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First published online January 13, 2009
doi: 10.1242/10.1242/dev.027706
Bone Research Program, ANZAC Research Institute, The University of Sydney, Sydney, NSW 2139, Australia.
Author for correspondence (e-mail:
hzhou{at}med.usyd.edu.au)
Accepted 4 December 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: Glucocorticoids, Osteoblasts, Wnt/β-catenin signaling, Cranial cartilage, Mmp14, Intramembranous bone formation, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). Hence,
endochondral ossification proceeds through stages of cartilage maturation and
mineralization before the cartilage template is remodelled into bone.
By contrast, the cranial bones are formed through intramembranous
ossification, where mesenchymal precursor cells derived from both the neural
crest and mesoderm convert directly into osteoblasts without the precondition
of a cartilage intermediate (Jiang et al.,
2002). Intramembranous ossification begins with centres of
condensing mesenchymal cells in which osteoblasts subsequently differentiate.
These centres then expand, and adjacent bones form sutures that consist of
mesenchymal cells providing a reservoir of stem cells for the further bone
formation and growth (the `osteogenic front')
(Opperman, 2000). Premature
closure of the sutures results in cranial dysmorphisms such as
craniosynostosis, one of the most common human craniofacial deformities
(Cohen and MacLean, 2000).
Genetic and molecular evidence suggests that factors such as bone
morphogenetic proteins (BMPs), fibroblast growth factors (FGFs) and the
Wnt/β-catenin signaling cascade play an important role in controlling
cranial bone formation (Byeong S. Yoon,
2004; Day et al.,
2005; Liu et al.,
2002; Nie et al.,
2006; Ohbayashi et al.,
2002; Spater et al.,
2006). However, the regulation of intramembranous skeletal
development upstream of these signaling pathways remains unknown.
Although the formation of calvarial bones does not follow the endochondral
ossification pattern, calvarial cartilage does exist during skull formation.
Embryonic cranial cartilage is prominent at mid-gestation and grows
substantially in the latter part of embryogenesis, reaching its most
conspicuous extent at E17.5 (Holmbeck et
al., 2003). However, unlike in endochondral bone formation, this
unmineralized cartilage does not become mineralized but gradually disappears
as intramembranous ossification proceeds. There is some evidence that the
cranial cartilage is removed, at least in part, through the action of matrix
metalloproteinase 14 (Mmp14) (Holmbeck et
al., 2003). As the calvarial cartilage is not directly involved in
intramembranous bone formation, the processing of calvarial cartilage has
often been ignored in studies of cranial bone development. As a result, the
mechanisms responsible for the patterning of cartilage processing during skull
development remain obscure. Specifically, it is unclear why and when the
cranial cartilage appears and disappears, which interactions occur between
cranial cartilage and other tissues, and which cellular signals eventually
induce cartilage removal.
Glucocorticoids (GC) play an important role in bone cell differentiation
and are known to influence both osteoblast and adipocyte lineage commitment
(Herbertson and Aubin, 1995;
Shalhoub et al., 1992;
Zhou et al., 2008). In
addition to the action of GC through its cognate receptor, specific enzymes
modulate GC metabolism within the cell at the pre-receptor level
(Draper and Stewart, 2005;
Stewart and Krozowski, 1999).
Within certain tissues, two isoforms of 11β-hydroxysteroid-dehydrogenase
(11βHSD) vary intracellular GC concentrations independently of
circulating GC levels: 11βHSD type 1 (11βHSD1) predominantly
converts inactive cortisone to active cortisol to increase intracellular GC
concentrations; by contrast, 11βHSD type 2 (11βHSD2)
unidirectionally catalyses the conversion of active GC to their inactive
metabolites (Stewart and Krozowski,
1999). Kream and colleagues generated a Col2.3-11βHSD2
transgenic mouse, in which the rat gene for 11βHSD2 was linked to the 2.3
kb collagen type I (Col2.3) promoter to target transgene expression to mature
osteoblasts (Kalajzic et al.,
2002b). The Col2.3 promoter has been well characterized and
specifically targets gene expression to mature osteoblasts and osteocytes in
the bones of transgenic mice, with no expression in bones derived from
wild-type littermates (Kalajzic et al.,
2002a; Kalajzic et al.,
2005). As overexpression of Col2.3-11βHSD2 in mice results in
the inactivation of cytoplasmic corticosterone, GC signaling is effectively
disrupted in the mature osteoblasts of Col2.3-11βHSD2 transgenic mice
(Sher et al., 2006;
Sher et al., 2004). Female
Col2.3-11βHSD2 transgenic mice exhibit vertebral osteopenia, reduced
femoral cortical bone area and thickness, and impaired mineralized nodule
formation in primary calvarial cultures
(Sher et al., 2004). We have
recently shown that cell cultures derived from Col2.3-11βHSD2 transgenic
mice show dominant adipogenesis and reduced osteoblastogenesis in vitro. This
phenotypic change is due to a failure in Wnt signaling, which normally allows
osteoblasts to exert direct control over the lineage commitment of their
mesenchymal progenitors (Zhou et al.,
2008).
