Docetaxel inhibits bone resorption through suppression of osteoclast formation and function in different manners

Osteoclasts are formed from the monocyte-macrophage lineage in response to receptor activator of nuclear factor κB ligand (RANKL) expressed by osteoblasts. Bone is the most common site of breast cancer metastasis, and osteoclasts play roles in the metastasis. The taxane-derived compounds paclitaxel and docetaxel are used for the treatment of malignant diseases, including breast cancer. Here we explored the effects of docetaxel on osteoclastic bone resorption in mouse culture systems. Osteoclasts were formed within 6 days in cocultures of osteoblasts and bone marrow cells treated with 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂. Docetaxel at 10⁻⁸ M inhibited osteoclast formation in the coculture when added for the entire culture period or for the first 3 days. Docetaxel, even at 10⁻⁶ M added for the final 3 days, failed to inhibit osteoclast formation. Osteoprotegerin, a decoy receptor of RANKL, completely inhibited osteoclast formation when added for the final 3 days. Docetaxel at 10⁻⁸ M inhibited the proliferation of osteoblasts and bone marrow cells. RANKL mRNA expression induced by 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂ in osteoblasts was not affected by docetaxel even at 10⁻⁶ M. Docetaxel at 10⁻⁶ M, but not at 10⁻⁸ M, inhibited pit-forming activity of osteoclasts cultured on dentine. Actin ring formation and l-glutamate secretion by osteoclasts were also inhibited by docetaxel at 10⁻⁶ M. Thus, docetaxel inhibits bone resorption in two different manners: inhibition of osteoclast formation at 10⁻⁸ M and of osteoclast function at 10⁻⁶ M. These results suggest that taxanes have beneficial effects in the treatment of bone metastatic cancers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Osteoclasts prefer aged bone

Summary We investigated whether the age of the bones endogenously exerts control over the bone resorption ability of the osteoclasts, and found that osteoclasts preferentially develop and resorb bone on aged bone. These findings indicate that the bone matrix itself plays a role in targeted remodeling of aged bones. Introduction Osteoclasts resorb aging bone in order to repair damage and maintain the quality of bone. The mechanism behind the targeting of aged bone for remodeling is not clear. We investigated whether bones endogenously possess the ability to control osteoclastic resorption. Methods To biochemically distinguish aged and young bones; we measured the ratio between the age-isomerized βCTX fragment and the non-isomerized αCTX fragment. By measurement of TRACP activity, CTX release, number of TRACP positive cells and pit area/pit number, we evaluated osteoclastogenesis as well as osteoclast resorption on aged and young bones. Results We found that the αCTX / βCTX ratio is 3:1 in young compared to aged bones, and we found that both α and βCTX are released by osteoclasts during resorption. Osteoclastogenesis was augmented on aged compared to young bones, and the difference was enhanced under low serum conditions. We found that mature osteoclasts resorb more on aged than on young bone, despite unchanged adhesion and morphology. Conclusions These data indicate that the age of the bone plays an important role in controlling osteoclast-mediated resorption, with significantly higher levels of osteoclast differentiation and resorption on aged bones when compared to young bones. Kim Henriksen and Diana J. Leeming contributed equally. Financial disclosure: Morten A. Karsdal, Per Qvist and Claus Christiansen own stock options in Nordic Bioscience A/S     

Characterization of the Bone Phenotype in ClC-7-Deficient Mice

Mice deficient in the chloride channel ClC-7, which is likely involved in acidification of the resorption lacuna, display severe osteopetrosis. To fully characterize the osteopetrotic phenotype, the phenotypes of osteoclasts and osteoblasts were evaluated. ClC-7^−/− mice and their corresponding wild-type littermates were killed at 4–5 weeks of age. Biochemical markers of bone resorption (CTX-I), osteoclast number (TRAP5b), and osteoblast activity (ALP) were evaluated in serum. Splenocytes were differentiated into osteoclasts using M-CSF and RANKL. Mature osteoclasts were seeded on calcified or decalcified bone slices, and CTX-I, Ca²⁺, and TRAP were measured. Acidification rates in membrane vesicles from bone cells were measured using acridine orange. Osteoblastogenesis and nodule formation in vitro were investigated using calvarial osteoblasts. ClC-7^−/− osteoclasts were unable to resorb calcified bone in vitro. However, osteoclasts were able to degrade decalcified bone. Acid influx in bone membrane vesicles was reduced by 70% in ClC-7^−/− mice. Serum ALP was increased by 30% and TRAP5b was increased by 250% in ClC-7^−/− mice, whereas the CTX/TRAP5b ratio was reduced to 50% of the wild-type level. Finally, evaluation of calvarial ClC-7^−/− osteoblasts showed normal osteoblastogenesis. In summary, we present evidence supporting a pivotal role for ClC-7 in acidification of the resorption lacuna and evidence indicating that bone formation and bone resorption are no longer balanced in ClC-7^−/− mice.     

