Human mesenchymal tumour-associated macrophages differentiate into osteoclastic bone-resorbing cells

The cellular mechanisms which account for the formation of osteoclasts and bone resorption associated with enlarging benign and malignant mesenchymal tumours of bone are uncertain. Osteoclasts are marrow-derived, multinucleated, bone-resorbing cells which express a macrophage phenotype. We have determined whether tumour-associated macrophages (TAMs) isolated from benign and malignant mesenchymal tumours are capable of differentiating into osteoclasts. Macrophages were cultured on both coverslips and dentine slices for up to 21 days with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and human macrophage colony-stimulating factor (M-CSF) or, in the absence of UMR 106 cells, with M-CSF and RANK ligand. In all tumours, the formation of osteoclasts from CD14-positive macrophages was shown by the formation of tartrate-resistant-acid-phosphatase and vitronectin-receptor-positive multinucleated cells which were capable of carrying out lacunar resorption. These results indicate that the tumour osteolysis associated with the growth of mesenchymal tumours in bone is likely to be due in part to the differentiation of mononuclear phagocyte osteoclast precursors which are present in the TAM population of these lesions.     

Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption.

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. INTRODUCTION: Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. MATERIALS AND METHODS: Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1. RESULTS: In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. CONCLUSIONS: We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.     

Afamin secreted from nonresorbing osteoclasts acts as a chemokine for preosteoblasts via the Akt-signaling pathway

Although it is well known that osteoclastic bone resorption is followed by osteoblastic bone formation, questions remain as to when coupling factors are produced during bone resorption and which stages of bone formation are affected by these factors. To clarify these mechanisms, we established an in vitro system to investigate the coupling phenomenon. We obtained conditioned media (CM) from osteoclasts in the early and late stages of differentiation and from bone resorption stages. The collected CM was used to treat primary mouse calvarial osteoblasts and preosteoblastic MC3T3-E1 cells and to evaluate its influence on the migration, viability, proliferation, and differentiation of osteoblasts. We found that CM from osteoclasts in the early stage of differentiation predominantly stimulated the migration of osteoblastic lineages. By further performing fractional analyses of the CM with liquid chromatography-tandem mass spectrometry, we identified afamin, which has binding activity with vitamin E, as a possible coupling factor. The CM collected from afamin siRNA-transfected osteoclasts significantly suppressed preosteoblast migration. Afamin activated Akt in preosteoblasts, and pretreatment with Akt inhibitor significantly blocked afamin-stimulated preosteoblast migration. In conclusion, these results indicate that osteoclasts themselves play a central role in the coupling of bone resorption and formation by stimulating preosteoblast migration. In addition, we identified afamin as one of osteoclast-derived chemokines that affect preosteoblasts through the activation of the Akt-signaling pathway.     

Osteoclast TGF‐β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

Osteoblast‐mediated bone formation is coupled to osteoclast‐mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age‐related bone loss. Osteoclasts release and activate TGF‐β from the bone matrix. Here we show that osteoclast‐specific inhibition of TGF‐β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF‐β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF‐β receptor signaling. Osteoclasts in aged murine bones had lower TGF‐β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF‐β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF‐β availability with age. Therefore, osteoclast responses to TGF‐β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age‐related bone loss. © 2015 American Society for Bone and Mineral Research.     

