Cloning and function of rabbit peroxisome proliferator-activated receptor delta/beta in mature osteoclasts

Osteoclasts modulate bone resorption under physiological and pathological conditions. Previously, we showed that both estrogens and retinoids regulated osteoclastic bone resorption and postulated that such regulation was directly mediated through their cognate receptors expressed in mature osteoclasts. In this study, we searched for expression of other members of the nuclear hormone receptor superfamily in osteoclasts. Using the low stringency homologous hybridization method, we isolated the peroxisome proliferator-activated receptor delta/beta (PPARdelta/beta) cDNA from mature rabbit osteoclasts. Northern blot analysis showed that PPARdelta/beta mRNA was highly expressed in highly enriched rabbit osteoclasts. Carbaprostacyclin, a prostacyclin analogue known to be a ligand for PPARdelta/beta, significantly induced both bone-resorbing activities of isolated mature rabbit osteoclasts and mRNA expression of the cathepsin K, carbonic anhydrase type II, and tartrate-resistant acid phosphatase genes in these cells. Moreover, the carbaprostacyclin-induced bone resorption was completely blocked by an antisense phosphothiorate oligodeoxynucleotide of PPARdelta/beta but not by the sense phosphothiorate oligodeoxynucleotide of the same DNA sequence. Our results suggest that PPARdelta/beta may be involved in direct modulation of osteoclastic bone resorption.     

Effects of fluid shear stress on mRNA expression of carbonic anhydrase II in polarized rat osteoclasts

The present study was designed to determine the effects of fluid shear stress on the mRNA expression of carbonic anhydrase II (CAII) in polarized rat osteoclasts. Cellular morphology of the polarized osteoclasts generated by a mechanical anatomical technique was examined by tartrate-resistant acid phosphatase (TRAP) staining and the osteoclastic resorption of dentine slices. The polarized osteoclasts were then stress-loaded by using a flow shear stress device newly developed by the osteoclast research group (patent number 200420034438; China), at 9 dyne/cm(2) for various time periods [0 (control group), 15, 30, 60, and 120 min], or at various stress levels [0 (control), 0.9, 2.9, 8.7, and 26.3 dyne/cm(2)] for 30 min. The mRNA expression of CAII was quantified using real-time fluorescent quantitative PCR (RT-PCR) and the data were analyzed with SPSS 12.0 software. The polarized osteoclasts were larger than regular monocytes (about 30 microm diameter) with irregular configuration, and the majority of polarized osteoclasts appeared to be spherical and had approximately 2-20 nuclei. The TRAP positive polarized osteoclasts showed asymmetrical red staining in the cytoplasm, and had many filaments and vacuoles. These cells formed resorptive pits in dentine slices. The levels of CAII mRNA expression were shown to be time-dependent, with the E+5 copy numbers being 7.88+/-0.09, 11.14+/-0.12, 15.83+/-0.18, 1.94+/-0.02, and 1.37+/-0.01 in cells treated at 9 dyne/cm(2) for 0, 15, 30, 60 and 120 min, respectively (P < 0.05). The levels of CAII mRNA expression (E+5 copy numbers) in cells treated with the stress levels of 0, 0.9, 2.9, 8.7 and 26.3 dyne/cm(2) were 7.97+/-0.201, 11.26+/-0.688, 15.94+/-0.201, 31.88+/-1.496, and 45.08+/-2.639, respectively (P < 0.05). These results indicate that there is a relationship between the fluid shear stress and the mRNA expression of CAII in polarized rat osteoclasts.     

Anthraquinone compounds from Morinda officinalis inhibit osteoclastic bone resorption in vitro