In the present study, we investigate the role of endogenous glucocorticoids on embryonic and postnatal murine skeletal development. Using a tissue-specific approach via osteoblast-targeted transgenic disruption of endogenous GC signaling, we delineate a novel paracrine mechanism in which osteoblasts - under the control of glucocorticoids - orchestrate the process of intramembranous bone formation by directing mesenchymal cell commitment towards osteoblastic differentiation while simultaneously initiating and controlling cartilage dissolution in the postnatal mouse. This pathway involves Wnt and Mmp14 as downstream effectors of GC action, and therefore may have relevance to wider areas of clinical concern, such as understanding the therapeutic and adverse effects glucocorticoid treatment, and the cellular mechanisms involved in disorders such as cancer and inflammatory joint disease.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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) and were a gift from Dr Barbara Kream (Department of
Medicine, University of Connecticut Health Center, Farmington, CT, USA).
Col2.3-GFP mice generated in the CD-1 outbred background
(Kalajzic et al., 2002b) were
provided as a gift by Dr David Rowe (Department of Genetics and Developmental
Biology, University of Connecticut Health Center, Farmington, CT, USA). Mice
were maintained at the animal facilities of ANZAC Research Institute (Sydney,
Australia) in accordance with Institutional Animal Welfare Guidelines and an
approved protocol. Mice were maintained under specific pathogen-free, temperature-controlled conditions throughout this study at the animal facilities of ANZAC Research Institute in accordance with Institutional Animal Welfare Guidelines and an approved protocol.
Skeletal preparation and histology staining
After sacrifice, mice were eviscerated and skin was removed. Following
fixation in 95% ethanol for 24 hours, mice were stained in Alcian Blue
solution (150 mg Alcian Blue, 800ml 98% ethanol, 200ml acetic acid) overnight.
After rinsing with 95% ethanol for several hours, the specimens were
transferred to 2% KOH for 24 hours and then further stained in Alizarin Red
solution (50 mg/l Alizarin Red in 2% KOH) for a further 24 hours. Skeletons
were kept in 1% KOH/20% glycerol until the skeletons became clearly visible
and then were stored in 50% ethanol/50% glycerol.
For histological analysis, tissues were harvested from Col2.3-11βHSD2 transgenic mice and their wild-type littermates and fixed with 4% paraformaldehyde in PBS for 24 hours, decalcified in 10% EDTA for 3-7 days (depending on animal age), embedded in paraffin and sectioned at 5 µm. Sections were then stained with Hematoxylin and Eosin for morphological studies, with Toluidine Blue for detection of cartilage, and with alkaline phosphatase (ALP) for detection of osteoblastic cells.
Microcomputed tomography (micro-CT)
Micro-CT of mouse heads was performed using a Skyscan 1172 scanner
(SkyScan, Kontich, Belgium). Scanning was carried out at 60 kV, 167 µA,
with no filter, and exposure set to 1180 ms. In total, 1125 projections were
collected at a resolution of 12.1 µm/pixel. Reconstruction of sections was
carried out with software associated with the scanner (Nrecon) with beam
hardening correction set to 50%. To obtain 3D visualization from reconstructed
sections we used VGStudio MAX 1.2 software (Volume Graphics GmbH, Heidelberg,
Germany). The sagittal suture area was measured on digitally recorded
projections of micro-CT 3D images using interactive image analysis software
(ImageJ, NIH).
TUNEL staining
Paraformaldehyde-fixed tissue sections on slides were deparaffinized and
rehydrated into distilled water through a series of decreasing ethanol washes.
TUNEL labeling of apoptotic cells was performed using an In Situ Cell Death
Detection Kit (Roche Diagnostics), according to the manufacturer's protocol.
