Human Molecular Genetics, 2012, Vol. 21, No. 18
doi:10.1093/hmg/dds181
Advance Access published on May 24, 2012
3941–3955
Intermittent PTH (1-34) injection rescues the
retarded skeletal development and postnatal
lethality of mice mimicking human achondroplasia
and thanatophoric dysplasia
Yangli Xie1, Nan Su1, Min Jin1, Huabing Qi1, Junbao Yang1, Can Li1, Xiaolan Du1, Fengtao Luo1,
Bo Chen1, Yue Shen1, Haiyang Huang1, Cory J. Xian2, Chuxia Deng3 and Lin Chen1,∗
1
State Key Laboratory of Trauma, Burns and Combined Injury, Center of Bone Metabolism and Repair, Institute
of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China, 2Sansom
Institute for Health Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide,
South Australia 5001, Australia and 3Genetics of Development and Disease Branch, National Institute of Diabetes,
Digestive and Kidney Diseases, US National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA
Received February 22, 2012; Revised April 19, 2012; Accepted May 10, 2012
Achondroplasia (ACH) and thanatophoric dysplasia (TD) are caused by gain-of-function mutations of fibroblast growth factor receptor 3 (FGFR3) and they are the most common forms of dwarfism and lethal dwarfism,
respectively. Currently, there are few effective treatments for ACH. For the neonatal lethality of TD patients,
no practical effective therapies are available. We here showed that systemic intermittent PTH (1-34) injection
can rescue the lethal phenotype of TD type II (TDII) mice and significantly alleviate the retarded skeleton development of ACH mice. PTH-treated ACH mice had longer naso-anal length than ACH control mice, and the
bone lengths of humeri and tibiae were rescued to be comparable with those of wild-type control mice. Our
study also found that the premature fusion of cranial synchondroses in ACH mice was partially corrected
after the PTH (1-34) treatment, suggesting that the PTH treatment may rescue the progressive narrowing of
neurocentral synchondroses that cannot be readily corrected by surgery. In addition, we found that the
PTH treatment can improve the osteopenia and bone structure of ACH mice. The increased expression of
PTHrP and down-regulated FGFR3 level may be responsible for the positive effects of PTH on bone phenotype of ACH and TDII mice.
INTRODUCTION
Fibroblast growth factor receptor 3 (FGFR3) has been proved
to be a negative regulator of endochondral bone growth (1).
Mutations in the coding sequence of the FGFR3 gene can
cause autosomal dominant human skeletal disorders, including
achondroplasia (ACH) (2), hypochondroplasia (HCH) (3), thanatophoric dysplasia (TD) (3), and some other related disorders (4). ACH is the most common non-lethal form of
dwarfism (5), which has the characteristic phenotypes of rhizomeric short limbs, macrocephaly, and lumbarlordosis. TD is a
severe and early postnatal lethal skeletal dysplasia, and can be
clinically divided into two types, TD type I and II (TDI and
TDII) (6,7).
Recently, the roles of activated FGFR3 signals in bone development of ACH have been studied in vivo using transgenic
(8) and knock-in (9,10) mouse models. Several studies suggested that the skeletal phenotypes in those mouse models
mimicking human ACH and TD resulted from disturbed proliferation and differentiation of growth plate chondrocytes
that lead to impaired endochondral bone growth (8,11). In
order to study the mechanisms for the pathogenesis of ACH
and TDII, we have generated several mouse models including
mice carrying Fgfr3 G369C and K644E mutations, which
∗
To whom correspondence should be addressed. Tel: +86 2368757041; Fax: +86 2368702991; Email: [email protected]
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Human Molecular Genetics, 2012, Vol. 21, No. 18
cause ACH and TDII in humans, respectively (9,12). As
expected, these mouse models display skeletal phenotypes
similar to the corresponding human conditions, including
short stature and dome-shaped skull. Specifically, mice carrying the Fgfr3 K644E mutation also exhibited underdeveloped
thoracic cage, which leads to lethality within several hours
after birth as a result of restricted respiratory function (12).
Studies with these models have generated valuable knowledge
regarding the mechanisms underlying the FGFR3-related skeletal dysplasias. FGFR3 is currently thought to inhibit chondrocyte proliferation, differentiation and matrix synthesis through
its downstream pathways including MAPK, PLCg, Stats and
cell-cycle inhibitors such as p21 (10,13 – 15). FGFR3 is also
found to promote chondrocyte apoptosis (15,16). Additionally,
FGF/FGFR3 signaling has been found to have crosstalk with
other signaling pathways critically involved in skeleton
development.
Although no effective clinical therapy is available for TD
patients, current treatments for ACH patients are mainly
growth hormone treatment and distraction osteogenesis
(17– 19), which have limitations in effectiveness. Although
the long-term effect of growth hormone therapy on ACH is
not significant (20), distraction osteogenesis can extend the
limbs of ACH patients only to some extent, and patients
usually suffer from marked physical and economical
burdens. Thus, there is urgent need to find new treatments
for ACH/TD patients. Indeed, our increased understanding
about the molecular mechanisms of ACH/TD has provided
us with more biological measures to treat these diseases. Theoretically, down-regulating the activity of FGFR3 and/or its
downstream signaling pathways may lead to a cure or alleviation of ACH/TD.
We have previously shown that ACH mice have decreased
expression of PTHR1 in their growth plates, and PTHrP treatment can partially rescue the retarded growth of the cultured
bone rudiments from ACH mice (21). Other studies have
also shown that PTH treatment can improve the growth of cultured chondrocytes of femurs from mice with transgenic expression of activated FGFR3 in the growth plates (16,22).
However, the actual in vivo effect of PTH signaling on the
skeletal development of mice mimicking human ACH/TD
and its action mechanism are still not fully clarified.
The purpose of this study was to elucidate whether the in
vivo systemic administration of PTH can rescue the underdeveloped skeleton of ACH and TDII mice. We found that
intermittent subcutaneous injection of PTH (1-34) can rescue
the retarded skeletal development and prevent early postnatal
lethality of ACH/TD mice, indicating that PTH can potentially
be used in clinic to treat ACH and/or TD patients.
