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First published online 25 October 2006
doi: 10.1242/dev.02680
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1 Department of Molecular, Cell and Developmental Biology, University of
California, Los Angeles, CA 90095, USA.
2 Department of Orthopaedic Surgery, David Geffen School of Medicine at the
University of California, Los Angeles, CA 90095, USA.
3 Department of Molecular Genetics, University of Texas MD Anderson Cancer
Center, Houston, TX 77030, USA.
4 Laboratory of Reproductive and Developmental Toxicology, National Institute of
Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
* Author for correspondence (e-mail: klyons{at}mednet.ucla.edu)
Accepted 22 September 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Bone morphogenetic protein, Cartilage, Chondrogenesis, Fibroblast growth factor, indian hedgehog, BMP receptors, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Olsen et al., 2000).
Longitudinal growth is driven by chondrocyte proliferation and enlargement.
Once growth-plate chondrocytes exit the cell cycle, they form a transient pool
of indian hedgehog (Ihh)-expressing prehypertrophic
chondrocytes (St-Jacques et al.,
1999). As they continue to differentiate, these cells upregulate
expression of collagen X and undergo hypertrophy. Ultimately, terminally
differentiated hypertrophic chondrocytes express osteopontin and matrix
metalloproteinase 13 (MMP13) prior to undergoing apoptosis. Hypertrophic
chondrocytes produce an extracellular matrix (ECM) that provides an
environment conducive to bone mineralization and blood-vessel invasion
(Kronenberg, 2003;
Long et al., 2001).
Several signaling pathways play vital roles in the growth plate. IHH
coordinates chondrocyte proliferation and differentiation, as well as
osteoblast formation in the periostium
(St-Jacques et al., 1999;
Long et al., 2004;
Chung et al., 2001).
Parathyroid hormone-related protein (PTHrP) forms a feedback loop with IHH
that regulates proliferation and the onset of hypertrophic differentiation
(Karp et al., 2000; Kawashima
et al., 1998; Lanske et al.,
1999). Fibroblast growth factor (FGF) signaling pathways
negatively regulate proliferation and differentiation via several mechanisms,
including activation of STAT and ERK1/2 MAPK
(Sahni et al., 2001;
Murakami et al., 2004).
Bone morphogenetic protein (BMP) signaling pathways are also crucial
regulators of chondrogenesis. BMPs transduce signals through complexes of type
I and type II serine/threonine kinase receptors. Upon BMP binding, type II
receptors phosphorylate serine/threonine residues in type I receptors.
Activated type I receptors phosphorylate, and thereby activate,
receptor-regulated SMAD (R-SMAD)1, R-SMAD5 and R-SMAD8. Subsequently, these
R-SMADs recruit and bind SMAD4. These SMAD complexes enter the nucleus and
regulate transcription of target genes
(ten Dijke et al., 2003;
Massagué and Gomis,
2006). BMPs also signal by activating TGFß-activated kinase 1
(TAK1). TAK1 activates p38 MAPK (Moustakas
and Heldin, 2005; Qiao et al.,
2005). Three type I receptors transduce BMP signals: BMP receptor
type IA (BMPRIA, also known as ALK3), BMP receptor type IB (BMPRIB, also known
as ALK6) and activin receptor type I (ACTRI, also known as ALK2)
(ten Dijke et al., 1994;
Rosenzweig et al., 1995;
Macías-Silva et al.,
1998).
Limb-culture studies have provided important insights into the diverse
roles that BMPs play in the growth plate
(De Luca et al., 2001;
Pathi et al., 1999;
Pizette and Niswander, 2000;
Minina et al., 2001).
Application of exogenous BMPs promotes proliferation, whereas exposure to the
BMP antagonist NOGGIN inhibits proliferation. Furthermore, these studies have
shown that BMP and FGF signaling have opposing actions in the growth plate
(Minina et al., 2002).
However, limb-culture studies have yielded contradictory results; some studies
suggest that BMPs exert stimulatory effects on differentiation, whereas others
support inhibitory effects. Whether these disparities arise as a result of
differences in (a) utilization of signaling pathways at the different stages
of development at which the studies were performed, (b) species-specific
effects, and/or (c) divergent pathways in different skeletal elements, is
unknown. Moreover, some of these studies involve application of BMPs at
non-physiological levels and to all tissues, including the surrounding
endothelial cells, perichondrium and osteoblasts, are exposed. The tissues
surrounding cartilage affect chondrogenesis in the growth plate, raising the
possibility of both direct and indirect effects
(Colnot et al., 2004;
Colnot et al., 2005).
