From the Department of Biotechnology, Faculty of
Engineering, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-ku,
Kyoto 603-8555, the § Department of Molecular Interaction
and Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto
University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, and
¶ Discovery Research Laboratories III, Pharmaceutical Discovery
Research Division, Takeda Chemical Industries, Ltd., 17-85
Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan
Received for publication, April 26, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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To determine the role of fibroblast growth factor
(FGF)·FGF receptor (FGFR) signaling in chondrogenesis, we
analyzed the gene expression of alternatively spliced FGFRs during
chondrogenic differentiation of ATDC5 cells in vitro. Two
isoforms of FGFR3 were expressed in these cells. One was the
complete form of FGFR3 (FGFR3) already reported, and the other was a
novel one that lacks the acid box domain (FGFR3 The fibroblast growth factor
(FGF)1 receptors (FGFRs) are
a family of transmembrane tyrosine kinases involved in signaling via
interactions with the FGF family (1-3). The FGFR family consists of
four closely related members, the amino acid sequences of which are
highly conserved both between different members of the family and
throughout evolution (1). They regulate a multitude of cellular
processes, including cell growth, differentiation, migration, and
survival (2, 3).
The structure of FGFRs has three different parts: an extracellular
portion with three Ig-like domains, a single transmembrane portion, and a split tyrosine kinase domain inside the cell. The overall complexity of the receptor is increased by the existence of
additional isoforms generated by alternative mRNA splicing (4-8).
These receptors possess an alternative sequence for the C-terminal half
of the third Ig domain (IgIII), encoded by a separate 5'-exon IIIa and
3'-exon, either IIIb or IIIc, which determines ligand
specificity (1, 7, 8). In the segment between the first Ig domain (IgI)
and the second Ig domain (IgII), there is a cluster of the acidic
residues, which is referred to as the "acid box," similar to that
found in adhesion molecules such as E-cadherin/uvomorulin (9, 10).
Another major alternative splicing event results in FGFR isoforms
differing in the IgI and acid box domains (4-6). FGFRs lacking IgI
have a higher ligand affinity for some FGF ligands, although it is not
known if ligand binding specificity is affected by removal of the IgI
domain (6). Distinct expression patterns of these isoforms have also
been reported in cell differentiation (11), embryonal development (12),
and tumor progression (13), suggesting that there are functional
differences between these isoforms.
Activation of three members of the FGFR family, FGFR1, FGFR2, and
FGFR3, by FGFs induces mitogenic responses in various cell types (14).
In addition, a gain of functional mutations in FGFR3 causes early
cessation of cell growth in chondrocytes (15, 16). These mutations
cause autosomal dominant disorders of skeletal development such as
hypochondroplasia (17), achondroplasia (18, 19), and thanatophoric
dysplasia types I and II (20, 21). These mutations activate
receptor signaling by either inducing ligand-independent receptor
dimerization (22, 23) or relieving the constraints for
autophosphorylation of receptor-tyrosine kinase (21, 24). Analysis of
FGFR3-deficient mice generated by targeted disruption of the FGFR3 gene
revealed that these mice have longer than average bones, expansion of
their growth plate, and increased chondrocyte proliferation (25, 26).
Moreover, the introduction of the achondroplasia mutation (G380R) into
the murine FGFR3 gene resulted in a dominant dwarf phenotype that
exhibited many of the features of human achondroplasia (27). These
results strongly suggested that FGFR3 is a negative regulator of bone
growth, in contrast to the common role of FGFRs in stimulation of cell
proliferation. The role of FGFR3 in growth inhibition is cell
type-specific, in which the receptors utilize signaling through STAT1
activation. Ligand stimulation of FGFR3 in a rat chondrosarcoma cell
line (28) and the thanatophoric dysplasia type II mutant (K650E) of
FGFR3 expressed in 293 cells (29) increased the phosphorylation of
STAT1 and its translocation to the nucleus. One of the consequences of
STAT1 activation is expression of p21 (30), a general inhibitor of
cyclin-dependent kinases, which causes growth arrest in chondrocytes.
To elucidate the role of the FGF·FGFR signaling system in normal bone
growth, we chose ATDC5 cells as an in vitro chondrogenesis model system. ATDC5 cells have been shown to be very useful for molecular analysis of early and late phase differentiation of chondrogenesis, because the cells display the sequential transitions of
their phenotype in a synchronous manner in vitro (31-33).
Taking advantage of the sequential differentiation of ATDC5 in culture, we studied the gene expression of FGFRs during early chondrocyte differentiation.
Our study showed temporal regulation of FGFR1, FGFR2, and FGFR3 gene
expression during chondrogenic differentiation. We also analyzed the
expression of alternatively spliced isoforms of these receptors
differing in IgI and acid box domains. During the study, we isolated
and cloned the cDNA of a novel splice variant of FGFR3, designated
"FGFR3 Materials--
Human recombinant FGF2 and FGF9 (N3) were gifts
from Takeda Chemical Industries (Osaka, Japan) (34, 35). Other human
recombinant FGFs were purchased from R & D Systems (Minneapolis, MN).
Heparin (Hepar Inc.) was a gift from Dr. J. Folkman. Bovine insulin was purchased from Sigma. BaF3 cells and WEHI-3 cells were obtained from the RIKEN cell bank (Tsukuba, Japan).