The root of Morinda officinalis has been claimed to have a protective effect against bone loss in sciatic neurectomized and ovariectomized osteoporotic rats, and this protective effect is supposed to be attributed to anthraquinone compounds in the plant. In the present study, we investigated the effects of three anthraquinones isolated from M. officinalis, including 1, 3, 8-trihydroxy-2-methoxy-anthraquinone (1), 2-hydroxy-1-methoxy-anthraquinone (2) and rubiadin (3) on bone resorption activity in vitro and the mechanism on osteoclasts derived from rat bone marrow cells. Compound 1, 2 and 3 decreased the formation of bone resorption pits, the number of multinucleated osteoclasts, and the activity of tartrate resistant acid phosphates (TRAP) and cathepsin K in the coculture system of osteoblasts and bone marrow cells in the presence of 1, 25-dihydroxyvitamine D(3) and dexamethasone. They also enhanced the apoptosis of osteoclasts induced from bone marrow cells with M-CSF and RANKL. In addition, Compound 1, 2 and 3 improved the ratio of mRNA and protein expression of OPG and RANKL in osteoblasts, interfered with the JNK and NF-κB signal pathway, and reduced the expression of calcitonin receptor (CTR) and carbonic anhydrase/II (CA II) in osteoclasts induced from bone marrow cells with M-CSF and RANKL. These findings indicate that the anthraquinone compounds from M. officinalis are potential inhibitors of bone resorption, and may also serve as evidence to explain the mechanism of the inhibitory effects of some other reported anthraquinones on bone loss.     

Cloning of mouse carbonic anhydrase mRNA and its induction in mouse erythroleukemic cells

Mouse carbonic anhydrase mRNA was detected in poly(A+) RNA of anemic spleens sedimenting as a RNA species at 14 S. Subsequently, poly(A+) RNA (12-16 S) was used as a template for the synthesis of double-stranded cDNA, which was inserted into the PstI site of pBR322 by oligo-dG:dC tailing. A recombinant plasmid containing carbonic anhydrase cDNA was identified by a positive hybridization selection assay and by partial DNA sequencing. Predicted amino acid sequences showed homology with the known sequences of rabbit and human carbonic anhydrase I and II. The clone contained sequences for most of the coding region and 600-700 base pairs at the 3' noncoding region of the mRNA. Hybridization analysis of poly(A+) RNA from uninduced and induced mouse erythroleukemic cells labeled for short and long time periods indicated that induction results in an increase of carbonic anhydrase mRNA in newly synthesized RNA.     

Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone,cartilage and giant cell tumor

Summary Carbonic anhydrase isoenzymes I and II have been localized in human bone and cartilage. Osteoclasts are strongly positive for carbonic anhydrase II but very little if any reaction is observed for carbonic anhydrase I. In tendon giant cell tumor osteoclastlike-giant cells contained high amounts of carbonic anhydrase II suggesting the close relation of these cells to normal osteoclasts. In growth plate cartilage strong staining was obtained in late proliferative and hypertrophic chondroxytes as well as in extracellular matrix of hypertrophic zone also only with anti-human carbonic anhydrase II.     

Carbonic anhydrase II associated with plasma membrane in a human pancreatic duct cell line (CAPAN-1).

The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH.     

Carbonic anhydrase II gene transcript in cultured osteoclasts from neonatal rats: effect of calcitonin

Carbonic anhydrase II (CA II), an enzyme catalyzing the interconversion of CO₂ and water to HCO ₃ ^? and protons, has a key role in osteoclastic bone resorption, but little is known of the regulation of CA II gene expression by calcitonin. Analysis of mRNA in osteoclasts has been difficult because of the problems of obtaining sufficient number of purified osteoclasts from bone. In this study, however, we have investigated the regulation of CA II mRNA in rat osteoclasts and their putative mononuclear precursors by using in situ hybridization. We have found that the CA II gene is expressed at high levels in osteoclasts and what are probably their maturing mononuclear precursors. Measurement of CA II mRNA in cultured osteoclasts and their putative mononuclear precursor cells by cytophotometry provided evidence that calcitonin, a direct inhibitor of mammalian osteoclast activity, reduces the levels of CA II mRNA in a dose dependent manner; maximum reduction was observed at a concentration of 100pM of calcitonin. In addition, calcitonin reduced the number of CA II mRNA-positive mononuclear precursor cells. The results also suggest that expression of the CA II gene is a feature of cells committed to the osteoclast lineage.     