Quantitative analyses of osteoclast changes in resorbing bone organ cultures

Summary The stimulation of bone resorption, assessed by the release of⁴⁵Ca from prelabeled bones, was associated with an increase in number of osteoclasts per bone section in parathyroid hormone (PTH)-treated bones, but not in lipopolysaccharide (LPS)-treated bones. By contrast the number of nuclei per osteoclast increased following LPS treatment, but was not affected by PTH. LPS-treated bones had more multinucleated cells, some having as many as 27 nuclei per osteoclast. More osteoclasts were adjacent to the bone collar in bones treated with LPS or PTH than in control bones. In LPS-treated bones this area also contained the largest osteoclasts, as determined by the greatest number of nuclei per osteoclast. The results suggest that LPS and PTH stimulate osteoclastic resorption by different mechanisms.     

Osteoclastic bone resorption: in vitro analysis of the rate of resorption and migration of individual osteoclasts

Osteoclasts isolated from the long bones of newborn rabbits were cultured on translucent devitalized bone slices and observed by phase-contrast time-lapse cinemicrography and scanning electron microscopy (SEM). This has allowed us to measure the rate of resorption and the rate of migration of individual osteoclasts. Our films show that osteoclasts do not resorb while migrating. When the osteoclastic resorption areas, which are easily recognizable with phase-contrast microscopy as areas delineated by refractile lines, were observed by SEM, such areas appeared as excavated areas lined with a network of collagen fibrils. The rate of migration was calculated using time lapse recordings, and varied from 30 micron/hr to 248 micron/hr, with a mean +/- SEM of 105 +/- 10 micron/hr. The rate of resorption by individual osteoclasts was calculated using both time lapse and SEM data, and varied from 43 micron 3/hr to 1225 micron 3/hr with a mean +/- SEM of 390 +/- 109 micron 3/hr. Additional observations indicated not only that the same osteoclast can resorb at a different rate at different times without any definable alteration of the culture conditions, but also that the same osteoclast can simultaneously resorb two lacunae at different rates. These observations provide, for the first time, data on the rate of resorption and the rate of migration of individual osteoclasts on a bone substratum.     

The effect of 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) on the resorptive function of osteoclasts of known nuclear number

Bisphosphonates (BP) are known to suppress osteoclastic resorption in vivo and in vitro, but doubt persists as to how, and the effect of BP on the resorptive capability of osteoclasts of known nuclear number is unknown. We aimed to find whether the addition of 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) changed the nuclear profile of an osteoclast population in vitro, and to measure the resorptive efficiency of individually characterized osteoclasts in the presence and absence of the BP. Prehatch chick bone cells were cultured for 24 hours on slices of dentine in medium with or without added APD at 10^-6 M or 10^-8 M, or in control medium on dentine presoaked with 10^-6 M APD for 48 hours. Total pit counts, and pit depths, areas and volumes for pits made by osteoclasts of known nuclear number, were found using confocal video-rate laser reflection microscopy and 3-D image analysis software. APD in the medium inhibited resorption and reduced the volume, area, and depth resorbed per nucleus per chick osteoclast. The nuclear number distribution did not shift significantly, suggesting that no preferential effect arose from the APD affecting one size of cell more than another. The large reduction found in pit numbers, depths, areas, and volumes in the APD dentine-pretreated group supports previous views that BP released during resorption act as metabolic inhibitors, altering protein synthesis by the cell. Larger cells made larger pits, but resorptive efficiency was similar for different cell sizes within the control or APD-treated groups. There was a strong negative correlation between the maximum depth resorbed per nucleus and the number of nuclei in the osteoclast in all groups.     

