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First published online 16 October 2008
doi: 10.1242/dev.025825
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1 Laboratory of Reproductive and Developmental Toxicology, NIEHS/NIH, Research
Triangle Park, NC 27709, USA.
2 School of Dentistry, University Michigan, Ann Arbor, MI 48109, USA.
3 School of Dentistry, University of Missouri-Kansas City, Kansas City, MO
64108, USA.
4 Endocrine Unit, Massachusetts General Hospital and Harvard Medical School,
Boston, MA 02114, USA.
5 Dental Research Center, University of North Carolina, Chapel Hill, NC 27599,
USA.
* Author for correspondence (e-mail: mishina{at}umich.edu)
Accepted 24 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: BMP receptor IA, Bone mass, Canonical Wnt signaling, Osteoblast, Osteoclastogenesis, Sclerostin, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Studies of human disorders chondrodysplasia
(Thomas et al., 1996) and
fibrodysplasia ossificans progressiva
(Shore et al., 2006) indicate
the importance of BMP signaling in cartilage and muscle, respectively,
suggesting that BMP signaling plays an important role in controlling
mineralization in musculoskeletogenesis. Bone is the primary component in
skeletogenesis, and osteoblasts are the predominant cell type in bone.
However, the mechanism by which BMP signaling in osteoblasts contributes the
skeletogenesis has not been fully described.
The majority of bones, including long and ectopic bone, are formed through
an endochondral process (Kronenberg,
2003). Condensed mesenchymal cells differentiate into chondrocytes
to form a cartilage template that is later replaced by osteoblasts
(Mackie et al., 2008;
Maes et al., 2007).
Mesenchymal cells and chondrocytes respond to BMP signaling to differentiate
and maintain their features in vivo
(Bandyopadhyay et al., 2006;
Tsuji et al., 2006;
Yoon et al., 2005). By
contrast, during the alternate process of intramembranous bone formation,
mesenchymal cells differentiate directly into osteoblasts without going
through a cartilaginous phase. In this study, we genetically altered BMP
signaling in osteoblasts using a mouse model. To avoid secondary effects from
chondrocytes on osteoblasts in the endochondral process, we primarily examined
intramembranous bone formation (e.g. calvaria) and demonstrated the direct
effects that BMP signaling has on osteoblasts.
BMP receptor type IA (BMPR1A), which is abundantly expressed in bone, is
activated by major BMP ligands BMP2 and BMP4. Conventional knockout of BMP2,
BMP4 and BMPR1A in mice leads to embryonic death before bone development
(Mishina et al., 1995;
Winnier et al., 1995;
Zhang and Bradley, 1996). We
previously disrupted Bmpr1a during adult stages in an
osteoblast-specific manner using Og2-Cre mice
(Mishina et al., 2004). This
study suggests that the response of osteoblasts to loss of BMP signaling is
age dependent, as bone volume decreased in young mice but increased in old
mice. Similarly, the mechanism by which BMP signaling regulates bone mass is
not straightforward, as loss-of-function of BMP2 and gain-of-function of BMP4
both reduce bone mass (Okamoto et al.,
2006; Tsuji et al.,
2006). Bone mass is determined by the balance of bone formation
and resorption, and osteoblasts regulate both processes. Thus, we focused on
osteoblasts and addressed the complicated effect of BMP signaling on bone
mass.
Human genetic studies have shown that loss-of-function mutations in
components of Wnt signaling, such as the Wnt co-receptor low-density
lipoprotein receptor-related protein 5 (LRP5), is associated with osteoporosis
(Gong et al., 2001;
Patel and Karsenty, 2002).
Dominant missense LRP5 mutations are associated with high bone mass (HBM)
diseases (Boyden et al., 2002;
Little et al., 2002;
Van Wesenbeeck et al., 2003),
indicating that canonical/β-catenin Wnt signaling enhances bone mass
(Baron et al., 2006;
Glass and Karsenty, 2006;
Krishnan et al., 2006). In
vitro, Wnt signaling induces BMP expression
(Bain et al., 2003;
Winkler et al., 2005), whereas
BMPs induce Wnt expression (Chen et al.,
2007; Rawadi et al.,
2003), suggesting that both BMP and Wnt signaling may
synergistically regulate each other in osteoblast, possibly through
autocrine/paracraine loop. Both BMP and Wnt signaling induce bone mass;
however, the mechanism by which BMP and Wnt signaling cooperate to affect bone
mass is not well understood, particularly during embryonic development when
bone mass dramatically increases.
