Titanium particles stimulate bone resorption by inducing differentiation of murine osteoclasts

BACKGROUND: Loosening of orthopaedic implants is mediated by cytokines that elicit bone resorption and are produced in response to phagocytosis of implant-derived wear particles. This accelerated bone resorption could be due to increased osteoclastic activity, survival, or differentiation. Although a number of in vitro studies have shown that wear particles increase osteoclastic activity, the increase was less than twofold in all cases. The objective of the current study was to test the hypothesis that wear particles stimulate bone resorption by inducing osteoclast differentiation. METHODS: Conditioned media were prepared from murine marrow cells or human peripheral blood monocytes incubated in the presence or absence of titanium particles. The effects of conditioned media on osteoclast differentiation were examined with use of a recently developed assay in which osteoclast precursors are co-cultured with mesenchymal support cells. RESULTS: The present study showed that titanium particles induced both murine marrow cells and human peripheral blood monocytes to produce factors that stimulated osteoclast differentiation. The mean increase in osteoclast differentiation was 29.3+/-9.4-fold. The stimulation of osteoclast differentiation led to a parallel increase in bone resorption. The amount of stimulation was regulated in a dose-dependent manner by the concentration of both titanium particles and conditioned media. The stimulation of osteoclast differentiation required interactions between the cells and the particles themselves and, therefore, was not due to metal ions, soluble contaminants released from the particles, or submicrometer particles. In contrast, conditioned media from control cells incubated in the absence of titanium particles had no detectable effect on any of the examined parameters. CONCLUSIONS: The present study showed that titanium particles stimulate in vitro bone resorption primarily by inducing osteoclast differentiation. In contrast, the titanium particles had only small effects on osteoclast activity or survival.     

Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts

Bone morphogenetic proteins (BMPs) play an important role in various kinds of pattern formation and organogenesis during vertebrate development. In the skeleton, BMPs induce the differentiation of cells of chondrocytic and osteoblastic cell lineage and enhance their function. However, the action of BMPs on osteoclastic bone resorption, a process essential for pathophysiological bone development and regeneration, is still controversial. In this study, we examine the direct effect of BMPs on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. BMP-2 caused a dose- and time-dependent increase in bone resorption pits excavated by the isolated osteoclasts. BMP-4 also stimulated osteoclastic bone resorption. The increase in osteoclastic bone resorption induced by BMP-2 was abolished by the simultaneous addition of follistatin, a BMP/activin binding protein that negates their biological activity. Just as it increased bone resorption, BMP-2 also elevated the messenger RNA expressions of cathepsin K and carbonic anhydrase II, which are key enzymes for the degradation of organic and inorganic bone matrices, respectively. Type IA and II BMP receptors (BMPRs), and their downstream signal transduction molecules, Smad1 and Smad5, were expressed in isolated osteoclasts as well as in osteoblastic cells, whereas type IB BMPR was undetectable. BMPs directly stimulate mature osteoclast function probably mediated by BMPR-IA and BMPR-II and their downstream molecules expressed in osteoclasts. The results presented here expand our understanding of the multifunctional roles of BMPs in bone development.     

Bisphosphonates suppress bone resorption by a direct effect on early osteoclast precursors without affecting the osteoclastogenic capacity of osteogenic cells: the role of protein geranylgeranylation in the action of nitrogen-containing bisphosphonates on osteoclast precursors.

Nitrogen-containing bisphosphonates (NBps) are taken up by osteoclasts and inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway. There is evidence, however, that cells other than mature osteoclasts, like osteoclast precursors and osteoblasts, are also involved in the action of Bps on bone resorption in vitro. To examine this issue further, we developed a new in vitro model, which allows the study of the effects of additives on early osteoclast precursors. In this model, osteogenic cells are essential for osteoclastogenesis. The model consists of 15-day-old fetal mouse metatarsals. At time of explantation, these bone rudiments do not yet contain a mineralized matrix or osteoclasts; only early osteoclast precursors are present in the perichondrium. During culture and after the addition of Nabeta-glycerolphosphate, the bones form a mineralized matrix that is consequently resorbed by osteoclasts that develop from their precursors. Short treatment of these explants with Bps, before the formation of a mineralized matrix, resulted in a subsequent dose-dependent inhibition of bone resorption. The relative potencies of eight Bps to suppress resorption were comparable with those observed after the addition of Bps after the formation of a mineralized matrix, the natural target of Bps. In addition, the effects of the NBp olpadronate, but not of clodronate, on osteoclastic resorption, could be partly reversed by geranylgeraniol. Results indicate that Bps can suppress osteoclastic resorption in vitro by a direct action on very early osteoclast precursors at the bone surface, and not by affecting the osteoclastogenic capacity of osteogenic cells. Moreover, the mechanism of action of the NBp olpadronate, but not clodronate, on early tartrate-resistant acid phosphatase-negative osteoclast precursors involves inhibition of protein geranylgeranylation, indicating a molecular mechanism similar to that established for mature osteoclasts.     