Cell Lines and Culture Conditions--
BaF3 cells were cultured
as described previously (36). BaF3 cells are a pro-B cell line that is
IL-3-dependent. BaF3 cells were cultured in RPMI 1640 containing 10% FBS and 10% conditioned medium from WEHI-3 cells,
which contained IL-3. ATDC5 cells were cultured in the maintenance
medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's
medium and Ham's F-12 medium (Life Technologies, Inc.)
containing 5% FBS (JRH Biosciences Co., Lenexa, KS), 10 µg/ml bovine
transferrin (Nacalai Tesque Co., Kyoto, Japan), and 3 × 10 Alcian Blue Staining of Cells--
Differentiated ATDC5 cells
were plated in 6-multiwell dishes and cultured in differentiation
medium. The cells were rinsed twice with ice-cold phosphate-buffered
saline and fixed with 100% methanol for 2 min at RNA Extraction and Northern Blot Analysis--
Rat chondrocytes
were isolated from rib cartilage as described by Shimomura et
al. (37). Total RNA was prepared from the chondrocytes by the
single step method of Chomczynski and Sacchi (38). ATDC5 cells were
inoculated in 100-mm plates and cultured in either differentiation
medium or maintenance medium. Total RNA was prepared from the cultures
using Isogen (Nippon Gene Co., Tokyo, Japan) following
instructions given by the provider. Total RNA (10 µg) was denatured
with 6.29% formaldehyde, separated by 1.2% agarose electrophoresis,
and capillary-transferred onto nylon membranes
(Hybond-N+, Amersham Pharmacia Biotech). Hybridization was
performed for 16 h at 42 °C with the appropriate probe
(106 dpm/ml) in hybridization solution containing 50%
formamide, 5× SSPE (20× SSPE contains 3.6 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA, pH 7.7), 5× Denhardt's solution, 0.5% (w/v) SDS, and 40 µg/ml sonicated denatured salmon sperm DNA (Stratagene, La Jolla,
CA). Probes were prepared to a specific activity of 5 × 108 cpm/µg with a random primer DNA labeling kit (Ready
to go labeling kit; Amersham Pharmacia Biotech) using
[ Reverse Transcription-PCR--
First strand cDNA was
synthesized using Moloney murine leukemia virus reverse
transcriptase (First strand cDNA synthesis kit; Amersham Pharmacia
Biotech). The RNA (5 µg in 8 µl) was heated at 65 °C for 10 min
and then cooled on ice. Then, 5 µl of the first strand cDNA
synthesis reaction mixture (135 mM Tris-HCl, pH 8.3, 204 mM KCl, 27 mM MgCl2, 5.4 mM each dNTP, and 0.24 mg/ml bovine serum albumin),
together with 1 µl of 0.2 M dithiothreitol and 0.2 µg/µl random d(N)6 primer were added to the RNA
solution. After incubation at 37 °C for 1 h the mixture was
heated to 95 °C for 5 min and then chilled on ice. PCR was performed
in a reaction mixture consisting of 25 µl of cDNA, 50 mM KCl, 10 mM Tris-HCl, pH 9.0, 0.1% Triton
X-100, 200 µM each dNTP, 0.625 units of Taq DNA polymerase (Promega), and 25 pmol each of the forward and reverse
sequence-specific primers. The reaction was carried out for 25-30
cycles of 1 min at 94 °C, 2 min at 60 °C, and 3 min at 72 °C,
with an extra 7-min extension at 72 °C for the last cycle
(RoboCycler Gradient40, Stratagene). The optimal
Mg2+ concentrations, cycles, and the forward and reverse
primers used for analysis of FGFR and other gene markers are summarized
in Table I. Aliquots of the PCR products (10 µl) were electrophoresed on 3.5% agarose gels (Nusieve GTG/Seakem) in Tris acetate EDTA buffer,
pH 8.0, and stained with 0.5 µg/ml ethidium bromide. A linear
amplification dependent on the amount of RNA was obtained under the
above conditions (from 2.5 to 40 ng of RNA). The amounts of mRNA
were adjusted in each RT-PCR reaction by checking amplification of the
glyceraldehyde-3-phosphate dehydrogenase transcripts. The identity of
all PCR products was confirmed by sequencing with the AmpliTaq cycle
sequence kit (Applied Biosystems, Inc., Foster City, CA) using the dye
terminator method and a 310 genetic analyzer (Applied Biosystems,
Inc.).
Cloning and Sequence of the Mouse Acid Box-deleted Form of FGFR3
cDNA--
3 µg of total RNA that was extracted from
undifferentiated ATDC5 cells was used for the reverse transcription,
which was performed with avian myeloblastosis virus reverse
transcriptase (Takara RNA LAPCR kit) and the oligo(dT) primer at
50 °C for 1 h. The cDNA was amplified using the forward
primer (5'-CCCGAGCTCTGGAGCCATGGTAGTC) and the reverse primer
(5'-CTAGGATCCGCGTAAACATTGCCTG) before being extracted with
phenol/chloroform and precipitated with cold ethanol. The DNA was then
ligated into the pT7Blue2 vector (Novagen), and competent
Escherichia coli cells (Nova Blue) were transformed with the ligated vector. The plasmids of positive clones were extracted, and the nucleotide sequences of their inserts were determined in both orientations with an AmpliTaq cycle sequence kit
(Applied Biosystems, Inc.), as described above.
FGFR Expression Plasmids and Stable Expression--
Full-length
cDNAs encoding the FGFR3 Immunoblotting--
The cells expressing each FGFR3 isoform were
homogenized in isotonic buffer containing 50 mM HEPES
buffer, pH 8.0, 200 mM sucrose, 2 mM EDTA, and
1 mM phenylmethylsulfonyl fluoride. The homogenate was
centrifuged at 100,000 × g for 1 h at 4 °C.
The cell membranes were then electrophoresed through SDS-7.0%
polyacrylamide gels and transferred to polyvinylidene difluoride
membranes (Immobilon-P, Millipore Co.). The membranes were incubated
with anti-C-terminal FGFR3 antibody (Santa Cruz Biotechnology,
Santa Cruz, CA) and then with horseradish peroxidase-conjugated second
antibody (Santa Cruz Biotechnology) before finally being developed with
enhanced chemiluminescence substrate solution (Super Signal West Pico; Pierce) and exposed to BioMax MS film (Kodak) to visualize
immunoreactive bands.
