo Alto, CA) and was used to amplify
human DTDST cDNA by polymerase chain reaction (PCR) with Ex
Taq 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).
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).
Cloning of Rat DTDST Genomic DNA--
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.
Nucleotide 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
(11) and MACAW (12).
Northern Blot Hybridization--
Total RNA was prepared from
various rat tissues by a single step method according to Chomczynski
and Sacchi (13). 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 [
-32P]dCTP (3,000 Ci/mmol; Amersham Pharmacia Biotech). Human
-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.
Detection of Splice Variants of DTDST mRNA by
RT-PCR--
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.
In Vitro Transcription and Injection into Oocytes--
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.
Sulfate Transport Assay--
The handling of Xenopus
laevis oocytes and the transport assay were carried out as
reported by Bissig (8). [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 (3) 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 mM
various salts, 1 mM cold sulfate, 10 mM
HEPES/Tris, pH 7.5). Radioactivity retained in the cells was
determined.
Thymidine Incorporation and Proteoglycan Synthesis in Growth
Plate Chondrocytes--
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. Humphries
et al. (14) 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 (15). 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 (16).
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 (16).
Immunohistochemistry--
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 (17) 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).
 |
RESULTS |
Structure of Rat DTDST Gene and Tissue Distribution of Its
Transcripts--
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 (Table I), 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) (3, 8). 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 (3). 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 (3). Rat Sat-1
cDNA was also amplified from total RNA isolated from rat kidney and
sequenced (8).
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.
1A. 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 (3). Furthermore, it was shown that two forms of cDNA were generated by
alternative utilization of exon III (Fig. 1B). As shown in Fig. 1C, 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.

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Fig. 1.
Structure of the transcripts for rat DTDST
gene expressed in chondrocytes. A, intron-exon organization
of the rat DTDST gene. Boxes represent exons, and the
solid areas indicate the coding region of the gene.
Nucleotide sequences at exon/intron junctions of the rat DTDST gene are
summarized in the table. The sequences corresponding to
introns are indicated by lowercase letters. /, a splice
site. B, two forms of DTDST transcripts were generated by
alternative splicing: one utilizes all five exons, and the other lacks
exon III. The coding regions are indicated by solid areas.
C, total RNA was extracted from cultured rat UMR-106
osteoblastic cells or rat growth plate chondrocytes in primary culture.
RT-PCR indicated the presence of two distinct transcripts for the DTDST
gene in both types of cell.
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Functional Analysis of Rat DTDST Expressed in Oocytes--
To
determine the anion transport function of DTDST gene product
unequivocally, we injected cRNAs of human and rat DTDST into Xenopus oocytes and compared them to cRNA prepared from rat
Sat-1 cDNA. First, we examined endogenous sulfate transport in
Xenopus 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 (8), demonstrating that DTDST
constitutes a unique transport system distinct from that of Sat-1 (Fig.
3).

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Fig. 3.
Effects of extracellular ions on sulfate
uptake of cRNA-injected oocytes. Xenopus oocytes were
injected with 0.6 ng of human or rat DTDST cRNA or rat sat-1
cRNA. Five days after injection, [35S]sulfate uptake was
measured for 1 h in the presence of various extracellular ions
(100 mM) at 20 °C. Values represent the means ± S.E. for 9 or 10 oocytes, depending on the experiment.
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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 (8), Sat-1-directed sulfate
uptake displayed an identical cis-inhibition pattern (Fig. 4).
Inhibition of sulfate transport by oxalate was also reported in sulfate
uptake directed by DRA, the gene of which is an intestine-specific
sulfate transporter and has a significant homology with DTDST (18).
Moreover, DRA has been demonstrated to transport oxalate (6). Thus, the
results may suggest that both DTDST and Sat-1 also transport
oxalate.

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Fig. 4.
Inhibition of cRNA-induced sulfate uptake by
various inhibitors. Oocytes were injected with 0.6 ng of human
DTDST or rat sat-1 cRNA. Five days after injection,
[35S]sulfate uptake was measured for 1 h in the
presence of extracellular sodium gluconate (for DTDST) or choline
chloride (for Sat-1) at 20 °C. The concentration of DIDS used in the
experiment was 1 mM, whereas other inhibitors were used at
the concentration of 5 mM. Values are the means ± S.E. for six or seven oocytes.
