1 Department of Anatomy and Biomedical Sciences Graduate Program, University of
California, San Francisco, CA 94143-0452, USA
2 Deutsches Krebsforschungszentrum Heidelberg (DKFZ), Division of Signal
Transduction and Growth Control (A100), Im Neuenheimer Feld 280, D-69120
Heidelberg, Germany
3 University of Melbourne, Department of Paediatrics, Royal Children's Hospital,
Parkville, Victoria, Australia
* Authors for correspondence (e-mail: domi{at}itsa.ucsf.edu; zena{at}itsa.ucsf.edu)
Accepted 25 September 2004
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SUMMARY |
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Key words: Collagenase, MMP, Homologous recombination, Knockout, Hypertrophic cartilage, Chondrocyte, Trabecular bone, Collagen, Aggrecan, Mouse
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Introduction |
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Recent studies have shown that chondrocyte apoptosis per se does not lead
to endochondral ossification (Colnot et
al., 2001). Angiogenesis has been implicated as a crucial step
(Maes et al., 2002
;
Vu et al., 1998
;
Zelzer et al., 2004
); however,
the accumulation of hypertrophic chondrocytes and their associated matrix in
models where angiogenic stimuli have been ablated indicate that ECM
degradation also fails to occur (Gerber et
al., 1999
). These observations lead to the hypothesis that
degradation and remodeling of the cartilage matrix is essential for vascular
invasion.
Remodeling of ECM components requires proteolytic breakdown, a process in
which a variety of proteases have been implicated. Of particular interest are
the members of the matrix metalloproteinase (MMP) family, owing to their
ability to cleave aggrecan and collagens, the two most abundant ECM components
of skeletal tissue. Collagen type II (Col2) is the primary fibrillar ECM
component secreted by resting and proliferating chondrocytes in the growth
plate, while collagen type I (Col1) is the primary ECM component secreted by
osteoblasts in the trabecular bone. Col2 is crucial in establishing correct
temporal and spatial organizational relationships with other matrix components
such as the proteoglycans. Aggrecan is the major proteoglycan of the
developing growth plate (Doege,
1999). Degradation of proteoglycans and Col2 occurs in the very
last stages of chondrocyte differentiation, just prior to vascular invasion
(Lee et al., 1999
).
Several MMPs are collagenolytic, cleaving fibrillar collagens between amino
acids 775 and 776 within their triple helical regions
(Fields et al., 1987;
Wu et al., 1990
). The
resulting 1/4 and 3/4 length cleavage fragments then denature and are degraded
further by gelatinases, including MMP9 and MMP2
(Werb, 1982
). Collagenolytic
MMPs expressed during endochondral ossification include MMP13, MMP8
(collagenase 2) and MMP14 (MT1-MMP). A number of MMPs are able to cleave
aggrecan, resulting in the exposure of cryptic epitopes within the protein.
Other proteinases, including ADAMTS1, ADAMTS4 and ADAMTS5
(Abbaszade et al., 1999
;
Kuno et al., 2000
;
Tortorella et al., 1999
), also
cleave aggrecan, albeit at different sites than those recognized by MMPs.
Studies with transgenic mice lacking collagenolytic MMP14
(Holmbeck et al., 1999) or
equipped with collagenase-resistant Col1
(Liu et al., 1995
;
Zhao et al., 1999
) suggest
that collagen cleavage and remodeling regulates bone homeostasis. However, the
roles of chondrocytes and osteoblasts versus other cells in ECM remodeling
have not been elucidated. Of the collagenolytic MMPs present in skeletal
tissue, MMP13 is of particular interest because a mutation in human
MMP13 causes the Missouri variant of spondyloepimetaphyseal dysplasia
(SEMD), a syndrome with abnormalities in development and growth of
endochondral skeletal elements (Kennedy et
al., 2003
). MMP13 is expressed by both terminal hypertrophic
chondrocytes and osteoblasts, and its substrates include both Col1 and Col2.
