Hill, Peter Alexander; (1994) Mechanisms regulating bone resorption in vitro. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis encompasses two separate aspects of the biology of osteoclasts. Firstly, proteins were purified from bone matrix that modulate osteoclast formation and function. The effects of two of the most abundant growth factors insulin-like growth factors-l (IGF-I) and -II (IGF-II) on osteoclast differentiation and function were also studied. The aim of the second area of research was to determine the roles of individual members of the cysteine proteinase (CP) and matrix metalloproteinase (MMP) family on bone resorption using selective enzyme inhibitors. Bone matrix is a rich source of polypeptide growth factors and evidence suggests that osteoclasts do not mature functionally until they reach bone. Since bone resorption occurs at discrete sites throughout the skeleton, it is likely that an osteoclast activating factor(s) could be sequestrated in bone matrix where it may regulate osteoclast formation from haemopoietic precursors. Matrix proteins were extracted from bovine cortical bone with EDTA/TrisHGI under non-dissociative conditions at neutral pH. Four distinct bone resorptive proteins of 14, 25, 29, and 40 kDa were purified and partially characterized using an in vitro neonatal calvarial assay. The 14 and 25 kDa proteins possessed insulin-like growth factor (IGF) and transforming growth factor-6 activity respectively, whilst the 40 kDa protein enhanced the formation of osteoclast-like cells in a mouse bone marrow àssay. The 29 kDa protein stimulated bone resorption in an isolated osteoclast assay, suggesting a direct action on osteoclasts. This is the first report of (i) a protein isolated from bone matrix that stimulates osteoclast differentiation and (ii) a protein that directly stimulates osteoclast function. IGF-I and -II were found to enhance both osteoclast differentiation and function but they were less effective than 1,25 Dihydroxyvitamin D3. The stimulatory effects of IGFs on osteoclast activity were mediated via an interaction with the type I IGF receptor present on osteoblastic cells. Osteoclasts solubilize mineralized bone by means of acid pH generation and proteinase action, notably enzymes of the métallo and cysteine classes. To determine the roles and sites of action of the individual proteinases in bone resorption both natural and synthetic enzyme inhibitors were utilized. Four CP inactivators were tested: Ep475, a compound with low membrane permeabilty that selectively inhibits cathepsins B,L,S and calpain; Ep453, the membrane permeant prodrug of Ep475; CA074, a compound with low membrane permeability that selectively inactivates cathepsin B; and CA074Me, the membrane permeant prodrug of CA074. Ep475, Ep453 and CA074Me inhibited both calvarial and osteoclast lacunar resorption, whilst CA074 was without effect. These results suggest that cathepsin L and/or S act extracellularly and possibly intracellularly whilst cathepsin B acts intracellularly perhaps through the activation of other proteinases involved in bone collagen degradation. Six MMP inhibitors were used: initially the tissue inhibitors of metalloproteinases (TIMP) -1 and -2 were tested followed by four synthetic inhibitors. These were CT435, a general MMP inhibitor; CT543 and CT1166, compounds that are concentration dependent selective inhibitors of gelatinases-A and -B; and Ro 31- 7467, a concentration-dependent selective inhibitor of collagenase. All the compounds produced a dose-dependent inhibition in calvarial bone resorption. However, CT1166 and CT543 only produced a 40% inhibition and Ro 31-7467 a 50% inhibition at concentration selective for the inhibition of gelatinase and collagenase respectively. The compounds also produced a partial reduction in the resorptive activity of interleukin-1 -stimulated osteoclast cultures. Immunofluoresence studies demonstrated that rabbit osteoclasts synthesize gelatinases-A and -B, collagenase, stromelysin and tissue inhibitor of metalloproteinases-1. Human recombinant TIMP-1 and TIMP-2 were as effective at preventing calvarial bone reorption as the synthetic MMP inhibitors. It is suggested that the MMPs are produced by osteoclasts and participate with the CPs in osteoclast lacunar resorption.

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