Osteoclast‐Derived Complement Component 3a Stimulates Osteoblast Differentiation

Bone remodeling is regulated by a coupling of resorption to subsequent formation; however, the “coupling factor” and underlying mechanism are not fully understood. Here, we found that the condition medium (CM) of mature osteoclasts contains a humoral factor that stimulates the differentiation of primary osteoblasts, as determined by alkaline phosphatase (ALP) activity. We purified osteoblastogenesis‐stimulating activity from 3 L of osteoclast CM through successive ion exchange chromatographies by monitoring the ALP activity of osteoblasts and identified complement component 3 (C3). Expression of the C3 gene increased during osteoclastogenesis, and the cleavage product C3a was detected by ELISA in the CM of osteoclasts but not in that of bone marrow macrophages. The osteoblastogenesis‐stimulating activity present in osteoclast CM was inhibited by a specific antagonist of the C3a receptor (C3aR), SB290157. Conversely, the retroviral expression of C3a as well as treatment with the C3aR agonist, benzeneacetamide, stimulated osteoblast differentiation. C3 gene expression in bone was increased in the high bone turnover states of ovariectomy (OVX) or a receptor activator of NF‐κB ligand (RANKL) injection, and blocking the action of C3a with the daily administration of SB290157 resulted in the attenuation of bone formation elevated by OVX and the exacerbation of bone loss. These results suggest that osteoclast‐derived C3a functions in the relay from bone resorption to formation and may be a candidate for a coupling factor. © 2014 American Society for Bone and Mineral Research.     

Coupling of resorption and formation on bone remodeling sequence in orthodontic tooth movement: A histochemical study

Rat's molars were submitted to orthodontic tooth movement. Bone formation areas were detected using lead-labeling technique. Osteoclasts and osteoblasts were detected by enzyme histochemistry using Tartrate resistant Acid phosphatase (TRACPase) and Alkaline phosphatase (ALPase) to determine simultaneously and mark the 2 types of cells on a same section. The sites selected for study were pressure/distal, tension/mesial and transitional areas of second molars. The results showed that: orthodontic force activated bone remodeling sequence throughout the alveolar bone; slight new bone formation was observed on the cement line on the pressure side. ALPase-positive cells were detected on the pressure side neighboring osteoclasts. On the tension side, bone formation was enhanced in the protrusions whereas both resorption and formation were observed in the depressions. In the transitional area, cellular sequence from osteoclastic bone resorption to bone formation was revealed over the cement line. These findings demonstrated that: coupling phenomena occur on the pressure side but with inhibited osteoblast activity. Bone formation on the tension side involves both promotion of bone formation by the traction force; bone remodeling sequence is established on the tension side by the interaction between osteoclastic bone resorption and bone formation that takes place in the depressions; Coupling phenomena occur in the transitional area as well. Our findings on the pressure side led to the consideration that osteoblastic cells in periodontal ligament would be involved in the regulation of osteoclastic bone resorption. Thus, there appears to be an interaction between osteoclastic and osteoblastic cells and an activated bone remodeling sequence involving the coupling phenomena as a mechanical adaptation to orthodontic force.     

A rat model for testing pharmacologic treatments of pressure-related bone loss

Fluid pressure, instability, or particles have been suggested to initiate the process leading to loosening of prosthetic implants. In a rat model where bone resorption is caused by oscillating fluid pressure, the resorptive response seems much stronger than the response that can be induced by particles or instability. Bone resorption is caused by osteoclasts. It has been suggested that the formation of osteoclasts is influenced by tumor necrosis factor-alpha, which can be blocked by etanercept. Osteoclasts can be inactivated with bisphosphonates, which bind to bone and inactivate osteoclasts when the bisphosphonate-containing bone is resorbed. Bone formation can be increased dramatically by intermittent parathyroid hormone treatment, especially at sites with high bone turnover. This might compensate for increased osteoclastic activity. Forty-two rats received a plate implant, by which fluid pressure was applied to a bone surface by compressing a soft tissue membrane. Eight rats were treated with etanercept 0.75 mg/kg/day, six rats were treated with alendronate 205 microg/kg/day, six rats received saline, and six rats were nonpressurized controls. Nine rats received intermittent parathyroid hormone treatment with nine separate controls. The area of bone resorption under the implant was evaluated by histomorphometry. Alendronate-treated rats showed less bone resorption, but etanercept, intermittent parathyroid hormone treatment, or saline did not reduce the fluid pressure-induced bone resorption. This model is a comparatively simple way of testing pharmacologic reduction of local bone resorption in vivo.     

