Effect of 1 alpha,25-dihydroxyvitamin D-3 and 24R,25-dihydroxyvitamin D-3 on metalloproteinase activity and cell maturation in growth plate cartilagein vivo

Recent studies indicate that 1 alpha ,25-dihydroxyvitamin D-3 (1(alpha),25[ OH](2)D-3) and 24R,25-dihydroxyvitamim D-3 (24R,25[OH](2)D-3) differentiall y regulate proliferation, differentiation, and matrix synthesis of growth p late chondrocytes. To determine whether both metabolites play the same or d ifferent roles in vivo, we used the vitamin D-deficient rat as a model. Ric kets was induced and then reversed by administering a single dose of ergoca lciferol, 1 alpha ,25 (OH)(2)D-3, or 24R,25 (OH)(2)D-3 and euthanizing the animals after 4, 24, 48, or 72 h. Growth plates were either processed for h istology and histomorphometry or extracted with buffered guanidine-HCl. Neu tral metalloproteinase activity in the extracts was measured by use of aggr ecan-containing beads, and collagenase activity was determined by use of ra dioactive type I collagen. The levels of tissue inhibitor of metalloprotein ases (TIMP) and plasminogen activator were also determined. The morphology of the growth plate varied as a function of treatment. While 24R,25(OH)(2)D -3 appeared to affect cell maturation and 1 alpha ,25(OH)(2)D-3 appeared to affect terminal differentiation and calcification, response to ergocalcife rol was indicative of the combined responses to the individual metabolites. Enzyme activity was regulated in a differential manner. Treatment with erg ocalciferol produced a rapid decline in both neutral metalloproteinase and collagenase activities that was statistically significant by 4 h. By contra st, 1 alpha ,25(OH)(2)D-3 had no effect on neutral metalloproteinase activi ty but caused a significant decrease in both active and total collagenase a ctivity by 4 h, while 24R,25(OH)(2)D-3 decreased neutral metailoproteinase activity by 48 h and had no effect on collagenase activity. Ergocalciferol had no effect on TIMP levels at any time examined, whereas 1 alpha ,25(OH)( 2)D-3 caused an increase at 48 and 72 h and 24R,25(OH)(2)D-3 completely blo cked TIMP production at 4 and 24 h. By contrast, plasminogen activator acti vity by ergocalciferol was decreased at 4 h, increased by 1 alpha ,25(OH)(2 )D-3 at 4 and 24 h, and decreased by 24R,25(OH)(2)D-3 at all time points ex amined. These in vivo results confirm our previous cell culture observation s showing that growth plate chondrocytes are differentially regulated by 1 alpha ,25(OH)(2)D-3 and 24R,25(OH)(2)D-3. Moreover, they show definitively that these two vitamin D metabolites play distinct roles not only in regula ting neutral metalloproteinase and collagenase activities in growth plate c artilage but in cell maturation and calcification of this tissue in vivo.