Echistatin is a potent inhibitor of bone resorption in culture

The venom protein, s-echistatin, originally derived from the saw-scaled viper Echis carinatus, was found to be a potent inhibitor of bone resorption by isolated osteoclasts. This Arg24-Gly25-Asp26-(RGD)-containing protein inhibited the excavation of bone slices by rat osteoclasts (IC50 = 0.1 nM). It also inhibited the release of [3H]proline from labeled bone particles by chicken osteoclasts (IC50 = 100 nM). By comparison, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited resorption by rat or chicken osteoclasts with an IC50 of 0.1 mM while ala24-echistatin was inactive. Video microscopy showed that rat osteoclast attachment to substrate was more sensitive to s-echistatin than was the attachment of mononuclear cells or chicken osteoclasts. The difference in sensitivity of rat and chicken osteoclasts to s-echistatin may be due to differences between receptors on rat and chicken osteoclasts for s-echistatin. Antibody localization of echistatin on these cells showed much greater echistatin binding to rat osteoclasts than to chicken osteoclasts. Laser scanning confocal microscopy after immunohistochemical staining showed that s-echistatin binds to osteoclasts, that s-echistatin receptors are most abundant at the osteoclast/glass interface, and that s-echistatin colocalizes with vinculin. Confocal interference reflection microscopy of osteoclasts incubated with s-echistatin, demonstrated colocalization of s-echistatin with the outer edges of clusters of grey contacts at the tips of some lamellipodia. Identification of the echistatin receptor as an integrin was confirmed by colocalization of echistatin fluorescence with staining for an alpha-like subunit. Attachment of bone particles labeled with [3H]proline to chicken osteoclasts confirmed that the mechanism of action of echistatin was to inhibit osteoclast binding to bone presumably by disrupting adhesion structures. These data demonstrate that osteoclasts bind to bone via an RGD-sequence as an obligatory step in bone resorption, that this RGD-binding integrin is at adhesion structures, and that it colocalizes with vinculin and has an alpha-like subunit.     

Immunohistochemical localization of membrane type 1-matrix metalloproteinase (MT1-MMP) in osteoclasts in vivo

Membrane type 1-matrix metalloproteinase (MT1-MMP) is capable of mediating proteolysis of extracellular matrix. The enzyme has been demonstrated in osteoclasts, in vitro. However, the precise localization in vivo, and therefore the function of the enzyme in osteoclasts, is still unclear. In this study, we immunohistochemically examined the localization of MT1-MMP in rat osteoclasts to clarify the role of MT1-MMP in osteoclastic bone resorption and bone turnover. The localization of MT1-MMP was visualized by the pre-embedding method using anti-MT1-MMP antibody and horseradish peroxidase (HRP) or gold-conjugated antibody. Immunoreactivity of anti-MT1-MMP was found in osteoclasts at the osteoclast-bone interface, but it was not uniform. Ultrastructurally, the immunoreactivity visualized by HRP was found in sealing zone. The plasma membrane at this site showed an irregular border and some invaginations. Immunoreactivity was also found on the surface of certain small vesicles in the cytoplasm. Enhanced silver granules were mainly associated with the sealing membrane. In this study, we demonstrated, for the first time, the localization of MT1-MMP in the sealing zone of osteoclast in vivo. Its distribution suggests that the enzyme modifies the bone surface to facilitate the migration and attachment of osteoclasts as well as scavenging the resorption lacunae.     

Involvement of calpain in osteoclastic bone resorption

There is increasing evidence that calpain contributes to the reorganization of the cytoskeleton in the integrin-mediated signaling pathway. Osteoclastic bone resorption requires cell-matrix contact, an event mediated by integrin alphavbeta3, and subsequent cytoskeletal reorganization to form characteristic membrane domains such as the sealing zone and ruffled border. In this study, therefore, we investigated whether calpain is involved in osteoclastic bone resorption. Membrane-permeable calpain inhibitors suppress the resorption activity of human osteoclasts, but an impermeable inhibitor does not. Upon the attachment of osteoclasts to bone, micro-calpain is translocated from the cytosolic to the cytoskeletal fraction and is autolytically activated. Both the activation of micro-calpain and the formation of actin-rings, the cytoskeletal structures essential for bone resorption, are inhibited by membrane-permeable calpain inhibitors. The activated micro-calpain in osteoclasts selectively cleaves talin, which links the matrix-recognizing integrin to the actin cytoskeleton. These findings suggest that calpain is a regulator of the bone resorption activity of osteoclasts through reorganization of the cytoskeleton related to actin-ring formation.     