Moreover, these studies do not reveal the endogenous roles of different type I
receptors.
A number of genetic approaches have been used to reduce BMP signaling in
developing skeletal elements in the mouse. Mice lacking Gdf5 and
Gdf6 exhibit defects in chondrogenesis and joint formation
(Storm et al., 1994;
Settle, Jr et al., 2003). Mice
lacking Bmpr1b exhibit defects in chondrogenesis
(Baur et al., 2000;
Yi et al., 2000). However, in
all of these cases, defects are restricted to a subset of skeletal elements,
most probably as a result of functional redundancy
(Yi et al., 2000;
Settle, Jr et al., 2003). The
idea that BMP signaling pathways play a much broader role than is suggested by
these phenotypes has been confirmed by analysis of transgenic mice
overexpressing noggin or Smad6
(Tsumaki et al., 2002;
Horiki et al., 2004). All
cartilages in these mice are hypoplastic and deficient in hypertrophic
chondrocytes, suggestive of a requirement for BMP pathways in chondrocyte
maturation. Recently, we demonstrated an essential role for BMP signaling at
early stages of chondrogenesis by generating chondrocyte-specific
Bmpr1a and Bmpr1b null mice
(Yoon et al., 2005).
Chondrogenesis in these double mutants is much more severely affected than in
the noggin-overexpressing transgenics
(Tsumaki et al., 2002).
We characterized the skeletal phenotypes of mice lacking Bmpr1A
(Bmpr1aCKO) and
Bmpr1aCKO;Bmpr1b+/-
(Bmpr1aCKO;1b+/-) in cartilage in order to
address the roles of BMP pathways in the growth plate. These mice displayed
generalized chondrodysplasia but, unlike
Bmpr1aCKO;1b-/- mice, in which chondrogenesis
is so severely disrupted that growth plates never form
(Yoon et al., 2005), all
skeletal elements were present, permitting an assessment of the role of BMP
pathways in the growth plate. This analysis demonstrated that BMP signaling is
essential for multiple aspects of growth-plate chondrogenesis, including
proliferation, and the completion of hypertrophic differentiation. We
demonstrated that the balance between outputs from BMP and FGF pathways plays
crucial roles in multiple aspects of chondrogenesis. Furthermore, these
studies indicated that BMP signaling is required to promote Ihh
expression and to inhibit FGFR1 expression. The increased level of
FGF-receptor expression appears to have functional consequences, as activation
of STAT and ERK1/2 MAPK, key effectors of FGF signaling, were elevated in
Bmpr1aCKO and
Bmpr1aCKO;1b+/- mutants. These results provide
a genetic demonstration that the progression of chondrocytes through the
growth plate is controlled by mutually antagonistic BMP and FGF signaling
pathways.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Mishina et al., 2002;
Ovchinnikov et al., 2000). To
generate cartilage-specific Bmpr1a null mice,
Bmpr1afx/+;Col2-Cre mice were intercrossed to generate
Bmpr1afx/fx;Col2-Cre (Bmpr1aCKO) mice.
Bmpr1afx/+;Bmpr1b+/-;Col2-Cre mice were
intercrossed to generate Bmpr1aCKO;Bmpr1b+/-
mice.
Skeletal preparation and histology
Skeletal preparations were performed as previously described
(Ivkovic et al., 2003).
Briefly, embryos were eviscerated and fixed in 95% EtOH overnight at 4°C,
followed by Alcian blue staining (30 mg/100 ml in 80% EtOH) at room
temperature. Samples were then stained in Alizarin red (5 mg/100 ml in 0.5%
KOH) and cleared in a series of graded KOH in glycerol. For histology, embryos
were fixed in 4% paraformaldehyde, decalcified and embedded in paraffin.
Sections were stained with Hematoxylin and Eosin or Alcian Blue and Nuclear
Fast Red (Luna, 1992).
