Authentic Matrix Vesicles Contain Active Metalloproteases (MMP)

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-β (TGF-β) and that MV-associated MMP-13 is capable of activating latent TGF-β. 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-β in the small latent complex, and MMP-13 associated with the MV surface was responsible for activation of TGF-β. Since the amount of TGF-β 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-β seen during late chondrocyte hypertrophy and mineralization of growth plate cartilage. 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-β (TGF-β) and that MV-associated MMP-13 is capable of activating latent TGF-β. 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-β in the small latent complex, and MMP-13 associated with the MV surface was responsible for activation of TGF-β. Since the amount of TGF-β 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-β seen during late chondrocyte hypertrophy and mineralization of growth plate cartilage. matrix vesicles metalloproteases transforming growth factor-β 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid transmission electron microscopy Matrix vesicles (MV)1are membrane-enclosed microstructures released from the plasma membrane of hypertrophic growth plate chondrocytes (1Hale J.E. Wuthier R.E. J. Biol. Chem. 1987; 262: 1916-1925Abstract Full Text PDF PubMed Google Scholar, 2Anderson H.C. J. Cell Biol. 1969; 41: 59-72Crossref PubMed Scopus (714) Google Scholar, 3Anderson H.C. Clin. Orthop. Relat. Res. 1995; : 266-280PubMed Google Scholar). These extracellular microstructures have the critical role of initiating the mineralization process in growth plate cartilage (1Hale J.E. Wuthier R.E. J. Biol. Chem. 1987; 262: 1916-1925Abstract Full Text PDF PubMed Google Scholar, 2Anderson H.C. J. Cell Biol. 1969; 41: 59-72Crossref PubMed Scopus (714) Google Scholar, 3Anderson H.C. Clin. Orthop. Relat. Res. 1995; : 266-280PubMed Google Scholar). They contain various proteins, including annexins II, V, and VI, and alkaline phosphatase (4Genge B.R. Wu L.N.Y. Wuthier R.E. J. Biol. Chem. 1989; 264: 10917-10921Abstract Full Text PDF PubMed Google Scholar, 5Kirsch T. Wuthier R.E. J. Biol. Chem. 1994; 269: 11462-11469Abstract Full Text PDF PubMed Google Scholar, 6Kirsch T. Nah H.-D. Shapiro I.M. Pacifici M. J. Cell Biol. 1997; 137: 1149-1160Crossref PubMed Scopus (187) Google Scholar) The annexins are Ca2+ channel-forming proteins, which enable Ca2+ influx into MV and the formation of the first crystal inside the vesicles (7Kirsch T. Harrison G. Golub E.E. Nah H.-D. J. Biol. Chem. 2000; 275: 35577-35583Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Alkaline phosphatase is an enzyme that generates inorganic phosphate from organic phosphate compounds (3Anderson H.C. Clin. Orthop. Relat. Res. 1995; : 266-280PubMed Google Scholar). In addition, extracellular matrix proteins, such as aggrecan, link protein, and types II and X collagen, are associated with the outer surface of MV (5Kirsch T. Wuthier R.E. J. Biol. Chem. 1994; 269: 11462-11469Abstract Full Text PDF PubMed Google Scholar, 8Wu L.N.Y. Genge B.R. Wuthier R.E. J. Biol. Chem. 1991; 266: 1187-1194Abstract Full Text PDF PubMed Google Scholar, 9Wu L.N.Y. Genge B.R. Lloyd G.C. Wuthier R.E. J. Biol. Chem. 1991; 266: 1195-1203Abstract Full Text PDF PubMed Google Scholar). 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 (3Anderson H.C. Clin. Orthop. Relat. Res. 1995; : 266-280PubMed Google Scholar). 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 (10Schmitz J.P. Dean D.D. Schwartz Z. Cochran D.L. Grant G.M. Klebe R.J. Nakaya H. Boyan B.D. J. Cell. Biochem. 1996; 61: 375-391Crossref PubMed Scopus (53) Google Scholar). Furthermore, we have reported previously an increased production of active TGF-β by hypertrophic chondrocytes and the involvement of MMP-13 in this process (11D'Angelo M. Pacifici M. J. Bone Miner. Res. 1997; 12: 1368-1377Crossref PubMed Scopus (48) Google Scholar, 12D'Angelo M. Sarment D.P. Billings P.C. Pacifici M. Trans. 44th Annu. Meet. Orthop. Res. Soc. 1998; 23: 497Google Scholar). 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-β in hypertrophic growth plate cartilage. Prehypertrophic chondrocytes were isolated from day 17 avian upper sterna utilizing microsurgical techniques as described previously (11D'Angelo M. Pacifici M. J. Bone Miner. Res. 1997; 12: 1368-1377Crossref PubMed Scopus (48) Google Scholar). 