Isolated osteoclasts resorb the organic and inorganic components of bone.

Abstract

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/~g of 25-50-/~m bone fragments within 5 d. Virtually all released tritium-labeled protein was of low molecular weight, 99% with Mr -10,000, and 65% with Mr _< 1,000. Moreover, when the 14% of resorbed [3H]proline-labeled peptides with Mr ~ 2,000 were examined for the presence of TC A and TC B, 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 TC A and TC B 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.

c-Src Control of Chloride Channel Support for Osteoclast HCl Transport and Bone Resorption

“…Antisense Treatment of Osteoclasts-Marrow cells from calcium-deprived hens were isolated as described and cultured in the presence of devitalized bone particles (15,32). After 24 h in culture, cells were exposed to phosphorothioate oligonucleotide using streptolysin-O permeabilization (33,34).…”

“…Vesicles-Osteoclasts and bone marrow cells were isolated from the long bones of calcium-deprived laying hens (Gallus domesticus), cultured and membranes prepared as described (10,15,32). Membrane pellets were stored at Ϫ80°C until analyzed.…”

Osteoclast Genetic Diseases

“…Bone resorption is a complex process requiring two different phases, the acidification of the extracellular lacuna to dissolve the inorganic bone matrix and the secretion of proteolytic www.intechopen.com enzymes to digest the organic components (Blair et al, 1986;Vaananen et al, 1998) (Figure 2). To achieve the acidification of the resorption lacunae and begin the process of bone demineralization, Carbonic Anhydrase II (CAII) generates carbonic acid from the hydration of CO 2 .…”

“…The chloride ion is then released in the bone resorption lacuna by a Cl -/H + antiport, ClC7, that, coupling with the proton pump activity, balances the ion charge across the membrane (Boyle et al, 2003;Graves et al, 2008;Teitelbaum & Patrick, 2003). The final goal of this process is to demineralise the bone and uncover the organic matrix ready to be digested by proteolytic enzymes, such as the metalloproteinase MMP9 released by endosomal vesicles, and the cathepsin K released by lysosomes (Blair et al, 1986;Bossard et al, 1996;Everts et al, 1992). …”

When developmental biology meets human pathology

“…Once differentiated, osteoclasts become polarized by forming a ruffled membrane on the bone surface where they are able to resorb the mineralized bone extracellular matrix. This degradation process comprises two phases: first, the inorganic phase of the bone matrix is chemically dissolved, and then the protein component of the bone extracellular matrix, i.e., mostly type I collagen, is enzymatically degraded (5). Bone demineralization results from secretion of HCl by the osteoclasts in the microenvironment delineated by their ruff led borders and the bone surface.…”