Immunohistochemistry and in situ hybridization
For immunohistochemistry (IHC), sections were microwaved in citrate buffer
for antigen demasking. Sections were blocked with 5% goat or donkey serum,
incubated with primary antibody (BMPRIA and BMPRIB: Orbigen; collagen II:
Research Diagnostics; collagen X: gift from Robin Poole; osteopontin:
Developmental Studies Hybridoma Bank; MMP13: Oncogene Research Products;
phospho-SMAD1, phospho-SMAD5 and phospho-SMAD8, phospho-ERK1/2 MAPK, and p27
Kip1: Cell Signaling Technology; phospho-STAT1: Zymed; STAT 5a: Lab Vision;
p16, p21 and PECAM: Santa Cruz Biotechnology; FGFR1: Sigma) overnight at
4°C, and then incubated with the appropriate secondary antibody at room
temperature. Color was developed with DAB or the Zymed kit chromogen, and
sections were counterstained with hematoxylin. For immunofluorescence, slides
were washed and incubated overnight at 4°C with Alexa-Fluor-555-conjugated
rabbit secondary antibody (Invitrogen).
In situ hybridization was performed with 35S-dUTP labeled RNA
probes and carried out as previously described
(Jamin et al., 2002).
Cell proliferation and TUNEL labeling
IHC for proliferating cell nuclear antigen (PCNA) was performed using
anti-PCNA antibody (Zymed), as described above for IHC. For TUNEL labeling,
the In Situ Cell Death Detection Kit, fluorescein (Roche), was used according
to the manufacturer's instructions. Cell proliferation was quantified as
previously described (Yi et al.,
2000).
Transient transfections and reporter assays
A 994 bp fragment of the mouse proximal Ihh promoter was a kind
gift from Toshihisa Komori (Yoshida et
al., 2004). 2HC8-luc contains 430 bp of the mouse proximal
Ihh promoter and was a kind gift from Akiko Hata
(Seki and Hata, 2004). The
Smad1 expression construct was a kind gift from Eddy DeRobertis
(Pera et al., 2003). RCS cells
(Mukhopadhyay et al., 1995)
were cultured in 10% FBS DMEM and transfected at 50-70% confluence using
GeneJuice (Novagen), according to the manufacturer's instructions, with 0.5
µg of 994-luc or 2HC8-luc, and either Smad1 or a lacZ
control plasmid. Cells were serum-starved for 24 hours post-transfection and
then treated with 60 ng/ml recombinant human BMP2 (R&D Systems). After an
additional 24 hours, luciferase activity was measured with the Dual-Luciferase
Promoter Assay System (Promega). Results were obtained in triplicate for each
experiment and were normalized with Renilla luciferase. Statistical
significance was assessed using a t-test for correlated samples.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). However,
the differences in relative expression levels in different regions raises the
possibility of a more predominant role of one receptor over the other in
specific aspects of chondrogenesis. IHC for phosphorylated BMP-specific R-SMADs (pSMAD1, pSMAD5 and pSMAD8) was performed to localize active BMP signaling. Activated SMAD signaling was found throughout the growth plate, consistent with the broad distribution of BMPRIA and BMPRIB (Fig. 1C). The percentage of cells responding to BMP signaling increased as chondrocytes progressed from the resting to the proliferative zone, suggesting a role for BMP signaling in this transition (Fig. 1D,E). Furthermore, levels of signaling increased steadily throughout the proliferative zone and peaked at the proliferative-prehypertrophic junction. Levels then appeared to decrease as cells became hypertrophic. These results indicate that proliferative and prehypertrophic chondrocytes are major targets of BMP signaling, and further suggest that canonical BMP pathways regulate the transition from proliferation to terminal differentiation.
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These mice were recovered at birth in Mendelian ratios, but died shortly afterwards as a result of respiratory failure caused by skeletal defects. Mutant ribs were shorter and thinner, resulting in a flattened, bell-shaped thoracic cavity. These defects reflect the generalized chondrodysplasia that occurs in Bmpr1aCKO mice (Fig. 2A,B).
Mice carrying various allelic combinations of Bmpr1afx
and Bmpr1b alleles were generated in order to examine whether BMPR1A
and BMPR1B exert overlapping functions.
