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© The Rockefeller University Press,
0021-9525/2000//879 $5.00
The Journal of Cell Biology, Volume 151, Number 4,
, 2000 879-890
Original Article |
Matrix Metalloproteinase 9 and Vascular Endothelial Growth Factor Are Essential for Osteoclast Recruitment into Developing Long Bones
Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737–744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development.
Key Words: matrix metalloproteinase • VEGF • osteoclast recruitment • endothelial cell • bone development
© 2000 The Rockefeller University Press
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Introduction |
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The molecular mechanisms directing osteoclastic resorptive activity to specific areas of the bone have generated much interest. Osteoclasts are generated from hematopoietic stem cells under the control of several systemic and local factors (Hofbauer et al. 2000). Osteoclast differentiation factor (Kong et al. 1999), osteoprotegerin (Simonet et al. 1997), macrophage colony-stimulating factor (M-CSF) (Felix et al. 1990; Wiktor-Jedrzejczak et al. 1990; Yoshida et al. 1990), vascular endothelial growth factor (VEGF) (Niida et al. 1999), interleukin-1 (Jimi et al. 1999), and tumor necrosis factor-
(Kobayashi et al. 2000) are involved in osteoclast differentiation. TGF-β (Dieudonné et al. 1991; Zheng et al. 1994), M-CSF (Fuller et al. 1993), hepatocyte growth factor (Fuller et al. 1995a), interleukin-8 and macrophage inflammatory protein-1 (Fuller et al. 1995b), C3 component of complement (Mangham et al. 1993), osteopontin (Faccio et al. 1998; Terai et al. 1999), and bone sialoprotein (Raynal et al. 1996) help target the resorptive activity of osteoclasts to specific sites of the matrix. In addition, proteinases of the matrix metalloproteinase (MMP) family are indispensable for the recruitment of osteoclasts in developing metatarsals (Blavier and Delaissé 1995), and this role is distinct from synergy with cysteine proteinases in solubilizing calcified matrix in the resorption zone (Everts et al. 1998; Saftig et al. 1998). The specific MMP(s) responsible for these osteoclastic activities, and its (their) mode of action have not been determined (Delaissé et al. 2000).
MMP-9 is a candidate for regulating these functions because it is highly expressed in osteoclasts (for review see Vu and Werb 1998). In embryonic bone, before the formation of the marrow cavity, MMP-9 is highly expressed in cells in the mesenchyme surrounding bone rudiments; as the marrow cavity develops, MMP-9 expression is detected in osteoclasts in the core of the diaphysis (Reponen et al. 1994; Blavier and Delaissé 1995; Jemtland et al. 1998). Subsequently, the marrow cavity expands longitudinally and at either end interacts with the epiphyseal area to form the growth plates and the metaphysis, areas that are very critical for bone growth and (re)modelling. MMP-9 is then expressed in typical (TRAP+) osteoclasts localized along the mineralized longitudinal septae of the metaphysis, as well as in osteoclast-like cells that are localized next to the nonmineralized transverse septae that separate them from the last row of hypertrophic chondrocytes (Jemtland et al. 1998; Vu et al. 1998). This is exactly at the invasion front of the growth plates, where endothelial cells are also abundant. Because these cells are localized against cartilage and differ from osteoclasts to some extent, they are sometimes called chondroclasts. Importantly, the histology of MMP-9 null mice shows that it is a key proteinase for the invasion of the growth plate, but does not support a role of MMP-9 in the actual solubilization of mineralized matrix by osteoclasts (Vu et al. 1998). In addition, a study combining in situ zymography, MMP-9 antagonists, and immunohistochemistry showed that MMP-9 acts on the nonmineralized transverse septae of the growth plate (Lee et al. 1999). It is not clear whether MMP-9 exerts a simple mechanical role, such as solubilizing the transversal septae, or plays a more subtle role, such as generating signals that render the cells invasive. Indeed, there are strong indications that MMP-9 exerts its role in invasion of the growth plate through the release of molecules sequestered in the extracellular matrix (Vu et al. 1998). VEGF is one such molecule, since blocking its function leads to impaired invasion of the growth plate, mimicking the phenotype of MMP-9–negative bones (Gerber et al. 1999).
In this study, we analyzed metatarsals of MMP-9–negative and MMP-9–positive embryos well before the formation of the growth plate, and found that the recruitment of the very first osteoclasts in these bones is affected by lack of MMP-9. By using a combination of histological analysis, bone resorption models, invasion chamber experiments, and MMP and VEGF antagonists, we show that the role of MMP-9 in this process is clearly distinct from the role other MMPs play in other steps of bone development. MMP-9 specifically regulates proteolysis of nonmineralized cartilage and release of ECM-bound VEGF, exerting direct chemotactic activity on osteoclasts.
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Rabbit anti–human MMP-9 antibody (Nielsen et al. 1997) was a gift from Dr. B.S. Nielsen and Dr. N. Borregaard (Finsen Laboratory and Rigshospitalet, Copenhagen). Rat anti–mouse CD34 antibody (Garlanda et al. 1997) was a gift from Dr. C. Garlanda (Pharmacology Institute Mario Negri, Milan, Italy). Recombinant human MMP-13 was a gift from Dr. C. Lopez-Otin (University of Oviedo, Oviedo, Spain). MMP-9 was purified to homogeneity from cultures of BHK cells transfected with mouse MMP-9 and provided by Dr. M. Ferreras (Center for Clinical and Basic Research). Recombinant human VEGF and recombinant human Flt-1/Fc chimera were provided by R&D Systems. Recombinant mouse mFlt-IgG chimera was a gift from Dr. Napoleone Ferrara (Genentech, Inc., San Francisco, CA). GM6001, a general MMP inhibitor, was from AMS Scientific. The other reagents were from suppliers described previously (Delaissé et al. 1985; Blavier and Delaissé 1995; Sato et al. 1998).