RESULTS
PTH (1-34) rescues the lethal phenotype of mice mimicking
human TDII
We generated mice heterozygous for the Fgfr3 K644E
mutation (Fgfr3+/K644E) (referred to as TDII) by crossing
Fgfr3+/K644Eneo mice with EIIa-Cre transgenic mice. All
Fgfr3+/K644E pups without PTH treatment (n ¼ 64) died
within a few hours after birth due to the restricted respiration
Table 1. Summary of survival of TDII mice with PTH treatment
ID
Sex distribution
Survival days
1 –7
9 –20
21– 28
29– 34
35– 40
41– 44
45– 48
49– 50
51
52
M 3, F 4
M 7, F 5
M 3, F 5
M 2, F 4
M 4, F 2
M 2, F 3
M 3, F 1
M2
F1
F1
5
7
14
28
57
90
120
127
140
150
All these mice received PTH (1-34) treatment (100 mg/kg body weight per day)
during gestation from E13.5 to newborn via subcutaneous injection. M, male, F,
female.
caused by retarded thoracic cage development, whereas all
wild-type (WT) littermates (n ¼ 79) developed normally. In
contrast, TDII mice (n ¼ 52) born by pregnant mice injected
with PTH (1-34) survived for at least 5 days after birth, and
12 TDII (23.1%) mice survived for .90 days (Table 1).
Before death, TDII mice without PTH treatment invariably
exhibited serious cyanosis and limited viability (Fig. 1B),
whereas PTH (1-34)-treated TDII mice (Fig. 1C) showed
ruddy skin and were viable, similar to that in WT pups. At
postnatal day 1 (P1), TDII mice (Fig. 1F), compared with
their WT littermate, showed decreased ossification in the
spine and epiphyses, significantly curved axial skeleton, as
well as notable round head (arrow in Fig. 1E). PTH-treated
TDII mice (Fig. 1F) had significantly improved skeletal development, and they had larger total length and body size than
those in untreated TDII mice, although they still exhibited
round head (arrows). TDII mice showed remarkable bowing
of tibia (Fig. 1K) and humerus (Fig. 1N) (arrows). The shortened ossified zone in TDII tibia was clearly exhibited
(bars). Hindlimbs and forelimbs were better developed in
PTH-treated TDII mice as evidenced by the longer ossified
zone and milder curvature. We examined the histology of
the P1 mouse long bones. The height of the whole epiphyseal
growth plate was similar between TDII and WT mice
(Fig. 1S– U). TDII mice had abnormal differentiation of
growth plates (Fig. 1T). Small and round-shaped resting
chondrocyte-like cells were randomly located in the proliferation and hypertrophic zones (arrow). TDII neonates died
from respiratory failure early after birth (23). We examined
the morphology of the postnatal lungs. WT mice had welldeveloped large alveoli. TDII pups displayed impaired lung
development at P1 with fewer and poorly formed alveoli
(Fig. 1W) compared with the WT controls (Fig. 1V), and
PTH (1-34)-treated TDII mice (Fig. 1X) had more alveoli in
their lungs compared with the untreated TDII mice.
At P5, TDII mice that survived after the PTH treatment had
notable round head (Fig. 2A), disorganized hypertrophic zone
(Fig. 2E and G) and less trabecular bone in lumbar vertebrae
(Fig. 2C) compared with those in WT littermates (Fig. 2B,
D and F). Although all those PTH-treated TDII mice survived
early postnatal lethality, at the age of 2 months, they still
exhibited small stature, short and round skull similar to
Human Molecular Genetics, 2012, Vol. 21, No. 18
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Figure 1. PTH (1-34) rescues the lethal phenotype of mice mimicking human TDII. (A–C) Morphology of the mutant (TDII) mice treated with PTH (1-34) and
the littermate controls (WT) at P1. TDII mice showed notable cyanosis. (D– R) Alizarin red S and Alcian blue staining of the skeletons in WT mice, TDII mice
and TDII mice treated with PTH at P1. (S– U) Histology (H&E staining) of the growth plates of proximal tibiae in WT mice and TDII mice treated with PTH at
P1. The growth plates of TDII mice were disorganized: resting chondrocyte-like cells were located in the proliferative and hypertrophic zones (arrow in T).
Dashed lines separate the resting, proliferative and hypertrophic zones (magnification ×40). (V– X) Morphology of the lungs (H&E staining, magnification ×200).
those of ACH mice (Fig. 2H). Soft X-ray analysis of
PTH-treated TDII mice showed impaired growth of bones
formed via endochondral ossification, including tibiae and vertebrae (Fig. 2I). PTH-treated TDII mice had shorter tibiae and
caudal vertebra, as well as decreased bone mass (Fig. 2I) compared with those of age-matched WT mice. Histological analysis revealed that PTH-treated TDII mice still had less
trabecular bone in tibia than that in WT mice (Fig. 2L and
M). All these data indicated that PTH (1-34) rescued the
lethal phenotype of TDII mice and improved their skeletal
phenotype.
PTH (1-34) rescues the retarded bone development
in ACH mice
In order to study the effects of the PTH (1-34) treatment on
ACH mouse growth and development, we measured the
body weight and naso-anal length of ACH mice and their littermates. Compared with vehicle-treated counterparts, ACH
mice treated with PTH (1-34) showed no remarkable differences in body weights and lengths at P7. With age, PTH
(1-34)-treated ACH mice grew markedly faster than ACH
control mice, but remained to have lower body weights than
their WT littermates (P , 0.05) (Table 2). The naso-anal
lengths of PTH (1-34)-treated ACH mice were longer than
those of ACH control mice at the age of 5 weeks (P ,
0.05), and were similar to the lengths of their WT littermates
after 6 weeks of age till 12 weeks (Table 3). The phenotype of
dome-shaped skull in ACH was also much milder after the
PTH treatment (Supplementary Material, Fig. S1).
PTH (1-34) partially rescues the premature fusion of
cranial synchondroses and alleviates the retarded long
bone growth and osteopenia in ACH mice
Since premature fusion of synchondroses in the cranial base is
a remarkable phenotype of ACH mice (10), we examined the
synchondroses in each group. PTH appeared to partially rescue
the premature fusion of the cranial synchondroses in ACH
mice (Fig. 3A –H). At P7, premature fusion of sphenooccipital
synchondroses was found obvious in ACH control mice
(Fig. 3C, arrow), and it was partially rescued by the PTH treatment (Fig. 3D, arrow). Sphenooccipital synchondroses were
completely closed and the anterior intraoccipital synchondroses (arrows) of ACH were encased in bone at P14
(Fig. 3G), whereas in PTH-treated ACH mice, premature
fusion of intraoccipital synchondroses was partially rescued
(Fig. 3H).