The proportion of apoptotic cells was quantified by counting two fields of
each section and two sections in each animal.
In situ hybridization
The 986 bp murine Mmp14 and 889 bp murine Wnt9a probes were generated by
reverse transcription PCR using RNA derived from mouse calvarial osteoblasts.
The resultant fragments (Mmp14, nucleotides 409-1394, GenBank Accession Number
X83536; Wnt9a, nucleotides 3-891, GenBank Accession Number AB072311) were
cloned into pGEM-T (Promega). The DIG-labeled riboprobes were transcribed with
either T7 or SP6 RNA polymerase to generate antisense and sense riboprobes
using a RNA labeling kit (Roche Diagnostics) according to the manufacturer's
instructions.
In situ hybridization was performed as previously described
(Kartsogiannis et al., 1998).
To ensure the sections of wild-type and transgenic mice were hybridized under
identical detection conditions, the wild-type and transgenic calvarial samples
were always sectioned as a pair and mounted on the same slide. After dewaxing,
sections were deproteinized with 0.2 M HCl followed by digestion with
proteinase K at 2 µg/ml in 0.1 M Tris buffer (pH 8.0)/50 mM EDTA for 30
minutes at 37°C. Tissues were then fixed in 4% paraformaldehyde for 15
minutes at room temperature before hybridization. Hybridization was performed
with hybridization buffer containing DIG-labeled antisense or sense probes at
a final concentration of 4-8 ng/µl for 16-18 hours. Slides were washed and
the hybridized probe was detected with the alkaline phosphatase-coupled
anti-DIG antibody (Roche Diagnostics).
Immunohistochemistry
Immunolocalization of Mmp14 was performed on 10 µm cryosections using
anti-Mmp14 rabbit polyclonal antibody (1:100; Chemicon International).
β-catenin immunohistochemical staining was performed on 5 µm paraffin
sections using anti-β-catenin rabbit polyclonal antibody (1:50; Cell
Signaling Technology) after heat-induced citrate buffer antigen retrieval. The
signals were detected using a biotinylated goat anti-rabbit secondary antibody
(1:150 dilution; Vector Laboratories) in combination with the ABC kit (Vector
Laboratories) and DAB substrate (Vector Laboratories). Wild-type and
transgenic calvarial samples were sectioned as a pair and mounted on the same
slide so they were incubated under identical detection conditions.
Wnt3a induced bone formation in vivo (calvarial injection)
One-day-old Col2.3-11βHSD2 transgenic mice were injected
subcutaneously with 100 ng of recombinant Wnt3a protein (Chemicon
International) in 0.01% CHAPS/PBS in a volume of 10 µl over their calvaria
above the sagittal suture between the parietal bones daily for 2 days. A group
of Col2.3-11βHSD2 transgenic mice received 0.01% CHAPS/PBS injections as
vehicle control. The mice were sacrificed 3 days after birth and were
subjected to micro-CT and histology analysis.
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Statistical analysis
All data were presented as the mean±s.e.m. and statistical analyses
were performed using Student's t-test. A P<0.05 was
considered statistically significant.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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To determine at what developmental stage this skeletal defect first occurred, embryos of Col2.3-11βHSD2 transgenic mice and their wild-type littermates were compared by whole body skeletal staining. In Col2.3-11βHSD2 transgenic embryos, a reduction in the size and mineralization of the frontal and parietal bones became apparent as early as E15.5 (data not shown). At E16.5, the cranial bones show reduced mineralization (Fig. 1H,K,L) and increased bony separation (Fig. 1L). In addition, a marked reduction in the size of the mineralized regions in the interparietal bones was seen (Fig. 1L).