Analysis of MMPs by Zymography
Calvariae and tibiae were extracted as described (Eeckhout et al. 1986) in a buffer containing 10 mM cacodylate-HCl, pH 6, 1 M NaCl, 0.1 mg/ml Triton X-100, 0.2 mg/ml NaN3, 10–6 M ZnCl2. The two metatarsal triads of each embryo were extracted together in 500 µl extraction buffer for 24 h, and extracts were concentrated four to five times, before analysis by zymography (Lefebvre et al. 1991). To determine whether there were compensatory changes in other proteolytic activities in the absence of MMP-9, a variety of zymography conditions were used: gelatin and casein as substrate, 7.5–12% acrylamide concentrations, and incubation times of 18–60 h.
Bone Resorption Models
Bone resorption was evaluated with well established models (Delaissé et al. 1985; Blavier and Delaissé 1995), on littermates of heterozygote parents. Timed pregnant mothers were injected at day 16 (day of vaginal plug discovery is designated day 0) with 100 µCi 45Ca. The middle three metatarsals and the tibiae were isolated at day 17, and calvariae were isolated at day 18. The middle three metatarsals were kept together as triads. The bone explants were placed on filter paper floating on 400 µl culture medium in 24-well plates. The medium consisted of BGJb solution supplemented with ascorbate (50 µg/ml), glutamax (500 µg/ml), and albumax (1 mg/ml). In addition, the media for long bones were supplemented with 1,25(OH)2D3 (10–8 M) (Blavier and Delaissé 1995), and those for calvariae with parathyroid hormones (2 x 10–8 M) (Delaissé et al. 1985). Cultures lasted usually for 4 d. We measured the 45Ca released every day in the media and that left in the tissue at the end of the culture. The demineralization of the bone explants during the cultures was then expressed cumulatively as percentage of total amount of radioactivity. The release of 45Ca from paired experimental groups was compared by the analysis of variance for multiple comparisons.
Histomorphometry
Metatarsal triads (consisting of the middle three metatarsals) were obtained from littermates of MMP-9+/– x MMP-9–/– parents. Histological procedures, the histochemical method for TRAP staining, and the determination of numbers and distributions of TRAP+ cells and of their nuclei in longitudinal sections of the diaphysis were performed as described previously (Blavier and Delaissé 1995). These analyses were performed by an investigator unaware of the genotype. Cells and their nuclei were scored as "in" upon their invasion into the calcified cartilage filling the core of the diaphysis, thus indicating their ability to invade. For each metatarsal triad, countings were typically performed in five sections at 20 µm from each other and the mean of these five counts was calculated. The results were expressed as mean number of cells or nuclei in one section of metatarsal triad. Counts in +/– and –/– bones were compared by using the analysis of variance. The analysis of cell and nucleus counts has always led to the same conclusions concerning invasion; however, for clarity, the illustrations in this work show only counts of nuclei because they are more representative of a process where new mononucleated cells are generated and progressively undergo fusion to multinucleated cells.
Immunohistochemistry
Paraffin sections were treated with 0.45% H2O2 in ethanol to inactivate endogenous peroxidase. For detection of CD34, the sections were digested with trypsin, blocked with casein, incubated overnight with a 1:100 dilution of anti-CD34 antibody, and treated successively with a 1:20 dilution of biotinylated anti–rat antibody and peroxidase-conjugated extravidin. For detection of MMP-9 (Nielsen et al. 1997), the sections were treated three times for 5 min in 10 mM Tris/1 mM EDTA, pH 9, in a microwave oven, blocked with BSA and 0.5% Triton, incubated overnight with a 1:400 dilution of anti–MMP-9 antibody, and finally reacted for 30 min with Dako EnVisionTM+ (Peroxidase, Rabbit, K4003). The CD34 and MMP-9 immunoreactivity was visualized by developing the slides in DAB, and H2O2 control MMP-9 immunostainings performed on MMP-9–deficient bones were negative. When CD34 immunostaining and TRAP activity staining were performed on the same sections, immunostainings were done first, followed by TRAP histochemistry. Sections were counterstained with Erlich's hematoxylin.
In Situ Hybridization
In situ hybridizations were performed on paraffin sections as reported (Graber et al. 1995). A fragment of the 3' end of mouse MMP-9 cDNA overlapping the stop codon (1633–1913) was subcloned into CR11-TOPO cloning vector (Invitrogen), and antisense and sense digoxygenin-labeled riboprobes (of 281 bp) were prepared with SP6 and T7 by using an RNA labeling kit (Boehringer). The hybridized probe was detected by using alkaline phosphatase–conjugated antidigoxygenin antibody and NBT–BCIP substrate mix (Boehringer). The sections were counterstained with methylene blue. For localization of VEGF expression, 35S-labeled antisense probes were prepared and used as described previously (Gerber et al. 1999).