Here, we have employed a tamoxifen-inducible Cre-loxP system under the control of a 3.2 kb type I collagen promoter and have disrupted or upregulated BMP signaling through BMPR1A in osteoblasts during embryonic bone development. We unexpectedly found increased bone mass in response to loss of BMPR1A in osteoblasts and a new interaction between BMP and Wnt signaling through sclerostin.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Hayashi and McMahon,
2002) under the control of a 3.2 kb mouse pro-collagen
1(I) promoter (Col1-CreERTM), which is active in
osteoblasts, odontoblasts and tendon fibroblasts
(Rossert et al., 1995), were
generated by pronuclear injection and crossed with floxed Bmpr1a mice
(Mishina et al., 2002). Mice
that conditionally express a constitutively active form of Bmpr1a
(caBmpr1a), which has a mutation in the GS domain of BMPR1A that
results in ligand-independent activation of Smad signaling after Cre
recombination, were generated using a transgenic construct similar to one
previously reported (Fukuda et al.,
2006) (see Fig. S4A in the supplementary material).
ROSA26 Cre reporter (R26R)
(Soriano, 1999) and
TOPGAL (DasGupta and Fuchs,
1999) mice were obtained from Dr Philippe Soriano and the Jackson
Laboratory, respectively. Tamoxifen (TM; Sigma) was dissolved in a small
volume of ethanol, diluted with corn oil at a concentration of 10 mg/ml. TM
(75 mg/kg) was injected intraperitoneally daily into pregnant mice (100 to 200
ml/mouse) for at least 3 days starting at E13.5.
Histological analysis and skeletal preparation
Whole-mount β-gal staining was performed as previously described
(Mishina et al., 2004). For
histological analysis, fetuses were fixed in 4% paraformaldehyde, embedded in
paraffin, and sectioned frontally for calvariae and sagittally for long bones
at 6 µm. Sections were stained with Hematoxylin and Eosin or Eosin alone
for β-gal stained samples. For von Kossa staining to detect mineral
deposition, sections were covered with filtered 5% silver nitrate (Sigma),
exposed to ultraviolet light for 45 minutes and placed in 5% sodium
thiosulfate (Sigma) for a few seconds. For BrdU (bromodeoxyuridine)
incorporation, 100 µM of BrdU (Roche) was injected into pregnant females
intraperitoneally 2 hours before collecting calvariae. TRAP (tartrate
resistant acid phosphatase) staining was performed using the leukocyte acid
phosphatase kit (Sigma). Immunostaining was performed using primary antibodies
against BMPR1A (Orbigen) (Yoon et al.,
2005) and phospho-Smad1, -Smad5, -Smad8 (Cell Signaling) and
sclerostin (R&D). Alexa Fluor (488, 594, Molecular Probes) and ABC kit
(Santa Cruz Biotechnology) were used for detection. Frozen frontal sections at
10 µm were prepared for phospho-Smad1, -Smad5 and -Smad8 antibodies. For
skeletal preparations, mice were dissected and fixed in 100% ethanol, and then
stained with Alcian Blue and Alizarin Red. To count total cell number in
sections, 1 µM of DAPI was treated for 10 minutes.
Quantitative real time RT-PCR (QRT-PCR)
RNA was isolated from calvariae using the Micro-FastTrack 2.0 Kit
(Invitrogen). cDNA was synthesized using the SuperScript Preamplification
System (GIBCO). PCR reactions, data quantification and analysis were performed
(Applied Biosystems). Values were normalized to Gapdh using the
2-
Ct method
(Livak and Schmittgen,
2001).
