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From INSERM U 349 Affiliated CNRS, Lariboisière Hospital, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß,
Fas, and Bax, and decreased Bcl-2 levels. Specific
neutralizing anti-IL-1 antibody reduced Fas levels, Bax
expression, effector caspases activity, and apoptosis
in mutant cells. Thus, the Apert S252W FGFR-2 mutation promotes
apoptosis in human osteoblasts through activation of protein kinase
C, overexpression of IL-1 and Fas, activation of
caspase-8, and increased Bax/Bcl-2 levels, leading to
increased effector caspases and DNA fragmentation. This identifies a
complex FGFR-2 signaling pathway involved in the premature apoptosis
induced by the Apert S252W FGFR-2 mutation in human calvaria
osteoblasts.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis is an important component involved in normal and pathological osteogenesis.17 Pathological abnormalities in cell death may result from alteration of diverse targets. In mammalian cells, apoptosis is a multiple step process implicating upstream induction phases and downstream execution stages.18-20 Upstream events involve inducing signal transduction cascades and activation of intracellular molecules. Downstream events in the apoptotic cascade involve release of proteins from mitochondria and activation of proteases and nucleases leading to DNA degradation and ultimately to cell death.21 One known upstream apoptotic pathway implicates Fas receptor (APO-1/CD95), a member of the tumor necrosis factor receptor family characterized by a death domain in the cytoplasmic region.22 Activation of Fas by Fas ligand results in receptor aggregation and triggers recruitment of Fas-associated death domain protein, allowing recruitment of caspase-8 pro-enzyme, activation of caspase-8, and subsequent downstream caspases, including caspase-3, leading to cell death.23,24 Caspases are a family of cysteine proteases that are activated in proteolytic cascades during cell death.25,26 Initiator caspases (caspases-2, -8, -9, -10) either directly or indirectly activate downstream effectors (caspases-3, -6, -7) that cleave intracellular substrates during the execution phase of apoptosis. Another upstream pathway that plays a central role in controlling cell death involves the apoptotic promoter Bax family and the inhibitory protein family Bcl-2.27,28 The heterodimerization of these molecules leads to balance apoptotic signals through activation of caspases. In osteoblasts, apoptosis was recently found to involve both Fas29,30 and Bax/Bcl-2 levels.12,31,32
The molecular mechanisms that are downstream of FGF/FGFR interactions and that cause apoptosis in osteoblasts remain primarily unknown. In differentiating murine calvaria osteoblasts, the apoptotic effect of FGF is associated with increased Bax level and delayed Bcl-2 accumulation.12 In other cell types, FGF-2 promotes apoptosis33,34 in part by down-regulating Bcl-2.34 Nothing is known about the signaling cascades that are involved in apoptosis in pathological cranial osteogenesis in humans. In this study, we have determined the effect of the activating Apert S252W FGFR-2 mutation on apoptotic cell death in human osteoblasts in vivo and in vitro, and we examined the mechanisms and signaling pathways involved in apoptosis in mutant cells. Our data indicate that the FGFR-2 Apert mutation triggers premature apoptosis in human mutant osteoblasts through PKC-dependent pathways involving interleukin (IL)-1, Fas, caspase-8, and Bax/Bcl-2 that converge to activate effector caspases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Calvaria samples were obtained from two aborted normal and two Apert 26-week-old fetuses in accordance with the French Ethical Committee recommendations. Mutation analyses performed by single-strand conformation polymorphism and restriction analyses of the coding sequence of the FGFR-2 gene revealed the S252W mutation, the most frequent mutation in Apert syndrome, in the two Apert fetuses.9 To examine apoptosis in situ, coronal sutures obtained from one Apert and one control fetus were fixed in 10% formaldehyde, decalcified in 4.1% ethylenediaminetetraacetic acid (EDTA) for 3 weeks at 37°C, and embedded in paraffin as described previously.35
Cell Cultures
To examine apoptosis and the underlying cellular mechanisms in vitro, normal and mutant calvaria cells, obtained by collagenase digestion from the coronal suture in one Apert and one control fetus as described previously,35 were immortalized by transfection with the original defective large T antigen of the SV-40 oncogene and called Apert (Ap) and control (Co) fetal cells.9 The phenotypic characteristics induced by the mutation in these cells have been recently described.9-11 Briefly, mutant Ap cells display increased expression of osteoblast marker genes and increased in vitro osteogenesis compared to normal Co cells, a phenotype that is similar to the pathological feature observed in the mutant suture in vivo.10 Ap and Co cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with glutamine (292 mg/L), 10% heat inactivated fetal calf serum, and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin).
