title: Inhibition of bone resorption in vitro by selective inhibitors of gelatinase and collagenase.
abstract: Two low-molecular-mass inhibitors of matrix metalloproteinases (MMPs), CT1166, a concentration-dependent selective inhibitor of gelatinases A and B, and Ro 31-7467, a concentration-dependent selective inhibitor of collagenase, were examined for their effects on bone resorption and type-I collagenolysis. The test systems consisted of measuring (1) the release of [3H]proline from prelabelled mouse calvarial explants; (2) the release of 14C from prelabelled type-I collagen films by mouse calvarial osteoblasts; and (3) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. In 24 h cultures, CT1166 and Ro 31-7467 inhibited both interleukin-1 alpha- (IL-1 alpha; 10(-10) M) and 1,25-dihydroxyvitamin D3 (10(-8) M)-stimulated bone resorption in cultured neonatal mouse calvariae at concentration selective for the inhibition of gelatinase (10(-9) M for CT1166) and collagenase (10(-8) M for Ro 31-7467) respectively. For each compound the inhibition was dose-dependent, reversible, and complete at a 10(-7) M concentration. However, CT1166 (10(-9) M) and Ro 31-7467 (10(-8) M) in combination were required to completely abolish IL-1 alpha-stimulated bone resorption in mouse calvariae throughout a 96 h culture period. Neither of the inhibitors affected protein synthesis, DNA synthesis nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme, beta-glucuronidase. Both CT1166 and Ro 31-7467 partially inhibited IL-1 alpha-stimulated lacunar resorption by isolated osteoclasts, but were without effect on unstimulated lacunar resorption. Rodent osteoclasts produced collagenase and gelatinases-A and -B activity. In contrast the substrate used to assess osteoclast lacunar resorption contained no detectable collagenase or gelatinase activity. Both compounds dose-dependently inhibited 1,25-dihydroxyvitamin D3 (10(-8) M)-stimulated degradation of type-I collagen by mouse calvarial osteoblasts; however, complete inhibition of collagenolysis was only achieved at concentrations at which CT1166 and Ro 31-7467 act as general MMP inhibitors. This study demonstrates that collagenase and gelatinases A and/or B participate in bone resorption. While these MMPs may be primarily involved in osteoid removal, we conclude that they may also be released by osteoclasts, where they participate in bone collagen degradation within the resorption lacunae.
authors.affiliation: Cell and Molecular Biology Department, Strangeways Research Laboratory, Worts Causeway, Cambridge, U.K.
authors.forename: P A
authors.initials: PA
authors.lastname: Hill
authors.forename: A J
authors.initials: AJ
authors.lastname: Docherty
authors.forename: K M
authors.initials: KM
authors.lastname: Bottomley
authors.forename: J P
authors.initials: JP
authors.lastname: O'Connell
authors.forename: J R
authors.initials: JR
authors.lastname: Morphy
authors.forename: J J
authors.initials: JJ
authors.lastname: Reynolds
authors.forename: M C
authors.initials: MC
authors.lastname: Meikle
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D001862:Bone Resorption; D003094:Collagen / Q000378:metabolism; D018093:Gelatinases / Q000037:antagonists & inhibitors*; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D009025:Morpholines / Q000494:pharmacology*; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010010:Osteoclasts / Q000201:enzymology*; D012886:Skull
chemical_list: D000577:Amides; C093640:CT 1166; D007375:Interleukin-1; D061965:Matrix Metalloproteinase Inhibitors; D009025:Morpholines; D009943:Organophosphorus Compounds; C078189:Ro 317467; D003094:Collagen; D018093:Gelatinases