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Keywords:

  • giant cell tumor;
  • ODF;
  • TRAIL;
  • OPG;
  • bone tumor

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

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-α. 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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Giant cell tumors (GCT), otherwise known as osteoclastomas, are rare primary neoplasms of the skeleton, associated with extensive localized bone loss. GCT contain within the tumor mass variable numbers of large, multinucleated cells that are phenotypically indistinguishable from osteoclasts,(1) the cell type involved in bone resorption during normal homeostasis of the skeleton. It is believed that the stromal cells are the tumor cells that induce osteoclastic bone resorption by recruiting osteoclast precursors and promoting their differentiation into functional osteoclasts.(2) The mechanisms by which they do so are not understood. Recently, stromal cells of GCT have been shown to release chemokines such as macrophage chemoattractant protein 1(3) and interleukin-8,(4) which could recruit precursors of the monocytic lineage into the stromal mass of the tumor, before their development into mature osteoclasts.

Considerable progress has been made toward an understanding of the mechanisms responsible for physiological osteoclastogenesis. A large number of hormones and cytokines have been identified that can exert direct and indirect stimulatory and antagonistic effects on the development of osteoclasts from hematopoietic precursors.(5) Principal among these are interleukin (IL) −1, −6, −11, −17, and −18; macrophage colony stimulating factor (M-CSF); PTHrP; and TNFα, as well as prostaglandin E2 (PGE2).

Most recently, a cell-surface TNF-ligand family member, termed osteoclast differentiation factor (ODF), previously identified as TRANCE(6) and RANKL,(7) was shown to be central to osteoclast development.(8) An engineered soluble form of ODF was shown to promote osteoclast development when added to peripheral blood mononuclear cells (PBMC) in stromal-free cultures, with the obligatory presence of M-CSF.(8) ODF binds to a previously characterized TNF receptor superfamily member, RANK,(9) on osteoclast precursors, and ligation with ODF has recently been shown to activate NF-κB by TNF-associated factors (TRAF) 2, 5, and 6.(10) A soluble TNF-receptor family member termed osteo-protegerin (OPG), which can inhibit osteoclast formation and bone resorption, was shown to be an ODF antagonist.(11) Overexpression of OPG in mice resulted in a muted osteopetrotic phenotype, with reduced cancellous and increased cortical bone, but normal elongation and development of the growth plate.(11) Conversely, OPG-knockout mice developed extensive osteoporosis associated with an increased number of otherwise normal osteoclasts.(12) Likewise, deletion of the ODF gene resulted in severe osteopetrosis and a complete lack of osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis.(13)

Another recently described member of the TNF-ligand superfamily, TRAIL/Apo2L,(14) has been shown to play a role in triggering apoptosis of cells bearing at least one of two receptors, DR4 and DR5. Two other receptors for TRAIL/Apo2L, DcR1 and DcR2, act as decoy receptors, protecting cells from apoptosis that also express one or more of DcR1 and DcR2.(15) TRAIL has also been shown to bind to OPG, reversing its inhibitory effect on osteoclastogenesis. Conversely, OPG is capable of inhibiting the apoptotic effect of TRAIL.(16) A role for TRAIL in physiological or pathological bone resorption has not been reported.

In light of these recently discovered molecules, it is plausible that their net local concentrations act to target osteoclast formation in the microenvironment of the bone, and that their relative concentrations, in turn, are regulated by circulating and local regulators of bone resorption, such as PGE2 and 1α,25(OH)2 vitamin D3.(17) In solid lytic tumors of the bone, the cytokines and growth factors produced by the tumor cells may disrupt the normal homeostatic balance of factors in the bone microenvironment, leading to increased local osteoclastogenesis and disregulation of osteoclastic activity. Alternatively, the GCT tumor cells themselves may express ODF and so directly support osteoclast formation.

