VITAMIN-D REGULATION OF METALLOPROTEINASE ACTIVITY IN MATRIX VESICLES

Matrix vesicles (MVs) are enriched in matrix metalloproteinases (MMPs) capable of degrading proteoglycans. The aim of the present study was to identify which MMPs are present in MVs and determine whether these MMPs are regulated by 1,25-(OH)(2)D-3 [1,25] and 24,25-(OH)(2)D-3 [24, 25]. To do this, growth zone (GC) and resting zone (RC) chondrocytes w ere isolated from rat costochondral cartilage and placed into culture. At confluence, GCs were treated with 1,25 and RCs with 24,25 for 24 h ours. MVs, plasma membranes (PMs), and conditioned media were then col lected from the cultures. RT-PCR demonstrated the presence of mRNA for stromelysin-1 and 72 kDa gelatinase in both RCs and GCs. Casein zymog raphy revealed activity at M(r) 48 and 28 kDa in MV, but not PM or con ditioned media; Western analysis confirmed that this activity was stro melysin-1. Gelatinolytic activity, at low levels, was also found in MV s, but not PMs or conditioned media. When enzyme activity was measured using a proteoglycan bead assay, it was found that both GCs and RCs p roduced MVs and PMs containing neutral metalloproteinase. Both cells a lso produced MVs and PMs containing plasminogen activator. The additio n of 1,25 to GCs caused a significant 4- to 5-fold increase in metallo proteinase activity in MVs, but not PMs. In contrast, MVs from culture s of RCs treated with 24,25 contained decreased metalloproteinase acti vity; enzyme activity in PMs was unaffected by 24,25. Plasminogen acti vator in MVs from RC was increased by treatment with 24,25, while MV e nzyme activity was decreased after treatment of GC cultures with 1,25. This study shows that both RCs and GCs produce stromelysin-1 and 72 k Da gelatinase and that these enzymes are preferentially localized in M Vs. Further, MMP and plasminogen activator activities in MVs and PMs a re regulated by vitamin D metabolites.