Generation of bone-resorbing osteoclasts from B220+ cells: its role in accelerated osteoclastogenesis due to estrogen deficiency.

Estrogen deficiency stimulates both osteoclastic bone resorption and pre-B lymphopoiesis, the interrelationships between which remain unknown. To investigate the involvement of an increase in the number of B220+ cells in accelerated osteoclastogenesis after estrogen deficiency, we first examined whether ovariectomy (OVX) increased the frequency of clonogenic osteoclast precursors in bone marrow. The results were that after OVX, the frequency of clonogenic osteoclast precursors is increased in bone marrow, suggesting that accumulated osteoclast precursors contribute to accelerated osteoclastogenesis. Further, we found that cocultures of B220+ cells purified from bone marrow cells and stromal ST2 cells in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] gave rise to osteoclasts that can resorb bone and express calcitonin receptors. When the frequencies of clonogenic osteoclast precursors in the purified B220+ and B220- cell fractions were compared, it was found that the fractions gave rise to osteoclasts at similar frequencies, which rules out the possibility of cross-contamination and suggests that the two fractions contain comparable numbers of osteoclast precursors. Furthermore, we identified cells that are positive for both tartrate-resistant acid phosphatase (TRAP) and B220, not only in cocultures of B220+ and ST2 cells, but also in freshly isolated unfractionated bone cells. Therefore, it is concluded that at least a subfraction of B220+ cells are capable of generating osteoclasts and that the increase in the number of B220+ cells caused by estrogen deficiency may contribute to accelerated bone resorption by this novel osteoclastogenesis pathway.     

Impaired bone resorption in cathepsin K-deficient mice is partially compensated for by enhanced osteoclastogenesis and increased expression of other proteases via an increased RANKL/OPG ratio

Previous reports indicate that mice deficient for cathepsin K (Ctsk), a key protease in osteoclastic bone resorption, develop osteopetrosis due to their inability to properly degrade organic bone matrix. Some features of the phenotype of Ctsk knockout mice, however, suggest the presence of mechanisms by which Ctsk-deficient mice compensate for the lack of cathepsin K. To study these mechanisms in detail, we generated Ctsk-deficient (Ctsk-/-) mice and analyzed them at the age of 2, 7, and 12 months using peripheral quantitative computed tomography, histomorphometry, resorption marker measurements, osteoclast and osteoblast differentiation cultures, and gene expression analyses. The present study verified the previously published osteopetrotic features of Ctsk-deficient mice. However, these changes did not exacerbate during aging indicating the absence of Ctsk to have its most severe effects during the rapid growth period. Resorption markers ICTP and CTX were decreased in the media of Ctsk-/- osteoclasts cultured on bone slices indicating impaired bone resorption. Ctsk-/- mice exhibited several mechanisms attempting to compensate for Ctsk deficiency. The number of osteoclasts in trabecular bone was significantly increased in Ctsk-/- mice compared to controls, as was the number of osteoclast precursors in bone marrow. The mRNA levels for receptor activator of nuclear factor (kappa)B ligand (RANKL) in Ctsk-/- bones were increased resulting in increased RANKL/OPG ratio favoring osteoclastogenesis. In addition, expression of mRNAs of osteoclastic enzymes (MMP-9, TRACP) and for osteoblastic proteases (MMP-13, MMP-14) were increased in Ctsk-/- mice compared to controls. Impaired osteoclastic bone resorption in Ctsk-/- mice results in activation of osteoblastic cells to produce increased amounts of other proteolytic enzymes and RANKL in vivo. We suggest that increased RANKL expression mediates enhanced osteoclastogenesis and increased protease expression by osteoclasts. These observations underline the important role of osteoblastic cells in regulation of osteoclast activity and bone turnover.     