Bmpr1afx/+;Col2Cre;Bmpr1b-/- mice were viable
and did not differ substantially from Bmpr1b-/- mice,
indicating that a single copy of Bmpr1a is sufficient to sustain
endochondral ossification (data not shown). Bmpr1b-/- mice
developed severe skeletal defects in distal phalanges and minor defects in the
radius/ulna and tibia/fibula (Yi et al.,
2000; Baur et al.,
2000). Bmpr1aCKO;1b+/- mice
exhibited a generalized chondrodysplasia of considerably greater severity than
that exhibited by Bmpr1aCKO mice. This finding is
consistent with the overlapping expression patterns of BMPRIA and BMPRIB in
the growth plate. In addition to rib malformations, axial defects were
apparent along the entire vertebral column
(Fig. 2C,D). The supraoccipital
bone was significantly narrower in Bmpr1aCKO mice, but was
completely absent in Bmpr1aCKO;1b+/- mice.
Similarly, Bmpr1aCKO vertebrae did not fuse and the dorsal
arches were broader; this phenotype was exacerbated in
Bmpr1aCKO;1b+/- mice. Skeletal defects were
also apparent throughout the appendicular skeleton
(Fig. 2E,F). The scapula became
increasingly hypoplastic as more alleles of Bmpr1a and
Bmpr1b were lost. Appendicular elements were narrower and shorter in
Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice, with the femur being most
severely affected. Interestingly, malformations were more severe at the distal
than at the proximal ends of mutant femurs and humeri, and at the proximal
rather than distal ends of the tibia and fibula. Thus, the growth plates most
severely affected were those that normally experience the greatest rates of
cell division and differential growth
(Wilsman et al., 1996a;
Wilsman et al., 1996b;
Pritchett, 1992).
Bmpr1aCKO and Bmpr1aCKO;Bmpr1b+/- mice exhibit defects in the growth plate
The above analysis reveals that, whereas chondrogenesis is essentially
absent in Bmpr1aCKO;1b-/- mice
(Yoon et al., 2005), a single
intact copy of Bmpr1b
(Bmpr1aCKO;1b+/-) restores endochondral
development to the extent that a recognizable, albeit highly abnormal,
skeleton forms. At E14.5, growth plates at the distal femurs of
Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice displayed numerous
histological defects (Fig. 3).
Although the sizes of the resting zones were comparable in
Bmpr1aCKO and wild-type mice, the proliferative zone was
reduced in mutants (arrows in Fig.
3A,B). This reduction was intensified in
Bmpr1aCKO;1b+/- mice. The severe constriction
in the width of the femur at the junction of the resting and proliferative
zones suggests that the initiation of column formation is impaired in the most
severely affected growth plates of
Bmpr1aCKO;1b+/- mice. Furthermore, the lengths
of the chondrocyte columns were significantly reduced. These phenotypes
persist throughout embryonic development, as they are also apparent at E16.5
and P0 (Fig. 3B, and data not
shown). Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice also displayed defects in
linear column formation. In wild-type growth plates, flattened proliferating
chondrocytes intercalate with one another immediately after cell division.
However, in Bmpr1aCKO mice, chondrocyte columns were
disorganized. Proliferating chondrocytes did not intercalate properly, as many
pairs of daughter cells remained juxtaposed
(Fig. 3C). This phenotype
became more exaggerated in Bmpr1aCKO;1b+/-
mice. Very few chondrocyte columns formed in the distal femur and distal
humerus, the most severely affected growth plates
(Fig. 3C), and chondrocytes had
a rounded morphology rather than the flattened shape observed in control
littermates.
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Progression through the cell cycle is negatively regulated by
cyclin-dependent kinase inhibitors (CKIs). Several CKIs are important
regulators of proliferation in chondrocytes, including p16, p21 and
p27Kip1 (Beier et al.,
1999). In control growth plates, these CKIs were strongly
expressed in the prehypertrophic and hypertrophic zones and only weakly in the
proliferative zone. However, there was a significant induction of p16, p21 and
p27 in the resting and proliferative zones in mutant growth plates
(Fig. 4D). Thus, proliferation
is inhibited in Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice, in part, because CKIs are
upregulated.
Defects in differentiation in mutant growth plates
Previous studies have shown that the onset of hypertrophic differentiation
is delayed in mice lacking all alleles of Bmpr1a and Bmpr1b
in cartilage (Yoon et al.,
2005). By contrast, histological analysis indicated that
hypertrophic chondrocytes were present in Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice by E14.5. At E14.5, the
lengths of the growth plates in the distal femur (presumptive joints to
centers of cartilage condensations) and the lengths of the hypertrophic zones
were smaller in Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice than in controls
(Fig. 3A). The initially
smaller hypertrophic zones in mutants at E14.5 may have arisen because of a
smaller pool of cells as a result of the reduced rate of proliferation and the
increased incidence of apoptosis.
|
). Osteopontin expression did become restricted to
terminal hypertrophic cells at later stages. MMP13 expression was restricted
to terminally differentiating hypertrophic chondrocytes in E14.5 control
growth plates, and its domain of expression was significantly reduced in
Bmpr1aCKO mice, suggesting that a smaller number of
chondrocytes had reached this stage of development by E14.5.