Cell Viability
To evaluate cell viability in vitro, trypan blue staining was used for determination of dead cells by dye exclusion. After addition of trypan blue [0.4% in phosphate-buffered saline (PBS)], the percentage of Ap cells and Co cells exhibiting both nuclear and cytoplasmic trypan blue staining (nonviable cells) was determined. A total of 1500 cells per well were counted for each cell type and the results were expressed as percentage of total cells.
Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling (TUNEL) Analysis in Vivo and in Vitro
To determine the effect of the FGFR-2 Apert mutation on apoptosis in vivo, DNA cleavage was assessed on normal and Apert coronal sutures by the TUNEL assay, as described by the manufacturer (Boehringer Mannheim, Mannheim, Germany). Briefly, paraffin-embedded tissues were deparaffinized in xylene and rehydrated through a graded series of ethanol. Sections were digested with 1 µg/ml proteinase K for 15 minutes. Endogenous peroxidase was quenched with 3% H2O2, permeabilized with 0.1% Triton X-100 at 4°C for 2 minutes, and incubated for 1 hour at 37°C with the TUNEL reaction mixture containing the terminal deoxynucleotidyl transferase. TUNEL signal was revealed with diaminobenzidine and mounted. To assess the effect of the mutation in vitro, apoptotic cells were detected on Ap and Co cells cultured on Labtek chambers in basal conditions and under the different treatments described below, using the TUNEL assay. Cells were washed with PBS, fixed with paraformaldehyde (4% in PBS), and endogenous peroxidase was quenched with 0.3% H2O2. Then TUNEL assay was performed as described above. TUNEL-positive cells were revealed by brown nuclei and apoptotic morphology, reflecting the specific dNTP transfer to 3'-hydroxy ends of DNA. The number of total and TUNEL-positive Ap and Co cells was then counted, and the results were expressed as percent of total cells. Positive controls consisted of sections and cells treated with DNase I for 10 minutes. Negative controls were obtained by omitting the transferase from the reaction.
Quantitation of DNA Fragmentation
Quantitative analysis of DNA fragmentation was performed as described.36 Briefly, Ap and Co cells were prelabeled with [3H]-thymidine (1 µCi/ml) in 1% bovine serum albumin (BSA) for 24 hours. Cells were washed, trypsinized, and then lysed in TTE [10 mmol/L Tris, pH 7.4, 10 mmol/L EDTA, and 0.2% (v/v) Triton X-100]. Fragmented DNA was separated from intact chromatin by centrifugation, and the pellet was suspended in TTE. [3H]-Thymidine incorporated into both soluble and unfragmented DNA was determined by liquid scintillation counting. Percentage of fragmented DNA was calculated as the ratio of fragmented/fragmented plus intact chromatin.
