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Short Communications |
B in Giant Cell Tumor of Bone
From the Department of Orthopaedic Surgery, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B ligand (RANKL)5
belong to the
membrane-associated TNF-ligand family. It has been shown, using an
in vitro culture system, that OPGL can both induce
osteoclastogenesis and activate mature osteoclasts.3,4
The
expression of OPGL in osteoblast/stromal cells parallels the formation
of osteoclasts in cocultures with bone marrow or spleen cell
populations. The recombinant OPGL can replace the requirements for
stroma cells in the in vitro model of
osteoclastogenesis.4
Mice with a disrupted opgl
gene show severe osteopetrosis and a defect in tooth eruption and
completely lack osteoclasts as a result of an inability of osteoblasts
to support osteoclastogenesis.7
It has been assumed that
OPGL acts as an osteoclastogenesis-inducing factor linked to
interaction between stromal cells and osteoclast progenitors. The cell
surface receptor that interacts with OPGL has recently been shown to be
the ligand for the TNFR-related protein receptor activator of NF-B
(RANK).8
Transgenic mice expressing a soluble RANK-Fc
fusion protein display osteopetrosis, a defect of osteoclast activity,
whereas polyclonal antibody against the RANK extracellular domain
promotes osteoclastogenesis in bone marrow cultures.8
On
the other hand, the decoy receptor of OPGL, OPG has been shown to
neutralize and interrupt stromal cell-derived OPGL signals, resulting
in the reduction of osteoclastogenesis.1,2
OPG, also known
as osteoclastogenesis inhibitory factor (OCIF),2
is a
soluble member of the TNF receptor family. OPG inhibits not only
formation of osteoclast-like cells in murine cultures in
vitro2
but also bone resorption in vitro
and in vivo.1,2
OPG knock-out mice exhibited
severe osteopenia due to accelerated bone resorption.1
In
short, it is conceivable that OPGL and OPG are key extracellular
regulators of osteoclastogenesis and bone resorption. RANK is receptor
necessary for the activation of OPGL. Giant cell tumor of bone (GCT), a rare primary osteolytic tumor of bone, is characterized by massive bone destruction at the epiphysis of long bones.9 Previous studies have shown that the spindle-shaped stromal-like mononuclear cells of GCT are the most likely candidate cells for the tumor’s neoplastic component.9-12 However, there is no evidence that tumor cells themselves are capable of bone resorption.13 Instead, tumor cells of GCT act by recruiting multinucleated osteoclast-like giant cells and hence promoting tumor-induced osteolysis.9,14,15 We previously showed that tumor cells were capable of recruiting circulating monocytes/osteoclast precursor cells and even osteoclasts through the production of transforming growth factor-ß1 (TGF-ß1)9 and monocyte chemoattractant protein 1 (MCP-1).10 Others have shown that the tumor cells of GCT also produce interleukins-1 and -6 and macrophage colony-stimulating factor, which are related to the induction of osteoclastogenesis and bone resorption.16-18 It is noteworthy that although individual cytokines play a role in the recruitment of osteoclasts and in osteoclastic bone resorption, none of them are capable of directly inducing the macrophage-like cells to form osteoclasts. In fact, cells from osteogenic tumors such as osteosarcomas can express a similar cytokine profile,19-21 as do cells from GCT, but induction of osteoclast formation is observed to a lesser degree. Thus the mechanism by which tumor cells of GCT generate osteoclast-like giant cells from recruited macrophage-like cells is unclear.
To determine whether OPGL is involved in the interaction of stromal-like tumor cells and macrophage-like mononuclear cells, which results in the generation of osteoclast-like giant cells in GCT, we have investigated the expression of OPGL, OPG, and RANK in all three cell types of GCT at the mRNA level and examined the regulation of gene expression in response to osteotropic hormones. A coculture system of stromal-like tumor cells and murine osteoclast precursor cell line RAW264.7 cells8 has been used to evaluate the ability of tumor cells to further induce osteoclastogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human GCT from five different cases and normal cancellous bone
containing no bone marrow cells were collected fresh from patients
after operations at Sir Charles Gairdner Hospital (Nedlands, WA,
Australia). The human osteoblast-like osteosarcoma cell line U2OS was
purchased from the American Type Culture Collection (Rockville, MD).