Specific antagonists of NMDA receptors prevent osteoclast sealing zone formation required for bone resorption

N-Methyl-d-aspartate (NMDA) glutamate receptors, widely distributed in the nervous system, have recently been identified in bone. They are expressed and are functional in osteoclasts. In the present work, we have studied the effects of specific antagonists of NMDA receptors on osteoclast activation and bone resorption. Using an in vitro assay of bone resorption, we showed that several antagonists of NMDA receptors binding to different sites of the receptor inhibit bone resorption. Osteoclast activation requires adhesion to the bone surface, cytoskeletal reorganization and survival. We demonstrated by autoradiography that the specific NMDA receptor channel blocker, MK 801, binds to osteoclasts. This antagonist had no effect on osteoclast attachment to bone and did not induce osteoclast apoptosis. In contrast, MK 801 rapidly decreased the percentage of osteoclasts with actin ring structures that are associated with actively resorbing osteoclasts. These results suggest that NMDA receptors expressed by osteoclasts may be involved in adhesion-induced formation of the sealing zone required for bone resorption.     

c-Src-mediated Phosphorylation of Thyroid Hormone Receptor-interacting Protein 6 (TRIP6) Promotes Osteoclast Sealing Zone Formation

Osteoclasts resorb bone through the formation of a unique attachment structure called the sealing zone. In this study, a role for thyroid hormone receptor-interacting protein 6 (TRIP6) in sealing zone formation and osteoclast activity was examined. TRIP6 was shown to reside in the sealing zone through its association with tropomyosin 4, an actin-binding protein that regulates sealing dimensions and bone resorptive capacity. Suppression of TRIP6 in mature osteoclasts by RNA interference altered sealing zone dimensions and inhibited bone resorption, whereas overexpression of TRIP6 increased the sealing zone perimeter and enhanced bone resorption. Treatment of osteoclasts with lysophosphatidic acid (LPA), which phosphorylates TRIP6 at tyrosine 55 through a c-Src-dependent mechanism, caused increased association of TRIP6 with the sealing zone, as did overexpression of a TRIP6 cDNA bearing a phosphomimetic mutation at tyrosine 55. Further, LPA treatment caused increases in osteoclast fusion, sealing zone perimeter, and bone resorptive capacity. In contrast, overexpression of TRIP6 containing a nonphosphorylatable amino acid residue at position 55 severely diminished sealing zone formation and bone resorption and suppressed the effects of LPA on the cytoskeleton. LPA effects were mediated through its receptor isoform LPA(2), as indicated by treatments with receptor-specific agonists and antagonists. Thus, these studies suggest that TRIP6 is a critical downstream regulator of c-Src signaling and that its phosphorylation is permissive for its presence in the sealing zone where it plays a positive role in osteoclast bone resorptive capacity.     

Ultrastructural localization and biochemical characterization of vitronectin in developing rat bone

This study has used light and electron microscope immunohistochemical and biochemical methods to localize and characterize vitronectin in early bone formation of developing rat mandible with rabbit antimurine vitronectin IgG. Developing jaws of foetuses were collected at embryonic day 15 (day 15) to day 18 from pregnant Wistar rats. After aldehyde fixation, specimens with and without osmium post-fixation were dehydrated and embedded in paraffin, Spurr's resin or LR gold resin for morphological and immunohistochemical examinations. At the light microscope level, in day 15 samples, positive vitronectin immunostaining was observed in small elongated areas of intercellular matrix and osteoblasts. Concomitant with initiation of matrix mineralization at day 16, vitronectin staining was similarly observed in small elongated areas containing intercellular matrix and osteoblasts but not clearly detected in fully mineralized bone matrix. The same staining profile was observed at days 17 and 18. At the ultrastructural level, immunogold particles were clearly detected over unmineralized matrix and cisterns of the rough-surfaced endoplasmic reticulum and the Golgi apparatus of osteoblasts as well as over demineralized bone matrix at day 16--18. In order to assess the presence of vitronectin in the mineral phase, mineral-binding bone proteins were extracted from fresh day 18 specimens using a three-step technique: 4 m guanidine HCl (G1 extract), aqueous EDTA without guanidine HCl (E extract), followed by guanidine HCl. Subsequent Western blot analysis of sodium dodecyl sulphate (SDS)--polyacrylamide gel electrophoresis revealed that the antibodies produced only a single band at an Mr of approximately 73 000 in both G1 and E extracts, indicating the presence of vitronectin in the mineralized bone matrix. These results indicate that, at the onset of bone formation, osteoblasts synthesize and release vitronectin, which is subsequently incorporated into the bone matrix and becomes a specific component of bone tissues. The observation of vitronectin in these critical stages of bone formation suggests that it may be involved in the regulation of bone formation. © 1998 Chapman & Hall     

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.     