Determination of Caspase Activity
To determine the implication of caspases involved in apoptosis in mutant cells, Ap and Co cells were plated in 6-well plates with 1% BSA in serum-free medium. After 24 hours, the cells were lysed in 400 µl of lysis buffer (10 mmol/L Tris, pH 7.4, 200 mmol/L NaCl, 5 mmol/L EDTA, 10% glycerol, 1% Nonidet P-40) for 30 minutes on ice and stored at -20°C. The activity of effector caspases (caspase-3, -6, -7) and initiator caspases (caspase-2, -8, -9) was determined by the cleavage of synthetic fluorogenic substrates containing the amino acid sequence recognized by specific caspases. The substrates were as follows: WEHD (Thr-Glu-His-Asp) for caspase 1, DEVD (Asp-Glu-Val-Asp) for caspase-3-like, IETD (Ile-Glu-Thr-Asp) for caspase-8, and were combined to a fluorophore (7-amino-4-methylcoumarin, AMC). On cleavage of the substrate by caspases, free AMC fluorescence emission was detected using a spectrofluorometer (F-2000, Hitachi, Japan). For the assay, aliquots of 100 µl were incubated for 2 hours at 37°C with 200 µl reaction buffer (0.1 mmol/L phenylmethyl sulfonyl fluoride, 10 mmol/L dithiothreitol, 10 mmol/L Hepes/NaOH, pH 7.4) containing 5-µl specific substrate (20 µmol/L final concentration). The fluorescence released in samples was measured by excitation at 367 nm and reading was made at 440 nm. The negative control was buffer mix and the positive control was free AMC (10 µmol/L in PBS). The free AMC fluorescence emission by caspases was related to protein level and was expressed as arbitrary units.
Immunocytochemistry of IL-1
To determine the expression of IL-1 that might be involved in
apoptosis, Ap and Co cells were cultured to confluence, fixed in 4%
paraformaldehyde at 4°C for 30 minutes, washed in PBS/0.01% Triton
X-100, incubated with 0.1% BSA/3% goat serum to block unspecific
binding, then exposed for 1 hour at room temperature to rabbit
polyclonal anti-human IL-1 or IL-1ß antibodies (Genzyme) diluted
1:100. Control cells were incubated with the appropriate solution
(rabbit IgG). After 1 hour exposure at room temperature, cells were
washed three times for 10 minutes in PBS and exposed to second
anti-rabbit antibody (1:50) linked to colloidal gold particles
(IntenSETMM; Amersham, Arlington Heights, IL) for 1 hour at room
temperature. The gold particle staining was enhanced by precipitation
of metallic silver (ImmunoGold Silver Staining), then washed before
visualization.
Western Blot Analysis
IL-1, CD95/Fas, Bax, and Bcl2 protein levels were
determined by Western blot analysis in Ap and Co cells. Co and Ap cells
were washed twice with cold PBS and scrapped into 300 µl of ice-cold
lysis buffer (10 mmol/L Tris-HCl, 5 mmol/L EDTA, 150 mmol/L NaCl, 30
mmol/L sodium pyrophosphate, 50 mmol/L NaF, and 1 mmol/L
Na3VO4) containing 10% glycerol and protease inhibitors
(Boehringer Mannheim). Protein samples were solubilized in 2x Laemmli
sodium dodecyl sulfate loading buffer and boiled at 95°C for 5
minutes. Fifty micrograms of proteins, determined using the DC protein
assay (Bio-Rad Laboratories, Hercules, CA), were resolved on 12%
acrylamide gel, then transferred onto polyvinylidene
difluoride-Hybond-P membranes (Amersham). Blots were saturated
overnight with 1% blocking solution (Boehringer Mannheim) in
Tris-buffered saline buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl)
containing 0.1% Tween-20. Membranes were then incubated with rabbit
polyclonal anti-human IL-1 or IL-1ß (1 µg/ml, Chemicon), mouse
monoclonal anti-human CD95/Fas (1 µg/ml, Immunotech), mouse
monoclonal anti-human Bax (0.5 µg/ml; Santa Cruz Biotechnology, Santa
Cruz, CA), mouse monoclonal anti-human Bcl2 (0.5 µg/ml, Santa Cruz),
or rabbit polyclonal anti-human ß-actin (1.5 µg/ml, Sigma) in 0.5%
blocking buffer. After 1 hour at room temperature, the membranes were
washed twice with Tris-buffered saline/0.1% Tween 20 and 0.5%
blocking buffer, and incubated for 1 hour with horseradish
peroxidase-conjugated secondary antibody for 1 hour at room
temperature. After incubation with the appropriate secondary antibodies
and washes, the signals were visualized with Boehringer Mannheim
chemiluminescence blotting substrate. The specific bands on the
autoradiograms were quantitated by densitometry.
