Human Osteoclast-like Cells Research Articles

A novel calcium sensor stimulating inositol phosphate formation and [Ca2+]i signaling expressed by GCT23 osteoclast-like cells.

Osteoclast activity is inhibited by elevated [Ca2+]o; however, the underlying molecular mechanism is unknown. We used the human osteoclast-like cells GCT23 to elucidate their cation-sensing properties. Cells responded to elevated [Ca2+]o with rapid concentration-dependent [Ca2+]i transients (EC50 = 7.8 mm, time to peak 44 +/- 4 sec) that were due to release from intracellular stores, followed by Ca2+ influx across the plasma membrane. Ca2+ store depletion by thapsigargin, endothelin-1, or bradykinin activated calcium entry pathways. Cells responded similarly to Ni2+ and Cd2+ with albeit slower kinetics (EC50 <10 microm and <100 microm, times to peak 140 +/- 25 sec and 150 +/- 24 sec, respectively). The three cations stimulated inositol phosphate production (two-fold, p <.02) similar to bradykinin (2.5-fold, p <. 002), which activates a phospholipase C (PLC)-coupled receptor in GCT23 cells. The cells did not respond to 0.1-1 mM Gd3+ or neomycin B, indicating that the parathyroid calcium receptor (PCaR) is not functionally expressed. In confirmation, PCaR could not be detected by reverse transcriptase polymerase chain reaction in GCT23 cells and in mouse osteoclasts, and the calcimimetic compound NPS R-568 failed to produce the left shift of the concentration-response curve characteristic for PCaR. Our data demonstrate for the first time that cation sensing by osteoclast-like GCT23 cells is mediated by a PLC-coupled receptor that is not identical to PCaR.

Multiple myeloma cells and cells of the human osteoclast lineage share morphological and cell surface markers

This study demonstrates that the multiple myeloma cell (MMC) in its plasma cell form is morphologically indistinguishable from human osteoclast-like cells that form in culture when peripheral blood mononuclear cells (PBMCs) are plated at high density in serum containing medium. MM has been described as a disease of B-cell lineage, monoclonal immunoglobulin (Ig) producing cells with unique properties: MM precursor cells lodge in bone, where they proliferate and differentiate into plasma cell tumors. Then, by some mechanism, presumably involving cytokines, these cells mediate an increase in neighboring osteoclast numbers and activity, leading to excessive bone erosion and hypercalcemia. Three days after plating PBMCs, tartrate resistant acid phosphatase- (TRAP-) blasts as well as TRAP+ cells, each with an eccentric nucleus, appear in culture. By day 10, TRAP+, vitronectin+ (VR+) cells, appear to be morphologically indistinguishable from multiple myeloma plasma cells (MMPCs) on cytocentrifuge preparations. These cells are CD19- and CD38++, as are MMCs reported by others. Other surface markers are also shared. Furthermore, Ig mRNA is demonstrated in the cytoplasm of cells at 8 days by in situ hybridization with the IgG FcA3 sequence. This novel finding is not unusual, in light of reports, demonstrating non-B-lineage Ig-producing cells. Thus, this study raises some serious questions about the true nature of MMCs.

Multiple myeloma cells and cells of the human osteoclast lineage share morphological and cell surface markers

This study demonstrates that the multiple myeloma cell (MMC) in its plasma cell form is morphologically indistinguishable from human osteoclast-like cells that form in culture when peripheral blood mononuclear cells (PBMCs) are plated at high density in serum containing medium. MM has been described as a disease of B-cell lineage, monoclonal immunoglobulin (Ig) producing cells with unique properties: MM precursor cells lodge in bone, where they proliferate and differentiate into plasma cell tumors. Then, by some mechanism, presumably involving cytokines, these cells mediate an increase in neighboring osteoclast numbers and activity, leading to excessive bone erosion and hypercalcemia. Three days after plating PBMCs, tartrate resistant acid phosphatase- (TRAP-) blasts as well as TRAP+ cells, each with an eccentric nucleus, appear in culture. By day 10, TRAP+, vitronectin+ (VR+) cells, appear to be morphologically indistinguishable from multiple myeloma plasma cells (MMPCs) on cytocentrifuge preparations. These cells are CD19- and CD38++, as are MMCs reported by others. Other surface markers are also shared. Furthermore, Ig mRNA is demonstrated in the cytoplasm of cells at 8 days by in situ hybridization with the IgG FcA3 sequence. This novel finding is not unusual, in light of reports, demonstrating non-B-lineage Ig-producing cells. Thus, this study raises some serious questions about the true nature of MMCs. J. Cell. Biochem. 71:559–568, 1998. © 1998 Wiley-Liss, Inc.

