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Vol. 13, No. 11, pp. 1361-1366, June 1, 1999
Departments of 1 Microbiology and 2 Pathology, New York University School of Medicine, New York, New York 10016 USA
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Abstract |
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 Top
 Abstract
 Introduction
Results and Discussion
Materials and methods
References
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Several genetic forms of human dwarfism have been linked to
activating mutations in FGF receptor 3, indicating that FGF signaling has a critical role in chondrocyte maturation and skeletal development. However, the mechanisms through which FGFs affect chondrocyte proliferation and differentiation remain poorly understood. We show
here that activation of FGF signaling inhibits chondrocyte proliferation both in a rat chondrosarcoma (RCS) cell line and in
primary murine chondrocytes. FGF treatment of RCS cells induces phosphorylation of STAT-1, its translocation to the nucleus, and an
increase in the expression of the cell-cycle inhibitor
p21WAF1/CIP1. We have used primary chondrocytes from
STAT-1 knock-out mice to provide genetic evidence that STAT-1 function
is required for the FGF mediated growth inhibition. Furthermore, FGF
treatment of metatarsal rudiments from wild-type and
STAT-1
/ murine embryos produces a
drastic impairment of chondrocyte proliferation and bone development in
wild-type, but not in STAT-1/
rudiments. We propose that STAT-1 mediated down regulation of chondrocyte proliferation by FGF signaling is an homeostatic mechanism which ensures harmonious bone development and morphogenesis.
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Introduction |
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Top
Abstract
Introduction
Results and Discussion
Materials and methods
References
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FGFs are a large family of fibroblast growth factors that signal
through their binding to specific tyrosine kinase receptors (FGFRs),
which also constitute a four-member gene family (Basilico and
Moscatelli 1992
). FGF signaling has a major role in a
variety of developmental processes, and recent results have highlighted its role in bone morphogenesis (for review, see Goldfarb 1996). Long
bone growth results from endochondral ossification, a strictly regulated process that requires proliferation and differentiation of
chondrocytes. Based on the evidence linking genetic forms of human
dwarfism such as achondroplasia (ACH), thanatophoric dysplasia (TD),
and hypochondroplasia to activating mutations in FGFR3, as well as from
the study of FGF transgenic (Coffin et al. 1995) or FGFR3 knockout mice
(Colvin et al. 1996; Deng et al. 1996), it has been suggested that FGFs
act as negative regulators of bone growth (Goldfarb 1996; Webster and
Donoghue 1997; Burke et al. 1998; Naski and Ornitz 1998). However, the
downstream events through which FGFs influence the proliferation or
differentiation of osteogenic chondrocytes remain to be elucidated. In
most cell types FGFs have a proliferative effect (Basilico and
Moscatelli 1992), and in vitro studies have shown that FGF treatment of
primary chondrocytes leads to an increase in cell proliferation and an inhibition of their differentiation (Kato and Iwamoto 1990; Hill et al.
1991; Wroblewski and Edwall-Arvidsson 1995; Legeai-Mallet et al. 1998),
a finding that appears to be at variance with the evidence from human
genetics. We therefore studied the biological response and the signal
transduction pathways activated by FGF treatment of primary murine
chondrocytes, as well as chondrocytic cell lines.
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Results and Discussion |
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Top
Abstract
Introduction
Results and Discussion
Materials and methods
References
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We used RCS cells, a rat
chondrosarcoma cell line that exhibits most
of the properties of proliferating chondrocytes. Among FGFRs these
cells express exclusively FGFR3 as well as other chondrocyte markers,
such as collagen II (not shown). We treated RCS cells with FGF1, a high
affinity ligand for all known FGFR isoforms (Ornitz et al. 1996).
Stimulation by FGF1 induces autophosphorylation of endogenous FGFR3
within 30 sec (Fig. 1a) and leads to the phosphorylation of downstream
molecules such as MAPK (Fig. 1c) and Shp-2 (not shown), which have been
reported to be activated in other cell types upon FGF stimulation
(Saxton et al. 1997). Surprisingly, FGF1 treatment
did not stimulate proliferation of these cells but resulted in a
drastic inhibition of growth, reflected in the frequency of
DNA-synthesizing cells observed in the cultures (Fig. 1b). DNA
synthesis inhibition was rapid and reached its maximum ~1 day after
treatment. No evidence of increased apoptosis was observed (not shown).