Inhibition of PKC Signaling and IL-1 Expression
To determine the signal transduction pathways involved in
apoptosis in mutant cells, Ap and Co cells were cultured in the
presence of the selective PKC inhibitor calphostin C (2 µmol/L).
After 24 hours, IL-1 and IL-1ß protein levels were determined by
Western blot, as described above. In parallel, apoptosis in Ap cells
was determined by the TUNEL assay, and the activity of caspases-1, -3,
-8, and -9 was determined as described above. To determine the
implication of IL-1 on apoptosis, Ap cells were treated with
neutralizing IL-1 or IL-1ß antibody (0 to 15 µg/ml), the
combined treatment (30 µg/ml) or IgG at equivalent amount, and cell
viability was determined by trypan blue exclusion. Apoptosis was also
determined by TUNEL analysis as described above. In parallel
experiments, the activity of caspase-3 and -8, and CD95/Fas protein
levels were determined as described above.
Data Analysis
The results are expressed as the mean ± SEM and were analyzed using the statistical package super-ANOVA (Macintosh, Abacus Concepts, Inc., Berkeley, CA). Differences between the mean values were evaluated with a minimal significance of P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze the effect of the Apert mutation on apoptosis in
situ, coronal sutures from Apert fetus with the S252W FGFR-2
mutation and normal fetus were subjected to TUNEL analysis. As shown in
Figure 1
, the fused Apert suture differed
from the normal age-matched suture. The fused suture is composed of
multiple and large bone trabeculae (
1/10 of the suture area is shown
here), whereas the normal suture is composed of a bone plate surrounded
by multiple mesenchymal cells (only 1/2 of the suture is shown here).
The pattern of TUNEL-positive apoptotic cells differed markedly n the
Apert sample compared to the age-matched normal suture. Specifically,
most differentiated osteoblasts and osteocytes were apoptotic in Apert
suture (Figure 1B)
. In contrast, in the normal suture, apoptosis was
found in mesenchymal cells and pre-osteoblasts whereas very few
osteoblasts and osteocytes were TUNEL-positive (Figure 1A)
. The TUNEL
labeling of cells was specific, as shown by the absence of TUNEL label
in control sections and the positive staining of all cells treated with
DNase I (not shown). These results revealed that the S252W FGFR-2
mutation induces premature apoptosis of differentiated osteoblasts and
osteocytes in vivo in the fused Apert suture.
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To further document the effect of the mutation on apoptosis,
mutant Ap cells and normal Co cell viability was studied by the trypan
blue exclusion assay. Figure 2A
shows
that most Co cells were viable in culture, with only a small percentage
of trypan blue-stained cells. In contrast, numerous trypan-blue stained
cells were found in Ap cultures, showing decreased cell viability
induced by the mutation (Figure 2A)
. To further determine the effect of
the mutation on osteoblast apoptosis, mutant Ap cells and normal
Co-cultured cells were subjected to TUNEL analysis to assess DNA
fragmentation by specific labeling of double-strand DNA. Figure 2B
shows that few Co cells were TUNEL-positive when cultured in 1% BSA
serum-free conditions. In contrast, numerous TUNEL-positive Ap cells
were found. Quantification of trypan blue-stained cells and
TUNEL-positive Ap and Co cells revealed that the FGFR-2 mutation
induced a twofold to threefold increase in the number of apoptotic
cells in vitro (Figure 2, A and B)
. Notably, the percentage
of Ap TUNEL-positive apoptotic cells did not significantly differ from
the percentage of trypan blue-positive Ap cells (representing apoptotic
and necrotic cells) (11.4 ± 0.43% versus 13.3 ±
0.40%, ns), indicating that the mutation affected apoptosis rather
than necrosis in mutant osteoblasts.