Intraosseous growth of human prostate cancer in implanted adult human bone: relationship of prostate cancer cells to osteoclasts in osteoblastic metastatic lesions

BACKGROUND: More than 80% of patients with advanced prostate cancer have skeletal involvement, but the biology of bone metastasis is poorly understood. This study investigated the in vivo formation and progression of bone metastases under conditions that resembled the human bone environment as closely as possible. METHODS: Adult human bone fragments were implanted subcutaneously into 120 male NOD/SCID mice. Four weeks later, 1 x 10(7) LNCaP prostate cancer cells or phosphate-buffered saline were injected intravenously into 80 or 40 mice, respectively. The implanted bone fragments were removed from 20 to 10 mice in each group at 2, 4, 6, and 8 weeks after injection. RESULTS: LNCaP colonized the bone marrow blood vessels within 2 weeks, and then gradually expanded into the entire medullary cavity. An osteoblastic response often occurred at the edges of metastatic foci (intertrabecular bone metaplasia). In addition, new bone formation was observed adjacent to mature lamellar bone (appositional bone formation). These two processes appeared to occur through different mechanisms, but might similarly cause osteosclerosis. Osteoclasts showed a marked increase in numbers at sites of early tumor invasion, whereas few osteoclasts were observed at sites where tumor invasion was complete. CONCLUSIONS: The predominance of osteoblastic change with resorption may lead to bone remodeling in metastatic lesions, and osteoclasts may play an important role in bone metastasis from prostate cancer.     

Involvement of vacuolar H+ -ATPase in incorporation of risedronate into osteoclasts

Although osteoclasts incorporate bisphosphonates during bone resorption, the mechanism of this incorporation by osteoclasts is not known. We previously reported that bisphosphonates disrupt the actin rings (clear zones) formed in normal osteoclasts, but did not disrupt actin rings in osteoclasts derived from osteosclerotic oc/oc mice, which have a defect in the gene encoding vacuolar H(+)-ATPase (V-ATPase). The present study showed that V-ATPase is directly involved in the incorporation of risedronate, a nitrogen containing bisphosphonate, into osteoclasts. Treatment of osteoclasts with risedronate disrupted actin rings and inhibited pit formation by osteoclasts on dentine slices. Bafilomycin A(1), a V-ATPase inhibitor, inhibited the pit-forming activity of osteoclasts but did not disrupt actin rings. Risedronate failed to disrupt actin rings in the presence of bafilomycin A(1). E-64, a lysosomal cysteine proteinase inhibitor, showed no inhibitory effect on the demineralization of dentine by osteoclasts but inhibited the digestion of dentine matrix proteins without disrupting actin rings. Risedronate disrupted actin rings even in the presence of E-64. Treatment of osteoclasts placed on plastic plates with risedronate also disrupted actin rings. Bafilomycin A(1) but not E64 prevented the disruption of actin rings in osteoclasts treated with risedronate on plastic plates. Inhibition of V-ATPase with bafilomycin A(1) also prevented disruption of actin rings by etidronate, a non-nitrogen-containing bisphosphonate. These results suggest that V-ATPase induced acidification beneath the ruffled borders of osteoclasts and subsequent bone demineralization triggers the incorporation of both nitrogen-containing and non-nitrogen-containing bisphosphonates into osteoclasts.     

Advances in osteoclast biology reveal potential new drug targets and new roles for osteoclasts

Osteoclasts are multinucleated myeloid lineage cells formed in response to macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) by fusion of bone marrow–derived precursors that circulate in the blood and are attracted to sites of bone resorption in response to factors, such as sphingosine‐1 phosphate signaling. Major advances in understanding of the molecular mechanisms regulating osteoclast functions have been made in the past 20 years, mainly from mouse and human genetic studies. These have revealed that osteoclasts express and respond to proinflammatory and anti‐inflammatory cytokines. Some of these cytokines activate NF‐κB and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling to induce osteoclast formation and activity and also regulate communication with neighboring cells through signaling proteins, including ephrins and semaphorins. Osteoclasts also positively and negatively regulate immune responses and osteoblastic bone formation. These advances have led to development of new inhibitors of bone resorption that are in clinical use or in clinical trials; and more should follow, based on these advances. This article reviews current understanding of how bone resorption is regulated both positively and negatively in normal and pathologic states. © 2013 American Society for Bone and Mineral Research.     

