Summary
To determine the possible involvement of cysteine-proteinases in bone matrix degradation by osteoclasts, the effects of the proteinase inhibitors leupeptin and E-64 were studied in anin vitro system using mouse bone explants. It was observed that in explants treated with the drugs, the amount of demineralized matrix opposing the ruffled border of the osteoclasts increased about 20-fold within 6 hours. This suggests that demineralization had proceeded whereas matrix degradation had been retarded. It was further noticed that in 12 of 287 osteoclasts, cytoplasmic vacuoles were present containing collagen fibrils that could not be distinguished from those in cartilage or bone. Their intracellular localization was proved by the study of serial sections. Finally, a significant reduction was shown as to the relative surface density of electrontranslucent vacuoles; this would seem to suggest reduced endocytic activity of the cells. Our observations support the view that cysteine-proteinases play an important role in osteoclastic bone resorption. It was further noticed that thein vitro effects of leupeptin and E-64 in certain respects resemble ultrastructural features of pycnodysostosis, an osteopetrosislike bone disorder. The data are in line with the hypothesis that this disease is caused by insufficient activity of osteoclastic cysteine-proteinases.
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References
Maroteaux P, Lamy M (1965) The malady of Toulouse-Lautrec. J Am Med Assn 191:715–717
Elmore SM (1967) Pycnodysostosis: a review. J Bone Joint Surg 49A:153–162
Everts V, Aronson DC, Beertsen W (1985) Phagocytosis of bone collagen by osteoclasts in two cases of pycnodysostosis. Calcif Tissue Int 37:25–31
Sakamoto S, Sakamoto M (1986) Bone collagenase, osteoblasts and cell-mediated bone resorption. In: Peck WA (ed) Bone and mineral research, vol. 4. Elsevier Scientific Publishers BV, Amsterdam, p 49
Vaes G (1980) Collagenase, lysosomes and osteoclastic bone resorption. In: Woolley DE, Evansson JM (eds) Collagenase in normal and pathological connective tissues. John Wiley and Sons, Chichester, p 185
Etherington DJ, Birkedal-Hansen H (1987) The influence of dissolved calcium salts on the degradation of hard-tissue collagens by lysosomal cathepsins. Collagen Rel Res 7:185–199
Delaissé JM, Eeckhout Y, Vaes G (1980) Inhibition of bone resorption in culture by inhibitors of thiol proteinases. Biochem J 192:365–368
Delaissé JM, Eeckhout Y, Vaes G (1984) In vivo and in vitro evidence for the involvement of cysteine proteinases in bone resorption. Biochem Biophys Res Commun 125:441–447
Everts V, Beertsen W, Tigchelaar-Gutter W (1985) The digestion of phagocytosed collagen is inhibited by the proteinase inhibitors leupeptin and E-64. Collagen Rel Res 5:315–336
Holtrop ME, King GJ (1977) The ultrastructure of the osteoclast and its functional implications. Clin Orthop Rel Res 123:177–196
Hancox NM, Boothroyd B (1961) Motion pictures and electron microscope studies on the embryonic avian osteoclast. J Biophys Biochem Cytol 11:651–661
Weibel ER (1969) Stereological principles for morphometry in electron microscopic cytology. Int Rev Cytol 26:235–302
Umezawa H (1976) Structures and activities of protease inhibitors of microbial origin. Meth Enzymol 45:678–695
Barrett AJ, Kembhavi AA, Hanada K (1981) E-64 (L-transepoxysuccinyl-leucyl-amido(4-guanidino)butane) and related epoxides as inhibitors of cysteine proteinases. Acta Biol Med Germ 40:1513–1517
Baron R, Neff L, Louvard D, Courtoy PJ (1985) Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border. J Cell Biol 101:2210–2222
Eeckhout Y, Delaissé JM, Vaes G (1986) Direct extraction and assay of bone tissue collagenase and its relation to parathyroid-hormone-induced bone resorption. Biochem J 239:793–796
Lucht U (1972) Cytoplasmic vacuoles and bodies of the osteoclast. Z Zellforsch 135:229–244
Marks SC Jr, Seifert MF (1985) The lifespan of osteoclasts: experimental studies using the giant granule marker characteristic of beige mice. Bone 6:451–455
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Everts, V., Beertsen, W. & Schröder, R. Effects of the proteinase inhibitors leupeptin and E-64 on osteoclastic bone resorption. Calcif Tissue Int 43, 172–178 (1988). https://doi.org/10.1007/BF02571316
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DOI: https://doi.org/10.1007/BF02571316