Effects of prostacyclin and prostaglandin E1 (PGE1) on bone resorption in the presence and absence of parathyroid hormone

Summary Prostaglandins have been shown to stimulate osteoclastic bone resorption in organ culture but morphologic studies of isolated osteoclasts have shown a transient calcitonin-like inhibiting effect of these agents. We looked for a dual effect on bone resorption by comparing the early and late effects of prostaglandin E₁ (PGE₁), prostacyclin (PGI₂), 6α-carbaprostaglandin I₂ (C-PGI₂), a carbon substituted analog of PGI₂, and salmon calcitonin (CT) on the release of previously incorporated⁴⁵Ca from fetal rat long bones cultured in the presence of an inhibitor of cyclooxygenase, RO-20-5720. Experiments were performed in both the presence and absence of PTH (400 ng/ml), which was administered 24 hours before addition of prostaglandins or CT. In control cultures not stimulated by PTH, CT (100 mU/ml) produced significant decreases in⁴⁵Ca release at 48, 72, and 96 hours while PGE₁ (10⁻⁶ M), PGI₂ (10⁻⁵), and C-PGI₂ (10⁻⁶ M) each produced significant increases in resorption at 24 through 96 hours. PGE₁ at 10⁻⁵ M, but not 10⁻⁶ M, caused a significant decrease in medium⁴⁵Ca of 21% at 1 and 2 hours. Medium calcium measurements suggest that the change in⁴⁵Ca was due to inhibition of release and not to increased uptake. PGI₂ (10⁻⁵ M) and C-PGI₂ (10⁻⁶ M) caused no significant inhibitory effect. In cultures stimulated by PTH, CT produced significant inhibition of bone resorption of 6 through 96 hours, but no inhibition of bone resorption was noted at either early or late time points with PGE₁, PGI₂, or C-PGI₂. Moreover, the addition of PGI₂ (10⁻⁵M) to PTH-treated cultures actually enhanced⁴⁵Ca release beginning to 6 hours, when PGI₂ alone had no effect upon bone resorption. These results confirm that high concentration of PGI₂ can stimulate bone resorption and show a similar response to a stable analog, C-PGI₂. Moreover, PGI₂ was found to enhance PTH-stimulated bone resorption. A small transient inhibition of⁴⁵Ca release was observed with a high concentration of PGE₁ (10⁻⁵M) in the absence of PTH, which could be due to a transient direct inhibitory action upon osteoclasts.     

Ibuprofen inhibits localized bone resorption in the middle ear

Summary Localized osteoclastic bone resorption plays a significant role in the pathogenesis of several diseases of the middle ear as well as orthodontic tooth movement and long bone remodeling. The mechanisms of control of localized bone loss and systemic bone resorption may be different but both may be mediated by a final common pathway which includes prostaglandins. Prostaglandins seem to have a predominantly stimulatory effect on bone resorption, although the exact mechanism is poorly understood. Ibuprofen, a nonsteroidal antiinflammatory drug, is known to inhibit the synthesis of prostaglandins. It is likely that ibuprofen, through its inhibition of prostaglandin synthesis, would decrease the localized osteoclastic bone resorption in a previously described animal model system. Mongolian gerbils were divided into three groups: low dose ibuprofen (10 mg/kg per day), high dose ibuprofen (30 mg/kg per day), and a control group. Following surgical implantation of catheters to the right bullae of each gerbil, pressure was applied for 8 days, stimulating osteoclastic bone resorption. After killing the animals and histomorphometric analysis of the bullae from each, comparisons were made between each group using osteoclast surface (percentage of bone area covered by osteoclasts), osteoclast number (number of osteoclasts/mm bone length), and osteoclast profile area (in μm²). Significantly lower osteoclast surface (Oc. S/BS) was found in pressurized bullae from both treatment groups when compared with pressurized bullae from controls (P<0.05) and significantly lower osteoclast number (N.Oc/T.L) in pressurized bullae from both treatment groups when compared with pressurized bullae from controls (P<0.05). These differences were found to be dose-dependent. No significant differences in individual osteoclast profile area were found in either treatment group when compared with controls.     