Modulation of Osteoclast Function by Adenovirus Vector-Induced Epidermal Growth Factor Receptor

We have explored the use of adenovirus vector-mediated gene transfer to introduce foreign genes into osteoclasts, terminally differentiated cells responsible for bone resorption. A replication-deficient adenovirus vector that contains a reporter gene encoding beta-galactosidase efficiently infected human osteoclast-like cells (OCLs) derived from human giant cell tumors and mouse OCLs formed in vitro. We then constructed an adenovirus vector carrying human epidermal growth factor receptor (EGFR) cDNA (Ax1CAhEGFR) and introduced the EGFR gene into mouse OCLs. Clear induction of EGF receptor was detected in Ax1CAhEGFR-infected OCLs (EGFR-OCLs) by immunocytochemistry and immunoblotting, and EGF stimulation induced rapid tyrosine phosphorylation of several proteins including EGF receptor itself. Large vacuoles appeared in EGFR-OCLs in response to EGF treatment, and pit-forming activity by EGFR-OCLs was dose-dependently suppressed by recombinant human EGF. In addition, survival of EGFR-OCLs was prolonged by EGF. No expression of EGF receptor or effects of EGF were observed in noninfected OCLs or control vector-infected OCLs. These results suggest that adenoviral vectors are useful for modulating osteoclast function by introducing foreign genes into osteoclasts and that they will be a good means of gene therapy of metabolic bone diseases.

Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption.

Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Paget's disease, bone metastasis, and hypercalcemia of malignancy. However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited. Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity. A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined. This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide. Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein. Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures. In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types. These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption.

Alendronate reduces adhesion of human osteoclast-like cells to bone and bone protein-coated surfaces.

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40-45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as alphaVbeta3 integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration.

Activation of αvβ3Integrin on Human Osteoclast-like Cells Stimulates Adhesion and Migration in Response to Osteopontin

Integrins mediate cell adhesion and can induce different cellular responses, including changes in intracellular pH, changes and oscillation in intracellular free calcium, and protein phosphorylation on tyrosine. During bone resorption, the integrin αvβ3regulates adhesion of osteoclasts to bone extracellular matrix proteins, such us osteopontin (Opn). Adhesion via αvβ3is followed by osteoclast polarization onto the bone surface and by the onset of bone resorption. To characterize these events at the molecular level, we investigated the state of activation of αvβ3on the human osteoclast-like cell line GCT23 using the monoclonal antibody AP5 which binds to and can induce, under low calcium conditions, activated αvβ3. By flow cytometry, approximately 50% of αvβ3on the surface of the osteoclast-like cell line GCT23 was reactive with AP5 and was therefore in the activated state. Incubation with AP5 in the presence of low calcium concentrations increased activated αvβ3to 90–100%. Activation of αvβ3increased the efficiency of GCT23 adhesion to Opn by 2-fold. Furthermore, haptotactic migration on Opn was also enhanced about 40% compared to control. We propose that changes in the activation state of αvβ3may be a regulation point for osteoclasts during bone resorption.

Cadherin-6 mediates the heterotypic interactions between the hemopoietic osteoclast cell lineage and stromal cells in a murine model of osteoclast differentiation.

Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell-cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell-cell contact caused evident morphological changes accompanied with tight cell-cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow-derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.

Retinoic Acid Induces Cell Proliferation and Modulates Gelatinases Activity in Human Osteoclast-like Cell Lines

The effect of Retinoic Acid (RA) on human osteoclast-like cell lines, obtained from Giant Cell tumors (GCT) of bone, has been investigated evaluating its action on bone resorption, cell proliferation, microtubular organization and gelatinases expression and activity. Increasing concentrations of RA significantly dose-dependently decreased GCTs bone resorption, while 10−7M RA promoted an increase of cell proliferation. By immunofluorescence we demonstrated that GCTs express A and B gelatinases and, by zymography, that their activity was enhanced in medium collected from GCTs cultured in the presence of 10−7M RA. These data indicate that RA increases cell proliferation and modulates metalloproteinases (MMPs) activity, crucial events during the migration of osteoclast precursors toward bone surfaces.

Matrix metalloproteinases activity in human osteoclast-like cells is modulated by bone seeking hormones and growth factors

Matrix metalloproteinases activity in human osteoclast-like cells is modulated by bone seeking hormones and growth factors

Matrix metalloproteinases activity in human osteoclast-like cells is modulated by bone seeking hormones and growth factors.

