Ion Transporters Involved in Acidification of the Resorption Lacuna in Osteoclasts

Osteoclasts possess a large amount of ion transporters, which participate in bone resorption; of these, the vacuolar-adenosine trisphosphatase (V-ATPase) and the chloride-proton antiporter ClC-7 acidify the resorption lacuna. However, whether other ion transporters participate in this process is currently not well understood. We used a battery of ion channel inhibitors, human osteoclasts, and their subcellular compartments to perform an unbiased analysis of the importance of the different ion transporters for acidification of the resorption lacuna in osteoclasts. CD14⁺ monocytes from human peripheral blood were isolated, and mature osteoclasts were generated using RANKL and M-CSF. The human osteoclasts were (1) used for acridine orange assays for evaluation of lysosomal acidification, (2) used for bone resorption assays, (3) used for generation of osteoclasts membranes for acid influx experiments, or (4) lysed in trizol for mRNA isolation for Affymetrix array analysis. Inhibitors targeted toward most of the ion transporters showed low potency in the acidification-based assays, although some inhibitors, such as carbonic anhydrase II and the sodium–hydrogen exchanger (NHE) inhibitors, reduced resorption potently. In contrast, inhibitors targeted at V-ATPase and ClC-7 potently inhibited both acidification and resorption, as expected. We here show evidence that acidification of the resorption lacuna is mainly mediated by V-ATPase and ClC-7. Furthermore, a group of other ion transporters, including carbonic anhydrase II, the NHEs, and potassium–chloride cotransporters, are all involved in resorption but do not seem to directly be involved in acidification of the lysosomes.     

Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

Background  

Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts.     

The chloride channel inhibitor NS3736 [corrected] prevents bone resorption in ovariectomized rats without changing bone formation.

Chloride channel activity is essential for osteoclast function. Consequently, inhibition of the osteoclastic chloride channel should prevent bone resorption. Accordingly, we tested a chloride channel inhibitor on bone turnover and found that it inhibits bone resorption without affecting bone formation. This study indicates that chloride channel inhibitors are highly promising for treatment of osteoporosis. INTRODUCTION: The chloride channel inhibitor, NS3736, blocked osteoclastic acidification and resorption in vitro with an IC50 value of 30 microM. When tested in the rat ovariectomy model for osteoporosis, daily treatment with 30 mg/kg orally protected bone strength and BMD by approximately 50% 6 weeks after surgery. Most interestingly, bone formation assessed by osteocalcin, mineral apposition rate, and mineralized surface index was not inhibited. MATERIALS AND METHODS: Analysis of chloride channels in human osteoclasts revealed that ClC-7 and CLIC1 were highly expressed. Furthermore, by electrophysiology, we detected a volume-activated anion channel on human osteoclasts. Screening 50 different human tissues showed a broad expression for CLIC1 and a restricted immunoreactivity for ClC-7, appearing mainly in osteoclasts, ovaries, appendix, and Purkinje cells. This highly selective distribution predicts that inhibition of ClC-7 should specifically target osteoclasts in vivo. We suggest that NS3736 is inhibiting ClC-7, leading to a bone-specific effect in vivo. RESULTS AND CONCLUSION: In conclusion, we show for the first time that chloride channel inhibitors can be used for prevention of ovariectomy-induced bone loss without impeding bone formation. We speculate that the coupling of bone resorption to bone formation is linked to the acidification of the resorption lacunae, thereby enabling compounds that directly interfere with this process to be able to positive uncouple this process resulting in a net bone gain.     

A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts

Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional or non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine with or without diphyllin, E64 or GM6001. Osteoclasts numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2T3 was treated with 50% of CM or non-CM for 12days. Bone formation was assessed by Alizarin Red extraction. CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed little resorption, but still an induction of bone formation by osteoblasts. Mimicking the reduction in bone resorption using the V-ATPase inhibitor Diphyllin, the cysteine proteinase inhibitor E64 and the MMP-inhibitor GM6001 showed that CM from diphyllin and E64 treated osteoclasts showed reduced ability to induce bone formation compared to CM from vehicle treated osteoclasts, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities. In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.     

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.     