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 mml-glutamine, 1 mm cysteine, and 1 mm sodium pyruvate (complete medium) (11D'Angelo M. Pacifici M. J. Bone Miner. Res. 1997; 12: 1368-1377Crossref PubMed Scopus (48) Google Scholar). For alginate bead cultures, cells were resuspended in 0.15 mm NaCl, pH 7.2, and alginate (Keltone LVCRTM, Kelco, NJ) (13Tschan T. Hoerler I. Houze Y. Winterhalter K.H. Richter C. Bruckner P. J. Cell Biol. 1990; 111: 257-260Crossref PubMed Scopus (80) Google Scholar, 14Hausselman H.J. Aydelotte M.B. Schumacher B.L. Keuttner K.E. Gitelis S.H. Thonar E.J.-M.A. Matrix. 1992; 12: 116-129Crossref PubMed Scopus (338) Google Scholar, 15Bonaventure J. Kadhom N. Cohen-Solal L. Ng K.H. Bourguignon J. Lasselin C. Freisinger P. Exp. Cell Res. 1994; 212: 97-104Crossref PubMed Scopus (448) Google Scholar). 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-β. MV were isolated from juvenile avian tibiotarsal growth plate cartilage by mild trypsin and collagenase digestion and purified by ultracentrifugation as described previously (5Kirsch T. Wuthier R.E. J. Biol. Chem. 1994; 269: 11462-11469Abstract Full Text PDF PubMed Google Scholar). 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. Alkaline phosphatase activity was measured in each fraction utilizingp-nitrophenyl phosphate as substrate (Sigma 104, Sigma) as described previously (5Kirsch T. Wuthier R.E. J. Biol. Chem. 1994; 269: 11462-11469Abstract Full Text PDF PubMed Google Scholar). Active TGF-β present in conditioned medium from chondrocytes cultures was determined as stimulation of a TGF-β-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) (16Abe M. Harpel J.C. Metz C.N. Nunes I. Loskutoff D.J. Rifkin D.B. Anal. Biochem. 1994; 216: 276-284Crossref PubMed Scopus (673) Google Scholar). 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-β (latent and active) present in conditioned medium, luciferase reporter gene activity was measured before and after heat inactivation of TGF-β present in conditioned medium at 85 °C for 12 min (17Miyazono K. Heldin C.-H. Bock G.R. Marsh J. Clinical Applications of TGF-β. Scholar). To the concentration of latent and active TGF-β present in conditioned medium, were by luciferase reporter gene activity after cells with of active For transmission electron microscopy 150 μg of each MV fraction was in in and and with a transmission electron as described previously T. Nah H.-D. Shapiro I.M. Pacifici M. J. Cell Biol. 1997; 137: 1149-1160Crossref PubMed Scopus (187) Google Scholar). concentration for each MV fraction was determined by the μg of each MV fraction were containing 0.1% as After with to remove the was incubated at 37 °C for 1 h in mm 1 pH in of to the of present in the For μg of each MV fraction were After to the were incubated with by were with the and specific for avian MMP-2 were a of specific for avian annexins II, V, and were as described previously M. Z. M. Pacifici M. Sarment Billings P.C. J. Cell. Biochem. 2000; PubMed Scopus Google Scholar, T. Harrison G. Golub E.E. J. Bone Miner. Res. 2000; PubMed Scopus Google specific for were from MV after hyaluronidase treatment μg of total were with a substrate with were incubated for 2 h at 37 and the was measured at an of and an of in a at specific for MMP-13 was by a of and and a of we that hypertrophic chondrocytes in serum-free cultures active TGF-β after in (11D'Angelo M. Pacifici M. J. Bone Miner. Res. 1997; 12: 1368-1377Crossref PubMed Scopus (48) Google Scholar). production of active TGF-β was cell types to TGF-β in a latent (17Miyazono K. Heldin C.-H. Bock G.R. Marsh J. Clinical Applications of TGF-β. Scholar, J. Cell. Biochem. 1994; PubMed Scopus Google Scholar, Harpel Nunes I. Rifkin D.B. 1997; Full Text PDF PubMed Scopus Google Scholar). This that an in active and total TGF-β by hypertrophic chondrocytes be of a role for TGF-β in chondrocyte To this chondrocytes were in serum-free and conditioned medium was isolated at various and assayed for TGF-β production using the assay. Since the active of TGF-β promoter we first incubated conditioned medium with cells to determine the amount of active TGF-β present in conditioned medium. To determine the total amount of TGF-β (latent and conditioned medium was first incubated at 85 °C for 12 min to latent TGF-β and incubated with was by cells with various of active TGF-β (16Abe M. Harpel J.C. Metz C.N. Nunes I. Loskutoff D.J. Rifkin D.B. Anal. Biochem. 