Chloroquine,hydroxystilbamidine, and dapsone inhibit resorption of fetal rat bone in organ culture

Summary Three potential inhibitors of lysosomal enzyme release, chloroquine, hydroxystilbamidine, and dapsone were tested for their effects on the release of previously incorporated⁴⁵Ca and beta (β)-glucuronidase from fetal rat long bones cultured in a chemically defined medium. At concentrations of 10⁻⁵ to 10⁻⁸M, all three agents were able to inhibit the stimulation of bone resorption by parathyroid hormone (PTH) or prostaglandin E₂ (PGE₂). Inhibition was seen at concentrations which did not alter the uptake of (³H)-2-deoxy-glucose or the incorporation of (³H)-thymidine in bone. While the inhibitors blocked the stimulation of β-glucuronidase release by PTH and PGE₂, they could also cause a direct increase in total β-glucuronidase content and release. Hence the usual strong correlation between the release of β-glucuronidase and⁴⁵Ca was no longer seen in the presence of inhibitors. These data indicate that chloroquine, hydroxystilbamidine, and dapsone are potent inhibitors of bone resorption which may act by blocking the release of lysosomal enzymes in cells stimulated by PTH or PGE₂, but may have a different effect on other cell populations.     

Inhibition of bone resorption by bisphosphonates: Interactions between bisphosphonates,osteoclasts, and bone

Summary Bisphosphonates are nonbiodegradable pyrophosphate analogues that are being used increasingly to inhibit bone resorption in disorders characterized by excessive bone loss. We have previously found that dichloromethylene bisphosphonate (Cl₂MBP) inhibits bone resorption through injury to the cells that resorb Cl₂MBP-contaminated surfaces. 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) is a more potent inhibitor of bone resorptionin vivo, and we have attempted to identify a step in the resorptive pathway that accounts for this increased potency. We found that when osteoclasts, isolated from neonatal rat long bones, were incubated on bone slices in the presence of bisphosphonates, AHPrBP was less, rather than more potent as a resorption-inhibitor than Cl₂MBP. The greater sensitivity of resorption to AHPrBPin vivo could neither be attributed to an effect of AHPrBP on the ability of osteoblastic cells to stimulate resorption in response to calcium-regulating hormonesin vitro nor to an effect on osteoclast generation: osteoclast formation was unaffected by concentrations of AHPrBP 10-fold higher than those of Cl₂MBP which inhibit bone resorption in the bone slice assay. We also found no evidence for impaired osteoclast generationin vivo in AHPrBP-treated rats. These results suggest that the comparisons of potencyin vitro do not include all the factors responsible for determining bisphosphonate potencyin vivo. Because bisphosphonates owe the specificity of their actions to their ability to bind to bone surfaces, we performed experiments using bone slices that had been immersed in bisphosphonates before use. Bone resorption was virtually abolished on bone slices preincubated in 10⁻³ M AHPrBP. Inhibition was associated with degenerative changes in osteoclasts and a more rapid decrease in the number remaining on the bone surface than occurred with Cl₂MBP. The effect was specific for osteoclasts, could be prevented if bone resorption was suppressed by calcitonin, and was not seen in osteoclasts incubated in AHPrBP on plastic coverslips. These observations suggest that AHPrBP inhibits bone resorption through injury to osteoclasts when they solubilize bisphosphonate-contaminated bone. We found that the concentration of AHPrBP used in the preincubation phase could be reduced by an order of magnitude if the volume of the AHPrBP solution was correspondingly increased. This implies that the concentration of bisphosphonate is less relevant to potency comparisons than the density of bisphosphonate on the bone surface. The latter will be strongly influencedin vivo not only by affinity for bone but by the pharmacokinetic and other properties of the compound.     