BMP signaling is required for terminal differentiation
By E16.5, additional defects in differentiation became apparent in
Bmpr1aCKO and
Bmpr1aCKO;1b+/- growth plates. Chondrocytes
appeared to undergo premature hypertrophy in mutants
(Fig. 3B), as evidenced by the
shorter distance between the articular surface and the beginning of the
hypertrophic zone. In addition, the hypertrophic zones of
Bmpr1aCKO and
Bmpr1aCKO;1b+/- femurs were expanded at this
stage. The expansion observed in Bmpr1aCKO mice was not
exacerbated by the loss of one Bmpr1b allele, and is not observed in
Bmpr1b-/- mice (Yi et
al., 2000). These results suggest a more prominent role for BMPRIA
than for BMPR1B in hypertrophic chondrocytes, consistent with the high level
of BMPRIA expression in this region. The expansion of the hypertrophic zone
could conceivably be caused by a decreased rate of transit through the
hypertrophic zone when BMP signaling is decreased, and/or by impaired
apoptosis of terminal hypertrophic chondrocytes. Cells with tightly packed
nuclei, a characteristic of terminally differentiated hypertrophic
chondrocytes, accumulated in mutant hypertrophic zones
(Fig. 3D)
(Colnot et al., 2004). This
finding suggests that the expanded hypertrophic zone may be a result, at least
in part, of a delay in clearance of late hypertrophic chondrocytes through
apoptotic mechanisms.
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Bmpr1aCKO growth plates exhibited aberrant angiogenesis
in the hypertrophic zone (Fig.
6). In control mice, angiogenesis proceeded evenly across the
chondro-osseous junction. However, in mutants, blood vessels invaded the
hypertrophic zone, forming a disorganized chondro-osseous junction. This
phenotype is possibly a consequence of the expansion in MMP13-expressing
chondrocytes in mutants. MMP13 is a potent angiogenic factor that promotes
growth-plate angiogenesis (D'Angelo et
al., 2000; Johansson et al.,
1997; Stickens et al.,
2004).
BMP signaling is an important regulator of IHH signaling
The IHH/PTHrP pathway forms a negative-feedback loop that regulates the
onset of hypertrophic differentiation. As in Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice,
Ihh-/- growth plates initially exhibit smaller
hypertrophic zones, followed by premature hypertrophic differentiation
(St-Jacques et al., 1999).
Therefore, we examined IHH/PTHrP pathway components in
Bmpr1aCKO mice (Fig.
7A,B). Decreased expression of Ihh was observed in
mutants, a finding consistent with the effects of the BMP antagonist NOGGIN in
limb-culture studies (Pathi et al.,
1999; Minina et al.,
2001). Furthermore, Bmpr1aCKO growth plates
had reduced expression of Ptc1, the receptor for IHH and a direct
target of IHH signaling. No differences were found in expression of
Pthr1, the receptor for PTHrP.
The existence of functional SMAD-responsive sites on the Ihh
promoter (Seki and Hata,
2004), and the high levels of pSMAD1, pSMAD5 and pSMAD8 activation
at the proliferative-prehypertrophic junction, suggest that IHH is a direct
target of SMAD-mediated BMP signaling in the growth plate. Promoter assays
using the Ihh promoter were performed in RCS chondrocytic cells to
test this possibility (Fig.
7C). Exogenous BMP2 treatment and SMAD1 transfection resulted in
an increase in promoter activity when a 994 bp Ihh promoter fragment
was used (Yoshida et al.,
2004), demonstrating that IHH is a target of BMP signaling in
chondrocytes. The addition of BMP2 and overexpression of Smad1 also
resulted in an increase in promoter activity when the Ihh
SMAD-responsive element identified by Seki and Hata
(Seki and Hata, 2004) was
examined. However, the fold induction was considerably less than with the 994
bp fragment, suggesting that additional BMP-responsive sites on the
Ihh promoter are utilized in chondrocytes.