1994; 216: 276-284Crossref PubMed Scopus (673) Google Scholar, K. Heldin C.-H. Bock G.R. Marsh J. Clinical Applications of TGF-β. Scholar). After mineralization in these cultures was microscopy in these cultures for mineral 1 these cultures were for an to the amount of total TGF-β increased and reached a and in the of TGF-β in an active increased to of the total TGF-β by chondrocytes after in This increased production of active TGF-β with the of mineralization seen in these cultures at day 1 was by in total TGF-β by the which at day and to × at in 1 at the of of of the total TGF-β was in an active 1 The of active TGF-β in these cultures high even the total TGF-β to 1 day These that the process of chondrocytes to hypertrophic chondrocytes is by increased production of active TGF-β (11D'Angelo M. Pacifici M. J. Bone Miner. Res. 1997; 12: 1368-1377Crossref PubMed Scopus (48) Google Scholar). previous has evidence that MV isolated from chondrocyte cultures contain MMPs (10Schmitz J.P. Dean D.D. Schwartz Z. Cochran D.L. Grant G.M. Klebe R.J. Nakaya H. Boyan B.D. J. Cell. Biochem. 1996; 61: 375-391Crossref PubMed Scopus (53) Google Scholar). suggested that MV be involved in TGF-β activation B.D. Schwartz Z. Dean D.D. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). it is that the in active which the initiation of be to the involvement of MV-associated To this we determined which MMPs are associated with authentic MV isolated from hypertrophic growth plate cartilage. Matrix vesicles were isolated from juvenile growth plate cartilage by mild trypsin and collagenase digestion and purified by ultracentrifugation as described previously (5Kirsch T. Wuthier R.E. J. Biol. Chem. 1994; 269: 11462-11469Abstract Full Text PDF PubMed Google Scholar). that MV were to in with and 2 of the MV the presence of annexins II, V, and 2 and high of alkaline phosphatase activity In addition, previous have evidence that MV are associated with extracellular matrix proteins, including types II and X (5Kirsch T. Wuthier R.E. J. Biol. Chem. 1994; 269: 11462-11469Abstract Full Text PDF PubMed Google Scholar, 8Wu L.N.Y. Genge B.R. Wuthier R.E. J. Biol. Chem. 1991; 266: 1187-1194Abstract Full Text PDF PubMed Google Scholar, 9Wu L.N.Y. Genge B.R. Lloyd G.C. Wuthier R.E. J. Biol. Chem. 1991; 266: 1195-1203Abstract Full Text PDF PubMed Google Scholar). These extracellular matrix components be removed from the vesicle surface vesicle treatment was to remove treatment with 1 m NaCl and pure bacterial collagenase types II and X (5Kirsch T. Wuthier R.E. J. Biol. Chem. 1994; 269: 11462-11469Abstract Full Text PDF PubMed Google Scholar, 8Wu L.N.Y. Genge B.R. Wuthier R.E. J. Biol. Chem. 1991; 266: 1187-1194Abstract Full Text PDF PubMed Google Scholar, 9Wu L.N.Y. Genge B.R. Lloyd G.C. Wuthier R.E. J. Biol. Chem. 1991; 266: 1195-1203Abstract Full Text PDF PubMed Google Scholar). in 2 treatment with hyaluronidase, NaCl, and collagenase the MV the presence of annexins II, V, and in the MV after the various 2 Furthermore, alkaline phosphatase activity was in the MV fraction and MV after the various acid a in after with hyaluronidase, NaCl, and collagenase with MV Finally, we incubated vesicles with CHAPS to rupture the MV membrane and expose the and other intralumenal The has been to contain and associated proteins, such as annexins 2 and alkaline phosphatase activity that MV were present after detergent treatment 2 MV were subjected to zymography, with with MMP-2, -9, and -13 were present The for MMP-13 is in the in a of the in was of high of activities of MMP-2 and -9 in the MV the of the in and to the MMP-13 Whereas MMP-13 activity was present in MV after hyaluronidase treatment MMP-13 activity was lost after combined treatment with hyaluronidase and NaCl In and -9 activities were still retained in the pellet fraction even after with hyaluronidase, NaCl, and detergent and The of activity and activity. these are the as bacterial collagenase which was to MV from growth plate cartilage. In bacterial collagenase of these in MV isolated by To the of active MMP-2, -9, and -13 present in with MV, activities present in MV were measured in the and presence of various using a In these MV were which were but still MMP-2, -9, and -13 activities which at a concentration of is a specific for MMP-13 