Multinucleated Tartrate-resistant Acid Phosphatase Research Articles

Heparin inhibits osteoclastic differentiation and function

We investigated the effects of Glycosaminoglycans (GAGs) on mouse monocytic cell line in regard to their differentiation, proliferation, and function in vitro. RAW 264.7 cells were cultured with receptor activator of NF-kappaB ligand (RANKL) and various GAGs. Osteoclastic cells were visualized by staining for tartrate-resistant acid phosphatase (TRAP) and detected using a phenyl-phosphate substrate method. RAW 264.7 cells were also cultured with stimulants contained in BD BioCoat OSTEOLOGIC(TM) kit, and bone resorption activity was assessed by counting the numbers of resorption pits. We also examined the effect of heparin on cell growth using MTT assay, while the expression level of c-Src protein was determined by immunoblot analysis. Heparin suppressed TRAP-positive multinucleated cell formation and TRAP activity induced by RANKL, whereas the other GAGs showed no effects on osteoclast differentiation. Heparin also inhibited the formation of resorption pits, while the others did not. In the MTT assay, none of the tested GAGs had an influence on RAW 264.7 cell proliferation. However, heparin reduced the level of c-Src protein in RAW 264.7 cells stimulated with RANKL. To determine the affinity of heparin and RANKL, they were subjected by HiTrap heparin column chromatography and each fraction was collected. Western blotting analysis revealed the expression of RANKL in the fraction bound to heparin. The binding of RANKL and heparin was confirmed by quartz-crystal microbalance. These results indicate that the inhibitory effect of heparin toward osteoclastogenesis induced by RANKL is due to the binding of heparin to RANKL.

Monocyte Chemotactic Protein-1 Mediates Prostate Cancer–Induced Bone Resorption

Prostate cancer preferentially metastasizes to bone, resulting in high mortality. Strategies to inhibit prostate cancer metastasis include targeting both tumor-induced osteoblastic lesions and underlying osteoclastic activities. We and others have previously shown that blocking receptor activator of nuclear factor-kappaB ligand (RANKL) partially blocks tumor establishment and progression in bone in murine models. However, levels of RANKL in the cell lines used in these studies were very low, suggesting that soluble factors other than RANKL may mediate the cancer-induced osteoclast activity. To identify these factors, a human cytokine antibody array was used to measure cytokine expression in conditioned medium collected from primary prostate epithelial cells (PrEC), prostate cancer LNCaP and its derivative C4-2B, and PC3 cells. All prostate cancer cells produced high amounts of monocyte chemotactic protein-1 (MCP-1) compared with PrEC cells. Furthermore, levels of interleukin (IL)-6, IL-8, GROalpha, ENA-78, and CXCL-16 were higher in PC3 than LNCaP. These results were confirmed by ELISA. Finally, human bone marrow mononuclear cells (HBMC) were cultured with PC3 conditioned medium. Although both recombinant human MCP-1 and IL-8 directly stimulated HBMC differentiation into osteoclast-like cells, IL-8, but not MCP-1, induced bone resorption on dentin slices with 21 days of culture in the absence of RANKL. However, the conditioned medium-induced bone resorption was inhibited by MCP-1 neutralizing antibody and was further synergistically inhibited with IL-8 antibody, indicating that MCP-1, in addition to IL-8, mediates tumor-induced osteoclastogenesis and bone resorption. MCP-1 may promote preosteoclast cell fusion, forming multinucleated tartrate-resistant acid phosphatase-positive osteoclast-like cells. This study may provide novel therapeutic targets for treatment of prostate cancer skeletal metastasis.

Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPα-interaction

Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPα) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPα-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPα strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)+ osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47−/− mice were strongly reduced, and bones of CD47−/− mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPα interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47−/− mice.

