title: 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.
abstract: During endochondral development, growth plate chondrocytes must remodel their matrix in a number of ways as they differentiate and mature. In previous studies, we have shown that matrix metalloproteinases (MMPs) extracted from matrix vesicles can extensively degrade aggrecan and that this is modulated by vitamin D metabolites in a manner involving protein kinase C (PKC). Matrix vesicles represent only a small component of the extracellular matrix, however, and it is unknown if the total metalloproteinase complement, including the MMPs and aggrecanases in the culture, is also regulated in a similar way. This study tested the hypothesis that vitamin D metabolites regulate the level of metalloproteinase activity in growth plate chondrocytes via a PKC-dependent mechanism and play a role in partitioning this proteinase activity between the media and cell layer (cells+matrix) in these cultures. 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)-10(-8) M 1alpha,25-(OH)(2)D(3). Cultures of both cell types were also treated with the PKC inhibitor chelerythrine in the presence and absence of vitamin D metabolites. At harvest, the media were either left untreated or treated to destroy metalloproteinase inhibitors, while enzyme activity in the cell layers was extracted with buffered guanidine and then treated like the media to destroy metalloproteinase inhibitors. Neutral metalloproteinase (aggrecan-degrading activity) activity was assayed on aggrecan-containing polyacrylamide gel beads and collagenase activity was measured on telopeptide-free type I collagen. Neutral metalloproteinase activity was found primarily in the cell layer of both cell types; however, activity was greater in extracts of GC cell layers. No collagenase activity could be detected in RC extracts 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 two-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 effect on collagenase activity. In contrast, 1,25-(OH)(2)D(3) caused a dose-dependent 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 metalloproteinases are involved in regulating the bulk turnover of collagen and aggrecan in growth plate chondrocytes and that the amount of metalloproteinase activity found is a function of the cell maturation state. Furthermore, 83-93% of neutral metalloproteinase activity and 100% of collagenase activity is localized to the cell layer. Moreover, the regulation of metalloproteinase activity by 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) involves a PKC-dependent pathway that is controlled by the target cell-specific vitamin D metabolite.
authors.affiliation: Department of Orthopaedics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
authors.forename: S
authors.initials: S
authors.lastname: Maeda
authors.forename: D D
authors.initials: DD
authors.lastname: Dean
authors.forename: V L
authors.initials: VL
authors.lastname: Sylvia
authors.forename: B D
authors.initials: BD
authors.lastname: Boyan
authors.forename: Z
authors.initials: Z
authors.lastname: Schwartz
mesh_terms: D015650:24,25-Dihydroxyvitamin D 3 / Q000378:metabolism*; D000818:Animals; D002117:Calcitriol / Q000378:metabolism*; D002358:Cartilage, Articular / Q000166:cytology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D006132:Growth Plate / Q000166:cytology; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D015650:24,25-Dihydroxyvitamin D 3; D011493:Protein Kinase C; D008666:Metalloendopeptidases; D002117:Calcitriol