T. N. K. Golub N. Y. PubMed Scopus Google in of the total activity present in MV of which other and in to MMP-13 T. N. K. Golub N. Y. PubMed Scopus Google the activity by an of MMP-2, -9 and of the total a and of of the activity These that the of MMPs associated with MV are MMPs associated with authentic MV isolated from growth plate vesicle 50 mm after hyaluronidase treatment μg of total were incubated with a substrate in the presence of various as described was measured at of were during the and of was as with MV. The are the of 2 MV in a MV after hyaluronidase treatment μg of total were incubated with a substrate in the presence of various as described was measured at of were during the and of was as with MV. The are the of 2 MV with avian MMP-13 and the of these MMPs in MV and the of by avian are size, substrate and in the the presence of in the MV the presence of the MMP-13 and degradation in MV after with hyaluronidase, NaCl, and collagenase activity was the and for MMP-13 was lost in MV treated with hyaluronidase, NaCl, collagenase and detergent suggesting that MMP-13 is directly associated with MV, but activity was lost and the was after the with hyaluronidase, NaCl, and This is with previous that the of MMP-13 by hypertrophic chondrocytes is to forms M. Z. M. Pacifici M. Sarment Billings P.C. J. Cell. Biochem. 2000; PubMed Scopus Google Scholar). In and the presence of MMP-2 in MV after and In addition, as in activity is retained in the MV pellet fraction after that MMP-2 and -9 to be integral components of MV and that the various their To determine whether MV are the of TGF-β we first whether TGF-β is associated with MV. The total amount of TGF-β present in the MV was assayed with the after of MV at 85 °C to TGF-β as described (16Abe M. Harpel J.C. Metz C.N. Nunes I. Loskutoff D.J. Rifkin D.B. Anal. Biochem. 1994; 216: 276-284Crossref PubMed Scopus (673) Google Scholar, K. Heldin C.-H. Bock G.R. Marsh J. Clinical Applications of TGF-β. Scholar). of TGF-β were present in the MV fraction before and after with hyaluronidase, NaCl, and collagenase MV MV detergent treatment the to MV of MV after with hyaluronidase, NaCl, and collagenase the presence of active TGF-β and the small latent of TGF-β After detergent treatment the for TGF-β were lost 5 These that the small latent TGF-β is an integral component of MV. The of TGF-β in the growth plate is as a latent J. Cell. Biochem. 1994; PubMed Scopus Google Scholar). In addition, in we that but MMP-2 -9, is capable of activating TGF-β from this M. Sarment D.P. Billings P.C. Pacifici M. Trans. 44th Annu. Meet. Orthop. Res. Soc. 1998; 23: 497Google Scholar). The of MV-associated MMPs to latent TGF-β was analyzed by the formation of active TGF-β from the latent present in conditioned medium from serum-free chondrocyte alginate bead cultures in the presence of MV Conditioned medium was with MV μg of total incubated at 37 and the amount of active TGF-β determined by the in of MV to conditioned medium a in the amount of active TGF-β with conditioned medium incubated MV. of MV to conditioned medium to a in the of active TGF-β. have that MV MMP-2 and -9 activity but MMP-13 activity. it is plausible that of the of MMP-13 MV are to latent TGF-β. To this we determined whether activation of latent TGF-β. in the of in of 5 and for of in a of activation of MMP-13 latent TGF-β. MV μg of total were incubated at 37 °C with conditioned medium from chondrocyte cultures containing latent TGF-β in the presence of various of an of The amount of active TGF-β present after 12 h of was determined by the assay. are the of with MV values are In this study, we that authentic MV isolated from growth plate cartilage contain active and active gelatinases, MMP-2 and Furthermore, authentic MV contain latent and active but these are to latent TGF-β present in conditioned medium from chondrocyte TGF-β activation by authentic MV was by at specific for MMP-13 suggesting that MV-associated MMP-13 is involved in activating TGF-β in hypertrophic growth plate cartilage. Although Dean D.D. Schwartz Z. Boyan B.D. Tissue 1992; PubMed Scopus Google Scholar) have that MV isolated from chondrocyte cultures contain MMP-2, -9, and the location of these MMPs within the vesicles was in their were isolated from avian chondrocytes and in D.D. Schwartz Z. Boyan B.D. Tissue 1992; PubMed Scopus Google and it is whether these MMPs are