Matrix metalloproteinases activity in human osteoclast-like cells is modulated by bone seeking hormones and growth factors.

Human osteoclast-like cells selectively recognize laminin isoforms, an event that induces migration and activates Ca2+ mediated signals.

Osteoclast precursors are chemotactically attracted to sites of bone resorption via migration pathways that include transendothelial crossing in blood capillaries. Transendothelial migration involves poorly understood interactions with basal lamina molecules, including laminins. To investigate osteoclast-laminin interactions, we used human osteoclast-like cell lines obtained from giant cell tumors of bone (GCT 23 and GCT 24). These cell lines are a well-characterized model for osteoclast functions, such as bone resorption and the behaviour of osteoclast precursors. Both GCT cell lines adhered to laminin-2 (merosin) coated wells in standard adhesion assays, but failed to adhere to laminin-1 (EHS-laminin). By light microscopy, GCT cells on laminin-2 were partially spread, with a motile morphology. None of the anti-integrin antibodies tested inhibited GCT cells adhesion to laminin-2. Peptides containing the integrin adhesion site RGD or the laminin adhesion sequence IKVAV did not inhibit GCT cell adhesion to laminin-2. By immunofluorescence, beta 1 integrins were organized in focal adhesions. However, in the presence of monensin this reorganization of beta 1 integrins was abolished, indicating that it was probably due to secretion of fibronectin by GCT cells subsequent to adhesion to laminin-2. GCT cells transmigrated through membranes coated with laminin-2, much more efficiently than through membranes coated with collagen. Migration was induced by osteocalcin, as a chemoattractant, in a dose-dependent manner. At low osteocalcin concentrations, transmigration was detectable on laminin-2 but not collagen. In cells loaded with fura-2, a sharp increase in intracellular Ca2+ was detected upon addition of soluble laminin-2, but not laminin-1, due to release from thapsigargin-dependent intracellular stores. In summary, osteoclasts may recognize laminin isoforms differentially. Initial adhesion to laminin-2 appears to be due to integrin-independent mechanisms. Such adhesion, though, may trigger secretion of fibronectin that could then support spreading and efficient chemotactic migration. These mechanisms may play an important role in facilitating chemotactic migration of osteoclast precursors toward the bone surface.

Production of interleukin-6 by human osteoclast-like cells from giant cell tumor of bone

Giant cell tumor (GCT) is a bone neoplasm which is characterized by the presence of large numbers of multinucleated osteoclast-like giant cells. Although GCT can be considered a benign lesion, it exhibits high local aggressiveness often associated with osteolytic properties. In this study, we used five different GCT primary cell cultures to evaluate whether osteoclast-like cells from GCT are able to produce interleukin-6 (IL-6), a cytokine strictly involved in the induction of osteoclast-mediated bone resorption. IL-6 assessment with ELISA revealed that osteoclast-like GCT cells produce low levels of this cytokine, which can be greatly increased after treatment with both lipopolysaccharide (LPS) or interleukin-1 beta (IL-1 beta). These data were confirmed by molecular analysis which revealed that GCT cells synthesize IL-6 mRNA and that the levels of IL-6 transcripts are greatly increased after treatment with both LPS and IL-1 beta. Moreover, by using a biologic assay with the 7TD1, a IL-6 dependent cell Line, we also determined that IL-6 synthesized by GCT cells is biologically active. This study supports the hypothesis that IL-6 locally released by GCT osteoclast-like cells may be involved in the induction of the osteolysis which is strictly associated with the biologic aggressiveness of GCT cells.

Human osteoclast-like cells in primary culture.

We describe a method for the isolation and culture of osteoclast-like cells from cancellous bone chips of iliac crests from patients undergoing reconstructive maxillofacial surgery. Under aseptic conditions, bone chips were cut into small pieces, incubated briefly with collagenase, and the isolated bone cells were separated from the bone chips by filtration using a nylon mesh. Bone cells were then cultured on a variety of surfaces for up to 10 days. Cell motility and fusion, together with the development of tartrate-resistant acid phosphatase activity, were seen in many cells soon after culture. The large osteoclast-like cells adhered to human cortical bone slices and produced resorption pits. These morphological and functional characteristics suggest that the cells we isolated and cultured were human osteoclasts and their precursors. Thus this method may provide a reliable means of obtaining human osteoclasts from normal tissue for short-term studies of their metabolism or from various skeletal diseases to study pathological aberrations and mechanisms.

