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 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 co-cultured 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-1 alpha (IL-1 alpha; 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-1 alpha-stimulated cultures, with maximal action at 10(-5) M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme beta-glucuronidase. 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-1 alpha 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.
Hill
P A
PA
Cell and Molecular Biology Department, Strangeways Research Laboratory, Worts Causeway, Cambridge, UK.
Murphy
G
G
Docherty
A J
AJ
Hembry
R M
RM
Millican
T A
TA
Reynolds
J J
JJ
Meikle
M C
MC
D000818:Animals; D001862:Bone Resorption / Q000209:etiology / Q000378:metabolism*; D002478:Cells, Cultured; D002645:Chickens; D018093:Gelatinases / Q000037:antagonists & inhibitors; D006023:Glycoproteins / Q000378:metabolism*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism*; D010627:Phenethylamines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D019714:Tissue Inhibitor of Metalloproteinases
C092859:CT 435; C092860:CT 543; D006023:Glycoproteins; D009025:Morpholines; D010627:Phenethylamines; D019714:Tissue Inhibitor of Metalloproteinases; D018093:Gelatinases; D008666:Metalloendopeptidases