osteoprotegerin and Giant-Cell-Tumor-of-Bone

Giant cell tumor is a relatively uncommon but painful tumor of bone, which can metastasize to the lungs. The RANK pathway is often reported to be involved in the pathogenesis of giant cell tumor of bone (GCTB). This pathway is a key signaling pathway of bone remodeling that plays a critical role in differentiation of precursors into multinucleated osteoclasts, and activation of osteoclasts leading to bone resorption. Dysregulation of RANK ligand (RANKL)-RANK-osteoprotegerin (OPG) signaling cascade induces the imbalance between bone formation and bone resorption, which leads to the changes in bone mass, increases osteoclast-mediated bone destruction, bone metastasis, and the progression of existing skeletal tumors. Recent evidences have shown that targeting the components of RANKL-RANK-OPG signaling pathway is a promising approach in the treatment of GCTB. This review study has focused on the association of RANKL-RANK-OPG pathway in the pathogenesis and progression of GCTB as well as discussed the possible therapeutic strategies by targeting this pathway.

Topics: Bone Resorption; Cell Differentiation; Gene Expression Regulation, Neoplastic; Giant Cell Tumor of Bone; Humans; Lung Neoplasms; Osteoclasts; Osteogenesis; Osteoprotegerin; RANK Ligand; Signal Transduction

Central giant cell granuloma (CGCG) is a benign lesion of the jaws with an unknown etiology. Clinically and radiologically, a differentiation between aggressive and non-aggressive lesions can be made. The incidence in the general population is very low and patients are generally younger than 30 years. Histologically identical lesions occur in patients with known genetic defects such as cherubism, Noonan syndrome, or neurofibromatosis type 1. Surgical curettage or, in aggressive lesions, resection, is the most common therapy. However, when using surgical curettage, undesirable damage to the jaw or teeth and tooth germs is often unavoidable and recurrences are frequent. Therefore, alternative therapies such as injection of corticosteroids in the lesion or subcutaneous administration of calcitonin or interferon alpha are described in several case reports with variable success. Unfortunately, randomized clinical trials are very rare or nonexistent. In the future, new and theoretically promising therapy options, such as imatinib and OPG/AMG 162, will be available for these patients.

Topics: Adrenal Cortex Hormones; Age Distribution; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Benzamides; Bone Density Conservation Agents; Calcitonin; Denosumab; Giant Cell Tumor of Bone; Granuloma, Giant Cell; Humans; Imatinib Mesylate; Interferons; Jaw Diseases; Osteoprotegerin; Piperazines; Pyrimidines; RANK Ligand; Subgingival Curettage

Giant cell tumor (GCT) of bone is a benign but locally aggressive neoplasm of bone. However, molecular mechanisms underlying osteolysis in GCT have not been deeply understood. The aim of this study was to investigate one of the possible mechanisms underlying the up-regulation of receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG) expression. First, we performed an immunohistochemical study on transforming growth factor-β1 (TGF-β1) expression in 83 cases with GCT and found that increased TGF-β1 staining was significantly correlated with Campanacci stages(Spearman's correlation = 0.335, p = 0.002). Next, we investigated the mechanism of the effect of TGF-β1 on osteolysis of GCT and examined the effects of TGF-β1 plus or minus specific inhibitor of Smad3 (SIS3) on the expression of RANKL/OPG ratio at the mRNA and protein levels in two primary GCT cell lines. The results clearly indicated that TGF-β1 is capable of significantly increasing RANKL/OPG ratio (p GCT1 = 0.000, p GCT2 = 0.000) and that SIS3 is capable of reversing the ratio, suggesting that Smad3 is the key to TGF-β1-induced increased the ratio. In the co-culture system, we found that SIS3 reversed the effects of TGF-β1-induced osteoclast formation in the co-culture system (p GCT1 = 0.000, p GCT2 = 0.000). Our findings indicate that TGF-β1 plays an important role in the osteolysis of GCT via Smad3.