Calvaria and osteoblast culture
For ex vivo bone culture, newborn calvariae were dissected at the sagittal
suture and cultured in modified BGJ (Invitrogen) supplemented with ascorbic
acid (Sigma, 50 mg/ml) and 5% fetal bovine serum for the first 24 hours in
culture. Hemicalvariae were treated with 4-hydroxyl TM (Sigma, 100 ng/ml) and
sclerostin (R&D, 50 ng/ml) in the absence of serum for 5 days. For in
vitro culture, osteoblasts were isolated from conditional knockout (cKO) and
wild-type newborn calvariae, and cultured in the same media with addition of
4-hydroxyl TM (Sigma, 100 ng/ml) every other day.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Developmental abnormalities in BMPR1A cKO bones
Col1-CreERTM mice were bred into mice homozygous for floxed
Bmpr1a. Bmpr1a cKO fetuses (cKO, Cre+; Bmpr1a
fx/fx) and wild-type controls (WT, Cre-; Bmpr1a fx/fx)
were collected after daily TM injection into pregnant female from E13.5. Gross
morphology of E18.5 cKO was normal (data not shown). Production of BMPR1A was
suppressed in osteoblasts and osteocytes in the cKO
(Fig. 1D), demonstrating
efficient and specific loss-of-function of BMPR1A in cKO osteoblasts.
Phosphorylated Smad 1/5/8 was modestly reduced in cKO
(Fig. 1E), presumably owing to
remaining signals from other type I receptors, such as BMPR1B and ACVR1.
Mineralization was increased in cKO parietal bones when assessed by Alizarin Red staining, although skeletal shape in the forelimb and hindlimb were unchanged (Fig. 2A). The ratio of bone volume to total tissue volume (BV/TV) was 30% higher in cKO calvariae and femora at E18.5 when assessed by micro computed tomography (µCT) (Fig. 2B). Hematoxylin and Eosin staining of E18.5 calvariae demonstrated that cKO parietal bones were markedly thicker than wild type (Fig. 2C). The cKO bony area positive for Eosin was loose, discontinuous and disorganized, whereas in wild type it was compact and lamellar in structure. Von Kossa staining for Ca2+ in mineralized tissue showed increases in mineralized area of cKO calvariae where bone continuity was disrupted (Fig. 2D). In cKO femora where bone mass was increased (Fig. 2B), woven bone was increased in the primary spongiosa (Fig. 2E). By BrdU incorporation for detection of proliferating cells in vivo, positive cells per total cells in bone were unchanged in cKO calvaria (Fig. 2F). These facts suggest that bone mass of cKO was increased both in calvariae and femora, which follow intramembranous and endochondral ossification, respectively.
Effects of BMPR1A signaling on bone formation and resorption markers
Bone mass is determined by the balance between bone formation and
resorption. We examined changes in bone formation markers using calvariae and
QRT-PCR. Expression of Runx2 and osterix (Sp7), genes that
are required for osteoblast differentiation, and bone sialoprotein
(Ibsp), which is induced rapidly prior to calcification, were reduced
in E16.5 cKO (Fig. 3A).
However, expression of these genes, as well as of alkaline phosphatase
(Akp2; Alpl - Mouse Genome Informatics) and osteocalcin
(Bglap2) was unchanged at E18.5. In addition, alkaline phosphatase
activity was also unchanged at E18.5 (data not shown). These results suggest
that bone formation is modestly reduced in E16.5 cKO calvariae, which could
impact the phenotype of E18.5.
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B
ligand), an osteoclast differentiation factor, and OPG (osteoprotegerin), a
decoy receptor for RANKL, both expressed by osteoblasts
(Simonet et al., 1997).
Rankl expression was reduced 30% at E19.5, and Opg was
increased 50% and 80% at E18.5 and E19.5, respectively
(Fig. 3B), resulting in the
reduction of the ratio, Rankl to Opg, by 30% and 60% at
E18.5 and E19.5, respectively (Fig.