We hypothesized that the stromal element of GCT expresses these newly discovered osteoclastogenic molecules, which could account for the presence of osteoclasts within the tumor mass in vivo, and the levels of expression of these positive and negative mediators may account for the degree of bone lysis observed in patients with GCT. We have identified within GCT samples expression of high levels of mRNA encoding ODF, RANK, and TRAIL. In a small series of tumors, we found a relationship between expression of the relative levels of ODF and TRAIL, in terms of the corresponding level of OPG, with the degree of bone lysis by these tumors in vivo. We provide evidence that GCT tumor cells provide all of the necessary differentiation signals required to support osteoclastogenesis consistent with the pathological destruction of bone associated with these tumors.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Tumor classification and isolation

The diagnosis of all tumors was established by biopsy before surgical management, and subsequently confirmed by repeat histology of the resected specimen. The clinical classification and bone lytic activity of all tumors used in this study are shown in Table 1. The giant cell tumor (GCT) samples included tumors representative of highly aggressive, moderately aggressive, and mildly aggressive types, based on clinical assessment and radiological analysis (Table 1). The clinical assessment included time since onset of symptoms, tumor size, extent of bone involvement, and the degree of extension into the soft tissue. The portion of each tumor selected for analysis was isolated from within the tumor mass, but excluded the central necrotic tissue and normal tissue surrounding the tumor.

Processing of tumor samples

Tumor samples were processed within 24 h of surgical removal. After being washed three times in sterile PBS, tissue was dissected in a petri dish using a scalpel blade, then either used directly for isolation of total RNA (see below) or enzymically digested for cell culture. For digestion, tissue was resuspended in 1 ml each of collagenase and dispase (10 mg/ml) and incubated for 1 h at 37°C. After this, the cell suspension was diluted 10-fold in αMEM medium containing 10% v/v FCS, filtered through a 40-μm cell strainer (Becton Dickinson, Franklin Lakes, NJ, U.S.A.), and then washed twice in the same medium. Cells were then either cryopreserved in a 20% solution of DMSO in medium containing 20% FCS or cultured in αMEM medium containing 10% FCS.

Subfractionation of GCT populations

GCT samples were used freshly or cryopreserved GCT samples were thawed and cultured for several days in DMEM medium containing 10% FCS in 75-cm2 flasks. Giant cells appeared in the stromal cell mass during this time. Stromal cells were removed by brief trypsin digestion, achieved by rinsing the cells once in sterile PBS before the addition of 2 ml trypsin solution (0.1% w/v in PBS), preheated to 37°C. After 30 s, most of the mononuclear stromal cells had detached, leaving giant cells and fan-shaped mononuclear cells attached. The trypsin was inactivated by the addition of 8 ml DMEM medium containing 10% FCS, and the stromal cells were then removed and placed into a fresh culture flask, maintaining a high-cell density. The remaining giant cells were subsequently referred to as giant cell–enriched cultures and were processed immediately for isolation of total RNA (see below). After 2–4 days, further giant cells appeared in the replated stromal mass and the preceding process was repeated. The subsequent stromal cell culture did not give rise to further giant cells.

Table Table 1.. Tumor Samples Used in This Study, Showing Clinical Classification, Lytic Properties of Tumor in the Bone, and Assessment of GCT Aggressiveness, as Defined by Criteria Described in the Text
CodeClassificationBone lytic activityAggressiveness
  1. aN.A. = not applicable.

GCT-Agiant cell tumorlytichigh
GCT-Bgiant cell tumorlyticmoderate
GCT-Cgiant cell tumorlytichigh
GCT-Dgiant cell tumorlyticmild
GCT-Egiant cell tumorlyticmoderate
GCT-Fgiant cell tumorlytichigh
GCT-Ggiant cell tumorlytichigh
GCT-Hgiant cell tumorlyticmoderate
NGC-BplasmacytomalyticN.A.a
NGC-Costeogenic sarcomamixed lytic/osteogenicN.A.
NGC-Dextraosseus chondrosarcomanot involving boneN.A.
NGC-FosteoblastomanonlyticN.A.
NGC-Gosteogenic sarcomanonlyticN.A.

Preparation of total RNA

Dissected tumor samples were dissolved in Trizol reagent (Life Technologies, Gaithersburg, MD, U.S.A.) at 2 ml/5 mm2 of original tissue. Freshly digested GCT samples or giant cell-enriched cultures and stromal cultures derived from successive rounds of the brief trypsinization protocol, described earlier, were lysed in 1 ml/1 × 106 cells of Trizol reagent. Total RNA was prepared from the dissolved tissues according to the manufacturer's instructions.