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Properties of the Sulfate Transport System in Growth Plate
Chondrocytes--
To explore the nature of sulfate transport in
chondrocytes, we cultured growth plate chondrocytes isolated from rat
ribs in DMEM/F-12 medium containing 5% FBS (9). Confluent cultures of
chondrocytes were rinsed three times with ion-free solution containing
300 mM sucrose and 10 mM HEPES/Tris (pH 7.5).
Then cells were incubated with 1 mM
[35S]sulfate for 5 min. [35S]Sulfate uptake
was measured by the method reported by Hästbacka (3). In the
presence of sodium gluconate, chondrocytes exhibited the active inward
transport of sulfate anion (Fig. 5).
However, the presence of extracellular chloride and bicarbonate
markedly inhibited sulfate uptake of the cells. This
Na+-independent transport profile of chondrocytes was
almost identical to that of the DTDST cRNA-injected oocytes (Figs. 3
and 5). The presence of extracellular chloride markedly facilitated
sulfate efflux from chondrocytes in which [35S]sulfate
had previously been loaded (Fig. 6).
Bicarbonate had no effect on efflux of sulfate anion, suggesting that
the cells possess a chloride/sulfate antiport system.

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Fig. 5.
Effects of extracellular ions on sulfate
uptake in primary rat growth plate chondrocytes. Rat growth plate
chondrocytes were grown to confluency in 24-multiwell plates. The cells
were rinsed three times with ion-free washing solution and then
incubated in 500 µl of uptake solution containing 1 mM
[35S]sulfate (40 µCi/ml). Uptake of
[35S]sulfate was measured for 5 min in the presence of
various extracellular ions (149 mM) at 37 °C. Values are
the means ± S.D. for triplicate wells.
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Fig. 6.
Effect of extracellular chloride ion on
sulfate efflux in primary rat growth plate chondrocytes. Confluent
rat growth plate chondrocytes in 24-multiwell plates were incubated for
5 min in 500 µl of uptake solution containing 1 mM
[35S]sulfate (40 µCi/ml). Efflux was initiated by
replacing the uptake solution with the efflux solution containing 1 mM cold sulfate and chloride or bicarbonate (149 mM). Osmolarity of uptake and efflux solutions was kept
constant. [35S]Sulfate retained in the cells was
determined at various times after efflux started. Values are the
means ± S.D. for triplicate wells.
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By Northern blot analysis, we characterized the tissue distribution of
DTDST mRNA using total RNA isolated from various rat tissues
including bone and cartilage (Fig. 7).
Since the hybridization signals with
-actin probe were significantly
weaker in rib cartilage than in other tissues, the result clearly
indicated that cartilage was one of the major expression sites of DTDST
mRNA (approximately 8 kilobase pairs in size). Compared with
cartilage, the level of DTDST mRNA in bone was very low.
Unexpectedly, DTDST mRNA was also clearly detected in small
intestine where intestine-specific sulfate transporter DRA is present
(6). Sat-1 mRNA was expressed in liver and kidney as previously
reported (8). Taken together, these results suggested that chondrocytes
evidently possess the DTDST sulfate/chloride antiport system.

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Fig. 7.
Northern blot analysis of rat DTDST mRNA
expression in various tissues. Total RNA was isolated from various
rat tissues. 10 µg of RNA was loaded in each lane and then hybridized
with rat DTDST, rat Sat-1, or -actin cDNA. The membrane was
exposed to a film for 2 weeks.
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Growth Response of Growth Plate Chondrocytes in Sulfate-deficient
Culture--
Undersulfation of proteoglycans is a major consequence of
impairment of the DTDST transport system in chondrocytes (7). Thus, we
studied the growth factor response of chondrocytes associated with
undersulfated proteoglycans. Rat growth plate chondrocytes in confluent
culture were maintained in sulfate-free DMEM/F-12 medium containing
0.3% FBS for 72 h. We first measured [35S]sulfate
and [3H]glucosamine incorporated into proteoglycans for
the last 24 h of incubation. Sulfate concentration in serum was
reported to be 0.3-2.5 mM, depending on the species and
lots of serum (15). Sulfate incorporation into proteoglycans in
sulfate-free medium containing 0.3% FBS was estimated to be reduced to
approximately 25% of the control cultures maintained in the standard
DMEM/F-12 medium containing 0.3% FBS, assuming the sulfate
concentration in FBS to be 2.5 mM (15) and a minor sulfate
contamination in sulfate-free medium to be 0.01 mM (14).
