From the Unité de Recherches sur les Handicaps
Génétiques de l'Enfant, INSERM U393, Institut Necker,
75743 Paris Cedex 15, France
Thanatophoric dysplasia (TD) is a lethal skeletal
disorder caused by recurrent mutations in the fibroblast growth factor
receptor 3 (FGFR 3) gene. The mitogenic response of fetal TD I
chondrocytes in primary cultures upon stimulation by either FGF 2 or
FGF 9 did not significantly differ from controls. Although the levels of FGFR 3 mRNAs in cultured TD chondrocytes were similar to
controls, an abundant immunoreactive material was observed at the
perinuclear level using an anti-FGFR 3 antibody in TD cells.
Transduction signaling via the mitogen-activated protein kinase pathway
was assessed by measuring extracellular signal-regulated kinase
activity (ERK 1 and ERK 2). Early ERKs activation following FGF 9 supplementation was observed in TD chondrocytes (2 min) as compared
with controls (5 min) but no signal was detected in the absence of
ligand. By contrast ligand-independent activation of the STAT signaling
pathway was demonstrated in cultured TD cells and confirmed by
immunodetection of Stat 1 in the nuclei of hypertrophic TD
chondrocytes. Moreover, the presence of an increased number of
apoptotic chondrocytes in TD fetuses was associated with a higher
expression of Bax and the simultaneous decrease of Bcl-2 levels. Taken
together, these results indicate that FGFR 3 mutations in TD I fetuses
do not hamper chondrocyte proliferation but rather alter their
differentiation by triggering premature apoptosis through activation of
the STAT signaling pathway.
 |
INTRODUCTION |
Fibroblast growth factor receptor 3 (FGFR
3)1 belongs to a class of
tyrosine kinase receptors involved in signal transduction (1). In the
presence of soluble or cell-surface heparan sulfate proteoglycans,
fibroblast growth factor (FGF) binding to FGFRs induces receptor
dimerization and autophosphorylation on tyrosine residues thus
triggering cell proliferation or differentiation through the
Ras-Raf-dependent and phospholipase
C
-dependent signal transduction pathways involving MAPK
stimulation (2-4).
FGFR 3 mutations have been recently shown to account for achondroplasia
(5, 6), hypochondroplasia (7), and thanatophoric dysplasia (TD I and TD
II; 8, 9). Based on expansion of the cartilage growth plate in
Fgfr 3 null mice, FGFR 3 was regarded as a negative regulator of long bone growth during endochondral ossification (10,
11). Subsequent transfection experiments in immortalized cell lines
have shown that FGFR 3 mutations trigger constitutive activation of the
receptor in a ligand-independent manner (12-14). Activation of the
receptor would result in the recruitment of two different Grb2·Sos
adapter complexes leading to activation of the Ras-MAPK signaling
pathway (4), but activation of the STAT pathway via the nuclear
translocation of Stat 1 has been also demonstrated in chondrocytes of
TD II patients (15). Yet, the mechanisms leading to disorganization of
the growth plate cartilage in TD remains unclear and the question of
whether TD mutations interfere with chondrocyte proliferation or alter
terminal differentiation is still opened. In an attempt to address this issue, chondrocytes isolated from cartilage of TD I fetuses were grown
in primary cultures and used for proliferation assays, transduction signaling, and programmed cell death analyses. The normal mitogenic response of TD chondrocytes along with the high figure of apoptotic cells in TD fetuses suggest that FGFR 3 mutations alter differentiation rather than proliferation by inducing premature apoptosis of
chondrocytes.
 |
MATERIALS AND METHODS |
Cartilage Samples--
Tibial and/or femoral cartilage fragments
were obtained from medically aborted TD fetuses following the informed
consent of parents. In all cases, pregnancy was legally terminated
after ultrasonographic and x-ray detection of lethal dwarfism.
Histological studies of cartilage sections supported the diagnosis of
TD I. The control group included spontaneously aborted fetuses showing no evidence of skeletal abnormalities.
Antibodies and Ligands--
Rabbit polyclonal anti-FGFR 3, anti-FGFR 1, anti-Bax, and mouse monoclonal anti-PCNA antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and a
polyclonal anti-FGFR 3 antibody was kindly provided by Dr. A. Yayon
(Rehovot, Israel). A mouse monoclonal anti-Bcl-2 antibody was purchased
from Dako (Glostrup, Denmark). A rabbit polyclonal anti-MAPK antibody
was purchased from Sigma and the anti-active MAPK polyclonal antibody
raised against the dually phosphorylated region of MAPK was from
Promega. An anti-lysosome-associated membrane protein monoclonal
antibody was obtained from Pharmigen (San Diego, CA).