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It has been reported that a mutated form of FGFR3, carrying the
strongly activating TDII mutation, can induce STAT-1 phosphorylation and DNA binding in transient transfection assays in 293 cells (Su et
al. 1997). STAT-1, originally identified as a signal transducing molecule in the IFN pathway, is activated by tyrosine phosphorylation and translocated to the nucleus where it then acts as a transcription factor (Darnell 1997). Because STAT-1 function has been linked to
anti-proliferative effects, we tested whether FGF treatment of RCS
cells activated the STAT-1 pathway. As shown in Figure 1, c and d, a
significant increase in STAT-1 phosphorylation, as well as increased
nuclear translocation of this factor, was observed following FGF
treatment. FGF treatment had no effect on STAT-3 phosphorylation and
nuclear translocation (data not shown). The activation of STAT-1 in
chondrocytes could lead to the induction of expression of STAT-1 target
genes such as IRF1 and
p21WAF/CIP1, which are involved in
inhibition of cell growth (Abdollahi et al. 1991; Chin et al. 1996). We
therefore studied the effect of FGF1 treatment of RCS cells on the
activity of a transiently transfected tk promoter-luciferase
reporter construct containing four copies of STAT binding sites derived
from the IRF1 gene. Treatment with FGF1 results in sevenfold
induction of luciferase activity within the first 12 hr (Fig. 1e). In
addition, analysis of the levels of expression of p21 by Western
blotting shows that FGF1 treatment results in an increase in p21
expression, while the levels of MAPK remained unchanged (Fig. 1f). The
p21 increase upon FGF-1 treatment correlates well with inhibition of
cell proliferation, in agreement with previous reports (Chin et al.
1996) showing that activation of STAT-1 leads to increase in p21
expression and to growth arrest in nonchondrocytic cell lines. Taken
together, our results indicate that activation of FGFR3 and possibly
STAT-1 activation mediate the inhibitory effect of FGF on chondrocyte proliferation in vitro.
It has been reported that signaling through F6FR1 cannot induce STAT-1
activation (Silvennoinen et al. 1993). Thus, we considered it possible
that STAT-1 activation with its consequent inhibition of proliferation
is specific to FGFR3. To address this question, we used NIH-3T3 cells,
which express FGFR1 and FGFR2, either untransfected or stably
transfected with the FGFR3 ACH mutant (NIH-3T3 ACH). FGF1 has a
proliferative effect on these cells (Li et al. 1997; data not shown).
Immunoprecipitation of FGFR3 from RCS and NIH-3T3 ACH cells shows that
the level of expression of FGFR3 is similar in both cell types (Fig.
2a). As expected, no FGFR3 could be
immunoprecipitated from untransfected NIH-3T3 cells.
Autophosphorylation of FGFR3 is partially constitutive but can be
increased further by ligand in NIH-3T3 ACH, whereas in RCS
autophosphorylation occurs only in the presence of the ligand (Fig.
2a). Although the level of phosphorylation of FGFR3 is similar in RCS
and NIH-3T3 ACH cells, the extent of phosphorylation of STAT-1 is quite
different (Fig. 2b). In untransfected NIH-3T3 cells that do not express
FGFR3, there is weak phosphorylation of immunoprecipitated STAT-1
following FGF1 addition, possibly because of the activation of FGFR1
and/or FGFR2. This weak degree of activation is not
enhanced in NIH-3T3 ACH cells (Fig. 2b). Similar results were obtained
with NIH-3T3 cells expressing wild-type FGFR3 (not shown). On the other
hand, STAT-1 was phosphorylated much more strongly in RCS cells than in
untransfected NIH-3T3 or NIH-3T3 cells expressing FGFR3 (Fig. 2b).
Thus, the introduction of FGFR3 in NIH-3T3 fibroblasts does not lead to
a significant increase in FGF-induced STAT-1 activation. This suggests
that rather than FGFR3 being an inhibitory receptor, the specific cell
environment of chondrocytes favors STAT-1 activation and growth
inhibition. A conclusive demonstration of this point will, however,
require further research.