RT-PCR analysis

First-strand complementary DNA (cDNA) was synthesized from 1.6 μg of total RNA from each sample, using a cDNA synthesis kit, according to the manufacturer's instructions (Promega Corp., Madison, WI, U.S.A.). cDNA was then amplified by PCR to generate products corresponding to mRNA encoding human gene products listed in Table 2. The 20-μl amplification mixture contained 1 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Norwalk, CT, U.S.A.), 100 ng each of the 5′ and 3′ primers, 0.2 mM dNTPs (Pharmacia Biotech, Uppsala, Sweden), 1.5 mM MgCl2, 2 μl 10 × reaction buffer, and sterile DEPC-H2O. PCR was performed for 23 cycles for GAPDH and 30–35 cycles for other primer pairs, such that all products could be assayed in the exponential phase of the amplification curve, in a thermal cycler (Corbett Research, Melbourne, Victoria, Australia). After an initial step at 95°C for 9 minutes to activate the polymerase, each cycle consisted of 1 minute of denaturation at 94°C, 1 minute of annealing at the temperatures indicated in Table 2, and 1 minute of extension at 72°C. This was followed by an additional extension step at 72°C for 1 minute. We designed human sequence-specific oligonucleotide primers on the basis of published sequences (Life Technologies). In all cases, the primers were mRNA-specific, in that the recognition sites of the upstream and downstream primers resided in separate exons or at intron/exon boundaries in the genomic sequence. Primer sequences and predicted PCR product sizes are shown in Table 2. Amplification products were resolved by electrophoresis on a 2% w/v agarose gel and poststained with SYBR-1 Green (Molecular Probes, Eugene, OR, U.S.A.). The relative amounts of the PCR products were determined by quantitating the intensity of bands using a FluorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA, U.S.A.). Amplified products corresponding to ODF, OPG, RANK, and TRAIL mRNA are represented as a ratio of the respective PCR product/GAPDH PCR product. To show that there were no false-positive results, PCR reactions were carried out using non-reverse-transcribed RNA, and on reaction mixtures in which no RNA was added. The specificity of the PCR reaction was confirmed by Southern transfer onto a nylon membrane (Hybond-N; Amersham Life Science Inc., Arlington Heights, IL, U.S.A.) and hybridization with digoxigenin (DIG)-labeled internal oligonucleotide probes (Table 1).

TRAP staining and calcitonin receptor autoradiography

Tumor cells cultured on glass coverslips were fixed and stained for tartrate-resistant acid phosphatase (TRAP), using a commercial staining kit (Sigma Chemical Company, Castle Hill, Australia), as recommended, and then counter-stained with methyl green. In some experiments, calcitonin receptor expression was investigated by autoradiography (results not shown). For this, cells on coverslips were incubated with 125I-sCT (2 × 105 cpm/ml; ∼0.2 nM) in the absence (total binding) or presence (nonspecific binding) of excess unlabeled sCT (1 μg/ml) for 1 h at 37°C. The coverslips were then washed and dipped in LM-1 nuclear emulsion for autoradiography (Amersham International, Buckinghamshire, U.K.) and exposed in the dark for 10 days at 4°C, before development.

Table Table 2.. RT-PCR Primers and Conditions for the Specific Amplification of Human mRNA
Target geneSenseaPrimer sequence (5′–3′)Annealing temperatureb (°C)Expected product size (BP)
  1. a S, “sense” primer; AS, “antisense” primer.

  2. b Annealing temperatures were determined empirically using AmpliTaq Gold (Perkin–Elmer) in a gradient thermal cycler (Corbett Research, Australia).