Monoclonal antibodies against the C-terminal domain of human Stat 1 and
against nucleoporin p62 were purchased from Transduction Laboratory.
The anti-mouse phospho-Stat-1 rabbit polyclonal antibody was from
Upstate Biotechnology.
The MAPKK/MEK-1 inhibitor, PD 98059, originated from New England
Biolabs (Beverly, MA). A monoclonal anti-
-tubulin antibody originated from Amersham. The anti-digoxygenin-AP, the substrate nitro
blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, and the in
situ cell death detection kit were purchased from Boehringer Mannheim. Human FGF 2, EGF, and mouse FGF 9 were purchased from Preprotech Inc. (Rocky Hill, NJ).
DNA Analysis--
Screening of FGFR 3 mutations on either white
blood cell or cultured skin fibroblast DNA was performed by single
strand conformation polymorphism and restriction analyses or by direct
sequencing of amplification products as described (16).
Chondrocyte Cultures and Proliferation Studies--
Cartilage
fragments were dissected free of perichondrium and cut into small
slices. Chondrocytes were released by hyaluronidase, trypsin, and
collagenase treatment (17). Primary cultures were initiated by plating
cells at high density to maintain their differentiated phenotype
(0.8-1.2 × 105 cells/cm2). For
proliferation studies, freshly isolated chondrocytes were plated on
96-well dishes (Falcon) in DMEM, supplemented with 10% fetal calf
serum and antibiotics and were allowed to reach subconfluency. Cells
were rinsed three times with serum-free DMEM containing 25 mM Hepes, pH 7.4, and incubated in the same medium at
37 °C for 16 h (or for times varying from 4 to 24 h) in
the presence of increasing concentrations of either FGF 2 or FGF 9 (10-100 ng/ml) at a final heparin concentration of 10 µg/ml.
Nonstimulated cells were incubated in serum-free DMEM with or without
heparin. In some experiments early confluent cells were synchronized by exposure to DMEM containing 0.1% fetal calf serum for 24 or 48 h.
Fresh serum-free DMEM supplemented with FGF 9 (100 ng/ml) was added and
chondrocytes were further incubated for 16 h before thymidine
supplementation. Cells were then washed twice with PBS and incubated
for 4 h in serum-free DMEM supplemented with
[3H]thymidine (ICN) at a concentration of 10 µCi/ml
(specific activity: 6.7 Ci/mmol) in a final volume of 100 µl. Cells
were harvested on a glass fiber filter paper and assayed for
radioactivity by liquid scintillation counting. Cells cultured under
identical conditions but in the absence of [3H]thymidine
were used to evaluate the number of cells per well.
Immunofluorescence Analyses--
Chondrocytes were plated on
tissue culture chamber slides (Nunc Inc.) at a density of 20,000 cells/chamber in DMEM (200 µl) supplemented with 10% fetal calf
serum, and allowed to attach for 4-5 days. Cells were rinsed twice
with PBS, fixed in 4% paraformaldehyde in PBS for 30 min, and
incubated for 30 min at 37 °C in the presence of 800 units/ml
hyaluronidase (Sigma). Permeabilization with Triton X-100 (0.1%) was
followed by incubation with the appropriate primary antibody for 1 h at room temperature. After washing three times with PBS-gelatin
(0.2%) and Triton X-100 (0.1%), the rabbit fluorescein isothiocyanate-labeled or mouse Cy3TM-conjugated second
antibody was added and the mixture was incubated for 1 h in a dark
room. Then cells were covered with mounting solution and examined with
a Leica microscope equipped for fluorescence.
Northern Blot and in Situ Hybridization Studies--
Total RNA
extraction from cultured chondrocytes, formaldehyde gel
electrophoresis, and blotting onto Hybond N membranes were performed as
described previously (17). Probes were labeled by random priming with
[
-32P]dCTP and hybridized to filters for 24 h at
42 °C. For in situ hybridization, sense and antisense
riboprobes were synthesized using either the Sp6 or the T3-T7 RNA
polymerases in the presence of dUTP-digoxygenin (Boehringer Mannheim).