Invasion and Chemotaxis Assay
For invasion experiments with MMP-9–positive and MMP-9–negative osteoclasts, osteoclasts were generated from marrow of MMP-9+/+ and MMP-9–/– mice, cultured in the presence of M-CSF and osteoclast differentiation factor (Lacey et al. 1998). For VEGF chemotaxis experiments, osteoclasts were generated from marrow of NMRI mice, cocultured with primary osteoblasts in the presence of 1,25(OH)2D3 (Akatsu et al. 1992). They were detached by sequential treatment with 0.1% bacterial collagenase (wt/vol) plus 0.1% dispase (wt/vol), and 0.5% trypsin (wt/vol), washed, resuspended in MEM with or without Flt-Fc, and seeded onto type I collagen-–coated membranes of culture inserts (Sato et al. 1998). These culture inserts were then placed in 12-well plates containing MEM supplemented with 0.1% albumax, with or without VEGF as indicated. After an overnight culture, the cells were stained for TRAP. Invasion was determined by counting before and after removal of the cells from the upper surface of the membranes, thereby rendering visible only osteoclasts or osteoclast extensions that had reached the lower surface of the membrane (Sato et al. 1998). Invasion scores were determined by taking the ratio of these two numbers. The scores of the different groups were compared with the analysis of the variance with Scheffe's F test.
Cartilage Degradation
6-mm cartilage discs were punched out of 10-d-old rabbit ears, devitalized, and further processed as reported (Hauser and Vaes 1978). They were then placed in 96-well plates in 200 µl MEM containing up to 50 nM MMP-13 and/or MMP-9 activated by 4-aminophenylmercuric acetate. The molar concentrations of active MMP-13 and MMP-9 were determined by active site titration with the tight-binding MMP inhibitor BB94 (Knight 1995). The discs were incubated at 37°C under 5% CO2 for up to 24 h. Hydroxyproline was measured in the media after hydrolysis in saturated Ba(OH)2 (Bergman and Loxley 1963). Under these conditions, hydroxyproline in the media increased linearly with incubation time.
Metatarsal Cultures with mFlt-IgG
Day 17 metatarsals (day of vaginal plug is designated as day 0) from wild-type mice were dissected and cultured for 3 d on Nucleopore membrane floating on 1 ml of BGJb culture medium supplemented with 10% fetal calf serum. Control IgG or the mouse chimeric VEGF receptor mFlt-Ig was added to a final concentration of 20 µg/ml. The medium was changed daily. After the culture, bones were fixed in 4% paraformaldehyde at 4°C overnight and processed for paraffin sections.
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MMP-9–/– bones from embryos of the same litter and of the same age differed from these MMP-9+/– bones mainly in two respects. First, the increase in the number of nuclei of TRAP+ cells was only 70% of that of MMP-9+/– bones (Fig. 5). The average number of nuclei per osteoclast, however, was not affected (Fig. 6). This observation suggests that the lack of MMP-9 does not affect cell fusion. Second, and more importantly, MMP-9 deficiency resulted in a delayed invasion of TRAP+ cells into the calcified cartilage (Fig. 5 and Fig. 7). At E18 and E19, the proportions of nuclei in the calcified cartilage were 20 and 40%, respectively, of those in MMP-9–positive bones. The osteoclasts of the MMP-9–negative bones tended then to accumulate at the interface between the osteoid (i.e., unmineralized collagen) of the bone collar and the calcified cartilage. During this period, the distribution of endothelial cells inside and outside the calcified cartilage was closely coordinated with that of TRAP+ cells, whether MMP-9 was lacking or not. Endothelial cells appeared inside the calcified cartilage only when TRAP+ cells had invaded the calcified cartilage (Fig. 7). Thus, the main result of MMP-9 deficiency was an impaired migration of TRAP+ cells and endothelial cells from the periosteum to the calcified cartilage.
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MMP-9 Is Not Involved in Solubilization of Mineralized Matrix by Osteoclasts
Osteoclasts that resorb mineralized matrix typically are polarized cells, forming a sealed-off resorption compartment upon attachment onto the surface of the mineralized matrix. Inhibitors of the cysteine proteinase cathepsin K and general MMP inhibitors inhibit collagen degradation in this zone (Everts et al. 1998). The effect of MMP inhibitors coupled with the high expression of MMP-9 in osteoclasts led to the hypothesis that MMP-9 is the critical MMP for bone matrix solubilization (Tezuka et al. 1994; Okada et al. 1995). However, bone histology of the MMP-9 knockout mice does not reveal a lack of osteoclastic resorption of mineralized matrix (Vu et al. 1998). In further support of the lack of a role of MMP-9 in solubilization of mineralized matrix, we show that specialized bone resorption models, which respond to general MMP inhibitors (Delaissé et al. 1985; Hill et al. 1994; Blavier and Delaissé 1995) (see Fig. 2), have the same bone degradation rates in MMP-9–negative and MMP-9–positive bones, even when bone resorption was stimulated with bone resorbing agents like parathyroid hormone and 1,25(OH)2D3. In calvariae, where MMPs have proved to be particularly important for matrix solubilization (Everts et al. 1999), MMP-9 deficiency was without effect. Cathepsin K, which is very efficient for matrix solubilization by osteoclasts (Saftig et al. 1998), did not compensate for MMP-9 because a cathepsin K inhibitor reduced resorption of tibiae to similar levels in MMP-9–negative and MMP-9–positive bones. Thus, the identity of the critical MMP involved in matrix solubilization in the resorption zone remains to be determined.