GAPDHSCACTGACACGTTGGCAGTGG60414
 ASCATGGAGAAGGCTGGGGCTC  
CTRSGCAATGCTTTCACTCCTGAGAAA58782
 ASAGTGCATCACGTAATCATATATC  
IL-1αSTTAGCTTCCTGAGCAATGTG60254
 ASGGTCTTCATCTTGGGCAGTC  
IL-1/3SAGGAAGATGCTGGTTCCCTGC60151
 ASCAGTTCAGTGATCGTACAGGTGC  
IL-1RTISAAGATGGTGACTCCCTCCTG62279
 ASGGCTTGTTCTGTAGATACAG  
IL-1RTIISTTACTCGTACACGATGTGG62277
 ASCTCTCTATGTGGGTGTCATTG  
IL-6SATGAACTCCTTCTCCACAAG62546
 ASGTGCCTGCAGCTTCGTCAGCA  
sIL-6RSCCCATGCAGGCACTTACTAC62350/256
 ASGTGGCTCGAGGTATTGTCAG  
IL-11SCACATGAACTGTGTTTGCCGCCTGG62291
 ASTTGTCAGCACACCTGGGAGCTGTAG  
IL-11 RSCAGCCAGATCAGCGGTTTACC62512
 ASCAGGTGCCAGCATCTAGAAAG  
IL-17STAGACTATGGAGAGCCGACC60450
 ASGGCAGAACTGATAATAGTGC  
IL-18SAACATAGGTCATGATCTCTGGACGTA58344
 ASGAAGATTCAAATTGCATCTTAT  
M-CSFSCAGTTGTCAAGGACAGCAC58670
 ASGCTGGAGGATCCCTCGGACTG  
ODFSAATAGAATATCAGAAGATGGCACTC62668
 ASTAAGGAGGGGTTGGAGACCTCG  
OPGSTGCTGTTCCTACAAAGTTTACG62435
 ASCTTTGAGTGCTTTAGTGCGTG  
PTHrPSTGGGCTACTCCGTGTCCCTG58547
 ASGACATTGGTCACACTTGTGTGG  
PTHrP RSGCTCCTCCATGACAAGGGGAAG60949
 ASCATAGAGCAATGGGGGAGACAG  
RANKSCCTACGCACAAGGCGAAGATGC62704
 ASCGTAGACCACGATGATGTCGCC  
TGF-βSCTAGACCCTTTCTCCTCCAGGAGACG62226
 ASGCTGGGGGTCTCCCGGCAAAAGGT  
TNF-αSTCAGATCATCTTCTCGAACC62361
 ASCAGATAGATGGGCTCATACC  
TRAILSACAGCAGTCAGACTCTGACAGG62838
 ASCATGGTCCATGTCTATCAAGTGC  
TRAPSCTGGCTGATGGTGCCACCCCTG65469
 ASCTCTCAGGCTGCAGGCTGAGG  

Identification of resorption pit formation

To assess the extent of lacunar bone resorption by GCT, freshly isolated or freshly thawed cells were plated onto discs of whale dentine and cultured for 4 days in αMEM medium containing 10% FCS. The dentine was then treated with 25% ammonia solution for 15 minutes, ultrasonicated for 5 minutes, followed by 15 minutes in trypsin at 37°C and a final 5 minutes' sonication to remove the adherent cells. The dentine was then dehydrated by passage through graded alcohol solutions from 70 to 100%, then dried under vacuum in a desiccator overnight before being mounted on stubs and carbon-coated for visualization using a Philips XL-20 scanning electron microscope (SEM).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Expression of osteoclast markers

Tumor samples (Table 1) were surgically removed and processed as described earlier. Cells isolated from giant cell tumors were routinely plated onto whale dentine slices and in all cases formed resorption lacunae within 48–72 h of plating. Cultured osteoclastoma cells were examined for the expression of calcitonin receptor (CTR), which is expressed in bone exclusively by osteoclasts. Consistent with previous reports,(18) only multinucleated giant cells within the tumor bound 125I-labeled sCT, whereas the stromal cells did not (not shown). RT-PCR also showed expression of the two predominant isoforms of the human CTR(19) in the giant cell-enriched fraction of segregated tumor cells (Fig. 2). Cultures of dispersed GCT cells, both unfractionated and fractionated, were stained for TRAP. Both giant cells and some mononuclear cells stained strongly positive for TRAP. In many of the stromal cells in unfractionated tumors, punctate staining for TRAP was observed, which may indicate internalization of exogenous TRAP by the stroma (Fig. 1). In addition, strong PCR signals for the osteoclast markers TRAP, carbonic anhydrase-II (CA-II) and cathepsin K, were routinely derived from all GCT samples tested and were predominantly expressed by the giant cell-enriched fractions of segregated tumor samples (Fig. 2).