Cells were prehybridized for 1 h at 70 °C, then hybridized
overnight with the purified probe at 70 °C. After washing, slides
were incubated for 1 h with an alkaline phosphatase-conjugated
anti-digoxygenin antibody. For visualization, the nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent was added and
incubated for 4-6 h until sufficient color develops. Probes included
(i) a COL2A1 cDNA cloned in pUC 18 (kindly provided by Dr. F. Ramirez, New York) and subcloned at the EcoRI site of a
Bluescript SK+ vector; (ii) a FGFR 3 cDNA probe
encompassing the Ig III loop and the transmembrane domain (nucleotides
867-1298) and cloned in a pCR 3 vector (In Vitrogen); (iii) a FGFR 1 cDNA probe (363 base pairs) encompassing part of the extracellular
and transmembrane domains (nucleotides 957-1320); and (iv) a rat
-actin cDNA clone (17).
MAPK and STAT Analyses--
Subconfluent primary chondrocyte
cultures were deprived of fetal calf serum overnight and incubated in
DMEM supplemented with 25 mM Hepes. FGF 2 (100 ng/ml) and
increasing concentrations of FGF 9 (1-100 ng/ml) were added in the
presence of heparin (10 µg/ml). Cells were incubated for 2 min to
4 h, then scrapped, resuspended in PBS, and centrifuged for 10 min
at 3,000 × g. Hepes lysis buffer (18) was added to
chondrocyte pellet (100 µl/106 cells). Samples were then
centrifuged at 20,000 × g for 30 min. The supernatant
containing the cytosolic extract were subjected to SDS-polyacrylamide
gel electrophoresis on 12% acrylamide, 0.12% bis-acrylamide gels
(19). Transfers to nylon membranes (Immobilon, Millipore Corp) were
performed according to standard procedures, and blots were sequentially
hybridized overnight at 4 °C in 5% nonfat milk and 0.1% Tween 20 with anti-MAPK polyclonal antibody and MAPK antibodies at a 1:5,000
final concentration. A second antibody coupled to peroxidase was added.
Bound protein was detected by chemiluminescence (ECL, Amersham). For
analysis of the STAT pathway, nylon membranes were sequentially
hybridized overnight at 4 °C with anti-phospho Stat 1 and anti-Stat
1 antibodies at respective dilutions of 1:1,000 and 1:5,000.
Analysis of Apoptotic Cells and Apoptotic
Factors--
Immunohistochemical detection of apoptosis in primary
cultured chondrocytes was achieved according to the terminal
deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)
assay using an in situ cell death detection kit (Boehringer
Mannheim). Briefly, fragmented DNA and apoptotic bodies were labeled
with fluorescein 12-dUTP using terminal deoxynucleotidyl transferase.
Incorporated fluorescein was visualized by incubation with an
anti-fluorescein antibody conjugated with alkaline phosphatase. After
chromogenic substrate reaction, apoptotic and non-apoptotic cells were
counted under a light microscope. For detection of anti- and
pro-apoptotic factors, Western blots were hybridized with anti-Bcl-2
and anti-Bax antibodies at respective dilutions of 1:200 and 1:500. The
blots were rehybridized with an anti-
-tubulin antibody for
quantitation (1:10,000 dilution).
Immunohistochemical Techniques--
Tibial cartilage fragments
from TD fetuses and age-matched controls were fixed with 4%
paraformaldehyde and embedded in paraffin. Serial sections were stained
with hematoxylin-eosin or pretreated with hyaluronidase (800 units/ml)
for 30 min at 37 °C, then incubated for 1 h with an antibody
against PCNA (a 36-kDa auxiliary protein of DNA polymerase
) at a
1:50 dilution for assessment of cells undergoing proliferation. Other
paraffin sections were incubated for 1 h with an anti-Stat 1 monoclonal antibody (1:100 dilution). Sections were then reacted with
horseradish peroxydase-conjugated secondary antibody and
diaminobenzidine was used as a substrate for visualization (Boehringer
Mannheim).
 |
RESULTS |
FGFR 3 Mutations--
Single strand conformation polymorphism and
restriction analyses of the coding sequence of the FGFR 3 gene led to
the detection of deleterious mutations in 14/14 TD I fetuses. Mutant
genotypes included the R248C (8/14), S249C (2/14), J807G (1/14), and
Y373C (3/14) mutations (Table I).
