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PubMed1991

Osteoclast differentiation.

The osteoclast is the primary bone resorbing cell. It is a highly specialized multinucleated cell whose primary function is to help in the control of calcium homeostasis. The osteoclast has been very difficult to study because of its relative inaccessability, low numbers, and fragility when isolated from bone. Recently, techniques have been developed to study the cell biology of the osteoclast that have expanded our ability to understand the biological and functional properties of osteoclasts. In this article, studies on the origin of the osteoclast are reviewed and the differentiation markers that are used to detect cells in the osteoclast lineage are discussed. Factors that affect osteoclast differentiation are presented and model systems currently in use for studying osteoclast differentiation are evaluated for their relative strengths and weaknesses. In addition, osteoclast differentiation during tooth eruption and root resorption and the effects of bone matrix elements on osteoclast differentiation are reviewed.

Cell Differentiation↗
PubMed1999

Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families.

Osteoblasts/stromal cells are essentially involved in osteoclast differentiation and function through cell-to-cell contact (Fig. 8). Although many attempts have been made to elucidate the mechanism of the so-called "microenvironment provided by osteoblasts/stromal cells," (5-8) it has remained an open question until OPG and its binding molecule were cloned. The serial discovery of the new members of the TNF receptor-ligand family members has confirmed the idea that osteoclast differentiation and function are regulated by osteoblasts/stromal cells. RANKL, which has also been called ODF, TRANCE, or OPGL, is a member of the TNF ligand family. Expression of RANKL mRNA in osteoblasts/stromal cells is up-regulated by osteotropic factors such as 1 alpha, 25(OH)2D3, PTH, and IL-11. Osteoclast precursors express RANK, a TNF receptor family member, recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into pOCs in the presence of M-CSF. RANKL is also involved in the survival and fusion of pOCs and activation of mature osteoclasts. OPG, which has also been called OCIF or TR1, is a soluble receptor for RANKL and acts as a decoy receptor in the RANK-RANKL signaling system (Fig. 8). In conclusion, osteoblasts/stromal cells are involved in all of the processes of osteoclast development, such as differentiation, survival, fusion, and activation of osteoclasts (Fig. 8). Osteoblasts/stromal cells can now be replaced with RANKL and M-CSF in dealing with the whole life of osteoclasts. RANKL, RANK, and OPG are three key molecules that regulate osteoclast recruitment and function. Further studies on these key molecules will elucidate the molecular mechanism of the regulation of osteoclastic bone resorption. This line of studies will establish new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment and functions such as osteopetrosis, osteoporosis, metastatic bone disease, Paget's disease, rheumatoid arthritis, and periodontal bone disease.

Animals↗
PubMed1991

Cyclosporin-A in vitro decreases bone resorption, osteoclast formation, and the fusion of cells of the monocyte-macrophage lineage.

We studied the in vitro effect of cyclosporin-A (CyA) on bone resorption using a fetal rat long bone-resorbing assay. CyA inhibited both PTH-stimulated and unstimulated bone resorption. The inhibitory effect of CyA on basal resorption was dose dependent, and it was more pronounced during the second period (less than or equal to 0.1 microgram/ml) of culture (days 5-7) than during the first period (days 2-4). A cytotoxic effect was ruled out by the absence of decrease in [3H]thymidine incorporation into bones up to a concentration of 5 micrograms/ml CyA. Histomorphometry performed after 4 and 7 days of culture showed that CyA (1 microgram/ml) decreased the number of osteoclasts per bone section after 7 days of culture (23.5 +/- 4.0 vs. 41.7 +/- 2.9 osteoclasts/bone section; P less than 0.05), but not after 4 days (25.6 +/- 3.3 vs. 23.0 +/- 2.5). These data suggested an effect of CyA on osteoclastic differentiation rather than on the function of mature osteoclasts. We further assessed the mechanisms of the inhibitory effect of CyA on osteoclastic differentiation in order to determine 1) the level of this action (proliferation and/or fusion of osteoclast precursors), and 2) if this action is direct or indirect. Autoradiographic studies were performed on bone sections after incubation of bones with [3H]thymidine for the last 48 h of culture. CyA decreased slightly but significantly the percentage of labeled nuclei per osteoclast and the number of osteoclasts containing at least one labeled nucleus (20.2 +/- 0.7 vs. 33.2 +/- 3.5; P less than 0.02). Moreover the number of nuclei per osteoclast was decreased after 7 days in CyA-treated bones (2.4 +/- 0.05 vs. 3.0 +/- 0.1; P less than 0.02). Taken together these results demonstrate that CyA slightly decreased the proliferation of osteoclast precursors, but markedly decreased their fusion. Similar effects were observed in cultures of rat marrow macrophages. CyA (1 microgram/ml) inhibited the fusion of macrophages into multinucleated cells elicited by 1 nM 1,25-dihydroxyvitamin D3, but had only a slight effect on the proliferation of these cells, as assessed by autoradiography. CyA also inhibited the formation of multinucleated cells and the fusion index in long term cultures of human cord blood monocytes, a cellular model for osteoclastic differentiation. By contrast, CyA had no effect on the formation of myotubes by fusion of cultured mononucleated rat myoblasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Endothelin inhibits osteoclastic bone resorption by a direct effect on cell motility: implications for the vascular control of bone resorption.