Hepatocyte growth factor can substitute for M-CSF to support osteoclastogenesis

Osteopetrotic mice lacking functional macrophage-colony stimulating factor (M-CSF) recover with ageing, suggesting that alternative osteoclastogenesis pathways exist. Hepatocyte growth factor (HGF) and M-CSF signal through tyrosine kinase receptors and phosphorylate common transducers and effectors such as Src, Grb2, and PI3-Kinase. HGF is known to play a role in osteoclast formation, and in this study we have determined whether HGF could replace M-CSF to support human osteoclastogenesis. We found that the HGF receptor, c-Met, is expressed by the CD14 + monocyte fraction of human peripheral blood mononuclear cells (PBMC). HGF was able to support monocyte-osteoclast differentiation in the presence of receptor activator for nuclear factor κB ligand as evidenced by the formation of numerous multinucleated tartrate-resistant acid phosphatase and vitronectin receptor positive cells which formed F-actin rings and were capable of lacunar resorption. The addition of a neutralising antibody to M-CSF did not inhibit osteoclast differentiation. HGF is a well-established survival factor and viability assays and live/dead staining showed that it promoted the survival and proliferation of monocytes and osteoclasts in a manner similar to M-CSF. Our findings indicate that HGF can substitute for M-CSF to support human osteoclast formation.

Stimulation of osteoclast formation by inflammatory synovial fluid

Peri-articular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. Under these conditions, the synovial fluid contains numerous inflammatory cells that produce cytokines and growth factors which promote osteoclast formation. The aim of this study was to determine whether inflammatory synovial fluid stimulates the formation of osteoclasts. Synovial fluid from rheumatoid arthritis (RA), pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients was added to cultures (n=8) of human peripheral blood mononuclear cells (PBMCs) in the presence and absence of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-kappaB ligand (RANKL). Osteoclast formation was assessed by the formation of cells positive for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and the extent of lacunar resorption. The addition of 10% OA, RA and PPA synovial fluid to PBMC cultures resulted in the formation of numerous multinucleated or mononuclear TRAP(+) and VNR(+) cells which were capable of lacunar resorption. In contrast to PBMC cultures incubated with OA synovial fluid, there was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoclast formation.

The Role of IGF‐I Signaling in Regulating Osteoclastogenesis

The proximal tibiae from IGF-I knockout mice (KO) show increased trabecular number and connectivity despite decreased bone formation, suggesting a concomitant reduction in bone resorption. Therefore, we examined the role of IGF-I on osteoclastogenesis. The number of tartrate-resistant acid phosphatase (TRAP) osteoclasts (N.OCL) in the KO mice were 76% of that observed in the wild-type (WT) mice in the proximal tibia. In vitro, the administration of RANKL and M-CSF to osteoclast (OCL) precursors (spleen cells) from WT mice stimulated the formation of multinucleated TRAP positive OCL. OCL precursors from the KO mice formed only mononuclear or binuclear TRAP positive OCL and in lesser numbers (55% of WT). IGF-I (10 ng/ml) further increased OCL number, number of nuclei and function in the WT cultures, but was much less effective in the cultures from KO mice. When KO osteoblasts (KOOB) and WT OCL precursors (spleen cells, WTSP) were co-cultured (KOOB:WTSP), the N.OCL formed was only 11% of that from co-cultures of WTOB and WTSP (WTOB:WTSP), consistent with the reduced expression of RANK and RANKL in bone from KO mice. Pre-treatment of KOOB by IGF-I 10 ng/ml for 9 days partially rescued OCL formation in KOOB and WTSP co-cultures (58% of WTOB:WTSP). In summary, our results indicate that IGF-I stimulates OCL multinucleation and increases their function. IGF-I is required for the normal interaction between the OB and OCL via its regulation of RANKL and RANK expression. The role of OBs in stimulating osteoclastogenesis is more impacted by lack of IGF-I than the ability of OCL precursors to respond to osteoblastic stimulation, although both OBs and OCL precursors appear to function subnormally in the absence of IGF-I.

Involvement of subchondral bone marrow in rheumatoid arthritis: Lymphoid neogenesis and in situ relationship to subchondral bone marrow osteoclast recruitment