3C). As RANKL stimulates osteoclastogenesis, whereas OPG inhibits
it, these changes were consistent with the reduced osteoclastogenesis in cKO
calvariae (Fig. 3B). Consistent
with these data, osteoclast activity evaluated by TRAP staining was
significantly reduced in cKO calvariae at E18.5
(Fig. 3D). In addition,
expressions of other BMP receptors type I (Bmpr1b, Acvr1), type II
(Bmpr2, Acvr2a, Acvr2b) and their ligands (Bmp2, Bmp4, Bmp6
and Bmp7), were not increased in cKO calvariae at E18.5 when assessed
using QRT-PCR (Fig. 3E),
suggesting that a compensation for the loss of BMPR1A by other BMP receptors
or ligands is unlikely. These results suggest that osteoclastogenesis in cKO
calvariae was decreased by loss of BMPR1A.
Effects of BMPR1A signaling on OPG and RANKL in vitro
Bmpr1a-deficient osteoblasts were reproduced by infecting primary
osteoblasts from Bmpr1a fx/fx calvariae with recombinant adenovirus
expressing Cre protein. Expressions of Bmpr1a and Rankl were
reduced 70% and 40%, respectively, whereas Opg increased 2.5-fold in
cKO osteoblasts (CRE) compared with control (Mock) (see Fig. S1A in the
supplementary material), resulting in reduced ratio of Rankl to
Opg by 80% (see Fig. S1A in the supplementary material). These
indicate reduced osteoclastogenesis in Bmpr1a-deficient osteoblasts
at least through the RANKL-OPG pathway, consistent with in vivo data
(Fig. 3).
Monocytic cells, precursors of osteoclasts, were isolated from adult cKO
and wild-type spleens, and were induced by RANKL and M-CSF. There was no
difference both in osteoclast number and Bmpr1a expression levels
between wild-type and cKO cells (see Fig. S1B,C in the supplementary
material), suggesting that cKO osteoclasts are intact and able to fully
accomplish osteoclastogenesis by responding to these cytokines. Next, a
mixture of osteoblasts and osteoclasts was isolated from adult cKO and
wild-type bone marrow cells independently, and induced by 1,
25-dihydroxyvitamin D3. The cKO mixture was defective in osteoclast
number and Bmpr1a expression levels (see Fig. S1B,C in the
supplementary material). There was no Cre activity in osteoclasts in bone
marrow (data not shown). These results suggest that osteoclasts in cKO bone
marrow are intact but cannot accomplish osteoclastogenesis, partly because of
a defect in RANKL-OPG signaling in Bmpr1a-deficient osteoblasts.
Taken together, these data suggest that the reduced osteoclastogenesis in cKO
calvaria (Fig. 3) is primarily
caused by defects in osteoblasts.
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; Glass and Karsenty,
2006; Hartmann,
2006; Krishnan et al.,
2006) regulate bone mass. However, there is no described
interaction between the two in either mouse models or human mutation. We first
investigated canonical Wnt signaling in cKO bones using TOPGAL Wnt
reporter mice. The Wnt signaling assessed by β-gal staining was increased
in cKO calvariae at E17.5 (Fig.
4A). Relative number of β-gal-positive cells was 8.5 times
higher in cKO calvariae compared with wild type, which was significant
(Fig. 4C). Although it is known
that endogenous levels of canonical Wnt signaling assessed using
TOPGAL mice are low in osteoblasts during embryonic bone development
(Hens et al., 2005), the
intensity of β-gal staining was dramatically increased in cKO long bones
compared with wild type (Fig.
4D,E). These suggest that canonical Wnt signaling in cKO mice was
stimulated during both endochondral and intramembranous ossification. Thus,
Bmpr1a deficiency in osteoblasts upregulates Wnt signaling at least
through the canonical pathway.
Downregulation of sclerostin in cKO bones
Alternation of Wnt-related genes were further examined. Sclerostin
(Sost) expression was consistently reduced by 95% in cKO calvariae
from E16.5 to E18.5 when assessed with QRT-PCR
(Fig. 5A) and was the most
severely downregulated at E18.5 on microarray data (-5.68-fold,
P=3.27E-24) (see Fig. S2 in the supplementary material).
Expression levels of Wnt target genes Axin2 and Ctgf were
significantly increased in cKO calvariae at E18.5 when assessed with QRT-PCR
(Fig. 5B), but those of Wnt
ligands (Wnt3a, Wnt5a, Wnt7a, Wnt7b and Wnt9a), other Wnt
inhibitors [dickkopf 1 (Dkk1), Dkk2 and secreted
frizzled-related proteins] and co-receptor Lrp5 were unchanged both
when assessed with microarray and QRT-PCR at E18.5
(Fig. 5A, data not shown).