The abundance of endothelin (ET)-producing endothelial cells in bone marrow and the proximity of these cells to bone-resorbing osteoclasts prompted us to evaluate the action of ET-1 on osteoclast function. Osteoclasts disaggregated from neonatal rat long bones were settled onto devitalized cortical bone substrate, and resorption was quantified by morphometry. The supernatant tartrate-resistant acid phosphatase activity was determined by a spectrophotometric method using paranitrophenol phosphate as substrate. Cell motility was quantified by time lapse video- and computer-assisted image processing using an empirical procedure for morphometric analysis. Cytosolic free calcium levels ([Ca2+]i) were measured in single cells by an indo 1-based microspectrofluorimetric method. Using the area of bone resorbed per slice as response, we found that ET-1 caused a significant (P = 0.011) concentration-dependent inhibition of osteoclastic bone resorption (EC50 = 2.5 nM) without inhibiting acid phosphatase secretion. Exposure of isolated osteoclasts to ET-1 also led to a marked concentration-dependent inhibition of osteoclast motility (EC50 = 7.9 nM; P = 0.013; t1/2 = 18 min) without significant effects on cell spread area. These effects of ET-1 were reversible after removing the peptide, and the cells remained viable during the experiments. In addition, ET-1 did not elevate [Ca2+]i at the concentrations tested. The results suggest that ET-1 specifically interacts with an osteoclast receptor to inhibit osteoclastic bone resorption and cell motility. As the concentration of ET-1 required for osteoclast inhibition was similar to that reported for smooth muscle contraction, it is possible that ET-1, produced locally from the bone marrow endothelial cell, might play a primary role in osteoclast regulation.

Acid Phosphatase↗
PubMed1992

Insulin-like growth factor-I supports formation and activation of osteoclasts.

Although the action of insulin-like growth factor-I (IGF-I) on bone formation has been extensively investigated, the effect of the factor on bone resorption is little known. We first examined the effect of IGF-I on bone resorption by preexistent osteoclasts by using unfractionated bone cells cultured on dentin slices. IGF-I had a dose-related effect of stimulating bone resorption by preexistent osteoclasts, whereas IGF-II did not. When IGF-I was added to cultures of bone cells after preexistent osteoclasts had degenerated on the dentin slices, IGF-I increased the number of osteoclastic multinucleate cells (MNCs) with tartrate-resistant acid phosphatase activity. Moreover, IGF-I augmented the area of pits produced by newly formed osteoclasts. These results suggest that IGF-I directly or indirectly stimulates osteoclast recruitment and activation. Therefore, we next examined the direct effect of IGF-I on osteoclastic MNC formation by using hemopoietic blast cells. In the presence of 1,25-dihydroxyvitamin D3, IGF-I, like granulocyte-macrophage colony-stimulating factor (GM-CSF), dose-dependently increased the number of TRAP-positive MNCs. This stimulatory effect of IGF-I was additive with that of GM-CSF. Both IGF-I and GM-CSF supported the survival of the blast cells, indicating that IGF-I as well as GM-CSF are supporting factors for osteoclast differentiation. In addition, the blast cells possessed high affinity binding sites for IGF-I, with a Kd of 0.8 nM. These data, thus, indicate that IGF-I stimulates osteoclastic bone resorption through its direct or indirect action of supporting the generation and activation of osteoclasts.

