Possible role of direct Rac1-Rab7 interaction in ruffled border formation of osteoclasts

Rab7 has been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts, it is involved in formation of the ruffled border, which is a late endosomal-like compartment in the plasma membrane. Here we report a new Rab7-interacting protein, Rac1, another small GTPase protein that binds to the GTP-form of Rab7 as found with a two-hybrid system. We demonstrate further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, partial colocalization is perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments enabling endosomal vesicles to switch tracks and may thus also regulate ruffled border formation in osteoclasts.     

Intracellular membrane trafficking pathways in bone-resorbing osteoclasts revealed by cloning and subcellular localization studies of small GTP-binding rab proteins

A variety of intracellular membrane trafficking pathways are involved in establishing the polarization of resorbing osteoclasts and regulating bone resorption activities. Small GTP-binding proteins of rab family have been implicated as key regulators of membrane trafficking in mammalian cells. Here we used a RT-PCR-based cloning method and confocal laser scanning microscopy to explore the expression array and subcellular localization of rab proteins in osteoclasts. Rab1B, rab4B, rab5C, rab7, rab9, rab11B, and rab35 were identified from rat osteoclasts in this study. Rab5C may be associated with early endosomes, while rab11B is localized at perinuclear recycling compartments and may function in the ruffled border membrane turnover and osteoclast motility. Interestingly, late endosomal rabs, rab7, and rab9, were found to localize at the ruffled border membrane indicating a late endosomal nature of this specialized plasma membrane domain in resorbing osteoclasts. This also suggests that late endocytotic pathways may play an important role in the secretion of lysosomal enzymes, such as cathepsin K, during bone resorption.     

Vesicular trafficking in osteoclasts

Bone-resorbing osteoclasts are highly dependent on vesicular trafficking pathways that are regulated by Rab GTPases. In particular, polarised transport of acidic vesicles of the endocytic/lysosomal pathway is required for formation of the ruffled border, the resorptive organelle of the osteoclast. The breakdown products of resorption are then transported through the osteoclast by transcytosis, enabling their excretion. In this review, we summarise these trafficking routes, highlight the emerging evidence that the bone disease osteopetrosis results from defects in vesicular trafficking in osteoclasts, and outline the similarities between the endocytic/lysosomal compartment in osteoclasts and secretory lysosomes in other cell types.     

Autophagy proteins regulate the secretory component of osteoclastic bone resorption

Osteoclasts resorb bone via the ruffled border, whose complex folds are generated by secretory lysosome fusion with bone-apposed plasma membrane. Lysosomal fusion with the plasmalemma results in acidification of the resorptive microenvironment and release of CatK to digest the organic matrix of bone. The means by which secretory lysosomes are directed to fuse with the ruffled border are enigmatic. We show that proteins essential for autophagy, including Atg5, Atg7, Atg4B, and LC3, are important for generating the osteoclast ruffled border, the secretory function of osteoclasts, and bone resorption in?vitro and in?vivo. Further, Rab7, which is required for osteoclast function, localizes to the ruffled border in an Atg5-dependent manner. Thus, autophagy proteins participate in polarized secretion of lysosomal contents into the extracellular space by directing lysosomes to fuse with the plasma membrane. These findings are in keeping with a putative link between autophagy genes and human skeletal homeostasis.     

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.     

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.     

Tctex-1, a novel interaction partner of Rab3D, is required for osteoclastic bone resorption

Vesicular transport along microtubules must be strictly regulated to sustain the unique structural and functional polarization of bone-resorbing osteoclasts. However, the molecular mechanisms bridging these vesicle-microtubule interactions remain largely obscure. Rab3D, a member of the Rab3 subfamily (Rab3A/B/C/D) of small exocytotic GTPases, represents a core component of the osteoclastic vesicle transport machinery. Here, we identify a new Rab3D-interacting partner, Tctex-1, a light chain of the cytoplasmic dynein microtubule motor complex, by a yeast two-hybrid screen. We demonstrate that Tctex-1 binds specifically to Rab3D in a GTP-dependent manner and co-occupies Rab3D-bearing vesicles in bone-resorbing osteoclasts. Furthermore, we provide evidence that Tctex-1 and Rab3D intimately associate with the dynein motor complex and microtubules in osteoclasts. Finally, targeted disruption of Tctex-1 by RNA interference significantly impairs bone resorption capacity and mislocalizes Rab3D vesicles in osteoclasts, attesting to the notion that components of the Rab3D-trafficking pathway contribute to the maintenance of osteoclastic resorptive function.     