However, glucosamine incorporation was not significantly affected by
the culture in sulfate-free medium compared with that in standard
medium, suggesting that there was no significant change in the rate of
proteoglycan synthesis in chondrocytes under the low sulfate culture
conditions. Treatment of cells with sodium chlorate, a reversible
inhibitor of glycosaminoglycan sulfation, resulted in further reduction of sulfate incorporation to several percent of the level in the cultures maintained in sulfate-free DMEM/F-12 medium containing 0.3%
FBS alone (Table II), whereas
[3H]glucosamine incorporation was not significantly
affected. FGF-2 is known to be a potent mitogen for chondrocytes (19).
As shown in Fig. 8, human recombinant
FGF-2 stimulated [3H]thymidine incorporation in
chondrocytes cultured in the standard DMEM/F-12 in a
dose-dependent manner. The addition of 10 µg/ml heparin
potentiated the growth response of the cells to FGF-2. When
chondrocytes, however, were maintained in undersulfation conditions,
the growth response of cells to FGF-2 was significantly abolished.
Cellular responsiveness to FGF-2 could not be recovered by the addition
of heparin in the severely undersulfated culture treated with sodium
chlorate.
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Table II
Effect of chlorate on sulfation of proteoglycans synthesized by rat
growth plate chondrocytes in culture
Rat growth plate chondrocytes grown in 96-multiwell plates were
metabolically labeled with [35S]sulfate or
[3H]glucosamine in the presence of indicated concentrations
of sodium chlorate in sulfate-free DMEM/F-12 medium containing 0.3%
FBS. The labeled proteoglycans were recovered from the culture by
coprecipitation with cold chondroitin sulfate and 0.1% CPC.
Radioactivities in the precipitates were determined. Values (cpm/well)
are the means ± S.D. for triplicate wells. Numbers in parentheses
represent percentages of control values.
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Fig. 8.
Effect of sodium chlorate on DNA synthesis in
rat growth plate chondrocytes stimulated by FGF-2. 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 in 96-multiwell plates. They were then 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 FGF-2 in the presence or
absence of 10 µg/ml heparin. Cells were incubated for another 26 h and labeled with 2 µCi/ml [3H]thymidine for the last
4 h. Values are the means ± S.D. for triplicate wells.
|
|
Localization of FGF-2 in Epiphyseal Cartilage of Developing
Bone--
FGF-2 is expressed in virtually all cell types in developing
bone, which includes cartilage, perichondrium and periosteum, bone, and
the surrounding connective tissues (20, 21). It is usually hard to
demonstrate extracellular localization of growth factors in bone and
cartilage by immunohistochemistry, since epitopes of the minor proteins
are masked by the abundant extracellular matrix components. After
unmasking the FGF-2 epitope with the hyaluronidase treatment, we
demonstrated extracellular localization of FGF-2 in the developing
bovine bone for the first time in detail by using a specific monoclonal
antibody against bovine FGF-2 (bFM-1) (17). All of the epiphyseal
cartilage zones and bone at the primary ossification center were
clearly stained by bFM-1 (Fig. 9A), whereas preimmune IgG
showed no positive staining (Fig. 9B). At higher
magnification, the cell surface or pericellular space was shown to be
stained intensely in the proliferating cartilage zone (Fig.
9C) as well as in the resting cartilage zone. Interestingly, virtually no immunoreactivity was detected in the interterritorial space (Fig. 9C). However, as cartilage differentiation
progressed toward the lower hypertrophic and calcified zone,
immunoreactivity gradually diffused out from the pericellular to the
interterritorial space of cartilage matrix (Fig. 9, A and
D). In the lowest layers of chondrocytes, although the cell
surface staining persisted, cells appeared to be separated by an
FGF-2-negative pericellular space from the intensely stained
interterritorial matrix. In addition, the extracellular FGF-2
immunoreactivity was also noted in osteoblasts (Fig. 9D,
arrows).

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Fig. 9.
Immunohistochemical localization of FGF-2 in
developing caudal vertebrae. A, anti-FGF-2 monoclonal
antibody, bFM-1, revealed localization of FGF-2 in epiphyseal cartilage
of bovine developing bone. B, no positive staining was
observed when mouse preimmune IgG was used as a negative control. A
high magnification shows intense staining for FGF-2 on the cell surface
(or pericellular space) of chondrocytes in the resting and
proliferating cartilage zones (C). FGF-2 is also noted in
both the cell membrane of hypertrophic chondrocytes and the
extracellular matrix of the lower hypertrophic and calcified cartilage
zones (D). Osteoblasts at the bone-forming front are
indicated by arrowheads. Bars, 50 µm.
|
|
 |
DISCUSSION |
The DTDST gene has been implicated in the pathogenesis of at least
three human chondrodysplasias (22) and encodes a membrane transporter
with 12 membrane-spanning domains, which is homologous to the
hepatocanalicular sulfate transporter Sat-1 (3, 8). However, the anion
transport activity of the gene product has not been directly
determined. In the present study, we have clearly demonstrated that the
gene product indeed functions as a Na+-independent sulfate
transporter by the use of an oocyte expression system (Fig. 3).