The mouse stromal cell line ST-2 was provided by Prof. G. C.
Nicholson at the Geelong Hospital (Geelong, VIC, Australia). The murine
myeloid cell line RAW264.7 was provided by Dr.
Ian Cassady at the University of Queensland (Queensland,
Australia). Dulbecco’s minimum essential medium (MEM), 
-MEM, and
fetal bovine serum (FBS) were purchased from TRACE (Sydney, Australia).
1,25-Dihydroxyvitamin D3
(1,25(OH)2D3) was obtained
from Calbiochem-Novabiochem (Alexandria, Australia);
dexamethasone was from Sigma (St. Louis, MO); recombinant human RANKL
(sRANKL) was from Pepro Tech (NJ, USA); RETROscript was from
Ambion (Austin, TX); the DIG RNA Labeling kit and the Fluorescent
Antibody Enhancer Set were from Boehringer Mannheim (Sydney,
Australia); the diagnostics tartrate resistant acid phosphatase (TRAP)
kit was from Sigma; rabbit anti-rat/mouse calcitonin receptor (CTR)
polyclonal antibody was provided by Dr. P. M. Sexton at the
Department of Pharmacology (University of Melbourne, Victoria,
Australia); Texas Red-X-conjugated swine anti-rabbit Ig secondary
antibody was purchased from Molecular Probes (Eugene, OR). All other
chemicals used were of the highest grade available.
Culture of Giant Cell Tumor of Bone
As described previously,4 tumor tissues were freshly chopped in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. The resultant cell suspension together with small pieces of tissue was transferred to 25-cm2 flasks for culture at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The pieces of tissue contained many multinuclear giant cells and mononuclear cells that had migrated out of tissue fragments across the culture flask surface and contributed to the cell population in culture. Half of the culture medium was changed every 3 days with fresh DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. On reaching confluence, primary cultures were subcultured, and each passaged culture was stored in liquid nitrogen. To examine the gene regulation of OPGL and OPG in tumor cells of GCT, GCT stromal-like tumor cells obtained after the ninth passage were cultured by the addition of 10-8 mol/L 1,25(OH)2D3 in the absence or presence of 10-7 mol/L dexamethasone. After 7 days of incubation, cells from each group were prepared for RNA extraction.
Culture of ST-2 Cells and Osteoblastic U2OS Cells
Both ST-2 and U2OS cells were cultured in -MEM containing 10%
FBS, 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100
µg/ml streptomycin, in a humidified atmosphere of 5%
CO2 and 95% air at 37°C. When the effect of
1,25(OH)2D3 and
dexamethasone was tested, dose dependence was studied by the addition
of 10-8 mol/L
1,25(OH)2D3 in the presence
or absence of dexamethasone at doses from 10-9
to 10-6 mol/L. All cultures were harvested and
used for total RNA extraction.
Coculture System with Stromal-Like Tumor Cells and RAW264.7 Cells
GCT stromal-like tumor cells at the ninth passage obtained from the liquid nitrogen store were seeded into culture chambers (flask style; Nunc) and treated with 10-8 mol/L 1,25(OH)2D3 in combination with 10-7 mol/L dexamethasone. To the stromal-like tumor cells reaching 50% confluence, 500 RAW264.7 cells were added to each chamber. The cocultures were continuously treated with both 1,25(OH)2D3 and dexamethasone. In separate experiments, RAW264.7 cells were cultured in medium alone or in medium with either 30 ng/ml sRANKL or a combination of 10-8 mol/L 1,25(OH)2D3 and 10-7 mol/L dexamethasone. All cultures were fed every 3 days with fresh medium. After 10 days, all of the cultures were fixed and proceeded to either TRAP histochemistry, CTR immunohistochemistry, or bone resorption pit assay (Zheng et al)9 to confirm the identity of osteoclasts. TRAP-positive multinuclear cells with more than three nuclei were scored and data were statistically analyzed by Student’s t-tests.