Animals↗
PubMed1993

Osteoclast differentiation in cocultures of a clonal chondrogenic cell line and mouse bone marrow cells.

Previous reports have demonstrated that hemopoietic progenitor cells derived from mouse bone marrow can form osteoclast-like cells when cultured in the presence of stromal cells and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We show here that in cocultures of mouse bone marrow cells and a clonal chondrogenic cell line (C5.18), a stimulation of the number of tartrate-resistant acid phosphatase-positive (TRAP+) colonies is seen with or without the addition of 1,25-(OH)2D3 to the cultures. A large proportion of the TRAP+ cells had calcitonin receptors. In addition resorption lacunae were observed on bone slices on which cocultures were maintained, thus these cells had the characteristics of osteoclasts. The number of osteoclast-containing colonies that formed in cocultures varied with the plating density of the C5.18 cells and the length of time the C5.18 cells were cultured before adding mouse bone marrow. These results suggested that osteoclast differentiation decreased with increasing cartilage differentiation. C5.18 cells treated with 1,25-(OH)2D3 before coculture stimulated TRAP+ osteoclast colony formation to a greater extent than untreated C5.18 cells, whereas C5.18 cells cultured in the presence of dexamethasone before coculture inhibited TRAP+ osteoclast colony formation relative to untreated C5.18 cells. Since 1,25-(OH)2D3 inhibits and dexamethasone stimulates cartilage differentiation in C5.18 cells, these results agree with the view that chondroprogenitor cells stimulate osteoclast colony formation, whereas cultures containing predominantly mature chondrocytes do not. Osteoclast-containing colonies were frequently associated with colonies of alkaline phosphatase-positive (AP+) cells. This raised the possibility that C5.18 cells stimulated osteoclast differentiation indirectly by increasing the numbers of AP+ stromal cells from the marrow population, which in turn could stimulate osteoclast differentiation from marrow hemopoietic progenitors. In cocultures in which the C5.18 cells were physically separated from the marrow cells, we also observed increased numbers of TRAP+ colonies growing in association with large colonies of AP+ cells, suggesting that C5.18 cells release a soluble factor that mediates these effects.

Acid Phosphatase↗
PubMed1997

Effects of retinol on activation of latent transforming growth factor-beta by isolated osteoclasts.

The multifunctional cytokine, transforming growth factor-beta (TGF beta), is found in many tissues in a latent or inactive form. The nature and composition of the latent complex can vary depending on tissue type. The release of active TGF beta from its latent complex is a potentially important mechanism for regulation of TGF beta activity. We have shown previously that osteoclasts activate latent TGF beta produced by bone and that bone cells produce a 100-kDa latent complex that lacks the latent TGF beta-binding protein. Here we investigated the effects of retinol on osteoclast activation of various forms of latent TGF beta. Two sources of osteoclasts were used that provide either mature avian osteoclasts or avian osteoclast precursors. Whereas both cell populations activate latent TGF beta, only mature osteoclasts respond to retinol with an increase in activation of latent TGF beta over basal levels. Activation could not be ascribed to pH changes in conditioned medium. Nonacid-dissociable 100-kDa latent complex, which is also produced by bone cells, was added to mature osteoclasts and to osteoclast precursors, but no activation was observed. Platelet latent TGF beta, which contains the 130-kDa latent TGF beta-binding protein, was activated by both osteoclast populations. Conditioned medium from the precursor population activated latent complex, whereas conditioned medium from mature cells did not. Activation of latent TGF beta by retinol-treated mature cells was not blocked by inhibitors of plasmin, nor was activation by conditioned medium from precursor cells. These data suggest that retinol-induced activation of latent TGF beta by osteoclasts is dependent on the stage of differentiation of these cells and the presence of other cell types, and that unlike other cell systems, the plasmin-plasminogen activator mechanism is not involved.