Cell Growth Assays--
Cell proliferation was assayed by
counting the number of BaF3 cells stably expressing FGFR3 Formation of Cartilage Nodules by ATDC5 in Vitro--
ATDC5 cells
were cultured in the medium containing 10 µg/ml insulin (the
differentiation medium) as described under "Experimental Procedures." As reported previously, the presence of insulin resulted in an increased number of chondrocytes in the culture, which promoted more efficient chondrogenic differentiation in ATDC5 cells compared with that in the absence of insulin (32). At the beginning of culture,
ATDC5 cells were undifferentiated and did not express chondrogenic
markers. The cells stopped growing at confluence, reached 4-5 days
after the culture was started, but remained undifferentiated with a
fibroblastic morphology. A transient condensation of cells with an
elongated spindle-like morphology preceded the formation of the
nodules. ATDC5 cells reentered the growth state through the cellular
condensation followed by the formation of cartilage nodules from day 7 to day 10, with a cell doubling time of 48 h. The cartilage
nodular structures seen from day 14 were composed of proliferating
chondrocytes with a round morphology. This postconfluent growth of
cells continued until day 21, when typical cartilage nodules were
formed all over the culture. This cell state is called early phase
differentiation (32). To visualize the chondrogenic differentiation,
Alcian blue staining of ATDC5 cells was performed as shown in Fig.
1. The Alcian blue positive cells produce
the proteoglycan aggrecan, which is a major component of cartilage. Alcian blue positive cells were not seen until day 10 (Fig. 1, a and b). During the postconfluent growth phase
between days 10 and 21, Alcian blue-positive cells appeared to expand
by the accumulation of cartilage matrix resulting from the ongoing
chondrogenesis (Fig. 1, c and d).
Expression of Cartilage-characteristic Extracellular Matrix Genes
and p21 Cyclin-dependent Kinase Inhibitor Gene during
Chondrogenic Differentiation of ATDC5 Cells--
Chondrogenic
differentiation of ATDC5 cells was further characterized by the
expression of cartilage-characteristic extracellular matrix genes such
as aggrecan and type II collagen. As shown in Fig.
2, transcripts of these genes were
undetectable by RT-PCR in the cells cultured with the differentiation
medium from day 3 until day 7. These transcripts became detectable
after day 10 and gradually increased until day 21. At the same time,
the cartilage nodules first appeared at day 10, and their number also
gradually increased until day 21. The mRNA of p21
cyclin-dependent kinase inhibitor was first detected on day
7, when the cells were in the condensation stage, and its expression
gradually increased until day 21. This correlates well with the
appearance of cartilage nodules and the increase in number of mature
chondrocytes in culture.
Expression of FGFR1, FGFR2, and FGFR3 Genes during Chondrogenic
Differentiation of ATDC5 Cells--
Fig.
3 shows the time course of changes in
FGFR mRNA by Northern blot analysis. FGFR1 (4.0 kb) and FGFR2 (4.5 kb) mRNAs were detected on all days tested. In contrast, FGFR3
mRNA was not detected in undifferentiated ATDC5 cells (day 3). By
means of Northern blot analysis, the 4.5 kb-FGFR3 transcript was weakly
detected on day 7, when the cells reached postconfluence, and the
levels of FGFR3 expression gradually increased until day 21. The
expression levels of FGFR1 and FGFR2 mRNAs appeared to greatly
increase until day 21 during formation of cartilage nodules (Fig. 3)
and continued even until day 42 during late phase differentiation, when
the number of hypertrophic chondrocytes increased in the culture (data not shown). FGFR4 transcripts were not detected at any phase in ATDC5
cells (data not shown).
Identification of the Acid Box-deleted Isoform of FGFR3--
Next,
the expression of the alternatively spliced isoforms of FGFRs that
differ in the presence or absence of IgI and the acid box was examined
by RT-PCR. We used the forward and reverse primers, which annealed to
the signal sequence region and to the 3'-half of IgII in each FGFR,
respectively (Table I). In FGFR1 and
FGFR2, several isoforms were detected, as reported previously (4, 5).
The two types of FGFR1 variants were detected, one of which contains
IgI (FGFR1 Sequence of the Mouse Acid Box-deleted Form of FGFR3
cDNA--
Because FGFR3 Mitogenic Activity of BaF3 Cell Lines Expressing FGFR3 Splice
Variants--
To test whether FGFR3 Gene Expression and Biological Roles of FGFRs in
Chondrogenesis--
Our results indicated that in cultures of ATDC5
cells a single population of progenitor cells undergoes chondrogenesis
without the influence of the environmental factors involved in
vivo. This system allowed us to investigate the molecular
mechanism of chondrogenic differentiation in vitro.