Furthermore, MMP13 can synergize with MMP9 in degradation of collagen
(Engsig et al., 2000
). In this
study, we generated mice deficient for MMP13 by homologous recombination in
all tissues, as well as mice lacking MMP13 in a tissue-specific manner. We
also generated mice deficient for both MMP13 and MMP9. Thus, we have been able
to define the specific contributions of MMP13 alone and in combination with
MMP9 to the rate-limiting processes during endochondral ossification.
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Materials and methods |
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To generate Mmp13/ mice, floxed MMP13
mice were crossed to ß-actin Cre mice, ensuring ubiquitous disruption of
the Mmp13 gene. The recombination of the floxed Mmp13
alleles was analyzed by Southern blotting and northern blotting.
Col2-Cre and Col1-Cre transgenic mice where the Cre
recombinase is expressed under the rat Col21 or the
Col1
1 promoter, respectively, were generated as
described elsewhere (Dacquin et al.,
2002
; Schipani et al.,
2001
).
All mice were housed in a specific pathogen free (SPF) environment and under light, temperature- and humidity-controlled conditions. Food and water were available ad libitum. The procedures for performing animal experiments were approved by the Institutional Animal Care and Use Committee, University of California, San Francisco.
Calvarial culture
Frontal and parietal calvariae from newborn to 5-day-old mice were cultured
and the conditioned media collected and assayed for the presence of MMP13 as
previously described (Peeters-Joris et
al., 1998).
Staining of whole skeletons
Whole skeletal preparations of 2-week-old mice were prepared and stained
with Alizarin Red and Alcian Blue as previously described
(McLeod, 1980).
BrdU labeling/histology
A 10 mg/ml stock of bromodeoxyuridine (BrdU; Sigma, St Louis, MO) was
injected intraperitoneally into 1-week-old mice at a dose of 100 µg BrdU
per gram of mouse. Mice were sacrificed 1 hour after injection and bones were
harvested. BrdU staining was carried out on paraffin sections using a kit
according to manufacturer's directions (Zymed, South San Francisco, CA).
In situ hybridization
Paraffin sections were placed on acid-etched, TESPA-treated slides and
prepared for in situ hybridization as described
(Albrecht et al., 1997).
Plasmids were linearized with the appropriate restriction enzymes to
transcribe either sense or antisense 35S-labeled riboprobes
[colIIA1, and colXA1 probes are described elsewhere
(Albrecht et al., 1997
);
Mmp2, Mmp13, Mmp14, VEGF probes are described elsewhere
(Colnot and Helms, 2001
);
Mmp8 is described elsewhere
(Sasano et al., 2002
)]. Slides
were washed at a final stringency of 65°C in 23 xSSC, dipped in
emulsion, and exposed for 1-2 weeks. Slides were counterstained with Hoechst
33342.
Histological analyses and immunohistochemistry
Tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline,
decalcified in EDTA, paraffin embedded, sectioned at 5 µm and stained with
von Kossa's stain, Safranin O/Fast Green or Picrosirius Red. Briefly, for
Safranin O/Fast Green staining, deparaffinized and rehydrated sections were
stained in Weigert's Iron Hematoxylin (Sigma, St Louis, MO), 0.02% aqueous
Fast Green (Sigma) followed by a rinse in 1% acetic acid and 0.1% aqueous
Safranin-O (Sigma). For Picrosirius Red staining, deparaffinized and
rehydrated sections were stained in a 0.1% solution of Direct Red 80 (Aldrich,
Milwaukee, WI) in saturated picric acid (Sigma) followed by washes in 0.5%
acetic acid. Additional 5 µm paraffin sections were reacted for TRAP
activity using a leukocyte acid phosphatase kit and counterstained with Methyl
Green (Sigma).
For immunohistochemistry, tissues were fixed, embedded and sectioned as
described above. For PECAM immunostaining, rat anti-mouse PECAM monoclonal
antibody (Pharmingen, San Diego, CA) was used at a dilution of 1:50. For
cleaved aggrecan immunostaining, deparaffinized, rehydrated sections were
deglycosylated in chondroitinase ABC (Seikagaku Corporation, Tokyo, Japan) and
rabbit anti-DIPEN polyclonal antibody
(Singer et al., 1995) was used
at a concentration of 0.6 µg/ml. For cleaved collagen staining, rabbit
anti-Col 3/4 polyclonal antibody (Hdm Diagnostics and Imaging, Toronto,
Canada) was used at a dilution of 1:800.