Immunohistochemistry using E17.5 cKO calvariae confirmed that Bmpr1a
deficiency in osteoblasts and osteocytes correlated with increased levels of
β-gal staining and reduced production of sclerostin at cellular levels
(Fig. 5C,D). The reduction of
sclerostin was reproduced in vitro using primary osteoblasts from newborn cKO
mice (Fig. 5E). Similarly,
Sost expression was 90% reduced by loss of BMPR1A when assessed using
adenoviral Cre infection in vitro (see Fig. S1A in the supplementary
material). These results suggest that sclerostin is a downstream effector of
BMPR1A on Wnt signaling, and that Bmpr1a deficiency in osteoblasts
increased canonical Wnt signaling at least by the suppression of
sclerostin.
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Smad-dependent BMPR1A signaling upregulates sclerostin expression in vivo
To reveal the effects of Smad-dependent BMPR1A signaling on sclerostin
expression and bone morphology in vivo, we generated inducible transgenic mice
expressing caBmpr1a (constitutively activated Bmpr1a) in
osteoblasts (see Fig. S4A in the supplementary material). After daily TM
injection from E13.5 to E17.5, gross morphology of caBmpr1a fetuses
was relatively normal at E18.5 (data not shown). Histology showed moderately
reduced thickness in caBmpr1a calvariae (Cre+,
caBmpr1a+) at E18.5, where levels of phosphorylated Smads were
enhanced compared with controls (Cre-, caBmpr1a+)
(Fig. 7A). Sost
expression when assessed by QRT-PCR increased approximately sevenfold in the
transgenic calvariae at E18.5, indicating that Smad signaling positively
controls Sost expression. Expressions of bone resorption markers were
all increased over fourfold, and Rankl levels increased four times
while Opg levels decreased 40%
(Fig. 7B), resulting in a
6.5-fold increase in the ratio of Rankl to Opg
(Fig. 7C). Expression of bone
formation markers was also increased in the caBmpr1a (see Fig. S4B in
the supplementary material). These changes observed in the caBmpr1a
were the opposite of those seen in Bmpr1a cKO calvariae
(Fig. 3). We next generated
rescue mice expressing caBmpr1a on a Bmpr1a cKO background
(Cre+, caBmpr1a+, Bmpr1afx/fx) and compared them
with littermate Bmpr1a cKO mice (cKO: Cre+,
caBmpr1a-, Bmpr1afx/fx). In E18.5 calvariae from rescued
mice, expression of Sost, Rankl and osteoclast markers increased over
four times (Fig. 7D), and the
ratio of Rankl to Opg increased about 2.5-fold
(Fig. 7E) when compared with
the cKO. There was also a reduction in morphological changes observed in
littermate Bmpr1a cKO mice (Fig.
7F). These results strongly suggest that Sost expression
is regulated by BMPR1A signaling at least through the Smad pathway.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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BMP signaling, bone formation and bone mass
By using a 3.2 kb mouse pro-collagen 1(I) promoter
(Rossert et al., 1995), we
demonstrate that reduction in BMP signaling through BMPR1A in osteoblasts
increased bone mass during embryonic stages. This is consistent with our
previous report that showed increased bone volume (BV/TV) at 10 months by loss
of BMP signaling in osteoblasts, using Og2-Cre mice in which
osteoblasts initiate Cre expression postnatally. However, it is not consistent
with 3-month-old mice, which showed decreased bone mass
(Mishina et al., 2004). The
bone phenotype observed in Bmpr1a-deficient mice with
Og2-Cre was milder than that with Col1-CreERTM. These
discrepancies may be due to differences in the timing of recombination between
promoters. As characteristics of osteoblasts change as they mature
(Aubin, 1998), the effects of
disrupting BMPR1A signaling may be influenced by the stages of osteoblast
maturation. In Col1-CreERTM mice, Cre activity was detected in
immature periosteum that wraps growth plates, in osteogenic centers and in
bone collars (Fig. 1B),
indicating that Cre activation occurs just after commitment of mesenchymal
cells towards osteoblastic cells. Therefore, Col1-CreERTM mice
can induce recombination earlier in osteoblastogenesis, including in
preosteoblasts, compared with Og2-Cre mice, which could explain why
the Bmpr1a-deficient mice using Og2-Cre mice did not change
expression levels of early osteogenic markers Runx2 and Bsp,
and showed mild bone phenotype (Mishina et
al., 2004).
