Osteoclasts and their relationship to bone as studied by electron microscopy

Summary Osteoclasts in metaphyses from young rats were systematically sectioned at different levels. Two types of osteoclasts were recognized. One type had no ruffled border while the other, and predominant type contained a ruffled border in a part of its length; some of the latter contained two ruffled borders. The closest contact between osteoclast and bone occurred at the level of the ruffled border and this bone under the border showed characteristic changes indicative of resorption. In some osteoclasts the ruffled border consisted of numerous slender cytoplasmic projections separated by very narrow spaces or channels while in other osteoclasts it was more open. The ruffled border was commonly surrounded by a transitional zone containing numerous thin filaments. The osteoclast usually had its greatest dimension at the level of the ruffled border and the cytoplasm here contained many bodies and vacuoles but a sparse endoplasmic reticulum. Away from the level of the ruffled border the cytoplasmic vacuoles and bodies were fewer while the endoplasmic reticulum was often more pronounced. Parts of the osteoclasts were usually situated close to a vessel. It is suggested that there is a correlation between the development of the ruffled border and the degree of bone resorption and that osteoclasts without a ruffled border are, at least temporarily, inactive with respect to bone resorption. The numerous cytoplasmic bodies, interpreted as lysosomes, are presumed to be important in the resorption process. The closely adjacent positioning of osteoclasts and vessels may facilitate the transport of resorption products to the blood.This research was supported by the Danish Research Council. Grant no. 512–727, 512–819 and 512–1545.I wish to thank Professor Arvid B. Maunsbach for valuable discussions.     

Immunocytochemical localization of cathepsin D in the rat osteoclast.

We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 micron) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsin-negative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Immunocytochemical localization of cathepsin D in the rat osteoclast

Summary We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 m) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections.At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsinnegative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface.These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Extensive Clear Zone and Defective Ruffled Border Formation in Osteoclasts of Osteopetrotic (ia/ia) Rats: Implications for Secretory Function

The cellular distribution of osteoclast integrin subunits α_v and β₃, the tissue distribution, and level of the apparent ligand osteopontin (OPN) as well as of the putative regulatory enzyme tartrate-resistant acid phosphatase (TRAP) were studied along with the intracellular distribution of the activation marker c-src in osteopetrotic ia/ia (incisors-absent) mutant rats and their normal littermates. In ia/ia rats, the osteoclasts are incapable of bone matrix resorption. Ultrastructurally the cells exhibit extended clear zones at the expense of ordinary ruffled borders. A secretory dysfunction in the mutant is strongly suggested by the absence of detectable extracellular TRAP, concomitant with an accumulation of the enzyme in abundant small cytoplasmic vesicles. Moreover, TRAP mRNA, protein content, as well as enzymatic activity were elevated. Furthermore, increased levels of integrin subunits α_v and β₃ were detected at the clear zone of mutant osteoclasts. OPN mRNA levels were elevated in long bones from mutants. In ia/ia rats, immunolabeling for OPN was homogeneously distributed at the surface facing osteoclasts, while in normal littermates it was concentrated at the clear zone area and barely detectable at ruffled borders. The absence of OPN labeling in the abundant, putative intracellular secretory vesicles in mutant osteoclasts suggests that these cells do not produce OPN. The osteoclasts of ia/ia rats appeared to produce and translocate the c-src protein to the cell membrane.In ia/ia a defect ruffled border-formation is observed along with extensive clear zone formation and decreased secretory function. The lesion may be due to a signaling defect, but in that case the defect seems to be located downstream to or not involving the c-src pathway. Our results illustrate the close relationship between secretory function and ruffled border formation in osteoclasts, a relationship that appears to be necessary for proper resorptive function.     

Extensive clear zone and defective ruffled border formation in osteoclasts of osteopetrotic (ia/ia) rats: implications for secretory function.

