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
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
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.