Alendronate acts on bone resorption of human osteoclast-like cells through the inhibition of cell attachment to bone surfaces

Alendronate acts on bone resorption of human osteoclast-like cells through the inhibition of cell attachment to bone surfaces

Metalloproteinases expression by human osteoclast-like cell lines

Metalloproteinases expression by human osteoclast-like cell lines

Alendronate acts on bone resorption of human osteoclast-like cells through the inhibition of cell attachment to bone surfaces

Alendronate acts on bone resorption of human osteoclast-like cells through the inhibition of cell attachment to bone surfaces

Translocation of protein kinase C isoenzymes by elevated extracellular Ca 2+ concentration in cells from a human giant cell tumor of bone

In this study we investigated the protein kinase C isoenzymes expressed by human osteoclast-like cells harvested from a giant cell tumor of bone (GCT23 cells), and by freshly isolated rat osteoclasts. Immunoblotting analysis revealed that the -α, -δ, and -ϵ, PKC isoforms, but not the -β isoenzyme, are expressed by GCT23 cells. Immunofluorescence studies demonstrated that PKC-α, -δ, and -ϵ are homogeneously expressed by both mononuclear and multinucleated GCT23 cells, as well as by rat osteoclasts. Similar to authentic osteoclasts, GCT23 cells responded to an increase of extracellular Ca 2+ concentration ([Ca 2+] 0) with a dose-dependent elevation of the cytosolic free Ca 2+ concentration ([Ca 2+] i). An increase of [Ca 2+] 0 stimulated the translocation of PKC-α from the cytosolic to the particulate fraction, suggesting the involvement of this isoenzyme in the signal transduction mechanism prompted by stimulation of the [Ca 2+] 0 sensing. By contrast, PKC-δ was not altered by exposure to elevated [Ca 2+] 0, whereas PKC-ϵ underwent reciprocal translocation, disappearing from the insoluble fraction and increasing in the cytosol. The effects of PKC on GCT23 cell functions were investigated by treatment with phorbol 12-myristate, 13-acetate (PMA). We observed that activation of PKC by PMA failed to affect adhesion onto the substrate, but down-regulated the [Ca 2+] 0-induced [Ca 2+] i increases. The latter effect was specific, since it was reversed by treatment with the PKC inhibitors staurosporine and chelerythrine.

Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism.

Calcitonin (CT) is a peptide hormone that interacts with the cAMP-and phospholipase C-associated CT receptor subtypes. We investigated whether CT modulates the interaction of human tumoral osteoclast-like (GCT23) cells with a protein of the bone matrix, bone sialoprotein-II (BSP-II). Single GCT23 cells loaded with the intracellular Ca2+ indicator fura-2 were treated with the maximal active dose (300 micrograms/ml) of the 18-mer Arg-Gly-Asp (RGD)-containing BSP-IIA fragment, and the cytosolic free Ca2+ concentration ([Ca2+]i) was measured by dual wavelength microfluorometry. BSP-IIA stimulated an elevation in [Ca2+]i, consisting mainly of a peak, followed by a rapid return toward baseline. Pretreatment with CT induced a modest elevation of [Ca2+]i. However, CT significantly inhibited the response to BSP-IIA in a dose-dependent manner. Maximal inhibition (90% vs. untreated) was observed in the micromolar range. The intracellular mechanisms leading to this effect were investigated by pretreatment of GCT23 cells with the cAMP permeant analog, (Bu2)cAMP, and the protein kinase-C-activating agent, 12-O-tetradecanoylphorbol 13-acetate. Similar to CT, both agents inhibited the response to 300 micrograms/ml BSP-IIA. The effect induced by CT was specific, because an increase in the extracellular Ca2+ concentration, which is also known to inhibit bone resorption, failed to modify the ability of BSP-IIA to alter [Ca2+]i in GCT23 cells. To investigate whether the CT-induced alteration of BSP-IIA-dependent cell signals was due to a modification in the synthesis of cell surface receptors (integrins) for the extracellular matrix macromolecules, 1-h CT-treated [35S]methionine metabolically labeled GCT23 cell lysates were immunoprecipitated with anti-alpha 3-, -alpha v-, -beta 1-, and -beta 3-integrin subunit antibodies. Autoradiography demonstrated that 10(-7)-10(-6) M CT did not alter new synthesis of the alpha v beta 3 and the alpha 3 beta 1 receptors. Similarly, CT did not affect surface expression of these receptors, assessed by enzyme-linked immunosorbent assay. Finally, no alteration of the adhesion rate and spreading of GCT23 cells onto BSP-IIA-coated substrates was observed. This indicates that CT-induced down-regulation of immediate cell signals prompted by BSP-IIA in GCT23 cells is a postintegrin receptor event.

Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism

Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism