Authentic Matrix Vesicles Contain Active Metalloproteases (MMP)

A ROLE FOR MATRIX VESICLE-ASSOCIATED MMP-13 IN ACTIVATION OF TRANSFORMING GROWTH FACTOR-beta *

Marina D'AngeloDagger, Paul C. Billings§, Maurizio Pacifici§, Phoebe S. Leboy, and Thorsten Kirsch§

From the Departments of Biochemistry and § Anatomy/Histology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 17033

Received for publication, October 24, 2000, and in revised form, December 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Matrix vesicles (MV) play a key role in the initiation of cartilage mineralization. Although many components in these microstructures have been identified, the specific function of each component is still poorly understood. In this study, we show that metalloproteases (MMP), MMP-2, -9, and -13 are associated with MV isolated from growth plate cartilage. In addition, we provide evidence that MV contain transforming growth factor-beta (TGF-beta ) and that MV-associated MMP-13 is capable of activating latent TGF-beta . To determine whether MMPs are associated directly with MV, vesicles isolated from growth plate cartilage were sequentially treated with hyaluronidase, NaCl, and bacterial collagenase to remove matrix proteins and other components attached to their outer surface. Finally, the vesicles were incubated with detergent to rupture the MV membrane and expose components that are inside the vesicles. Each treated MV fraction was subjected to substrate zymography, immunoblotting, and substrate activity assay. Whereas active MMP-13 was lost after combined treatment with hyaluronidase and NaCl, MMP-2 and -9 activities were still retained in the pellet fraction even after detergent treatment, suggesting that the gelatinases, MMP-2 and -9, are integral components of MV. In addition, MV contain TGF-beta in the small latent complex, and MMP-13 associated with the MV surface was responsible for activation of TGF-beta . Since the amount of TGF-beta activated by hypertrophic chondrocytes increased with mineral appearance in serum-free chondrocyte cultures, a role for active MV-associated MMPs is suggested in activation of TGF-beta seen during late chondrocyte hypertrophy and mineralization of growth plate cartilage.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Matrix vesicles (MV)1 are membrane-enclosed microstructures released from the plasma membrane of hypertrophic growth plate chondrocytes (1-3). These extracellular microstructures have the critical role of initiating the mineralization process in growth plate cartilage (1-3). They contain various proteins, including annexins II, V, and VI, and alkaline phosphatase (4-6) The annexins are Ca2+ channel-forming proteins, which enable Ca2+ influx into MV and the formation of the first crystal inside the vesicles (7). Alkaline phosphatase is an enzyme that generates inorganic phosphate from organic phosphate compounds (3). In addition, extracellular matrix proteins, such as aggrecan, link protein, and types II and X collagen, are associated with the outer surface of MV (5, 8, 9).

During mineralization of the growth plate, the first crystal phase forms and grows inside the vesicle lumen. Once these intralumenal crystals have reached a certain size, they rupture the membrane and grow out into the extracellular matrix (3). The process by which this occurs has yet to be defined, but it is plausible to assume that MMPs are likely involved in the degradation and remodeling of the extracellular matrix for subsequent mineralization. This hypothesis is supported by a previous report suggesting the presence of MMPs in MV isolated from chondrocyte cultures (10). Furthermore, we have reported previously an increased production of active TGF-beta by hypertrophic chondrocytes and the involvement of MMP-13 in this process (11, 12). In this report we isolated authentic MV from chick growth plate cartilage and analyzed them for the presence of MMPs and their location within the vesicles. Furthermore, we tested the role of MV, especially MV-associated MMPs, in activation of TGF-beta in hypertrophic growth plate cartilage.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chondrocyte Isolation and Serum-free Cell Culture-- Prehypertrophic chondrocytes were isolated from day 17 avian upper sterna utilizing microsurgical techniques as described previously (11). Tissue fragments were incubated in 0.25% trypsin and 0.1% crude collagenase mixture in Hanks' buffered saline solution for 3-4 h at 37 °C. Resulting cell suspensions were filtered through a Nitex filter, counted, and resuspended at a density of 5 × 106 cells/ml. Agarose cultures were prepared as described earlier in 2 ml of serum-free Dulbecco's modified Eagle's medium, high glucose (Life Technologies, Inc.), containing 50 µg/ml penicillin/streptomycin, 2 mM L-glutamine, 1 mM cysteine, and 1 mM sodium pyruvate (complete medium) (11). For alginate bead cultures, cells were resuspended in 0.15 mM NaCl, pH 7.2, and alginate (Keltone LVCRTM, Kelco, NJ) (13-15). Bead cultures were prepared by extruding the alginate/chondrocyte suspension dropwise through an 18-gauge needle into 102 mM CaCl2 resulting in ~1 × 105 chondrocytes per bead. Beads were rinsed with 0.15 M NaCl, placed into 35-mm Petri dishes, and covered with 2 ml of complete medium. Conditioned medium was collected and stored at -70 °C until assayed for TGF-beta .