Animals↗
PubMed2001

Androgens suppress osteoclast formation induced by RANKL and macrophage-colony stimulating factor.

Androgen deficiency in males leads to an increase in osteoclastic bone resorption and a progressive decrease in bone mineral density. In the current studies, we examined the ability of 5 alpha-dihydrotestosterone to suppress osteoclast formation induced by receptor activator of NF-kB ligand (RANKL) and macrophage-colony stimulating factor in vitro. 5 alpha-Dihydrotestosterone suppressed the differentiation of bone marrow monocytes into osteoclasts from both sham-operated and orchidectomized mice. Androgen deficiency also led to an increase in the number of hematopoietic precursors capable of forming osteoclasts and increased the relative responsiveness of these cells to androgens in vitro. Interestingly, E2 was as effective as 5 alpha-dihydrotestosterone in suppressing osteoclast formation in bone marrow monocytes from both sham and orchidectomized mice. As with bone marrow monocytes, 5 alpha-dihydrotestosterone also suppressed RANKL-induced osteoclast formation in the monocyte-macrophagic cell line RAW264.7. In RAW264.7 cells, androgens appear to block RANKL-induced osteoclast formation through selective regulation of c-JUN: Accordingly, 5 alpha-dihydrotestosterone suppressed RANKL-induced c-Jun N-terminal kinase activation and reduced c-Jun expression levels. These effects resulted in a reduction in RANKL-induced activator protein-1 DNA binding activity and a corresponding suppression in activator protein-1-mediated transcriptional activation. These studies indicate that both E and androgens can suppress osteoclast formation via a direct, stromal cell-independent action on osteoclast precursors to block key transcription factors such as c-Jun essential for osteoclast differentiation.

Androgens↗
PubMed2002

TNFalpha potently activates osteoclasts, through a direct action independent of and strongly synergistic with RANKL.

TNFalpha is pivotal to the pathogenesis of inflammatory and possibly postmenopausal osteolysis. Much recent work has clarified mechanisms by which TNFalpha promotes osteoclastogenesis, but the means by which it activates osteoclasts to resorb bone remain uncertain. We found that very low concentrations of TNFalpha promoted actin ring formation, which correlates with functional activation in osteoclasts, both in osteoclasts formed in vitro and extracted from newborn rats. TNFalpha was equipotent with RANKL for this action. Activation by TNFalpha was unaffected by blockade of RANKL by OPG, its soluble decoy receptor, suggesting that this was due to a direct action on osteoclasts. Bone resorption was similarly directly and potently stimulated, in a RANKL-independent manner in osteoclasts, whether these were formed in vitro or in vivo. Interestingly, TNFalpha promoted actin ring formation at concentrations an order of magnitude below those required for osteoclastic differentiation. Moreover, TNFalpha strongly synergized with RANKL, such that miniscule concentrations of TNFalpha were sufficient to substantially augment osteoclast activation. The extreme sensitivity of osteoclasts to activation by TNFalpha suggests that the most sensitive osteolytic response of bone to TNFalpha is through activation of existing osteoclasts; and the strong synergy with RANKL provides a mechanism whereby increased osteolysis can be achieved without disturbance to the underlying pattern of osteoclastic localization.

Actins↗
PubMed1996

The osteoclast generation: an in vitro and in vivo study with a genetically labelled avian monocytic cell line.

Osteoclasts are multinucleate giant cells responsible for bone resorption. Osteoclast precursors are hematopoietic mononucleate cells, which give rise to osteoclasts after fusion. Nevertheless, the precise stage of differentiation where osteoclast precursors diverge from other hematopoietic lineages is still debated. We describe here both in vitro and in vivo approaches to the study of the osteoclast differentiation pathway. We used cells of the BM2 avian monocytic cell line, which are able to differentiate into macrophages both in vitro and in vivo. In order to follow the progeny of BM2 monocytes, we have derived a BM2 cell clone expressing the nlslacZ gene (BM2nlslacZ) which has still retained the main features of the parental cell line. In vitro, when BM2nlslacZ cells were triggered toward macrophages, they participated in the formation of multinucleate osteoclast-like cells as seen by their blue nuclei. Furthermore, when BM2nlslacZ cells were injected into the blood stream of chicken embryos, they could give rise to blue nucleate macrophages in the bone marrow, as well as to osteoclasts with blue nuclei in bone. Finally, we have shown that fusion of tagged mononucleate precursor cells not only occurs with other mononucleate precursor cells but also with mature multinucleate osteoclasts. This work shows that cells already engaged in the late stages of the monocytic differentiation pathway are able to differentiate into osteoclasts and that osteoclast divergence takes place after the monocyte stage.