Micro-CT
Tibiae were dissected from 1-year-old mice and analyzed using a micro-CT
system (µCT40, Scanco Medical, Bassersdorf, Switzerland). The trabeculae of
tibiae bone were scanned using a Cone-Beam type scan into 240 slices with a
voxel of 7 x7 x7 µm. Three-dimensional trabecular structural
parameters were measured directly, as previously described
(Jiang et al., 2003).
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Results |
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To assess which cells of the skeletal elements express Mmp13, in situ hybridization was performed on bones at various stages of the endochondral ossification process. At early stages (15 days post coitus, dpc), Mmp13 was expressed in hypertrophic chondrocytes and newly recruited bone cells in the primary ossification center (Fig. 1D). Following the establishment of the growth plates, expression of Mmp13 became restricted to only the very last rows of hypertrophic chondrocytes and the osteoblasts of the trabecular bone (Fig. 1E).
MMP13 deficiency causes altered growth plate architecture and increased trabecular bone during endochondral ossification
Staining of skeletons from Mmp13/ animals
with Alizarin Red and Alcian Blue showed that all elements appeared normal and
were equivalent in length compared with wild-type littermates
(Fig. 2A). For microscopic
analyses, we focused on the tibiae and metatarsals, which differ with respect
to the kinetics of growth plate closure. In contrast to metatarsals, which
close their growth plates at about 4 weeks of age, a small part of residual
growth plate cartilage remains in fully developed tibiae, separating the
epiphysis from the diaphysis. At 15 dpc, metatarsals and tibiae show the first
signs of the formation of a primary ossification center
(Fig. 1D). At this stage,
Mmp13 mRNA is highly expressed in the late hypertrophic chondrocytes,
where it colocalizes with collagen type X
(Wu et al., 2001).
Mmp13 is also expressed in the newly recruited osteoblasts that
migrate along the residual mineralized cartilage matrix
(Gack et al., 1995
;
Johansson et al., 1997
). Long
bones of 15 dpc Mmp13/ mice showed no
obvious differences when compared with those from wild-type littermates
(Fig. 2B,C). Thus, it did not
appear that the initial stages of endochondral ossification were affected in
the absence of MMP13.
|
A second notable phenotype in the long bones of Mmp13/ mice was an increase in trabecular bone. Although tibiae from 1-week-old Mmp13/ mice showed no apparent change in trabecular bone compared with wild type, the shape of the bone spicules was irregular (Fig. 3A,B). At 3 weeks, tibiae from Mmp13/ mice had a visible increase in trabecular bone (Fig. 3C,D). A similar increase was also visible in the metatarsals after 1 week of age (data not shown). The severity of the trabecular bone phenotype progressed with age. In contrast to the expansion of the hypertrophic cartilage, however, the increase in trabecular bone persisted for months in tibiae as well as femurs (Fig. 3E-H). To confirm these histological observations, we performed micro-computed tomography (micro-CT) on tibiae from wild-type and Mmp13/ mice. We found a dramatic and significant increase [Mmp13/, 0.295±0.026 (n=4); wild type, 0.069±0.022 (n=3), as total bone volume per tissue volume (mean±s.e.m., P=0.0015, two-tailed t-test)] in trabecular bone volume in the Mmp13/ mice compared with wild-type littermates (Fig. 3I,J). Interestingly, the cortical bone volume was not significantly different. By one year of age, the trabecular volume in Mmp13/ was comparable with wild type (data not shown). These data suggest that a role for Mmp13 is confined to the most active stages of trabecular bone remodeling.