Expression levels of bone formation markers (Runx2, Sp7, Ibsp,
Akp2 and Bglap2) were increased more than twofold in
caBmpr1a mice (see Fig. S4B in the supplementary material),
indicating that BMP signaling through Smad1/5/8 induces osteoblastogenesis, as
is well known in vitro (Chen et al.,
2004). Some of these markers (Runx2, Sp7 and
Ibsp) were reduced in cKO bones as expected
(Fig. 3A), consistent with the
modest decrease in Smad phosphorylation levels
(Fig. 1E) and our previous
report that loss of BMP signaling decreases bone formation rate over bone
surface (BFR/BS) during adulthood (Mishina
et al., 2004). It is also suggested that normal bones respond to
endogenous BMPs by phosphorylating Smads at a very low level, which is
difficult to detect by immunostaining (Fig.
7A). It is possible that proliferation of osteoblasts is increased
in cKO bones, which could influence increased bone mass; however, this is less
likely because the number of BrdU-positive cells per total cells in bone was
unchanged (Fig. 2E).
Histomorphometric analysis of bone is an established method to assess bone
formation but is technically unfeasible in fetuses. Thus, we applied
histomorphometry to adult cKO mice, where we confirmed osteoblasts failed to
support osteoclastogenesis (see Fig. S1B in the supplementary material). Bone
volume (BV/TV) was significantly increased, but formation rate (BFR/BS) and
osteoclast number per bone area (N.Oc/T.Ar) were significantly decreased and
osteoblast surface per bone surface (Ob.S/BS) were unchanged in the adult cKO
(Kamiya et al., 2008). Similar
to embryonic cKO data (Fig. 3),
expression levels of osteoclast markers and the ratio of Rankl to
Opg were significantly reduced when assessed by QRT-PCR in the adult
cKO (Kamiya et al., 2008).
These results suggest that osteoblast number is unchanged and bone formation
is modestly reduced, while resorption markedly decreases in cKO bones,
resulting in a net increase in bone mass. In addition, BMP signaling can
control bone formation and resorption by inducing both osteoblastogenesis and
osteoclastogenesis in vivo.
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; Usui et al.,
2008). However, the link is not detected in those mice
overexpressing Noggin or Bmp4 in osteoblasts using a 2.3 kb
type I collagen promoter (Okamoto et al.,
2006) or by knocking out BMPR1A using an osteocalcin promoter
(Og2-Cre) (Mishina et al.,
2004), whereas osteoclastogenesis is upregulated by BMP signaling.
As the 2.3 kb type I collagen and osteocalcin promoter are first activated in
mature osteoblasts (Ducy and Karsenty,
1995; Kalajzic et al.,
2002), immature osteoblasts, where the 3.2 kb type I collagen
promoter is active, may be crucial for regulating osteoclastogenesis through
the RANKL-OPG pathway. Furthermore, it is possible that BMP signaling directly
controls osteoclastogenesis through the RANKL-OPG signaling pathway probably
by downregulating Opg expression in addition to upregulating
Rankl, because BMP response elements were identified in the
Opg promoter by Evolutionary Conserved Regions (ECR) browser search
(data not shown). Further analysis is necessary to clarify the direct
regulation of RANKL-OPG pathway by BMP signaling.
It is also possible that secondary mediators such as Wnts regulate
expression of RANKL and OPG. Two recent studies have suggested that Wnt
signaling in osteoblasts through the canonical β-catenin pathway not only
boosts bone formation by fostering osteoblast activity but can also inhibit
bone resorption by affecting osteoclasts
(Goldring and Goldring, 2007).