Topics: Adult; Bone Neoplasms; Cell Differentiation; Cell Line, Tumor; Coculture Techniques; Female; Giant Cell Tumor of Bone; Humans; Male; Osteoclasts; Osteolysis; Osteoprotegerin; RANK Ligand; RNA, Messenger; Smad3 Protein; Transforming Growth Factor beta1; Up-Regulation; Young Adult

Giant cell tumor of bone (GCTB) is a benign, locally destructive neoplasm, with tumors comprised of mesenchymal fibroblast-like stromal cells; monocytic, mononuclear cells of myeloid lineage; and the characteristic osteoclast-like, multinucleated giant cells. Hampering the study of the complex interaction of its constituent cell types is the propensity of longstanding, repeatedly passaged cell cultures to undergo phenotypic alteration and loss of osteoclast-inducing capacities. In this study, we employed a novel, single-step technique to purify freshly harvested stromal cells using a CD14-negative selection column. Using 9 freshly harvested GCTB specimens and the purified stromal cell component, we performed analyses for markers of osteoblast lineage and analyzed the capacity of the stromal cells to undergo osteoblastic differentiation and induce osteoclastogenesis in co-cultures with monocytic cells. Successful purification of the CD14-negative stromal cells was confirmed via flow cytometric analysis and immunocytochemistry. Osteogenic media upregulated the expression of osteocalcin, suggesting an osteoblastic lineage of the GCTB stromal cells. The effects of the Wnt pathway agonist, SB415286, and recombinant human bone morphogenetic protein (BMP)-2 on osteoblastogenesis varied among samples. Notably, osteogenic media and SB415286 reversed the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) expression ratio resulting in diminished osteoclastogenic capacity. Recombinant human BMP2 had the opposite effect, resulting in enhanced and sustained support of osteoclastogenesis. Targeting the giant cell tumor stromal cell may be an effective adjunct to existing anti-resorptive strategies.

Topics: Adolescent; Adult; Aminophenols; Bone Morphogenetic Proteins; Cell Differentiation; Cell Separation; Culture Media; Female; Giant Cell Tumor of Bone; Humans; Ligands; Male; Maleimides; Middle Aged; Osteoblasts; Osteoclasts; Osteogenesis; Osteoprotegerin; Polymerase Chain Reaction; RANK Ligand; Stromal Cells; Wnt Signaling Pathway; Young Adult

Denosumab and Zoledronic acid (ZOL) are two antiresorptive drugs currently in use for treating osteoporosis. They have different mechanisms of action but both have been shown to delay the onset of skeletal-related events in patients with giant cell tumor of bone (GCT). However, the anti-tumor mechanisms of denosumab on the neoplastic GCT stromal cells remain unknown. In this study, we focused on the direct effects of denosumab on the neoplastic GCT stromal cells and compared with ZOL. The microscopic view demonstrated a reduced cell growth in ZOL-treated but not in denosumab-treated GCT stromal cells. ZOL was found to exhibit a dose-dependent inhibition in cell growth in all GCT stromal cell lines tested and cause apoptosis in two out of three cell lines. In contrast, denosumab only exerted a minimal inhibitory effect in one cell line and did not induce any apoptosis. ZOL significantly inhibited the mRNA expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in two GCT stromal cell lines whereas their protein levels remained unchanged. On the contrary, denosumab did not regulate RANKL and OPG expression at both mRNA and protein levels. Moreover, the protein expression of Macrophage Colony-Stimulating Factor (M-CSF), Alkaline Phosphatase (ALP), and Collagen α1 Type I were not regulated by denosumab and ZOL either. Our findings provide new insights in the anti-tumor effect of denosumab on GCT stromal cells and raise a concern that tumor recurrence may occur after the withdrawal of the drug.