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|
Mice with conditional deletion of Mmp13 have specific hypertrophic cartilage and trabecular bone phenotypes
The increased trabecular bone density observed in the
Mmp13/ mice could be the consequence of
improper cartilage matrix degradation caused by MMP13 deficiency in late
hypertrophic chondrocytes. Alternatively, a defect in trabecular bone
formation could be the result of the MMP13 deficiency in osteoblasts. To test
which of these mechanisms is responsible for the trabecular bone defects, we
conditionally inactivated Mmp13 in chondrocytes or osteoblasts. For
this purpose, we crossed floxed Mmp13 mice (MMP13fl/fl) to mice
carrying the Cre recombinase transgene under control of the Col2A1
promoter (Col2-Cre) or by the Col11 promoter
(Col1-Cre). MMP13+/fl;Col2-Cre+/ or
MMP13+/fl;Col1-Cre+/ mice were then crossed to MMP13fl/fl
mice.
As expected, MMP13fl/fl;Col2-Cre+/ mice displayed a similar increase in hypertrophic chondrocytes as observed in Mmp13/ mice (Fig. 5A,B). In contrast to Mmp13/ mice, MMP13fl/fl;Col2-Cre+/ mice did not show a noticeable increase in trabecular bone volume (Fig. 5C,D). However, we noted that the spicules of trabecular bone in MMP13fl/fl;Col2-Cre+/ were irregular in shape and length when compared with wild-type mice. This is similar to the irregular organization of trabecular bone seen initially in the Mmp13/ mice (Fig. 3), and suggests that ablation of MMP13 from chondrocytes has only a mild modeling affect on trabecular bone. The absence of an overt change in trabecular bone in MMP13fl/fl;Col2-Cre+/ mice indicates that the increased trabecular bone observed in Mmp13/ mice is the result of absence of Mmp13 expression in osteoblasts.
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|
|
Mmp9/; Mmp13/ mice display a dramatic alteration in growth plate architecture and skeletal development
MMP13 is able to perform the initial fibrillar collagen cleavage and MMP9
can then degrade the denatured collagen cleavage fragments. To test the
possible synergy between MMP9 and MMP13, we crossed
Mmp13/ and
Mmp9/ mice
(Vu et al., 1998) to obtain
animals that were deficient for both proteinases
(Mmp9/;Mmp13/).
Mmp9/;Mmp13/
mice were viable, but were visibly runted. The long bones of postnatal
Mmp9/;Mmp13/
mice exhibited a dramatic phenotype in the growth plate, namely an expanded
hypertrophic chondrocyte zone, that was more severe than the growth plate
phenotype of either the Mmp9/ or the
Mmp13/ mice. The normal columnar
architecture was lost in the overpopulated
Mmp9/;Mmp13/
hypertrophic zone, while the front of ossification took on a conformation
similar to, but more severe than, that observed in the
Mmp9/ mice
(Fig. 8A). mRNA in situ
analyses using an Osp-specific probe showed an increase in the number
of terminally differentiated hypertrophic cells in the MMP9/MMP13 knockout
mice. This shows that exit from the growth plate is delayed, but also that the
cells are viable and not necrotic (data not shown). In addition to the
phenotype observed at the primary site of ossification, development of the
secondary site of ossification was delayed in
Mmp9/;Mmp13/
bones. This delay was first visible at 2 weeks of age and persisted throughout
the rapid growth phase (Fig.
8A, insets). Examination of 5-month-old
Mmp9/;Mmp13/
mice revealed that the growth plates in metatarsals did eventually close and
developed secondary sites of ossification, although the structure of the
residual endosteum and bone marrow cavity was slightly altered from wild type
(Fig. 7B) and the mice had
dramatically shortened skeletal elements
(Fig. 8C).