Isolation and Treatment of MV-- MV were isolated from juvenile avian tibiotarsal growth plate cartilage by mild trypsin and collagenase digestion and purified by ultracentrifugation as described previously (5). To remove matrix proteins and other components attached to the outer surface, MV were sequentially treated with 0.25 units of hyaluronidase (Type IV-S, Sigma), 1 M NaCl and 5 units of pure bacterial collagenase from Clostridium histolyticum (Form III, Advanced Biofacture, Lynbrook, NJ) per 150 µg of total MV proteins. To disrupt the vesicle membrane, vesicles were treated with phosphate-buffered saline containing 0.1% CHAPS (Sigma). After each treatment, MV were pelleted by ultracentrifugation, and aliquots were removed for analysis.

Determination of Alkaline Phosphatase Activity-- Alkaline phosphatase activity was measured in each fraction utilizing p-nitrophenyl phosphate as substrate (Sigma 104, Sigma) as described previously (5).

Determination of TGF-beta Present in Conditioned Medium from Chondrocytes Cultures-- Active TGF-beta present in conditioned medium from chondrocytes cultures was determined as stimulation of a TGF-beta -responsive plasminogen activator inhibitor-1 promoter construct linked to a luciferase reporter, and values represent luciferase reporter gene activity. This promoter construct was stably transfected in mink lung epithelial cells (PAI/L cells) (16). Amounts of luciferase activity were determined using Promega Luciferase Assay System (Promega, Madison, WI) with the Optocomp I luminometer (GEM Instruments, Pineville, NC). To determine the concentration of total TGF-beta (latent and active) present in conditioned medium, luciferase reporter gene activity was measured before and after heat inactivation of TGF-beta present in conditioned medium at 85 °C for 12 min (17). To calculate the concentration of latent and active TGF-beta present in conditioned medium, standard curves were generated by measuring luciferase reporter gene activity after treating cells with defined concentrations of commercially available active TGF-beta 2 (R & D Systems, Minneapolis, MN).

Electron Microscopy-- For transmission electron microscopy (TEM), 150 µg of each MV fraction was pelleted, fixed in Karnovsky fixative, embedded in plastic, sectioned, and viewed and photographed with a JEM100CXII transmission electron microscope as described previously (6).