Topics: Alkaline Phosphatase; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Cell Count; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Collagen Type I; Denosumab; Diphosphonates; Gene Expression Regulation, Neoplastic; Giant Cell Tumor of Bone; Humans; Imidazoles; Macrophage Colony-Stimulating Factor; Osteoprotegerin; RANK Ligand; RNA, Messenger; Solubility; Stromal Cells; Zoledronic Acid

Giant cell tumor (GCT) is the most common nonmalignant primary bone tumor reported in Hong Kong. It usually affects young adults between the ages of 20 and 40. This tumor is well known for its potential to recur following treatment. To date no effective adjuvant therapy exists for GCT. Our project aimed to study the effects of pamidronate (PAM), farnesyl transferase inhibitor (FTI-277), geranylgeranyl transferase inhibitor (GGTI-298), and their combinations on GCT stromal cells (SC). Individual treatment with PAM, FTI-277, and GGTI-298, inhibited the cell viability and proliferation of GCT SC in a dose-dependent way. Combination of FTI-277 with GGTI-298 caused synergistic effects in reducing cell viability, and its combination index was 0.49, indicating a strong synergism. Moreover, the combination of FTI-277 with GGTI-298 synergistically enhanced cell apoptosis and activated caspase-3/7, -8, and -9 activities. PAM induced cell-cycle arrest at the S-phase. The combination of PAM with GGTI-298 significantly increased OPG/RANKL mRNA ratio and activated caspase-3/7 activity. Our findings support that the combination of bisphosphonates with GGTIs or FTIs with GGTIs may be used as potential adjuvants in the treatment of GCT of bone.

Topics: Alkyl and Aryl Transferases; Apoptosis; Benzamides; Bone Density Conservation Agents; Bone Neoplasms; Caspases; Cell Division; Cell Survival; Diphosphonates; Drug Synergism; Enzyme Inhibitors; Farnesyltranstransferase; Gene Expression; Giant Cell Tumor of Bone; Humans; Methionine; Osteoprotegerin; Pamidronate; Prenylation; RANK Ligand; RNA, Messenger; S Phase; Tumor Cells, Cultured

Water-soluble phosphorylated chitosan (P-chitosan) and disodium (1 → 4)-2-deoxy-2-sulfoamino-β-D-glucopyranuronan (S-chitosan) are two chemically modified chitosans. In this study, we found that P-chitosan significantly promotes cell proliferation of both human primary osteoblasts (OBs) and the OB like stromal cell component of the giant cell tumor of bone (GCTB) cells at the concentration from 125 to 1000 µg ml⁻¹ at all time points of 1, 3, 5 and 7 days after treatment. Further investigation of the osteogenic effect of the P-chitosan suggested that it regulates the levels of osteoclastogenic factors, receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression. An interesting finding is that S-chitosan at lower concentration (100 µg ml⁻¹) stimulates cell proliferation while a higher dose (1000 µg ml⁻¹) of S-chitosan inhibits it. The inhibitory effect of S-chitosan on human primary GCT stromal cells was greater than that of OBs (p < 0.05). Taken together, our findings elucidated the osteogenic effect of P-chitosan and the varying effects of S-chitosan on the proliferation of human primary OBs and GCT stromal cells and provided us the rationale for the construction of novel bone repair biomaterials with the dual properties of bone induction and bone tumor inhibition.

Topics: Base Sequence; Biocompatible Materials; Cell Proliferation; Cell Survival; Cells, Cultured; Chitosan; DNA Primers; Gene Expression; Giant Cell Tumor of Bone; Humans; Materials Testing; Osteoblasts; Osteogenesis; Osteoprotegerin; Phosphorylation; RANK Ligand; RNA, Messenger; RNA, Neoplasm; Solubility; Stromal Cells; Tumor Cells, Cultured; Water