To evaluate the presence and immunohistochemical characteristics of subchondral bone marrow inflammatory infiltrate in rheumatoid arthritis (RA) and to determine the in situ relationship between marrow inflammation and osteoclast recruitment. Bone samples and paired synovia from 8 RA patients undergoing joint surgery were analyzed by immunohistochemistry and in situ hybridization for specific lymphoid neogenetic features, such as T and B cell composition, follicular dendritic cell (FDC) networks, peripheral lymph node addressin (PNAd)-positive high endothelial venules, and lymphoid chemokine expression. Osteoclasts were identified as multinucleated tartrate-resistant acid phosphatase (TRAP)-positive and cathepsin K-positive cells adherent to the bone surface. An inflammatory infiltrate with perivascular aggregates of variable size was detected in 7 (87.5%) of 8 synovial samples and in paired bone samples. Lymphoid neogenetic features typical of rheumatoid synovium were also recognized in the bone marrow. PNAd+ blood vessels were found in 4 of 8 patients, CD21+ FDC networks in 2 patients, CXCL13+ cells in 5 patients, and CCL21+ cells in 6 patients. TRAP-positive and cathepsin K-positive osteoclasts were identified on both the synovial and marrow sides of the bone surface. Bone marrow samples showing a higher degree of inflammation were characterized by a significantly increased number of osteoclasts adherent to the subchondral bone. Our data demonstrate that lymphoid aggregates with lymphoid neogenetic features are detectable on the subchondral side of the joint in established RA. Moreover, the local inflammation/aggregation process appears to be related to osteoclast differentiation on the marrow side of subchondral bone, supporting a functional role of the bone compartment in local damage.

Mechanisms Involved in Enhancement of Osteoclast Formation and Function by Low Molecular Weight Hyaluronic Acid

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

Effect of CD44 deficiency on in vitro and in vivo osteoclast formation

In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock-out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild-type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild-type and CD44 k.o. mice. Bone marrow cells from wild-type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild-type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild-type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild-type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild-type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment.

Molecular basis of osteoclastogenesis induced by osteoblasts exposed to wear particles

In this study, we investigate the molecular mechanisms by which human osteoblasts (HOB) challenged with wear debris promote the differentiation of osteoclast precursors. HOB were obtained from trabecular bone and exposed to alumina (Al2O3) or ‘ultra-high molecular weight polyethylene’ (UHMWPE) particles for 24h. The supernatant (HOB-CM) was used for the immunoenzymatic detection of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), as well as for inducing the osteoclast differentiation from peripheral blood mononuclear cells (PBMC). The OPG-to-RANKL ratio was significantly decreased in the conditioned medium of UHMWPE-challenged HOB. Morphological and cytochemical analysis showed that HOB-CM induced by itself the osteoclast formation, but a large amount of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive giant cells were obtained when PBMCs were cultured with 1μg/mL UHMWPE HOB-CM. The expression of genes involved in osteoclast differentiation and activation was evaluated, i.e. c-fms, RANK, c-src, c-fos, cathepsin-K (CATK), TRAP, and calcitonin R (CTR). The UHMWPE HOB-CM increases c-src expression, suggesting that polyethylene debris favour the paracrine activity of HOB in inducing the pathway involved in osteoclast polarization and adhesion. On the contrary, Al2O3 HOB-CM downregulates c-fos expression, suggesting that the passage from macrophages into the osteoclast lineage is deviated. These results show that Al2O3 wear debris is less active than UHMWPE in inducing osteoclast differentiation. Moreover, they provide new insight into the molecular basis of particle-induced osteoclastogenesis, that is the starting point for planning mode-specific targeting of periprosthetic osteolysis.

Effects of the phytoestrogen coumestrol on RANK-ligand-induced differentiation of osteoclasts.

Phytoestrogens, which have structural similarity to 17beta-estradiol, have been reported to act as agonists/antagonists of estrogen in animals and humans. Estrogen is known to have an important role in maintaining bone mass, because the concentration of serum estrogen decreases after menopause and the estrogen deficiency causes bone loss. In this study, we investigated the effects of coumestrol and other phytoestrogens on osteoclast differentiation using estrogen receptor alpha-transfected RAW264.7 (RAW264.7-ERalpha) cells. When the cells were cultured with the receptor activator of nuclear factor kappa B-ligand (RANKL), both formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and TRAP activity were increased compared with control cells that were cultured in the absence of RANKL. Coumestrol decreased RANKL-induced formation of TRAP-positive multinucleated cells and TRAP activity dose-dependently. RANKL-stimulated RAW264.7-ERalpha cells formed resorption pits on calcium phosphate films and the pit formation was inhibited by coumestrol in a dose-dependent manner. RT-PCR analyses revealed that coumestrol (10 microM) decreased mRNA levels of calcitonin receptor (CTR) and matrix metalloproteinase-9 (MMP9) in RANKL-treated cells. In addition, pretreatment of coumestrol decreased RANKL-induced phosphorylation of extracellular signal-regulated kinases/p44/42 (ERK1/2). These results suggest that coumestrol has an inhibitory effect on the differentiation of osteoclasts, at least partially via ERK1/2 pathway.