Expression of hematopoietic cytokine mRNA

Fractionated stromal and giant cell-enriched populations of two GCT samples were tested for expression of a range of hematopoietic cytokines that have been implicated in osteoclastogenesis (Fig. 2). The tumorigenic stromal cells of GCT-E were found to express mRNA encoding a number of osteoclastogenic cytokines, including IL-6, IL-11, IL-17, PTHrP, and TGF-β. In addition, the stromal cells expressed the functional IL-1 receptor type I (IL-1RTI). The stromal cells also expressed mRNA encoding M-CSF, an essential cofactor in osteoclastogenesis.(8) The giant cell-enriched fraction of GCT-E, however, expressed abundant IL-1α and -β, the nonfunctional decoy receptor for IL-1 (IL-1RTII), soluble IL-6 receptor (sIL-6R), TNF-α, GM-CSF, and the inhibitory IL-18. There were some differences in the expression pattern observed between the tumors. For example, the stromal cells of GCT-A expressed abundant mRNA for both forms of IL-1, sIL-6R, IL-11R, IL-18, and TNF-α, in addition to those described for GCT-E (data not shown). We cannot rule out that the expression of the above-mentioned cytokines detected in the giant cell–enriched fractions of GCT may represent minor contamination of this fraction with stromal cells. Because the purpose of this study was to investigate expression of cytokines in the tumorigenic stromal element in particular, we did not attempt to remove completely all of the stromal cells from the strongly adherent giant cells. The continued expression of the cytokine profile, described here by GCT samples grown in vitro for up to 2 weeks, suggests that the stromal element of GCT is capable of providing many of the recognized growth and survival factors for osteoclasts, in the absence of normal osteoblast and bone marrow stroma.

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Figure FIG. 1.. Photomicrographs showing giant cell tumor cells (GCT-A) cultured in vitro for 1 week and stained for TRAP. (A) Shows the unfractionated tumor and (B) shows an enriched population of giant cells after tryptic removal of the stromal cell layer, as described in Materials and Methods.

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Expression of ODF, OPG, RANK, and TRAIL mRNA

ODF has been shown to be a central factor in osteoclast development. We were able to detect abundant ODF mRNA by RT-PCR in unfractionated GCT samples (Fig. 3). To our knowledge, this is the first report of ODF expression in any tumor type. To ascertain which cell type was likely to express which molecule, we fractionated GCT samples into preparations of stromal cells alone and cultures highly enriched for giant cells. As shown for GCT-E in Fig. 4, ODF mRNA was detected in the stromal fraction. ODF mRNA expression was weakly detectable in the giant cell– enriched fraction, although this may be the result of contamination with stromal cells. Similar results were obtained in fractionated populations of GCT-A (data not shown).

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Figure FIG. 2.. Hematopoietic cytokine expression by fractionated subpopulations of tumor stroma and giant cell–enriched (GC-enriched) populations of GCT-E. RT-PCR products were loaded onto 2% agarose gels, electrophoresed, and stained with SYBR-1 Green. PCR products were then visualized using a FluorImager (Molecular Dynamics), as described in Materials and Methods.

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The receptor for ODF on osteoclast precursor cells is a novel member of the TNF receptor superfamily, RANK.(9) RANK mRNA was found to be present in the primary GCT samples (Fig. 3). Subfractionation of the GCT showed that RANK mRNA was exclusively present in the giant cell–enriched fraction (Fig. 4), consistent with a role for ODF/RANK ligation in osteoclast function and survival.

We also examined the GCT for expression of another recently described member of the TNF ligand superfamily, TRAIL/Apo2L.(14) This molecule has been found to play a role in tumor apoptosis.(15) In addition, TRAIL can bind to OPG, reversing its inhibitory effect on osteoclastogenesis. Conversely, OPG is capable of inhibiting the apoptotic effect of TRAIL.(16) We found high-level expression of TRAIL mRNA in all GCT samples tested (Fig. 3), suggesting a functional role for TRAIL, perhaps binding OPG and increasing the available ODF for ligation to RANK on the osteoclast precursors. TRAIL mRNA was detected in both giant cell and stromal fractions of segregated GCT (Fig. 4).