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, the
percentage of DNA fragmentation, measured by the ratio of
fragmented/fragmented plus intact chromatin, was increased in Ap cells
compared to Co cells. Overall, these data show that the S252W FGFR-2
mutation constitutively increases apoptosis in mutant osteoblasts. Increased Caspase-8 and -3 Activity in Mutant Apert Osteoblasts
To begin investigating mechanisms involved in the constitutive
increase in apoptosis in mutant cells, Ap and Co cells were cultured in
basal conditions, and the activity of caspase-1, -3, and -8 was
determined using specific substrates. Figure 3
shows that, in basal conditions,
caspase-1 activity was similar in normal and mutant osteoblasts (Figure 3A)
. In contrast, the activity of the initiator caspase-8 was increased
threefold in Ap cells compared to Co cells (Figure 3B)
. The activity of
the effector caspases (caspase-3-like) was also dramatically increased
in mutant cells (Figure 3C)
. Moreover, suppression of caspase-8
activity, using the specific inhibitor z-IETD-fmk, abolished
caspase-3-like activity in normal Co cells and mutant Ap cells (Figure 3D)
. This reveals that apoptosis is associated with constitutive
activation of caspase-8, leading to activation of downstream effector
caspases in human mutant osteoblasts.
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We recently reported that the S252W FGFR-2 mutation constitutively
activates PKC signaling whereas erk1,2 and p38 MAP kinases are not
affected in mutant Ap cells.11
To assess whether PKC
activity may be involved in the increased apoptosis in mutant cells, Ap
cells were treated with calphostin C, a selective inhibitor of PKC, and
apoptosis was determined by TUNEL analysis. As shown in Figure 4A
, the number of TUNEL-positive Ap cells
was greater than in Co cells, and treatment of Ap cells with 2 µmol/L
calphostin C restored the number of TUNEL-positive Ap cells to normal
levels (Figure 4A)
. Moreover, suppression of PKC activity by calphostin
C reduced both caspase-8 and caspase-3 activity to the basal level in
Co cells (Figure 4, B and C)
. Thus, inhibition of PKC activity
abolished the constitutive increase in caspase-8 and -3 activity as
well as the increased apoptosis induced by the mutation in Ap mutant
osteoblasts.
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and IL-1ß in Mutant
Osteoblasts
Until now, the results showed that the increased apoptosis induced
by the S252W FGFR-2 Apert mutation in osteoblasts involved increased
PKC activity and, subsequently, increased effector caspase activity.
Another possible mechanism may be CD95/Fas-mediated apoptosis, which is
promoted by IL-1 in osteoblasts. We addressed this hypothesis by first
investigating the in vitro expression of IL-1 and IL-1ß
in cultured Ap and Co cells. As shown in Figure 5A
, Co cells showed weak IL-1 and
IL-1ß immunostaining. By contrast, Ap cells showed high IL-1 and
IL-1ß immunoreactivity compared to Co cells (Figure 5A)
. The
expression of IL-1 and IL-1ß protein levels was further analyzed
by Western blot. Figure 5B
shows that IL-1 and IL-1ß protein
levels were increased in Ap cells compared to Co cells, confirming the
immunocytochemical analysis (Figure 5A)
. In parallel experiments, we
found increased IL-1 and IL-1ß immunolabeling in osteoblasts in
the Apert suture compared to normal coronal suture (not shown). These
results show that the FGFR-2 mutation induces a constitutive increase
in IL-1 and IL-1ß expression in mutant osteoblasts. To assess
whether the increased IL-1 and IL-1ß expression may result from
PKC activation in mutant cells, Ap cells were treated with the PKC
inhibitor calphostin C and IL-1 and IL-1ß levels were analyzed by
Western blot. As shown in Figure 5, B and C
, treatment of Ap cells with
calphostin C restored IL-1 and IL-1ß protein levels to control
levels in Co cells. Thus, PKC activation induced by the mutation is
responsible for the increased IL-1 and IL-1ß expression in mutant
osteoblasts.
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and IL-1ß in Apoptosis in Mutant Osteoblasts
To assess whether the increased IL-1 and IL-1ß expression is
involved in apoptosis induced by the mutation, we tested the effects of
anti-IL-1 and anti-IL-1ß neutralizing antibodies or IgG on
apoptosis and caspase-3 activity. Apoptosis was determined by trypan
blue exclusion because necrosis is low in these conditions. As shown in
Figure 6A
, incubation of Ap cells with
IL-1 antibody dose-dependently decreased the number of trypan
blue-stained Ap cells. A similar effect of anti-IL-1ß was found
(Figure 6A)
. The number of trypan blue-stained Ap cells did not
significantly differ from normal levels in Co cells in the presence of
either IL-1 antibody or IL-1ß antibody (15 µg/ml) (Figure 6A)
.