Hydroxyapatite particles are capable of inducing osteoclast formation.

Hydroxyapatite (HA) coatings have been used to improve implant fixation by promoting bone formation around the prosthesis. A macrophage response to HA particulates has been noted around loosened HA-coated prostheses. As biomaterial wear particle-associated macrophages are known to be capable of differentiating into osteoclasts that are capable of bone resorption, we examined whether particulate HA could similarly induce macrophage-osteoclast differentiation. HA-associated macrophages were isolated from granulomas, formed by subcutaneous implantation of HA, and co-cultured with UMR 106 osteoblast-like cells in the presence of 1,25-dihydroxyvitamin D(3) for up to 14 days on glass coverslips and bone slices. HA-associated macrophage-osteoclast differentiation was evidenced by the formation of numerous multinucleated tartrate resistant acid phosphatase (TRAP)-positive cells which formed lacunar resorption pits on bone slices. Polymethylmethacrylate (PMMA) particle-associated macrophages, isolated from subcutaneous PMMA-containing granulomas, caused significantly more osteoclast formation and bone resorption than HA-associated macrophages. These results indicate that macrophages responding to HA particles are capable of osteoclast differentiation. They also suggest that particles derived from uncemented (HA-coated) implants are likely to induce less osteoclast formation and osteolysis than cemented implants.

Substance P Activates Osteoclast Formation and Osteoclastic Bone Resorption through the Neurokinin-1 Receptor.

Axons containing substance P (SP) serve bone tissue, however, the role of SP in bone metabolism, particularly on osteoclastic bone resorption, is unknown. Therefore, we examined the distribution of neurokinin 1-receptors (NK1-R), which have a high affinity to SP, in rat osteoclasts in vivo, and investigated the effects of SP on osteoclast formation and osteoclastic bone resorption in vitro. Using electron microscopy, immunoreactive products of NK1-R were seen in the plasma membrane and cytoplasm of osteoclasts, and were also observed in preosteoclast-like cells. Cell suspensions containing osteoclasts were prepared from neonatal rats and cultured on ivory slices. The addition of 10-10-10-6 M SP caused the number of mono- and multi-nucleated tartrate-resistant acid phosphatase positive (TRAP(+)) cells to increase. The increase in TRAP(+) cells with the addition of 10-8 M SP was inhibited by treatment with the SP receptor antagonist. In cultures on glass coverslips, timelapse studies show that SP induced cell spreading within 5 min and maintained the spreading. The number of resorption pits excavated by the osteoclasts and the resorption area per osteoclast increased in a 48-hour incubation with 10-8 M SP. These results suggest that SP stimulates osteoclast formation and activates osteoclastic bone resorption through NK1-R.

Rho-A Is Critical for Osteoclast Podosome Organization, Motility, and Bone Resorption

Rho plays a regulatory role in the formation of actin stress fibers and focal adhesions, and it is also involved in integrin-mediated signaling events. To study the role of Rho in alpha(v)beta(3)/gelsolin-dependent signaling, the HIV-Tat peptide, hemagglutinin (HA)-tagged Rho(Val-14) (constitutively active) and Rho(Asn-19) (dominant negative) were transduced into avian osteoclasts. Protein transduction by HA-Tat was highly efficient, and 90-100% of the cells were transduced with HA-tagged proteins. We demonstrate here that Rho(Val-14) transduction (100 nM) stimulated gelsolin-associated phosphatidylinositol 3-kinase activity, podosome assembly, stress fiber formation, osteoclast motility, and bone resorption, mimicking osteoclast stimulation by osteopontin/alpha(v)beta(3.) The effects of Rho(Val-14) transduction stimulation was time-dependent. C3 exoenzyme blocked the effects of Rho(Val-14) and induced podosome disassembly, loss of motility, and inhibition of bone resorption. Transduction of Rho(Asn-19) produced podosome disassembly, and blocked osteopontin stimulation. These data demonstrate that integrin-dependent activation of phosphoinositide synthesis, actin stress fiber formation, podosome reorganization for osteoclast motility, and bone resorption require Rho stimulation.