The RANKL/RANK/OPG pathway

Understanding of osteoclast formation and activation has advanced considerably since the discovery of the RANKL/RANK/OPG system in the mid 1990s. Osteoblasts and stromal stem cells express receptor activator of NF-jB ligand (RANKL), which binds to its receptor, RANK, on the surface of osteoclasts and their precursors. This regulates the differentiation of precursors into multinucleated osteoclasts and osteoclast activation and survival both normally and in most pathologic conditions associated with increased bone resorption. Osteoprotegerin (OPG) is secreted by osteoblasts and osteogenic stromal stem cells and protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from interacting with RANK. The RANKL/OPG ratio in bone marrow is thus an important determinant of bone mass in normal and disease states. RANKL/RANK signaling also regulates lymph node formation and mammary gland lactational hyperplasia in mice, and OPG protects large arteries of mice from medial calcification. This article reviews the roles of the RANKL/RANK/OPG system in bone and other tissues.     

Stimulatory effect of insulin-like growth factor binding protein-5 on mouse osteoclast formation and osteoclastic bone-resorbing activity.

Insulin-like growth factor binding protein-5 (IGFBP-5) stimulates osteoblast proliferation directly or indirectly through IGF-I action, but its effects on osteoclast formation and osteoclastic activity are unknown. We tested the effects of IGFBP-5 on osteoclastic activity and osteoclast formation. IGFBP-5 significantly stimulated pit formation by pre-existent osteoclasts in mouse bone cell cultures and its stimulatory effect was completely blocked by IGF-I antibody (Ab). However, IGFBP-5 did not affect the bone-resorbing activity of isolated rabbit osteoclasts. When IGFBP-5 was added to unfractionated bone cells after degeneration of pre-existent osteoclasts, IGFBP-5 (77 pM-7.7 nM) dose-dependently stimulated osteoclast-like cell formation, irrespective of the presence of IGF-I Ab. Moreover, osteoclast-like cells newly formed by IGFBP-5 from unfractionated bone cells possessed the ability to form pits on dentine slices. We next examined the direct effect of IGFBP-5 on osteoclast precursors in the absence of stromal cells, using hemopoietic blast cells derived from spleen cells. IGFBP-5 dose-dependently stimulated osteoclast-like cell formation from osteoclast precursors, irrespective of the presence of IGF-I Ab. Growth hormone (GH) as well as IGF-I significantly stimulated bone resorption by pre-existent osteoclasts in mouse bone cell cultures and these stimulatory effects were completely blocked by IGF-I Ab. GH as well as IGF-I stimulated osteoclast-like cell formation from unfractionated bone cells and this stimulatory effect of GH was significantly but partially blocked by IGF-I Ab. The direct stimulatory effect of GH on osteoclast-like cell formation from hemopoietic blast cells was not affected by IGF-I Ab. The present data indicate that IGFBP-5 stimulates bone resorption both by stimulation of osteoclast formation in an IGF-I-independent fashion and by IGF-I-dependent activation of mature osteoclasts, possibly via osteoblasts, in vitro.     

Macrophage colony stimulating factor increases bone resorption in dispersed osteoclast cultures by increasing osteoclast size.

Several reports indicate that macrophage colony stimulating factor (MCSF) is one of the major factors required for osteoclast proliferation and differentiation. Paradoxically, it has also been reported that MCSF inhibits osteoclastic activity. We therefore decided to investigate in detail the effects of MCSF on resorption and osteoclast formation to try and clarify this issue. Osteoclast-containing cultures were obtained from rabbit long bones and cultured on plastic culture dishes or devitalized bovine bone slices. MCSF (4-400 ng/ml) stimulated osteoclastic bone resorption in a time-dependent manner and at all doses examined. After 48 h of culture in the presence of MCSF, we observed a 2-fold increase in the total area of bone resorbed, as well as a significant increase in the area of bone resorbed per osteoclast and the number of resorption pits per osteoclast. This effect was paralleled by an increase in the number of larger osteoclasts (as determined by the number of nuclei per cell) and an increase in the size and depth of the resorption pits. Since the total number of osteoclasts remained the same, the MCSF-induced increase in resorptive activity appeared to be related to an increase in the average size of the osteoclasts. When resorption was expressed as the amount of bone resorbed per osteoclast nucleus, larger osteoclasts resorbed more per nucleus, suggesting that large osteoclasts, as a population, are more effective resorbers than small osteoclasts. Interestingly, when osteoclasts were plated at one-fifth the standard density, the amount of bone resorbed per osteoclast decreased considerably, indicating that resorptive activity is also affected by cell density of osteoclasts and/or of other cells present. However, at this lower density MCSF still increased osteoclast size and resorption by the same fold increase over control, suggesting that the effect of MCSF was independent of factors related to cell density.     