|
MMP9 and MMP13 contribute to ECM remodeling in endochondral bone formation
Our data have shown that chondrocyte proliferation, differentiation,
chondrocyte apoptosis, angiogenesis and bone deposition are not rate limiting
during endochondral bone formation in the
Mmp13/ mice. However, accumulation of
cartilage matrix caused by expansion of the hypertrophic zone and increased
trabecular bone mass are robust features of the phenotype. MMP9 and MMP13 are
able to cleave aggrecan and collagen, the most abundant molecules in the
developing bones, at least in vitro
(Billinghurst et al., 1997;
Fosang et al., 1996
;
Mercuri et al., 2000
;
Mitchell et al., 1996
;
Vu et al., 1998
). This points
to ECM remodeling as a crucial event. Accordingly, we asked whether cleavage
of these two potential targets of MMP9 and/or MMP13 in the developing growth
plate and trabecular bone is altered during endochondral ossification in mice
deficient for MMP13 or MMP9, or both. We first used an antibody directed
toward DIPEN, a cryptic epitope in aggrecan that is exposed specifically upon
MMP cleavage (Singer et al.,
1995
) to assess the state of aggrecan degradation. This
MMP-specific aggrecan cleavage epitope was present along the front of
ossification in wild-type, Mmp9/ and
Mmp13/ tibiae, but not in
Mmp9/;
Mmp13/ tibiae
(Fig. 9A). In all cases, this
neoepitope was confined to the last transverse septa of the growth plate
(LTS). These results indicate that MMP13 and MMP9 together are responsible for
MMP-dependent aggrecan cleavage.
|
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Discussion |
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Cartilage degradation depends on degradation of its most abundant ECM
component, fibrillar collagen (Alini et
al., 1992; Mwale et al.,
2000
), which can be cleaved by collagenolytic MMPs
(Sternlicht and Werb, 2001
;
Werb, 1997
). In this study we
have demonstrated, for the first time in vivo, a specific role for MMP13 in
the processes of endochondral ossification. Our data, together with the
observations made in MMP14 (MT1-Mmp)/ mice
(Holmbeck et al., 1999
),
indicate that MMP13 is the major collagenase for Col2 cleavage. In the face of
continued VEGF expression in the expanded hypertrophic cartilage zone of
Mmp13/ mice, it is apparent that degradation
of Col2 may be required for vascular invasion, even in the presence of
abundant angiogenic stimuli. An interesting recent study demonstrates that
migration of endothelial cells and vessel formation can be prevented or
substantially diminished when a mutant type I collagen that is resistant to
cleavage by MMPs is used as the matrix for the explants
(Zijlstra et al., 2004
). In
our study, the ablation of Mmp13 did not affect blood vessel
morphology at the bone-cartilage junction. However, to maintain the normal
appearance of the blood vessels without an accumulation of endothelial cells
in the Mmp13/ growth plate, it follows that
the vascular development would have to be diminished in proportion to the
increased persistence of the hypertrophic cartilage. A logical extension of
this hypothesis is that increasing levels of MMP13 should accelerate
vascularization, which it does (Zijlstra
et al., 2004
). Taken together, these observations place MMP13
upstream of angiogenesis and suggest that degradation of the cartilage ECM
creates a permissive environment for blood vessels to invade.
What are the critical events on the cartilage side?
As MMP13 is the dominant collagenase in cartilage, it is not surprising
that its absence caused a delay in endochondral ossification. At the
cartilage-bone interface, only a thin layer of cartilage matrix separates the
hypertrophic chondrocytes from invading capillaries. This layer is known as
the last transverse septa (LTS) (Lee et
al., 1999). The LTS is an active site of matrix remodeling, marked
by degradation of fibrillar collagen. Our studies have shown that MMP13 is the
major collagenase involved in degradation of Col2 at this site. The region of
Col2 cleavage is restricted to the LTS, while MMP13 is expressed in a slightly
larger area of the hypertrophic chondrocyte zone. As MMP13 is secreted in a
latent form, it is possible that its activation occurs only at the LTS,
proximal to the expression domain of its major activator MMP14
(Jimenez et al., 2001
;
Knauper et al., 1996
).
The other major component of the cartilage matrix is aggrecan. Recent
studies have shown that aggrecan protects cartilage collagen from degradation
by proteinases, including MMPs (Pratta et
al., 2003), and that there is an inverse relationship between
collagenolytic activity and glycosaminoglycan content in hypertrophic
cartilage (Byers et al., 1997
;
Mwale et al., 2000
). This
suggests that for collagenases to cleave fibrillar collagens effectively,
aggrecan would first have to be degraded in this environment. This is
interesting to consider, as our studies show that MMP13 and MMP9 synergize to
cleave aggrecan and Col2 in the most terminal hypertrophic zone of the growth
plate. Thus, it is possible that MMP9 and/or MMP13 are first responsible for
clearing aggrecan molecules in this microenvironment, and that this
degradation is then followed by cleavage of Col2 by MMP13.