Everolimus suppresses cancellous bone loss, bone resorption, and cathepsin K expression by osteoclasts

The proliferation inhibitor of the macrolide class, everolimus, is a drug shown to be effective in the prevention of organ transplant rejection and to have a potential in the treatment of rheumatoid arthritis and certain cancers. As these diseases or their current treatments are associated with bone loss, we examined the effect of everolimus on mouse and human bone cells in vitro and on bone in an ovariectomized (OVX) rat model. Everolimus potently inhibited primary mouse and human osteoclast activity in the pit assay (IC50 values of 0.6-4.0 nM), as well as osteoclast formation, measured as the number of tartrate-resistant acid phosphatase (TRAP) multinucleated cells (IC50 values of 7.7-10.5 nM). Inhibition of osteoblastic differentiation was also observed (IC50 value of 13.5 nM). As expected, everolimus inhibited proliferation of osteoclast precursors and stimulated apoptosis, albeit with insufficient potency and efficacy to explain inhibition of osteoclast activity. Thus, everolimus appeared to directly inhibit bone resorption, which is in accord with the detected inhibition of mRNA and protein expression of cathepsin K; the main collagen-degrading protease in osteoclasts. Despite the in vitro antiproliferative activity of everolimus and the observed inhibition of osteoblast differentiation, no detrimental effects were detected at different skeletal sites in mature OVX rats at doses up to 3 mg/kg/day. This everolimus dose also prevented the OVX-induced loss of cancellous bone by 60%, an effect predominantly associated with decreased osteoclast-mediated bone resorption, resulting in a partial preservation of the cancellous bone network. Everolimus inhibited S6 kinase 1 activity in rat blood cells, skin, and bone, at doses equivalent to those used for efficacy experiments in the OVX rat model, which demonstrated in vivo targeting of the expected molecular pathway. In conclusion, everolimus directly inhibits bone resorption by osteoclasts and thus could at least be neutral or protective for bone in vivo, which would favor its use in disease indications associated with bone loss.     

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.     

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

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

Potent and selective inhibition of human cathepsin K leads to inhibition of bone resorption in vivo in a nonhuman primate.

Cathepsin K is a cysteine protease that plays an essential role in osteoclast-mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB-357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB-357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin-releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB-357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N-terminal telopeptides (NTx) and C-terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.     

A Rationale for Osteoclast Selectivity of Inhibiting the Lysosomal V-ATPase a3 Isoform

Osteoclastic bone resorption can be completely abolished by inhibiting the vacuolar H⁺-ATPase (V-ATPase), a proton pump composed of at least 12 different subunits. However, V-ATPases are ubiquitous and it is unclear whether the osteoclast V-ATPase has a unique composition that would allow its selective inhibition. Aiming to answer this question, we compared human osteoclasts and monocytic THP.1 cells with respect to the localization of the a3 isoform of the 116-kDa subunit, which is indispensable for bone resorption, and sensitivity to SB242784, a V-ATPase inhibitor that prevents experimentally induced osteoporosis. By immunofluorescence, a3 was essentially nondetectable in THP.1 cells, while in osteoclasts a3 was highly upregulated and localized to lysosomes in nonresorbing osteoclasts. We isolated the lysosomal compartment from both sources as latex bead-containing phagolysosomes and compared them. Osteoclast phagolysosomes and THP.1 phagolysosomes both contained a3 and a1; however, the a3/a1 ratio was 3.8- to 11.2-fold higher in osteoclast phagolysosomes. Importantly, the V-ATPase-dependent acidification of phagolysosomes from both sources was essentially equally sensitive to SB242784. Thus, we observed no indication of a qualitative uniqueness of the osteoclast V-ATPase; rather, the high a3-level in osteoclasts may represent an upregulation of the common lysosomal V-ATPase. Our results, together with the reported phenotype of a3 deficiency and the reported efficacy of SB242784 in vivo, suggest that V-ATPase structure-independent mechanisms render bone resorption more sensitive than lysosomal function to V-ATPase inhibition. One such mechanism may be compensation of a3 by a1, which may be sufficient for retaining lysosomal function but not bone resorption.     

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.     