DTDST-directed sulfate transport was markedly inhibited by thiosulfate
and oxalate, but not by succinate. DIDS completely blocked
DTDST-directed sulfate uptake (Fig. 4). Thus, DTDST exhibited a
cis-inhibition pattern identical to that of Sat-1.
Despite a striking sequence similarity to Sat-1, DTDST-directed sulfate
uptake was significantly repressed by outside chloride anion (Fig. 3).
In contrast, Sat-1-directed uptake was greatly facilitated by the
presence of outside chloride anion but repressed by outside
bicarbonate. These findings were compatible with the notion that
canalicular transporter Sat-1 is a sulfate/bicarbonate antiporter (8,
23). Hästbacka et al. (3) presumed DTDST to encode a
sulfate/chloride antiporter on the basis of their observation that skin
fibroblasts from a DTD patient lacked a sulfate/chloride antiport
activity previously characterized in human lung fibroblasts (IMR-90)
(24). The fact that DTDST-directed sulfate uptake depended on outside
chloride gives further support to the notion that DTDST functions as a
sulfate/chloride antiporter.
Previously, Hästbacka (3) reported by Northern blot analysis
using poly(A)+ RNA that expression of DTDST mRNA could
be detected widely in multiple tissues in humans. However, taking into
consideration that mutations in the gene participate in the
pathogenesis of osteochondrodysplasia, cartilage and bone must be
important target tissues to examine. In the present study, Northern
blot analysis using total RNA revealed that DTDST mRNA is
predominantly expressed in cartilage (Fig. 7). No obvious hybridization
signal was detected in bone. Since cartilage synthesizes a large amount
of sulfated proteoglycans and deposits them in their extracellular
matrix to maintain its biological functions, cartilage-specific
expression of the DTDST gene must reflect a greater requirement of
cartilage for proteoglycan sulfation via the DTDST transport system.
Interestingly, we also found that small intestine was one of the major
expression sites of DTDST mRNA (Fig. 7). Mutations in the
intestine-specific sulfate transporter gene DRA are
implicated in congenital chloride diarrhea (25). Congenital chloride
diarrhea is characterized by large watery stools with a high chloride
concentration, possibly due to a defect of the chloride/bicarbonate
exchange system in the ileum and colon (26, 27). Thus, DTDST together
with DRA may participate in the intestinal chloride/bicarbonate
exchange system. However, the physiological functions of DTDST
expressed in the intestines remain to be elucidated.
Since carrier-mediated sulfate transport at the plasma membrane is
rate-limiting for macromolecular sulfation (24), proper DTDST functions
must be critical for sulfation of proteoglycans and matrix organization
in cartilage. In the course of our study, we for the first time
established the structure of DTDST transcripts in cartilage and found
that there exist two types of transcripts derived from an alternative
splicing of the third exon in the 498-base pair 5'-untranslated region
(Figs. 1 and 2). It is not known at present what the functional
significance is of these alternative transcripts in cartilage. However,
common ion dependence of sulfate transport in DTDST cRNA-injected
oocytes and cultured chondrocytes strongly indicated that the DTDST
transport system plays a crucial role in cartilaginous sulfate uptake
(Figs. 3 and 5). Induction of sulfate efflux by outside chloride
further suggested the presence of DTDST-like sulfate/chloride
antiporter (Fig. 6). Although the possibility that some unknown sulfate
transport systems are responsible for sulfate uptake into chondrocytes
cannot be ruled out, epiphyseal cartilage cells isolated from an ACG-1B fetus completely lacked sulfate transport activity (7). The cells
produced severely undersulfated proteoglycans in culture. Rossi
et al. have shown that this particular patient bore a
mutation in the coding region of the DTDST gene causing one amino acid substitution in the transport protein (7). These results suggested that
the DTDST sulfate transporter is a major sulfate transporter in
chondrocytes. The tissue distribution of DTDST mRNA (Fig. 7) was
also compatible with this notion.
