Activation of latent transforming growth factor beta 1 by stromelysin 1 inextracts of growth plate Chondrocyte-derived matrix vesicles

Previous studies have shown that matrix vesicles isolated from cultures of costochondral growth zone chondrocytes and treated with 1 alpha ,25-dihydro xyvitamin D-3[1 alpha ;25(OH)(2)D-3] can activate recombinant human latent transforming growth factor beta1 (rhTGF-beta1). It is unknown what enzyme o r other factor in the extracellular organelles is responsible for the activ ation. This study tested the hypothesis that enzymes present in matrix vesi cles can activate latent TGF-beta1 and that this is regulated by 1 alpha ,2 5(OH)(2)D-3. To do this, we examined the ability of matrix vesicle extracts to activate small latent rhTGF-beta1. In addition, enzymes previously dete rmined to be present in matrix vesicles were screened for their ability to activate small latent rhTGF-beta1. Recombinant human matrix metallo-protein ase 2 (rhMMP3; 72 kDa gelatinase), rhMMP3 (stromelysin 1), purified human p lasminogen, and purified urokinase (plasminogen activator) were each tested at varying concentrations. To assess the role of cell maturation, we used a cell culture model in which chondrocytes are derived from two distinct zo nes of rat costochondral cartilage, the resting zone and the growth zone. M atrix vesicles were isolated from these cultures and then tested. The resul ts showed that extracts of matrix vesicles produced by both growth zone and resting zone chondrocytes were able to activate small latent rhTGF-beta1. The effects were dose and time dependent, with greater activity being found in extracts of matrix vesicles from the growth zone chondrocyte cultures. Only rhMMP3 was able to activate small latent rhTGF-beta1, indicating that stromelysin-1, but not MMP-2, plasminogen, or urokinase, was involved. As o bserved in the extracts, the effect of rhMMP3 was time and dose dependent. When anti-MMP-3 antibody was added to matrix vesicle extracts from both cel l types, activation of small latent rhTGF-beta1. was dose-dependently block ed. Neither 1 alpha ,25(OH)(2)D-3 nor 24R,25(OH)(2)D-3 had a direct effect on activation of small latent rhTGF-beta1 by the extracts. However, when in tact matrix vesicles were treated with 1 alpha ,25(OH)(2)D-3, their ability to activate small latent rhTGF-beta1 was increased. Inhibition of phosphol ipase A,with quinacrine blocked the la,25(OH),D,dependent effect. These res ults suggest that the ability of 1 alpha ,25(OH)(2)D-3-treated matrix vesic les to activate small latent TGF-PI is via action of the secosteroid on the matrix vesicle membrane, not on the enzymes responsible for activating lat ent TGF-beta1. Because matrix vesicles isolated from growth zone chondrocyt es have been shown to contain increased phospholipase A(2) activity after t reatment with la;25(OH),D,, it is likely that this secosteroid promotes los s of membrane integrity through phospholipase A(2)-dependent formation of l ysophospholipids, resulting in the release of MMP3 into the matrix, where l atent TGF-beta1 is stored. Taken together, the results of the current study show that matrix vesicles produced by growth plate chondrocytes contain MM P-3, that this enzyme is at least partially responsible for activation of s mall latent TGF-beta1 in the matrix, and that 1 alpha ,25(OH)(2)D-3 regulat es MMP release from matrix vesicles.