Defective microtubule-dependent podosome organization in osteoclasts leads to increased bone density in Pyk2(-/-) mice

The protein tyrosine kinase Pyk2 is highly expressed in osteoclasts, where it is primarily localized in podosomes. Deletion of Pyk2 in mice leads to mild osteopetrosis due to impairment in osteoclast function. Pyk2-null osteoclasts were unable to transform podosome clusters into a podosome belt at the cell periphery; instead of a sealing zone only small actin rings were formed, resulting in impaired bone resorption. Furthermore, in Pyk2-null osteoclasts, Rho activity was enhanced while microtubule acetylation and stability were significantly reduced. Rescue experiments by ectopic expression of wild-type or a variety of Pyk2 mutants in osteoclasts from Pyk2(-/-) mice have shown that the FAT domain of Pyk2 is essential for podosome belt and sealing zone formation as well as for bone resorption. These experiments underscore an important role of Pyk2 in microtubule-dependent podosome organization, bone resorption, and other osteoclast functions.     

Bone acidic glycoprotein 75 inhibits resorption activity of isolated rat and chicken osteoclasts.

Matrix protein effects on the differentiated activity of osteoclasts were examined in order to understand the functional significance of bone protein interactions with osteoclasts. Bone acidic glycoprotein 75 (BAG 75) from rat calvariae inhibited the resorption of bone by isolated rat osteoclasts with IC50 = 1 nM compared to IC50 = 10 nM for chicken osteoclasts. By contrast, other phosphoproteins similarly isolated from bone were less effective in inhibiting resorption with IC50 = 100 nM osteopontin and IC50 greater than 100 nM bone sialoprotein. Likewise, RGD-containing matrix proteins vitronectin, thrombospondin, and fibronectin all displayed IC50 greater than or equal to 100 nM. Mechanistically, 10 nM BAG 75 marginally slowed, but did not block, the association of bone particles with chicken osteoclasts compared with osteopontin or control media. Pretreatment of osteoclasts with 50 nM BAG 75 had no effect on subsequent bone resorption; however, pretreatment of bone with BAG 75 before incubation with osteoclasts reduced the extent of resorption by 55%. These data suggest that a BAG 75/bone surface complex, rather than BAG 75 alone, represents the inhibitory form. Consistent with this hypothesis, direct binding studies provided no evidence of specific, high-affinity receptors on osteoclasts for BAG 75, nor was an excess of BAG 75 (100 nM) able to compete with 0.3 nM sechistatin for osteoclastic avB3-like receptors. However, BAG 75 displayed cooperative binding to tissue fragments and bone particles at concentrations greater than 10 nM, suggesting that BAG 75 self-associates into higher-order species on bone surfaces. Electron microscopy confirmed the time-dependent polymerization of BAG 75 into interconnecting filaments. These data suggest a novel, inhibitory activity for surface-bound BAG 75 on bone resorption that does not appear to involve the osteoclastic avB3-like integrin.     

Inhibition of osteoclast function by adenovirus expressing antisense protein-tyrosine kinase 2

Osteoclast activation is initiated by adhesion to bone, cytoskeletal rearrangement, formation of the sealing zone, and formation of the polarized ruffled membrane. Previous findings suggest that protein-tyrosine kinase 2 (PYK2), a cytoplasmic kinase related to focal adhesion kinase, participates in these events. This study examines the role of PYK2 in adhesion-mediated signaling and osteoclast function, using PYK2 antisense. We produced a recombinant adenovirus containing a 300-base pair reversed 5'-coding region of PYK2 and used full-length PYK2 as a control. Murine osteoclast-like cells or their mononuclear precursors were generated in a co-culture of bone marrow and osteoblasts. Infection with antisense adenovirus significantly reduced the expression of endogenous PYK2 protein relative to uninfected cells or to cells infected with sense PYK2 and caused: 1) a reduction in osteoclast formation in vitro; 2) inhibition of cell spreading and of actin ring formation in osteoclasts plated on glass or bone and of attachment and spreading of osteoclast precursors plated on vitronectin; 3) inhibition of bone resorption in vitro; 4) marked reduction in p130(Cas) tyrosine phosphorylation; and 5) no change in alpha(v)beta(3) integrin expression or c-Src tyrosine phosphorylation. Taken together, these findings support the hypothesis that PYK2 plays a central role in the adhesion-dependent cytoskeletal organization and sealing zone formation required for osteoclastic bone resorption.     