One study provided evidence that the Wnt pathway positively regulates
osteoblast expression of osteoprotegerin (OPG) by overexpressing stabilized
β-catenin in osteoblasts in mice, resulting in decreased osteoclast
differentiation and increased bone volume
(Glass et al., 2005). Another
study showed that osteoblasts lacking the β-catenin gene exhibited
impaired maturation and mineralization with elevated expression of
Rankl and diminished Opg, suggesting that the Wnt pathway
can suppress osteoclast-mediated bone resorption
(Holmen et al., 2005). In our
study, expression levels of Opg and Rankl as well as
osteoclast markers (Mmp9, Ctsk and Trap) were partially
restored by exogenous sclerostin ex vivo with a concomitant reduction in Wnt
signaling (Fig. 6). Similarly,
Wnt inhibitors Dkk1 and Dkk2 can induce osteoclastogenesis by changing the
RANKL-OPG pathway in vitro (Fujita and
Janz, 2007). These facts suggest that Wnt inhibitors in the
canonical/β-catenin pathway enhance osteoclastogenesis. In addition,
Wnt/β-catenin-responsive LEF1-binding sites were identified both in the
Opg promoter and the Rankl promoter by an ECR browser search
(data not shown). These facts strongly suggest that the changes in
Bmpr1a-deficient bones are due to decreased bone resorption and
osteoclastogenesis through the RANKL-OPG pathway, and that sclerostin is
presumably involved in the pathway as a Wnt signaling inhibitor
(Fig. 8).
|
; Itasaki et al.,
2003; Piccolo et al.,
1999). However, although sclerostin directly binds BMPs
(Kusu et al., 2003;
Winkler et al., 2003), its
antagonistic effects on BMP activity in mammals are still controversial.
Resent in vitro studies showed that sclerostin is not a BMP antagonist
(van Bezooijen et al., 2004),
as it binds only weakly to BMPs and does not inhibit direct BMP-induced
responses (ten Dijke et al.,
2008). Together with the evidence from human mutations in
SOST or its co-receptor LRP5, in vitro studies revealed that
sclerostin is a Wnt inhibitor that binds LRP5
(Li et al., 2005;
Semenov et al., 2005;
van Bezooijen et al., 2007).
Loss-of-function and hypomorphic mutations in SOST cause
sclerosteosis (Balemans et al.,
2001; Brunkow et al.,
2001) and Van Buchem disease
(Balemans et al., 2002;
Staehling-Hampton et al.,
2002), respectively, and bone mass is increased in these
disorders, similar to Sost KO mouse
(Li et al., 2008). In human
and mouse, these Sost mutants share the high bone mass (HBM) phenotype with
humans who have gain-of-function mutations in LRP5
(Boyden et al., 2002;
Little et al., 2002;
Van Wesenbeeck et al., 2003)
and by overexpression of Lrp5 in mice
(Babij et al., 2003). By
contrast, loss-of-function of LRP5 leads to osteoporosis pseudoglioma
syndrome in humans with low bone mass
(Gong et al., 2001;
Patel and Karsenty, 2002),
which is similar to the bone phenotype of mice that overexpress Sost
(Winkler et al., 2003).
Consistent with these facts, Bmpr1a cKO mice show a HBM phenotype
with lack of sclerostin expression. In addition, sclerostin differs from the
BMP antagonist Noggin with respect to effects on Wnt signaling, as sclerostin
inhibits Wnt (Fig. 6) whereas
Noggin upregulates it (see Fig. S3A in the supplementary material). Thus, our
study strongly suggests that sclerostin physiologically acts as an inhibitor
of canonical Wnt signaling in vivo during embryonic bone development, and loss
of sclerostin largely directs the Bmpr1a cKO bone phenotype.
The phenotype of Sost KO mice
(Li et al., 2008) is different
from that observed in Bmpr1a cKO with respect to the morphology of
the bone, as Bmpr1a cKO mice showed disorganized bone structure
(Fig. 2), which is not
described in Sost KO mice (Li et
al., 2008) or SOST human disorders. This fact implies
that BMPR1A is not only an upstream effector of sclerostin but also has other
roles beyond regulating Sost in the osteoblasts. In the Sost
KO there is no increase in osteoclast number or erosion surface, which is
consistent with mice overexpressing LRP5 with HBM phenotype
(Babij et al., 2003),
suggesting that bone formation and resorption are uncoupled in these mutants.