The number of BrdU-positive chondrocytes in the proliferating zone of P7 growth plates was found to be remarkably
enhanced by the PTH treatment in both ACH and WT mice
(Fig. 3I– L). The PTH (1-34)-treated ACH mice showed a significant increase in the thickness of hypertrophic zones compared with those in untreated ACH mice (Fig. 3M – P and
W). At P8, the secondary ossification center began to be
observed in WT mice and PTH-treated WT mice (Fig. 3Q
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Figure 2. TDII mice treated with PTH have shorter stature, dome-shaped skull and osteopenia. (A) Morphology of the mutant (TDII) mice treated with PTH and
the littermate controls (WT) at P5. Survived TDII mice had dome-shaped skulls. (B and C) Micrographs of decalcified paraffin sections of vertebrae stained with
H&E at P5 (magnification ×100). (D– G) Histology (H&E staining) of the growth plates of proximal tibiae in WT mice and TDII mice treated with PTH at P5
showed PTH-treated TDII mice still had a marked abnormal hypertrophic zone and less trabecular bone (magnification: D and E ×100, F and G ×200).
(H) Morphology of the mutant (TDII) mice treated with PTH and the littermate controls (WT) at 2 months. (I) X-ray pictures of WT mice and TDII mice
treated with PTH (1-34) at 2 months showed TDII mice had osteopenia. (J –M) Histology (H&E staining) of the growth plates of proximal tibiae in WT
mice and TDII mice treated with PTH at 2 months. PTH-treated TDII mice have less trabecular bone in tibiae (magnification: J and K ×40; L and
M ×100; J and L, WT mice; K and M, PTH-treated TDII mice). (N and O) Growth curves of TDII mice and littermate controls. TDII mice were smaller
than age-matched control. Graphs show mean values + SD (Student’s t-test, ∗∗∗ P , 0.001).
and S), whereas it was not formed in untreated ACH control
littermates (Fig. 3T) in the proximal tibia. In the ACH
control group, the location of the future secondary ossification
center consisted of hypertrophic chondrocytes and resting
chondrocyte-like cells, whereas in PTH-treated ACH mice,
the secondary ossification center can be observed (Fig. 3U).
At P14, the secondary ossification centers were well formed
in each mouse group (data not shown), and the thickness of
the growth plate proliferative zone was increased by the
PTH treatment in ACH mice (Supplementary Material,
Fig. S2A). At 2 months, the lengths of humeri and that of
femurs of ACH mice were shorter than those of WT littermates, whereas in PTH-treated ACH group, the humeri and
femurs were statistically longer when compared with ACH
control (Fig. 3V). These results suggest that intermittent
PTH (1-34) administration partially rescued the retarded
Human Molecular Genetics, 2012, Vol. 21, No. 18
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Table 2. Body weights of WT and ACH mice treated with or without PTH (1-34) (g) (n ¼ 6 –8)
Group
WT control
PTH-treated WT
ACH control
PTH-treated ACH
Age (weeks)
1
2
3
4
5
6
7
8
4.7 + 0.1
4.6 + 0.2
4.7 + 0.1
4.6 + 0.1
5.6 + 0.4
6.0 + 0.5
5.7 + 0.2
5.6 + 0.3
6.7 + 0.5
7.5 + 0.6
6.6 + 0.3
6.6 + 0.4
10.1 + 0.5
10.8 + 0.6
9.5 + 0.2
10.0 + 0.4
14.9 + 0.6
15.3 + 0.7
11.1 + 0.5a
12.1 + 0.6a,b
17.4 + 1.0
17.7 + 1.0
11.4 + 0.9a
13.2 + 0.8a,b
17.8 + 1.0
18.3 + 1.0
13.4 + 1.0a
15.8 + 0.9a,b
18.2 + 1.3
19.4 + 1.2
15.2 + 1.1a
16.5 + 1.2a,b
a
Versus control WT, P , 0.05.
Versus control ACH, P , 0.05.
b
Table 3. Naso-anal lengths of WT and ACH mice treated with or without PTH (1-34) (cm) (n ¼ 6– 8)
Group
WT control
PTH-treated WT
ACH control
PTH-treated ACH
Age (weeks)
1
2
3
4
5
6
7
8
2.8 + 0.2
2.7 + 0.1
2.3 + 0.1
2.3 + 0.1
3.7 + 0.2
3.7 + 0.2
3.5 + 0.1
3.6 + 0.2
5.0 + 0.2
5.1 + 0.2
4.6 + 0.3
5 + 0.2
6.2 + 0.3
6.3 + 0.3
5.1 + 0.2
6.0 + 0.3
6.4 + 0.3
6.8 + 0.3
5.7 + 0.2a
6.3 + 0.3b
6.8 + 0.3
7.1 + 0.3
5.9 + 0.3a
6.7 + 0.4b
7.2 + 0.4
7.3 + 0.4
6.6 + 0.4a
7.1 + 0.4b
8.5 + 0.6
8.6 + 0.5
7.6 + 0.5a
8.2 + 0.5b
a
Versus control WT, P , 0.05.
Versus control ACH, P , 0.05.
b
long bone growth of ACH mice by increasing the chondrocyte
proliferation and promoting the formation of the secondary ossification center.
Since we observed previously that ACH mice have osteopenia (24), we further examined the bone phenotype at the adult
stage. In both genotypes, bone mass of femurs was increased
in 2-month-old mice after the PTH (1-34) treatment
(Fig. 4A). Quantification of BMD by dual-energy X-ray absorptiometry (DEXA) revealed that the PTH treatment
increased the total femoral BMD of ACH mice significantly
by 16.1% (Fig. 4B). Micro-computed tomography
(micro-CT) analysis of the distal metaphyses of femurs was
used to assess the structural parameters, including bone
volume/tissue volume (%, BV/TV), trabecular number
(Tb.N), trabecular thickness (Tb.Th) and separation (Tb.Sp)
in 2-month-old mice. The ACH control group displayed an increase of Tb.Sp, and decrease of BV/TV, Tb.N and Tb.Th,
which indicated osteopenia as we previously reported (24).
In ACH mice, the PTH treatment increased BV/TV, Tb.N
and Tb.Th markedly by 74.0, 29.4 and 23.1%, respectively,
and decreased the Tb.Sp by 24.8%, indicating improvement
of osteopenia in ACH mice after the PTH treatment. In WT
littermates, PTH administration increased the Tb.N by
26.1% and decreased the Tb.Sp by 11.6% (Fig. 4C – G). The
trabecular bone in the proximal tibia from 2-month-old ACH
mice was shorter and sparser than that of WT mice, and the
PTH treatment increased the trabecular number (Supplementary Material, Fig. S2B). These results are consistent with
the results obtained by micro-CT examination. Mildly
enhanced TRAP staining in the tibia was observed in ACH
mice compared with WT mice, and no notable changes were
observed in 2-month-old mice after the PTH treatment at the
development stage (Supplementary Material, Fig. S2C).
These observations indicate that the decreased bone mass
and compromised architecture resulting from activated
mutation in FGFR3 in adult mice can be ameliorated by the
PTH treatment at the development stage.