MMP-9 Is a Major MMP for Osteoclast Recruitment in Primitive Long Bones, and Is Specifically Required for the Passage of the Cells through Their Osteoid–Cartilage Interface
The first destructive task of osteoclasts in primitive long bones is the formation of the marrow cavity. Preosteoclasts are recruited into the bone from the surrounding mesenchyme, a process that is completely inhibited by general MMP inhibitors (Blavier and Delaissé 1995). We have now identified MMP-9 as a major MMP for this recruitment process. This role of MMP-9 is also supported by observations in bones of wild-type mice, since there is an excellent correlation between MMP-9 and invasion of osteoclasts into the core of the diaphysis. However, MMPs distinct from MMP-9 also participate in this recruitment, since the inhibition induced by the absence of MMP-9 is only partial whereas the inhibition generated by general MMP inhibitors is complete.
The biological events required by this recruitment are the generation of preosteoclasts in the mesenchyme surrounding the bone, their maturation, and their migration into the core of the diaphysis. It is clear that the main role of MMP-9 (and/or other MMPs) relates to the latter migration because lack of MMP-9 (or of MMP activity) leads to an accumulation of TRAP+ cells around the diaphysis, and does not prevent the formation of multinucleated cells. The generation of TRAP+ cells in E18 and E19 in the absence of MMPs is somewhat smaller (Blavier and Delaissé 1995; this work). This may be due to the modulating effect that resorption may exert on the recruitment of new osteoclasts (Van De Wijngaert et al. 1988), or to decreased VEGF bioavailability which may participate in osteoclast differentiation (Niida et al. 1999).
There are several critical steps along the migration route of the TRAP+ cells. The preosteoclasts must leave the periosteal cell layer, move through a 12–25-µm seam of osteoid whose inner edge is slightly and discontinuously mineralized, and finally invade the outer cartilage septae that are also discontinuously mineralized (Blavier and Delaissé 1995). In the absence of MMP-9, the TRAP+ cells tend to accumulate at the osteoid–cartilage interface, suggesting that their movement through osteoid is not affected. Experiments with type I collagen gels in invasion chambers confirmed that the intrinsic ability of osteoclasts to move through collagen is not significantly affected by the absence of MMP-9. This is in contrast to the effects of general MMP inhibitors, which block the migration of the (pre)osteoclasts through osteoid in E17 metatarsals (Blavier and Delaissé 1995) or through type I collagen in invasion chambers (Sato et al. 1998; this study). This shows that other (or another) MMPs are especially important for the migration through osteoid, which involves only focal lysis (Blavier and Delaissé 1995). A good candidate is MT1-MMP, a membrane-bound collagenolytic proteinase that has been identified in invadopodia and at the leading edge of moving osteoclasts (Sato et al. 1997). MT1-MMP null mice show bone and cartilage defects that have not been investigated in detail (Holmbeck et al. 1999).
What Is the Molecular Mode of Action of MMP-9 for Cartilage Invasion?
Our data are compatible with several molecular modes of action of MMP-9 in cartilage invasion. First, MMP-9 may speed up cartilage solubilization. This is interesting because such proteolytic action is required exactly at the point of the migration route where MMP-9 becomes necessary. At E17 this cartilage matrix is discontinuously mineralized (Blavier and Delaissé 1995). The classical resorptive machinery of the osteoclast is responsible for solubilizing the mineralized cartilage, as shown by the effects of classical inhibitors of their resorptive activity, including bisphosphonates and cysteine proteinase inhibitors (Blavier and Delaissé 1995). In contrast, these inhibitors do not inhibit invasion of the osteoclasts into the core of the diaphysis, and this invasion largely proceeds by passage through nonmineralized septae (Blavier and Delaissé 1995). Since this passage requires MMP-9, it appears that the role of MMP-9 relates to solubilization of nonmineralized septae. This is reminiscent of cartilage invasion at the growth plate: the transversal septae are nonmineralized and their invasion does not require resorptive activity of osteoclasts (Schenk et al. 1967), but requires MMP-9 (Vu et al. 1998). MMP-9 is present at the edge of the last transversal septa and in debris of type II collagen at various states of digestion (Vu et al. 1998; Lee et al. 1999). Our test tube assays show that MMP-9 stimulates unmineralized cartilage degradation in the presence of MMP-13. Interestingly, MMP-13 is highly expressed in the hypertrophic chondrocytes filling the diaphysis of embryonic metatarsals, as well as in the growth plates (Blavier and Delaissé 1995; Gack et al. 1995), and binds cartilage septae (Lee et al. 1999). It has been proposed that MMP-9 and MMP-13 cooperate for degrading the unmineralized cartilage of these septae (Lee et al. 1999). Our experiments provide direct support to this hypothesis. One may speculate that MMP-9 speeds up the degradation of proteoglycans, thereby rendering the collagen more accessible for degradation by MMP-13. Alternatively, MMP-9 may promote the digestion of the collagen that was destabilized by the attack of MMP-13. However, it is unclear to what extent the effect of MMP-9 on collagenolysis is essential for cartilage invasion, since cartilage invasion at the secondary site of ossification is only minimally affected by the lack of MMP-9 (Vu et al. 1998), although this invasion also requires breaching of cartilage septae. Both MMP-9 and MMP-13 are normally expressed at these sites (Delaissé, unpublished results).