Inhibition of bone resorption by inorganic phosphate is mediated by both reduced osteoclast formation and decreased activity of mature osteoclasts

High concentrations of inorganic phosphate (Pi) are known to inhibit bone resorption, although the mechanism(s) underlying this effect is unclear. To investigate whether Pi can inhibit the formation of osteoclasts we studied the effects of changes in Pi concentration between 1 and 4 mM on osteoclast-like cell formation in 1 week cultures of mouse bone marrow. Osteoclast-like cells were identified by multinuclearity, positive staining for tartrate-resistant acid phosphatase (TRAP), and contraction in response to calcitonin. Increasing concentrations of Pi inhibited formation of these cells in a dose-dependent manner. To study effects of Pi on the bone-resorbing activity of mature osteoclasts we isolated osteoclasts from calcium-deficient egg-laying hens or rat pups and incubated them on sperm whale dentine slices. High Pi concentrations markedly reduced both the number of resorption pits formed per dentine slice and the mean area of each pit in both avian and mammalian systems. These data indicate that high concentrations of Pi act on bone directly, both to inhibit generation of new osteoclasts from their precursor cells and to inhibit bone resorption by mature osteoclasts. These effects of extracellular Pi concentration may play an important modulatory role on bone turnover in vivo and have potential importance in several disease states in which Pi metabolism is perturbed.     

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)     

The relative contribution of cysteine proteinases and matrix metalloproteinases to the resorption process in osteoclasts derived from long bone and scapula

It has been suggested that functional heterogeneity exists between osteoclasts from different bone sites. This could be exploited to design therapeutics that would selectively inhibit bone resorption only at compromised sites. To further investigate the existence of functional differences between osteoclasts from different bone sites we assessed whether osteoclasts isolated from intramembranous bone differ from osteoclasts isolated from endochondral bone in the extent that they utilize cysteine proteinases and matrix metalloproteinases to degrade the organic matrix of bone. The differential involvement of the two classes of proteases was assessed by analyzing dose-dependent effects of the matrix metalloproteinase inhibitor, CT-1746, and of the cathepsin inhibitor, E64, on bone resorption. Osteoclasts isolated from the scapula (intramembranous) and long bones (endochondral) of newborn New Zealand white rabbits were seeded on cortical bovine bone slices in the presence or absence of inhibitors. Resorptive activity was evaluated by measuring the number and area of resorption pits and by measuring the release of collagen degradation products in the culture medium. In the absence of inhibitors, scapular osteoclasts and long bone osteoclasts had similar activity based on these criteria. The resorptive activity of scapular osteoclasts was inhibited to a greater extent by the MMP inhibitor CT-1746 than by the cysteine proteinase inhibitor E64. Conversely, resorption by osteoclasts derived from long bones was inhibited to a greater degree by the cysteine proteinase inhibitor. These results strongly suggest that there are functional differences between dispersed osteoclasts derived from the scapula and long bones, with scapular osteoclasts utilizing matrix metalloproteinases to a greater extent than cysteine proteinases and long bone osteoclasts using cysteine proteinases to a greater extent than matrix metalloproteinases.     

Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells

Bisphosphonates (BPs) target bone due to their high affinity for calcium ions. During osteoclastic resorption, these drugs are released from the acidified bone surface and taken up by osteoclasts, where they act by inhibiting the prenylation of small GTPases essential for osteoclast function. However, it remains unclear exactly how osteoclasts internalise BPs from bone and whether other cells in the bone microenvironment can also take up BPs from the bone surface. We have investigated this using a novel fluorescently-labelled alendronate analogue (FL-ALN), and by examining changes in protein prenylation following treatment of cells with risedronate (RIS). Confocal microscopic analysis showed that FL-ALN was efficiently internalised from solution or from the surface of dentine by resorbing osteoclasts into intracellular vesicles. Accordingly, unprenylated Rap1A accumulated to the same extent whether osteoclasts were cultured on RIS-coated dentine or with RIS in solution. By contrast, J774 macrophages internalised FL-ALN and RIS from solution, but took up comparatively little from dentine, due to their inability to resorb the mineral. Calvarial osteoblasts and MCF-7 tumour cells internalised even less FL-ALN and RIS, both from solution and from the surface of dentine. Accordingly, the viability of J774 and MCF-7 cells was drastically reduced when cultured with RIS in solution, but not when cultured on dentine pre-coated with RIS. However, when J774 macrophages were co-cultured with rabbit osteoclasts, J774 cells that were adjacent to resorbing osteoclasts frequently internalised more FL-ALN than J774 cells more distant from osteoclasts. This was possibly a result of increased availability of BP to these J774 cells due to transcytosis through osteoclasts, since FL-ALN partially co-localised with trancytosed, resorbed matrix protein within osteoclasts. In addition, J774 cells occupying resorption pits internalised more FL-ALN than those on unresorbed surfaces. These data demonstrate that osteoclasts are able to take up large amounts of BP, due to their ability to release the BP from the dentine surface during resorption. By contrast, non-resorbing cells take up only small amounts of BP that becomes available due to natural desorption from the dentine surface. However, BP uptake by non-resorbing cells can be increased when cultured in the presence of resorbing osteoclasts.     

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.     

Signaling axis in osteoclast biology and therapeutic targeting in the RANKL/RANK/OPG system

Bone integrity is maintained through a balance between bone formation and bone resorption, and osteoclasts are primary cells involved in bone resorption. Recent studies have revealed an essential role of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) in the development of osteoclasts, and detailed molecular cascades that induce osteoclast differentiation, activation and apoptosis have been clarified. Osteoclasts are involved in various pathologic conditions, such as osteoporosis, rheumatoid arthritis and tumor-induced bone disease, which are characterized by abnormal bone resorption, and the finding of RANKL has provided us a good therapeutic target for such pathologic conditions.     

Intercellular Cross-Talk Among Bone Cells: New Factors and Pathways

Intercellular communication within the bone microenvironment is critical for the maintenance of normal bone structure. Osteoblast-lineage cells at all stages of differentiation, from pluripotent precursors to matrix-embedded osteocytes, produce regulatory factors that modulate the differentiation and activity of both bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoclasts can also release factors that feed back to regulate osteoblast activity. Intercellular cross-talk within the bone microenvironment is not restricted only to these bone cells. Other cells within the bone marrow microenvironment, including adipocytes, T cells, and macrophages, play key roles that influence the processes of bone formation and resorption. This review discusses recent work that provides new insights into some of these communication networks and the factors involved, including osteocytic production of receptor activator of nuclear factor-κB ligand (RANKL) and sclerostin, osteoblastic production of interleukin-33, osteoclast-derived Semaphorin 4D, ephrin signaling, and signals from T helper cells and resident osteal macrophages (osteomacs).     

Bovine dentine organic matrix down-regulates osteoclast activity

Physiological root resorption is a phenomenon that normally takes place in deciduous teeth; root resorption of permanent teeth occurs only under pathological conditions. The molecular mechanisms underlying these processes are still unclear. Our previous study showed that osteoclasts cultured on deciduous dentine exhibited a higher degree of resorption and higher levels of cathepsin K and MMP-9 mRNA than osteoclasts cultured on permanent dentine. These results could be because of different susceptibilities to acid and the different organic matrices between deciduous and permanent dentine. Thus, the purpose of this study was to investigate the effect of dentine extracts from bovine deciduous and permanent dentine on osteoclast activity. Osteoclasts, obtained from mouse bone marrow cells co-cultured with an osteoblast-rich fraction in the presence of 1,25-(OH)₂-vitamin D3 and PGE2, were incubated with or without 0.6 M HCl extracts from bovine deciduous or permanent dentine for 48 h. TRAP positive cell number, TRAP activity, the areas of resorption pits, and mRNA levels of TRAP, v-ATPase, calcitonin receptor, cathepsin K, and MMP-9 were examined. The results illustrated that TRAP activity, the resorbed area, and the mRNA levels of osteoclast marker genes seemed to be suppressed by both deciduous and permanent dentine extracts. These findings indicate that some factors that suppress osteoclast activity are contained in both deciduous and permanent dentine extracts. Although there was no significant difference in osteoclast activity between deciduous and permanent dentine extracts, osteoclasts incubated with permanent dentine extracts tend to exhibit less resorption activity than those incubated with deciduous dentine extracts. However, we could not clearly explain the causes of this.     