PTH treatment rescues the retarded growth of cultured
tibiae from embryonic ACH mice
PTH may have effects on cells other than skeletal cells, which
may be responsible for the in vivo effects of the PTH (1-34)
treatment on skeletons of ACH and TDII mice observed
above. To clarify this issue, we then utilized an in vitro
bone culture system to examine the direct effects of PTH
(1-34) on bone growth. We found that the total lengths of cultured tibiae from both ACH and WT mice were gradually
increased during the culture period (Fig. 5A). ACH tibiae
had a significantly reduced longitudinal growth rate when
compared with tibiae from WT mice (15.6 + 0.6 and 19.3 +
1.2%, respectively; P , 0.001). The PTH treatment increased
the growth rate of WT tibiae (Fig. 5B) and partially but significantly improved the bone growth of ACH tibiae when compared with those ACH tibiae treated with vehicle control
(21.55 + 2%), which was comparable with those of WT
mice. Analysis of the growth of different zones of cultured
bones revealed a greater inhibition on the growth of calcified
bone in untreated ACH mice than that in WT controls
(Fig. 5C). The expansion of the cartilage stimulated by the
PTH (1-34) treatment markedly overrode the inhibitor effect
of activated FGFR3 on the growth of calcified bone, and
thus the total lengths of the tibiae were increased following
the PTH treatment.
PTH (1-34) stimulates the proliferation and differentiation
of cultured chondrocytes
To further explore the mechanism for PTH rescue, the effect
of PTH on the proliferation and differentiation of
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Figure 3. PTH (1-34) increases the lengths of tibiae and femurs and alleviates the disorganized growth plates in ACH mice. (A –H) Alizarin red S and Alcian
blue staining of the skeletons of mice in each group at P7 and P14. PTH treatment improved the premature fusion of cranial synchondroses in ACH mice. (I– L)
Labeling of BrdU in the growth plates of each group, with PTH-treated mice showing more BrdU-positive cells in growth plates (magnification ×100). (M –P)
Histological analysis of the growth plates of ACH mice in comparison with WT mice. The hypertrophic zone is denoted with bars (Safranin-O and fast green
staining; magnification ×100). (Q–U) Comparison of secondary ossification centers of the proximal tibia from 8-day-old mice. The asterisk shows secondary
ossification (H&E staining; magnification ×200). (V) Bone lengths of 2-month-old mice (n ¼ 6). (W) Quantification of widths of hypertrophic zones in growth
plates (n ¼ 6 –8). Graphs show mean values + SD (two-way ANOVA, ∗∗ P , 0.01; ∗∗∗ P , 0.001).
chondrocytes was evaluated in cultured primary chondrocytes.
Although proliferation was decreased in chondrocytes derived
from ACH mice compared with that of WT control (P ,
0.01), intermittent PTH (1-34) treatments could promote the
proliferation of chondrocytes from both WT (P , 0.01
versus WT control) and ACH mice (P , 0.01 versus ACH
control) despite the fact that PTH-treated chondrocytes from
ACH mice still had a lower proliferation compared with WT
Human Molecular Genetics, 2012, Vol. 21, No. 18
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Figure 4. PTH (1-34) ameliorates osteopenia in ACH mice. (A) Faxitron X-ray analysis of total femurs at 2 months. (B) BMD of femurs from ACH mice was
significantly increased by the PTH treatment (n ¼ 8). (C– G) Quantitative micro-CT analyses of distal femoral metaphysis at 2 months. Three-dimensional
images show reduced trabecular bone in ACH mice and increased trabecular bone with the PTH treatment group (C). Quantification of the structural parameters
showed that BV/TV, Tb.N and Tb.Th were significantly improved by the PTH treatment in ACH mice (D –G). Graphs show mean values + SD (two-way
ANOVA, ∗ P , 0.05; ∗∗ P , 0.01; ∗∗∗ P , 0.001).
control (P , 0.01) (Fig. 6A). The changes in the differentiation of chondrocytes were evaluated by measuring the
mRNA expression of genes related to chondrogenic differentiation using real-time PCR. The results showed that the expression levels of Col 2a1, Col 10a1 and PTHrP mRNA of
chondrocytes from ACH mice were all increased after the
PTH treatment (Fig. 6B). In addition, Alcian blue staining of
chondrocytes showed that ACH chondrocytes secreted less
cartilaginous matrix compared with WT control, whereas
PTH (1-34) increased the secretion of extracellular matrix
(ECM) in ACH chondrocytes (Fig. 6C), which may be
involved in the rescuing effect of PTH on the retarded bone
growth in ACH mice. To gain insights into the mechanism
of PTH (1-34)’s effect on FGFR3-related skeletal dysplasia,
the expression of FGFR3 in primary mouse chondrocytes
was examined after exposure to PTH. The result showed
that the levels of FGFR3 were higher in ACH chondrocytes
than those in WT controls, and were down-regulated following
1028 M PTH (1-34) treatment for 12 h in both WT and ACH
cells (Fig. 6D).
PTH (1-34) promotes differentiation of mesenchyme cells
of limb buds to chondrocytes
Because the PTH treatment significantly increased limb formation, we next performed micro-mass culture assay to
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Human Molecular Genetics, 2012, Vol. 21, No. 18
Figure 5. PTH (1-34) rescues FGFR3-mediated inhibition of growth in cultured murine tibiae. (A) Representative photographs showing the effects of the PTH
treatment on the growth of cultured tibiae (cultured for 7 days) from ACH mice or WT mice (WT). (B) Quantification of increased percentage in total lengths of
bones from WT and ACH mice cultured for 7 days in the absence or presence of PTH (1-34). (C) Changes in bone lengths of the calcified bone (CB) or cartilage
(C) after 7-day cultures. ACH specimens had lower growth velocity compared with WT ones. PTH treatment promoted the growth of cartilage and had an inhibition of growth in calcified bone in both WT and ACH specimens (△, increased length). Graphs show mean values + SD (two-way ANOVA, n ¼ 6 –8, ∗∗ P ,
0.05; ∗∗∗ P , 0.001).
study the effect of the PTH (1-34) treatment on the chondrogenic differentiation of mesenchyme cells from limb buds.
Condensations of mesenchymal cells released from the limb
buds of E11.5 WT and ACH mice plated at high density
first appeared at day 2. The size and density of the cultured
cell mass were increased up to day 6. The small, shallow condensations observed on day 2 were differentiated into nodules
of rounded chondrocytes surrounded by flattened undifferentiated cells. At day 6 (5 days after the addition of
PTH (1-34)), although there were no remarkable differences
in the number of nodules between PTH-treated and -untreated
ACH cells (Fig. 6E), the areas of nodules in the presence of
PTH (1-34) were larger. The nodules in the PTH-treated
group contained more cells and were much deeper and
better formed than those in the non-treated group (Fig. 6F).