The cellular distribution of osteoclast integrin subunits alpha(v) and beta(3), the tissue distribution, and level of the apparent ligand osteopontin (OPN) as well as of the putative regulatory enzyme tartrate-resistant acid phosphatase (TRAP) were studied along with the intracellular distribution of the activation marker c-src in osteopetrotic ia/ia (incisors-absent) mutant rats and their normal littermates. In ia/ia rats, the osteoclasts are incapable of bone matrix resorption. Ultrastructurally the cells exhibit extended clear zones at the expense of ordinary ruffled borders. A secretory dysfunction in the mutant is strongly suggested by the absence of detectable extracellular TRAP, concomitant with an accumulation of the enzyme in abundant small cytoplasmic vesicles. Moreover, TRAP mRNA, protein content, as well as enzymatic activity were elevated. Furthermore, increased levels of integrin subunits alpha(v) and beta(3) were detected at the clear zone of mutant osteoclasts. OPN mRNA levels were elevated in long bones from mutants. In ia/ia rats, immunolabeling for OPN was homogeneously distributed at the surface facing osteoclasts, while in normal littermates it was concentrated at the clear zone area and barely detectable at ruffled borders. The absence of OPN labeling in the abundant, putative intracellular secretory vesicles in mutant osteoclasts suggests that these cells do not produce OPN. The osteoclasts of ia/ia rats appeared to produce and translocate the c-src protein to the cell membrane. In ia/ia a defect ruffled border-formation is observed along with extensive clear zone formation and decreased secretory function. The lesion may be due to a signaling defect, but in that case the defect seems to be located downstream to or not involving the c-src pathway. Our results illustrate the close relationship between secretory function and ruffled border formation in osteoclasts, a relationship that appears to be necessary for proper resorptive function.     

Three-dimensional distribution of the clear zone of migrating osteoclasts on dentin slices in vitro

Osteoclasts are cells that dynamically alternate resorption and migration on bone surfaces, and have the special structure called ruffled borders and clear zones by transmission electron microscopy (TEM). However, TEM features, especially the distribution of the clear zone of osteoclasts during migration, remains unclear. This study aimed to examine osteoclasts cultured on dentin slices by TEM and clarify the features of migrating osteoclasts, especially the three-dimensional distribution of clear zones. Osteoclasts obtained from mice were cultured with dentin slices for 72 h, and then cells were fixed and the tartrate-resistant acid phosphatase (TRAP) activity was detected. Specimens were embedded in Epon, then TRAP-positive cells were serially sectioned by alternating semithin and ultrathin sections. The cells were examined by TEM and the three-dimensional structures were reconstructed by computer. By TEM, most TRAP-positive cells were resorbing osteoclasts with ruffled borders and a clear zone. There were osteoclasts without ruffled borders, and these cells had clear zone-like structures and lamellipodia. The three-dimensional reconstruction showed that resorbing osteoclasts had rounded contours and ring-shaped clear zones encircling ruffled borders, and that osteoclasts without ruffled borders had irregular and flat shapes; the clear zone-like structures showed a dot or patch-like distribution. The presence of lamellipodia of the osteoclasts without ruffled borders shows that the cells are migrating osteoclasts. These results suggest that dot or patch-like distribution is the feature of the clear zone of osteoclasts during migration, and that these structures play the role of focal contacts and adhesion to the dentin surfaces during cell migration.     

Morphological evidence of reduced bone resorption in the osteosclerotic (oc) mouse

Osteopetrosis, a metabolic bone disease characterized by a generalized sclerosis of the skeleton, is inherited as an autosomal recessive in a number of mammalian species. The pathogenesis of congenital osteopetrosis is mediated by a reduction in bone resorption as a result of decreased osteoclast function. This hypothesis is based on both functional and structural evidence of reduced bone resorption in all mutations examined to date. The present study examined the histology of cartilage and bone, the ultrastructure of osteoclasts, and the morphology of mineralized bone surfaces in a lethal osteopetrotic mutation, the osteosclerotic (oc) mouse. Histologically, epiphyseal cartilage growth plates, especially the hypertrophic zone, are markedly thickened in oc mice and metaphyses contain excessive osteoid, features characteristic of rickets. Transmission electron microscopy revealed that less than one-quarter of osteoclasts in oc mice demonstrated evidence of ruffled border formation compared with three-quarters of the osteoclasts in normal littermates. In mutants, ruffled borders were less elaborate and cytoplasmic processes penetrated into bone surfaces, suggesting that bone may be removed by mechanical rather than by enzymatic means. There was little morphological evidence of cartilage degradation and broad laminae limitantes persisted in mutants. Mineralized surfaces that undergo resorption in normal mice showed no evidence of bone resorption by scanning EM in mutants. The presence of a rachitic condition, the observations of reduced bone resorption, and the possible contribution of undermineralized matrices to decreased bone resorption are characteristics of the osteosclerotic mutation which suggest that it is a unique osteopetrotic mutant in which to study both the development and regulation of skeletal metabolism.     