However, as we discussed earlier, loss of BMP signaling reduced both bone
formation and resorption together. In addition, the diameter of collagen
fibrils in Bmpr1a cKO bones were heterogeneous, aggregations of
collagen fibers were disorganized and MMP expression levels were reduced (data
not shown), suggesting that BMP signaling additionally regulates proper bone
structure and turnover. In support of our speculation, reduction in
osteoclastogenesis was not fully restored by sclerostin treatment ex vivo
(Fig. 6C). These facts suggest
that BMP signaling in osteoblasts directly regulates bone resorption
independently of Wnt signaling through sclerostin, which in turn maintains
proper bone structure and bone mass.
|
|
; Sutherland et al.,
2004). Although sclerostin is produced primarily by osteocytes in
adult bones (Poole et al.,
2005; van Bezooijen et al.,
2004), it is also detected in osteoblasts before birth
(Fig. 5), suggesting the
positive regulatory role of BMPR1A signaling in Sost expression both
in osteoblasts and osteocytes during embryonic bone development.
Interestingly, the promoter region of the Sost gene contains putative
BMP response elements that were detected by an ECR browser search (data not
shown), suggesting possible regulation of Sost by BMP signaling at
the transcriptional level.
Interaction of BMP and Wnt signaling in bone
Accumulating evidence suggests that both BMP and Wnt signaling may regulate
each other in a context and age-dependent manner
(Barrow et al., 2003;
Guo et al., 2004;
He et al., 2004;
Huelsken et al., 2001). Only a
few cascades such as Pten/Akt (Zhang et
al., 2006) and Smad1/Dvl1 (Liu
et al., 2006) are reported in intracellular crosstalk between the
BMP and Wnt pathways. However, as in bone, the interaction between BMP and Wnt
signaling is not well described generally in vivo, partly because alteration
of BMP signaling has not been studied in Wnt signaling mutants and vice versa.
Some in vitro studies have demonstrated that both BMP and Wnt pathways
synergistically regulate each other possibly through autocrine/paracraine
loop, as BMPs induce Wnts in C2C12 cells and primary osteoblasts
(Chen et al., 2007;
Rawadi et al., 2003), and Wnt
signaling, on the contrary, enhances BMPs expression in C3H10T1/2 cells
(Bain et al., 2003;
Winkler et al., 2005). These
studies indicate that BMP signaling may upregulate Wnt signaling.
By contrast, our in vivo study suggests that BMP signaling downregulates
Wnt signaling, as loss of BMPR1A signaling upregulates Wnt signaling by
inhibiting Sost expression. These discrepancies are partly due to the
differences of cell type examined, as both osteoblasts and osteoclasts always
affect each other as coupling factors in vivo, and coupling effects on the two
signaling pathways are difficult to address using osteoblasts alone in vitro.
Furthermore, it is also difficult for in vitro studies to address
age-dependent changes in both signaling pathways and their interactions.
Similar to our results, other studies have shown antagonistic interaction of
BMP and Wnt signaling in lung (Dean et
al., 2005), intestine (He et
al., 2004), hair (Zhang et
al., 2006) and joints (Guo et
al., 2004). Moreover, Noggin treatment ex vivo upregulated
canonical Wnt signaling and downregulated Sost expression (see Fig.
S3A,B in the supplementary material), presumably by antagonizing BMP2 and
BMP4, ligands for BMPR1A. Thus, our study strongly suggests that BMPR1A
signaling in osteoblasts regulates negatively canonical Wnt signaling through
downstream effector sclerostin during embryonic bone development.
In conclusion, we demonstrate a new interaction between the BMP and Wnt signaling pathways in osteoblasts through sclerostin, a Wnt inhibitor and a bone mass regulator. BMP signaling via BMPR1A directs osteoblasts to reduce bone mass in part by upregulating sclerostin expression and supporting osteoclastogenesis through the RANKL-OPG pathway.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/22/3801/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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