c-fos antisense DNA inhibits proliferation of osteoclast progenitors in osteoclast development but not macrophage differentiation in vitro

We previously reported that osteoclast formation in vitro, by coculture of mouse bone marrow and primary osteoblastic cells, occurs in two phases: proliferation of osteoclast progenitors followed by terminal differentiation into mature osteoclasts. Using this coculture system, we examined the effects of c-fos antisense and sense phosphorothioate oligonucleotides on osteoclast development and macrophage differentiation. Treatment with c-fos antisense for the first 4 days of coculture inhibited osteoclast formation in a dosedependent fashion. However, when c fos antisense was added during the second phase of coculture (4–6 days), osteoclast formation was unaffected. In contrast, c-fos antisense treatment had no effect on the appearance of F4/80 antigen-positive cells of the macrophage lineage in these cultures or on the induction by colony stimulating factor-1 of macrophage colony formation in cultures of mouse bone marrow cells in agar. Neither osteoclast differentiation nor macrophage appearance was inhibited by adding control c-fos sense in the cocultures. When c-fos antisense was added into an assay of bone resorption by mature osteoclasts, pit formation on dentine slices was unaffected. These results indicate that c-fos plays an important role in the proliferative phase of osteoclast progenitors in osteoclast development, but not in the terminal differentiation phase or in the bone resorbing activity of mature osteoclasts. c-fos antisense specifically inhibited osteoclast formation but had no effect on macrophage development.     

Transmigration: a new property of mature multinucleated osteoclasts.

Even though it is assumed that multinucleated osteoclasts are migrating cells on the bone surface to be resorbed, we show that they can also selectively transmigrate through layers of cells usually found in the bone microenvironment. This activity is associated with c-src and MMPs and can be stimulated by bone metastatic breast cancer cells, a process blocked by bisphosphonate treatment. INTRODUCTION: Osteoclasts have an hematopoietic origin and are bone-resorbing cells. Monocytic precursors migrate to the bone surface where they fuse to form multinucleated osteoclasts able to migrate over the bone surface. We studied whether multinucleated osteoclasts were also able to transmigrate through tissues. MATERIALS AND METHODS: Murine spleen-derived and green fluorescent protein (GFP)-Raw derived osteoclasts were seeded on osteoblasts and several other cell types. The cells were fixed for 20 minutes, 4 or 12 h after osteoclast seeding, and stained with phalloidin to visualize actin using confocal microscopy. Drugs such as PP2 and GM6001, inhibitors of c-src and matrix metalloproteinases (MMPs), respectively, and risedronate were used to determine osteoclast transmigration regulating factors. RESULTS: We observed by confocal microscopy that multinucleated osteoclasts specifically transmigrate through confluent layers of various cell types present in the bone microenvironment in vitro. This is an efficient process associated with c-src and MMPs but is independent of podosomes. Moreover, conditioned medium from bone metastatic breast cancer cells stimulates osteoclast transmigration in vitro, a process inhibited by bisphosphonate treatment. CONCLUSIONS: Our data describe a new property of mature multinucleated osteoclasts to transmigrate through various cell types. The ability to control this highly regulated osteoclast transmigration process may offer new therapeutic strategies for bone diseases associated with an imbalance in bone remodeling caused by excessive osteoclast resorption.