THE EFFECTS OF SELECTIVE INHIBITORS OF MATRIX METALLOPROTEINASES (MMPS) ON BONE-RESORPTION AND THE IDENTIFICATION OF MMPS AND TIMP-1 IN ISOLATED OSTEOCLASTS

We have compared the effects of a general matrix metallo-proteinase (M MP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; K-i<20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of Ca-45(2+) from prelabelled mouse calvarial explants; (ii) the release of Ca-45(2 +) from prelabelled osteoid-free calvarial explants co-cultured with p urified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. Both CT435 and CT543 dose-de pendently inhibited the release of Ca-45(2+) from neonatal calvarial b ones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D-3. Moreover, CT543 produced a 40% inhibition at a concentration (10 (-8) M) selective for the inhibition of human gelatinases A and B. CT4 35 (10(-5) M) and CT543 (10(-5) M) partially inhibited the release of Ca-45(2+) from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and app roximately 36% for cultures stimulated by interleukin-1 alpha (IL-1 al pha; 10(-10) M). Neither inhibitor prevented lacunar resorption on ivo ry by unstimulated rat osteoclasts, but the compounds produced a parti al reduction in both the number and total surface area of lacunae in I L-1 alpha-stimulated cultures, with maximal action at 10(-5) M, Neithe r of the inhibitors affected protein or DNA synthesis, nor the IL-1 al pha-stimulated secretion of the lysosomal enzyme beta-glucuronidase. I mmunocytochemistry demonstrated that isolated rabbit osteoclasts const itutively expressed gelatinase A and synthesized gelatinase B, collage nase and stromelysin, as well as the tissue inhibitor of matrix metall oproteinases-1 (TIMP-1) following IL-1 alpha stimulation. These experi ments have shown that in addition to collagenase, gelatinases A and B are likely to play a significant role in bone resorption, They further suggest that MMPs produced by osteoclasts are released into the sub-o steoclastic resorption zone where they participate in bone collagen de gradation.