Animals↗
PubMed1988

Localization of platelet antigens and fibrinogen on osteoclasts.

The antigenic phenotype of the human osteoclast, which is known to be derived from a circulating mononuclear precursor cell of haemopoietic origin, is controversial. Recent studies have shown that macrophage as well as megakaryocyte/platelet antigens are expressed by osteoclasts. In this study, we have sought to define, by immunohistochemistry, the nature and possible function of platelet antigens expressed by human osteoclasts in foetal and adult bone specimens. Monoclonal antibodies to platelet glycoprotein IIIa (gpIIIa) and CD9 antibodies stained osteoclasts in all bone specimens examined. Fibrinogen was also localized to the osteoclast membrane in foetal bone imprints. In addition, we found that CD9 and gpIIIa antibodies reacted weakly with monocytes in buffy coat smears. Antibodies to factor 8 and glycoproteins Ib and IIb/IIIa did not react with osteoclasts. These results show that osteoclasts, monocytes, macrophages, megakaryocytes and platelets possess common antigens and that fibrinogen is present on the surface of osteoclasts. By analogy with platelets, CD9 and gpIIIa may play a role in fibrinogen binding by osteoclasts. Possible mechanisms by which platelet antigens and fibrinogen binding could affect osteoclast function are proposed.

Antibodies, Monoclonal↗
PubMedDate not supplied

Cloning and characterization of osteoclast precursors from the RAW264.7 cell line.

Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-kappaB ligand (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell cultures, which are poorly suited to molecular and transgene studies because of the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study, we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP-positive multinuclear cells. Clones capable of forming large TRAP-positive multinuclear cells also expressed beta3 integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-kappaB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation.

Acid Phosphatase↗
PubMed2005

Degradation of the organic phase of bone by osteoclasts: a secondary role for lysosomal acidification.

UNLABELLED: Osteoclasts degrade bone matrix by secretion of hydrochloric acid and proteases. We studied the processes involved in the degradation of the organic matrix of bone in detail and found that lysosomal acidification is involved in this process and that MMPs are capable of degrading the organic matrix in the absence of cathepsin K. INTRODUCTION: Osteoclasts resorb bone by secretion of acid by the vacuolar H+-adenosine triphosphatase (V-ATPase) and the chloride channel ClC-7, followed by degradation of the matrix, mainly collagen type I, by cathepsin K and possibly by matrix metalloproteinases (MMPs). However, the switch from acidification to proteolysis and the exact roles of both the ion transporters and the proteinases still remain to be studied. MATERIALS AND METHODS: We isolated CD14+ monocytes from human peripheral blood from either controls or patients with autosomal dominant osteopetrosis type II (ADOII) caused by defective ClC-7 function and cultured them in the presence of RANKL and macrophage-colony stimulating factor (M-CSF) to generate osteoclasts. We decalcified cortical bovine bone slices and studied the osteoclasts with respect to morphology, markers, and degradation of the decalcified matrix in the presence of various inhibitors of osteoclast acidification and proteolysis, using normal calcified bone as a reference. RESULTS: We found that ADOII osteoclasts not only have reduced resorption of the calcified matrix, but also 40% reduced degradation of the organic phase of bone. We found that both acidification inhibitors and cathepsin K inhibitors reduced degradation of the organic matrix by 40% in normal osteoclasts, but had no effect in the ADOII osteoclasts. Furthermore, we showed that inhibition of MMPs leads to a 70% reduction in the degradation of the organic bone matrix and that MMPs and cathepsin K have additive effects. Finally, we show that osteoclastic MMPs mediate release of the carboxyterminal telopeptide of type I collagen (ICTP) fragment in the absence of cathepsin K activity, and therefore, to some extent, are able to compensate for the loss of cathepsin K activity. CONCLUSIONS: These data clearly show that osteoclastic acidification of the lysosomes plays a hitherto nonrecognized role in degradation of the organic matrix. Furthermore, these data shed light on the complicated interplay between acidification dependent and independent proteolytic processes, mediated by cathepsin K and the MMPs, respectively.

Adult↗
PubMed1998

Wortmannin inhibits spreading and chemotaxis of rat osteoclasts in vitro.

Wortmannin (WT) and 17beta-hydroxywortmannin (HWT), which are inhibitors of phosphatidylinositol-3(OH)-kinase (PI3K), have been shown previously to inhibit bone resorption in vitro and in vivo, possibly by interfering with formation of the osteoclast ruffled border. Since migration of osteoclasts also plays an important role in the process of bone resorption, we investigated the effects of these inhibitors on osteoclast morphology and motility. Both HWT and WT caused a sustained decrease in the planar area of osteoclasts in vitro (half maximal effect at 25 and 165 nM, respectively), with the effect of HWT on cell area more readily reversible than WT. These agents also caused accumulation of intracellular vesicles. Time-lapse video microscopy was used to record the migration of osteoclasts in response to macrophage colony-stimulating factor (M-CSF) or vehicle, flowing passively from a micropipette positioned 200-400 microm from the cell. M-CSF caused directed migration of osteoclasts, indicating chemotaxis (over 3 h osteoclasts migrated 96 +/- 14 microm in response to M-CSF vs. 11 +/- 2 microm in control experiments). Both WT (100 or 500 nM) and LY294002 (100 microM), a specific PI3K inhibitor structurally unrelated to WT, significantly inhibited osteoclast chemotaxis in response to M-CSF. Taken together, these effects of WT, HWT, and LY294002 are consistent with an important role for PI3K in regulating cytoskeletal function in osteoclasts. The inhibitory effects of WT and HWT on bone resorption may be due, in part, to impairment of osteoclast motility.

Androstadienes↗
PubMed2000

Protein geranylgeranylation is required for osteoclast formation, function, and survival: inhibition by bisphosphonates and GGTI-298.

Bisphosphonates are the important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Although their molecular mechanism of action has not been fully elucidated, recent studies have shown that the nitrogen-containing bisphosphonates can inhibit protein prenylation in macrophages in vitro. In this study, we show that the nitrogen-containing bisphosphonates risedronate, zoledronate, ibandronate, alendronate, and pamidronate (but not the non nitrogen-containing bisphosphonates clodronate, etidronate, and tiludronate) prevent the incorporation of [14C]mevalonate into prenylated (farnesylated and geranylgeranylated) proteins in purified rabbit osteoclasts. The inhibitory effect of nitrogen-containing bisphosphonates on bone resorption is likely to result largely from the loss of geranylgeranylated proteins rather than loss of farnesylated proteins in osteoclasts, because concentrations of GGTI-298 (a specific inhibitor of geranylgeranyl transferase I) that inhibited protein geranylgeranylation in purified rabbit osteoclasts prevented osteoclast formation in murine bone marrow cultures, disrupted the osteoclast cytoskeleton, inhibited bone resorption, and induced apoptosis in isolated chick and rabbit osteoclasts in vitro. By contrast, concentrations of FTI-277 (a specific inhibitor of farnesyl transferase) that prevented protein farnesylation in purified rabbit osteoclasts had little effect on osteoclast morphology or apoptosis and did not inhibit bone resorption. These results therefore show the molecular mechanism of action of nitrogen-containing bisphosphonate drugs in osteoclasts and highlight the fundamental importance of geranylgeranylated proteins in osteoclast formation and function.

Animals↗
PubMed2002

Colocalization of intracellular osteopontin with CD44 is associated with migration, cell fusion, and resorption in osteoclasts.