Interstitial Collagenase Activity Stimulates the Formation of Actin Rings and Ruffled Membranes in Mouse Marrow Osteoclasts

Interstitial collagenase activity stimulates bone resorption by mouse marrow osteoclasts [1]. Here, we show that collagenase activity promotes bone resorption by activating adherent osteoclasts to resorb bone. Inhibition of interstitial collagenase activity, either with peptidomimetic hydroxymates or with a specific anti-interstitial collagenase inhibiting antibody, reduced bone resorption by 73-92%. Equal numbers of osteoclasts adhered to bone in the presence of collagenase inhibitors and osteoclast survival was unaffected. In contrast, formation of actin rings and polarization of the vacuolar-H+-ATPase (V-ATPase) to ruffled membranes, two indicators of osteoclast activation, were decreased by inhibiting collagenase activity and stimulated in the presence of cleaved or heat-denatured type I collagen in proportion to increases and decreases of bone resorptive activity. Addition of excess recombinant osteoprotegerin-ligand to cultures did not restore bone resorption in the presence of interstitial collagenase inhibitors. These data support the hypothesis that cleaved collagen stimulates osteoclastic bone resorption by triggering cytoskeletal reorganization and transport of V-ATPase from cytoplasmic stores to ruffled membranes.     

Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood

Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Osteoclast formation was only observed in the CD14+ population, not in the CD14− population, and only in the presence of both M-CSF and RANKL, confirming that the CD14+ system is a pure population of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells demonstrated that actin rings were only formed in the presence of RANKL. Moreover, the osteoclasts were capable of forming acidic resorption lacunae, and inhibitors of lysosomal acidification attenuated this process. Finally, we measured the response to known bone resorption inhibitors, and found that the osteoclasts were sensitive to these and thereby provided a robust and valid method for interpretation of the effect of antiresorptive compounds. In conclusion, we have established a robust assay for developing osteoclasts that can be used to study several biological aspects of the osteoclasts and which in combination with the resorption marker CTX-I provides a useful tool for evaluating osteoclast function in vitro.     

Alterations in osteoclast function and phenotype induced by different inhibitors of bone resorption - implications for osteoclast quality

Background  

Normal osteoclasts resorb bone by secretion of acid and proteases. Recent studies of patients with loss of function mutations affecting either of these processes have indicated a divergence in osteoclastic phenotypes. These difference in osteoclast phenotypes may directly or indirectly have secondary effects on bone remodeling, a process which is of importance for the pathogenesis of both osteoporosis and osteoarthritis. We treated human osteoclasts with different inhibitors and characterized their resulting function.     

Characterization of Circulating Human Osteoclast Progenitors: Development of In Vitro Resorption Assay

Several cell surface markers were used to isolate monocytes as osteoclast progenitors with an immunomagnetic cell separation system. Use of this system with specific monocyte antibodies produced 99% pure monocytes. When purified monocytes were cultured on bovine bone slices in the presence of receptor activator of nuclear factor-B (RANKL), macrophage-colony stimulating factor (M-CSF), tumor necrocis factor alpha (TNF-), and dexamethasone for 14 days, CD14⁺ CD11b⁺, and CD61⁺ monocytes had approximately 90-, 30- and 20-fold higher osteoclast formation capacities/plated cells compared to the control culture. CD15⁺ monocytes generated few tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP+ MNC), and CD169⁺ monocytes generated no TRACP+ MNC. This suggests, that there are various subsets of monocytes in the blood circulation and that they have different capacities in osteoclast formation. These results show that circulating human osteoclast progenitors can be efficiently purified by immunomagnetic cell separation system using anti-CD14, -CD11b, and -CD61 antibodies. These purified monocyte fractions had different ability to give rise to osteoclasts. CD169 was not found to be suitable for osteoclast progenitor isolation. Optimal concentration of dexamethasone for osteoclast formation and bone resorption was 10 nM. To develop a human resorption assay, osteoclasts were first induced for 7 days, whole media were replaced, cultures were continued for additional 3 days and C-terminal telopeptide of type I collagen was determined from culture media. This assay was shown to be functional, since two well-known resorption inhibitors, bafilomycin A₁ and calcitonin, dose-dependently inhibited the resorption activity of osteoclasts.     