Interestingly, gene expression of the cyclin-dependent
kinase inhibitor p21 was first seen during cell condensation (around
days 7-10), which was followed by increased levels of collagen type II
and aggrecan gene expression. These results demonstrated that ATDC5
cells gradually stop proliferating as they differentiate into
proliferating chondrocytes and finally into mature chondrocytes in a
manner typical of growth plate chondrocytes. The levels of FGFR1 and
FGFR2 gene expression were high in undifferentiated ATDC5 cells, and
the expression levels of these FGFRs increased markedly during
chondrogenic differentiation (Fig. 3). In contrast, the level of FGFR3
expression was very low in undifferentiated ATDC5 cells, compared with
levels of FGFR1 and FGFR2 expression, but gradually increased
during cell condensation. FGFR1 and FGFR2 were thought to stimulate
cell proliferation prior to cell condensation. After 2 weeks in
culture, the level of FGFR3 transcripts was increased, indicating that
FGFR3 is expressed in proliferating and mature chondrocytes. This was
consistent with the high levels of FGFR3 expression observed in
developing mice: in the prebone cartilage rudiments as well as in
cartilage during endochondral ossification (48). The increased level of
FGFR3 transcripts correlated well with the increase in
cyclin-dependent kinase inhibitor p21 gene expression, as
the proliferating chondrocytes were further differentiating and
maturing to form cartilage nodules. Our findings supported a model in
which the expression of FGFR3 may lead to growth inhibition in
chondrocytes, despite the higher levels of FGFR1 and FGFR2 expression.
Because the levels of FGFR1 and FGFR2 expression increased markedly
after cell condensation, FGFR1 and FGFR2 also must have received FGF
signals during the differentiation process. Thus, these receptors may
share the ligands available during chondrogenesis. This raises several
questions, including what the consequences of signaling from FGFR1 and
FGFR2 are, whether this signaling stimulates growth of chondrocytes,
and whether this signaling accelerates the differentiation of
chondrocytes and growth arrest. It will also be interesting to further
investigate the cross-talk between the signals from FGFR1, FGFR2, and FGFR3.
Expression of Alternatively Spliced Isoforms of FGFRs Differing in
IgI, Acid Box, and IgIII Domains during Chondrogenesis--
Various
alternatively spliced isoforms of FGFR1 and FGFR2 differing in IgI and
acid box domains were expressed during chondrogenesis, but the
expression ratios of these isoforms were unchanged (Fig. 4). In
contrast, that of FGFR3 isoforms was differentially regulated. Whether
these isoforms have different roles in chondrogenesis has not yet been
clarified. The temporal expression of FGFR3 is consistent with its role
in mediating growth arrest of mature chondrocytes. FGFR1 isoforms have
different roles in mesodermal cell migration and growth during limb
development (13). FGFR2 isoforms differing in the IgIII domain mediate
a reciprocal regulation loop between FGFs expressed in mesenchyme and
ectoderm during the early development of vertebrate limbs (3, 49, 50). This regulation was shown to be dependent on the cell type-specific differential expression of the FGFR2 isoforms IIIb (FGFR2b) and IIIc
(FGFR2c) (39, 51, 52). FGFR1, FGFR2, and FGFR3 isoforms expressed in
this system had the IIIc exon, which is specific for mesenchymal cells.
During this study, we found that a novel isoform of FGFR3, FGFR3 Biological Activities and Heparin Requirements of FGFR3
Using FGF1 as an internal standard, we found that FGFR3 Biological Significance of FGF and FGFR Expression during
Chondrogenesis--
FGF2 is abundant in cartilage. It was originally
extracted from bovine scapular cartilage and purified as a
cartilage-derived growth factor (59). FGFR3
Further studies are required to examine the native ligands of the FGFR3
isoforms as well as the signaling cascade activated upon FGF·FGFR
binding in chondrogenesis. Similar studies on FGFR1 and FGFR2 isoforms
and the cross-talk between FGFR1, FGFR2, and FGFR3 signaling
are also important. These studies will provide insight into whether
FGFR3AB). The gene of
FGFR3
AB was expressed in undifferentiated ATDC5 cells. In contrast,
the transcripts of FGFR3 were not detectable in undifferentiated cells
but increased during cellular condensation, which is an obligatory step
for chondrogenic differentiation. FGFR1 and FGFR2 expression was higher than that of FGFR3 in undifferentiated cells. The gene expression of
cell cycle inhibitor p21 was induced during cell condensation and
correlated best with the expression of FGFR3 among the FGFR isoforms
expressed. The differential expression of FGFR3 isoforms during
chondrogenesis suggests that these isoforms may play different roles in
the regulation of growth and differentiation in chondrocytes. To define
the mitogenic response of FGFR3
AB and FGFR3 to FGFs, their cDNAs
were stably transfected into mouse BaF3 pro-B cells. FGFR3
preferentially mediates the mitogenic response to FGF1 and poor
response to FGF2. In contrast, FGFR3
AB mediated a higher mitogenic
response to FGF2 as well as to FGF1. In addition, FGFR3
AB responds
to FGF1 at lower concentrations of heparin than FGFR3 does. These
results suggest that the acid box plays an important role in the
regulation of FGFR3 to mediate biological activities in response to FGFs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
AB," which lacks the acid box domain in the extracellular
part of FGFR3. To determine whether FGFR3
AB has the potential to
stimulate cell proliferation, and if so, whether it has a different
ligand specificity than that of FGFR3, we compared the mitogenic
response of BaF3 cells stably expressing FGFR3
AB or FGFR3 to various FGFs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
8 M sodium selenite (Sigma), as
described previously (32). Cells were maintained at 37 °C in a
humidified 5% CO2, 95% air atmosphere. The
inoculum size of the cells was 6 × 103 cells/well in
a 24-multiwell plate and 2.5 × 105 cells/plate in a
100-mm plate. For induction of chondrogenesis, the cells were
cultured in maintenance medium supplemented with 10 µg/ml bovine
insulin (differentiation medium).
20 °C. They were
stained with 0.1% Alcian blue 8GS (Fluka, Buchs, Switzerland) in 0.1 M HCl for 2 h at 25 °C and then washed three times
with distilled water, as previously described (32).