Although osteopontin (OPN) is recognized generally as a secreted protein, an intracellular form of osteopontin (iOPN), associated with the CD44 complex, has been identified in migrating fibroblastic cells. Because both OPN and CD44 are expressed at high levels in osteoclasts, we have used double immunofluorescence analysis and confocal microscopy to determine whether colocalization of these proteins has functional significance in the formation and activity of osteoclasts. Analysis of rat bone marrow-derived osteoclasts revealed strong surface staining for CD44 and beta1- and beta3-integrins, whereas little or no staining for OPN or bone sialoprotein (BSP) was observed in nonpermeabilized cells. In permeabilized perfusion osteoclasts and multinucleated osteoclasts, staining for OPN and CD44 was prominent in cell processes, including filopodia and pseudopodia. Confocal microscopy revealed a high degree of colocalization of OPN with CD44 in motile osteoclasts. In cells treated with cycloheximide (CHX), perinuclear staining for OPN and BSP was lost, but iOPN staining was retained within cell processes. In osteoclasts generated from the OPN-null and CD44-null mice, cell spreading and protrusion of pseudopodia were reduced and cell fusion was impaired. Moreover, osteoclast motility and resorptive activity were significantly compromised. Although the area resorbed by OPN-null osteoclasts could be rescued partially by exogenous OPN, the resorption depth was not affected. These studies have identified an intracellular form of OPN, colocalizing with CD44 in cell processes, that appears to function in the formation and activity of osteoclasts.

Animals↗
PubMed2003

Leupaxin is a critical adaptor protein in the adhesion zone of the osteoclast.

Leupaxin is a cytoskeleton adaptor protein that was first identified in human macrophages and was found to share homology with the focal adhesion protein, paxillin. Leupaxin possesses several protein-binding domains that have been implicated in targeting proteins such as focal adhesion kinase (pp125FAK) to focal adhesions. Leupaxin can be detected in monocytes and osteoclasts, both cells of hematopoietic origin. We have identified leupaxin to be a component of the osteoclast podosomal signaling complex. We have found that leupaxin in murine osteoclasts is associated with both PYK2 and pp125FAK in the osteoclast. Treatment of osteoclasts with TNF-alpha and soluble osteopontin were found to stimulate tyrosine phosphorylation of both leupaxin and leupaxin-associated PYK2. Leupaxin was found to co-immunoprecipitate with the protein tyrosine phosphatase PTP-PEST. The cellular distribution of leupaxin, PYK2, and protein tyrosine phosphorylation-PEST co-localized at or near the osteoclast podosomal complex. Leupaxin was also found to associate with the ARF-GTPase-activating protein, paxillin kinase linker p95PKL, thereby providing a link to regulators of cytoskeletal dynamics in the osteoclast. Overexpression of leupaxin by transduction into osteoclasts evoked numerous cytoplasmic projections at the leading edge of the cell, resembling a motile phenotype. Finally, in vitro inhibition of leupaxin expression in the osteoclast led to a decrease in resorptive capacity. Our data suggest that leupaxin may be a critical nucleating component of the osteoclast podosomal signaling complex.

Animals↗
PubMed1998

Control of osteoclast differentiation.

The osteoclast is the primary bone-resorbing cell and is derived from the monocyte/macrophage lineage. Bipotent osteoclast precursors, which can form both osteoclasts and monocyte-macrophages, proliferate and differentiate to become unipotent post-mitotic committed osteoclast precursors. These post-mitotic committed precursors fuse to form the multinucleated osteoclast, which is then activated to resorb bone. A variety of soluble and membrane-bound factors play a critical role in regulating osteoclast formation, including growth factors, systemic hormones, and cells in the marrow microenvironment, such as osteoblasts and marrow stromal cells. Cell-to-cell interactions are important in both the formation and activity of the osteoclast. Recent molecular biological studies have identified transcription factors, such as c-fos and PU.1, which are required for osteoclast differentiation. In this review, we discuss the phenotypic changes that are induced as the cells mature from bipotent early precursors to mature osteoclasts; factors that have been identified that are involved in this process; and the role of marrow stromal cells and osteoblasts in osteoclast differentiation.

Animals↗