Calcitonin Receptor Immunofluorescence Confocal Microscopy
Calcitonin receptor in cocultures of stromal-like cells and RAW264.7 cells was detected by the method described by Quinn et al.22 In brief, cocultures prepared as described above were first incubated with 5% sheep serum in 0.5% BSA/PBS for 10 minutes. The cells were then incubated in PBS/BSA containing rabbit anti-rat/mouse CTR antibody (diluted 1:50) preincubated with MBP-CTR antigen. After a 1-hour incubation at room temperature, the cells were rinsed in PBS, and then PBS/BSA containing Texas Red-X-conjugated swine anti-rabbit Ig antibody (diluted 1:100) was added. After another 1-hour incubation in the dark, the antibodies were rinsed away by washing in PBS. Cells were examined under a confocal laser scanning microscope (Bio-Red 10000) for the immunofluorescence of calcitonin receptor.
RNA Extraction and RT-PCR
By the use of RNAzol B (Tel-Test) protocols, total cellular RNA
was isolated from solid tumors, cultured cells derived from human GCT,
normal bone, U2OS cells, ST-2 cells, RAW264.7
cells, and the coculture system and then reverse-transcribed into cDNA,
using 100 units of Moloney-Murine Leukemia Virus (M-MLV) reverse
transcriptase (Ambion) according to the manufacturer’s instructions.
Primers used for the detection of OPGL, OPG, and RANK are listed in
Table 1
. Housekeeping genes
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and acidic ribosomal
phosphoprotein (36B4) were used as internal controls in the
examinations of human and mouse gene expressions, respectively.
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Fluorescence in Situ Hybridization
The cDNA fragments of human OPGL, OPG, and RANK, which were 486, 324, and 497 bp, respectively, were generated by RT-PCR of total RNA from GCT solid tumor and then separately inserted into pCR2.1 with the Original TA Cloning Kit (Invitrogen). Recombinant plasmids with correct orientation were then purified and transcribed into digoxigenin-labeled antisense riboprobes with T7 RNA polymerase, using a DIG RNA labeling kit (Boehringer Mannheim). All of the clones were sequenced for the confirmation of authentic genes. In situ hybridization was performed as previously described.23,24 The final concentration of each probe in hybridization solution was 0.3 ng/µl, and RNase treatment (100 µg/ml) before hybridization was used as the negative control. Detection of hybridization products was performed with a fluorescent antibody enhancer set (Boehringer Mannheim). We counterstained slides with 2 µg/ml of propidium iodide for 30 minutes at room temperature to view nuclei or with TRAP histochemistry to confirm the characteristics of both osteoclast-like giant cells and macrophage-like cells. Signals were detected by confocal microscopy (BioRed, 1000).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To allow estimation of the level of OPG, OPGL, and RANK gene
transcripts in solid tumor and cultures of GCT, cycle-dependent PCR
reactions were performed to generate amplification curves for each
gene, using specific primers (Figure 1A)
.
A total of 28 cycles was selected for semiquantitation of OPGL and OPG
gene transcripts, and 33 cycles for RANK and 26 cycles for GAPDH gene
expression. As shown in Figure 1B
, all GCT solid tumors expressed OPGL,
OPG, and RANK mRNAs, whereas both normal bone and U2OS cells contained
much less OPGL gene transcript and no RANK gene transcript. It appears
that the ratio of OPGL/OPG in GCT was higher than that in normal bone
and osteosarcoma. No correlation of OPGL, OPG, and RANK expression with
the Enneking Clinical Stage25
of GCT was found at
presentation. Two cases of GCT at stage III (Figure 1B
, lanes 2 and 3)
expressed almost the same level of these gene transcripts as others in
GCT at stage I or II (Figure 1B
, lanes 1, 4, and 5).
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To further examine the localization of OPGL, OPG, and RANK gene
transcripts in GCT at the cellular level, fluorescence in
situ hybridization (FISH) was performed by using
digoxigenin-labeled specific riboprobes (Figure 2)
. Signals for each gene transcript were
distributed differently in the cytoplasm of various cell types. OPG
signals were detected in spindle-shaped stromal-like tumor cells,
macrophage-like mononuclear cells, and multinuclear osteoclast-like
giant cells. However, OPGL signals were mainly in stromal-like tumor
cells, whereas RANK signals were mainly in macrophage-like mononuclear
cells and multinuclear osteoclast-like giant cells. It is noteworthy
that the RANK-positive macrophage-like mononuclear cells were also
positive for TRAP when double staining of RANK and TRAP was used (data
not shown). Treatment with RNase resulted in the absence of signals in
all cells, indicating the specificity of the probes for target mRNA
sequence (Figure 2)
.