integral components of MV whether these MMPs are associated with extracellular matrix proteins that are present in the vesicle In this study, we isolated authentic MV from in growth plate cartilage and the components and their location within these MV of these MMPs was determined using with hyaluronidase, NaCl, and pure bacterial These were to remove extracellular matrix proteins from the vesicles (5Kirsch T. Wuthier R.E. J. Biol. Chem. 1994; 269: 11462-11469Abstract Full Text PDF PubMed Google Scholar). MMP-2 and -9 activity was retained in the pellet fraction even after detergent treatment that these MMPs are integral components of MV. MMP-13 activity was lost after hyaluronidase and NaCl the presence of of MMP-13 in the pellet fraction after treatment with hyaluronidase, NaCl, and suggesting that MMP-13 is an integral component of MV. The during the which is with previous from and other that MMP-13 is to in and is to forms M. Z. M. Pacifici M. Sarment Billings P.C. J. Cell. Biochem. 2000; PubMed Scopus Google Scholar, Matrix Google Scholar). that MMP-2, -9, and -13 are the MMPs present in MV and that they are integral components of the vesicles. MV have been to have the critical role of initiating the mineralization process in growth plate cartilage (3Anderson H.C. Clin. Orthop. Relat. Res. 1995; : 266-280PubMed Google Scholar). The first crystal phase forms and grows inside the vesicle in a before the crystals rupture the MV membrane and grow out into the extracellular matrix (3Anderson H.C. Clin. Orthop. Relat. Res. 1995; : 266-280PubMed Google Scholar). matrix remodeling is for the of the crystals from the vesicles into the extracellular MMP-13 is an collagenase with high for II and was to II and X Matrix Google Scholar). MV-associated MMP-2 and -9 the resulting The combined of these MV-associated collagenase and matrix remodeling and the of mineralization of the extracellular have previously that MMP-13 is by hypertrophic but active MMP-13 is in late hypertrophic chondrocytes M. Z. M. Pacifici M. Sarment Billings P.C. J. Cell. Biochem. 2000; PubMed Scopus Google Scholar). MMP-2 is involved in a resulting in the activation of H. C. H. G. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google K. G. 1996; PubMed Scopus Google Scholar). it is that MV after released from the plasma membrane of hypertrophic chondrocytes contain MMP-2 and MV-associated MMP-2 be involved in activating the MV-associated of the and active of MMP-13 be activated at the activity is Boyan B.D. Schwartz Z. Dean D.D. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) have reported that MV isolated from chondrocyte cultures were capable of activating latent TGF-β. In this study, we and their and that authentic MV isolated from growth plate cartilage are capable of activating latent TGF-β by hypertrophic Furthermore, that active and of the small latent are present in authentic MV even after treatment with hyaluronidase, NaCl, and suggesting that small latent TGF-β be directly associated with the outer surface of authentic MV and that the be in with MV-associated MMP-2 and In addition, we provide evidence that at which MMP-13 activation of suggesting that MMP-13 a role in activating TGF-β from small latent MV be the for initiation of but they be the for activation of TGF-β in hypertrophic growth plate cartilage. Active TGF-β has been to be an that of J. Cell. Biochem. 1996; 61: PubMed Scopus Google Scholar, Y. Y. Y. H. J. Biochem. PubMed Scopus Google Scholar, H. K. K. K. K. K. Y. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). MV-associated MMP-2 is involved in activating MV-associated The active of MMP-13 active TGF-β from the MV-associated small latent of TGF-β. The active TGF-β the of cells to to the of cartilage by In this the presence of active MMP-2, -9, and TGF-β in authentic MV isolated from hypertrophic growth plate cartilage and for these MMPs in matrix remodeling and TGF-β activation during mineralization and MV released from chondrocytes contain components for initiation of matrix and subsequent For they contain the Ca2+ channel-forming annexins II, V, and VI, components for formation of the first intralumenal crystal MMPs, for matrix and latent TGF-β which after activated by MV-associated MMPs be for and the of cartilage by The of these and other MV components and their still have to be Harrison for with of authentic MV, for and for with