Acid secretagogue-induced stimulation of gastric parietal cell gene expression

Carbamoylcholine (carbachol), histamine, and gastrin are three principal stimulants of gastric acid secretion. To explore the mechanisms by which these agents exert their actions in parietal cells, we examined their effects on the gene expression of the enzymes responsible for H+ generation. Each secretagogue induced rapid and coordinate increases in steady-state levels of mRNAs encoding carbonic anhydrase II and H+,K+-ATPase in isolated canine gastric parietal cells. Furthermore, pronounced increases, with different kinetics, in expression of beta-actin mRNA were observed. With increasing time after cell isolation, carbonic anhydrase II and H+,K+-ATPase, but not beta-actin, mRNA levels were attenuated, suggesting that parietal cell-specific genes may be dependent upon maintenance of parietal cell contacts within intact mucosal tissue. Pretreatment of the cells with competitive inhibitors of each secretagogue blocked the increases. Our results indicate that acid secretagogue-specific receptor activation in parietal cells triggers coordinate gene expression of the two enzymes involved in H+ ion generation and that beta-actin may be an important regulator of acid secretion.     

IL-17A suppresses the expression of bone resorption-related proteinases and osteoclast differentiation via IL-17RA or IL-17RC receptors in RAW264.7 cells

Interleukin-17 (IL-17) is produced exclusively by activated T cells and neutrophils, and stimulates osteoclastic bone resorption via osteoblasts by inducing the expression of “receptor activator of NF-κB (RANK) ligand” (RANKL). However, the direct effects of IL-17 on the differentiation of osteoclast precursors into osteoclasts and on the function of osteoclasts have not been clarified. Therefore, we examined the effects of IL-17A on the differentiation of osteoclast precursors using RAW264.7 cells and also on the expression of carbonic anhydrase II (CA II), cathepsin K, matrix metalloproteinases-9 (MMP-9), RANK, c-fms, and IL-17 receptors in these cells. The cells were cultured with or without 0.1, 1.0, 10 or 50 ng/mL IL-17 in the presence of soluble RANKL for up to 10 days. The CA II, cathepsin K, and MMP-9 mRNA and protein expression levels were examined using real-time PCR and Western blotting, respectively. The mRNA expression levels of RANK, c-fms, and IL-17 receptors were monitored by real-time PCR. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of the cells. TRAP-positive cells were observed after day 5 of culture, and the number of cells decreased in the presence of 10 and 50 ng/mL IL-17A at days 5 and 7. In the presence of IL-17A, the expressions of cathepsin K, MMP-9 and c-fms decreased markedly on days 5 and/or 7 of culture, whereas the expression of CA II and IL-17 receptor (type A) increased remarkably at days 3 and 7, respectively. The expression of RANK and IL-17 receptor (type C) was not affected by the addition of IL-17A. These results suggest that the differentiation of osteoclast precursors into osteoclasts is suppressed at high concentrations of IL-17A. Furthermore, IL-17A suppresses the hydrolysis of matrix proteins during bone resorption by decreasing the production of cathepsin K and MMP-9 in osteoclasts.     

Mitochondrial carbonic anhydrase in osteoclasts and two different epithelial acid-secreting cells

Summary Acid secreting cells are rich in mitochondria and contain high levels of cytoplasmic carbonic anhydrase II. We have studied the ultrastructural distribution of a mitochondrial isoenzyme, carbonic anhydrase V, in two different acid-secreting epithelial cells, gastric parietal cells and kidney intercalated cells as well as in osteoclasts, which are the main bone resorbing cells. The mitochondria differ in carbonic anhydrase V content in these three acid-producing cells: gastric parietal cell mitochondria show strong immunolabelling for this isoenzyme, osteoclast mitochondria faint labelling and kidney intercalated cell mitochondria no labelling. The immunolabelling was located in the mitochondrial matrix, often in close contact with the inner mitochondrial membrane. These results show that mitochondrial carbonic anhydrase levels are not related to acid-transporting activity.     