Blockade of osteoclast-mediated bone resorption through occupancy of the integrin receptor: A potential approach to the therapy of osteoporosis

Bone resorption requires the tight attachment of the bone-resorbing cells, the osteoclasts, to the bone mineralized matric. Integrins, a class of cell surface adhesion glycoproteins, play a key role in the attachment process. Most integrins bind to their ligands via the arginyl-glycyl-aspartyl (R-G-D) tripeptide present within the ligand sequence. The interaction between integrins and ligaands results in bidirectional transfer of signals across the plasma Membrane. Tyrosine phosphorylaation occurs within cells as a result of integrin binding to ligaands and probably plays a role in the formation of the osteoclast clear zone, a specialized region of the osteoclast membraane maintained by cytoskeletal structure and involved in bone resorption. Human osteoclasts express α₂β₃ and α_vβ₃ integrins on their surface. Such signaling may also lead to “inside-out” effects, like increased expression of integrin receptors on the cell surface, or increased affinity of the integrin to its ligand. The α_vβ₃ integrin, a vitronectin receptor, plays an essential role in bone resorption. Antibodies to this integrin and short synthetic RGD-containing peptides are able to block bone resorption in vitro. Echistatin, an RGD-containing protein from a snake venom, binds to the α_vβ₃ integrin and blocks bone resssorption both in vitro and in vivo. Peptides containing the RGD motif are potential competitive “antagonists” a of the osteoclast integrins and may have utility in the blockage of bone resorption. Agonists may be identified by stimulation of intracellularsignaling. In theory, tissue spacificity can be achieved by (1) introducing specific amino acids in positions adjacent to the RGD sequence, (2) identifying non-RGD integrin binding domains, or (3) modulating the affinity of integrins for their endogenous ligands.     

Distinctive Subdomains in the Resorbing Surface of Osteoclasts

We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a ‘sealing zone’, encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but also to facilitate resorption by acting as a ‘functional sealing zone’.     

Glutamatergic regulation of bone remodeling

L-glutamate (Glu) is the predominant neuromediator in the mammalian central nervous system (CNS). Bone is highly innervated and there is growing evidence of a neural control of bone cell metabolism. The recent discovery of Glu-containing nerve fibers in bone and Glu receptors (GluR) and transporters in bone cells suggest that this neuromediator may also act as a signaling molecule in bone and regulate bone cell function. Our previous studies have demonstrated that ionotropic N-Methyl-D-Aspartate (NMDA) GluR are highly expressed by mammalian osteoclasts. NMDA receptors (NMDAR) are heteromers associating the NR1 subunit and one of the four types of NR2 subunits (NR2A to D). We showed that osteoclasts express NR1, NR2B and NR2D subunits, suggesting a molecular diversity of NMDAR in these cells. Electrophysiological studies have confirmed that NMDAR are functional in mature osteoclasts, and features of Glu-induced current recorded in these cells indicate a major NR2D subunit composition. Using an in vitro assay of bone resorption, we showed that several antagonists of NMDAR binding to different sites of the receptor inhibit bone resorption. In particular, the specific NMDAR channel blocker MK801 had no effect on osteoclast attachment to bone and survival while it rapidly decreased the percentage of osteoclasts with actin ring structures that are associated with actively resorbing osteoclasts. NMDAR may thus be involved in adhesion-induced formation of the sealing zone required for bone resorption. NMDAR are also expressed by osteoclast precursors isolated from mouse bone marrow. We recently confirmed the presence of NR1, NR2B and NR2D in these cells and demonstrated their expression at all differentiation stages from osteoclast precursors to mature resorbing osteoclasts. No regulation of these subunits mRNA expression levels was observed throughout the osteoclastic differentiation sequence. Activation of NMDAR may therefore represent a new mechanism for regulating osteoclast formation and activity. While the origin of Glu in bone is still unknown, the possibility of a glutamatergic neurotransmission in this tissue is suggested by the detection of Glu in nerve fibers in close contact to bone cells. Furthermore, we recently demonstrated that sciatic neurectomy in growing rats induces a bone loss associated with a reduction of nerve profiles immunostained for Glu. These results suggest that Glu may be released from glutamatergic nerve profiles present in bone and therefore contribute to the local regulation of bone cell function.     