-33P]dCTP (3000 Ci/mmol, PerkinElmer Life
Sciences). Templates were as follows: a 364-bp HincII
fragment of pT7Blue 2-mouse FGFR1 for FGFR1 mRNA, a 1.11-kb
BamHI fragment of pT7Blue2-mouse FGFR2 for FGFR2 mRNA, a
0.97-kb BalI fragment of pT7Blue2-mouse FGFR3 for FGFR3
mRNA, and a 452-bp purified PCR fragment for
glyceraldehyde-3-phosphate dehydrogenase mRNA. The nylon
membranes were washed once for 15 min at 42 °C in 2× SSPE and 0.1%
SDS, once for 30 min at 42 °C in 1× SSPE and 0.1% SDS, and twice
for 15 min at room temperature in 0.1× SSPE and 0.1% SDS. The
membranes were exposed to BioMax MS film (Eastman Kodak Co.) at
80 °C with a TranScreen L-E intensifying screen (Kodak).
AB and FGFR3 were cloned into the pBKRSV
expression vector (Stratagene). First strand cDNA was
synthesized using the total RNA derived from ATDC5 cells. The
full-length cDNAs encoding FGFR3 were amplified by LA PCR, using a pair of primers (forward, 5'-CGACACTAGTGGAGCCATGGTAGTCC-3'; reverse, 5'-CTAGCGGCCGCGTAAACATTGCCTG-3'). The resulting DNA was then
cloned into the pT7Blue2 T-vector and sequenced. The plasmid pT7Blue2
T-FGFR3
AB was obtained as described above. The following fragments
were isolated and ligated to yield the two pBKRSV plasmids: an
SpeI-BamHI restriction fragment of FGFR3 from the
pT7Blue2 T plasmids or NheI-BamHI fragment of
FGFR3
AB with an NheI-BamHI fragment of the
pBKRSV
lac expression vector, in which the lac promoter was removed.
To express these FGFRs in BaF3 cells, 4 × 106 cells
were incubated with 20-30 µg of pBKRSV-FGFR3 or pBKRSV-FGFR3
AB plasmids for 10 min at 4 °C. The BaF3 cells were then electroporated in a Bio-Rad gene pulser at 400 V and 960 microfarads before
being plated. After 2-3 days of incubation in RPMI 1640 containing
10% FBS and 10% of the conditioned medium from WEHI-3 cells, the BaF3 cells were recovered. After washing the medium, the BaF3 cells were
selected with 10 ng/ml FGF1 and 10 µg/ml heparin in RPMI 1640 containing 10% FBS without the conditioned medium from WEHI-3 cells.
AB or
FGFR3. Cells were washed twice with RPMI 1640 containing 10% FBS but
lacking IL-3. The cells were plated at 1 × 104
cells per well in a total volume of 500 µl of medium in
24-multiwell plates. Various concentrations of FGFs were added in the
presence or absence of the indicated concentrations of heparin. After 3 days, the viable cells were harvested and counted by a Coulter Counter
ZM type, Beckman Coulter Co.). Experiments were performed at
least in triplicate, and the results are expressed as means ± standard deviations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alcian blue staining of ATDC5
cells. Alcian blue staining of ATDC5 cells showing the
accumulation of cartilage matrix. ATDC5 cells were cultured in
6-multiwell plates with differentiation medium for 7 (a), 10 (b), 14 (c), and 21 (d) days. Cultures
were stained with 0.1% Alcian blue as described under "Experimental
Procedures".
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Fig. 2.
Expression of cartilage-characteristic
extracellular matrix genes and the p21 cyclin-dependent
kinase inhibitor gene. ATDC5 cells were grown in the presence of
10 µg/ml insulin (the differentiation medium) for 1, 3, 5, 7, 10, 14, and 21 days. Total RNA was then isolated. 5 µg of total RNA was used
for reverse transcription in 15-µl reaction mixtures, and an aliquot
was used for PCR as described under "Experimental Procedures." The
primer pairs used were specific for collagen type II, aggrecan,
p21 cyclin-dependent kinase inhibitor, and
glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 3.
Northern hybridization analysis of FGFR1,
FGFR2, and FGFR3 mRNA levels. 10 µg of total RNA was
denatured and electrophoresed on a 1.2% agarose gel. After blotting to
Hybond-N+ membranes, each membrane was hybridized
with 33P-labeled FGFR1, FGFR2, FGFR3, or
glyceraldehyde-3-phosphate dehydrogenase cDNA.
; 605 bp) and the other of which lacks IgI (FGFR1
; 338 bp) (Fig. 4). In contrast, three types of
FGFR2 variants were detected. These are the complete form (633 bp), an
isoform that lacks IgI (366 bp), and an isoform that lacks IgI
and the acid box (288 bp) (Fig. 4). We extracted all the DNA fragments
and confirmed their sequences. The expression levels of these FGFR1 and
FGFR2 isoforms were increased during chondrogenic differentiation of
ATDC5 cells, but the ratio of them remained unchanged throughout. It
was found that all isoforms expressed in ATDC5 cells had only the IIIc
exon (data not shown). This result corresponds to the previous
observation that the IIIc isoform of FGFRs is expressed mainly in
mesenchymal lineages, whereas the IIIb isoform expression is restricted
to epithelial lineages (39). In the case of FGFR3, two PCR bands (446 bp and 392 bp) were detected (Fig.
5A). It revealed that the
446-bp fragment was derived from the previously reported complete form
of FGFR3 that contains IgI (36) and that the 392-bp fragment lacked the acid box between IgI and IgII in the extracellular domain. The 392-bp
fragments were thought to be derived from an alternatively spliced
variant of FGFR3, and we designated it "FGFR3
AB." Interestingly, only the 392-bp PCR band was detected by RT-PCR using cDNA from the
undifferentiated ATDC5 cells (day 2). This PCR band was also detected
using cDNA from the rat rib cartilage-derived chondrocytes, suggesting that FGFR3
AB is expressed not only in ATDC5 cells but
also in chondrocytes in vivo (Fig. 5B).