Furthermore, quantification of TUNEL-positive (apoptotic) Ap cells
showed that IL-1 and IL-1ß antibodies at optimal dosage (total
dose, 30 µg/ml) decreased apoptosis in Ap mutant cells. However, the
number of Ap apoptotic cells remained higher than in Co cells (Figure 6B)
, suggesting that suppression of IL-1 did not completely abolish
apoptosis in mutant cells.
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, CD95/Fas protein levels were
increased in Ap cells compared to Co cells. The addition of
neutralizing IL-1 and IL-1ß antibodies (total dose, 30 µg/ml)
decreased CD95/Fas expression in mutant osteoblasts compared to a
nonspecific IgG (Figure 6C)
. This suggests a role of IL-1 in Fas
overexpression in Ap cells. However, Fas levels in Ap cells were not
restored to normal levels in Co cells. To further determine the role of
IL-1 and IL-1ß in the apoptotic process in mutant cells, Ap cells
were treated with IL-1 and IL-1ß antibodies (total dose, 30
µg/ml) or IgG and caspase-3 activity was determined. As shown in
Figure 6D
, treatment of Ap cells with neutralizing IL-1 and IL-1ß
significantly inhibited caspase-3 activity in Ap cells (Figure 6D)
.
Again, caspase-3-like activity was not completely restored to normal.
Similar results were obtained with caspase-8 activity (data not shown).
These results indicate that suppression of IL-1 and IL-1ß
expression leads to reduced CD95/Fas overexpression, caspase-3 activity
and apoptosis in mutant osteoblasts. However, caspase-3 activity and
apoptosis were not completely abolished by neutralizing IL-1
antibodies, suggesting that another pathway may be involved. Bax and Bcl2 Expression in Mutant Apert Osteoblasts
Apoptosis is governed in part by Bax and Bcl-2 that are
pro-apoptotic and prosurvival proteins, respectively. To determine the
implication of these proteins in apoptosis induced by the Apert FGFR-2
mutation, we examined their expression in Ap mutant cells. Western blot
analysis showed that Bax protein levels were increased in Ap cells
compared to normal Co cells cultured in basal conditions (Figure 7A)
. In contrast, Bcl2 levels were lower
in Ap cells than in Co cells, which resulted in a huge increase in the
Bax/Bcl2 ratio in mutant cells (Figure 7A)
. Furthermore, we found that
IL-1 and IL-1ß neutralizing antibodies (total dose, 30 µg/ml)
reduced by 50% Bax expression in Ap cells (Figure 7B)
. These results
suggest that the increase in Bax and decreased Bcl-2 may contribute to
apoptosis in Ap cells, and that IL-1 overexpression mediates in part
the increased Bax level in Ap mutant cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß, CD95/Fas,
caspase-8 activity, and Bax/Bcl2 levels. We first showed that premature
apoptosis is a hallmark of human osteoblast abnormalities induced by
the Apert S252W FGFR-2 mutation in vivo. Increased apoptosis
was restricted to more mature osteoblasts and osteocytes in the Apert
suture, showing that apoptosis induced by the mutation affects more
differentiated cells. This is supported by the observation that
apoptosis increases with osteoblast maturation in vitro and
is a prominent feature during the late development of the mature
osteoblast phenotype.16
This is also consistent with the
recent finding that transfection with the S252W FGFR-2 mutation induces
apoptosis in mouse differentiating calvaria cells.12
The
present data in human Apert osteoblasts supports the previous
hypothesis that FGFR activating mutations affect apoptosis in human
skeletal cells. Indeed, recurrent mutations in FGFR-3 in thanatophoric
dysplasia were found to induce apoptosis in