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To determine whether STAT-1 activation was directly related to the
inhibitory effects of FGFs on chondrocyte proliferation, we studied
primary growth plate chondrocytes isolated from 10-day-old wild-type
and STAT-1 knockout (
/) mice (Durbin et
al. 1996). These cells express both FGFR1 and FGFR3 (Fig.
3a). Primary chondrocytes were treated with FGF1 and
the rate of DNA synthesis measured by the frequency of cells
incorporating BrdU. Double staining with Alcian blue, a marker for
chondrocytic cells, allowed us to distinguish chondrocytes from
nonchondrocytic cells such as fibroblasts and osteoblasts. FGF1
treatment of wild-type chondrocyte resulted in a significant decrease
in chondrocyte DNA synthesis within 24 hr (Fig. 3b). In contrast,
contaminating cells such as fibroblasts, which typically represent
~15% of cultures, responded to FGF treatment by an increase in
their proliferation rate (data not shown). DNA synthesis in STAT-1
knockout chondrocytes was unaffected by FGF treatment, although these
cells express the same FGFRs as wild-type cells (Fig. 3).
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These results therefore indicate that FGF treatment of primary
chondrocytes results in inhibition of cell proliferation as observed in
RCS cells and that STAT-1 function is required to produce the observed
growth inhibitory effect. To further confirm the involvement of STAT-1
in chondrocyte proliferation, we performed organ culture of metatarsal
cartilage rudiments from E15 wild-type and
STAT-1/ mouse embryos. When
FGF1 was added to the metatarsals, only wild-type chondrocytes
exhibited a dramatic decrease in DNA synthesis, whereas the
chondrocytes from STAT-1/
showed a similar rate of DNA synthesis in the absence or presence of
FGF1 (Fig. 4). Furthermore, we show that long-term
treatment (7 days) of E15 metatarsals from wild-type embryos with FGF1
leads to a dramatic perturbance of their development (Fig.
5). When E15 wild-type metatarsals, which are
composed primarily of undifferentiated chondrocytes, are cultured for 7 days they undergo considerable longitudinal growth and development.
They show a well-organized growth plate composed of orderly columns of
proliferating chondrocytes that have undergone differentiation to
prehypertrophic and hypertrophic cells positive for type X collagen
(Col X), a specific marker for hypertrophic chondrocytes (Fig. 5b). In
contrast to the control metatarsals, the treatment of E15 cartilage
rudiments with FGF1 for 7 days results in the formation of bones that
are shorter and wider in their proximal and distal extremities (Fig.
5c). These metatarsals exhibit no organization of the growth plate, an
absence of columnar chondrocytes, and a considerable reduction of the
hypertrophic zone, as shown by staining for Col X. This demonstrates
that in addition to inhibiting proliferation, FGF affects the process
of the differentiation of chondrocytes.
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To verify whether STAT-1 also mediates the effect of FGF signaling on
bone development, we cultured E15 metatarsals from
STAT-1/ embryos for the same
length of time. In contrast to wild-type metatarsals, FGF treatment had
no effect on growth plate chondrocytes. In both untreated and treated
metatarsals, chondrocytes underwent proliferation, prehypertrophy, and
terminal differentiation to hypertrophic cells that expressed Col X
(Fig. 5e,f). However, FGF-treated
STAT-1/ metatarsals exhibited
some increase in length and width compared to controls, which could be
due to the unmasking of the growth stimulatory effects of FGF signaling
when the STAT-1-mediated growth inhibitory pathway is blocked. Thus,
treatment with FGF of E15 bone rudiments appears to mimic the
inhibition of endochondral ossification and bone development observed
in human fetuses with homozygous ACH or TD and in mouse models (Shah et
al. 1973; Stanescu et al. 1990; Naski et al. 1998; Li et al. 1999). We
show that this effect also requires STAT-1 function.
It is interesting to note that in these long-term experiments, FGF
treatment had no significant inhibitory effect on cells of the
osteoblastic lineage and, rather, seemed to increase their proliferation. This is quite evident in the FGF-treated metatarsals shown in Figure 5c, which display a dramatic thickening of the periosteum. This phenomenon was not observed in
STAT-1/ metatarsals (Fig. 5f),
suggesting either that the increased osteoblast proliferation is
mediated by STAT-1 or, more likely, this effect is at least partially
indirect and may require inhibition of formation of the hypertrophic zone.