Immunolocalization of beta 3 subunit of integrins in osteoclast membrane

Utilizing isolated and cultured osteoclasts it has been possible to establish that they adhere to the substrate through specialized close contact areas, the podosomes, that in fully spread osteoclasts in vitro or in vivo are located within the clear zone. The cytochemical organization of podosomes has further been investigated in order to elucidate their possible involvement in the control of substrate recognition, that precedes bone resorption. An immunofluorescence investigation, performed utilizing human osteoclasts, shows that the beta 2 integrin subunit that in human monocytes is expressed and located in podosomes is absent in human osteoclasts, while the beta 3 subunit of the vitronectin receptor is expressed by osteoclasts, but not by other monocyte-derived cells and colocalizes with vinculin around the actin core of the podosome. The beta 1 subunit of the fibronectin receptors is also found, but with a diffuse pattern, in the osteoclast membrane. These results indicate that podosomes, while present in different cell types, may have in the osteoclast an unique cytochemical organization related to the peculiar function of this cell.     

Evidence that failure of osteoid bone matrix resorption is caused by perturbation of osteoclast polarization

Osteoclasts resorb bone by a complex dynamic process that initially involves attachment, polarization and enzyme secretion, followed by their detachment and migration to new sites. In this study, we postulated that mineralized and osteoid bone matrix signal osteoclasts differently, resulting in the resorption of mineralized bone matrix only. We, therefore, compared the cytoplasmic distribution of cytoskeletal proteins F-actin and vinculin using confocal laser-scanning microscopy in osteoclasts cultured on mineralized and demineralized bone slices and correlated the observations with their functional activity. Our results have demonstrated significant differences in F-actin and vinculin staining patterns between osteoclasts cultured on mineralized bone matrix and those on demineralized bone matrix. In addition, the structural variations were accompanied by significant differences in bone resorbing activity between osteoclasts grown on mineralized bone matrix and those on demineralized bone matrix after 24 h of culture -- resorption only occurring in mineralized bone but not in demineralized bone. These results indicated that failure of osteoid bone resorption is caused by perturbation of osteoclast polarization. © 1998 Chapman & Hall     

Apatite-mediated actin dynamics in resorbing osteoclasts

The actin cytoskeleton is essential for osteoclasts main function, bone resorption. Two different organizations of actin have been described in osteoclasts, the podosomes belt corresponding to numerous F-actin columns arranged at the cell periphery, and the sealing zone defined as a unique large band of actin. To compare the role of these two different actin organizations, we imaged osteoclasts on various substrata: glass, dentin, and apatite. Using primary osteoclasts expressing GFP-actin, we found that podosome belts and sealing zones, both very dynamic actin structures, were present in mature osteoclasts; podosome belts were observed only in spread osteoclasts adhering onto glass, whereas sealing zone were seen in apico-basal polarized osteoclasts adherent on mineralized matrix. Dynamic observations of several resorption cycles of osteoclasts seeded on apatite revealed that 1) podosomes do not fuse together to form the sealing zone; 2) osteoclasts alternate successive stationary polarized resorption phases with a sealing zone and migration, nonresorption phases without any specific actin structure; and 3) apatite itself promotes sealing zone formation though c-src and Rho signaling. Finally, our work suggests that apatite-mediated sealing zone formation is dependent on both c-src and Rho whereas apico-basal polarization requires only Rho.     

Arg-Gly-Asp (RGD) peptides and the anti-vitronectin receptor antibody 23C6 inhibit dentine resorption and cell spreading by osteoclasts.