CARTILAGE RESORPTION IN THE TIBIAL EPIPHYSEAL PLATE OF GROWING RATS

An electron microscopic study of the tibial epiphyseal plates of growing rats reveals that the resorption of unmineralized and mineralized cartilage occurs by two different mechanisms. During resorption the unmineralized transverse cartilaginous walls between chondrocytes are invaded by capillary sprouts. At the resorption zone, numerous cytoplasmic processes derived primarily from the perivascular cells and, to a lesser extent, from the endothelial cells of the sprouts penetrate and appear to lyse the unmineralized transverse cartilaginous walls. Hydrolases released from the degenerating chondrocytes and/or capillary sprouts may also participate in this process. The second resorption mechanism involves the mineralized longitudinal cartilaginous septa. Resorption of these septa is mediated by chondroclasts whose fine structure is identical with that of osteoclasts. The active surface of the chondroclasts has a ruffled border. The surface membrane of the chondroclasts is relatively smooth on either side of the ruffled border and lies in direct apposition with the underlying mineralized cartilage. This observation suggests that the microenvironment in the zone of resorption may be maintained by the neighboring unruffled surfaces of the chondroclasts, which thus seal off and segregate the active portions of these cells.     

Increased resorptive activity and accompanying morphological alterations in osteoclasts derived from the oim/oim mouse model of osteogenesis imperfecta

The current study addresses whether alterations in osteoclasts (OCs) derived from oim/oim mice, an established model of moderate-to-severe OI, are present. Bone marrow cells from oim/oim and wildtype (+/+) mice were cultured on bone slices in the presence of MCSF and RANKL and evaluated at days 0, 1, 2, 4, and 7. OCs were identified by tartrate-resistant acid phosphatase (TRAP) staining, and bone slice resorption pits were analyzed by reflection microscopy. Flow cytometry was used to examine CD51 (integrin alphaV) and CD61 (integrin beta3) markers. Confocal microscopy was used to assess changes in OC morphology and resorption. There was no difference between the OC precursors of the two genotypes in expression of CD51 and CD61 markers. At day 2, the bone slices seeded with oim/oim cells had a greater percentage of mononuclear cells associated with resorption pits compared to +/+ bone slices. At day 4, the diameter and area of oim/oim OCs were larger compared to the +/+ OCs, and the number of nuclei per OC was also greater for the oim/oim group. At day 7, the oim/oim OCs contained more F-actin rings compared to the +/+ OCs, and the number of OCs in the oim/oim group was greater compared to the +/+ group. The resorbed area of bone slices for the oim/oim group was also greater compared to the +/+ group at day 7. In conclusion, oim/oim mononuclear resorbing cells and OCs showed cellular changes and greater resorptive activity compared to +/+ cells, features that likely contribute to dysregulated bone remodeling in OI.     

Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption

During skeletal growth and remodeling the mineralized bone matrix is resorbed by osteoclasts through the constant secretion of protons and proteases to the bone surface. This relies on the formation of specialized plasma membrane domains, the sealing zone and the ruffled border, and vectorial transportation of intracellular vesicles in bone-resorbing osteoclasts. Here we show that Rab7, a small GTPase that is associated with late endosomes, is highly expressed and is predominantly localized at the ruffled border in bone-resorbing osteoclasts. The decreased expression of Rab7 in cultured osteoclasts by antisense oligodeoxynucleotides disrupted the polarization of the osteoclasts and the targeting of vesicles to the ruffled border. These impairments caused a significant inhibition of bone resorption in vitro. The results indicate that the late endocytotic pathway is involved in the osteoclast polarization and bone resorption and underscore the importance of Rab7 in osteoclast function.     