Second, MMP-9 may favor cartilage invasion by releasing growth and differentiation factors. Proteolytic release of matrix-bound components from E17 metatarsals and growth plates appears to be involved in recruitment of TRAP+ cells and endothelial cells, respectively (Van De Wijngaert et al. 1988; Vu et al. 1998). Among the factors affecting osteoclast recruitment in both growth plate and E17 metatarsals are M-CSF (Felix et al. 1990; Hofstetter et al. 1995; Jemtland et al. 1998), TGF-β (Dieudonné et al. 1991), and VEGF (Gerber et al. 1999). That active TGF-β and VEGF are made bioavailable by MMP-9 (Bergers et al. 2000; Yu and Stamenkovic 2000) may be particularly relevant to the recruitment process. VEGF function is required in growth plate invasion (Gerber et al. 1999) and diaphysis invasion (this work); however, it is absent at the secondary site of ossification of the epiphysis (Werb, unpublished results) where the role of MMP-9 is also less important (Vu et al. 1998). A hypothesis that unifies these observations is that the major mode of action of MMP-9 is to make VEGF functional as a chemoattractant and promote invasion.
Still other modes of action of MMP-9 in diaphysis invasion are possible. Several molecules appear to be positioned at the osteoid–cartilage interface, i.e., precisely at the site where MMP-9 becomes important. These include molecules with strong effects on cell motility, such as transferrin, bone sialoprotein, osteopontin, and osteonectin (Gentili et al. 1994; Sommer et al. 1996). Interestingly, MMP-9 cleaves osteopontin and osteonectin molecules in their functional domain (Sasaki et al. 1997; Delaissé, unpublished results). The adhesive properties of osteopontin are strongly affected by this fragmentation (Senger et al. 1994; Xuan et al. 1994). Finally, osteoclasts express CD44 (Nakamura et al. 1995), an adhesion molecule with a strong affinity for hyaluronic acid that is abundant in cartilage. Hyaluronic acid may induce clustering of CD44 and MMP-9 on their plasma membrane, thereby promoting invasiveness, as reported for cancer cells (Yu and Stamenkovic 1999).
Relative Importance of Osteoclasts, Chondroclasts, and Endothelial Cells in Cartilage Invasion
Several cell types that are able to express MMP-9 are present in the area of the invasion front of the growth plate. They include endothelial cells, chondroclasts, and osteoclasts. Chondroclasts refer to the cells against the nonmineralized transversal septae, whereas osteoclasts refer to cells along the mineralized longitudinal trabeculae (Vu et al. 1998). These two cell types show inverse amounts of MMP-9 and TRAP at early developmental stages, chondroclasts being richer in MMP-9 and poorer in TRAP (Jemtland et al. 1998). Moreover, chondroclasts do not form a ruffled border to the same extent as typically polarized, resorbing osteoclasts (Nordahl et al. 1998). We did not detect distinct osteoclast and chondroclast populations in diaphysis invasion. This may be due to the random distribution of mineralized and nonmineralized portions of cartilage in the primitive diaphysis (Blavier and Delaissé 1995), in contrast to the situation in the growth plate (Schenk et al. 1967). At least some chondroclasts may derive from a cell lineage other than osteoclasts, because MMP-9–positive cells are present and growth ossification takes place in osteopetrotic mice (Fos–/–; Grigoriadis et al. 1994). However, osteoclasts can exhibit distinct phenotypes depending on their microenvironment, such as mineral (Gravallese et al. 1998), and distinct osteoclast activities do require functional and structural reorganizations, typical of epithelial–mesenchymal transitions (Nakamura et al. 1996). In particular, the polarized resorbing osteoclast secretes the cysteine proteinase cathepsin K into the acidic resorption zone to solubilize the mineralized matrix, whereas the rounded, nonpolarized osteoclasts depend on MMPs and not on cysteine proteinases for migration in nonmineralized collagen (Sato et al. 1998). The nonpolarized osteoclast-related cell that invades nonmineralized cartilage in the growth plate and diaphysis depends on MMP-9 for this invasion (Vu et al. 1998; this work).
Another important question is the identity of the target cells of the VEGF. VEGF effects have often been interpreted in the context of chemotaxis on endothelial cells. However, osteoclasts express VEGF receptor 1 (Flt-1), and VEGF can support osteoclast generation (Niida et al. 1999). Our work also shows that VEGF acts directly on osteoclasts and promotes their invasive activity. The fact that both endothelial cells and osteoclasts are responsive to VEGF fits well the observation that invasion of the diaphysis of the primitive metatarsals is approximately simultaneous for both cell types. Through these mechanisms VEGF serves to coordinate ECM degradation and recruitment of endothelial cells and osteoclasts.
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This work was supported by a grant from the National Institutes of Health (AR46238 to Z. Werb and T. Vu), by a mentored Physician Scientist Award (HL03880 to T. Vu), and by the Danish Cancer Society and the Danish Research Council (to L. Lund).
Submitted: 30 June 2000
Revised: 8 September 2000
Accepted: 12 September 2000
Abbreviations used in this paper: M-CSF, macrophage colony-stimulation factor; MMP, matrix metalloproteinase; TRAP, tartrate-resistant acid phosphatase; VEGF, vascular endothelial growth factor.