Studies with a range of monoclonal and polyclonal antisera to components of the human, rat, and chick vitronectin receptor, alpha V beta 3, and the VLA beta 1 chain show that chick and rat osteoclasts express similar integrin receptors to those described in man. Biochemical analysis with monoclonal antibody 23C6 confirmed the presence on chick osteoclasts of a vitronectin receptor heterodimer of similar size (110/95 kDa reduced) to that immunoprecipitated from human osteoclastoma giant cells. The synthetic peptide GRGDSP, corresponding to the cell adhesion sequence in fibronectin, but not GRGESP peptide, induced significant (P less than 0.005) osteoclast retraction in chick and rat osteoclasts at IC50s (+/- SEM) of 210.0 +/- 14.4 and 191.4 +/- 13.7 microM, respectively; monoclonal anti-vitronectin receptor alpha V beta 3 complex antibody, 23C6, produced similar changes in chick osteoclasts (IC50 = 1.45 +/- 0.22 microM). Antibody 23C6 inhibited the number of pits resorbed in dentine by chick osteoclasts over a concentration range of 4.4 to 88 micrograms/ml; a significant 76% reduction (P = 0.03) was observed at a final concentration of 88 micrograms/ml (6 microM). The effect of peptides upon dentine resorption was less dramatic. No consistent inhibition was seen using chick osteoclasts. Inhibitory effects on resorption by rat osteoclasts were, however, observed; significant reduction in resorption occurred with both GRGDSP (78%; P less than 0.01) and GRGESP (67%; P = 0.02) peptides at 400 microM peptide concentration. These data demonstrate that osteoclast function can be disrupted by low concentrations of the anti-vitronectin receptor antibody, 23C6. The inhibitory effects of the peptides used in this study produced effects on dentine resorption which were generally weaker and variable, although osteoclast cell adhesion was consistently inhibited in an Arg-Gly-Asp (RGD)-dependent manner. We conclude that the vitronectin receptor may play an important role in effecting resorption of mineralized tissues by osteoclasts.     

Sex differences in bone resorption in the mouse femur

Summary In male and female dd-mice at 4, 7, and 14 weeks of age and in 7 and 14-week-old mice gonadectomized at 4 weeks of age, the number of osteoclasts and the number and size of bone resorption areas along the surface of bone trabeculae in the distal metaphysis of the femur were determined. Osteoclasts were counted at the light-microscopic level in paraffin sections of decalcified femora. The number and size of the bone resorption areas were examined by scanning electron microscopy of femora after removing organic material by means of KOH and NaOCl treatment. In untreated mice, the number of osteoclasts and the number and size of bone resorption areas showed no sex differences at 4 weeks of age but were larger in females than males at 7 and 14 weeks of age. In gonadectomized mice, the number of osteoclasts and the bone resorption areas increased in males and decreased in females. The results of the gonadectomy experiments suggest that bone resorption in young adult mice is stimulated by female sex hormone and inhibited by male sex hormone.     

Osteoclast and its roles in calcium metabolism and bone development and remodeling

Osteoclasts are multinucleated cells responsible for bone resorption and play important roles in normal skeletal development, in the maintenance of its integrity throughout life, and in calcium metabolism. During bone resorption, the cytoskeleton of osteoclasts undergoes extensive reorganization, with polarization and formation of ruffled borders to secrete acid and formation of sealing zone to prevent leakage. The differentiation and function of osteoclasts are in turn regulated by osteoblasts, stromal cells, and bone. They are also subjected to negative feedback regulation by extracellular and intracellular calcium concentrations.     

The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation.

Recent identification in bone of transporters, receptors, and components of synaptic signaling suggests a role for glutamate in the skeleton. We investigated effects of glutamate and its antagonist MK801 on osteoclasts in vitro. Glutamate applied to patch clamped osteoclasts induced significant increases in whole-cell membrane currents (P<0.01) in the presence of the coagonist glycine. Agonist-elicited currents were significantly decreased after application of MK801 (100 microM, P<0.01), but MK801 had no effect on actin ring formation necessary for osteoclast polarization, attachment, and resorption. In cocultures of bone marrow cells and osteoblasts in which osteoclasts develop, MK801 inhibited osteoclast differentiation and reduced resorption of pits in dentine (3 to 100 microM; P<0.001). MK801 added early in the culture (for as little as 2-4 days) was as effective as addition for the entire culture period. Addition of MK801 for any time after day 7 of culture was ineffective in reducing osteoclast activity. Using rat and rabbit mature osteoclasts cultured on dentine or explants of mouse calvariae prelabeled with (45)Ca, we could not detect significant effects of MK801 on osteoclastic resorption. These data show clearly that glutamate receptor function is critical during osteoclastogenesis and suggest that glutamate is less important in regulating mature osteoclast activity.-Peet, N. M., Grabowski, P. S., Laketic-Ljubojevic, I., Skerry, T. M. The glutamate receptor antagonist MK801 modulates bone resorption in vitro by a mechanism predominantly involving osteoclast differentiation.