Clinical correlation

It has been proposed that the ratio of ODF:OPG in the bone microenvironment may be an indicator of osteoclastogenic potential. We were therefore interested to compare this ratio between a number of GCT samples and also to compare this parameter with the extent of observed osteolysis associated with particular tumors. In this small series of tumors, clear differences in the expression of ODF, OPG, TRAIL, and RANK mRNA between tumor types were observable. All giant cell tumors examined exhibited high levels of ODF mRNA, which were generally greater than observed with the other tumors examined, including a lytic osteogenic sarcoma, an osteoblastoma, a sclerotic osteogenic sarcoma, and an extraosseus chondrosarcoma (Fig. 3A). A lytic plasmacytoma also expressed appreciable ODF mRNA (Fig. 3A). mRNA encoding TRAIL was also generally abundant in the GCT samples and was less abundant in the non-GCT samples. The lowest levels of TRAIL mRNA were recorded in the chondrosarcoma and the sclerotic osteogenic sarcoma. High levels of both ODF and TRAIL are likely to provide an environment that stimulates resorption, ODF increasing osteoclast differentiation and activation, and TRAIL, perhaps reducing the local effect of OPG; therefore, high levels of either of these relative to OPG could potentially be a marker of tumor aggressiveness. We quantitated the levels of ODF and TRAIL mRNA relative to OPG mRNA and expressed these as their respective ratios (Fig. 3D). As a group, the GCT exhibited higher ratios of ODF:OPG than did the non-GCT tested. Generally, the ratios of TRAIL:OPG were higher for the GCT than the non-GCT. Although larger numbers of tumors will need to be examined to confirm these apparent clinical correlations, these data suggest that levels of these mRNA species may prove useful in tumor identification.

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Figure FIG. 3.. (A) RT-PCR analysis of freshly resected tumor samples for expression of mRNA species encoding GAPDH, ODF, OPG, RANK, and TRAIL. RT-PCR products were loaded onto 2% agarose gels, electrophoresed, and stained with SYBR-1 Green. PCR products were then visualized using a FluorImager (Molecular Dynamics), as described in Materials and Methods. Quantitation of RT-PCR products, shown in (A), expressed as (B) ratios of ODF and OPG mRNAs normalized to the level of GAPDH mRNA, (C) ratios of TRAIL and OPG mRNA normalized to GAPDH mRNA, and (D) ratios of ODF and TRAIL mRNAs relative to the level of OPG mRNA. * represents actual numerical value of relative expression.

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Figure FIG. 4.. (A) Expression of ODF, OPG, RANK, and TRAIL mRNA in the fractionated subpopulations of tumor GCT-E, and (B) quantitation of these with respect to the levels of mRNA encoding the housekeeping gene GAPDH.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

ODF has been shown to be a central factor in osteoclast development, although to date, little information is available regarding its role in pathological bone destruction by tumors. To our knowledge, this is the first report of the expression of ODF in a pathological/oncological context. We examined a number of GCT, as well as other non-GCT of the bone associated with or without bone lysis, for their expression of ODF, RANK, OPG and TRAIL mRNA. We were able to detect abundant ODF mRNA by RT-PCR in all unfractionated GCT samples tested. Fractionation of one GCT sample into stromal and giant cell fractions indicated that ODF mRNA expression is predominant in the stromal element of the GCT (Fig. 4). The stromal elements of GCT, therefore, are equipped with a fundamental requirement for osteoclast development. In a physiological setting, the potency of ODF in its ability to stimulate osteoclast development is probably balanced by the local concentrations of its inhibitor OPG, which is a potent inhibitor of osteoclastogenesis.(11) It has been proposed that the ratio of ODF:OPG in the bone microenvironment may determine the local osteoclastogenic potential. Recently, the levels of ODF mRNA relative to OPG mRNA were shown to correlate with the previously observed osteoclastogenic potential of several osteoblastic cell lines.(20) We were interested to compare this ratio, between a number of human bone tumors (including GCT and lytic and nonlytic non-GCT), with the extent of observed osteolysis in the corresponding individuals. All GCT samples tested showed high ratios of ODF:OPG when compared with other nonlytic tumors (Fig. 3B). The highest ratio of ODF:OPG was found in the stromal fraction of separated GCT, although our data do not rule out that giant cells themselves express ODF mRNA (Fig. 4).

The receptor for ODF on osteoclast precursor cells is a novel member of TNF receptor superfamily, RANK.(7,9) Although mRNA for this molecule does not always correlate with expression of protein at the cell surface,(7) abundant RANK mRNA was found to be expressed in most of the primary GCT samples tested. Subfractionation of the GCT showed that most RANK mRNA was present in the giant cell–enriched fraction, consistent with its presence in the hematopoietic element of the tumor, and with a role for ODF/RANK ligation in osteoclast function and survival.(9) In stroma passaged sufficiently to remove any further giant cells, we could no longer detect mRNA for RANK, suggesting the exclusive expression of RANK mRNA by giant cells in these tumors. Supportive of the above-mentioned concepts, very little RANK mRNA was found in the non-lytic non-GCT examined here, whereas appreciable levels were found in one of two lytic non-GCT.