Substrate Gel Zymography and Immunoblot Analysis-- Total protein concentration for each MV fraction was determined by the BCA assay (Pierce). 100 µg of each MV fraction were run on 8% SDS-polyacrylamide gels containing 0.1% gelatin as substrate. After washing with 1% Triton X-100 to remove SDS, the gel was incubated at 37 °C for 1 h in 100 mM Tris, 1 mM CaCl2, pH 8.0. Coomassie staining resulted in areas of clear bands corresponding to the molecular weight sizes of proteases present in the sample. For immunoblot analysis, 100 µg of each MV fraction were separated on 8-16 or 4-20% Tris glycine, SDS-polyacrylamide gradient gels (NOVEX, San Diego, CA). After electrotransfer to nitrocellulose membranes, the membranes were incubated with primary antibodies followed by secondary peroxidase-conjugated antibodies. Specific reactive bands were viewed with the Renaissance Chemiluminescence kit (PerkinElmer Life Sciences) and Kodak X-Omat Blue film. Polyclonal antibodies specific for avian MMP-2 were a kind gift of Dr. James Quigley; polyclonal antibodies specific for avian MMP-13, annexins II, V, and VI were produced as described previously (18, 19); polyclonal antibodies specific for TGF-beta 2 were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Fluorescence-labeled Gelatin Substrate Assay-- MV fractions after hyaluronidase treatment (200 µg of total protein) were mixed with a fluorescence-labeled gelatin substrate (Molecular Probes, Eugene, OR). Samples with or without inhibitors were incubated for 2 h at 37 °C, and the fluorescence was measured at an excitation wavelength of 495 nm and an emission wavelength of 515 nm in a Photon Technology International fluorimeter (South Brunswick, NJ). Inhibitors used include CMT-8, at inhibitor concentrations specific for MMP-13 (this inhibitor was kindly provided by Dr. Brad Zerler, CollaGenex Pharmaceuticals, Newtown, PA)); doxycycline (Sigma), a general inhibitor of collagenases and gelatinases; and 1,10 phenanthroline (Molecular Probes, Eugene, OR), a general inhibitor of MMP activities.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hypertrophic Chondrocytes Increased the Production of Active TGF-beta Prior to Mineralization-- Previously, we demonstrated that hypertrophic chondrocytes maintained in serum-free agarose cultures produced ~20% active TGF-beta after 7 days in culture (11). Increased production of active TGF-beta was unexpected since most cell types studied to date produce TGF-beta in a latent form (17, 20, 21). This result suggests that an increase in active and total TGF-beta produced by hypertrophic chondrocytes may be indicative of a role for TGF-beta in chondrocyte maturation. To test this hypothesis, pre-hypertrophic chondrocytes were grown in long term serum-free agarose culture, and conditioned medium was isolated at various time points and assayed for TGF-beta production using the PAI/L assay. Since only the active form of TGF-beta stimulates PAI-1 promoter activity, we first incubated conditioned medium with PAI/L cells to determine the amount of active TGF-beta present in conditioned medium. To determine the total amount of TGF-beta (latent and active), conditioned medium was first incubated at 85 °C for 12 min to heat-activate latent TGF-beta and then incubated with PAI/L cells. A standard curve was generated by incubating PAI/L cells with various defined concentrations of active TGF-beta (16, 17). After 42 days, mineralization in these cultures was evident; bright field microscopy revealed dark areas in these cultures indicative for mineral deposits (Fig. 1A). When these cultures were maintained for an extended period (up to 82 days), the amount of total TGF-beta produced steadily increased and reached a maximum between days 42 and 56 in culture (Fig. 1B). Notably, the proportion of TGF-beta produced in an active form steadily increased to reach 100% of the total produced TGF-beta by chondrocytes after 49 days in culture. This increased production of active TGF-beta also coincides with the induction of mineralization seen in these cultures at day 42 (Fig. 1A). Mineralization was accompanied by greater than 2-fold increase in total TGF-beta produced by the chondrocytes, which peaked at day 56 and slowly returned to basal levels (~ 1.5 ng TGF-beta /5 × 106 cells/48 h) at 82 days in culture (Fig. 1B). Interestingly, at the time of onset of mineralization, ~100% of the total TGF-beta was produced in an active form (Fig. 1B). The percentage of active TGF-beta in these cultures remained high even when the total TGF-beta levels returned to basal levels (Fig. 1B, day 82). These data indicate that the maturation process of prehypertrophic chondrocytes to hypertrophic mineralizing chondrocytes is accompanied by increased production of active TGF-beta (11).



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Fig. 1.   Increase of active TGF-beta during mineralization of hypertrophic growth plate chondrocytes. Prehypertrophic chondrocytes were cultured at high density under serum-free agarose conditions as described under "Experimental Procedures." Conditioned medium from these cultures was collected every 48 h through an 82-day culture period. A, phase contrast micrograph showing the presence of dark areas indicative of mineral deposits (arrows) in chondrocyte cultures grown under serum-free agarose conditions for 56 days (magnification × 200). B, the concentration of active and latent TGF-beta present in conditioned medium of these cultures after various days was determined by measuring luciferase reporter gene activity after treating PAI/L cells with non-heat-activated and heat-activated conditioned medium and measuring luciferase reporter gene activity as described under "Experimental Procedures." Standard curves were generated by measuring luciferase reporter gene activity after treating PAI/L cells with defined concentrations of commercially available active TGF-beta . Data are the average of four different experiments.