An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors.

The present study provides a novel assay system to examine the differentiation of osteoclast progenitors on devitalized bone slices. We used the population of bone cells liberated enzymatically from 14-day-old mouse embryonal calvariae as a source of osteoclast progenitors. The analysis of differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts was assessed in terms of the formation of TRAP-positive cells and pits or resorption lacunae, respectively, on devitalized bone slices. Osteoclasts having bone-resorbing activity appeared when the calvarial cell population was cultured in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. The resorbing activity increased in a 1 alpha,25-(OH)2D3 dose-related manner. However, calcitonin, a potent inhibitor of differentiation and activation of osteoclast lineage cells, reduced the area of the resorption lacunae in a dose-dependent fashion. The bone-resorbing cells on the bone slices expressed an obvious ruffled border and clear zone, structures specific to mature osteoclasts. These results suggest that osteoclast progenitors in the mouse calvarial population examined differentiated into mature osteoclasts in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. Further, using this assay system we assessed the effect of some other osteotropic factors on the differentiation of osteoclast progenitors to mature osteoclasts. IL-1, IL-6, and PTH increased the formation of TRAP-positive cells and pits and the area of resorption lacunae in a dose-dependent fashion. However, prostaglandin E2 was unable to induce the formation of resorption lacunae, although a significant appearance of TRAP-positive cells was observed at a concentration of 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sesquiterpene lactone parthenolide blocks lipopolysaccharide-induced osteolysis through the suppression of NF-kappaB activity.

Effective treatment for bacteria-induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF-kappaB inhibitor found in medicinal herbs, can block LPS-induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-kappaB activity. INTRODUCTION: Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-kappaB activity. MATERIALS AND METHODS: The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo. RANKL-induced osteoclast differentiation from RAW264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro. Assays for NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors. Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis. Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6. RESULTS: We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model. In vitro studies showed that low concentrations of PAR (<1 microM) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (>5 microM) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner. Furthermore, PAR inhibited LPS-induced NF-kappaB activation, p65 translocation, and IkappaB-alpha degradation both in mature osteoclasts and their precursors in a time- and dose-dependent manner. In addition, PAR inhibited NF-kappaB activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1beta, or TNF-alpha to varying degrees and reduced the gene expression of RANK and TRAF6. CONCLUSION: The NF-kappaB pathway is known to mediate both osteoclast differentiation and survival. These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-kappaB activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.     

Identification of biphenylcarboxylic acid derivatives as a novel class of bone resorption inhibitors.

A novel class of biphenylcarboxylic acid derivatives are described that inhibit osteoclastic bone resorption in vitro by promoting osteoclast apoptosis and that prevent ovariectomy-induced bone loss in vivo. The compounds act by a novel mechanism that seems to be distinct from existing antiresorptive drugs. INTRODUCTION: Many common bone diseases such as osteoporosis, Paget's disease, and cancer-associated bone disease are characterized by excessive bone loss caused by increased osteoclastic activity. Successful treatment of these diseases is based on osteoclast inhibition. The osteoclast inhibitory drugs that are currently available fall into relatively few mechanistic classes, indicating the need to identify novel antiresorptives. Here we describe a series of biphenylcarboxylic acid derivatives that have potent inhibitory effects on osteoclastic bone resorption in vitro and on ovariectomy-induced bone loss in vivo. MATERIALS AND METHODS: Compounds were tested for inhibitory effects on bone resorption in vitro using mouse osteoblast-bone marrow co-cultures, isolated rabbit osteoclasts, and mouse osteoclasts generated from bone marrow. Some experiments were also performed on human osteoclasts generated from peripheral blood mononuclear cells. We also investigated the effects of specific compounds on ovariectomy-induced bone loss in vivo in mice. RESULTS: One of the most potent compounds identified was the butanediol ester of biphenyl carboxylic acid (ABD056), which inhibited osteoclast formation in mouse osteoblast-bone marrow co-cultures by 50% (IC50) at a concentration of 26 microM and in macrophage-colony stimulating factor (M-CSF)- and RANKL-stimulated mouse bone marrow cultures with an IC50 of 8 microM. Mechanistic studies showed that ABD056 caused osteoclast apoptosis and inhibited TNFalpha-induced NF-kappaB activation. No inhibitory effects on osteoblast growth or differentiation were observed at concentrations of up to 100 microM. When administered to mice at doses of 5 and 10 mg/kg/day, ABD056 prevented ovariectomy-induced bone loss. CONCLUSIONS: Butanediol biphenylcarboxylic acid derivatives represent a new class of antiresorptive drug that might be of therapeutic value in the prevention and treatment of diseases characterized by osteoclast activation such as osteoporosis, cancer-associated bone disease, and Paget's disease of bone.     

Human bone-derived cells support formation of human osteoclasts from arthroplasty-derived cells in vitro

Mononuclear osteoclast precursors are present in the wear-particle-associated macrophage infiltrate found in the membrane surrounding loose implants. These cells are capable of differentiating into osteoclastic bone-resorbing cells when co-cultured with the rat osteoblast-like cell line, UMR 106, in the presence of 1,25(OH)2 vitamin D3. In order to develop an in vitro model of osteoclast differentiation which more closely parallels the cellular microenvironment at the bone-implant interface in situ, we determined whether osteoblast-like human bone-derived cells were capable of supporting the differentiation of osteoclasts from arthroplasty-derived cells and analysed the humoral conditions required for this to occur. Long-term co-culture of arthroplasty-derived cells and human trabecular-bone-derived cells (HBDCs) resulted in the formation of numerous tartrate-resistant-acid-phosphatase (TRAP) and vitronectin-receptor (VNR)-positive multinucleated cells capable of extensive resorption of lacunar bone. The addition of 1,25(OH)2 vitamin D3 was not required for the formation of osteoclasts and bone resorption. During the formation there was release of substantial levels of M-CSF and PGE2. Exogenous PGE2 (10(-8) to 10(-6) M) was found to stimulate strongly the resorption of osteoclastic bone. Our study has shown that HBDCs are capable of supporting the formation of osteoclasts from mononuclear phagocyte precursors present in the periprosthetic tissues surrounding a loose implant. The release of M-CSF and PGE2 by activated cells at the bone-implant interface may be important for the formation of osteoclasts at sites of pathological bone resorption associated with aseptic loosening.     

Suppression of adjuvant-induced arthritic bone destruction by cyclooxygenase-2 selective agents with and without inhibitory potency against carbonic anhydrase II.

In vitro assays revealed that COX-2 inhibitors with CA II inhibitory potency suppressed both differentiation and activity of osteoclasts, whereas that without the potency reduced only osteoclast differentiation. However, all COX-2 inhibitors similarly suppressed bone destruction in adjuvant-induced arthritic rats, indicating that suppression of osteoclast differentiation is more effective than that of osteoclast activity for the treatment. INTRODUCTION: Cyclooxygenase (COX)-2 and carbonic anhydrase II (CA II) are known to play important roles in the differentiation of osteoclasts and the activity of mature osteoclasts, respectively. Because several COX-2 selective agents were recently found to possess an inhibitory potency against CA II, this study compared the bone sparing effects of COX-2 selective agents with and without the CA II inhibitory potency. MATERIALS AND METHODS: Osteoclast differentiation was determined by the mouse co-culture system of osteoblasts and bone marrow cells, and mature osteoclast activity was measured by the pit area on a dentine slice resorbed by osteoclasts generated and isolated from bone marrow cells. In vivo effects on arthritic bone destruction were determined by radiological and histological analyses of hind-paws of adjuvant-induced arthritic (AIA) rats. RESULTS: CA II was expressed predominantly in mature osteoclasts, but not in the precursors. CA II activity was inhibited by sulfonamide-type COX-2 selective agents celecoxib and JTE-522 similarly to a CA II inhibitor acetazolamide, but not by a methylsulfone-type COX-2 inhibitor rofecoxib. In vitro assays clearly revealed that celecoxib and JTE-522 suppressed both differentiation and activity of osteoclasts, whereas rofecoxib and acetazolamide suppressed only osteoclast differentiation and activation, respectively. However, bone destruction in AIA rats was potently and similarly suppressed by all COX-2 selective agents whether with or without CA II inhibitory potency, although only moderately by acetazolamide. CONCLUSIONS: Suppression of osteoclast differentiation by COX-2 inhibition is more effective than suppression of mature osteoclast activity by CA II inhibition for the treatment of arthritic bone destruction.     