Rab3D regulates a novel vesicular trafficking pathway that is required for osteoclastic bone resorption

Rab3 proteins are a subfamily of GTPases, known to mediate membrane transport in eukaryotic cells and play a role in exocytosis. Our data indicate that Rab3D is the major Rab3 species expressed in osteoclasts. To investigate the role of Rab3D in osteoclast physiology we examined the skeletal architecture of Rab3D-deficient mice and found an osteosclerotic phenotype. Although basal osteoclast number in null animals is normal the total eroded surface is significantly reduced, suggesting that the resorptive defect is due to attenuated osteoclast activity. Consistent with this hypothesis, ultrastructural analysis reveals that Rab3D(-/-) osteoclasts exhibit irregular ruffled borders. Furthermore, while overexpression of wild-type, constitutively active, or prenylation-deficient Rab3D has no significant effects, overexpression of GTP-binding-deficient Rab3D impairs bone resorption in vitro. Finally, subcellular localization studies reveal that, unlike wild-type or constitutively active Rab3D, which associate with a nonendosomal/lysosomal subset of post-trans-Golgi network (TGN) vesicles, inactive Rab3D localizes to the TGN and inhibits biogenesis of Rab3D-bearing vesicles. Collectively, our data suggest that Rab3D modulates a post-TGN trafficking step that is required for osteoclastic bone resorption.     

THE EFFECTS OF PARATHYROID HORMONE, COLCHICINE, AND CALCITONIN ON THE ULTRASTRUCTURE AND THE ACTIVITY OF OSTEOCLASTS IN ORGAN CULTURE

The ultrastructure of osteoclasts was examined in fetal rat bones after stimulation or inhibition of resorption in culture. A central ruffled border area completely encircled by a clear zone was considered to represent the resorbing system of the cell. The proportion of ruffled border and clear zone in osteoclast cross sections was compared with changes in bone resorption as measured by the release of previously incorporated radioactive calcium (⁴⁵Ca). In control cultures 55% of the osteoclast cross sections showed an area closely apposed to bone and this consisted mainly of clear zone; only 11% showed ruffled borders. Treatment with parathyroid hormone (PTH) increased ⁴⁵Ca release, increased the frequency of finding areas closely apposed to bone (79%), and markedly increased the frequency of the ruffled border area (64%). Colchicine given concurrently with PTH decreased the number of osteoclasts. Colchicine or calcitonin treatment after PTH stimulation decreased the proportion of ruffled border area significantly by 1 h; this was followed by a decrease in ⁴⁵Ca release. These inhibited osteoclasts resembled osteoclasts from control, unstimulated cultures, suggesting that the cells had returned to their inactive state. Colchicine-treated osteoclasts also showed a loss of microtubules and a massive accumulation of 100 Å filaments, suggesting that synthesis of microtubular subunits had increased.     

Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail

The medullary bone serves as a source of labile calcium mobilized during calcification of the egg shell in birds. Quantitative histological methods demonstrate that the numbers of medullary bone osteoclasts and nuclei per osteoclast remain unchanged during the egg cycle in the Japanese quail (Coturnix). Therefore, cyclic changes in bone resorption cannot be explained by modulations of osteoclasts from and into other bone cells, a mechanism previously suggested for certain species of birds. Rather, dramatic changes in osteoclast cell-surface features occur during the egg cycle, which might account for cyclic variations in resorptive activity. During egg shell calcification, osteoclasts with ruffled borders are closely apposed to bone surfaces; the cytoplasm is rich in vacuoles that contain mineral crystals and seem to derive from the ruffled border. At the completion of egg shell calcification, the ruffled borders and vacuoles move away from the bone surface, although the osteoclast remains attached to the bone along the filamentous or "clear" zone. Associated with the disappearance of the ruffled borders is the appearance of extensive interdigitated cell processes along the peripheral surface of the osteoclast away from the bone. These unusual structures, which may serve as a reservoir of membrane, largely disappear when ruffled borders and associated structures reappear. Therefore, in these hens, the osteoclasts modulate their cell surface rather than their population during the egg cycle.     

Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts

Bone resorption relies on the extracellular acidification function of V-ATPase (vacuolar-type proton-translocating ATPase) proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of the osteoclast-specific ruffled border V-ATPases remains largely unknown. In the present study, we found that the V-ATPase subunit Atp6v1c1 (C1) is highly expressed in osteoclasts, whereas subunits Atp6v1c2a (C2a) and Atp6v1c2b (C2b) are not. The expression level of C1 is highly induced by RANKL [receptor activator for NF-kappaB (nuclear factor kappaB) ligand] during osteoclast differentiation; C1 interacts with Atp6v0a3 (a3) and is mainly localized on the ruffled border of activated osteoclasts. The results of the present study show for the first time that C1-silencing by lentivirus-mediated RNA interference severely impaired osteoclast acidification activity and bone resorption, whereas cell differentiation did not appear to be affected, which is similar to a3 silencing. The F-actin (filamentous actin) ring formation was severely defected in C1-depleted osteoclasts but not in a3-depleted and a3(-/-) osteoclasts. C1 co-localized with microtubules in the plasma membrane and its vicinity in mature osteoclasts. In addition, C1 co-localized with F-actin in the cytoplasm; however, the co-localization chiefly shifted to the cell periphery of mature osteoclasts. The present study demonstrates that Atp6v1c1 is an essential component of the osteoclast proton pump at the osteoclast ruffled border and that it may regulate F-actin ring formation in osteoclast activation.     

A histomorphometric, structural, and immunocytochemical study of the effects of diet-induced hypocalcemia on bone in growing rats.

Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal-metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell-matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone-calcified cartilage interface in some cases were thickened but generally did not appear affected at bone-bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption.     

Atherogenic diet-induced bone loss is primarily due to increased osteoclastogenesis in mice

Atherogenic diet (AD) decreased bone density and increased serum cholesterol level in male mice, implying that cholesterol participates in bone loss. The aim of the present study was to identify the cells responsible for bone loss and evaluate the involved mechanism. AD resulted in increased number and surface of osteoclasts (OCs) with in vivo tartrate-resistant acid phosphatase (TRAP) staining, suggesting a critical role of OCs in cholesterol-induced bone loss. In vitro, cholesterol loading by oxidized low-density lipoprotein (oxLDL) increased the size and number of OCs as well as bone resorption activity, suggesting that cholesterol loading affects the number and activity of OCs. In contrast, cholesterol depletion by simvastatin decreased osteoclastogenesis and bone resorption. oxLDL stimulated osteoblasts (OBs) to increase expression of receptor activator of nuclear factor kappa-Β ligand (RANKL), resulting in increased OC formation when OBs were co-cultured with bone marrow derived macrophages. oxLDL increased expression of CD36 and liver X receptors (LXRα) in OCs as well as low density lipoprotein receptor (LDLR) and LXRα in OBs. These results suggest that CD36 and LXRα mediate the effect of oxLDL in OCs, whereas LDLR and LXRα mediate the effect of oxLDL in OBs. These findings demonstrate cholesterol-induced bone loss with increasing number and activity of OCs in mice, suggesting another harmful effect of cholesterol, a major cause of atherosclerosis.     

Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling

Endochondral ossification in the epiphyseal growth plate of long bones is associated with programmed cell death (PCD) of a major portion of the chondrocytes. Here we tested the hypothesis that at the ossification front of the epiphyseal growth plate osteoclasts preferentially phagocytose chondrocytes that are undergoing PCD. We injected biotin-labelled annexin-V (anx-V-biotin, an early marker of PCD) intravenously in young adult mice. After 30 min of labelling, long bones were recovered and the tissue distribution examined of anx-V-biotin-labelled cells in the growth plate using ABC-peroxidase histochemistry. Positive staining for anx-V-biotin was detected in hypertrophic chondrocytes still present in closed lacunae at some distance from the ossification front. At the ossification front, chondrocyte lacunae were opened and close contacts were seen between tartrate-resistant acid phosphatase-positive osteoclasts and hypertrophic cartilage cells. Osteoclasts were significantly more frequently in contact with anx-V-biotin-labelled chondrocytes than with unlabelled chondrocytes. Osteoclasts also contained labelled and unlabelled phagocytic fragments within their cytoplasm. We conclude that in the growth plate osteoclasts preferentially phagocytose hypertrophic chondrocytes that are dying, suggesting these dying cells may signal osteoclasts for their removal.     