Localization of the expression of types I, III, and IV collagen, TGF-beta 1 and c-fos genes in developing human calvarial bones

Total RNA extracted from developing calvarial bones of 15- to 18-week human fetuses was studied by Northern hybridization: in addition to high levels of type I collagen mRNAs, the presence of mRNAs for type III and type IV collagen, TGF-beta and c-fos was observed. In situ hybridization of sections containing calvarial bone, overlying connective tissues, and skin was employed to identify the cells containing these mRNAs. Considerable variation was observed in the distribution of pro alpha 1(I) collagen mRNA in osteoblasts: the amount of the mRNA in cells at or near the upper surface of calvarial bone was distinctly greater than that in cells at the lower surface, indicating the direction of bone growth. High levels of type I collagen mRNAs were also detected in fibroblasts of periosteum, dura mater, and skin. Type III collagen mRNA revealed a considerably different distribution: the highest levels were detected in upper dermis, lower levels were seen in fibroblasts of the periosteum and the fibrous mesenchyme between bone spiculas, and none was seen in osteoblasts. Type IV collagen mRNAs were only observed in the endothelial cells of blood capillaries. Immunohistochemical localization of type III and IV collagens agreed well with these observations. The distribution of TGF-beta mRNA resembled that of type I collagen mRNA. In addition, high levels of TGF-beta mRNA were observed in osteoclasts of the calvarial bone. These cells, responsible for bone resorption, were also found to contain high levels of c-fos mRNA. Production of TGF-beta by osteoclasts and its activation by the acidic environment could form a link between bone resorption and new matrix formation.     

Recovery of biologically functional messenger RNA from agarose gels by passive elution

A general method for isolating biologically active messenger RNA (mRNA) from agarose gels is reported. Purified cellular RNA is resolved by preparative agarose gel electrophoresis and recovered in high yields (80%) by passive diffusion. Polyadenylated mRNA isolated from the eluted RNA is functionally intact based on the ability of the RNA to serve as a template in cell-free translation systems and complementary DNA synthesis reactions. The entire procedure is simple and rapid. A substantial purification of the mRNAs coding for skeletal muscle myosin heavy chain, light chain subunits and carbonic anhydrase III has been achieved employing this method.     

Histomorphometric identification of carbonic anhydrase in fetal rat bone embedded in glycolmethacrylate

Carbonic anhydrase was identified in bone-resorbing cells present in sections of fetal rat femur embedded in glycolmethacrylate. Using a slight modification of the Hansson's histochemical method, we demonstrated that most chondroclasts (91.8-95.4%) and osteoclasts (95.1-96.3%) display a positive histochemical reaction for carbonic anhydrase. This staining was consistently inhibited in the presence of very low concentrations (10(-6), 10(-7) M) of the specific inhibitor acetazolamide. The number of chondroclasts reacting for carbonic anhydrase was identical to the number of acid phosphatase-stained chondroclasts determined on adjacent sections. A large majority of osteoclasts (96.3%) stained for carbonic anhydrase and for acid phosphatase (97.2%), with more osteoclasts reacting for the latter enzyme than the former (76.8 +/- 8.5 (SD) vs 85.3 +/- 9.2 cells/mm2 of endosteal bone; p less than 0.01). The observation that acetazolamide at a concentration as low as 10(-7) M inhibited Hansson's reaction, together with our histomorphometric results, validates the use of histochemical staining for carbonic anhydrase to evaluate activity of bone-resorbing cells identified in plastic-embedded fetal bone tissue.     

The Foreign Body Giant Cell Cannot Resorb Bone,But Dissolves Hydroxyapatite Like Osteoclasts

Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14⁺ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones—cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H⁺-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K.     

Messenger RNA sorting in enterocytes. Co-localization with encoded proteins.

This study describes the intracellular compartmentalization of three different mRNAs in the polarized rat fetal enterocyte. They encode proteins that are known to be localized within different regions of the epithelial cell namely (i) the apical, membrane-bound glycoprotein, lactase-phlorizin hydrolase (lactase), (ii) the mitochondrially localized enzyme, carbamoylphosphate synthetase (CPS), and (iii) the cytoplasmically localized enzyme, phosphoenolpyruvate carboxykinase (PEPCK). These mRNAs are found in close proximity to their respective protein products, i.e. the apical membrane, mitochondria and cytoplasm, respectively. The significance of these observations is twofold; (i) they indicate that mRNAs are sorted into specific domains of the cytosol of intestinal epithelial cells; and (ii) they imply the presence of two distinct pathways of mRNA targeting one that allows transport of mRNAs that are translated on ribosomes associated with the rough endoplasmic reticulum (lactase mRNA), and the other that allows sorting of mRNAs that are translated on free polysomes (CPS and PEPCK mRNA).     