Impaired BMP signaling leads to increased activation of FGF signaling pathways
Impaired BMP signaling in Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice leads to phenotypes
resembling those observed in mouse models of FGF-induced achondroplasia. These
observations prompted us to examine whether effectors of FGF signaling
pathways are activated in BMP receptor mutants. At least two signaling
pathways mediate FGF signaling in chondrocytes; STATs and ERK1/2 MAPK
(Sahni et al., 2001;
Murakami et al., 2004). STATs
mediate the ability of FGFs to promote apoptosis and CKI expression
(Sahni et al., 2001;
Aszodi et al., 2003).
Activation of ERK1/2 MAPK has been suggested to mediate the ability of FGFs to
prevent chondrocyte differentiation
(Murakami et al., 2004). In
control mice, activated phospho-STAT1 and STAT5a were predominantly localized
to the prehypertrophic and hypertrophic zones
(Fig. 8A,B, and data not
shown). By contrast, there was a significant increase in activated STAT1- and
STAT5a-positive chondrocytes in the proliferative zones in
Bmpr1aCKO;1b+/- mice. Furthermore, a higher
percentage of chondrocytes in mutant hypertrophic zones exhibited active STAT
signaling.
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FGFR1 levels are increased in Bmpr1aCKO and Bmpr1aCKO;1b+/- growth plates
One mechanism that could account for the activation of multiple mediators
of FGF signaling would be the elevated expression of FGF receptors. FGFR1 is
expressed most highly in prehypertrophic and hypertrophic chondrocytes,
regions where pSMAD1, pSMAD5, and pSMAD8 levels are also at their highest
(Fig. 1)
(Peters et al., 1992;
Jacob et al., 2006).
Therefore, we examined levels of FGFR1 expression
(Fig. 9). The domain of
expression of FGFR1 was expanded considerably in mutant growth plates at
E16.5; in addition to expression in the enlarged hypertrophic zones of
Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice, FGFR1 expression could
now be detected throughout the columnar and epiphyseal regions. This finding
suggests that BMP pathways antagonize FGF pathways in part by inhibiting
expression of FGF receptors.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) (our unpublished data), phenotypes that could potentially
arise from defects in the formation of the epiphysis and/or articular
cartilage.
|
The contribution of the remaining type I receptor, ACTRI, in the growth
plate remains to be addressed. Overexpression of CA-ACTRI delays
differentiation and inhibits collagen X expression, effects opposite to those
observed when growth plates are treated with BMPs. In agreement, CA-ACTRI
induces expression of PTHrP, an inhibitor of hypertrophic differentiation
(Zhang et al., 2003). These
results, although unexpected, correlate with sites of ACTRI expression in the
resting and proliferative zones; areas where chondrocytes need to prevent
premature differentiation (Zhang et al.,
2003). Future genetic studies are necessary to examine whether
ACTRI receptors are redundant with BMPRIA and BMPRIB in chondrogenesis.
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;
Minina et al., 2001). We
further demonstrate that BMPs regulate proliferation, at least in part, by
inhibiting CKIs, such as p16, p21 and p27. Interestingly, at the
resting/proliferative junction, where the highest rates of proliferation are
normally found (Smits et al.,
2004), there is a significant increase in apoptosis in
Bmpr1aCKO;1b+/- mice, suggesting that BMPs may
regulate the decision to proliferate or die. In a recent study, ID3 was
identified as an important regulator of this decision in neural-crest
precursors (Kee and Bronner-Fraser,
2005). IDs are direct targets of BMP signaling in multiple cell
types, and promote proliferation (Kowanetz
et al., 2004). Consequently, BMPs, acting through IDs, may have a
similar role at the resting/proliferative junction.
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;
Zhang et al., 2003). A high
percentage of chondrocytes fail to intercalate and form columns in mutant
growth plates, and in the more severely affected
Bmpr1aCKO;1b+/- mice, fail to take on the
characteristic flattened morphology. Similar features have been noted in
Ihh-/- growth plates
(St-Jacques et al., 1999),
further strengthening the argument for a functional connection between BMP and
IHH signaling.
As shown previously, loss of BMP signaling leads to a delay in the onset of
hypertrophic differentiation (Yoon et al.,
2005). At E14.5, Bmpr1aCKO;1b+/-
mutant growth plates have reduced hypertrophic zones and have only just begun
to express the late hypertrophic marker MMP13. This initial delay may be due
to a reduced pool of cells as a result of impaired proliferation and increased
apoptosis. This feature is also indicative of decreased signaling output from
the PTHrP/IHH feedback loop (St-Jacques et
al., 1999).