Authentic MV Contain MMP-2, -9, and -13-- A previous study has provided evidence that MV isolated from chondrocyte cultures contain MMPs (10). An additional study suggested that MV might be involved in TGF-beta activation (22). Thus, it is possible that the persistent increase in active TGF-beta , which accompanies the initiation of mineralization, may be due to the involvement of MV-associated proteases. To test this hypothesis, we determined which MMPs are associated with authentic MV isolated from hypertrophic growth plate cartilage.

Matrix vesicles were isolated from juvenile chicken growth plate cartilage by mild trypsin and collagenase digestion and purified by ultracentrifugation as described previously (5). TEM analysis revealed that MV were round to oval in size with diameters between 100 and 300 nm (Fig. 2A, untreated MV). Immunoblot analysis of the MV fractions confirmed the presence of annexins II, V, and VI (Fig. 2B, untreated MV) and high levels of alkaline phosphatase activity (Fig. 2C, untreated MV). In addition, previous studies have provided evidence that MV are associated with extracellular matrix proteins, including proteoglycans, types II and X collagen (5, 8, 9). These extracellular matrix components can be selectively removed from the vesicle surface without destroying vesicle integrity. Hyaluronidase treatment was shown to remove surface-attached proteoglycans, whereas treatment with 1 M NaCl and pure bacterial collagenase removes surface-attached types II and X collagen (5, 8, 9).



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Fig. 2.   Characterization of authentic MV after various treatments. MV isolated from juvenile avian growth plate cartilage were sequentially treated with hyaluronidase, 1 M NaCl, and bacterial collagenase to remove attached extracellular matrix components. Finally, MV were treated with CHAPS to disrupt the MV membrane and to expose intravesicular components. A, each MV fraction was pelleted and fixed in Karnovsky fixative for transmission electron microscopy. Electron micrographs show untreated MV, MV following sequential treatments with hyaluronidase, NaCl, and collagenase (Hyl/NaCl/Coll MV), and MV preparations following sequential treatment with hyaluronidase, NaCl, collagenase, and CHAPS (Hyl/NaCl/Coll/CHAPS MV). Magnification × 87,000. B, 100 µg of total protein of each MV fraction were run on 8-16% Tris glycine gradient gels for immunoblot analysis of annexins II, V, and VI. Untreated, untreated MV preparations; Hyl, MV after hyaluronidase treatment; Hyl/NaCl, MV after sequential treatment with hyaluronidase and NaCl; Hyl/NaCl/Coll, MV after sequential treatment with hyaluronidase, NaCl, and collagenase; Hyl/NaCl/Coll/CHAPS, MV after sequential treatment with hyaluronidase, NaCl, collagenase, and CHAPS. C, specific alkaline phosphatase activity of each MV fraction.

As shown in Fig. 2A, sequential treatment with hyaluronidase, NaCl, and collagenase left the MV structure intact. Immunoblot analysis revealed the presence of annexins II, V, and VI in the MV fractions after the various treatments (Fig. 2B). Furthermore, alkaline phosphatase activity was similar in the untreated MV fraction and MV fractions after the various treatments (Fig. 2C). However, amino acid analysis revealed a significant reduction in hydroxyproline content (0.074 nM) after sequential treatments with hyaluronidase, NaCl, and collagenase compared with untreated MV (0.1324 nM). Finally, we incubated hyaluronidase/NaCl/collagenase-treated vesicles with CHAPS to rupture the MV membrane and expose the nucleational core complex and other intralumenal components. The nucleational core complex has been shown to contain Ca2+, Pi, acidic phospholipids, and associated proteins, such as annexins (24; see also Fig. 2B), and some alkaline phosphatase activity (see Fig. 2C). TEM analysis confirmed that no intact MV were present after detergent treatment (Fig. 2A, Hyl/NaCl/Coll/CHAPS MV).

