The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts

Abstract

We have compared the effects of a general matrix metalloproteinase (MMP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; Ki<20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of 45Ca2+ from prelabelled mouse calvarial explants; (ii) the release of 45Ca2+ from prelabelled osteoid-free calvarial explants cocultured with purified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. Both CT435 and CT543 dose-dependently inhibited the release of 45Ca2+ from neonatal calvarial bones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D3. Moreover, CT543 produced a 40% inhibition at a concentration (10—8 M) selective for the inhibition of human gelatinases A and B. CT435 (10−5 M) and CT543 (10−5 M) partially inhibited the release of 45Ca2+ from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and approximately 36% for cultures stimulated by interleukin-1α (IL-1α; 10−10 M). Neither inhibitor prevented lacunar resorption on ivory by unstimulated rat osteoclasts, but the compounds produced a partial reduction in both the number and total surface area of lacunae in IL-1α-stimulated cultures, with maximal action at 10−5 M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1α-stimulated secretion of the lysosomal enzyme β-glu-curonidase. Immunocytochemistry demonstrated that isolated rabbit osteoclasts constitutively expressed gelatinase A and synthesized gelatinase B, collagenase and stromelysin, as well as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) following IL-1α stimulation. These experiments 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-osteoclastic resorption zone where they participate in bone collagen degradation.