The data presented in this report show that activation of FGF signaling
in primary chondrocytes, chondrocytic cell lines, and cartilage bone
rudiments from murine embryos results in significant inhibition of cell
proliferation and bone development. This result is in line with the
effect of activating FGFR3 mutations in several forms of human
dwarfism, particularly ACH and TD, but contrasts with previous reports
(Kato and Iwamoto 1990; Hill et al. 1991; Wroblewski and
Edwall-Arvidsson 1995; Legeai-Mallet et al. 1998) indicating that FGF
treatment of primary chondrocytes led to stimulation of cell
proliferation. This discrepancy could be due to the heterogeneous nature of the cell cultures studied. A recent report (Mancilla et al.
1998) showed that FGF2 treatment of rat E20 metatarsal rudiments
resulted in inhibition of DNA synthesis of proliferative and epiphyseal
chondrocytes. Furthermore, we show that FGF-mediated inhibition of
proliferation in chondrocytes requires STAT-1 function and that the
STAT-1 requirement may be related to its ability to induce
antiproliferative genes, such as
p21WAF/CIP1. Studies of signal
transduction in a variety of cell systems have shown that although many
signal transduction pathways are activated simultaneously by receptor
stimulation, it has been difficult to link a specific downstream target
with a specific biological response. Thus, this report presents one of
the few examples in which a signal transduction pathway, STAT-1
activation, has been shown to be required for a specific cellular
response, inhibition of chondrocyte proliferation. The mechanism of
STAT-1 activation by FGFR3 is currently being investigated. Our data suggest that the ability to activate STAT-1 depends on the cellular context in which FGFR3 is expressed, as the introduction of FGFR3 or of
its activating ACH mutation in fibroblasts did not lead to growth
inhibition or increased ability to activate STAT-1 in response to FGF.
It is interesting to note that
STAT-1/ mice have not been
reported to have bone defects, but this aspect has never been studied
in detail, particularly during embryonic development or during early
life, and is currently under investigation. These and related studies
on how FGF affects the differentiation program of cells of the
chondrocytic lineage should provide important information on the
mechanisms of bone morphogenesis and on the way by which unregulated
FGF signaling causes bone morphogenetic disorders.
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Materials and methods |
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Top
Abstract
Introduction
Results and Discussion
Materials and methods
References
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Cell culture and proliferation assay
RCS cells were maintained as monolayer cultures under conditions
described previously (Mukhopadhyay et al. 1995). Primary chondrocytes
were isolated from long bone cartilages of 10-day-old wild-type and
STAT-1/ mice. Growth plates
were dissected and the chondrocytes isolated as described previously
(Amling et al. 1997). The growth plates were incubated with 0.1%
collagenase type A (Sigma) for 30 min at room temperature with constant
shaking. Thereafter, the supernatant was discarded, a fresh solution of
0.2% collagenase was added, and the incubation was carried out for 3 hr at 37°C. Cells were filtered through a sterile nylon 0.45-µm
mesh, centrifuged for 10 min at 1000 rpm. Then cells were incubated at
37°C, in DMEM/F12 (1:1) medium supplemented with
10% FCS, 50 µg/ml ascorbic acid (Sigma), and 100 µg/ml sodium pyruvate. For proliferation assays, 5 × 104 cells/400 µl were seeded on
coverslips; for RNA extraction, 8 × 105
cells/3 ml were cultured in a 6-cm tissue culture dish.
The medium was changed every other day and FGF treatment was started at
day 4 of the culture. For DNA synthesis, RCS cells and primary
chondrocytes were incubated in the presence of 1 µg/ml BrdU for 6 hr. Cells were fixed, permealized,
and incubated with anti-BrdU antibody according to the manufacturer's
instructions (Boehringer Mannheim). Cells incorporating BrdU were
visualized and counted using fluorescence microscopy.