Monocytes mediate osteoclastic bone resorption by prostaglandin production

Summary Monocytes are frequently found adjacent to active bone resorption surfaces in both physiological and pathological situations and may play a key role in bone resorption. There is strong circumstantial evidence that monocytes are precursors for osteoclasts in vivo, and recently they have been shown to resorb devitalized bone directly. The present study shows that monocytes can also resorb bone by stimulation of osteoclasts. Live fetal rodent bones prelabeled with⁴⁵Ca and cultured for 48–96 h in the presence of human monocytes or monocyte-conditioned medium released 80% more mineral than bones cultured in control medium. Bone matrix sustained comparable resorption as demonstrated by a 2-fold decrement in the extracted dry weights of the bones cultured in monocyte-conditioned medium. Histological examination of the bones cultured with monocytes or monocyte-conditioned medium showed increased osteoclast number and activity when compared with bones cultured in control medium. Known inhibitors of osteoclastic activity (phosphate 6 × 10⁻³M, cortisol 10⁻⁶M, and calcitonin 50 mU/ml) inhibited monocyte-conditioned medium-mediated bone resorption. The monocyte-conditioned medium contained sufficient prostaglandin E to account for the bone resorption. Indomethacin 10⁻⁵M added to the monocyte cultures blocked monocyte-conditioned media-induced bone resorption and prostaglandin release. These experiments suggest that monocytes stimulate osteoclastic bone resorption by prostaglandin production. Monocyte-induced bone resorption is partly reversed by inhibitors of osteoclast function. Monocyte-induced osteoclastic bone resorption may play an important role in physiologic bone remodeling and in bone destruction that occurs in chronic inflammatory diseases such as rheumatoid arthritis and periodontal disease.     

Regulation of RANKL‐induced osteoclastogenesis by RING finger protein RNF114

Normal bone remodeling is a continuous process orchestrated by bone‐resorbing osteoclasts and bone‐forming osteoblasts, which an imbalance in bone remodeling results in metabolic bone diseases. RANKL, a member of the TNF cytokine family, functions as a key stimulator for osteoclast differentiation and maturation. Here, we report that RNF114, previously identified as a psoriasis susceptibility gene, plays a regulatory role in the RANKL/RANK/TRAF6 signaling pathway that mediates osteoclastogenesis. Our results demonstrated that RNF114 expression was significantly down‐regulated in mouse osteoclast precursor cells undergoing RANKL‐induced osteoclast differentiation. RNF114 knockout did not affect development or viability of the subpopulation of bone marrow macrophages capable of differentiating into osteoclasts in culture. However, in the presence of RANKL, RNF114 knockout bone marrow macrophages exhibited enhanced cell proliferation and augmented osteoclast differentiation, as shown by an increased expression of mature osteoclast markers, increased osteoclastic TRAP activity and bone resorption. Conversely, ectopic expression of RNF114 inhibited CTSK expression, TRAP activity, and bone resorption in RANKL‐treated pre‐osteoclasts. RNF114 also suppressed RANKL‐activated NFATc1 expression and NFAT‐regulated promoter activity. RNF114 suppressed TRAF6‐, but not TAK1/TAB2‐mediated NF‐κB activation downstream of RANKL/RANK. In particular, TRAF6 protein levels were down‐regulated by RNF114, possibly via K48‐mediated proteasome‐dependent degradation. These data suggested that RNF114's inhibitory effect on RANKL‐stimulated osteoclastogenesis was mediated by blocking RANK/TRAF6/NF‐κB signal transduction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:159–166, 2018.     

Atp6v0d2 Is an Essential Component of the Osteoclast‐Specific Proton Pump That Mediates Extracellular Acidification in Bone Resorption