What are the critical events on the bone side?
In addition to the expansion of the hypertrophic zone, long bones of
Mmp13/ mice have increased trabecular bone.
In contrast to the expansion of the hypertrophic cartilage observed in these
animals, the increase in trabecular bone persists for months. Conditional
inactivation of Mmp13 in chondrocytes shows that the increased
trabecular bone mass observed in Mmp13/ mice
occurs independently of the reduced cartilage ECM degradation caused by loss
of this proteinase in late hypertrophic chondrocytes. The absence of MMP13
does not abolish collagen degradation in the trabeculae, but it is unlikely
that this is due to a compensatory mechanism of upregulation of other MMPs
that cleave collagen, as no change in expression levels of other MMPs present
in bone and cartilage was observed (see Fig. S1 in the supplementary
material). We favor a scenario in which other MMPs that generally play minor
roles in this area, most probably MMP14, compensate sufficiently to allow
endochondral ossification to proceed. Interestingly, cortical bone is
unaffected by the lack of MMP13, suggesting other MMPs contribute to its
remodeling.
Formation of trabecular bone involves modeling as well as remodeling
processes, and it appears that MMP13 is involved in both. Initially,
Mmp13/ mice do not display increased
trabecular bone volume, but the spicules are irregular in shape. An increase
in trabecular bone becomes apparent when mice reach 3 weeks of age, peaks at
about 6 months of age and then gradually normalizes. By contrast, conditional
inactivation of Mmp13 in cartilage leads to irregularities in
trabecular bone spicules similar to those seen in
Mmp13/ mice early in development. It is not
surprising that a defect in cartilage matrix remodeling would affect
trabecular bone formation, as trabecular bone is laid down upon the remnants
of calcified cartilage. Upon vascularization of the hypertrophic cartilage,
osteoprogenitor cells reach the interior of this zone and differentiate on the
plates of mineralized cartilage between the columns of hypertrophic
chondrocytes (Maes et al.,
2002). Therefore, it is possible that osteoblast differentiation
and function depends on the correct remodeling of cartilage, and that the
altered residual cartilage matrix in Mmp13/
endochondral bones causes impaired osteoblast differentiation and subsequent
improper bone deposition (Maes et al.,
2002
). In keeping with these observations, conditional
inactivation of MMP13 in osteoblasts maintained proper hypertrophic cartilage
remodeling, but showed increased trabecular bone. Thus, the action of MMP13 in
cartilage contributes to modeling of trabecular bone, while the action of
MMP13 in osteoblasts contributes to remodeling of trabecular bone.
It is important to note that the Mmp13/
bone phenotype is not the same as that produced by altering the susceptibility
of the collagen substrate. Mice with targeted mutations in
Col11 that render Col1 collagenase-resistant show
increased bone deposition (Zhao et al.,
2000
). However, the resistant collagen phenotype is late and
occurs mainly in the cortical bone, unlike the early trabecular bone phenotype
of the Mmp13/ mice. Second, mice with
collagenase-resistant Col1 have increased apoptosis of osteocytes
(Zhao et al., 2000
), lending
further support to the evidence that collagen cleavage products may be
anti-apoptotic (Montgomery et al.,
1994
). We did not observe increased apoptosis in the
Mmp13/ mice, as other MMPs expressed in bone
and cartilage (Sasano et al.,
2002
) still cleave collagens at low rates in
Mmp13/ mice, and this may be sufficient to
prolong cell survival and prevent accumulation of cortical bone with age in
these mice.