Another recently cloned TNF-ligand family member, termed TRAIL, was characterized initially for its role in apoptosis.(15) TRAIL has been shown to be an antagonist of OPG and can reverse the inhibitory effect of OPG on osteoclast formation.(16) The expression of TRAIL in the bone and its role in normal osteoclast physiology has not been established. We found high levels of expression of TRAIL mRNA in all GCT samples tested (Fig. 3A), suggesting a functional role for TRAIL, perhaps binding local OPG and increasing the available ODF for ligation to RANK on the osteoclast precursors. In fractionated tumor samples, TRAIL mRNA was clearly expressed by the stromal cells and equally strong signals for TRAIL were detected in the GC-enriched fraction (Fig. 4). The two lytic non-GCT samples examined here expressed intermediate levels of TRAIL mRNA, whereas the two nonlytic GCT expressed the least. It will be of interest to examine the expression of other TRAIL receptors(15) in these samples, to explore a potential apoptotic/survival role for them in these tumors.

In terms of ratios of ODF, TRAIL, and RANK mRNA to OPG mRNA, there was a clear distinction between giant cell tumors and other lytic tumors and tumors not associated with bone destruction. The nonlytic tumors did not express abundant mRNA for ODF, RANK, or TRAIL, but did express abundant mRNA for the ODF inhibitor OPG. The lytic non-GCT had increased levels of mRNA for ODF and TRAIL to OPG mRNA ratios but they remained markedly less than what is observed for GCT. It is possible that the production of these factors in cases of non-GCT is a result of stimulation of normal stroma, whereas in the giant cell tumor it is thought that the stroma is tumorigenic, and is likely to be constitutively producing these mRNA species as a function of the transformed phenotype. It will be of interest to explore the molecular mechanisms responsible for this, particularly the role of potential autocrine factors also produced by the tumor cells, such as IL-1/IL-1RTI and IL-6/sIL-6R. Elevated PTHrP expression has been implicated in the molecular mechanism responsible for the establishment and osteolysis associated with breast(21) and prostate(22) skeletal metastases, and the hypercalcemia of humoral malignancy such as multiple myeloma.(23) Interestingly, however, we observed low levels of PTHrP mRNA in this study, suggesting that PTHrP may not play a major role in GCT.

The expression of ODF by the stromal fraction of GCT is consistent with the current body of evidence that suggests that these cells are mesenchymal in origin and osteoblast-like. Previously, it was shown that the stromal cells could be induced to form mineralized nodules in vitro and formation of bone in SCID mice.(24) It was also shown that the osteoclast-like cells in GCT resorb bone in the presence, but not the absence, of the stromal element of the tumor.(2) This is consistent with the recognized requirement for cell-cell contact of the osteoclast with the osteoblast or stroma for function. We have shown evidence here that the mechanism by which GCT stromal cells induce the bone-resorbing function of giant cells is likely to be by ODF/RANK ligation, consistent with the normal situation of mature osteoblasts and osteoclasts. To date, it has not been shown that GCT stromal cells are capable of supporting osteoclastogenesis from immature precursors, for example, CTR and vitronectin receptor-negative mononuclear cells. However, in addition to ODF, we have shown here the expression of mRNA encoding a wide range of hematopoietic cytokines implicated in osteoclast formation and survival, including IL-1α and -β, IL-1 receptor, IL-6, soluble IL-6 receptor, IL-17, TNF-α, and importantly, M-CSF. Although cytokine mRNA levels do not always correlate with secreted protein, we believe that these data are supportive of the overall osteoclastogenic nature of the GCT.

In summary, these data provide important new insights into the molecular biology of an osteolytic tumor type. The expression of molecules, thought to play a role in normal osteoclastogenesis, in GCT suggests the exciting possibility that they also play a role in pathological bone loss because of the stimulation of inappropriate osteoclastic activity. It will be important to determine the implications of these findings for other destructive primary bone and metastatic tumors.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by grants from the Anti-Cancer Foundation of Australia, The National Health and Medical Research Council of Australia, The Adelaide Bone and Joint Research Foundation, Merck Sharp and Dohme (Australia Pty Ltd Research Foundation), the Royal Adelaide Hospital Research review committee, and Bristol-Myers Squibb/Zimmer. The authors thank Dr. Mark Clayer, M.D., Department of Orthopaedic Surgery, Queen Elizabeth Hospital, Woodville, South Australia, for providing one of the tumor samples examined in this study.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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