Organ Cultures
Metatarsal long bone rudiments from E15 wild-type and
STAT/ mouse embryos were dissected
under sterile conditions. The cartilaginous long bones were left
intact. Organ culture was carried out in 
-MEM without nucleosides
(GIBCO) supplemented with 50 µg/ml ascorbic acid, 300 µg/ml L-glutamine, 50 µg/ml gentamicine, 250 µg/ml
Fungizone, 1 mM 
-glycerophosphate, and 0.2% BSA Cohn
fraction V (Sigma) (complete medium). Each long bone was cultured
individually in 24-well plates containing 400 µl of complete medium
in the presence or absence of 200 ng/ml of recombinant
human FGF1 and 10 µg/ml heparin. The metatarsal
cultures were maintained at 37°C for either 48 hr or 7 days and the
medium changed every other day. The experiments were set up for
left/right paired observations, with one metatarsal serving as a control to the other. For the proliferation assay, the
long bones were treated with FGF for 48 hr and BrdU was added during
the last 6 hr of treatment. The long bones were fixed at day 0, 2, and
7 of the organ culture.
Immunohistochemistry
Metatarsals were fixed in 4% paraformaldehyde overnight at 4°C and embedded in paraffin, and 4-µm tissue sections were performed. Sections were deparaffinized by treatment with xylene plus 100%, 95%, and 70% ethanol, followed by washes in TS buffer (100 mM Tris/HCl at pH 7.4, 150 mM NaCl) and permealization with 0.25% Triton X-100. For localization of Col X, sections were treated with 1 mg/ml testicular hyaluronidase at 37°C for 45 min in a humidified chamber. After overnight blocking with 3% goat serum, the endogenous peroxidase was inactivated by incubating in 5% H2O2 in methanol for 10 min. Anti-BrdU monoclonal (Boehringer Mannheim) or anti-Col X polyclonal antibody was used with a Vectastain Elite ABC Kit (Vector Labs) to stain the cells. The color reaction (dark brown) was deduced with Sigma Fast DAB peroxidase substrate according to the manufacturer's manual.
Immunoprecipitation and Western blotting
Cells were stimulated with 100 ng/ml FGF1 and 10 µg/ml heparin at 37°C for various times, and cells were lysed either in RIPA buffer or HNTG buffer (50 mM HEPES at pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, and 1% Triton X-100) in the presence of protease and phosphatase inhibitors. Immunoprecipitations were performed with polyclonal anti-FGFR3 and anti-STAT-1 antibodies (Santa Cruz). Immunoprecipitates and total protein lysates were separated by SDS-PAGE and analyzed by ECL detection system (Amersham). A polyclonal antibody specific for the tyrosine phosphorylated form of STAT-1 (New England Biolabs) was used in Western blot analysis when indicated.
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Acknowledgments |
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We thank Drs. G. Inghirami and F. Gonzalez for their help in histological preparation, Dr. B.R. Olsen for providing the Col X antibody and Dr. R. Baron for the RCS cells. We thank Drs. M. Mohammadi and J. Schlessinger for providing human recombinant FGF1. This investigation was supported by a fellowship from the Arthritis Foundation to M.S. and by U.S. Public Health Service grant CA42568 from the National Cancer Institute.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked `advertisement' in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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[Key Words: FGF signaling; chondrocytic maturation; bone development; STAT-1]
Received March 17, 1999; revised version accepted April 13, 1999.
3 Corresponding author.
E-MAIL basilc01{at}mcrcr.med.nyu.edu; FAX (212) 263-8714.
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References |
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Top
Abstract
Introduction
Results and Discussion
Materials and methods
References
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1(II) collagen gene.
J. Biol. Chem.
270:
27711-27719
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) or absence (
) of FGF1 (10 ng/ml) and labeled
with BrdU for the times indicated after FGF addition. Each histogram
represents the average of three experiments; error bars represent
S.D.. (c) RCS cells were stimulated with FGF1 (100 ng/ml) and phosphorylation of STAT-1
(
was used as
control for STAT-1 nuclear translocation (top). (e)
Expression of an IRF promoter-driven plasmid is up-regulated by FGF in
RCS cells. Cells were transfected with 0.4 µg/106 cells of a plasmid expressing the
luciferase gene under the control of the tk promoter and four
copies of the STAT-1 binding sequences derived from the IRF-1
promoter (