Na+/H+ antiporter is the primary proton transport system used by osteoclasts during bone resorption

We have examined the effects of inhibitors of proton transport systems on osteoclastic bone resorption using an in vitro bone slice assay, where osteoclasts (OCs) are free from the influence of other bone cells. Amiloride (AM) and dimethylamiloride (DMA), inhibitors of the Na+/H+ antiporter, were potent inhibitors of bone resorption (IC50 approximately 9 and 0.7 microM for AM and DMA, respectively). Omeprazole (OM), a potent inhibitor of parietal cell K+/H+(-)ATPase, was a poor inhibitor of OC bone resorption (IC50 approximately 100 microM). These results strongly suggest that the Na+/H+ antiporter is the primary proton system used by OCs during bone resorption.     

Integrin subunit β3 plays a crucial role in the movement of osteoclasts from the periosteum to the bone surface

We have shown that, when mouse parietal bones were incubated in culture medium containing indomethacin, the number of tartrate-resistant acid phosphatase-positive osteoclasts (TRAP + OCs) on the bone surface was drastically reduced (down-regulation), and the number on the periosteal membrane adjacent to the resorbing surface was increased. Subsequent incubation of bones with prostaglandin E2 (PGE2) rapidly reversed these changes (up-regulation). In the work reported here, the osteoclast-associated integrin subunit β3 was stained by immunohistochemistry. The β3-positive osteoclast (β3 + OC) population on freshly isolated bone was comprised of about 67% TRAP + OCs and 33% TRAP − OCs. Like TRAP + OCs, β3 + OCs were reduced in number on the surface of bones incubated with indomethacin, but, in contrast to the TRAP + OCs, β3 + OCs were not seen on the periosteal membrane. Following up-regulation of TRAP + OCs with PGE2, large numbers of β3 + OCs appeared on the bone surface and, again, were not seen on the periosteal membrane. Echistatin, a peptide that binds to the αvβ3 integrin on osteoclasts, was found to inhibit the up-regulation of TRAP + OCs in a dose-dependent manner but had no effect on the down-regulation of TRAP + OCs. Similarly, echistatin inhibited the up-regulation of β3 + OCs on the bone surface, and, under these conditions, β3 + OCs were observed on the periosteal membrane. The addition of anti-β3 antibody also inhibited the up-regulation of TRAP + OCs in response to PGE2. The association of β3 protein expression with the up-regulated osteoclast and the inhibition of up-regulation by echistatin and by anti-β3 antibody provide strong evidence that β3 plays an essential role in the movement of osteoclasts from the membrane to the bone. J. Cell. Physiol. 175:1–9, 1998. © 1998 Wiley-Liss, Inc.     

Synaptotagmin VII regulates bone remodeling by modulating osteoclast and osteoblast secretion

Maintenance of bone mass and integrity requires a tight balance between resorption by osteoclasts and formation by osteoblasts. Exocytosis of functional proteins is a prerequisite for the activity of both cells. In the present study, we show that synaptotagmin VII, a calcium sensor protein that regulates exocytosis, is associated with lysosomes in osteoclasts and bone matrix protein-containing vesicles in osteoblasts. Absence of synaptotagmin VII inhibits cathepsin K secretion and formation of the ruffled border in osteoclasts and bone matrix protein deposition in osteoblasts, without affecting the differentiation of either cell. Reflecting these in vitro findings, synaptotagmin VII-deficient mice are osteopenic due to impaired bone resorption and formation. Therefore, synaptotagmin VII plays an important role in bone remodeling and homeostasis by modulating secretory pathways functionally important in osteoclasts and osteoblasts.