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Various osteotropic hormones have been shown to be the regulators
of osteoclastogenesis.3,25,26
Because GCTs are considered
to arise from mesenchymal stromal cells that have the capacity to
recruit and harbor osteoclasts,9-16
we attempted to
determine whether
1,25(OH)2D3 and
dexamethasone were capable of regulating OPGL and OPG expression in the
stromal-like tumor cells of GCT and the mouse bone marrow-derived
mesenchymal stromal cell line ST-2 cells. Cells were treated with
1,25(OH)2D3 at
10-8 mol/L and dexamethasone at
10-7 mol/L for a period of 7 days. As shown in
Figure 3A
,
1,25(OH)2D3 significantly
increased OPGL mRNA levels while reducing OPG levels in ST-2 cells.
Dexamethasone alone had no effect on OPGL and OPG gene expression (data
not shown) but enhanced those effects of
1,25(OH)2D3 on both gene
expression in a dose-dependent manner (Figure 3A
, bottom). On the other
hand, the effect of
1,25(OH)2D3 and
dexamethasone on OPGL and OPG gene expression in primary cultures of
GCT tumor cells varied between cases (Figure 3B)
. In case 2 GCT primary
culture (Figure 3B
, top), both agents had no obvious effect on the
reduction of OPG gene transcription but increased OPGL mRNA levels.
On the other hand, dexamethasone increased OPGL levels and
decreased OPG levels in a dose-dependent manner when combined with
1,25(OH)2D3 in case 4 GCT
primary culture (Figure 3B
, bottom). It appears that the balance
between the levels of OPGL and OPG gene transcripts in these cells was
significantly altered by the addition of both osteotropic agents.
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Given that there is no human macrophage cell line that is capable
of differentiating into osteoclasts, whereas mouse
RAW264.7 cells have been demonstrated to be able
to generate osteoclast-like cells in the presence of OPGL but not other
cytokines,8
we have attempted to determine whether tumor
cells of GCT are capable of inducing the differentiation of
RAW264.7 cells into osteoclast-like cells. As
shown in Figure 4
,
RAW264.7 cells alone expressed high levels of
RANK (Figure 4
, top) but did not differentiate into osteoclasts that
express TRAP (Figure 4
, middle, a). RAW264.7
cells cocultured with tumor cells in the presence of
1,25(OH)2D3 and
dexamethasone differentiated into TRAP-positive osteoclast-like cells
after 10 days (Figure 4
, middle, d). Treatment of
RAW264.7 cells with human sRANKL also induced the
formation of TRAP-positive osteoclast-like cells (Figure 4
, middle, c).
These multinuclear cells formed by the fusion of
RAW264.7 cells were shown to express calcitonin
receptor (Figure 4
, middle, e) and are capable of bone resorption
(Figure 4
, middle, f). On the other hand, there is no evidence of
osteoclast formation when RAW264.7 cells are
treated with 1,25(OH)2D3
and dexamethasone in the absence of tumor cells (Figure 4
, middle, b).