The interactions between the receptor activator of NF-κB (RANK), its ligand (RANKL), and the decoy receptor for RANKL, osteoprotegerin (OPG), play a pivotal role in promoting osteoclast differentiation and activation leading to bone resorption. Giant cell tumours, chondroblastomas, and aneurysmal bone cysts harbour osteolytic lesions containing osteoclast-like giant cells. We investigated the characteristics of the RANKL signalling pathway in each of these bone lesions.. We evaluated 44 cases of giant cell tumour, 12 cases of chondroblastoma, six cases of aneurysmal bone cyst, and five cases of metastatic giant cell tumour (including paired primary giant cell tumours). We assessed RANK, RANKL, and OPG expression in chondroblastomas, giant cell tumours, and aneurysmal bone cysts using immunohistochemical methods.. Our findings revealed that RANK, RANKL, and OPG expression differed significantly among disease types. Giant cells of chondroblastomas showed significantly higher RANK expression than the giant cells of giant cell tumours and aneurysmal bone cysts; similarly, stromal cells of chondroblastomas showed significantly higher OPG expression than the stromal cells of giant cell tumours and aneurysmal bone cysts. Furthermore, giant cells of giant cell tumours expressed significantly more RANK than the giant cells of aneurysmal bone cysts.. The expression of RANK, RANKL, and OPG in osteoclast-like giant cells differs significantly by disease; OPG expression differs significantly between giant cell tumours and chondroblastomas.

Topics: Bone and Bones; Bone Cysts, Aneurysmal; Bone Neoplasms; Chondroblastoma; Giant Cell Tumor of Bone; Giant Cells; Humans; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction

Giant-cell tumor of bone (GCTB) and giant-cell tumor of soft tissue (GCTST) are tumors that contain a prominent osteoclastlike giant-cell component. The precise relationship between these morphologically similar tumors is unclear, and the cellular mechanism whereby giant cells accumulate within these and other locally aggressive tumors is uncertain. In this study, we have examined the cytochemical, functional, and molecular phenotype of the mononuclear and multinucleated components of GCTB and GCTST. Giant cells in GCTB and GCTST exhibited an osteoclast phenotype expressing tartrate-resistant acid phosphatase and vitronectin receptor and being capable of lacunar resorption. The mononuclear stromal cells derived from GCTB and GCTST exhibited an osteoblast phenotype, expressing alkaline phosphatase, and the receptor activator for nuclear factor kappaB ligand (RANKL), a factor that is essential for osteoclast formation. These cells also expressed osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, and TRAIL, a receptor that binds OPG. Lacunar resorption by giant cells isolated from GCTB and GCTST was inhibited by OPG, zoledronate, and calcitonin. These findings indicate that the mononuclear and giant-cell components of GCTB and GCTST have similar phenotypic features and that the accumulation of osteoclasts in these giant-cell-rich tumors occurs by a RANKL-dependent process. RANKL expression by osteoblastlike mononuclear stromal cells in these tumors stimulates osteoclast formation and resorption; this would account for the osteolysis associated with these giant-cell-rich tumors. Inhibitors of osteoclast formation and activity are likely to be effective in controlling the osteolysis associated with GCTB and possibly other giant-cell-rich lesions.

Topics: Apoptosis Regulatory Proteins; Bone Neoplasms; Bone Resorption; Carrier Proteins; Coculture Techniques; Giant Cell Tumor of Bone; Giant Cell Tumors; Glycoproteins; Humans; Immunohistochemistry; Membrane Glycoproteins; Monocytes; Osteoclasts; Osteolysis; Osteoprotegerin; Phenotype; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Soft Tissue Neoplasms; Stromal Cells; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Matrix metalloproteinases (MMPs) are regarded as a significant regulator in tumor invasion and metastasis. Previous studies have shown that extracellular matrix metalloproteinase inducer (EMMPRIN) in tumor cells induces the synthesis of MMPs. EMMPRIN is abundantly present on the surface of tumor cells and stimulate adjacent stromal cells to synthesize MMPs to induce tumor progression. Giant cell tumor (GCT) of bone is a benign but locally aggressive primary neoplasm of bone. The spindle-shaped mononuclear stromal cells are considered to be the tumor components of GCT, which are capable of inducing osteoclast formation by recruiting the circulating monocyte and macrophage. In this study, we proposed that EMMPRIN is associated with the biological progression and aggressiveness of GCT. We have conducted semi-quantitative RT-PCR to determine the correlation of EMMPRIN expression with the clinical stage of GCT. We have also examined the cellular localization of EMMPRIN in GCT using in-situ hybridization (ISH) and Immunohistochemistry (IH). The results showed that EMMPRIN was present in GCT and its mRNA levels were associated with the clinical stage of GCT. Higher expression level of EMMPRIN was observed in GCT with advanced stage (stage III). There was a great significance (P < 0.05) of EMMPRIN expression between stage I & II and stage III GCTs. Both ISH and IH demonstrated that EMMPRIN is present at the multinuclear osteoclast-like giant cells of GCT, with strong immunostaining on the cell membrane. The stromal-like tumor cells were also positively stained but the intensity was weaker. Interestingly, the production of EMMPRIN in osteoclast-like cells of GCT seems to be regulated by stromal-like tumor cells. Receptor activator of NF-kappaB ligand (RANKL), which has been previously shown to be produced by the stromal-like tumor cells for the recruitment of osteoclast-like giant cells in GCT, enhanced the expression of EMMPRIN mRNA during the differentiation of macrophage-like RAW(264.7) cells into osteoclasts. In short, our studies suggest that EMMPRIN may be an important regulatory factor involved in the biological behaviors of GCT.

Topics: Animals; Antigens, CD; Antigens, Neoplasm; Antigens, Surface; Avian Proteins; Basigin; Blood Proteins; Bone Neoplasms; Cell Differentiation; Cell Line; Female; Gene Expression; Giant Cell Tumor of Bone; Glycoproteins; Humans; Immunohistochemistry; In Situ Hybridization; Macrophages; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Membrane Glycoproteins; Mice; Osteoclasts; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2

To investigate the gene expression of osteoprotegerin (OPG) and osteoclast differentiation factor (ODF/TRANCE/RANKL), two new members of the TNF-receptor superfamily, in giant cell tumor (GCT); to discuss the molecular mechanism of extensive bone resorption caused by GCT.. Using TRIzol reagent to prepare total RNA from GCT sample and normal bone tissue. By a first-strand complementary DNA (cDNA) synthesis kit, cDNA was synthesized from 2.0 micro g RNA according to the manufacturer's instructions. cDNA was then amplified by PCR. Amplification products were resolved by electrophoresis on a 1.5% agarose gel and stained with EB. The relative quantity of the PCR products were determined and the mRNA levels of OPG, ODF, M-CSF (cofactor of ODF), and RANK (receptor of ODF) were compared with that of the normal bone.. GCT contained highly expressed mRNA of ODF, OPG, M-CSF and RANK. There was mRNA expression of OPG, M-CSF and RANK and less expression of ODF in normal bone. The ODF mRNA and RANK mRNA in GCT were more abundant than that in normal bone. In GCT, the ratio of ODF mRNA exceeded OPG expression. But in normal bone, the OPG mRNA exceeded ODF expression.. The results suggest that GCT contains all signals including OPG, ODF, M-CSF and RANK that are essential for inducing osteoclastogenesis and promoting bone resorption.