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Akatsu T., Tamura T., Takahashi N., Udagawa N., Tanaka S., Sasaki T., Yamaguchi A., Nagata N. & Suda T.. Preparation and characterization of a mouse osteoclast-like multinucleated cell population, J. Bone Miner. Res., 7, 1992, 1297–1306.[Medline]
Bergers G., Brekken R., McMahon G., Vu T.H., Itoh T., Tamaki K., Tanzawa K., Thorpe P., Itohara S., Werb Z. & Hanahan D.. Gelatinase B triggers the angiogenic switch during carcinogenesis, Nat. Cell Biol., 2, 2000, 737–744.[Medline]
Bergman D. & Loxley R.. Two improved and simplified methods for the spectrophotometric determination of hydroxyproline, Anal. Chem, 35, 1963, 1961–1965.
Blavier L. & Delaissé J.-M.. Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primitive long bones, J. Cell Sci., 108, 1995, 3649–3659.[Abstract]
Delaissé J.-M., Eeckhout Y., Sear C., Galloway A., McCullagh K. & Vaes G.. A new synthetic inhibitor of mammalian tissue collagenase inhibits bone resorption in culture, Biochem. Biophys. Res. Commun, 133, 1985, 483–490.[Medline]
Delaissé J.-M., Eeckhout Y., Neff L., Francois-Gillet C., Henriet P., Su Y., Vaes G. & Baron R.. Pro)collagenase (matrix metalloproteinase-1) is present in rodent osteoclasts and in the underlying bone-resorbing compartment, J. Cell Sci., 106, 1993, 1071–1082.[Abstract]
Delaissé J.-M., Engsig M.T., Everts V., Ovejero M.C., Ferreras M., Lund L., Vu T.H., Werb Z., Winding B. & Lochter A.. Proteinases in bone resorptionobvious and less obvious roles, Clin. Chim. Acta., 291, 2000, 223–234.[Medline]
Dieudonné S.C., Foo P., van Zoelen E.J. & Burger E.H.. Inhibiting and stimulating effects of TGF-beta 1 on osteoclastic bone resorption in fetal mouse bone organ cultures, J. Bone Miner. Res., 5, 1991, 479–487.
Eeckhout Y., Delaissé J.-M. & Vaes G.. Direct extraction and assay of bone tissue collagenase and its relation to parathyroid-hormone-induced bone resorption, Biochem. J., 239, 1986, 793–796.[Medline]
Everts V., Delaissé J.-M., Korper W. & Beertsen W.. Cysteine proteinases and matrix metalloproteinases play distinct roles in the subosteoclastic resorption zone, J. Bone Miner. Res, 13, 1998, 1420–1430.[Medline]
Everts V., Korper W., Jansen D.C., Steinfort J., Lammerse I., Heera S., Docherty A.J. & Beertsen W.. Functional heterogeneity of osteoclastsmatrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone, FASEB J, 13, 1999, 1219–1230.
Faccio R., Grano M., Colucci S., Zallone A.Z., Quaranta V. & Pelletier A.J.. Activation of alphav beta3 integrin on human osteoclast-like cells stimulates adhesion and migration in response to osteopontin, Biochem. Biophys. Res. Commun., 249, 1998, 522–525.[Medline]
Felix R., Cecchini M.G. & Fleisch H.. Macrophage colony stimulating factor restores in vivo bone resorption in the op/op osteopetrotic mouse, Endocrinology., 127, 1990, 2592–2594.
Fuller K., Owens J.M., Jagger C.J., Wilson A., Moss R. & Chambers T.J.. Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts, J. Exp. Med., 178, 1993, 1733–1744.
Fuller K., Owens J. & Chambers T.J.. The effect of hepatocyte growth factor on the behaviour of osteoclasts, Biochem. Biophys. Res. Commun, 212, 1995, 334–340a.[Medline]
Fuller K., Owens J.M. & Chambers T.J.. Macrophage inflammatory protein–1 alpha and IL-8 stimulate the motility but suppress the resorption of isolated rat osteoclasts, J. Immunol, 154, 1995, 6065–6072b.[Abstract]
Gack S., Vallon R., Schmidt J., Grigoriadis A., Tuckermann J., Schenkel J., Weiher H., Wagner E.F. & Angel P.. Expression of interstitial collagenase during skeletal development of the mouse is restricted to osteoblast-like cells and hypertrophic chondrocytes, Cell Growth Differ, 6, 1995, 759–767.[Abstract]
Garlanda C., Berthier R., Garin J., Stoppacciaro A., Ruco L., Vittet D., Gulino D., Matteucci C., Mantovani A., Vecchi A. & Dejana E.. Characterization of MEC 14.7, a new monoclonal antibody recognizing mouse CD34a useful reagent for identifying and characterizing blood vessels and hematopoietic precursors, Eur. J. Cell Biol., 73, 1997, 368–377.[Medline]
Gentili C., Doliana R., Bet P., Campanile G., Colombatti A., Cancedda F.D. & Cancedda R.. Ovotransferrin and ovotransferrin receptor expression during chondrogenesis and endochondral bone formation in developing chick embryo, J. Cell Biol, 124, 1994, 579–588.