PTH (1-34) improves the proliferation of chondrocytes
in ACH mice partially through the down-regulation
of FGFR3 and up-regulation of PTHrP
FGFR3 can inhibit chondrocyte proliferation, differentiation
and matrix synthesis through its downstream pathways including MAPK and cell-cycle inhibitors such as p21 (10,13,15).
We found that the PTH treatment decreased the p21 level in
ATDC5 cells (Fig. 7A). WT FGFR3 and constitutively
active FGFR3 (FGFR3 K650M, causing TD in humans)
were transfected in 293T cells, then the cells were treated
with or without 1028 M PTH (1-34) for 6 h. Our data
showed that the PTH treatment significantly inhibited the
phosphorylation of both WT FGFR3 and FGFR3 K650M
(Fig. 7B), indicating that PTH can inhibit the activation of
FGFR3. PTHrP has been found to increase the proliferation
of chondrocytes (25), and we observed that the PTH (1-34)
treatment increased the expression of PTHrP mRNA in chondrocytes (Fig. 6C), which suggests that PTHrP may be related
to the improved proliferation in PTH-treated chondrocytes
from ACH mice. To examine whether PTH promotes the proliferation of chondrocytes through PTHrP, we utilized small
interfering RNA (siRNA) to knockdown the endogenous
PTHrP and investigated its effects on chondrocyte proliferation. The transfection of PTHrP-targeted siRNA (Si PTHrP)
significantly decreased PTHrP expression by 80% in ATDC5
cells when compared with the scrambled control siRNA (Si
control) (Fig. 7C). PTHrP protein expression was also suppressed by Si PTHrP (Fig. 7D). After transfection with Si
PTHrP or Si control and WT or FGFR3 K650M in six-well
plates for 24 h, cells were trypsinized and plated in 96-well
plates at 5000 cells per well. After overnight culture and the
cells were exposed to 1028 M PTH (1-34) for 6 h; and 24 h
later, the proliferation of ATDC5 cells was analyzed by
MTT assay. Our data showed that although both WT FGFR3
and FGFR3 K650M mutants suppressed the proliferation of
ATDC5 cells, the FGFR3 K650M group exhibited stronger inhibition on ATDC5 proliferation when compared with that in
the WT FGFR3 group. We further showed that the PTH (1-34)
treatment improved the proliferation in both the WT and
FGFR3 K650M groups. RNAi, against PTHrP, partially
blunted the promoting effects of the PTH (1-34) treatment
on the proliferation of ATDC5 cells (Fig. 7D and E).
DISCUSSION
ACH and TD are the most common forms of dwarfism and
lethal dwarfism, respectively, for which at present there are
Human Molecular Genetics, 2012, Vol. 21, No. 18
3949
Figure 6. PTH (1-34) stimulates proliferation and differentiation of cultured chondrocytes and also promotes limb bud mesenchymal cell differentiation to chondrocytes. (A) MTT proliferation assay showed decreased proliferation of ACH chondrocytes, and the PTH (1-34) treatment increased the proliferation in both
WT and ACH chondrocytes on day 4. (B) mRNA expression of COL2A1, COL10A1 and PTHrP was evaluated by qRT-PCR in cultured chondrocytes from WT
and ACH mice on day 4. Cyclophilin A was the internal standard and data are expressed as fold changes in relation to WT control (n ¼ 5– 6). (C) Alcian blue
staining showed decreased cartilaginous matrix on day 4 in chondrocytes from ACH compared with that of WT cells, and the PTH (1-34) treatment increased
ECM secretion in cultured chondrocytes from both WT and ACH mice. (E) There were no significant differences in the number of nodules between PTH-treated
and -untreated cells on day 2, day 4 and day 6. (D) Effects of 1028 M PTH (1-34) treatment for 12 h on the expression levels of FGFR3 as assessed by western
blotting in primary chondrocytes isolated from WT and ACH mice. (F) The nodules formed at day 6 after 5-day culture in the presence of PTH (1-34) were larger
in size and had more cells than those in the non-treated ACH group. Alcian blue staining was much darker in the PTH-treated group. Dashed circles show areas of
nodules (magnification: left panels ×100, middle panels ×400, right panels ×400). Expressions of chondrocyte differentiation markers. Graphs show mean
values + SD (two-way ANOVA, ∗∗ P , 0.01; ∗∗∗ P , 0.001).
few effective treatments. ACH patients can be treated with
growth hormone and the surgical limb-lengthening technique.
While the recombinant hGH treatment can increase the shortterm growth velocity in children with ACH/HCH, the increase
cannot be readily achieved during the long-term treatment
(20,26). Surgical limb lengthening may produce cosmetic
and psychological benefits to some extent, but the extremely
invasive nature, expense, risks and complications may make
patients not to choose this surgery (18,27). Moreover, the stenosis of the foramen magnum and spinal canals cannot be
readily corrected by surgery and may finally cause serious
neurological complications, including hydrocephalus and
sudden death in infancy as well as headaches in older ACH
patients (28 –30). As for the neonatal lethality of TD patients,
no practical, effective therapies are available in clinics presently. TD patients usually already have development abnormality
during gestation, which may lead to death before birth (31).
The growth-promoting treatment and surgery are difficult to
be carried out during pregnancy. So far, there are few reports
about the treatment of postnatal TD patients, either (32).
3950
Human Molecular Genetics, 2012, Vol. 21, No. 18
Figure 7. PTH (1-34) improves cell proliferation through the down-regulation of FGFR3 activity and the up-regulation of PTHrP. (A) PTH treatment downregulated the protein level of p21 in the FGFR3-expressing chondrogenic cell line ATDC5. (B) PTH treatment inhibited tyrosine kinase activity of FGFR3.
WT and FGFR3 K650M mutants were transiently expressed in 293T cells, lysed and immunoprecipitated with anti-FGFR3 antibody. Blots were developed
using an anti-FGFR3 antibody or an anti-phosphotyrosine antibody (4G10). (C– D) Real-time PCR and western blotting show that Si PTHrP decreased the expression of PTHrP in both RNA and protein levels. (D and E) Intermittent PTH (1-34) treatments improved the blunted ATDC5 proliferation resulting from
FGFR3 transfection, and the SiRNA against PTHrP partially offset this effect. Viability in ATDC5 cells, measured by MTT assay. Cell viability is plotted
as a percentage of cells treated with Si Control in the absence of PTH. Experiments were performed in triplicate, with standard deviations shown.