Biology of the calcium phosphate integration in human long bones

Calcium phosphate ceramics implanted in bones are integrated following a sequence of biological events which has already been described. We had the opportunity to analyze histologically some HA-ceramic samples that had been implanted in humans for various periods of time. Eight biopsies were obtained from calcium phosphate ceramics implanted in different sites. All the samples were implanted for periods ranging from 4 to 15 months. The samples were fixed in ethanol and embedded in hydroxyethylmetacrylate or polymethyl methacrylate. 5 μm thick sections were prepared and stained with Giemsa or by Von Kossa method. Alkaline phosphatase and tartrate resistant acid phosphatase (TRAP) were revealed by histochemical methods. Different osteointegration stages were evidenced. In early stages, hematoma remnants were still visible. These implants were invaded by a loose connective tissue in which some cell condensations were apparent. They lead to the formation of bone trabeculae by direct ossification. Some osteoblasts differentiated at the ceramic surface and synthesized osteoid tissue on the material. The fibroblast-like cells of the loose connective tissue were TRAP+. Most of them had TRAP+ nucleus and contained no cytoplasm vesicles. Multinucleated TRAP+ osteoclasts showing TRAP- negative nuclei were found in Howship's lacunae at the bone surface. Certain implants were integrated within mature bone in a later stage.

A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse.

Adult marrow contains mesenchymal progenitor cells (MPCs) that have multiple differentiation potentials. A conditionally immortalized MPC clone, BMC9, has been identified that exhibits four mesenchymal cell phenotypes: chondrocyte, adipocyte, stromal (support osteoclast formation), and osteoblast. The BMC9 clone, control brain fibroblasts and another marrow-derived clone, BMC10, were isolated from a transgenic mouse (H-2Kb-tsA58) containing a gene for conditional immortality. To test for chondrogenic potential, cells were cultured in defined medium containing 10 ng/ml transforming growth factor beta and 10-7 M dexamethasone in 15-ml polypropylene tubes ("aggregate cultures"). Adipogenic potential was quantitated by flow cytometry of Nile Red-stained cells cultured for 1 and 2 weeks in medium containing isobutyl methylxanthine, indomethacin, insulin, and dexamethasone. Support of osteoclast formation was measured by quantitating multinucleated tartrate-resistant acid phosphatase-positive cells in spleen cell cocultures of test clones (immortomouse clones and positive control ST2 cells) cultured in the presence of 10-7 M vitamin D3 and 150 mM ascorbate-2-phosphate. In vivo osteogenic potential was assayed by histologic examination of bone formation in subcutaneous implants, into athymic mouse hosts, of a composite of cells combined with porous calcium phosphate ceramics. The bone marrow-derived clone BMC9 has the potential to express each of the four mesenchymal characteristics tested, while brain fibroblasts, tested under identical conditions, did not exhibit any of these four mesenchymal characteristics. BMC10 cells exhibited osteogenic and chondrogenic phenotypes, but showed only minimal expression of adipocytic or osteoclast-supportive phenotypes. Clone BMC9 is, minimally, a quadripotential MPC isolated from the marrow of an adult mouse that can differentiate into cartilage and adipose, support osteoclast formation, and form bone. The BMC9 clone is an example of an adult-derived multipotential progenitor cell that is situated early in the mesenchymal lineage.

Carbonic Anhydrase II Plays a Major Role in Osteoclast Differentiation and Bone Resorption by Effecting the Steady State Intracellular pH and Ca 2+

Carbonic anhydrase II (CA II) expression is characteristic for the early stage of osteoclast differentiation. To study how CA II, which is crucial in proton generation in mature osteoclasts, influences the osteoclast differentiation process we performed rat bone marrow cultures. In this model, acetazolamide, a specific CA inhibitor, decreased the 1,25(OH) 2D 3-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells, in a dose-dependent manner. We then performed intracellular pH (pH i) and Ca 2+(Ca 2+ i) measurements for cultured osteoclasts and noticed that addition of acetazolamide caused a rapid, transient increase of both parameters. The increase in pH iwas dependent neither on the culture substrate nor on the extracellular pH (pH e) but the increase could be diminished by DIDS or by bicarbonate removal. Membrane-impermeable CA inhibitors (benzolamide and pd5000) did not have this effect. Addition of CA II antisense oligonucleotides into the cultures reduced the pH iincrease significantly. CA II inhibition was also found to neutralize the intracellular vesicles at extracellular pH (pH e) of 7.4, but at less extent at pH e7.0. In mouse calvaria cultures, bone resorption was inhibited dose dependently by acetazolamide at pH e7.4 while inhibition was smaller at pH e7.0. We conclude that CA II is essential not only in bone resorption but also in osteoclast differentiation. In both processes, however, the crucial role of CA II is at least partially due to the effect on the osteoclast pH iregulation.