When MV fractions were subjected to gelatin zymography, bands with molecular weights consistent with MMP-2, -9, and -13 were present (Fig. 3, untreated MV). The band for MMP-13 is rather weak in the zymogram shown in Fig. 3A. However, a longer incubation of the gels in Tris/NaCl buffer was not possible because of high levels of activities of MMP-2 and -9 in the MV fractions. Thus, Fig. 3B represents the same area of the gel shown in Fig. 3A enlarged and contrast adjusted to help visualize the MMP-13 band. Whereas MMP-13 activity was present in MV fractions after hyaluronidase treatment (Fig. 3B, Hyl), MMP-13 activity was lost after combined treatment with hyaluronidase and NaCl (Fig. 3B, Hyl/NaCl). In contrast, MMP -2 and -9 activities were still retained in the pellet fraction even after sequential treatments with hyaluronidase, NaCl, collagenase, and detergent (Fig. 3A, Hyl, Hyl/NaCl, Hyl/NaCl/Coll, and Hyl/NaCl/Coll/CHAPS). The zymograms also revealed one band of activity above and one band below MMP-9 activity. By molecular weight, these bands are the same size as bacterial collagenase which was used to isolate MV from growth plate cartilage. In fact, antibodies against bacterial collagenase recognized bands of these exact molecular weights in MV isolated by our protocol.2



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Fig. 3.   Metalloproteases associated with authentic MV. 100 µg of total protein of each MV fraction were run on an 8% SDS-polyacrylamide gel containing 0.1% gelatin substrate and analyzed by zymography. Coomassie staining revealed clear areas corresponding to active protease in the sample. A, untreated, untreated MV preparations; Hyl, MV after hyaluronidase treatment; Hyl/NaCl, MV after sequential treatment with hyaluronidase and NaCl; Hyl/NaCl/Coll, MV after sequential treatment with hyaluronidase, NaCl, and collagenase; Hyl/NaCl/Coll/CHAPS, MV after sequential treatment with hyaluronidase, NaCl, collagenase, and CHAPS. B, the area of the gel represented with the bracket in A was rescanned to improve the contrast and visualization of the band representing MMP-13 activity.

To assess the proportion of active MMP-2, -9, and -13 present in association with MV, MMP activities present in MV fractions were measured in the absence and presence of various MMP inhibitors using a fluorescent gelatin substrate. In these experiments hyaluronidase-treated MV were used which were proteoglycan-depleted, but still contained MMP-2, -9, and -13 activities (see Fig. 3). CMT-8, which at a concentration of 15 µM is a specific inhibitor for MMP-13 (25), resulted in 9% inhibition of the total activity present in MV (Table I). A higher dose of CMT-8, which inhibits other collagenases and gelatinases in addition to MMP-13 (25), reduced the MMP activity by 23%. Doxycycline, an inhibitor of MMP-2, -9 and -13, inhibited 56% of the total activity, whereas 1,10-phenanthroline, a metal ion chelator and general inhibitor of MMP activity, depleted 61% of the activity (Table I). These data demonstrate that the majority of MMPs associated with MV are active.


                              
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Table I
Active MMPs associated with authentic MV isolated from growth plate cartilage
MV fractions after hyaluronidase treatment (control Hyl-treated MV; 200 µg of total protein) were incubated with a fluorescence-labeled gelatin substrate in the absence or presence of various MMP inhibitors as described under "Experimental Procedures." Fluorescence was measured at excitation/emission wavelengths of 495/515 nm. Inhibitors were added during the incubation period, and percent of inhibition was calculated as compared with control Hyl-treated MV. The data are the average of n = 2 separate MV preparations.

Immunoblot analysis with antibodies against avian MMP-13 and -2 confirmed the existence of these MMPs in MV (Fig. 4, A and B, untreated MV). We did not confirm the identity of MMP-9 by immunoblot analysis because antibodies against avian MMP-9 are not currently available. However, size, substrate specificity, and EDTA sensitivity in the zymograms clearly indicate the presence of MMP-9 in the MV preparations.



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Fig. 4.   Immunoblot analysis for MMPs associated with authentic MV. 100 µg of total protein of each MV fraction were run on a 4-20 or 8-16% Tris glycine SDS-polyacrylamide gradient gels and electrotransferred onto nitrocellulose membranes, and the membranes were immunostained with antibodies specific for MMP-13 (A) and MMP-2 (B). Untreated, untreated MV preparations; Hyl, MV after hyaluronidase treatment; Hyl/NaCl, MV after sequential treatment with hyaluronidase and NaCl; Hyl/NaCl/Coll, MV after sequential treatment with hyaluronidase, NaCl, and collagenase; Hyl/NaCl/Coll/CHAPS, MV after sequential treatment with hyaluronidase, NaCl, collagenase, and CHAPS.