Osteoclasts from mice deficient in tartrate-resistant acid phosphatase have altered ruffled borders and disturbed intracellular vesicular transport

Tartrate-resistant acid phosphatase (TRAP) is an enzyme highly expressed in osteoclasts (OC) and chondroclasts. As an approach to pinpoint the function of TRAP in bone-resorbing osteoclasts, the morphological phenotypic alterations of bone and osteoclasts in mice with targeted disruption of the TRAP gene were assessed by quantitative histomorphometry and immunocytochemistry at the light microscopic and ultrastructural levels. TRAP-deficient mice display alterations in the epiphyseal growth plates as evidenced by increased height with disorganized columns of chondrocytes, in particular affecting the zone of hypertrophic chondrocytes, consistent with a disturbance of chondrocyte maturation and chondroclastic resorption at the epiphyseal/metaphyseal junction. TRAP -/- mice express an early onset osteopetrotic bone phenotype, apparent already at 4 weeks of age. The differentiation of OCs was apparently normal; however, the osteoclasts in TRAP-deficient mice were less active in terms of degradation or release of the resorption marker C-terminal type I collagen cross-linked peptide, indicative of an intrinsic defect. Ultrastructural morphometry disclosed that OCs from TRAP-deficient young mice exhibited an increased relative area of ruffled borders. Moreover, mutant OC accumulated cytoplasmic vesicles 200-500 nm in size in both ruffled border and basolateral parts of the cytoplasm, reflecting disturbed intracellular transport. The accumulated vesicles were not likely derived from the secretory pathway, since cathepsin K was detected at normal levels in the ruffled border area and matrix in TRAP -/- mice. In summary, the resorptive defect in TRAP-deficient OCs is reflected by a disturbance at the level of ruffled borders and intracellular transport vesicles. Consequently, accumulation of vesicles in the cytoplasm of mutant OCs indicates a novel function for TRAP in modulating intracellular vesicular transport in osteoclasts.     

Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man

Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H(+)-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H(+)-ATPase of the osteoclast ruffled membrane.     

Rab13 is upregulated during osteoclast differentiation and associates with small vesicles revealing polarized distribution in resorbing cells

Osteoclasts are bone-resorbing multinucleated cells that undergo drastic changes in their polarization due to heavy vesicular trafficking during the resorption cycle. These events require the precise orchestration of membrane traffic in order to maintain the unique characteristics of the different membrane domains in osteoclasts. Rab proteins are small GTPases involved in regulation of most, if not all, steps of vesicle trafficking. The investigators studied RAB genes in human osteoclasts and found that at least 26 RABs were expressed in osteoclasts. Out of these, RAB13 gene expression was highly upregulated during differentiation of human peripheral blood monocytic cells into osteoclasts. To study its possible function in osteoclasts, the investigators performed immunolocalization studies for Rab13 and various known markers of osteoclast vesicular trafficking. Rab13 localized to small vesicular structures at the superior parts of the osteoclast between the trans-Golgi network and basolateral membrane domain. Rab13 localization suggests that it is not involved in endocytosis or transcytosis of bone degradation products. In addition, Rab13 did not associate with early endosomes or recycling endosomes labeled with EEA1 or TRITC-conjugated transferrin, respectively. Its involvement in glucose transporter traffic was excluded as well. It is suggested that Rab13 is associated with a putative secretory function in osteoclasts.     

Limited and selective localization of the lysosomal membrane glycoproteins LGP85 and LGP96 in rat osteoclasts

 Monospecific antibodies against two major glycoproteins of rat lysosomal membranes with apparent molecular masses of 96 and 85 kDa, termed LGP96 and LGP85, respectively, were used as probes to determine the expression and distribution of lysosomal membranes in rat osteoclasts. At the light microscopic level, the preferential immunoreactivity for both proteins was found at high levels at the side facing bone of actively bone-resorbing osteoclasts. Osteoclasts detached from bone surface were devoid of immunoreactivity for each protein. At the electron microscopic level, both proteins were exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts with well-developed ruffled border membrane. No immunolabeling for both proteins was observed in the basolateral membrane and the clear zone of bone-resorbing osteoclasts. The plasma membrane of preosteoclasts and post- and/or resting osteoclasts showed little or no reactivity against these two antibodies. The results indicate that lysosomal membrane glycoproteins are actively synthesized in active osteoclasts, rapidly transported to the ruffled border area, and contribute to the formation and maintenance of the acidic resorption lacuna of osteoclasts. Accepted: 9 December 1998     

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.     