Another striking abnormality apparent in Col2.3-11βHSD2 transgenic mice was the persistence of intact cartilage underneath the calvarial bones (Fig. 2B). These cartilage remnants were present in all P1 Col2.3-11βHSD2 transgenic mice and significantly larger than those seen in their wild-type littermates (Fig. 2A,B). Histology of the parietal bones revealed a chaotic bone matrix and disorganized osteoblasts in Col2.3-11βHSD2 transgenic mice (Fig. 2C and 2D). By contrast, Toluidine Blue staining confirmed the presence of a broad and vital band of cartilage directly underneath the poorly developed parietal bones in all transgenic mice (Fig. 2F), whereas in wild-type mice only small islets of cartilage were seen, and bone was well organized and almost completely formed (Fig. 2E). Alkaline phosphatase (ALP) staining and evidence of mineralization were absent in the remnant cartilage plates (Fig. 2G,H), indicating this cartilage differs from endochondral cartilage, which usually stains strongly for ALP (data not shown). In addition, numerous ALP+ preosteoblastic cells were seen at the advancing edge of developing calvarial bone above the cartilage plate in wild-type mice (Fig. 2I) but only a few ALP+ osteoblastic cells were present at a similar position in transgenic mice (Fig. 2J). These observations suggest that osteoblast differentiation is suppressed during calvaria development in Col2.3-11βHSD2 transgenic mice.
A further abnormality noted in Col2.3-11βHSD2 transgenic mice is the appearance of ectopic cartilage below the sutures. In 7-day-old (P7) Col2.3-11βHSD2 transgenic mice, the region around the sagittal and lambdoid sutures stained clearly with Alcian Blue, indicating the presence of cartilage (Fig. 3B). Histology of the same regions revealed the presence of ectopic cartilage in the sagittal sutures with greater separation of parietal bones in Col2.3-11βHSD2 transgenic mice (Fig. 3D) when compared with wild-type animals (Fig. 3C). These observations correlate well with the results of the micro-CT studies as described above (Fig. 1E,F). In addition, thicker cartilage plates remained lateral to the parietal bone regions, suggesting that the removal of parietal cartilage is further delayed (Fig. 3F). Whereas on day 7, the parietal bones had formed to their full extent, these bones remained thinner, the osteoblasts stayed disorganized and the sub-parietal cartilage remnants were retained (Fig. 3F,H).
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Disruption of GC signaling in mature osteoblasts leads to reduced Mmp14 expression in the cranial skeleton
Mmp14 (or MT1-Mmp) has been shown to be essential for calvarial cartilage
removal in rodents. Mice genetically deficient in Mmp14 exhibit impaired
cranial cartilage degradation and reduced apoptosis of non-hypertrophic
chondrocytes (Holmbeck et al.,
2003). Interestingly, these Mmp14 knockout mice seem to share a
similar calvarial cartilage phenotype to that seen in Col2.3-HSD2 transgenic
mice. This led us to hypothesize that the delayed cranial cartilage removal in
Col2.3-11βHSD2 transgenic mice may result from impaired Mmp14
expression.
To this aim, we compared cranial bone sections of neonatal (P0) Col2.3-11βHSD2 transgenic mice and their wild-type littermates. At this age, the cranial cartilage has not been fully removed in wild-type animals, allowing for assessment of Mmp14 expression and activity in wild-type and Col2.3-11βHSD2 transgenic animals. In situ hybridization and immunohistochemistry revealed that both mRNA and protein levels of Mmp14 were lower in cranial cartilage chondrocytes of Col2.3-11βHSD2 transgenic (Fig. 4B,D) compared with their wild-type littermates (Fig. 4A,C). Real time RT-PCR demonstrated that mRNA expression for Col2a1, a cartilage marker, was significantly higher (P=0.003) in transgenic than in wild-type mice, whereas mRNA levels of Mmp14 were four times lower (P=0.01) in transgenic parietal bone compared with their wild-type counterparts (Fig. 4K). In wild-type mice, removal of the calvarial cartilage commenced with proteoglycan loss from the cartilage matrix, followed by a gradual dissolution of the matrix itself (Fig. 4E) and the apoptotic demise of chondrocytes (Fig. 4G,L). By contrast, those processes were not seen in Col2.3-11βHSD2 transgenic mice (Fig. 4F,H). TUNEL staining demonstrated near-complete absence of apoptotic chondrocytes in the cartilage of Col2.3-11βHSD2 transgenic mice (Fig. 4H,L).
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Canonical Wnt signaling: a potential paracrine signal in calvarial bone development
Previously, by culturing primary osteoblasts derived from
Col2.3-11βHSD2 transgenic and their wild-type littermates, we have
discovered that mature osteoblasts provide a GC-dependent paracrine Wnt
signaling to control mesenchymal progenitor cell lineage commitment through
the active secretion of Wnt7b and Wnt10b proteins
(Zhou et al., 2008).