Interestingly, immunoblot analysis revealed the presence of both the 58-kDa MMP-13 and smaller degradation products in MV fractions after sequential treatments with hyaluronidase, NaCl, and collagenase where no detectable activity was visible on the zymograms (compare Figs. 3A and 4A, Hyl/NaCl and Hyl/NaCl/Coll). Immunoreactivity for MMP-13 was lost in MV fractions treated with hyaluronidase, NaCl, collagenase and detergent (Fig. 4A, Hyl/NaCl/Coll/CHAPS) suggesting that MMP-13 is directly associated with MV, but its activity was lost and the protein was partially degraded after the sequential treatments with hyaluronidase, NaCl, and collagenase. This is consistent with our previous findings showing that the bulk of MMP-13 produced by hypertrophic chondrocytes is rapidly degraded to inactive smaller forms (18). In contrast, immunoblot analysis and zymography revealed the presence of MMP-2 in MV pellets after all treatments (compare Figs. 3A and 4B). In addition, as shown in Fig. 3A, MMP-9 activity is retained in the MV pellet fraction after all treatments, indicating that MMP-2 and -9 appear to be integral components of MV and that the various treatments do not affect their activities.

MV-associated MMPs Activate TGF-beta -- To determine whether MV are the site of TGF-beta activation, we first investigated whether TGF-beta is associated with MV. The total amount of TGF-beta present in the MV fractions was assayed with the PAI/L assay after incubation of MV fractions at 85 °C to heat-activate TGF-beta as described above (16, 17). Equivalent amounts of TGF-beta were present in the MV fraction before and after sequential treatments with hyaluronidase, NaCl, and collagenase (0.164 pg/µg MV protein versus 0.183 pg/µg MV protein), whereas following detergent treatment the levels decreased to 0.05 pg/µg MV protein (Fig. 5A). Immunoblot analysis of MV fractions after sequential treatments with hyaluronidase, NaCl, and collagenase revealed the presence of active TGF-beta 25-kDa homodimer and the small latent N-terminal peptide of TGF-beta (Fig. 5B, lane 1). After detergent treatment the immunoreactive bands for TGF-beta were lost (Fig. 5B, lane 2). These findings suggest that the small latent TGF-beta complex is an integral component of MV.



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Fig. 5.   Presence of TGF-beta in authentic MV. A, the amount of total TGF-beta present in MV was determined by a modified mink lung epithelial cell line stably transfected with a plasminogen activator inhibitor promoter construct driving a luciferase reporter (PAI/L). MV fractions were incubated at 85 °C for 12 min to heat-activate any TGF-beta present, and the concentration of total TGF-beta was calculated from a standard curve derived from various defined concentrations of active human recombinant TGF-beta 2 as described under "Experimental Procedures." The concentration of total TGF-beta is reported in pg per µg of total MV protein. B, 100 µg of total protein of each MV fraction were run on 4-20% SDS-polyacrylamide gradient gels. After electrotransfer to nitrocellulose membranes, the membranes were immunostained with primary antibodies against TGF-beta 2. Lane 1, MV after sequential treatments with hyaluronidase, NaCl, and collagenase; lane 2, MV after sequential treatments with hyaluronidase, NaCl, collagenase, and CHAPS. LAP,= latency-associated peptide of the small latent TGF-beta complex.