The relationships between this loop and BMP pathways have not been fully
elucidated. Based on BMP-receptor overexpression studies in the chick, it was
proposed that BMPs mediate the ability of IHH to induce PTHrP expression
(Zou et al., 1997). However,
in the mouse, it has been shown that BMPs do not directly upregulate PTHrP
(Minina et al., 2001;
Kobayashi et al., 2005).
Similarly, some studies have suggested that BMP pathways induce Ihh
expression in chondrocytes (Zhang et al.,
2003), whereas other studies do not support this hypothesis
(Kobayashi et al., 2005). We
show that the Ihh promoter is responsive to SMAD1 in chondrocytes,
suggesting that BMPs induce Ihh, and that this regulation may be
direct.
By E16.5, Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice develop expanded
hypertrophic zones. The expanded domains of osteopontin and MMP13 expression,
and the accumulation of cells with tightly packed nuclei, a characteristic of
late hypertrophic chondrocytes, suggest that the expansion of the hypertrophic
zone is due, at least in part, to delayed differentiation and/or failure to
complete terminal differentiation. Our conclusions differ somewhat from those
found in limb-culture studies following inhibition of BMP pathways by
treatment with NOGGIN (Minina et al.,
2001). In those studies, exposure to NOGGIN led to an expanded
domain of osteopontin expression; in contrast to our results, a reduced rather
than an expanded domain of collagen X expression was observed. These results
led the researchers to conclude that differentiation is accelerated when BMP
signaling is inhibited (Minina et al.,
2001). In Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice, although the length of
the hypertrophic zone is expanded, the onset of ossification is delayed rather
than accelerated, as would be predicted from the limb-culture models. The
expansion of collagen X expression, accumulation of terminal hypertrophic
chondrocytes and impaired ossification in Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice suggest that BMP signaling
is required for completion of terminal hypertrophic differentiation. These
different outcomes may reflect differences in responses to acute (as in
limb-culture studies) versus chronic (as in our genetic study) loss of BMP
signaling, or differences in target tissues (all tissues are exposed to
exogenous factors in limb culture whereas our genetic study utilized
chondrocyte-specific loss of BMP signaling).
|
). Growth
plates of these mice exhibit both decreased proliferation and expression of
Ihh, and increased expression of CKIs
(Naski et al., 1998;
Chen et al., 1999;
Sahni et al., 1999),
phenotypes recapitulated in Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice. Limb-culture studies have
shown that BMP and FGF signaling pathways have opposing functions in the
growth plate (Minina et al.,
2002). However, the mechanism of this antagonism has not been
elucidated. Our analysis of Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice demonstrates that BMPs
antagonize FGF signaling by inhibiting at least two of the intracellular
pathways activated by FGFs: STAT and ERK1/2 MAPK. One possible mechanism by
which BMPs may inhibit STAT and ERK1/2 is by negatively regulating the
expression of FGF signaling components. However, some limb-culture studies
suggest that BMPs promote the expression of FGF signaling components in the
growth plate, rather then inhibiting them. Future studies are needed to
clarify the mechanisms by which BMPs inhibit FGF signaling, but our studies
suggest that BMPs inhibit FGF pathways at least in part by limiting the domain
of expression of FGFR1. Given the essential roles for FGFR3 and FGF18 in
chondrogenesis (reviewed in Ornitz,
2005), examination of the expression levels of these signaling
components will be of great interest in future studies.
Interesting, loss of Fgfr1 in chondrocytes leads to a delay in
terminal maturation (Jacob et al.,
2006). This finding is seemingly at odds with our hypothesis that
BMP and FGF pathways exert opposing functions in the growth plate, as loss of
BMP signaling also leads to a delay in terminal maturation. There are several
possible explanations for this apparent discrepancy. First, it is conceivable
that BMP and FGF pathways have opposing roles in some aspects of
chondrogenesis, but additive or synergistic roles in others. Thus FGF and BMP
pathways may collaborate to promote aspects of hypertrophic chondrocyte
maturation. Second, FGFR1 and FGFR3 exhibit some overlapping expression, and
both receptors can transduce similar signals (reviewed in
Ornitz, 2005). Thus, it is
possible that functional redundancy and/or compensatory upregulation of other
FGF receptors occurs in Fgfr1CKO mice. Third, the delays
in maturation observed in Fgfr1CKO and Bmpr
mutant mice may occur through distinct mechanisms. In support of this latter
proposal, expression of OPN, MMP9 and MMP13 is reduced or unaltered in
Fgfr1CKO mice, whereas the domains of expression of these
markers are increased in Bmpr mutants
(Fig. 8, data not shown).