chondrocytes.37
Conversely, the overexpression of a mouse
dominant-negative FGFR-1 mutation was found to suppress
apoptosis.38
We sought mechanistic insight into the pro-apoptotic effect of the
S252W FGFR-2 mutation in human calvaria osteoblasts by examining the
signal transduction pathways and the intracellular mechanisms activated
by the mutation. Several serine/threonine protein kinases have been
proposed to control apoptotic mechanisms, including Erk1,2
mitogen-activated protein kinase (MAPK), p38 Map kinase, c-Jun N
terminal kinase (JNK), and protein kinases A, B, and
C.39,40
Recent reports indicate that FGFR-2 mutations
increase PKC expression and activity in human mutant
osteoblasts.8,11,41
More specifically, we found that the
S252W FGFR-2 mutation activates PLC
and PKC whereas Erk 1,2 or p38
kinases are not activated in Apert human osteoblasts.11
Our finding that the PKC inhibitor calphostin C decreased apoptosis
suggests an important role for PKC in apoptosis induced by the mutation
in Ap cells. Although activation of PKC has been previously observed to
contribute to apoptotic signaling,39
novel PKC isoforms
seem to be pro-apoptotic whereas classical and atypical PKC isoenzymes
are associated with cell survival.40
Human osteoblasts
with FGFR-2 mutations express numerous PKC isoforms.8,11
However, the precise role of each isoenzyme in the apoptotic effect of
FGFR-2 mutations in Apert syndrome remains to be clarified. In this
regard, Apert osteoblasts display increased PKC
activity8,11
and expression,41
suggesting a
role for the PKC isoform in apoptosis in mutant cells.
Having shown that the increased apoptosis induced by the FGFR-2
mutation is PKC-dependent, we assessed the downstream events involved
in apoptosis in mutant cells. Evidence that apoptosis in Apert
osteoblasts involves IL-1 and Fas pathways is supported by several
findings. Mutant osteoblasts constitutively overexpress IL-1 and
IL-1ß protein levels that were corrected by the PKC inhibitor
calphostin C, indicating a role for PKC in IL-1 overexpression in
mutant cells. Our finding that neutralizing anti-IL-1 antibodies
reduced Fas overexpression and apoptosis suggests that Fas-mediated
apoptosis is mediated by IL-1 overexpression in mutant cells. Thus,
apoptosis in Apert osteoblasts seems to be primarily mediated by a
PKC-dependent overexpression of IL-1 and subsequent activation of
Fas-mediated apoptosis. Because Fas expression may be directly
activated by PKC,42
it is also possible that PKC
activation may directly increase Fas expression in mutant cells. A role
for Fas and IL-1 in apoptosis induced by the Apert S252W FGFR-2
mutation is consistent with the finding that IL-1 and Fas-mediated
apoptosis controls cell death in osteoblasts.29,30
Fas-mediated apoptosis is known to activate a series of complex
mechanisms, leading to procaspase cleavage and formation of
caspases.43,44
Procaspase-8 is cleaved after ligation of
specific transmembrane death receptors such as Fas,25
and
activation of caspase-8 is one of the signaling pathways leading from
Fas to apoptosis.45,46
Our finding that
neutralizing IL-1 antibodies reduced the constitutive increase in
caspase-8 and caspase-3 activities indicates that the IL-1 and
Fas-mediated pathway activates caspase-8 and ultimately, effector
caspases and DNA degradation in Apert osteoblasts. Although numerous
diseases have been previously associated with increased Fas
levels,47
this study is the first to present
evidence for increased Fas-mediated apoptosis in abnormal (premature)
membranous ossification induced by a genetic FGFR mutation in humans.