The preferred storage form of TGF-beta in the growth plate is as a latent complex (20). In addition, in preliminary studies we showed that MMP-13, but not MMP-2 or -9, is capable of activating TGF-beta from this complex (12). The ability of MV-associated MMPs to activate latent TGF-beta was analyzed by assaying the formation of active TGF-beta from the latent complex present in conditioned medium from serum-free chondrocyte alginate bead cultures in the presence of MV fractions. Conditioned medium was mixed with MV fractions (150 µg of total protein), incubated overnight at 37 °C, and the amount of active TGF-beta determined by the PAI/L method. As shown in Fig. 6, addition of MV to conditioned medium caused a 1.8-fold increase in the amount of active TGF-beta compared with conditioned medium incubated without MV. However, addition of hyaluronidase/NaCl-treated MV to conditioned medium did not lead to a significant increase in the levels of active TGF-beta . We have demonstrated above that hyaluronidase/NaCl-treated MV showed MMP-2 and -9 activity but no MMP-13 activity. Thus, it is plausible that because of the loss of MMP-13 activity, hyaluronidase/NaCl-treated MV are not able to activate latent TGF-beta . To explore this possibility further, we determined whether CMT-8 inhibits MV-mediated activation of latent TGF-beta . As shown in Fig. 7, the addition of CMT-8 in concentrations of 5 and 15 µM (specific for inhibition of MMP-13) resulted in a significant inhibition of MV-mediated activation of TGF-beta .



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Fig. 6.   TGF-beta activation by authentic MV. 150 µg of total protein of untreated MV, hyaluronidase, 1 M NaCl-treated MV (Hyl/NaCl MV), and hyaluronidase, 1 M NaCl/bacterial collagenase/CHAPS-treated MV (Hyl/NaCl/Coll/CHAPS MV) were incubated at 37 °C with conditioned medium (Cond.Med., CM) from chondrocyte cultures containing latent TGF-beta . The amount of active TGF-beta after 12 h of incubation was measured by the PAI/L assay. Data are the average of three experiments with three different MV preparations; values are means ± S.D.



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Fig. 7.   MV-associated MMP-13 activates chondrocyte-produced latent TGF-beta . MV (150 µg of total protein) were incubated at 37 °C overnight with conditioned medium from chondrocyte cultures containing latent TGF-beta in the presence of various concentrations of CMT-8, an inhibitor of MMP-13. The amount of active TGF-beta present after 12 h of incubation was determined by the PAI/L assay. Data are the average of four experiments with two different MV preparations; values are means ± S.D.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrate that authentic MV isolated from chicken growth plate cartilage contain active collagenase, MMP-13, and active gelatinases, MMP-2 and -9. Furthermore, authentic MV not only contain latent and active TGF-beta , but these particles are able to activate latent TGF-beta present in conditioned medium from chondrocyte cultures. TGF-beta activation by authentic MV was inhibited by CMT-8, at concentrations specific for MMP-13 inhibition, suggesting that MV-associated MMP-13 is involved in activating TGF-beta in hypertrophic growth plate cartilage.

Although Dean et al. (26) have shown that MV isolated from chondrocyte cultures contain MMP-2, -9, and -3, the location of these MMPs within the vesicles was not discerned. Vesicles in their study were isolated from avian chondrocytes passaged several times and cultured in monolayer (26), and thus it is not clear whether these MMPs are integral components of MV or whether these MMPs are associated with extracellular matrix proteins that are present in the vesicle preparations. In this study, we isolated authentic MV from in vivo growth plate cartilage and characterized the major MMP components and their location within these MV fractions. Location of these MMPs was determined using sequential treatments with hyaluronidase, NaCl, and pure bacterial collagenase. These treatments were shown to selectively remove extracellular matrix proteins from the vesicles (5). MMP-2 and -9 activity was retained in the pellet fraction even after detergent treatment revealing that these MMPs are integral components of MV. MMP-13 activity was lost after hyaluronidase and NaCl treatment. However, immunoblot analysis revealed the presence of degraded products of MMP-13 in the pellet fraction after sequential treatment with hyaluronidase, NaCl, and collagenase, suggesting that MMP-13 is also an integral component of MV. The protein probably got degraded during the sequential treatments, which is consistent with previous findings from our laboratory and other laboratories showing that MMP-13 is very sensitive to in vitro manipulation and is rapidly degraded to inactive smaller forms (18, 27). Thus, our data clearly indicate that MMP-2, -9, and -13 are the major MMPs present in MV and that they are integral components of the vesicles.

MV have been shown to have the critical role of initiating the mineralization process in growth plate cartilage (3). The first crystal phase forms and grows inside the vesicle lumen in a protected environment before the crystals rupture the MV membrane and grow out into the extracellular matrix (3). Extensive matrix remodeling is required for the outgrowth of the crystals from the vesicles into the extracellular matrix. MMP-13 is an interstitial collagenase with high affinity for type II collagen and was shown to cleave type II and X collagen (27). MV-associated MMP-2 and -9 can then further degrade the resulting collagen fragments. The combined action of these MV-associated collagenase and gelatinases might allow rapid matrix remodeling and the release of matrix-bound Ca2+, thereby enabling rapid mineralization of the extracellular matrix.