Gerber H.-P., Vu T.H., Ryan A.M., Kowalski J., Werb Z. & Ferrara N.. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation, Nat. Med, 5, 1999, 623–628.[Medline]
Gillet C., Eeckhout Y. & Vaes G.. Purification of procollagenase and collagenase by affinity chromatography on Sepharose-collagen, FEBS Lett., 74, 1977, 126–128.[Medline]
Graber H.U., Muller C.F., Vandevelde M. & Zurbriggen A.. Restricted infection with canine distemper virus leads to downregulation of myelin gene transcription in cultured oligodendrocytes, Acta Neuropathol., 90, 1995, 312–318.[Medline]
Gravallese E.M., Harada Y., Wang J.T., Gorn A.H., Thornhill T.S. & Goldring S.R.. Identification of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis, Am. J. Pathol., 152, 1998, 943–951.[Abstract]
Grigoriadis A.E., Wang Z.-Q., Cecchini M.G., Hofstetter W., Felix R., Fleisch H.A. & Wagner E.F.. c-Fosa key regulator of osteoclast-macrophage lineage determination and bone remodeling, Science., 266, 1994, 443–448.
Hauser P. & Vaes G.. Degradation of cartilage proteoglycans by a neutral proteinase secreted by rabbit bone marrow macrophages in culture, Biochem. J., 172, 1978, 275–284.[Medline]
Hill P.A., Murphy G., Docherty A.J., Hembry R.M., Millican T.A., Reynolds J.J. & Meikle M.C.. The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts, J. Cell Sci, 107, 1994, 3055–3064.[Abstract]
Hofbauer L.C., Khosla S., Dunstan C.R., Lacey D.L., Boyle W.J. & Riggs B.L.. The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption, J. Bone Miner. Res, 15, 2000, 2–12.[Medline]
Hofstetter W., Wetterwald A., Cecchini M.G., Mueller C. & Felix R.. Detection of transcripts and binding sites for colony-stimulating factor-1 during bone development, Bone., 17, 1995, 145–151.[Medline]
Holmbeck K., Bianco P., Caterina J., Yamada S., Kromer M., Kuznetsov S.A., Mankani M., Robey P.G., Poole A.R., Pidoux I., Ward J.M. & Birkedal-Hansen H.. MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover, Cell., 99, 1999, 81–92.[Medline]
Jemtland R., Lee K. & Segre G.V.. Heterogeneity among cells that express osteoclast associated genes in developing bone, Endocrinology., 139, 1998, 340–349.
Jimi E., Nakamura I., Duong L.T., Ikebe T., Takahashi N., Rodan G.A. & Suda T.. Interleukin 1 induces multinucleation and bone-resorbing activity of osteoclasts in the absence of osteoblasts/stromal cells, Exp. Cell Res., 247, 1999, 84–93.[Medline]
Knight G.. Active site titration of peptidases, Methods Enzymol, 248, 1995, 85–101.[Medline]
Kobayashi K., Takahashi N., Jimi E., Udagawa N., Takami M., Kotake S., Nakagawa N., Kinosaki M., Yamaguchi K. & Shima N.. Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL–RANK interaction, J. Exp. Med, 11991, 2000, 275–286.[Medline]
Kong Y.Y., Yoshida H., Sarosi I., Tan H.L., Timms E., Capparelli C., Morony S., Oliveira-dos-Santos A.J., Van G. & Itie A.. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph node organogenesis, Nature., 397, 1999, 315–323.[Medline]
Lacey D.L., Timms E., Tan H.L., Kelley M.J., Dunstan C.R., Burgess T., Elliott R., Colombero A., Elliott G. & Scully S.. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation, Cell., 93, 1998, 165–176.[Medline]
Lee E.R., Murphy G., El-Alfi M., Davoli M.A., Lamplugh L., Docherty A.J. & Leblond C.P.. Active gelatinase B is identified by histozymography in the cartilage resorption sites of developing long bones, Dev. Dyn., 215, 1999, 190–205.[Medline]
Lefebvre V., Peters-Joris C. & Vaes G.. Production of gelatin-degrading matrix metalloproteinase (type IV collagenases) and inhibitors by articular chondrocytes during their differentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor alpha, Biochim. Biophys. Acta., 1094, 1991, 8–18.[Medline]
Mangham D.C., Scoones D.J. & Drayson M.T.. Complement and the recruitment of mononuclear osteoclasts, J. Clin. Pathol., 46, 1993, 517–521.
Nakamura H., Kenmotsu S., Sakai H. & Ozawa H.. Localization of CD44, the hyaluronate receptor, on the plasma membrane of osteocytes and osteoclasts in rat tibiae, Cell. Tissue Res., 280, 1995, 225–233.[Medline]
Nakamura I., Takahashi N., Sasaki T., Jimi E., Kurokawa T. & Suda T.. Chemical and physical properties of the extracellular matrix are required for the actin ring formation in osteoclasts, J. Bone Miner. Res., 11, 1996, 1873–1879.[Medline]
Nielsen B.S., Sehested M., Kjeldsen L., Borregaard N., Rygaard J. & Danø K.. Expression of matrix-metalloproteinase-9 in vascular pericytes in human breast cancer, Lab. Invest., 77, 1997, 345–355.[Medline]
Niida B.S., Kaku M., Amano H., Yoshida H., Kataoka H., Nishikawa S., Tanne K., Maeda N., Nishikawa S.I. & Kodama H.. Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption, J. Exp. Med., 190, 1999, 293–298.