The effects of parathyroid hormone or 1,25-dihydroxyvitamin D3 on monocyte-osteoclast fusion

Summary ³H-thymidine-labeled blood monocytes were cultured with osteoclasts in the presence or absence of parathyroid hormone or 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in order to evaluate (1) the percentage of monocytes capable of fusing with osteoclasts, (2) if parathyroid hormone or 1,25(OH)₂D₃ influences the contribution of blood monocytes to osteoclast nuclear turnover. We found that within 24 hours of culture, about 8% of blood monocytes fuse with osteoclasts regardless of the presence of parathyroid hormone (PTH) or 1,25(OH)₂D₃. On the other hand, formation of nonosteoclastic giant cells by fusion of monocytes is enhanced by 5×10⁻⁹ M 1,25(OH)₂D₃ but only in the presence of the bone resorptive cells.     

Deletion of FGFR3 in Osteoclast Lineage Cells Results in Increased Bone Mass in Mice by Inhibiting Osteoclastic Bone Resorption

Fibroblast growth factor receptor 3 (FGFR3) participates in bone remodeling. Both Fgfr3 global knockout and activated mice showed decreased bone mass with increased osteoclast formation or bone resorption activity. To clarify the direct effect of FGFR3 on osteoclasts, we specifically deleted Fgfr3 in osteoclast lineage cells. Adult mice with Fgfr3 deficiency in osteoclast lineage cells (mutant [MUT]) showed increased bone mass. In a drilled‐hole defect model, the bone remodeling of the holed area in cortical bone was also impaired with delayed resorption of residual woven bone in MUT mice. In vitro assay demonstrated that there was no significant difference between the number of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts derived from wild‐type and Fgfr3‐deficient bone marrow monocytes, suggesting that FGFR3 had no remarkable effect on osteoclast formation. The bone resorption activity of Fgfr3‐deficient osteoclasts was markedly decreased accompanying with downregulated expressions of Trap, Ctsk, and Mmp 9. The upregulated activity of osteoclastic bone resorption by FGF2 in vitro was also impaired in Fgfr3‐deficient osteoclasts, indicating that FGFR3 may participate in the regulation of bone resorption activity of osteoclasts by FGF2. Reduced adhesion but not migration in osteoclasts with Fgfr3 deficiency may be responsible for the impaired bone resorption activity. Our study for the first time genetically shows the direct positive regulation of FGFR3 on osteoclastic bone resorption. © 2016 American Society for Bone and Mineral Research.     

In Vitro Generation of Mature Human Osteoclasts

Mononuclear precursors of human osteoclasts are found in the CD14⁺ monocyte fraction of circulating peripheral blood mononuclear cells (PBMCs). It is possible to generate osteoclasts in vitro from PBMCs cultured with macrophage colony-stimulating factor and receptor activator for nuclear factor κB ligand. In these cultures, however, it is not possible to distinguish the effect of a specific agent on osteoclast resorption activity as opposed to osteoclast differentiation. To produce a population of mature human osteoclasts to study osteoclast lacunar resorption specifically, we cultured CD14⁺ human monocytes on hydrophobic dishes in order to generate and maintain osteoclasts in suspension prior to culturing them on coverslips and dentine slices. Multinucleated cells formed in these cultures expressed vitronectin receptor, tartrate-resistant acid phosphatase, and cathepsin K. These cells also produced F-actin rings and were capable of extensive lacunar resorption on dentine slices after 24 h in culture. Lacunar resorption was inhibited by calcitonin and zoledronate but not by osteoprotegerin. This method of generating a highly enriched population of mature human osteoclasts should provide a valuable means of specifically assessing the effect of molecular factors (e.g., cytokines, growth factors, hormones) and therapeutic agents on osteoclast resorption activity.     