PCR primers for amplification of FGFR and other cDNAs
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Fig. 4.
RT-PCR analysis of FGFR1 and FGFR2 isoforms
alternatively spliced in their IgI and IgII domains. ATDC5 cells
were grown in 100-mm plates with differentiation medium. Total RNA was
isolated on days 3, 5, 7, 10, 14, and 21. 5 µg of total RNA was used
for reverse transcription in 15 µl of reaction mixture, and aliquots
were used for PCR as described under "Experimental Procedures." The
primer pairs used are summarized in Table I.
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Fig. 5.
RT-PCR analysis of the alternatively spliced
isoform of FGFR3 expressed during chondrogenic differentiation of ATDC5
cells and rat rib cartilage-derived chondrocytes. A,
ATDC5 cells were grown in the differentiation medium for 21 days. Total
RNA was isolated on days 2, 7, 14, and 21 before the RT-PCR was
performed as described above. B, the rat chondrocytes were
harvested from rib cartilage, and total RNA was isolated as described
under "Experimental Procedures." Lane M, molecular
weight marker, X174/HinfI; lane 1, rat
chondrocytes; lane 2, undifferentiated ATDC5 cells (day
2).
AB appears to be novel, the full-length
cDNA of FGFR3
AB from RNA of undifferentiated ATDC5 cells was
cloned further. The forward and reverse primers for amplification of the cDNA by RT-PCR were based on the mouse FGFR3 sequence (36) (Fig. 6A). FGFR3
AB encoded
a polypeptide consisting of 782 amino acids, with an amino acid
sequence identical to that of mouse FGFR3 (800 amino acids) except for
the absence of the acid box region in the extracellular domain (Fig.
6B). Sequence comparison studies between FGFR3
AB and
genomic FGFR3 suggested that there is alternative splicing of the acid
box coding exon and the 5'-half of the IgII coding exon (40) (data not
shown). It has been previously reported that FGFR3 has two
alternatively spliced isoforms in the 3'-half of IgIII in the
extracellular domain. These are IIIc and IIIb isoforms, which
differ in the ligand specificity (41, 42). The novel FGFR3
AB and
FGFR3 expressed in ATDC5 cells have the IgIIIc domain (Fig.
6C).
View larger version (109K):
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Fig. 6.
Nucleotide sequence of mouse
FGFR3 AB cDNA. A,
nucleotide sequence of mouse FGFR3
AB cDNA. Nucleotides are
numbered above the line, and translated amino acids are numbered on the
right-hand side. The locations of the PCR primers
used to amplify FGFR3
AB are indicated by underlines.
B, amino acid sequence comparison of the extracellular
domains of mouse FGFR3
AB and FGFR3 (GenBankTM accession
numbers M81342, M61881, and A48991). Periods represent gaps
indicating the deletion of the acid box in mouse FGFR3
AB. The
extracellular domains are indicated with arrows. Open
box, the basic heparin binding site; shaded boxes,
cysteine residues at the base of the Ig loop. C, a schematic
diagram of mouse FGFR3
AB and mouse FGFR3. IgI, IgII, and IgIII,
Ig-like domains I, II, and III. The C-terminal half of the IgIII domain
is encoded by the IIIc exon.
AB has the potential to
stimulate the cell proliferation, and if so, whether it has different
ligand specificity from that of FGFR3, both isoforms were stably
expressed in BaF3 cells. BaF3 cells are a pro-B cell line that is
IL-3-dependent. They do not express endogenous FGFRs, but
when transfected with an FGFR cDNA, they exhibit a
dose-dependent proliferative response to FGFs (8, 36).
Following selection, clonal cell lines expressing FGFR3 or FGFR3
AB
were analyzed for their receptor expression and mitogenic response to
various FGF ligands. Receptor expression levels were assessed by
Western blotting (Fig. 7). The size of FGFR3
AB was not detectably different from that of FGFR3 (Fig. 7).
The predicted molecular mass of the mature receptors (FGFR3, 88 kDa;
FGFR3
AB, 86 kDa) differ only by ~2 kDa. Because FGFR3 has been
shown to be a glycosyl protein (43, 44), the molecular mass
(125 kDa) of both FGFR3 isoforms appeared to be larger than the
estimated one. From this experiment, we concluded that both cell lines
express comparable numbers of receptors. Both BaF3 cell lines could
proliferate in response to FGFs in culture conditions, despite the
absence of IL-3. The mitogenic response of both BaF3 cell lines was
compared at various concentrations of FGFs in the presence of 10 µg/ml heparin (Fig. 8). Heparin was
added to the cultures to substitute for the heparan sulfate
proteoglycans that determine the ligand specificity of each FGFR. In
the absence of exogenously added heparin, FGF cannot stimulate a
mitogenic response in BaF3 cells expressing FGFRs (8, 36, 42), because BaF3 cells do not express heparan sulfate proteoglycans, which support
the FGF·FGFR complex formation (45) and are expressed in a tissue- or
cell-specific manner (46). The parental BaF3 cells did not proliferate
in the presence of FGFs and heparin (data not shown). Among the FGFs
tested, FGFR3 preferentially responded to FGF1, whereas FGFR3
AB
exhibited a higher response to FGF9, FGF4, and FGF2 than to FGF1. To
assess the relative mitogenic activity of FGFs on each FGFR3 isoform
and to make comparisons between two isoforms, we normalized the data in
Fig. 8, A and B to that of FGF1. Because FGF1 is
known as a universal FGFR ligand, the mitogenic response to FGF1 was
used as a 100% control in each cell line (42). The relative mitogenic
response to each FGF was averaged at two different concentrations
(0.312 and 1.25 nM) to reduce the influence of experimental
error (Table II). FGFR3 shows a
22.7-fold, 2.9-fold, and 3.6-fold lower response to FGF2, FGF4, and
FGF8, respectively, than to FGF1. The differential response of FGFR3 to
FGF1 and FGF2 is in agreement with the previous paper, which showed
that mouse FGFR3 preferentially binds to FGF1 (36). In our study, the
response of FGFR3
AB to FGF2 and FGF4 was equal to that of FGF1
(105.2 and 112.4%, respectively). The response to FGF9 and FGF8 was
also increased compared with FGFR3 (FGF9, from 27.6 to 70.0%; FGF8,
from 63.2 to 119.2%). Next, we determined the heparin requirement of
FGFR3 and FGFR3
AB. Both FGFR3- and FGFR3
AB-expressing BaF3 cells
demonstrate an absolute requirement for heparin in their response to
FGF1 (Fig. 9). After 3 days of incubation
in the absence of heparin, the number of FGFR3
AB- and
FGFR3-expressing BaF3 cells decreased from 10,000 cells/well to
627 ± 142 cells/well and 1,187 ± 83 cells/well,
respectively. FGFR3
AB-expressing cells showed the half-maximum
response to FGF1 and FGF2 at a heparin concentration of 0.6 and 0.2 µg/ml, respectively. These results are consistent with the fact that FGF1 has a lower affinity for heparin than does FGF2 (47). In contrast,
FGFR3 cells showed the half-maximal response to FGF1 at a concentration of 2.8 µg/ml, and their response to FGF2 was poor
at these concentrations of heparin.
