Authentic matrix vesicles contain active metalloproteases (MMP) - A role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-beta

Matrix vesicles (MV) play a key role in the initiation of cartilage mineral ization. Although many components in these microstructures have been identi fied, the specific function of each component is still poorly understood. I n this study, we show that metalloproteases (MMP), MMP-2, -9, and -13 are a ssociated with MV isolated from growth plate cartilage. In addition, we pro vide evidence that MV contain transforming growth factor-beta (TGF-beta) an d that MV-associated MMP-13 is capable of activating latent TGF-beta. To de termine whether MMPs are associated directly with MV, vesicles isolated fro m 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 MCV membrane and expose components that are inside the vesicles. Each treated MV fraction was subjected to substrate zymograp hy, immunoblotting, and substrate activity assay. Whereas active MMP-13 was lost after combined treatment with hyaluronidase and Nacl, MMP-8 and -9 ac tivities were still retained in the pellet fraction even after detergent tr eatment, suggesting that the gelatinases, MMP-2 and -9, are integral compon ents of MV. In addition, MV contain TGF-beta in the small latent complex, a nd MMP-13 associated with the MV surface was responsible for activation of TGF-beta. Since the amount of TGF-beta activated by hypertrophic chondrocyt es increased with mineral appearance in serum-free chondrocyte cultures, a role for active MV-associated MMPs is suggested in activation of TGF-beta s een during late chondrocyte hypertrophy and mineralization of growth plate cartilage.