V-ATPase subunit ATP6AP1 (Ac45) regulates osteoclast differentiation, extracellular acidification, lysosomal trafficking, and protease exocytosis in osteoclast-mediated bone resorption

Lysosomal trafficking and protease exocytosis in osteoclasts are essential for ruffled border formation and bone resorption. Yet the mechanism underlying lysosomal trafficking and the related process of exocytosis remains largely unknown. We found ATP6ap1 (Ac45), an accessory subunit of vacuolar-type H(+)-ATPases (V-ATPases), to be highly induced by receptor activator for nuclear factor kappa B ligand (RANKL) in osteoclast differentiation. Ac45 knockdown osteoclasts formed normal actin rings, but had severely impaired extracellular acidification and bone resorption. Ac45 knockdown significantly reduced osteoclast formation. The decrease in the number of osteoclasts does not result from abnormal apoptosis; rather, it results from decreased osteoclast precursor cell proliferation and fusion, which may be partially due to the downregulation of extracellular signal-regulated kinase (ERK) phosphorylation and FBJ osteosarcoma oncogene (c-fos), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and "transmembrane 7 superfamily member 4" (Tm7sf4) expression. Notably, Ac45 knockdown osteoclasts exhibited impaired lysosomal trafficking and exocytosis, as indicated by the absence of lysosomal trafficking to the ruffled border and a lack of cathepsin K exocytosis into the resorption lacuna. Our data revealed that the impaired exocytosis is specifically due to Ac45 deficiency, and not the general consequence of a defective V-ATPase. Together, our results demonstrate the essential role of Ac45 in osteoclast-mediated extracellular acidification and protease exocytosis, as well as the ability of Ac45 to guide lysosomal intracellular trafficking to the ruffled border, potentially through its interaction with the small guanosine-5'-triphosphatase (GTPase) Rab7. Our work indicates that Ac45 may be a novel therapeutic target for osteolytic disease.     

Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclasts cultured on collagen-hydroxyapatite films

Diseases exhibiting excessive bone loss are often characterized by an increase in the size and number of osteoclasts in affected areas, suggesting that osteoclast size is associated with increased resorptive activity or efficiency. Because osteoclastic bone resorption depends on proton extrusion via a bafilomycin A1-sensitive vacuolar type H+ ATPase (V-ATPase), we investigated the relationship between osteoclast size and state of activity on the one hand, and proton-extruding mechanisms (bafilomycin A1-sensitive V-ATPase and amiloride-sensitive Na+/H+ exchange) on the other. In determining resorptive activities of individual osteoclasts, osteoclast-containing cell suspensions obtained from newborn rabbit long bones were cultured on apatite-collagen complex (ACC)-coated coverslips. Large osteoclasts resorbed 2.5 times more per cell than small osteoclasts, but the amount resorbed per nucleus was the same for the two categories. However, a much larger percentage of large osteoclasts was resorbing compared with small osteoclasts. To study pH regulatory mechanisms in individual large and small osteoclasts, the cells were loaded with the pH-sensitive indicator BCECF and analyzed by single-cell fluorescence. Small and large resorbing osteoclasts had significantly higher basal pH(i) than their nonresorbing counterparts. Also, small nonresorbing osteoclasts were insensitive to bafilomycin A1 addition or Na+ removal from the medium, large nonresorbing osteoclasts responded slightly, and all resorbing osteoclasts (small and large) responded strongly. Differences were also seen in the recovery from an acid load: both small and large nonresorbing osteoclasts were more sensitive to amiloride inhibition, while large resorbing cells were more sensitive to bafilomycin A1 inhibition. Small resorbing cells were inhibited equally by bafilomycin A1 and amiloride. These results clearly show that a greater proportion of large osteoclasts are active in resorption and that pH(i) regulation is associated with enhanced proton pump activity in actively resorbing osteoclasts. Thus, large and small osteoclasts differ in the proportion of cells that are resorbing, while pH regulatory mechanisms differ mainly between resorbing and nonresorbing cells.     

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.     

Heparin enhances osteoclastic bone resorption by inhibiting osteoprotegerin activity