Achondrogenesis type 1B is a lethal chondrodysplasia characterized by
severe underdevelopment of the skeleton and extreme micromelia.
Histologic sections of the patient's epiphyseal cartilage showed
collagenous rings surrounding chondrocytes with coarse collagen fibrils
(7). Skeletal underdevelopment and micromelia in the ACG-1B patients
imply a causative role of undersulfated proteoglycans during
development. Fibroblast growth factors are known to be a potent mitogen
for chondrocytes (28) and participate in limb morphogenesis (29).
Sulfated proteoglycans are also important for transmission of growth
signals (30). We prepared cultures of rat growth plate chondrocytes
associated with undersulfated proteoglycans by treatment of the cells
with sodium chlorate in sulfate-free medium (Table II). Our
sulfate-free culture conditions did not affect the rate of proteoglycan
synthesis of the cells as estimated by [3H]glucosamine
incorporation, but [35S]sulfate incorporation into
proteoglycans was greatly reduced. Under the normal culture conditions,
FGF-2 clearly stimulated DNA synthesis in quiescent chondrocytes in
confluent cultures (Fig. 8). The addition of heparin to the cultures
evidently potentiated FGF-2-stimulated [3H]thymidine
incorporation (Fig. 8). However, transmission of FGF-2 signal to
chondrocytes was markedly impaired in the undersulfated cultures. Thus,
chondrocytes require sulfated proteoglycans for transmission of FGF
signaling.
Ishihara et al. (31, 32) reported a loss of response to
FGF-2 in endothelial cells upon treatment with sodium chlorate, but
cellular responses to the factor were completely recovered by the
addition of heparin to the culture medium even in the presence of
chlorate. In contrast, the addition of heparin to the chondrocyte cultures could not rescue a loss of cellular response to FGF-2 in the
severely undersulfated culture, depending on the dose of chlorate (Fig.
8). It is known that extracellular space in cartilage has a highly
organized structure due to the complex interactions between
extracellular matrix components. A chondrocyte and its pericellular
microenvironment in hyaline cartilage, a metabolic unit of cartilage
called a chondron, are separated by a fibrillar capsule from the
weight-bearing interterritorial matrix (33, 34). As shown in Fig. 9,
A and C, FGF-2 is expressed and localized in the
pericellular space near the cell surface in cartilage except for the
hypertrophic cartilage zone at the cartilage/bone junction. There is
much evidence that heparan sulfate proteoglycans play a role in
sequestering FGF by providing matrix binding sites and cell surface
receptors (35, 36). This type of sequestering mechanism probably
underlies the distribution pattern of FGF in cartilage. Isolated
chondrocytes are capable of reconstructing these extracellular
structures in vitro
(33).2 We speculate that
polyanionic heparin molecules could not be readily accessible to the
signal transmission machinery in the pericellular microenvironment
through the barrier of abundant extracellular matrix in the chondrocyte
cultures. Thus, unlike endothelial cells, the proper organization of
sulfated proteoglycans in the cartilage matrix may be an important
requirement for chondrocytes to grow. The DTDST-mediated sulfate
transport system in chondrocytes may be of crucial importance to
endochondral bone development.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Y. Koga (Research Director) and
Dr. T. Katsumata (Discovery Research Laboratories 3, Sumitomo
Pharmaceuticals Research Center) for encouragement. We also thank Dr.
Y. Ishiduka (Discovery Research Laboratories 3, Sumitomo
Pharmaceuticals Research Center) for technical support and to M. Toda
for excellent technical assistance. Anti-FGF-2 monoclonal antibody,
bFM-1, was kindly provided by Dr. Y. Yoshitake (Kanazawa Medical
University).
 |
FOOTNOTES |
*
This work was supported in part by the Special Coordination
Funds for Promoting Science and Technology from the Science and Technology Agency of Japan (to Y. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D82883 (rat DTDST) and U14528, L02785, and L23413 (human
DTDST, human DRA, and rat Sat-1, respectively).
§
Research Fellow of the Japan Society for the Promotion of
Science.
To whom correspondence should be addressed: Dept. of
Biochemistry, Osaka University Faculty of Dentistry, 1-8 Yamadaoka,
Suita, Osaka 565, Japan. Tel./Fax.: 81-6-879-2889; E-mail:
hiraki{at}dent.osaka-u.ac.jp.
1
The 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.
2
Y. Hiraki, unpublished data.
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