Topics: Carrier Proteins; Gene Expression Regulation, Neoplastic; Giant Cell Tumor of Bone; Glycoproteins; Humans; Membrane Glycoproteins; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger

In giant cell tumors of bone (GCTBs), the mesenchymal stromal cells are the neoplastic cells and induce recruitment and formation of osteoclasts (OCs). Studies on recently discovered members of the tumor necrosis factor receptor-ligand family have demonstrated a crucial role of RANKL (receptor activator of nuclear factor kappa B [RANK] ligand) expressed by osteoblast/stromal cells and of its receptor RANK expressed by OCs during OC differentiation and activation. OCs typically are present in large numbers in GCTBs, suggesting that these tumors may contain cells expressing factors that stimulate OC precursor recruitment and differentiation. We used immunohistochemical analysis to study RANKL and RANK expression in 5 GCTBs. Multinucleated cells and some mononuclear cells showed strong positive staining with anti-RANK antibodies; RANKL was present in a subset of mononuclear cells that did not express the hematopoietic lineage cell marker CD45, a feature that identified them as mesenchymal tumor cells. Our results suggest that RANKL expression may have a role in the pathogenesis of GCTBs and in the formation of the large OC population present in these tumors.

Topics: Animals; Antibody Specificity; Carrier Proteins; Cell Count; COS Cells; DNA, Complementary; Gene Expression; Giant Cell Tumor of Bone; Glycoproteins; Humans; Immunohistochemistry; Leukocyte Common Antigens; Leukocytes, Mononuclear; Membrane Glycoproteins; Mesoderm; Osteoblasts; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Transfection

Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.

Topics: Bone Neoplasms; Bone Resorption; Carrier Proteins; Fluorescent Antibody Technique; Giant Cell Tumor of Bone; Glycoproteins; Humans; Membrane Glycoproteins; Microscopy, Electron, Scanning; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

Giant cell tumor of bone (GCT) is a rare primary osteolytic tumor of bone that is characterized by massive tissue destruction at the epiphysis of long bones. There is no evidence that tumor cells themselves are capable of bone destruction; instead, it appears that the tumor cells of GCT act by promoting osteoclastogenesis and, as a consequence, osteoclastic bone resorption. However, the mechanism by which this is achieved is not understood. Here we attempted to determine whether osteoprotegerin ligand (OPGL), the factor that is necessary and essential for osteoclastogenesis, is involved in tumor cell-recruited osteoclast-like giant cell formation in GCT. Using fluorescence in situ hybridization, we sought to determine mRNA expression of OPGL, its receptor RANK, and its decoy receptor OPG in three major cell types of GCT. We demonstrated that OPG mRNA was expressed in all three cell types of GCT, OPGL transcripts were mainly detected in spindle-shaped stromal-like tumor cells, whereas RANK was expressed only in macrophage-like mononuclear cells and multinuclear osteoclast-like giant cells. By semiquantitative RT-PCR, we also showed that the level of OPGL mRNA in GCT is much higher than that in normal bone and osteogenic osteosarcoma. In contrast, a similar level of OPG transcripts was detected in these three kinds of tissues, and RANK mRNA was detectable only in GCT tissues. We have further examined the regulation of gene expression of OPGL and OPG in tumor cells in response to osteotropic hormones. Administration of 1,25(OH)(2)D(3) and dexamethasone resulted in maximum up-regulation of OPGL level and down-regulation of OPG level in cultured GCT stromal-like tumor cells and the mouse bone marrow-derived ST-2 stromal cell line. Furthermore, we have shown that tumor cells of GCT induce differentiation of RANK-expressing myeloid RAW(264.7) cells into osteoclast-like cells in the presence of 1,25(OH)(2)D(3) and dexamethasone. Our findings suggest that OPGL is involved in the tumor cell-induced osteoclast-like cell formation in GCT. The ratio of OPGL/OPG by tumor cells may contribute to the degree of osteoclastogenesis and bone resorption.