View this table:
[in this window]
[in a new window]
|
Table I
Summary of FGFR 3 mutations and analytical methods used on primary
cultured TD chondrocytes and cartilage samples
Twenty age-matched control fetuses (gestational age ranging from 16 to
30 weeks) with no clinical or radiological signs of skeletal defects
were used for comparison.
|
|
Phenotypic and Genotypic Characterization of Human Cultured
Chondrocytes--
When plated at a density of 8 × 104 cells/cm2, control and TD chondrocytes
reached subconfluency in 5 days. In situ hybridization of
cultured chondrocytes with an antisense COL2A1 riboprobe gave a strong
signal in most control and TD cells (Fig.
1, a and b), demonstrating that growing normal or TD chondrocytes at a high density
on plastic support did not result in a rapid cell dedifferentiation to
a mesenchymal state. Consistently, Northern blot analysis of chondrocyte mRNAs with
1(II) cDNA probe showed high levels
of collagen type II gene expression in both control and TD chondrocytes (Fig. 2). Subsequent hybridization of TD
cells with an antisense FGFR 3 riboprobe revealed a faint signal which
matched that found in control chondrocytes (Fig. 1, c and
d). Northern blot analysis revealed low levels of FGFR 3 IIIc mRNAs and high levels of FGFR 1 mRNA transcripts in both
control and TD chondrocytes (Fig. 2), while FGFR 3 IIIb and FGFR 2 mRNAs were undetectable (not shown).

View larger version (148K):
[in this window]
[in a new window]
|
Fig. 1.
In situ hybridization of control and TD
chondrocytes with riboprobes specific of COL2A1 and FGFR 3 IIIc
mRNAs. Primary cultured chondrocytes of control (a
and c) and TD I fetuses (b and d) were
grown to subconfluency and hybridized with a COL2A1 (a and
b) or a FGFR 3 IIIc antisense riboprobe (c and
d). Sense probes gave no signal (not shown).
Bars = 20 µm.
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2.
Northern blot analysis of cultured
chondrocyte RNAs with COL2A1, FGFR 1, FGFR 3 (IIIc isoform), and
-actin cDNA probes. a, RNA from 15- (lane
1) or 22-week-old control fetal chondrocytes (lane 3),
19- (lane 2) or 23-week-old TD chondrocytes (lane
4). b, RNA from control chondrocytes in primary
cultures (lanes 1 and 3) or after one passage
(lane 2) and RNA from TD chondrocytes in primary culture
(lanes 4 and 6) or after one passage (lane
5).
|
|
Localization of FGFR 3 Proteins in TD Chondrocytes--
The
subcellular localization of FGFR 3 proteins in cultured chondrocytes
was investigated by using an antibody raised against the C-terminal end
of the protein. In control cells, immunofluorescence was mostly visible
in the cytoplasm and a faint stain ring was occasionally seen around
the nucleus. Surprisingly, counterstaining of nuclei with
4',6-diamidino-2'-phenylindole dihydrochloride revealed a strong
perinuclear staining in 40-50% of TD I chondrocytes regardless of the
FGFR 3 mutations (Fig. 3,
d-f). This distribution was not altered by a overnight serum
deprivation or by FGF 2 or FGF 9 stimulation (5-30 min). In an attempt
to confirm the perinuclear localization of FGFR 3 in TD cells, double
immunostaining experiments were performed with both anti-FGFR 3 (green) and anti-nucleoporin antibodies (red).
Co-localization of the receptor with nucleoporin, a marker of the
nuclear membrane, was demonstrated by the appearance of an intermediate
color (yellow) around the nucleus (Fig. 3, g-i).
By contrast, double immunostaining with antibodies against FGFR 3 and
lysosome-associated membrane protein 2 (a lysosomal marker), only
revealed a partial overlap of the signals suggesting that mutant FGFR 3 was not preferentially retained in lysosomes for degradation (Fig. 3,
j-l). Finally, staining with an antibody directed against
the mannose 6-phosphate receptor, a specific marker of the late
endosomes showed no evidence for co-localization of the two receptors
(not shown).

View larger version (82K):
[in this window]
[in a new window]
|
Fig. 3.
Immunofluorescent staining of control and TD
chondrocytes in primary culture. a, b, and c,
staining of control (a) and TD chondrocytes (b
and c) using a rabbit polyclonal anti-FGFR 3 antibody
(dilution 1:50) at a low magnification (a and b;
bar = 20 µm) and at a higher magnification
(bar = 80 µm). d-f, staining and
counterstaining of chondrocytes from a TD I fetus (patient 2) with an
anti-FGFR 3 antibody (d), with
4',6-diamidino-2'-phenylindole dihydrochloride (e) or both
(f), bar = 80 µm. g-i,
immunostaining of TD chondrocytes with an anti-FGFR 3 antibody
(g), an anti-nucleoporin antibody at a 1:300 dilution
(h), and both (i). Overlapping of
green and red signals results in a
yellow pattern (i), bar = 80 µm. j-l, immunostaining of TD chondrocytes with an
anti-FGFR 3 antibody (j), an anti-lysosome-associated
membrane protein-2 antibody at a 1:400 dilution (k) and both
(l), bar = 80 µm.
|
|
Mitogenic Response of TD Chondrocytes--
Our observation that
cultured chondrocytes expressed both FGFR 1 and FGFR 3 suggested that
either FGF 2 or FGF 9 could be used to measure cell proliferation,
especially as they efficiently bind FGFR 1 and FGFR 3 IIIc,
respectively (20-22). Consequently, subconfluent control chondrocytes
were first incubated in the presence of FGF 9. A peak of thymidine
incorporation was noted after a 16-24-h FGF 9 stimulation (Fig.
4a). In subsequent
proliferation studies, FGF-supplemented cells were incubated for
16 h prior to thymidine uptake measurement. Then we analyzed the
influence of synchronization on the proliferative activity of control
and TD chondrocytes. Cells were serum-deprived for 24 or 48 h
before supplementation of FGF 9 ligand. While FGF 9 had a significant mitogenic effect on non-deprived cells, serum depletion for 24 or
48 h resulted in a marked reduction of thymidine incorporation (Fig. 4b). In the presence of heparin, cells exhibited a
dose-dependent proliferative response which revealed a
maximum stimulation for FGF 2 or FGF 9 concentrations ranging from 50 to 100 ng/ml (Fig. 4c). The proliferation of chondrocytes
from TD I fetuses harboring various FGFR 3 mutations was compared with
age-matched control cells. TD chondrocytes showed a magnitude of
stimulation by FGF 2 and FGF 9 that did not significantly differ from
that of controls (Fig. 4d). Binding experiments with
125I-FGF 2 indicated that dissociation constants
(Kd) of control or mutant cells were in the same
range (1.2 ± 0.4 × 10
9 M and
0.8 ± 0.2 × 10
9 M for control and
TD chondrocytes, respectively).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
Mitogenic effects of FGF 2 and FGF 9 on cultured chondrocytes from control and TD I fetuses. a,
effect of increasing incubation times with FGF 9 (100 ng/ml) on the
[3H]thymidine incorporation of control chondrocytes (C1,
22 weeks). b, effect of serum deprivation on the mitogenic
response of control to FGF 9 stimulation (C2, 24 weeks) and TD
chondrocytes (patient number 14). Cells were nondepleted or
serum-depleted for 24 or 48 h, then incubated with FGF 9 (100 ng/ml) for 16 h before thymidine supplementation. c,
dose-response effect of FGF 2 and FGF 9 on control chondrocytes (C3, 17 weeks). d, mitogenic activities of FGF 2 and FGF 9 on
chondrocytes from two different TD fetuses (patients numbers 11 and 12)
and two age-matched controls (C4, 24 weeks; C5, 25 weeks). Heparin was
supplemented to culture medium at a final concentration of 10 µg/ml
and cells were counted at the end of the incubations. Bars
represent the mean ± S.D. for eight replicate incubations. ,
DMEM; , heparin; , FGF 2; , FGF 9.
|
|
Activation of the MAP Kinase Pathway in TD Chondrocytes--
Since
FGFR 3 mutations are expected to produce a constitutive receptor
activation and signal transduction in TD, phosphorylation of the MAPK
(ERK 1 and 2) via the MAPK activation pathway was tested using an
antibody against phospho-ERKs. In the absence of ligand, no
phosphorylated MAPK was detected in both control and TD chondrocytes.
Addition of FGF 9 to the culture medium (5-30 min) resulted in the
detection of phosphorylated ERK 1 and 2 in both cell types but the
intensity of the signal in TD was stronger than in control chondrocytes
(Fig. 5, a and b).
Indeed, a short FGF 9 stimulation (100 ng/ml for 2 min) allowed to
detect a specific signal in mutant but not in control chondrocytes
(Fig. 5c). Although ERK phosphorylation peaked only 5 min
after ligand supplementation, the specific signal was sustained for
more than 2 h in TD cells (Fig. 5d). By contrast,
stimulation with EGF resulted in a rapid ERK phosphorylation in both
cell types which rapidly decreased after 30 min and was almost
undetectable after 1 h (results not shown). Further evidence of
MAPK pathway involvement upon binding of FGF 9 to FGFR 3 was given by
abolition of the FGF 9-dependent MAPK phosphorylation by a
MEK 1 inhibitor, PD98059, both in control and mutant chondrocytes (not
shown). Finally, evidence that MAPK stimulation in TD cells was
mediated by mutated FGFR 3 proteins stemmed from experiments using EGF.
While adding FGF 9 to the culture medium increased MAPK
phosphorylation, stimulating the EGF receptor with EGF in either
control or TD chondrocytes had similar effects on the level of
MAPK phosphorylation (Fig. 5e).

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 5.
Immunodetection of the activated MAP kinase
ERK 1 (p44) and ERK 2 (p42) in control and TD chondrocyte cell
lysates. a, activation of MAP Kinases by increasing
concentrations of FGF 9 in control chondrocytes. b,
comparison of MAP kinase activation by FGF 9 in chondrocytes from
control and TD fetuses carrying two different FGFR 3 mutations (patient
numbers 2 and 5). FGF 9 was supplemented for 30 min at a final
concentration of 100 ng/ml. c, time course of MAP kinase
activation by FGF 9 (100 ng/ml) in control and TD chondrocytes (patient
number 3). d, duration of MAPK activation in control and TD
chondrocytes (patient number 1) by FGF 9 (100 ng/ml). e,
activation of MAPK by FGF 2 and EGF as compared with FGF 9 in control
and TD chondrocytes (patient number 6).
|
|
Activation of the STAT Pathway in Cultured TD
Chondrocytes--
The possible involvement of the STAT pathway in the
signal transduction of mutant chondrocytes was tested by immunoblotting methods. Whole cell extracts were incubated with an anti-phospho-Stat 1 antibody so as to detect the activated form of the transcription factor. A significant 92-kDa band was observed in the absence of ligand
stimulation in TD cells while no detectable signal was noted in control
cells (Fig. 6a). FGF 9 supplementation had no effect on Stat 1 phosphorylation.
Rehybridization of nylon membranes with anti-Stat 1 and
anti-
-tubulin antibodies as internal standard revealed slightly
higher amounts of Stat 1 in TD cells than in control cells (Fig. 6,
b and c).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 6.
Immunoblot analysis of Stat 1 activation in
TD chondrocyte lysates. A, detection of phosphorylated Stat
1 with anti-phospho-Stat 1 antibody in TD (lanes 3-6,
patients numbers 1 and 5) and control chondrocytes (lanes 1, 2, and 7). B, Stat 1 levels in control and
TD chondrocytes. C, detection of -tubulin for protein
quantitation.
|
|
Immunohistochemical Analyses of TD Cartilage Growth
Plates--
The cartilage growth plates of control and TD I fetuses
were tested using immunohistochemical methods. Staining of control cartilage with an anti-Stat 1 antibody disclosed a faint signal in the
cytoplasm of hypertrophic cells while a positive staining of the nuclei
was noted in TD hypertrophic chondrocytes thus confirming Stat 1 activation in TD cells (Fig. 7,
c-f). Control chondrocytes in the resting and proliferative
zones stained positively for PCNA, a marker of S-phase cells, whereas
normal hypertrophic cells displayed no immunoreactivity. In contrast,
the nuclei of some TD chondrocytes located in the hypertrophic zone
stained positively with PCNA identifying them as proliferative cells
(Fig. 7, a and b).

View larger version (95K):
[in this window]
[in a new window]
|
Fig. 7.
Detection of PCNA (a and
b) and Stat 1 (c-f) by
immunohistochemical staining of hypertrophic chondrocytes from control
and TD cartilage. a, absence of PCNA expression in the
hypertrophic zone of control cartilage. b, positive PCNA
staining of the nuclei in hypertrophic TD chondrocytes (patient number
3); bars = 70 µm. c and e, Stat 1 expression in the cytoplasm (brown staining) of control
hypertrophic cells at a low (c) and high (e)
magnification. d and f, Stat 1 expression in the
nuclei of hypertrophic TD cells (patient number 2) at low
(d) and high magnification (f); the brown
nuclear staining is indicated by white arrows.
bars: c and d = 50 µm; e
and f = 15 µm.
|
|
Analysis of Apoptotic Cells--
Subconfluent chondrocytes were
labeled for DNA fragmentation using the TUNEL assay. Control cells from
three different fetuses gave no staining while approximately 1-2% of
TD cells derived from three different patients stained positively,
regardless of the location of mutations (Fig.