Isolation and Characterization of Murine Clonogenic Osteoclast Progenitors by Cell Surface Phenotype Analysis

AbstractOsteoclasts are bone resorbing cells of hematopoietic origin; however, a progenitor cell population that gives rise to mature osteoclasts remains elusive. We have characterized a unique cell surface phenotype of clonogenic osteoclast progenitors (colony-forming unit–osteoclast [CFU-O]) and obtained a marrow cell population selectively enriched for these progenitors. Whole bone marrow cells were sequentially separated based on physical and cell surface characteristics, and the presence of CFU-O and other hematopoietic progenitors was examined. CFU-O was enriched in a nonadherent, low-density, lineage-marker–negative (Lin−), Thy1.2-negative (Thy1.2−), Sca1-negative (Sca1−), and c-kit–positive (c-kit+) population, as were the progenitors that were responsive to macrophage–colony-stimulating factor(CSF; CFU-M), granulocyte-macrophage-CSF (CFU-GM), and stem cell factor (CFU-SCF). When the Lin−Thy1.2−Sca1−population was divided into c-kithigh and c-kitlow populations based on c-kit fluorescence, over 88% of CFU-M, CFU-GM, and CFU-SCF were found in the c-kithighpopulation. In relation to the above mentioned hematopoietic progenitors, CFU-O was significantly higher in the c-kitlowpopulation: 80% of progenitors present in the c-kitlowpopulation were CFU-O. The CFU-O in both c-kithigh and c-kitlow populations showed key features of the osteoclast: multinucleated tartrate-resistant acid phosphatase–positive cell formation, expressions of vitronectin receptors, c-src and calcitonin receptors, and bone resorption. We have identified a progenitor cell population in the earliest stage of the osteoclast lineage so far described and developed a method to isolate it from other hematopoietic progenitors. This should help pave the way to understand the molecular mechanisms of osteoclast differentiation.

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.

Inhibitory effects of bisphosphonate (YM175) on bone resorption induced by a metastatic bone tumor.

The effects of a third-generation bisphosphonate, YM175 (disodium dihydrogen (cycloheptylamino)-methylene-1,1-bisphosphonate), on bone resorption induced by a metastatic human melanoma cell line (A375) were investigated morphologically using an experimental model of bone metastases in nude mice. An injection of A375 in the left cardiac ventricle produced multiple osteolytic lesions. Then, 4 weeks after the cell injection, we administrated YM175 (1 mg/kg) intravenously once and sacrificed the animals 3 days later. On histochemical observation, there was a layer of stromal cells with numerous mononuclear and multinucleated tartrate-resistant acid phosphatase (TRAPase)-positive cells in the untreated control group. In contrast, this layer was extensively reduced in most areas, and only a few TRAPase-positive cells were seen around tumor nests and on the bone surface in the experimental group. Most of the TRAPase-positive cells were stained only weakly and/or homogeneously, and there was little evidence of cell polarity. Some of them were vacuolated. Ultrastructurally, they were round and devoid of ruffled borders and clear zones. The findings suggest that YM175 decreases the number and activity of osteoclasts. In addition, a few showed the morphology of cell death, which seemed to be one of the reasons leading to the decrease of osteoclasts. There was no substantial change in the morphological relationships or ultrastructure of osteoclast precursor cells, stromal cells, extracellular matrices, and tumor cells between the experimental and the control groups. In the experimental group, the distribution of extracellular matrices (heparan sulfate proteoglycan and fibronectin) was less conspicuous, but the localization of osteotropic cytokines (interleukin-6 and prostaglandin E2) was essentially similar to that of the control group. The cause leading to the decrease of osteoclast precursor cells remains to be clarified. In conclusion, YM175 inhibits bone resorption induced by tumor, by decreasing the activity of mature osteoclasts and possibly affecting the production of osteoclast precursor cells.