Researchers have been working to find other approaches to
treat or alleviate the skeletal phenotypes of FGFR3-related
skeletal dysplasia. Current therapeutic strategies have
focused on reducing signals activated by FGFR3 (33).
Aviezer et al. (34) reported an inhibitor selective for FGFR3
reconstituted normal growth in cultured limb bones from
ACH mice, but had little effect on live mice. Rauchenberger
et al. (35) found an antibody against FGFR3 which can effectively block ligand-induced FGFR3 activation; however, there
have been no subsequent reports about its in vivo effect on
bone development. In addition, BMP signaling was found to
rescue the reduced regions of proliferating and hypertrophic
chondrocytes in growth plates of cultured limbs from ACH
mice (36), but whether BMP can increase the bone length of
ACH mice has not been reported. Furthermore, since overexpression of Snail1 in the developing bone has been shown to
lead to ACH-like phenotype in mice by acting as a downstream signaling molecule of FGFR3 in chondrocytes that regulates both Stats and MAPK pathways (37), it is suggested that
Snail1 may be a new encouraging non-surgical therapeutic
target (31). Similarly, CNP has been found to prevent the
shortening of achondroplastic bones by correcting the
decreased ECM synthesis in the growth plates through its inhibition on the MAPK pathway of FGF signaling; however,
CNP had no effects on the Stat1 pathway of FGF signaling
that mediates the decreased proliferation and the delayed differentiation of achondroplastic chondrocytes (38– 40). Targeted overexpression of CNP in cartilage or systemic
administration of CNP was shown to partially rescue the
impaired skeletal growth of ACH mice (39). Since all these
molecules have not been used clinically or have not been
examined systematically, further work is needed to study
their dosage, clinical safety and compliance before their eventual clinical application.
We previously revealed that the expression of PTHR1 in
growth plates of ACH mice was decreased (21), and Li
et al. (41) found that FGFR3 signaling down-regulated the expression of PTHR1 gene through the Jak/Stat pathway in
chondrogenic cells (ATDC5). We and Ueda et al. (21,22)
further found that PTHrP or PTH (1-34) can partially
reverse the retarded long bone growth of the cultured bone
rudiments from knock-in and transgenic ACH mice. As recombinant human PTH (1-34) (teriparatide) is the
FDA-approved drug to treat osteoporosis in humans by promoting bone formation, these results indicate that the clinically available drug, PTH (1-34), may potentially be used to treat
ACH or TD patients. However, so far there are no data about
the in vivo effect of PTH on ACH/TD patients or mice. In the
current study, the in vivo effects and action mechanisms of the
PTH (1-34) treatment on skeletal development and survival
have been explored in the ACH and TDII mice and cells isolated from these mice. We found that systemically administered PTH (1-34) can rescue the lethal phenotype of TDII
mice. All TDII offspring born from pregnant mice injected
with PTH (1-34) survived for 5 days or longer after birth,
whereas all untreated TDII mice died several hours after
Human Molecular Genetics, 2012, Vol. 21, No. 18
birth, which is consistent with our previous report (12). Since
the lungs of PTH (1-34)-treated TDII mice were improved to
have grossly normal alveolarization and the PTH treatment
significantly promoted skeletal growth of TDII mice, it is
likely that the prevention of the early postnatal lethality of
TDII mice by PTH (1-34) is related to the improved lung
alveolarization secondary to the ameliorated development of
thoracic cavity.
In ACH mice, our study found that the PTH (1-34) treatment had significant positive effects. PTH-treated ACH mice
had longer naso-anal lengths than ACH control mice, and
the bone lengths of humeri and tibiae were rescued to be comparable with those of WT control mice. Interestingly, our
study also found that the premature fusion of cranial synchondroses in ACH mice was delayed after the PTH (1-34) treatment, suggesting that the PTH treatment may alleviate the
progressive narrowing of neurocentral synchondroses that
cannot be readily corrected by surgery, and thus reduce the
complications in the nervous system caused by the limited
volume of the skull. In addition, as we observed previously
that ACH mice have osteopenia (24), in the current study
we further found that the PTH treatment also improved the
bone mass and structure, which indicates that the PTH treatment at the development stage not only promotes the skeletal
growth during the development stage, but also improves the
bone quality at the adult stage. The mechanism for improved
bone quality is not clear presently, although it may be secondary to the improved development of the skeleton.
The cellular mechanism for the rescue of ACH and TD mice
by PTH is not clear. Impaired chondrocyte proliferation and
differentiation, as well as increased chondrocyte apoptosis,
have been attributed to the retarded growth of long bones in
ACH/TD patients and mice (21,42). PTH/PTHrP/PTHR1 signaling can promote the proliferation of chondrocytes and
inhibit the differentiation of chondrocytes through its downstream signaling molecules (25,43). Whereas PTHrP signaling
plays a dominant role in regulating the pool of proliferating
cells during limb development, FGFR3 signaling has a more
prominent role in cartilage maturation (44). In the current
study, we revealed that PTH (1-34)-treated ACH mice had
an increased chondrocyte proliferative index and a thicker
hypertrophic zone. These data suggest that the PTH treatment
leads to increased chondrocyte proliferation and differentiation, which was further supported by results from studies
on cultured primary chondrocytes. However, although the
increased chondrocyte proliferation was expected as PTH/
PTHrP/PTHR1 signaling can stimulate chondrocyte proliferation, the enhanced chondrocyte differentiation following the
PTH treatment was unexpected as PTHrP/PTHR1 signaling
is known to block chondrocyte differentiation (25). Since
chondrocyte proliferation is tightly followed by differentiation, we speculate that the enhanced proliferation in
PTH-treated ACH mice may override the inhibition of PTH
on chondrocyte differentiation. Alternatively, the different
effects of the continuous and intermittent PTH treatments
may be responsible for the results. In fact, the inhibitory
effects of PTH signaling on chondrocyte differentiation have
been mainly concluded from patients or mice with
gain-of-function or loss-of-function mutations of PTHR1 or
PTHrP (45– 49), in which the functional status of PTH
3951
pathway lasts for the life time, which differs with the intermittent action of PTH that was injected to mice in our current
study. In bone metabolism, the effects of intermittent and continuous activation of PTH signaling are known to be different.
Although intermittent PTH treatments have anabolic effects on
bone remodeling by promoting osteoblast proliferation, differentiation and mineralization, continuous usage has opposite
effects through its relatively stronger stimulation on osteoclasts (50,51). However, whether the enhanced chondrocyte
differentiation observed in the current study following intermittent PTH treatments is related to the manner of PTH
(1-34) administration needs to be further studied. Overall,
we found that intermittent PTH (1-34) administration can
promote chondrocyte proliferation and differentiation and
rescue the bone growth retardation of ACH mice and postnatal
lethality of TDII mice.