Bone resorption relies on the extracellular acidification function of vacuolar (V‐) ATPase proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of osteoclast‐specific V‐ATPases remains largely unknown. In this study, we found that Atp6v0d2 (d2), an isoform of the d subunit in the V‐ATPase, showed 5‐fold higher expression than that of Atp6v0d1 (d1) in mature osteoclasts, indicating a potential function in osteoclastic bone resorption. When d2 was depleted at an early stage of RANKL‐induced osteoclast differentiation in vitro, formation of multinucleated cells was severely impaired. However, depletion of d2 at a late differentiation stage did not affect osteoclast fusion but did abolish the activity of extracellular acidification and bone resorption of mature osteoclasts. We also showed the association of the two tagged‐proteins d2 and a3 when co‐expressed in mammalian cells with a co‐immunoprecipitation assay. Moreover, glutathione‐S‐transferase (GST) pull‐down assay showed the direct interaction of d2 with the N terminus of Atp6v0a3 (a3), which is the functionally identified osteoclast‐specific component of V‐ATPase. Therefore, our results show the dual function of d2 as a regulator of cell fusion in osteoclast differentiation and as an essential component of the osteoclast‐specific proton pump that mediates extracellular acidification in bone resorption.     

Macrophage colony-stimulating factor and interleukin-6 release by periprosthetic cells stimulates osteoclast formation and bone resorption.

Periprosthetic bone loss is an important contributory factor for aseptic loosening of total joint replacements. It has recently been shown that osteoclast precursor cells are present in the wear particle-associated macrophage infiltrate found in the membrane surrounding loose implants and that these cells are capable of differentiating into osteoclastic bone-resorbing cells. Long-term co-culture of arthroplasty-derived macrophages and the rat osteoblast-like cell line, UMR-106, in the presence of 1,25(OH)2D3 results in the formation of numerous multinucleated cells that are positive for tartrate-resistant acid phosphatase and vitronectin receptor and capable of extensive lacunar bone resorption. The aim of this study was to determine the effect of cytokines/growth factors, known to be present in the arthroplasty membrane, on this process of osteoclast differentiation. During osteoclast formation, increased levels of macrophage colony-stimulating factor, interleukin-6, and to a lesser extent, interleukin-1beta, but not tumour necrosis factor alpha, were detected in the co-culture supernatants. Addition of neutralising antibodies to human interleukin-1beta or tumour necrosis factor alpha to the co-culture system did not inhibit osteoclast formation. In contrast, co-cultures to which neutralising antibodies to human macrophage colony-stimulating factor or interleukin-6 were added contained fewer cells positive for tartrate-resistant acid phosphatase and vitronectin receptor and formed significantly fewer resorption pits. Time-course studies showed that macrophage colony-stimulating factor and interleukin-6 increase osteoclast formation mainly in the early stages of osteoclast differentiation. These results indicate that the release of macrophage colony-stimulating factor and interleukin-6 by activated cells in the arthroplasty membrane is likely to contribute to pathological bone resorption associated with aseptic loosening by stimulating differentiation of mononuclear phagocyte osteoclast precursors into mature bone-resorbing cells.     

Netrin‐1 Is a Critical Autocrine/Paracrine Factor for Osteoclast Differentiation

Bone metabolism is a vital process that involves resorption by osteoclasts and formation by osteoblasts, which is closely regulated by immune cells. The neuronal guidance protein Netrin‐1 regulates immune cell migration and inflammatory reactions, but its role in bone metabolism is unknown. During osteoclast differentiation, osteoclast precursors increase expression of Netrin‐1 and its receptor Unc5b. Netrin‐1 binds, in an autocrine and paracrine manner, to Unc5b to promote osteoclast differentiation in vitro, and absence of Netrin‐1 or antibody‐mediated blockade of Netrin‐1 or Unc5b prevents osteoclast differentiation of both murine and human precursors. We confirmed the functional relationship of Netrin‐1 in osteoclast differentiation in vivo using Netrin‐1‐deficient (Ntn1^‐/‐) or wild‐type (WT) bone marrow transplanted mice. Notably, Ntn1^‐/‐ chimeras have markedly diminished osteoclasts, as well as increased cortical and trabecular bone density and volume compared with WT mice. Mechanistic studies revealed that Netrin‐1 regulates osteoclast differentiation by altering cytoskeletal assembly. Netrin‐1 increases regulator of Rho‐GEF subfamily (LARG) and repulsive guidance molecule (RGMa) association with Unc5b, which increases expression and activation of cytoskeletal regulators RhoA and focal adhesion kinase (FAK). Netrin‐1 and its receptor Unc5b likely play a role in fusion of osteoclast precursors because Netrin‐1 and DC‐STAMP are tightly linked. These results identify Netrin‐1 as a key regulator of osteoclast differentiation that may be a new target for bone therapies. © 2015 American Society for Bone and Mineral Research.