Metalloproteinase activity in growth plate chondrocyte cultures is regulated by 1,25-( OH)(2)D-3 and 24,25-( OH)(2)D-3 and mediated through protein kinase C

Citation
S. Maeda et al., Metalloproteinase activity in growth plate chondrocyte cultures is regulated by 1,25-( OH)(2)D-3 and 24,25-( OH)(2)D-3 and mediated through protein kinase C, MATRIX BIOL, 20(2), 2001, pp. 87-97
Citations number
67
Categorie Soggetti
Biochemistry & Biophysics
Journal title
MATRIX BIOLOGY
ISSN journal
0945053X → ACNP
Volume
20
Issue
2
Year of publication
2001
Pages
87 - 97
Database
ISI
SICI code
0945-053X(200104)20:2<87:MAIGPC>2.0.ZU;2-O
Abstract
During endochondral development, growth plate chondrocytes must remodel the ir matrix in a number of ways as they differentiate and mature. In previous studies, we have shown that matrix metalloproteinases (MMPs) extracted fro m matrix vesicles can extensively degrade aggrecan and that this is modulat ed by vitamin D metabolites in a manner involving protein kinase C (PKC). M atrix vesicles represent only a small component of the extracellular matrix , however, and it is unknown if the total metalloproteinase complement, inc luding the MMPs and aggrecanases in the culture, is also regulated in a sim ilar way. This study tested the hypothesis that. vitamin D metabolites regu late the level of metalloproteinase activity in growth plate chondrocytes v ia a PKC-dependent mechanism and play a role in partitioning this proteinas e activity between the media and cell layer (cells + matrix) in these cultu res. To do this, resting zone cells (RC) were treated with 10(-9)-10(-7) M 24R,25-(OH)(2)D-3, while growth zone cells (GC) were treated with 10(-10)-1 0(-8) M 1 alpha ,25-(OH)(2)D-3. Cultures of both cell types were also treat ed with the PKC inhibitor chelerythrine in the presence and absence of vita min D metabolites. At harvest, the media were either left untreated or trea ted to destroy metalloproteinase inhibitors, while enzyme activity in the c ell layers was extracted with buffered guanidine and then treated like the media to destroy metalloproteinase inhibitors. Neutral metalloproteinase (a ggrecan-degrading activity) activity was assayed on aggrecan-containing pol yacrylamide gel beads and collagenase activity was measured on telopeptide- free type I collagen. Neutral metalloproteinase activity was found primaril y in the cell layer of both cell types; however, activity was greater in ex tracts of GC cell layers. No collagenase activity could be detected in RC e xtracts until the metalloproteinase inhibitors were destroyed. In contrast, extracts of GC cell layers contained measurable activity without removing the inhibitors, and destroying the inhibitors resulted in a greater than tw o-fold increase in activity. No collagenase activity was found in the media of either cell type. 24,25-(OH)(2)D-3 caused a dose-dependent increase in neutral metalloproteinase activity in extracts of RC cells, but had no effe ct on collagenase activity. In contrast, 1,25-(OH)(2)D-3 caused a dose-depe ndent decrease in collagenase activity in extracts of GC cells, but had no effect on neutral metalloproteinase activity. In both cases, the effect of the vitamin D metabolite was mediated through the activation of PKC. These results support the hypothesis that metallopreteinases are involved in regu lating the bulk turnover of collagen and aggrecan in growth plate chondrocy tes and that the amount of metalloproteinase activity found is a function o f the cell maturation state.