Interestingly, Mmp14 is a direct downstream target gene for the canonical Wnt
signaling pathway, as the Mmp14 promoter is directly targeted by
β-catenin/Tcf4 complex (Takahashi et
al., 2002). Accumulation of β-catenin by recombinant Wnt3a or
LiCl treatment resulted in upregulation of Mmp14 expression in human
mesenchymal stem cells (Neth et al.,
2006). This led us to examine, by immunohistochemical staining,
potential changes in the accumulation of β-catenin protein in calvaria of
neonatal Col2.3-11βHSD2 transgenic mice and their wild-type littermates.
In wild-type animals, high levels of β-catenin protein were found in both
calvarial osteoblasts and chondrocytes
(Fig. 5A). By contrast, no
accumulated β-catenin protein was detected in Col2.3-11βHSD2
transgenic mice in either cranial osteoblasts or chondrocytes, despite intense
staining of neuronal cells (Fig.
5B). These findings indicate that Wnt signaling was suppressed in
the calvaria of transgenic mice. Interestingly, loss of Wnt9a leads to ectopic
differentiation of cartilage in the sagittal suture and at the base of the
parietal bones (Spater et al.,
2006), a phenotype similar to that of Col2.3-11βHSD2
transgenic mice. We therefore investigated Wnt9a mRNA expression in parietal
bones. In situ hybridization revealed that Wnt9a mRNA was localized in
osteoblasts of the calvarial bone surfaces of wild-type mice
(Fig. 5E). By contrast, Wnt9a
mRNA was not detected in transgenic littermates
(Fig. 5F). Interestingly, Wnt9a
mRNA was not detectable in parietal cartilage of either wild-type or
Col2.3-11βHSD2 transgenic mice (Fig.
5E,F), indicating that Wnt9a is mainly expressed in osteoblastic
cells. As there was no transgene detected in cranial chondrocytes, the reduced
accumulation of β-catenin protein in the chondrocytes of
Col2.3-11βHSD2 transgenic mice is likely to be due to reduced stimulation
by Wnt proteins secreted by neighbouring osteoblasts, which are known to lack
normal intracellular GC signaling.
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), with Col2.3-11βHSD2 transgenic mice to generate
Col2.3-11βHSD2-GFP-transgenic and Col2.3-GFP wild-type littermates.
Col2.3-11βHSD2-GFP transgenic mice carry the same phenotype as
Col2.3-11βHSD2 transgenic mice. We then assessed, in the parietal bones
of Col2.3-11βHSD2-GFP transgenic and Col2.3-GFP wild-type mice, the mRNA
expression levels for Wnt7b, Wnt10b and Wnt9a relative to GFP mRNA expression
levels. The relative expression in the parietal bones of both Wnt10b and Wnt9a
mRNA, which are predominantly transcribed in osteoblasts, was significantly
lower in Col2.3-11βHSD2 transgenic than in Col2.3-GFP littermates
(Fig. 5G). Wnt7b mRNA was
expressed at a very low level in Col2.3-GFP-wild-type animals (just detectable
after 40 cycles of RT-PCR) and was not significantly lower in
Col2.3-11βHSD2 transgenic mice (data not shown).
Treatment with exogenous Wnt3a rescues the phenotype in transgenic mice.
If endogenous glucocorticoids regulate parietal cartilage removal and
suture expansion through Wnt/β-catenin signaling, the phenotype in
transgenic mice should be rescued through activation of β-catenin by
exogenous Wnt3a treatment. Recombinant Wnt3a (100 ng) was applied daily for 2
days by subcutaneous injection over the calvaria of 1-day-old mice. At day 3,
the parietal bone volume was significantly increased and, as a consequence,
suture areas were significantly reduced in Wnt3a-treated transgenic mice as
shown by micro-CT analysis (Fig.
6C,D). The cranial bones of Wnt3a-treated mice were well
mineralized as evidenced by a more solid appearance and reduced patchy
radio-opacity (Fig. 6C).