The molecular mechanisms for the effects of the PTH treatment on FGFR3-related skeletal dysplasia are not fully clarified currently. PTH signaling is transmitted through the
G-protein-coupled receptor, which has classical downstream
signaling pathways including PKA, PKC and MAPK/ERK
(25,52). Whether these pathways are involved in the effects
of PTH on the ACH/TD skeleton is not known at this
moment. CNP has been found to rescue the skeletal phenotype
of ACH mice by inhibiting the ERK pathway, a major downstream pathway of FGFR3 partially responsible for the
retarded skeletal development of ACH (53). Since PTH can
activate ERK (54), the PTH-activated ERK pathway may
not likely be involved in the effects of PTH on the ACH skeleton. We found that the PTH treatment can decrease the expression of FGFR3 and the phosphorylation of FGFR3,
which may be an important reason for the rescue effects of
PTH on the ACH skeleton. The molecular mechanisms for
the down-regulation of FGFR3 expression by PTH remain to
be further explored. p21 is a cyclin-dependent kinase inhibitor
and can cause cell-cycle arrest in the G1 phase (55). Besides
the increased expression of p21 in mice mimicking human
ACH (15), growth plate chondrocytes from ACH children
and TDII fetuses were found to have increased expression of
p21 (56,57). Since we found that the PTH treatment suppressed the expression of p21 in ATDC5 cells, we speculate
that the decreased FGFR3 activity and p21 expression resulting from the PTH treatment are related to the rescue effect
of PTH (1-34) on the retarded bone growth of TD and ACH
mice. In addition, we also found that the PTH treatment led
to increased expression of PTHrP in cultured chondrocytes.
Since PTHrP promotes chondrocyte proliferation (25), our
results suggest the increased PTHrP expression may also be
responsible for the positive effect of PTH on bone development. Furthermore, we utilized siRNA approach to knockdown the PTHrP expression and found that Si PTHrP
blunted the promoting effect of PTH (1-34) on the proliferation of ATDC5 cells. Thus, the increased expression of
PTHrP level may also be involved in the promoting effects
of PTH (1-34) on the retarded bone growth in ACH and
TDII mice.
Although we revealed the beneficial effects of in
vivo-administered PTH on the repressed skeletal development
of ACH/TD mice, the effects are still limited, as the
PTH-treated TDII mice still displayed short and round skull,
3952
Human Molecular Genetics, 2012, Vol. 21, No. 18
and the structure of long bone growth plates, although
improved, was still disorganized with a morphology similar
to that found in ACH mice. More research is needed to optimize the outcome of the PTH treatment.
Our preliminary data showed that additional postnatal PTH
(1-34) treatment can lead to longer survival of TDII mice, but
the mutants still have skeleton dysplasia. To further improve
treatment effects, future studies are needed to investigate the
timing and dosage of PTH administration, as well as the combination treatment of PTH with other measures such as growth
hormone, BMP, CNP and/or surgical approach.
In addition, more research into long-term safety of the PTH
treatment is needed. PTH was found to have the potential to
cause osteosarcoma in rats administered daily up to 2 years
(58), and human data showed that 2 patients in more than
430 000 osteoporotic persons who have received the PTH
(1-34) treatment developed osteosarcoma (59). In the current
study, the tibial and femoral metaphyses were checked attentively, where the primary osteosarcoma commonly occurs
(58), and were found to have no malignancy in PTH
(1-34)-treated mice. Additionally, since somatic gain-offunction mutations of FGFR3 have been found in cervical
cancer, bladder cancer and multiple myeloma (60,61), we
have also examined this issue carefully in PTH (1-34)treated ACH mice, and found no obvious differences in the
histology of important internal organs (heart, lung, liver,
kidney and small intestine) between TDII and WT mice
born from pregnant mice treated with PTH (1-34) (data not
shown).
In summary, in our present study, we demonstrated that the
PTH (1-34) treatment can rescue the retarded skeletal development and early postnatal lethality and improve the bone
quality of ACH/TD mice possibly through its down-regulation
of FGFR3 activity and up-regulation of PTHrP expression.
Our study suggests that PTH can potentially be used in
clinic to treat ACH and/or TD patients.
MATERIALS AND METHODS
Animals
Fgfr3+/G369C and Fgfr3+/K644Eneo mice were generated by us
before (9,12), and EIIa-Cre transgenic mice were kindly provided by Dr H. Westphal (62). To generate mice heterozygous
for the mutation (Fgfr3+/K644E) (TDII), the neo gene was
excised from Fgfr3+/K644Eneo construct, using Cre-loxP recombination by crossing with EIIa-Cre transgenic mice. All
experiments were performed in accordance with protocols
approved by the Institutional Animal Care and Use Committee
of Daping Hospital.
Administration of PTH to mice
Recombinant human PTH (1-34) was purchased from the
Anaspec Institute (Fremont, Canada) and dissolved in
vehicle (sterile water for injection). For Fgfr3+/G369C mice
(ACH mice) and their WT littermates, PTH was administered
subcutaneously at the dose of 100 mg/kg body weight per day
for 4 weeks after birth. PTH was also administered subcutaneously at the dose of 100 mg/kg body weight in pregnant
Fgfr3+/K644Eneo mice crossed with EIIa-Cre mice at E13.5.
Untreated animals were injected with the same volume of
vehicle alone as control.
Skeletal analysis and histology
Briefly, whole-skeletons were harvested, stored in 70%
ethanol and were subjected to high-resolution X-ray examination using Faxitron MX20, and the lengths of the bones
were measured on the soft X-ray film. BMD was measured
by DEXA (PIXimus Mouse 11 densitometer, GE Medical
System, Madison, WI, USA). The whole-skeleton staining
with Alizarin red S and Alcian blue was performed as
described (63). For histological analysis, the right tibiae
were fixed in 4% paraformaldehyde in 0.01 M PBS (pH 7.4),
decalcified in 15% EDTA (pH 7.4) and embedded in paraffin
as described (24). Six-micrometer-thick sections were sliced
and stained with Safranin-O, Fast Green, hematoxylin and
eosin (H&E). Analyses of the resting zone, proliferation
zone and hypertrophic zone were conducted based on zonal
definition as described by Reinholt et al. (64).