Heparin is a highly sulfated glycosaminoglycan and has been shown to activate osteoclastic bone resorption though how is not yet clear. Here we investigate the molecule involved in heparin-induced activation of osteoclasts using an in vitro osteoclast culture assay. The formation and activation of osteoclasts are induced by receptor activator of NFkappaB ligand (RANKL) on osteoblasts, and inhibited by osteoprotegerin (OPG), a decoy receptor of RANKL, which is secreted from osteoblasts. In a coculture of mouse bone marrow cells and osteoblasts treated with 1,25-dihydroxyvitamin D(3) and prostaglandin E(2) on dentin slices, the bone marrow cells differentiate into osteoclasts, and resorption pits are formed on the dentin slices. Addition of heparin, various glycosaminoglycans, and chemically modified heparins to the coculture reveals that heparin enhances the pit-forming activity of osteoclasts, and this effect of heparin on the activation of osteoclasts is dependent on its sugar chain structure. By contrast, mRNA expression levels of RANKL, RANK, and OPG in the coculture are not altered by heparin treatment. Furthermore, neither RANK nor RANKL binds to heparin, suggesting that heparin does not directly interact with these proteins. Instead, heparin specifically binds to OPG and prevents OPG-mediated inhibition of osteoclastic bone resorption in the coculture. Heparin treatment does not enhance osteoclastic bone resorption in a monoculture of osteoclasts derived from bone marrow cells, and in the coculture using osteoblasts from OPG-deficient mice. A (125)I-OPG binding assay showed that OPG binds to osteoblasts and that this binding is inhibited by the addition of heparin, suggesting that OPG binds to RANKL on the osteoblast membrane and that heparin blocks this interaction. These results demonstrate that heparin enhances osteoclastic bone resorption by inhibiting OPG activity.     

Development and characterization of a human in vitro resorption assay: demonstration of utility using novel antiresorptive agents.

A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the alphavbeta3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase. The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human alphavbeta3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype. The cryopreserved cells can be cultured on bovine cortical bone for 24-48 h and resorption can be measured by either confocal microscopy or biochemical assays. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds. In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay. Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.     

FR177995, a novel vacuolar ATPase inhibitor, exerts not only an inhibitory effect on bone destruction but also anti-immunoinflammatory effects in adjuvant-induced arthritic rats

There is considerable evidence that osteoclasts are involved in the pathogenesis of juxta-articular bone destruction in rheumatoid arthritis. Vacuolar ATPases (V-ATPases), which are highly expressed in the ruffled border membrane of osteoclasts, play a central role in the process of bone resorption, and V-ATPase inhibitors are effective in preventing bone destruction in several animal models of lytic bone diseases. Here, we evaluated for the first time the effects of V-ATPase inhibition in rats with adjuvant-induced arthritis (AIA) using FR177995, a novel V-ATPase inhibitor. FR177995 completely inhibited H(+) transport driven by V-ATPase, but exerted no effect on the H(+) transport activities of F- and P-ATPase, indicating that FR177995 is a specific inhibitor of V-ATPase. FR177995 acted directly on osteoclastic bone resorption and equally inhibited in vitro bone resorption stimulated by IL-1, IL-6 or PTH. In addition, FR177995 dose-dependently reduced retinoic acid-induced hypercalcemia in thyroparathyroidectomized-ovariectomized rats. When FR177995 was administered to AIA rats once a day, the loss of femoral bone mineral density was significantly improved. Moreover, indicators of cartilage damage (arthritis score and glycosaminoglycan content in the femoral condyles) and inflammation parameters (paw swelling volume, erythrocyte sedimentation rate and plasma sialic acid level) were found to be unexpectedly ameliorated. These results strongly suggest that V-ATPase may be an interesting drug target in the treatment of rheumatoid arthritis.     

The N-terminal fragment of osteocalcin is released during osteoclastic bone resorption in vitro

As a novel method for measuring bone resorption, an immunoassay system for human N-terminal osteocalcin (N-OC) was developed to determine if osteocalcin molecules are released from bone degraded by osteoclasts in vitro. The assay system employed a monoclonal antibody to the first 20 N-terminal residues of osteocalcin as the solid phase and polyclonal antibodies against these same residues as an enzyme conjugate. This assay system could detect the N-terminal portion of osteocalcin formed during the degradation of the human osteocalcin molecule by trypsin or cathepsin D. Osteoclasts were isolated from human alveolar bone and cultured on human bone slices; the osteocalcin content in the media from these cultures was measured by this N-OC assay method. The N-terminal fragment of osteocalcin was the major form of osteocalcin released during osteoclastic bone resorption along with small amounts of intact osteocalcin. Interleukin-1 (IL-1β) and interleukin-6 (IL-6), stimulators of osteoclastic bone resorption, increased the N-OC level by 1.5 fold compared with the level of N-OC in osteoclast cultures in the absence of stimulators. In contrast, treatment of the cultures with calcitonin or an inhibitor of cathepsin D (E-64), both of which inhibit osteoclastic bone resorption, decreased the amount of N-OC in the culture supernatants. These results suggest that the N-terminal fragment of osteocalcin is released during osteoclastic bone resorption and that its level may serve as an index of bone resorption in vitro. Received: June 2, 1997 / Accepted: Oct. 1, 1997     

A vacuolar ATPase inhibitor, FR167356, prevents bone resorption in ovariectomized rats with high potency and specificity: potential for clinical application.