Increased synthesis and specific localization of a major lysosomal membrane sialoglycoprotein (LGP107) at the ruffled border membrane of active osteoclasts

The immunocytochemical localization was investigated of a major lysosomal membrane sialoglycoprotein with a molecular mass of 107 kDa, which was designated as LGP107. The study utilized rat osteoclasts with different bone resorbing activity and osteoclast precursors at various stages of differentiation and maturation together with monospecific antibodies to this protein. Despite its localization primarily in lysosomes and endosomes in the other cell types examined, LGP107 was exclusively confined to the apical plasma membrane at the ruffled border of the active osteoclast, where the osteoclast is in contact with the bone surface. The protein was also concentrated in a number of endocytic vacuoles in the vicinity of the ruffled border membrane. However the labeling was not found in the basolateral membranes of the active osteoclast. The ruffled border membrane detached from the bone surface showed a marked decrease in the extent of the immunolabeling. The post-and/or resting osteoclasts, which were located away from the bone surface, were totally devoid of the membraneous localization of LGP107. No definite immunolabeling was found in the immature preosteoclasts. These results indicate that the protein is largely synthesized in the active osteoclast and rapidly translocated to the ruffled border membrane by vectorial vesicle transport. LGP107 is suggested to contribute to the formation and maintenance of the specialized acidic environment for bone resorption.     

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.     

CELLULAR BIOLOGY OF BONE RESORPTION

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged.
1. The resorption of bone is the result of successive steps that can be regulated individually.
2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone.
3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation.
4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells.
5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO, by carbonic anhydrase and secreted by an ATP-driven vacuolar H⁺-K⁺-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone.
6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca²⁺) generated as a result of osteoclastic bone resorption.
7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.     

Absorption of peroxidase by osteoclasts as studied by electron microscope histochemistry

Summary The ability of osteoclasts to take up protein by endocytosis was examined using peroxidase as a tracer. 5 minutes after intravenous injection the tracer was located around the osteoclast and in the space between its ruffled border and the bone. Inside the cell peroxidase was located in some cytoplasmic vacuoles behind the ruffled border and along the cell membrane. 40 minutes after injection there was a large increase in the number of membrane limited cytoplasmic structures containing reaction product, these being distributed in general throughout the cell but with a high concentration behind the ruffled border. These structures which were filled throughout with peroxidase represented either vacuoles or bodies.The study demonstrates, first that the osteoclast is able to absorb peroxidase, second that a transport of material occurs from the periphery towards the central part of the cell. From the extensive endocytosis along the ruffled border, where the bone resorption takes place, it is suggested that also organic components of the bone may be taken up by the osteoclast under bone resorption in a manner similar to that for peroxidase.This research was supported by the Danish Research Council. Grant. no. 512-819. I wish to thank Mrs. Ruth Nielsen for skilful technical assistance during this work.     

Degradation of hydroxyapatite in vivo and in vitro requires osteoclastic sodium-bicarbonate co-transporter NBCn1

Dissolution of the inorganic bone matrix releases not only calcium and phosphate ions, but also bicarbonate. Electroneutral sodium-bicarbonate co-transporter (NBCn1) is expressed in inactive osteoclasts, but its physiological role in bone resorption has remained unknown. We show here that NBCn1, encoded by the SLC4A7 gene, is directly involved in bone resorption. NBCn1 protein was specifically found at the bone-facing ruffled border areas, and metabolic acidosis increased NBCn1 expression in rats in vivo. In human hematopoietic stem cell cultures, NBCn1 mRNA expression was observed only after formation of resorbing osteoclasts. To further confirm the critical role of NBCn1 during bone resorption, human hematopoietic stem cells were transduced with SLC4A7 shRNA lentiviral particles. Downregulation of NBCn1 both on mRNA and protein level by lentiviral shRNAs significantly inhibited bone resorption and increased intracellular acidification in osteoclasts. The lentiviral particles did not impair osteoclast survival, or differentiation of the hematopoietic or mesenchymal precursor cells into osteoclasts or osteoblasts in vitro. Inhibition of NBCn1 activity may thus provide a new way to regulate osteoclast activity during pathological bone resorption.     

Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.     

Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.