Micro-CT scanning and analysis
The bone structure and the volume of the femoral distal metaphysis were scanned with micro-CT (viva CT-40, Scanco
Medical AG, Switzerland). Image acquisition was performed
at 70 kV and 114 mA. Every measurement used the same
filtering and segmentation values. Bone structural parameters
including BV/TV, Tb.Th, Tb.N and Tb.Sp were automatically
determined after manually selecting the trabecular bone
area (65).
BrdU labeling assay
Mice were injected 100 mg/g body weight BrdU intraperitoneally (Sigma-Aldrich) 2 h before sacrifice. Sections of knee
joints were incubated overnight with anti-BrdU antibody
(Sigma-Aldrich), followed by a secondary antibody and DAB
color development, and counterstaining with hematoxylin.
Mesenchymal cell micro-mass cultures
Cells released from E11.5 limb buds were plated in 20 spots
containing 1 × 107 cells/ml on 60 mm dishes and left for 1 h
at 378C before adding 4 ml of Dulbecco’s modified Eagle’s
medium and F-12 (1:1) medium (Hyclone) containing 10%
FBS and incubated overnight. Cultures were then rinsed
once with serum-free medium before adding medium supplemented with 2% FBS with or without 1028 M PTH (1-34)
and incubating for 4 days with medium changes every
second day. PTH (1-34) was added for 6 h, after which the
medium was changed and the culture was maintained for
another 42 h. Thus, every 48 h acts as a cycle to mimic the
intermittent administration of PTH (1-34) (50). At the indicated times, dishes were rinsed three times with PBS (pH
7.4) before fixing for 1 h in 4% paraformaldehyde in PBS.
Fixed cultures were rinsed three times for 5 min each time
with PBS and stained with Alcian blue.
Human Molecular Genetics, 2012, Vol. 21, No. 18
Primary chondrocyte cultures
Primary chondrocytes were isolated from cartilage of knee
joints of P3 mice, using enzyme digestion (66). Cells were
plated at a density of 2 × 106 cells/ml, cultured in DMEM/
F-12 (1:1) medium (Hyclone) supplemented with 100 U/ml
penicillin, 100 mg/ml streptomycin and 10% FBS until reaching subconfluence. PTH (1-34) (1028 M) was added for 6 h in
every 48 h cycle as described above.
3953
normalized to their corresponding values of endogenous
control gene Cyclophilin A. PCR data are expressed as fold
change in relation to WT control. The primer sequences are
as follows: Collagen II (Col2a1): 5′ -CTGGTGGAGCAG
CAAGAGCAA-3′ and 5′ -CAGTGGACAGTAGA CGGAG
GAAAG-3′ ; Collagen X (col10a1): 5′ -GCAGCATTACGAC
CCAAGAT-3′ and 5′ -CATGA TTGCACTCCCTGAAG-3′ ;
PTHrP: 5′ -CATCAGCTACTGCATGACAAGG-3′ and 5′ -GG
TGGTTTTTGGTGTTGGGAG-3; Cyclophilin A: 5′ -CGAGCTTA
GCACTGGAGA-3′ and 5′ -TGGCGTGTAAAGTCACCACC-3.
Cell proliferation assay
Cell proliferation was detected using an in vitro colorimetric
assay (Sigma-Aldrich). Chondrocytes were washed with PBS
and incubated in 200 ml of medium containing 20 ml of
MTT solution (5 mg/ml dissolved in 2% NaHCO3) for 4 h
at 378C. Subsequently, MTT solution was removed and
150 ml of dimethyl sulfoxide (DMSO) was added. After
being mixed for 10 min at room temperature, the purple formazan salt product resulting from the reduction of MTT was
solubilized by DMSO and quantified spectrophotometrically
at 490 nm.
In vitro culture of embryonic tibia bones
Culture of the embryonic tibia bone was conducted according
to the protocol described before (22,67). Tibiae were harvested
from E15.5 mice and were cultured in 48-well plates for 7 days
with BGJb medium (Fitton-Jackson Modified) supplemented
with 0.05 mg/ml ascorbic acid, 0.5 mg/ml L-glutamine, 10 mg/
ml streptomycin, 10 U/ml penicillin, 1 mM b-glycerophosphate
and 0.2% BSA. The culture medium was changed every other
day. The total lengths and longitudinal lengths of the cartilage
and calcified bone were measured using Image-Pro Plus 7.0
(22). Changes in lengths were expressed as percentage increase
relative to the value before the treatment [percentage increase ¼
(length at day 7 2 length at day 0)/length at day 0].
Transfection with siRNA targeting PTHrP
siRNAs targeting PTHrP mRNA (Si PTHrP) and negative Si
Control were designed and synthesized by Ribio Corporation
(Guangzhou, China) and transiently transfected using the
LipofectamineTM 2000 (Invitrogen), according to the manufacturer’s instructions. Forty-eight hours later, PTHrP
mRNA levels were determined by real-time PCR after 48 h
and the levels of PTHrP protein were analyzed by western
blotting after (see what follows) 72 h.
Real-time PCR
Total RNA of cells was isolated using the Trizol reagent
according to the manufacturer’s instructions (Invitrogen),
which was used to assess effects of treatments on mRNA
expressions of genes of interest. All reactions were performed
in an Mx3000P PCR machine (Stratagene) using the Two-Step
QuantiTect SYBR Green RT-PCR Kit (Takara), and the reaction conditions were optimized for each of the genes by changing the annealing temperature. Each run was replicated three
times. Expression levels for each gene of interest were
Western blotting
Mouse primary chondrocytes and ATDC5 were lysed in an
ice-cold buffer containing 50 mM Tris– HCl (pH 7.4),
150 mM NaCl, 1% Nonidet P-40, 0.1% SDS and a cocktail
of protease inhibitors (Roche). Protein samples were resolved
on a 10% SDS– PAGE gel and transferred onto a PVDF membrane (Millipore). Then antibodies against FGFR3, PTHrP and
p21 (Santa Cruz, USA) were used to probe the blots followed
by ECL signal detection.
Statistical analysis
Data were evaluated statistically in SPSS (Version 10.0 for
Windows). Results are shown as mean value + SD. Statistics
were analysed by Student’s t-test or two-way analysis of variance (ANOVA) with Turkey’s post hoc test. Significance was
accepted at P-values ,0.05.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG online.
ACKNOWLEDGEMENTS
We thank Tujun Weng, Jing Yang and Siyu Chen (Daping
Hospital, Third Military Medical University, Chongqing,
China) for technical support.
Conflict of Interest statement. None declared.
FUNDING
The work was supported by the Special Funds for Major State
Basic Research Program of China (973 program) (grant
number 2012CB518106), and grants from the National
Natural Science Foundation of China (grant numbers
81030036, 30530410, 30901527 and 30971607).
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