These results demonstrated that tumor cells of GCT are capable of
inducing osteoclast formation by RAW264.7 cells
in the presence of
1,25(OH)2D3 and
dexamethasone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GCT is characterized by abundant multinuclear osteoclast-like giant cells scattered among mononuclear cells.15 Although the histogenesis of GCT is not fully elucidated, it is generally believed that it is the stromal-like tumor cells that have the ability to recruit circulating monocytes to become multinuclear osteoclast-like giant cells in GCT.9-16 We reported here the novel findings that OPGL, the osteoclastogenesis-inducing factor, was abundantly expressed in stromal-like tumor cells of GCT, and that RANK, the receptor for OPGL, was expressed in macrophage-like cells and osteoclast-like multinuclear giant cells. On the other hand, OPG, the decoy receptor for OPGL, was also ubiquitously expressed in the stromal-like tumor cells, indicating that a negative feedback loop may exist in which the tumor cells of GCT themselves may modulate the presentation of OPGL molecules on their surface, which in turn can be inhibited by OPG. Thus the ratio of OPGL and OPG gene expression in tumor cells may determine local osteoclastogenesis and osteoclastic bone resorption in the lesion. If the level of OPGL gene expression exceeds that of OPG in the microenvironment, osteoclast formation may be effectively induced. Conversely, if OPG gene expression is at higher levels than OPGL, osteoclast formation may be suppressed. In this study, we have shown a higher ratio of OPGL to OPG mRNA in all cases of GCT than that in normal bone and osteoblast-like osteosarcoma cell lines. This suggests that the production of OPGL may be of great importance for the tumor cell-induced formation of osteoclast-like giant cells in GCT. Bearing in mind that there is still a limitation of using mRNA to assess the expression of OPGL and OPG, further study should be conducted to elucidate their ratio at the protein level.
Previous studies have shown that the combination of 1,25(OH)2D3 and dexamethasone significantly stimulates osteoclast-like cell formation in cocultures of mouse spleen cells or in mouse bone marrow cultures.29-31 In general accord with these findings, our results demonstrated that the combination of these two agents resulted in maximum up-regulation of OPGL gene expression and down-regulation of OPG expression in the mouse mesenchymal stromal cells tested. It is noteworthy that OPGL mRNA was also remarkably increased in primary cultured tumor cells of GCT when treated with 1,25(OH)2D3 and dexamethasone. Although tumor cells of GCT did not show a consistent decrease in OPG mRNA levels in the presence of both agents, it is reasonable to presume that these tumor cells, which express increased OPGL molecules on their membrane in the presence of 1,25(OH)2D3 and dexamethasone, would induce the differentiation of osteoclast progenitors into osteoclasts. In our present study, we have showed that the murine myeloid RAW264.7 cells differentiated into multinucleated osteoclast-like cells when cocultured with stromal-like tumor cells of GCT in the presence of 1,25(OH)2D3 and dexamethasone. The osteoclast-like cells formed in the coculture satisfied the major criteria of osteoclasts, including multinucleation and the presence of TRAP activity and calcitonin receptor. Given that the induction of osteoclast formation is required by the addition of 1,25(OH)2D3 and dexamethasone, which are the agents for up-regulation of OPGL expression, it is possible that tumor cell-induced osteoclast-like giant cell formation is mediated through the OPGL molecule expressed in stromal-like tumor cells.
In summary, we have shown for the first time that stromal-like tumor cells of GCT express OPGL, whereas macrophage-like cells and multinuclear osteoclast-like giant cells express the receptor for OPGL, the RANK. OPG, the decoy receptor for OPGL, was also found in GCT. However, the ratio of OPGL to OPG mRNA was much higher in GCT than that in normal bone and osteosarcoma cells. Osteotropic agents 1,25(OH)2D3 and dexamethasone up-regulate gene expression of OPGL in tumor cells of GCT. RAW264.7 cells, which express high levels of RANK mRNA, can differentiate into osteoclast-like cells when cocultured with stromal-like tumor cells in the presence of1,25(OH)2D3 and dexamethasone. Our findings suggest that OPGL may be essential for the tumor cell to induce the formation of osteoclast-like giant cells in GCT. It appears that the ratio of OPGL/OPG produced by stromal-like tumor cells may contribute to the degree of osteoclast-like giant cell formation and bone destruction in GCT.
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Footnotes |
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Supported by grants from the National Health and Medical Research Council, the Orthopaedic Research and Education Fund and the Medical Research Fund of Western Australia (to M. H. Z.).
Drs. Huang and Xu contributed equally to this work.
Accepted for publication November 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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but not IL-1 and IL-6 modifies the susceptibility of human osteosarcoma cells to NK lysis. Int J Oncol 1998, 13:349-353[Medline]
and basic fibroblast growth factor induction of matrix metalloproteinases and their inhibitors in osteosarcoma cells is modulated by the metastasis associated protein CAPL. Anticancer Res 1998, 18:3299-3303[Medline]
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