8). Comparison of Bcl-2 and Bax levels
using Western blotting indicated that Bax expression in chondrocytes
derived from five TD patients was higher than in controls. On the other
hand, the Bcl-2 was relatively abundant in control chondrocytes but
almost undetectable in TD cells (Fig. 9).
Although variations were noted between TD samples, the Bcl-2/Bax ratio
was consistently reduced.

View larger version (131K):
[in this window]
[in a new window]
|
Fig. 8.
TUNEL staining of control and TD cultured
chondrocytes. a, phase-contrast microscopy of TD
chondrocytes (patient number 5) at subconfluency (bar = 20 µm). b, TUNEL-stained control chondrocytes
(bar = 20 µ,). c, TUNEL-stained TD
chondrocytes (bar = 20 µm). d, higher
magnification of an apoptotic TD chondrocyte (bar = 80 µm).
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 9.
Immunoblot analysis of the apoptotic factors
Bax and Bcl-2 in TDI and control chondrocyte cell lysates. Blots
were probed with anti-Bcl-2, anti-Bax, and anti- -tubulin antibodies.
Cell lysates were prepared from five different TD chondrocyte cultures
(lane 1, patient number 1; lane 2, patient number
2; lane 5, patient number 3; lane 6, patient
number 5; lane 7, patient number 6) and three age-matched
control chondrocyte cultures (lanes 3, 4, and
8).
|
|
 |
DISCUSSION |
In an attempt to elucidate the functional consequences of FGFR 3 mutations on endochondral ossification, primary cultured chondrocytes
from control and TD I fetuses and the growth plate of cartilage
sections were studied. We first investigated the pattern of FGFR gene
expression in cultured fetal chondrocytes. Northern blot and in
situ hybridization experiments on normal and TD chondrocytes
detected comparable levels of FGFR 3 gene expression. Similar results
have been obtained with fetal cartilage sections (23). FGFR 1 was more
strongly but equally expressed in both cell types, while no FGFR 2 gene
product was detected. These results are consistent with previous
in situ hybridization studies which have shown that (i) the
mouse Fgfr 3 gene is mainly expressed in the resting and
proliferative zones of cartilage (10, 24), (ii) Fgfr 1 predominates in hypertrophic chondrocytes, and (iii) Fgfr 2 is absent from cartilage and expressed in the periosteum and
perichondrium only (25, 26).
We subsequently investigated the proliferative capacities of primary
cultured chondrocytes from TD I fetuses carrying the R248C and S249C
mutations. Mean TD chondrocyte density at subconfluency did not
significantly differ from that of age-matched controls. Similarly,
mitogenic responses of control and mutant cells were in the same range,
irrespective of the ligand tested. Serum deprivation equally affected
the mitogenic response of both control and TD cells. Taken together,
our data suggested that FGFR 3 mutations did not alter the
proliferative capacities of resting chondrocytes and that the defective
growth of the long bones in TD is more likely related to an abnormal
cell differentiation than to a lack of chondrocyte proliferation during
fetal development. These results are at variance with in
vitro studies on hematopoietic BaF3 cells transfected with a
mutant FGFR 3 cDNA carrying the R248C mutation. Indeed, the
transfected cells failed to respond to mitogenic stimulation by FGF 1, while they actively proliferated in the absence of FGF (13). These
discrepancies might be ascribed to (i) differences between the cell
types and/or to (ii) homozygosity for the mutation in transfected cells
(while heterozygous mutations are present in TD chondrocytes).