Nordahl J., Andersson G. & Reinholt F.P.. Chondroclasts and osteoclasts in bones of young ratscomparison of ultrastructural and functional features, Calcif. Tissue Int., 63, 1998, 401–408.[Medline]
Okada Y., Naka K., Kawamura K., Matsumoto T., Nakanishi I., Fujimoto N., Sato H. & Seiki M.. Localization of matrix metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase = gelatinase B) in osteoclastsimplications for bone resorption, Lab. Invest., 72, 1995, 311–322.[Medline]
Raynal C., Delmas P.D. & Chenu C.. Bone sialoprotein stimulates in vitro bone resorption, Endocrinology., 137, 1996, 2347–2354.[Abstract]
Reponen P., Sahlberg C., Munaut C., Thesleff I. & Tryggvason K.. High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development, J. Cell Biol., 124, 1994, 1091–1102.
Saftig P., Unziker E., Wehmeyer O., Jones S., Boyde A., Rommerskirch W., Moritz J.D., Schu P. & Von Figura K.. Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin K deficient mice, Proc. Natl. Acad. Sci. USA., 95, 1998, 13453–13458.
Sasaki T., Gohring W., Mann K., Maurer P., Hohenester E., Knauper V., Murphy G. & Timpl R.. Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens, J. Biol. Chem., 272, 1997, 9237–9243.
Sato T., Ovejero M.C., Hou P., Heegaard A.M., Kumegawa M., Foged N.T. & Delaissé J.-M.. Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts, J. Cell Sci., 110, 1997, 589–596.[Abstract]
Sato T., Foged N.T. & Delaissé J.-M.. The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors, J. Bone Miner. Res., 13, 1998, 59–66.[Medline]
Schenk R.K., Spiro D. & Wiener J.. Cartilage resorption in the tibial epiphyseal plate of growing rats, J. Cell Biol., 34, 1967, 275–291.
Scheven B.A., Kawilarang-De Haas E.W., Wassenaar A.M. & Nijweide P.J.. Differentiation kinetics of osteoclasts in the periosteum of embryonic bones in vivo and in vitro, Anat. Rec., 214, 1986, 418–423.[Medline]
Senger D.R., Perruzzi C.A., Papadopoulos-Sergiou A. & Van de Water L.. Adhesive properties of osteopontinregulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain, Mol. Biol. Cell., 5, 1994, 565–574.[Abstract]
Simonet W.S., Lacey D.L., Dunstan C.R., Kelley M., Chang M.S., Luthy R., Nguyen H.Q., Wooden S., Bennett L. & Boone T.. Osteoprotegerina novel secreted protein involved in the regulation of bone density, Cell., 89, 1997, 309–319.[Medline]
Sommer B., Bickel M., Hofstetter W. & Wetterwald A.. Expression of matrix proteins during the development of mineralized tissues, Bone., 19, 1996, 371–380.[Medline]
Terai K., Takano-Yamamoto T., Ohba Y., Hiura K., Sugimoto M., Sato M., Kawahata H., Inaguma N., Kitamura Y. & Nomura S.. Role of osteopontin in bone remodeling caused by mechanical stress, J. Bone Miner. Res., 14, 1999, 839–849.[Medline]
Tezuka K., Nemoto K., Tezuka Y., Sato T., Ikeda Y., Kobori M., Kawashima H., Eguchi H., Hakeda Y. & Kumegawa M.. Identification of matrix metalloproteinase 9 in rabbit osteoclasts, J. Biol. Chem, 269, 1994, 15006–15009.
Van De Wijngaert F.P., Schipper C.A., Tas M.C. & Burger E.H.. Role of mineralizing cartilage in osteoclast and osteoblast recruitment, Bone., 9, 1988, 81–88.[Medline]
Vu T. & Werb Z.. Gelatinase Bstructure, regulation and function, Parks W.C. & Mecham R.P., Matrix Metalloproteinases, 1998, 115–148, Academic Press, San Diego..
Vu T., Shipley H.J.M., Bergers G., Berger J.E., Helms J.A., Hanahan D., Shapiro S.D., Senior R.M. & Werb Z.. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes, Cell., 93, 1998, 411–422.[Medline]
Wiktor-Jedrzejczak W., Bartocci A., Ferrante A.W. Jr., Ahmed-Ansari A., Sell K.W., Pollard J.W. & Stanley E.R.. Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse, Proc. Natl. Acad. Sci. USA., 87, 1990, 4828–4832.
Xuan J.W., Hota C. & Chambers A.F.. Recombinant GST-human osteopontin fusion protein is functional in RGD-dependent cell adhesion, J. Cell. Biochem., 54, 1994, 247–255.[Medline]
Yoshida H., Hayashi S., Kunisada T., Ogawa M., Nishikawa S., Okamura H., Sudo T., Shultz L.D. & Nishikawa S.. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene, Nature., 345, 1990, 442–444.[Medline]
Yu Q. & Stamenkovic I.. Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated tumor invasion, Genes Dev., 13, 1999, 35–48.
Yu Q. & Stamenkovic I.. Cell surface-localized matrix metalloproteinase-9 prototypically activates TGF-beta and promotes tumor invasion and angiogenesis, Genes Dev., 14, 2000, 163–176.
Zheng M.H., Fan Y., Wysocki S.J., Lau A.T., Robertson T., Beilharz M., Wood D.J. & Papadimitriou J.M.. Gene expression of transforming growth factor-beta 1 and its type II receptor in giant cell tumors of bone. Possible involvement in osteoclast-like cell migration, Am. J. Pathol., 145, 1994, 101995–102104.
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