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Reduced Mmp14 expression by cranial chondrocytes results in impaired cartilage removal
During skull formation, embryonic parietal cartilage is formed as
primordium at day E16.5, mostly in the area of the parietal and interparietal
bones. This cartilage then grows substantially during the later part of
embryogenesis and the early neonatal period, but gradually disappears as
ossification proceeds concurrent with the degradation of the extracellular
matrix of cartilage and chondrocyte apoptosis. This process is arrested in
Mmp14-deficient mice (Holmbeck et al.,
1999; Holmbeck et al.,
2003; Holmbeck et al.,
2005). As Mmp14 is a type I membrane-bound protein, it needs to be
expressed by resident chondrocytes to catalyse cartilage degradation. In
Col2.3-11βHSD2 transgenic mice, in which GC signaling has been rendered
dysfunctional in mature osteoblasts only, cranial cartilage removal was
delayed concurrent with a reduction in the expression of chondrocytic Mmp14
mRNA and protein, and decreased chondrocyte apoptosis. These observations
indicate that there are paracrine upstream molecules secreted by osteoblasts
that regulate Mmp14 expression in calvarial cartilage.
Osteoblast-derived Wnt9a and Wnt10b activate paracrine signaling in parietal chondrocytes to initiate Mmp14-mediated cartilage removal
Using calvarial cells in an in vitro culture system, we have previously
shown that mature osteoblasts direct mesenchymal progenitor cells to
differentiate away from the adipogenic towards the osteoblastic lineage by a
glucocorticoid-dependent mechanism (Zhou
et al., 2008). Dominant adipogenesis and greatly reduced
osteoblastogenesis were observed in calvarial cell cultures from
Col2.3-11βHSD2 transgenic mice when compared with wild-type mice. This
phenotypic shift in mesenchymal progenitor cell commitment coincided with a
reduction in Wnt7b and Wnt10b mRNA and β-catenin protein levels in
transgenic versus wild-type cultures. In addition, transwell co-culture of
transgenic mesenchymal progenitor cells with wild-type osteoblasts restored
commitment to the osteoblast lineage, as did treatment of transgenic cultures
with exogenous Wnt3a. The ability of wild-type osteoblasts to restore
commitment to the osteoblast lineage was blocked by sFRP1, a Wnt
inhibitor.
Given that MMP14 is a canonical Wnt target gene
(Neth et al., 2006;
Takahashi et al., 2002) and
mature osteoblasts are a dominant source of Wnt proteins
(Zhou et al., 2008), we
proceeded to investigate the nature of canonical Wnt signaling pathways
between osteoblasts and neighbouring chondrocytes in calvarial bone. Thus, we
demonstrate that Wnt10b and Wnt9a mRNA levels were lower in the parietal bones
of Col2.3-11βHSD2 transgenic mice compared with their wild-type
littermates. Using in situ hybridization, we further show that in wild-type
mice, Wnt9a mRNA localizes only to calvarial osteoblasts but not to
chondrocytes. By contrast, large amounts of β-catenin protein were found
both in mature osteoblasts and nearby chondrocytes of wild-type mice. Taken
together, these findings indicate that chondrocytes can be stimulated by
osteoblast-derived Wnt proteins to initiate the intracellular canonical Wnt
signaling cascade. By contrast, Wnt9a was not detectable in
Col2.3-11βHSD2 transgenic mice by in situ hybridization and, accordingly,
β-catenin protein was absent from calvarial chondrocytes and present at
reduced levels in only a few nearby osteoblasts. These observations suggest
that activation of β-catenin in chondrocytes of parietal cartilage is
dependent on Wnts produced by the neighbouring osteoblasts under the control
of endogenous glucocorticoids. The primary role of these secreted canonical
Wnts is to control calvarial bone and cartilage development. The reason why
Wnt7b expression was observed in cultured calvarial osteoblasts
(Zhou et al., 2008), instead
of Wnt 9a as seen in our in vivo experiments, is currently unclear but is
probably due to differences in the local regulation of Wnt expression
occurring in vivo. Importantly, applying recombinant Wnt3a, a canonical Wnt
protein, by supracalvarial injection to transgenic mice resulted in complete
cartilage degradation. This finding adds further proof to the concept that Wnt
signaling acts as an upstream regulator of Mmp14 expression during murine
cranial development.
Osteoblast-derived Wnt proteins activate paracrine signaling in mesenchymal cells to initiate bone formation and growth
During skull growth, the sutures serve as growth centres, where mesenchymal
cells reside as a reservoir for postnatal osteogenesis and new bone formation.