Topics: Animals; Bone Neoplasms; Calcitriol; Carrier Proteins; Coculture Techniques; Dexamethasone; DNA Primers; Gene Expression; Giant Cell Tumor of Bone; Glycoproteins; Humans; In Situ Hybridization, Fluorescence; Membrane Glycoproteins; Mice; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

The mechanisms by which primary tumors of the bone cause bone destruction have not been elucidated. Unlike most other lytic bone tumors, osteoclastomas, otherwise known as giant cell tumors (GCT), contain osteoclast-like cells within the tumor stroma. A new member of the TNF-ligand superfamily member, osteoclast differentiation factor (ODF/OPGL/RANKL/TRANCE), was recently identified. ODF was shown to directly stimulate osteoclastogenesis, in the presence of M-CSF. In this study, the expression of ODF was examined in a number of tumor samples associated with bone lysis in vivo. In addition, we investigated expression of the ODF receptor on osteoclast precursors, RANK, as well as the ODF inhibitor osteoprotegerin (OPG), and another TNF-ligand superfamily member, TRAIL, previously shown to abrogate the inhibitory effects of OPG. We report here the novel finding that GCT stromal cells contain abundant ODF mRNA, whereas the giant cell population exclusively expresses RANK mRNA. These results are consistent with the osteoclast-mediated bone destruction by these tumors. We also report the expression of OPG and TRAIL mRNA in GCT samples. A comparison with other lytic and nonlytic tumors of bone showed that GCT express more ODF and TRAIL mRNA relative to OPG mRNA. In addition, GCT were found to express a number of cytokines previously reported to play central roles in osteoclastogenesis, namely, IL-1, -6, -11, -17, as well as TNF-alpha. Importantly, GCT were also found to express high levels of M-CSF mRNA, a cytokine shown to be an essential cofactor of ODF, and a survival factor for mature and developing osteoclasts. Furthermore, expression of these molecules by stromal cells isolated from GCT continued in vitro. Thus GCT constitutively express all of the signals that are currently understood to be necessary for the differentiation of osteoclasts from precursor cells.

Topics: Apoptosis Regulatory Proteins; Biomarkers; Bone Neoplasms; Carrier Proteins; Cell Differentiation; Cytokines; Gene Expression; Giant Cell Tumor of Bone; Glycoproteins; Hematopoiesis; Ligands; Membrane Glycoproteins; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; RNA, Messenger; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Spindle-shaped cells were established from four giant-cell tumors of bone. When human blood monocytes were co-cultured with these cells, multinucleated giant-cell formation of monocytes was induced. Intriguingly, even when a filter (pore size: 0.45 microm) was interposed between monocytes and the spindle-shaped cells, polykaryocytes also appeared. These multinucleated giant cells were positive for tartrate-resistant acid phosphatase, expressed calcitonin receptor, and showed bone-resorption activity, characteristics of osteoclast-like cells. These findings indicate that soluble factors secreted from these cells play an important role in osteoclast-like cell formation from blood monocytes. These data additionally suggest that these cells support osteoclast-like cell formation in giant-cell tumors of bone. The cells also expressed mannose receptor, fibronectin, receptor activator of nuclear factorkappaB, and several cytokine mRNAs, including interleukin-6, receptor activator of nuclear factorkappaB ligand/osteoclast differentiation factor/osteoprotegerin ligand, and macrophage colony-stimulating factor. However, all of these molecules except receptor activator of nuclear factorkappaB ligand mRNA could also be detected in control HeLa and CV-1 cells. Although the soluble receptor activator of nuclear factorkappaB ligand has not been found under physiological conditions, it is possible that it is cleaved by cellular proteases and the truncated receptor activator of nuclear factorkappaB is released from cells. Identification of the soluble factors capable of inducing osteoclast formation from blood monocytes is a pressing problem to be solved.

Topics: Bone Neoplasms; Cell Differentiation; Cell Size; DNA Primers; Gene Expression; Giant Cell Tumor of Bone; Glycoproteins; HeLa Cells; Humans; Interleukin-6; Lipopolysaccharide Receptors; Macrophage Colony-Stimulating Factor; Monocytes; Osteoclasts; Osteoprotegerin; Phenotype; Receptors, Calcitonin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor