Abstract
Osteoclasts are the principal resorptive cells of bone, yet their capacity to degrade collagen, the major organic component of bone matrix, remains unexplored. Accordingly, we have studied the bone resorptive activity of highly enriched populations of isolated chicken osteoclasts, using as substrate devitalized rat bone which had been labeled in vivo with L-[5-3H]proline or 45Ca, and bone-like matrix produced and mineralized in vitro by osteoblast-like rat osteosarcoma cells. When co-cultured with a radiolabeled substrate, osteoclast- mediated mineral mobilization reached a maximal rate within 2 h, whereas organic matrix degradation appeared more slowly, reaching maximal rate by 12-24 h. Thereafter, the rates of organic and inorganic matrix resorption were essentially linear and parallel for at least 6 d when excess substrate was available. Osteoclast-mediated degradation of bone collagen was confirmed by amino acid analysis. 39% of the solubilized tritium was recovered as trans-4-hydroxyproline, 47% as proline. 10,000 osteoclasts solubilized 70% of the total radioactivity and 65% of the [3H]-trans-4-hydroxyproline from 100 micrograms of 25-50 micron bone fragments within 5 d. Virtually all released tritium- labeled protein was of low molecular weight, 99% with Mr less than or equal to 10,000, and 65% with Mr less than or equal to 1,000. Moreover, when the 14% of resorbed [3H]proline-labeled peptides with Mr greater than or equal to 2,000 were examined for the presence of TCA and TCB, the characteristic initial products of mammalian collagenase activity, none was detected by SDS PAGE. In addition, osteoclast-conditioned medium had no collagenolytic activity, and exogenous TCA and TCB fragments were not degraded by osteoclasts. On the other hand, osteoclast lysates have collagenolytic enzyme activity in acidic but not in neutral buffer, with maximum activity at pH 4.0. These data indicate that osteoclasts have the capacity to resorb the organic phase of bone by a process localized to the osteoclast and its attachment site. This process appears to be independent of secretion of neutral collagenase and probably reflects acid protease activity.
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- Ash P., Loutit J. F., Townsend K. M. Osteoclasts derived from haematopoietic stem cells. Nature. 1980 Feb 14;283(5748):669–670. doi: 10.1038/283669a0. [DOI] [PubMed] [Google Scholar]
- Bonucci E. The organic-inorganic relationships in bone matrix undergoing osteoclastic resorption. Calcif Tissue Res. 1974;16(1):13–36. doi: 10.1007/BF02008210. [DOI] [PubMed] [Google Scholar]
- Bornstein P., Sage H. Structurally distinct collagen types. Annu Rev Biochem. 1980;49:957–1003. doi: 10.1146/annurev.bi.49.070180.004521. [DOI] [PubMed] [Google Scholar]
- Breul S. D., Bradley K. H., Hance A. J., Schafer M. P., Berg R. A., Crystal R. G. Control of collagen production by human diploid lung fibroblasts. J Biol Chem. 1980 Jun 10;255(11):5250–5260. [PubMed] [Google Scholar]
- Burleigh M. C., Barrett A. J., Lazarus G. S. Cathepsin B1. A lysosomal enzyme that degrades native collagen. Biochem J. 1974 Feb;137(2):387–398. doi: 10.1042/bj1370387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Delaissé J. M., Eeckhout Y., Vaes G. Inhibition of bone resorption in culture by inhibitors of thiol proteinases. Biochem J. 1980 Oct 15;192(1):365–368. doi: 10.1042/bj1920365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eilon G., Raisz L. G. Comparison of the effects of stimulators and inhibitors of resorption on the release of lysosomal enzymes and radioactive calcium from fetal bone in organ culture. Endocrinology. 1978 Dec;103(6):1969–1975. doi: 10.1210/endo-103-6-1969. [DOI] [PubMed] [Google Scholar]