Synergy between MMP13 and MMP9
Our data show that MMP9 and MMP13 synergize during endochondral
ossification. These MMPs may first act to degrade aggrecan in the hypertrophic
zone of the growth plate, making Col2 accessible to cleavage by MMP13. MMP9
may then act downstream to clear denatured collagen cleavage products
generated by MMP13. Although MMPs have not been considered to be the major
aggrecanases (Arner, 2002;
Malfait et al., 2002
), our
data suggest that they do contribute to the physiological and developmental
turnover of aggrecan in this region. Our studies in mice that lack both MMP9
and MMP13 support the hypothesis that MMP9 and MMP13 act synergistically to
degrade the LTS. And although cathepsins also produce degradation fragments
identical to those created by MMP-mediated cleavage of aggrecan
(Mort et al., 1998
), our data
from the
Mmp9/;Mmp13/
mice show that these MMPs are major proteinases involved in degradation of
aggrecan in the growth plate.
Double null mice have a dramatic accumulation of hypertrophic cartilage
accompanied by loss of normal growth plate architecture. The bone phenotype of
Mmp9/ mice was dominant in the
Mmp9/;Mmp13/
mice; these animals displayed less trabecular bone and delayed bone marrow
cavity formation. MMP9 has important roles in osteoclast and osteoblast
recruitment and function (Colnot et al.,
2003), as well endothelial progenitor mobilization
(Hattori et al., 2003
;
Heissig et al., 2003
).
Nevertheless, the resolution of the phenotypes that occurs after several
months indicates other processes are able to compensate in the absence of MMP9
and MMP13. However, this compensation is stochastic and incomplete, as the
bones that do form are shorter and misshapen when compared with wild type.
How does ECM turnover relate to angiogenesis?
Chondrocyte differentiation and bone formation are tightly coupled in a
process that involves chondrocyte apoptosis, cartilage matrix remodeling and
vascular invasion. A number of previous studies have focused on angiogenesis
as the critical step in the process. However, chondrocyte apoptosis can be
uncoupled from vascularization, as inactivation of galectin 3 leads to
precocious hypertrophic chondrocyte death without an alteration in
angiogenesis (Colnot et al.,
2001). Removal of angiogenic stimuli by ablating VEGF from
hypertrophic chondrocytes (Maes et al.,
2002
; Zelzer et al.,
2004
) or by blocking VEGF receptors
(Gerber et al., 1999
) results
in expansion of the hypertrophic cartilage zone. Likewise, angiogenesis may be
uncoupled from ECM remodeling because the process of inhibiting VEGF function
also inhibits VEGF-dependent recruitment of the MMP9-expressing osteoclasts
needed to promote ECM remodeling (Gerber
et al., 1999
). However, in both the
Mmp13/ and
Mmp9/ mice, osteoclast recruitment still
occurs, but, as the ECM degradation is decreased, in parallel the vasculature
recruitment keeps pace with the slower rate of endochondral ossification.
These observations suggest that degradation of the cartilage ECM creates a
permissive environment for blood vessels to invade. Cleaving of ECM molecules
could merely create a passage way for invading blood vessels that otherwise
would not be able to penetrate the dense cartilage matrix. Alternatively,
cleaving of matrix molecules could make angiogenic factors accessible.
Remodeling of the cartilage matrix is likely the rate-limiting step as it
depends on degradation of its most abundant component, fibrillar collagen.
Insights from human skeletal disorders
The importance of matrix components in maintaining structural integrity and
function of cartilage, as well as the role of the cartilage matrix in the
differentiation process of its cellular components is well known. Genetic
defects in humans and targeted mutagenesis in mice have demonstrated that
aberrant assembly of cartilage matrix can lead to severe impairment of
endochondral ossification (for a review, see
Aszodi et al., 2000). SEMD is a
heterogeneous group of human skeletal disorders characterized by defective
growth and modeling of the long bones, which in most cases is caused by
mutations in cartilage matrix proteins such as Col2
(Borochowitz et al., 2004
;
Tiller et al., 1995
). The
Missouri variant of SEMD (Gertner et al.,
1997
) is caused by to a point mutation in MMP13
(Kennedy et al., 2003
). As is
the case in our Mmp13/ mice, the human
phenotype is developmental and is ameliorated with age. This is evidence that
proper endochondral ossification requires not only assembly of cartilage
collagens but also their degradation.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/23/5883/DC1
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