Clearly, additional in vivo studies are needed to clarify the nature of the
interactions between BMP and FGF signaling pathways in distinct regions of the
growth plate.
Our analysis of Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice also demonstrates that BMP
signaling partially inhibits ERK1/2 MAPK. Although the increased levels of
FGFR1 expression suggest that ERK1/2 levels may be elevated in
Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice because of activation of
FGF pathways, it is possible that additional pathways that transduce their
signals through ERK1/2 are perturbed in mutants. In wild-type growth plates,
ERK1/2 MAPK is activated in periarticular, prehypertrophic and hypertrophic
chondrocytes. The localization of active signaling in the prehypertrophic and
hypertrophic zones is consistent with the role that has been ascribed to
ERK1/2 MAPK signaling in chondrocyte differentiation
(Murakami et al., 2004). There
is no detectable difference in the level of ERK1/2 MAPK signaling in the
prehypertrophic and hypertrophic zones of Bmpr1aCKO and
Bmpr1aCKO;1b+/- mice. However, we cannot rule
out the possibility that the overall level of ERK1/2 MAPK activation is
altered in prehypertrophic and/or hypertrophic chondrocytes in mutants, as IHC
more readily reveals spatial rather than quantitative changes. The percentage
of cells engaged in ERK1/2 MAPK signaling is increased in the periarticular
regions of mutant growth plates. The role of ERK1/2 MAPK in periarticular
cells has not been fully described. However, ERK1/2 MAPK may have an important
role in promoting articular cartilage and/or joint formation
(Bastow et al., 2005); our
studies thus suggest that the ability of excess BMP signaling to lead to joint
fusions (e.g. Brunet et al.,
1998) may in part be a consequence of inhibition of ERK1/2
activity.
It is likely that multiple signal transduction pathways, both downstream
and independent of FGF signaling, are altered upon loss of BMP signaling in
the growth plate. FGFs transduce their effects on chondrocytes through Rb
proteins, p38, PLC
and AKT, in addition to STAT and ERK1/2
(Cobrinik et al., 1996;
Laplantine et al., 2002;
Krejci et al., 2004;
Raucci et al., 2004;
Sugimori et al., 2005;
Priore et al., 2006;
Zhang et al., 2006). Further
studies will be required to determine the extent to which alterations in each
of these pathways contribute to the phenotypes observed when BMP signaling is
decreased.
Taken together, our studies reveal that BMP pathways are essential for
multiple aspects of chondrogenesis, and that BMPs regulate IHH levels at least
in part via canonical SMAD pathways. To what extent do BMPs utilize canonical
(SMAD-mediated) versus non-canonical pathways in cartilage remains an
important unanswered question. Current models suggest that SMAD4 is required
for transduction of canonical signals via BMP SMAD1, SMAD5 and SMAD8
(ten Dijke et al., 2003;
Massagué and Gomis,
2006). Given the severity of the phenotypes seen in mice lacking
Bmpr1a and Bmpr1b in cartilage
(Yoon et al., 2005) (see also
the results described here), it is thus surprising that mice lacking
Smad4 in cartilage exhibit fairly normal skeletal development, prior
to developing postnatal chondrodysplasia
(Zhang et al., 2005). This
disparity may reflect a more important role for non-canonical than canonical
pathways in cartilage. Alternatively, several recent studies suggest that not
all SMAD-mediated responses to TGFß require SMAD4
(Ijichi et al., 2004;
He et al., 2006;
Liu et al., 2006). Analyses of
the role of the Drosophila SMAD4 homolog Medea indicate that BMP
SMADs can also function independently of SMAD4
(Wisotzkey et al., 1998).
Analyses of mice lacking BMP SMADs in cartilage will thus be required to
resolve whether the different phenotypes of mice lacking SMAD4 and those
lacking BMP receptors reflect roles for non-canonical pathways, or canonical
pathways independent of SMAD4.
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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