FR167356, a novel inhibitor of vacuolar ATPase, has high potency against osteoclast V-ATPase and low potency against lysosomal V-ATPase. FR167356 is the first compound of this nature to be tested. It has the potential to be useful for clinical application. INTRODUCTION: It has been suggested that the key issue regarding the therapeutic usefulness of V-ATPase inhibitors is their selectivity. MATERIALS AND METHODS: In in vitro and in vivo studies, we compared FR167356 with other vacuolar ATPase (V-ATPase) inhibitors, bafilomycin A1 and SB242784. H+ transport by various membrane vesicles was assayed by measuring uptake of acridine orange. Inhibitory activity against in vitro bone resorption was examined by measuring the Ca2+ release from cultured calvariae. In vivo, hypercalcemia was induced by retinoic acid in thyroparathyroidectomized-ovariectomized rats, and the effect on serum Ca2+ level was assessed. Ovariectomized rats were treated with FR167356 or SB242784. One week after surgery, free deoxypyridinoline levels in 24-h urine samples, which were collected from 6 h after administration of FR167356, were measured by ELISA. After 4 weeks of treatment, plasma biochemical parameters were analyzed. BMD of the distal femur metaphysis was measured with pQCT. Histomorphometric analysis of the proximal tibias was performed. Blood gases of rats treated with FR167356 were measured with a blood gas analyzer for estimating the effect of FR167356 on in vivo function of renal V-ATPase. RESULTS: FR167356, which is distinctly different from other V-ATPase inhibitors, has a high potency against osteoclast V-ATPase and low potency against lysosomal V-ATPase. Similarly, FR167356 inhibited bone resorption in vitro when stimulated by PTH, IL-1, and IL-6. FR167356 reduced retinoic acid-induced hypercalcemia in thyroparathyroidectomized-ovariectomized rats in a dose-dependent manner. Moreover, FR167356 was shown to restore BMD of ovariectomized rats caused by the inhibition of bone resorption. Ovariectomized rats treated with FR167356 did not show adverse symptoms, whereas SB242784 caused a decrease in body weight gain and significant changes in two plasma biochemical parameters. Interestingly, FR167356 treatment did not affect blood acid-base balance; however, FR167356 inhibited renal V-ATPase with a similar potency as for osteoclast V-ATPase inhibition. CONCLUSION: Comparison of FR167356 with SB242784 implies that the characteristics of FR167356 may be more appropriate for clinical application as a V-ATPase inhibitor.     

Glucocorticoids stimulate resorption in fetal rat parietal bones in vitro

The effect of glucocorticoids on bone resorption was examined in a serum-free mineralizing organ culture system derived from 20 day fetal rat parietal bones. Bone resorption was assessed by prelabeling the fetal rats in utero with 45Ca and determining the daily release of 45Ca into the medium of cultured bones. During the first 24 h of treatment a transient stimulation of bone resorption was found; 4.5 +/- 0.3% of the total 45Ca was released into the medium with 1 nM corticosterone and 4.1 +/- 0.2% with 10 nM corticosterone compared to 2.9 +/- 0.2% in control bones. Treatment with 1 and 10 nM dexamethasone for 24 h also showed an increase in 45Ca release compared to control bones. During the same time period 45Ca release was 6.9 +/- 1.4% with 10 nM parathyroid hormone. At later time points 100 and 1000 nM corticosterone inhibited 45Ca release, but 1 and 10 nM corticosterone values were similar to controls. At 24 h the number of osteoclasts per mm2 tissue in bone lacunae was significantly elevated with 1-100 nM corticosterone and 10 nM parathyroid hormone compared to control bones. In control bones 0.10 +/- 0.05 osteoclasts per mm2 of tissue were found, but 0.59 +/- 0.21 osteoclasts per mm2 were seen with 10 nM corticosterone and 1.50 +/- 0.34 with 10 nM parathyroid hormone. An additional assay of bone resorption, the release of lysosomal beta-glucuronidase into the medium was also elevated in glucocorticoid and parathyroid hormone-treated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)