- Francois-Gillet C., Delaissé J. M., Eeckhout Y., Vaes G. Immunoreactive collagenase and bone resorption. Biochim Biophys Acta. 1981 Feb 18;673(1):1–9. [PubMed] [Google Scholar]
- GROSS J. Studies on the formation of collagen. I. Properties and fractionation of neutral salt extracts of normal guinea pig connective tissue. J Exp Med. 1958 Feb 1;107(2):247–263. doi: 10.1084/jem.107.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heath J. K., Atkinson S. J., Meikle M. C., Reynolds J. J. Mouse osteoblasts synthesize collagenase in response to bone resorbing agents. Biochim Biophys Acta. 1984 Nov 6;802(1):151–154. doi: 10.1016/0304-4165(84)90046-1. [DOI] [PubMed] [Google Scholar]
- Heersche J. N. Mechanism of osteoclastic bone resorption: a new hypothesis. Calcif Tissue Res. 1978 Nov 10;26(1):81–84. doi: 10.1007/BF02013238. [DOI] [PubMed] [Google Scholar]
- Holtrop M. E., Raisz L. G., Simmons H. A. The effects of parathyroid hormone, colchicine, and calcitonin on the ultrastructure and the activity of osteoclasts in organ culture. J Cell Biol. 1974 Feb;60(2):346–355. doi: 10.1083/jcb.60.2.346. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
- Loutit J. F., Nisbet N. W. Resorption of bone. Lancet. 1979 Jul 7;2(8132):26–27. doi: 10.1016/s0140-6736(79)90186-7. [DOI] [PubMed] [Google Scholar]
- Mecham R. P., Lange G. Antigenicity of elastin: characterization of major antigenic determinants on purified insoluble elastin. Biochemistry. 1982 Feb 16;21(4):669–673. doi: 10.1021/bi00533a013. [DOI] [PubMed] [Google Scholar]
- Osdoby P., Martini M. C., Caplan A. I. Isolated osteoclasts and their presumed progenitor cells, the monocyte, in culture. J Exp Zool. 1982 Dec 30;224(3):331–344. doi: 10.1002/jez.1402240306. [DOI] [PubMed] [Google Scholar]
- Oursler M. J., Bell L. V., Clevinger B., Osdoby P. Identification of osteoclast-specific monoclonal antibodies. J Cell Biol. 1985 May;100(5):1592–1600. doi: 10.1083/jcb.100.5.1592. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raisz L. G. Physiologic and pharmacologic regulation of bone resorption. N Engl J Med. 1970 Apr 16;282(16):909–916. doi: 10.1056/NEJM197004162821608. [DOI] [PubMed] [Google Scholar]
- Roswit W. T., Halme J., Jeffrey J. J. Purification and properties of rat uterine procollagenase. Arch Biochem Biophys. 1983 Aug;225(1):285–295. doi: 10.1016/0003-9861(83)90032-2. [DOI] [PubMed] [Google Scholar]
- Stricklin G. P., Bauer E. A., Jeffrey J. J., Eisen A. Z. Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures. Biochemistry. 1977 Apr 19;16(8):1607–1615. doi: 10.1021/bi00627a013. [DOI] [PubMed] [Google Scholar]
- Teitelbaum S. L., Stewart C. C., Kahn A. J. Rodent peritoneal macrophages as bone resorbing cells. Calcif Tissue Int. 1979 Jul 3;27(3):255–261. doi: 10.1007/BF02441194. [DOI] [PubMed] [Google Scholar]
- Wright S. D., Silverstein S. C. Phagocytosing macrophages exclude proteins from the zones of contact with opsonized targets. Nature. 1984 May 24;309(5966):359–361. doi: 10.1038/309359a0. [DOI] [PubMed] [Google Scholar]
- Zambonin Zallone A., Teti A., Primavera M. V. Isolated osteoclasts in primary culture: first observations on structure and survival in culture media. Anat Embryol (Berl) 1982 Dec;165(3):405–413. doi: 10.1007/BF00305576. [DOI] [PubMed] [Google Scholar]