Insulin-like Growth Factor I Does Not Stimulate Bone Resorption in Cultured Neonatal Mouse Calvarial Bones

Insulin-like growth factor I (IGF-I) has documented anabolic effects on osteoblasts, whereas its influence on osteoclasts and on bone resorption is unclear. We have investigated the effects of IGF-I on osteoclast recruitment and bone resorption in vitro. IGF-I (at and above 1 nM) stimulated the formation of multinucleated tartrate-resistant acid phosphatase positive cells in murine bone marrow cultures, incubated for 9 days. The number of multinucleated cells increased to 540 ± 160% of control (mean ± SEM) in cultures treated with 10 nM IGF-I. IGF-I (0.1–100 nM) had no effect by itself on ⁴⁵Ca-release from prelabelled neonatal mouse calvarial bones. However, IGF-I (100 nM) had an inhibitory effect on bone resorption induced by prostaglandin E₂ and 1,25(OH)₂D₃. These findings indicate that IGF-I enhances the formation of osteoclasts-like cells in long-term bone marrow cultures. In bone organ cultures, however, IGF-I has an inhibitory effect on stimulated bone resorption, suggesting that IGF-I inhibits existing osteoclasts and, alternatively, that IGF-I interferes with the osteoblast-derived factor(s) that stimulate existing osteoclasts. Received: 15 August 1995 / Accepted: 1 April 1996     

Bisphosphonate Inhibits Bone Turnover in OPG−/− Mice Via a Depressive Effect on Both Osteoclasts and Osteoblasts

Osteoclast differentiation and functioning are strictly controlled by RANKL expressed on osteoblast membrane surfaces, but whether osteoclasts exert control over osteoblasts remains unclear. In the present study, we examined the effect of an osteoclast inhibitor, a bisphosphonate (BP), on the response of maxillary bone to mechanical stress in a high-turnover osteoporosis model (OPG^−/− mice, a model of juvenile Paget disease). Mechanical stress was induced by use of orthodontic elastics to move the maxillary first molar. BP was administered once per day beginning 5 days before elastic insertion. Relative to wild type (WT), in the OPG^−/− mice tooth movement distance was greater, resorption of the interradicular septum occurred to a greater extent, the osteoclast count was higher, and serum alkaline phosphatase (ALP) was higher. However, administration of BP to OPG^−/− mice reduced tooth movement distance, increased bone volume at the interradicular septum, decreased the osteoclast count, and reduced serum ALP. BP administration also caused a temporal shift in peak Runx2 staining in OPG^−/− mice, such that the overall staining time course was similar to that observed for WT mice. We conclude that BP administration not only inhibited osteoclast activity in OPG^−/− mice but also systemically and locally inhibited osteoblast activity. It is possible that osteoclasts are able to exert some negative control over osteoblasts.     

Inhibition of parathyroid hormone-stimulated bone resorption by monovalent cation ionophores

Summary The actions of divalent cation ionophores on bone resorption in vitro are complex; both enhancement of resorption and inhibition of stimulated resorption have been observed with the ionophore A23187. We have found in neonatal mouse calvaria, in which divalent ionophores were only inhibitory, that monovalent cation ionophores were even more potent inhibitors of stimulated bone resorption. Nigericin, monensin, and X206 each inhibited the release of calcium (Ca) from calvaria that were stimulated to resorb by 0.1 U/ml parathyroid hormone (PTH). Actions of the three ionophores were dose dependent and were maximal at 10⁻⁷M, 3×10⁻⁷M, and 1.2×10⁻⁷M, respectively, compared to A23187, which was maximally inhibitory at 2×10⁻⁶M. After pretreatment with nigericin alone or together with PTH for 24 h, inhibition of stimulated resorption was partially reversible. Prolonged (48 h) treatment, either with ionophore alone or together with PTH, caused irreversible inhibition of stimulated Ca release. However, the ionophore was only partially inhibitory if it was added to calvaria stimulated by pretreatment with PTH alone for 24 or 48 h. Resorption stimulated by prostaglandin E₂, 1,25-dihydroxyvitamin D₃, and epidermal growth factor was also inhibited by monovalent ionophores, indicating that the inhibition was not at the level of the PTH receptor. In addition, ionophores did not lower basal or PTH-stimulated production of cyclic AMP by calvaria. Submaximal doses of nigericin were synergistic with calcitonin or ouabain in inhibiting PTH-stimulated resorption. These results are consistent with the hypothesis that stimulated release of Ca from bone occurs by a Na/Ca exchange mechanism. Thus monovalent cation ionophores would increase intracellular Na⁺, thereby decreasing the Na gradient across bone cell membranes, leading to conditions unfavorable for Ca efflux coupled to further Na influx.