We have shown previously that MMP-13 mRNA is already produced by early hypertrophic chondrocytes, but active MMP-13 protein is only found in late stage hypertrophic chondrocytes (18). MMP-2 is involved in a cascade resulting in the activation of pro-MMP-13 zymogen (28, 29). Thus, it is possible that MV after being released from the plasma membrane of hypertrophic chondrocytes contain MMP-2 and pro-MMP-13 zymogen. MV-associated MMP-2 might then be involved in activating the MV-associated pro-form of MMP-13. Thus, the sensitive and easily degradable active form of MMP-13 would be only activated at the site where its activity is required.

Boyan et al. (22) have reported that MV isolated from chondrocyte cultures were capable of activating latent TGF-beta . In this study, we confirm and expand their findings and demonstrate that authentic MV isolated from growth plate cartilage are capable of activating latent TGF-beta produced by hypertrophic chondrocytes. Furthermore, our data reveal that active TGF-beta 2 and latency-associated peptide of the small latent TGF-beta 2 complex are present in authentic MV fractions even after sequential treatment with hyaluronidase, NaCl, and collagenase, suggesting that small latent TGF-beta may be directly associated with the outer surface of authentic MV and that the complex may be in close association with MV-associated MMP-2 and -13. In addition, we provide evidence that CMT-8 at concentrations, which specifically inhibit MMP-13 activity, significantly suppressed MV-mediated activation of TGF-beta , suggesting that MMP-13 plays a major role in activating TGF-beta from its small latent complex. Thus, MV might not only be the site for initiation of mineralization, but they might also be the site for activation of TGF-beta in hypertrophic mineralizing growth plate cartilage. Active TGF-beta has been shown to be an important factor that promotes early stages of osteoblast differentiation (30-32). Thus, MV-associated MMP-2 is involved in activating MV-associated MMP-13. The active form of MMP-13 releases active TGF-beta from the MV-associated small latent form of TGF-beta . The active TGF-beta may then facilitate the differentiation of cartilage-invading preosteoblastic cells to osteoblastic cells, leading to the replacement of cartilage by bone.

In conclusion, this study reveals the presence of active MMP-2, -9, -13, and TGF-beta in authentic MV isolated from hypertrophic growth plate cartilage and suggests potential roles for these MMPs in matrix remodeling and TGF-beta activation during mineralization and bone formation. Thus, MV released from terminal differentiated chondrocytes contain essential components required for initiation of mineralization, matrix remodeling, and subsequent bone formation. For example, they contain the Ca2+ channel-forming annexins II, V, and VI, essential components for formation of the first intralumenal crystal phase; MMPs, required for matrix remodeling; and latent TGF-beta which after being activated by MV-associated MMPs may be required for osteoblastic differentiation and the replacement of cartilage by bone. The exact roles of these and other MV components and their potential interactions, however, still have to be elucidated.


    ACKNOWLEDGEMENTS

We thank Gerald Harrison for help with isolation of authentic MV, Sylvia Decker for TEM expertise, and Dr. Brad Zerler (Collagenex) for providing us with CMT-8.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants AR 43732, AR 46245 (to T. K.), DE 11876, DE 05676 (to M. D'A.), and AR 40075 (to P. S. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: Anatomy and Cell Biology, Temple University Medical School, 3400 North Broad St., Philadelphia, PA 19140.

To whom correspondence should be addressed: Dept. of Orthopaedics and Rehabilitation, H089, Penn State College of Medicine, Hershey Medical Center, 500 University Drive, Hershey, PA 17033. Tel.: 717-531-7788; Fax.: 717-531-1607; E-mail: tkirsch@psu.edu.

Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M009725200

2 G. Harrison, personal communication.


    ABBREVIATIONS

The abbreviations used are: MV, matrix vesicles; MMP, metalloproteases; TGF-beta , transforming growth factor-beta ; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TEM, transmission electron microscopy.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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