Rat skeletal muscle membrane associated carbonic anhydrase is 39-kDa, glycosylated, GPI-anchored CA IV.

Sarcolemmal membrane vesicle preparations from white and red muscles of rat were found to contain a carbonic anhydrase which was indistinguishable from carbonic anhydrase IV from rat lung. This isozyme appears to account for all of the carbonic anhydrase activity in the sarcolemmal vesicle preparations. Digestion of 39-kDa CA IV with endoglycosidase F reduced the Mr to 36 kDa, suggesting that it contains one N-linked oligosaccharide. Treatment of sarcolemmal vesicles with phosphatidylinositol-specific phospholipase C released all of the activity, indicating that the enzyme is anchored to membranes by a phosphatidylinositol-glycan linkage. White muscle sarcoplasmic reticulum vesicles also contain a small amount of 39-kDa CA IV-type enzyme. A 52-kDa polypeptide in sarcoplasmic reticulum membranes cross-reacts with anti-human CA II and anti-rat CA II antisera, but does not bind to the sulfonamide affinity column. This cross-reacting polypeptide has no detectable CA activity.     

CELLULAR BIOLOGY OF BONE RESORPTION

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged.
1. The resorption of bone is the result of successive steps that can be regulated individually.
2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone.
3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation.
4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells.
5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO, by carbonic anhydrase and secreted by an ATP-driven vacuolar H⁺-K⁺-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone.
6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca²⁺) generated as a result of osteoclastic bone resorption.
7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.     

Studies of OC-STAMP in Osteoclast Fusion: A New Knockout Mouse Model,Rescue of Cell Fusion,and Transmembrane Topology

The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized cell fusion mechanisms.     

Histochemical detection of carbonic anhydrase with diemthylaminonaphthalene-5-sulfonamide.

A new specific method for the detection of carbonic anhydrase, EC 4.2.1.1, in tissues is described. The reaction of carbonic anhydrase with dimethylaminonaphthalene-5-sulfonamide (DNSA) forms a highly fluorescent complex. The specificity of the method is proved by the quenching of this fluorescence with ethoxzolamide (6-ethoxybenzothiazole-5-sulfonamide). The difference in the wavelength makes it possible to absorb the fluorescence of the unbound dimethylaminonaphthalene-5-sulfonamide by filters. Kidney, proventriculus, and bone from chicken have been examined. Carbonic anhydrase has been detected in the cytoplasm of the columnar lining cells, proximal tubule cells, and osteoclasts.     

Expression of typical osteoclast markers by PBMCs after PEG-induced fusion as a model for studying osteoclast differentiation

Bone is a metabolically active organ subjected to continuous remodeling process that involves resorption by osteoclast and subsequent formation by osteoblasts. Osteoclast involvement in this physiological event is regulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). Fusion of mono-nuclear pre-osteoclasts is a critical event for osteoclast differentiation and for bone resorption. Here we show that PBMCs can be successfully fused with polyethylenglicol (PEG) in order to generated viable osteoclast-like cells that exhibit tartrate-resistant acid phosphatase (TRAP) and bone resorptive activities. PEG-fused PBMCs expressed additional markers compatible with osteoclastogenic differentiation such as carbonic anhydrase II (CAII), calcitonin receptor (CR), cathepsin K (Cat K), vacuolar ATPase (V-ATPase) subunit C1 (V-ATPase), integrin β3, RANK and cell surface aminopeptidase N/CD13. Actin redistribution in PEG-fused cells was found to be affected by cell cycle synchronization at G0/G1 or G2/M phases. PEG-induced fusion also led to expression of tyrosine kinases c-Src and Syk in their phosphorylated state. Scanning electron microscopy images showed morphological features typical of osteoclast-like cells. The results here shown allow concluding that PEG-induced fusion of PBMCs provides a suitable model system for understanding the mechanisms involved in osteoclastogenesis and for assaying new therapeutic strategies.