In normal chondrocytes as in transfected cells, FGF-mediated FGFR
activation induced signal transduction and triggered stimulation of the
MAPK pathway (3, 4). At variance with the transient MAPK activation by
EGF, FGF 9 stimulation resulted in a sustained MAPK activation,
suggesting that differentiation rather than proliferation is involved
(27). Studying the possible constitutive activation of the receptor in
TD chondrocytes indicated that FGF 9 supplementation produced a faster
phosphorylation of MAPK/ERKs in mutant cells than in controls. However,
no stimulation was detected in the absence of ligand as observed in
PC12 cells transfected with a chimeric mutant receptor carrying the
G375C achondroplasia mutation (28). Our inability to detect ERK
phosphorylation in the absence of FGF should be ascribed to (i) the low
receptor number, (ii) the low kinase activity of FGFR 3 in primary
cultured chondrocytes, and/or (iii) the ability of FGFR 3 to
heterodimerize with FGFR 1. Alternatively, one can hypothesize that the
MAPK pathway is not the only FGFR signaling pathway. A highly
controlled balance between the MAPK and STAT pathways has been recently
demonstrated in growth factor-stimulated cells (29). The observation of
a Stat 1 activation by the recurrent K650E FGFR 3 mutation in TD II
gives support to the view that this pathway might also play a key role
in growth retardation (15). In keeping with this, we have observed a
significant ligand-independent Stat 1 phosphorylation in cultured TD I
chondrocytes and a Stat 1 nuclear staining in hypertrophic chondrocytes
of TD I cartilage growth plate while a cytoplasmic staining only
occurred in control cartilage cells.
Although FGFRs transfected cells have shown immunostaining patterns
consistent with a plasma membrane localization of receptors (12, 30), a
perinuclear localization of FGFR 1 in FGF 2-stimulated ovine epiphyseal
growth plate chondrocytes or FGF 1-stimulated NIH 3T3 cells has been
also reported (31, 32). Moreover, nuclear localization of FGFR 1 in FGF
2-stimulated Swiss 3T3 fibroblasts and accumulation of a truncated FGFR
3 form in the nucleus of breast epithelial cells have been demonstrated
(30, 33). Thus, our detection of a perinuclear staining in cultured TD
I chondrocytes with anti-FGFR 3 antibodies is not totally unexpected
and seems consistent with the perinuclear localization of FGFR 3 in the cartilage growth plate of TD patients (23). If one hypothesizes that
ligand-independent FGFR 3 homodimerization in TD cells mimics the
ligand stimulation occurring in 3T3 cells, it is conceivable that FGFR
3 mutations triggered the translocation of the receptor from the cell
surface to the juxtanuclear region (32, 34). Indeed, the
co-localization of anti-FGFR 3 and anti-nucleoporin antibodies in the
perinuclear compartment of TD chondrocytes gives support to this
assertion and tends to exclude accumulation of the mutant receptor in
the lysosome/endosome system for degradation.
The growth of tubular bones normally involves chondrocyte proliferation
and differentiation into hypertrophic cells followed by replacement of
cartilage by bone tissue. Indeed, terminally differentiating
hypertrophic chondrocytes are known to undergo cell death through
apoptosis (35, 36). The reduced length growth of long bones in TD
fetuses could result from defective chondrocyte proliferation,
premature terminal differentiation, or both. Our observation that
chondrocytes from TD fetuses proliferated normally suggests that
defective terminal differentiation of chondrocytes is involved. In
keeping with this, a marked reduction in the proportion of hypertrophic
chondrocytes expressing collagen type X, a specific marker of terminal
differentiation, has been observed in the growth plate of TD I patients
(23) and chondrocytes expressing PCNA were found in the hypertrophic
zone of TD cartilage. Interestingly, activation of the STAT signaling
pathway in EGF-treated cancer cell lines has been proved to induce
apoptosis (29). Hence, the Stat 1 activation in TD I hypertrophic
chondrocytes along with the occurrence of apoptotic cells (1-2%) and
the reduced Bcl-2/Bax ratio in TD I chondrocytes strongly suggest that
apoptosis is involved in defective endochondral ossification. Indeed,
programmed cell death of hypertrophic cells in normal mouse cartilage
has been recently shown to be controlled by balanced levels of Bcl-2 and Bax, two members of a survival gene family with opposite effects (37). The decrease in Bcl-2 levels are expected to promote cell death,
possibly through Bax homodimerization (38). Alternatively, stimulated
expression of caspases (the ICE family of proteases, Ref. 39) through
Stat 1 activation could also participate to the apoptotic pathway (15,
40). In any case, increased chondrocyte apoptosis would result in
reduced bone elongation. The previous demonstration that overexpression
of a dominant negative mouse FGFR 1 mutation suppressed apoptosis (41)
gives support to the view that FGFR mutations producing a gain of
receptor function increase apoptosis.
We thank Dr A. Yayon for providing anti-FGFR
3 antibodies, Drs. P. Freisinger, M. Gonzales, and J. Roume for
providing fetal cartilage samples, and Monique Dailhat for help in
preparing this manuscript.