In this process, Wnt/β-catenin signaling is required to suppress
chondrogenesis and to allow osteoblasts to form
(Day et al., 2005;
Hill et al., 2005). It has been
shown that either knockout of Wnt9a or inactivation of β-catenin in
mesenchymal cells induces ectopic cartilage formation in the developing
calvaria, particularly below the sutures
(Day et al., 2005;
Spater et al., 2006). By
contrast, mice with mutant Axin2, a negative regulator of the canonical Wnt
pathway that promotes degradation of β-catenin, display premature closure
of sutures as a result of excessive β-catenin accumulation
(Yu et al., 2005). Thus, it
seems clear that canonical Wnt signaling is essential for intramembranous
ossification. However, so far the source of Wnt in this signaling cascade has
remained obscure. In the present study, we observed that Col2.3-11βHSD2
transgenic mice show a developmental phenotype similar to that of Wnt9a mutant
mice (Spater et al., 2006)
with calvarial bone hypoplasia and osteopenia, increased suture patency, and
ectopic differentiation of cartilage in the sagittal suture. Of note, the
phenotype in Col2.3-11βHSD2 transgenic mice was associated with a
dramatic reduction in β-catenin protein accumulation in calvarial
osteoblasts and progenitor cells located in the sutures, indicating that
canonical Wnt signaling was attenuated. Treatment with exogenous Wnt3a by
supracalvarial injection rescued the transgenic phenotype to resemble the wild
type with reduced suture areas, well mineralized cranial bones and complete
cranial cartilage removal. Hence, depending on the cells' local environment,
canonical Wnt signaling may act as a molecular switch between osteoblast,
chondrocyte and adipocyte cell fates when mesenchymal progenitor cells are
differentiating.
Autocrine canonical Wnt signaling in osteoblasts
The pronounced disorganization of both the resident osteoblasts and the
collagenous bone matrix in Col2.3-11βHSD2 transgenic calvaria indicate
that endogenous GC, via osteoblastic Wnt signaling, may have a direct effect
on bone formation through modulating mature osteoblast function. We have found
reduced β-catenin protein accumulation and Mmp14 expression not only in
calvarial chondrocytes but also in Col2.3-11βHSD2 transgenic calvarial
osteoblasts. In addition, Wnt3a treatment not only rescued the abnormal
cartilage phenotype, but also significantly improved parietal bone formation,
mineralization and, consequently, suture narrowing. It is interesting to note
that Mmp14-deficient mice develop a similar phenotype of disturbed cranial
intramembranous ossification, characterized by reduced calvarial bone
formation in association with chaotic bone matrix organization and disordered
osteoblasts (Holmbeck et al.,
1999). These findings indicate that Mmp14 may play an important
role in normal bone formation. We therefore hypothesize that osteoblasts are
controlled through a GC-dependent autocrine Wnt signaling loop. Therefore, in
addition to the lack of paracrine Wnt signaling to mesenchymal progenitor
cells, reduced autocrine Wnt signaling in transgenic osteoblasts may impair
osteoblastic Mmp14 expression, contributing to the phenotype of disturbed
calvarial bone formation as observed in Col2.3-11βHSD2 transgenic
mice.
Proposed model of GC dependent canonical Wnt signaling in cranial development
In this model (Fig. 7), we
suggest that endogenous glucocorticoids stimulate the expression and secretion
of Wnt proteins in mature cranial osteoblasts, in keeping with our previous
results (Zhou et al., 2008).
The ensuing canonical Wnt signaling cascade induces: (1) mesenchymal
progenitor cells to differentiate away from the chondrocyte towards the
osteoblast lineage to form bone; (2) osteoblasts to initiate Mmp14-mediated
remodeling of the collagenous matrix surrounding osteoblasts; and (3) parietal
cartilage chondrocytes to initiate Mmp14-mediated cartilage degradation. These
concurrent and tightly interconnected pathways establish a novel role for both
glucocorticoids and osteoblasts in the intricate process of intramembranous
bone development.
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). This
difference may explain why long bone development (i.e. endochondral
ossification) is not affected in Col2.3-11βHSD2 transgenic mice. Our study highlights an important role for glucocorticoids in skeletal function that is mediated through regulation of Wnt signaling with downstream effects on Mmp14, and therefore may have relevance to wider areas such as understanding the therapeutic and adverse effects GC treatment. This study also highlights the importance of the interaction of glucocorticoids and Wnt proteins with Mmp14, the latter being a significant mediator of adverse outcomes in cancer and chronic arthritis. Thus, our findings may open novel avenues to advance research into the mechanism of malignant and inflammatory joint disease.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/3/427/DC1
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Footnotes |
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* These authors contributed equally to this work 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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