Abstract
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.
Highlights
Matrix vesicles (MV) play a key role in the initiation of cartilage mineralization
Hypertrophic Chondrocytes Increased the Production of Active TGF- Prior to Mineralization—Previously, we demonstrated that hypertrophic chondrocytes maintained in serumfree agarose cultures produced ϳ20% active TGF- after 7 days in culture [11]
We demonstrate that authentic MV isolated from chicken growth plate cartilage contain active collagenase, MMP-13, and active gelatinases, MMP-2 and -9
Summary
Chondrocyte Isolation and Serum-free Cell Culture—-Prehypertrophic chondrocytes were isolated from day 17 avian upper sterna utilizing microsurgical techniques as described previously [11]. 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- Present in Conditioned Medium from Chondrocytes Cultures—Active TGF- present in conditioned medium from chondrocytes cultures was determined as stimulation of a TGF--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]. 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
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