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Fig. 7.
Expression of FGFR3 and
FGFR3 AB in BaF3 cells. The cell membrane
fractions were electrophoresed on 6.0% SDS-polyacrylamide gel
electrophoresis. The expression of receptor proteins was determined by
Western blotting with anti-FGFR3 C terminus antibodies. C,
BaF3 cells untransfected;
AB, BaF3 cells expressing
FGFR
AB; FGFR3, BaF3 cells expressing FGFR3.
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Fig. 8.
Mitogenic response of BaF3 cells expressing
FGFR3 or FGFR3 AB stimulated with FGFs.
BaF3 cells expressing FGFR3 (A) or FGFR3
AB (B)
were plated in 24-well plates at a density of 1 × 104
cells/500 µl in RPMI 1640 containing 10% fetal bovine serum, and
then increasing concentrations of FGFs and 10 µg/ml heparin were
added. After 3 days, the cells were harvested, and the cell number was
counted by a Coulter Counter. In the graph, all data are the mean of
triplicate samples, and the number with no addition is subtracted from
all the data. The S.D. is shown with bars. Filled
squares, FGF1; filled circles, FGF2; filled
triangles, FGF4; filled diamonds, FGF6; open
squares, FGF8; and open circles, FGF9. The number of
BaF3 cells expressing FGFR3 obtained after the growth assay was as
follows: no addition, 987 ± 70 cells/well; heparin, 1,040 ± 131 cells/well; 5 nM FGF1 and heparin, 47,407 ± 395 cells/well. The number of BaF3 cells expressing FGFR3
AB was as
follows: no addition, 960 ± 35 cells/well; 10 µg/ml heparin,
1,427 ± 237 cells/well; 5 nM FGF1 and heparin,
38,487 ± 2,284 cells/well.
Relative mitogenic activity of FGFs 1, 2, 4, 6, 8, and 9
View larger version (14K):
[in a new window]
Fig. 9.
Heparin dependence for FGF1 and FGF2
mitogenic activity. BaF3 cells expressing FGFR3 (A) or
FGFR3 AB (B) were plated in 24-well plates at a density of
1 × 104 cells/500 µl with RPMI 1640 containing 10%
fetal bovine serum, and then increasing concentrations of heparin and
10 ng/ml (0.645 nM) FGF1 (filled squares) or 10 ng/ml (0.541 nM) FGF2 (filled circles) were
added. After 3 days, the cells were harvested, and the cell number was
counted by a Coulter Counter. The results are presented as a percentage
of the cell number in the presence of FGF1 and 10 µg/ml heparin. The
number in the absence of heparin is subtracted from all the data. The
numbers of BaF3 expressing FGFR3 obtained after the growth assay were
as follows: FGF1 without heparin, 1,187 ± 83 cells/well; FGF1 and
10 µg/ml heparin, 29,380 ± 1,512 cells/well; FGF2 and 10 µg/ml heparin, 1,247 ± 163 cells/well. The numbers of BaF3
cells expressing FGFR3
AB were as follows: FGF1 without heparin,
627 ± 142 cells/well; FGF1 and 10 µg/ml heparin, 44,173 ± 1,714 cells/well; FGF2 and 10 µg/ml heparin, 42,180 ± 1,367 cells/well.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
AB,
was expressed in ATDC5 cells. FGFR3
AB seemed to be generated by
alternative splicing that deletes exon 4 encoding the acid box (40).
FGFR3
AB was shown to be expressed in rat chondrocytes taken from
cartilage tissue, suggesting that this form is present in
vivo (Fig. 5B). It is interesting to determine whether
FGFR3 and FGFR3
AB have distinct biological activities and signal
transduction pathways in chondrocytes. The factors that control the
alternative splicing of FGFRs in a cell- and time-specific manner
should be identified in the near future.
AB and
FGFR3--
Binding of FGF and heparin to the receptor induces
FGF·FGFR dimerization and receptor-tyrosine kinase
autophosphorylation that initiates signal transduction events inside
the cell (24). The biological activity of FGFR3 is cell type-specific.