Because Bax and Bcl-2 are known to play critical roles in programmed cell death in several cell types and are influenced by FGFs33,34 we hypothesized that apoptosis in Apert osteoblasts may involve alteration in the balance between Bax and Bcl-2. The increased Bax levels and decreased Bcl-2 levels in mutant cells indicates that the alteration of Bax/Bcl-2 may contribute to apoptosis induced by the FGFR-2 mutation. This is consistent with the previous finding that apoptosis induced by FGF-1 is associated with increased Bax and delayed Bcl-2 in differentiating murine calvaria cells.12 The increased Bax in Ap cells seems to result from IL-1 overexpression, which indicates that IL-1 overexpression may induce premature cell death in mutant cells in part by altering the Bax/Bcl-2 ratio. Because apoptosis in Ap mutant cells was not completely corrected by anti-IL-1 antibodies, apoptosis may also arise from IL-1-independent mechanisms. The present data are compatible with a model in which the increased apoptosis induced by the S252W FGFR-2 mutation is triggered by a PKC-dependent pathway involving IL-1, Fas, caspase-8, and also Bax/Bcl-2 that ultimately converge to increase effector caspases in mutant osteoblasts.
The mechanisms by which FGF signaling controls cranial suture ossification are still unclear. Although FGFs are important factors controlling osteoblast proliferation and differentiation,48 recent data indicate that the effects of FGF depend on the maturation stage of osteoblasts. Indeed, FGFs induce opposite effects on calvaria cell proliferation, differentiation, and apoptosis depending on the differentiation stage, and distinct responses to FGF were found in immature and mature osteoblasts.12,49 Thus, the apparently different alterations of cell proliferation and differentiation reported in FGFR-2 mutant human and murine osteoblasts8,9,12 may arise from variable FGF signaling mechanisms leading to distinct effects in mature and immature calvaria osteoblasts. One possible mechanism for the variable effects of FGF signaling may be a distinct expression of FGFRs in immature and mature calvaria cells. In this regard, we found that FGFR-2 is down-regulated in differentiated mutant Apert osteoblasts in vitro and in vivo.10,48 FGFR-2 down-regulation in more differentiated mutant osteoblasts may limit the proliferative activity of the cells and contribute to the premature osteoblast differentiation.50 Thus, a combination of increased differentiation and apoptosis in mature osteoblasts may accelerate the osteogenic process and contribute to premature cranial ossification in Apert syndrome. Further analysis of FGF/FGFR signaling pathways in relation to cell differentiation and apoptosis, now in progress in our laboratory, may shed more light on the mechanisms leading to the premature suture ossification in Apert syndrome.
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Acknowledgements |
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Footnotes |
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Accepted for publication February 8, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3 inhibits programmed cell death in HL-60 cells by activation of sphingosine kinase. Cancer Res 1998, 58:1817-1824, IL-1 and RhoA GTPase. J Bone Miner Res (in press)
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 Y. Wang, R. Xiao, F. Yang, B. O. Karim, A. J. Iacovelli, J. Cai, C. P. Lerner, J. T. Richtsmeier, J. M. Leszl, C. A. Hill, et al. Abnormalities in cartilage and bone development in the Apert syndrome FGFR2+/S252W mouse Development, August 1, 2005; 132(15): 3537 - 3548. [Abstract] [Full Text] [PDF]
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 K. Kaabeche, H. Guenou, D. Bouvard, N. Didelot, A. Listrat, and P. J. Marie Cbl-mediated ubiquitination of {alpha}5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation J. Cell Sci., March 15, 2005; 118(6): 1223 - 1232. [Abstract] [Full Text] [PDF]
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 D. M. Ornitz and P. J. Marie FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease Genes & Dev., June 15, 2002; 16(12): 1446 - 1465. [Full Text] [PDF]
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 M. Yousfi, F. Lasmoles, V. El Ghouzzi, and P. J. Marie Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvarial osteoblast apoptosis due to increased TNF{alpha} expression and caspase-2 activation Hum. Mol. Genet., February 1, 2002; 11(4): 359 - 369. [Abstract] [Full Text] [PDF]
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E. Hay, J. Lemonnier, O. Fromigue, and P. J. Marie Bone Morphogenetic Protein-2 Promotes Osteoblast Apoptosis through a Smad-independent, Protein Kinase C-dependent Signaling Pathway J. Biol. Chem., July 27, 2001; 276(31): 29028 - 29036. [Abstract] [Full Text] [PDF]
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