Activation of FGFR3 leads to cell proliferation (e.g. BaF3
cells and NIH3T3 cells), cell transformation (53), and inhibition of
chondrocyte proliferation (28). In this study, we chose BaF3 cells in
which to compare the biological activities of FGFR3
AB and FGFR3.
Both receptors mitogenically respond well to FGF1 and FGF9 in the
presence of 10 µg/ml heparin (Fig. 8). Heparin is required for high
affinity binding of FGF to the FGFR in cells that are unable to
synthesize cell surface heparan sulfate (45). Heparin interacts with
FGFs and independently of FGF ligand with a specific sequence of FGFR in the N-terminal region of the IgII domain, which is composed of a
cluster of basic and hydrophobic residues (K18K in FGFR1) (54). The
basic heparin binding site is well conserved among the four FGFRs and
corresponds to the sequence from Arg134 to
Arg151 in FGFR3 (Fig. 6B). The acid box is
located next to the heparin binding site. A dimeric model has been
deduced from the crystal structure of the bacterially expressed
extracellular domains IgII and IgIII of FGFR1 in complex with FGF2
(55). In this model, a positively charged canyon on the surface of the
dimeric receptors is formed by the heparin binding site on IgII.
Binding of heparin to the canyon stabilizes the dimeric structure of
the FGF·FGFR complex. This hypothesis is consistent with the results
of previous studies demonstrating that FGFR1 lacking IgI and the acid
box exhibited higher binding affinity for FGF and heparin (56). This
also agreed with our results indicating that FGFR3
AB requires lower
concentrations of heparin compared with FGFR3. The acid box may bind to
the basic heparin binding region on the IgII domain, thereby competing
with heparin for FGFR binding. It may also affect FGF·FGFR
interactions. These results suggest that the binding activity of FGFR3
to heparin may be modulated by the presence of the acid box and
that as a consequence the mitogenic activity of the receptor is regulated.
AB showed
higher responses to FGF2, FGF4, and FGF8 than did FGFR3 (Table II). The
low mitogenic response of FGFR3 to FGF2 is consistent with the results
of a previous study using BaF3 cells expressing the mouse full-length
FGFR3 (11). It has also been reported that a soluble FGFR3c did not
bind to FGF2 but bound to FGF1 (36). However, the mitogenic responses
of a chimeric receptor, which was engineered to contain the entire
extracellular domain of FGFR3 and the tyrosine kinase domain of FGFR1,
were equivalent to FGF1 and FGF2 (42). The mitogenic responses of
FGFR3
AB to FGF2 and other FGFs in our study were similar to those of
the chimeric receptor. Many studies have suggested that the ligand
binding domains reside in IgII and IgIII but do not include the acid
box (8, 56, 57). The increased responsiveness of FGFR3
AB to FGF2 may
be due to a conformational change caused by the complete deletion of
the acid box, because the protein folding of the ligand binding region
in FGFR3 may be strongly affected by the presence of the acid box.
Mapping ligand binding studies in chimeric FGFR1 and FGFR3 suggested a
two-binding region model, i.e. proximal and distal binding
sites (58). In this model, the distal binding site, the C-terminal half
of FGFR1 Ig domain IIIc, is important for FGF2 binding, and the
proximal binding site in FGFR1, the IgII loop and the IgI-IgII linker
region, is cooperatively linked to the distal binding site. In
contrast, the distal binding site in FGFR3 was poor for binding to
FGF2. This may account for the decreased binding of FGFR3 to FGF2. In
the crystal model mentioned above (55), FGF2 was shown to interact with
seven amino acids (Asp154, Lys155, and
Leu157-Pro161) within the basic heparin binding
site that were thought to be the responsible residues in the proximal
binding site. In addition, FGF8 and FGF9 were also shown to bind to a
broad region spanning the IgII loop and IgI-IgII linker sequence in
FGFR3. Thus, deletion of the acid box may enhance not only heparin
binding but also the ligands binding to the proximal binding site in
FGFR3. It is necessary to determine whether the increased mitogenic
responses of FGFR3
AB to FGF2 and other FGFs are due to the increased
ligand binding ability of this isoform, and if so, we should analyze the mechanism by which the acid box regulates FGF binding.
AB showed higher
sensitivity to FGF2 than did FGFR3. FGFR3
AB may stimulate the signal
cascades in mesenchymal cells during early development of the limb bud,
utilizing FGF2 that is also expressed in the mesenchyme (60). During
chondrogenesis, FGFR3
AB may modulate FGFR3 signaling by forming a
heterodimer between these two isoforms. Ligand specificity of FGFR
isoforms may also be regulated in a cell type-dependent
manner by the expression of specific cell surface heparan sulfate
proteoglycans (46, 61). Our results suggest that FGFR3
AB is likely
to be the preferred receptor for FGF under restricted heparan sulfate
proteoglycan conditions and that the acid box plays an important role
in the regulation of FGFR3 to mediate biological activities in response to FGFs. Therefore, it will be important to compare the functions of
these isoforms when they are expressed in prechondrocytes and chondrocytes.
AB and FGFR3 play different roles in regulation of the growth
and differentiation of chondrocytes.
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ACKNOWLEDGEMENTS |
---|
We thank M. Nitta for providing WEHI-3 medium and V. Silva for helpful discussion. V. Silva has joined our research group as an extramural year student from King's College of London, Department of Life Science.
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FOOTNOTES |
---|
* 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) AF024638.
To whom correspondence should be addressed. E-mail:
mseo@cc. kyoto-su.ac.jp.
Published, JBC Papers in Press, December 28, 2000, DOI 10.1074/jbc.M003535200
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ABBREVIATIONS |
---|
The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; IL-3, interleukin 3; FBS, fetal bovine serum; bp, base pair(s); kb, kilobase; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR.
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