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RESEARCH REPORT |
1 Dept. of Oral and Maxillofacial Surgery, National Dental Centre, 5 Second Hospital Avenue, S168938, Singapore;
2 Dept. of Anatomy, National University of Singapore;
3 Dept. of Oral and Maxillofacial Surgery, National University of Singapore;
4 Dept. of Oral and Maxillofacial Surgery, St Bartholomew’s and the Royal London School of Medicine and Dentistry, United Kingdom; and
5 Eastman Institute of Oral Science, UK;
* corresponding author, juliet_tay{at}yahoo.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B ligand) promotes osteoclast differentiation, stimulates osteoclast activity, and prolongs osteoclast survival and adherence to bone. Abnormalities of the RANKL/RANK/osteoprotegerin system have been implicated in a range of diseases, including osteoporosis. To date, no work has been done in osteolytic lesions of the facial skeleton. In this study, specimens of ameloblastomas, dentigerous cysts, odontogenic keratocysts, and radicular cysts were subjected to immunohistochemical analysis for RANKL and tartrate-resistant acid phosphatase (TRAP). Immunofluorescence staining for TRAP was visualized under confocal microscopy. All specimens demonstrated distinct positive immunoreactivity to RANKL and TRAP. The TRAP-positive cells also stained with in situ hybridization for human calcitonin receptor, a definitive marker for osteoclasts. Mononuclear pre-osteoclasts were observed to migrate from blood to the connective tissue stroma and multinucleate toward the bone surface. It can be concluded that RANKL plays a role in bone resorption in osteolytic lesions of the facial skeleton.
KEY WORDS: RANKL • immunohistochemistry • confocal microscopy • osteoclast • bone resorption
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B ligand (RANKL), a cell-bound receptor, receptor activator of nuclear factor B (RANK), and a secreted decoy receptor, osteoprotegerin (OPG). RANKL is also known as osteoclast differentiation factor (ODF) (Yasuda et al., 1998), OPG ligand (Lacey et al., 1998), and TNF-related activation-induced cytokine (TRANCE) (Wong et al., 1997). It is expressed in 3 forms—a cell-bound peptide (Lacey et al., 1998; Yasuda et al., 1998), a truncated ectodomain created from the cell-bound form by enzymatic cleavage (Lacey et al., 1998), and a primary secreted form (Kong et al., 1999). The cell-bound form is commonly expressed by stromal cells, osteoblasts, mesenchymal periosteal cells, chondrocytes, and endothelial cells (Wong et al., 1997; Anderson et al., 1998; Lacey et al., 1998; Yasuda et al., 1998; Kartsogiannis et al., 1999). The primary secreted form is limited to activated T-cells (Kong et al., 1999) and a squamous cell carcinoma cell line (Nagai et al., 2000). The actions of RANKL include promotion of osteoclast differentiation (Lacey et al., 1998), stimulation of osteoclast activation (Lacey et al., 1998; Yasuda et al., 1998), survival (Fuller et al., 1998), and adherence to bone surface (O’Brien et al., 2000). Some osteoclastogenic cytokines, such as prostaglandin E2 and TGF-ß, may facilitate and cooperate with RANKL-induced osteoclast formation and activation (Sells Galvin et al., 1999; Wani et al., 1999). The effects of RANKL are counteracted by OPG, which acts as a soluble neutralizing receptor.
With the characterization of the RANKL/RANK/OPG cytokine system, several studies have implicated RANKL and OPG as the essential cytokine system that regulates tumor-bone interactions, including lymphomas, multiple myeloma, leukemias, giant cell granulomas, and chondromas, as well as bone metastasis involving breast and prostate cancer (Hofbauer et al., 2001). The aim of this study was to elucidate if osteolytic processes in bone-resorbing cysts and tumors of the facial skeleton are mediated via the RANKL pathway.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemical Staining for RANKL
Five-micron sections were mounted on 3-aminopropyltriethoxy-silane-coated slides. These specimens were de-waxed by being passed through a series of xylol and alcohol baths. To inhibit endogenous peroxidase activity, we treated de-waxed sections with 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 5 min. Non-specific reactivity was blocked with the use of normal goat serum for 30 min. The sections were reacted with mouse monoclonal antibodies against human RANKL/ODF (Sigma, St. Louis, MO, USA) at a dilution of 1:200 overnight at 4°C. The sections were incubated with biotinylated monoclonal anti-mouse IgG (Sigma, St. Louis, MO, USA) diluted in PBS containing 1% bovine serum albumin (BSA) at a concentration of 1:15 for 30 min and ExtraAvidin Perioxidase (Sigma, St. Louis, MO, USA) diluted with PBS containing 1% BSA to a concentration of 1:15 for another 30 min. To visualize the immunoreactant, we flooded the sections with diaminobenzidine solution for peroxidase reactivity (Sigma, St. Louis, MO, USA). Counterstaining was done with hematoxylin for light microscopy. As a negative control, 1% BSA diluted in PBS was used instead of the primary antibody.
Immunohistochemical Staining for TRAP
The procedure was similar to RANKL immunohistochemistry except that we carried out high-temperature antigen unmasking by heating the slides in citrate buffer. The primary antibody used was mouse monoclonal antibody against human TRAP (Novocastra, Newcastle Upon Tyne, UK) at a dilution of 1:50 overnight at 4°C.
Immunofluorescence Staining
We also subjected the specimens to immunofluorescence staining to localize TRAP activity using FITC-conjugated secondary anti-mouse antibody. After being blocked with normal goat serum for 30 min, specimens were incubated with mouse monoclonal antibodies against human TRAP (Novocastra, Newcastle Upon Tyne, UK) at a dilution of 1:50 for 2 hrs at room temperature (RT). FITC-conjugated secondary antibody (Sigma, St. Louis, MO, USA) was then applied for 1 hr at room temperature. Propidium iodide (PI) counterstaining was carried out by incubation of the sections in PI/Triton X-100 staining solution with RNAse for 15 min. The coverslips were mounted on slides with fluorescence mounting media (DAKO, Carpinteria, CA, USA), and sections were viewed under a LSM 510 Carl Zeiss confocal laser scanning microscope.
In situ Hybridization for Human Calcitonin Receptor
We carried out in situ hybridization using antisense oligonucleotides labeled with digoxigenin-dUTP (Boehringer Mannheim). The antisense oligonucleotides specific for human calcitonin receptor (CTR) were synthesized according to the following sequence (Yoshida et al., 2003): 5' ATG GTC GCA ACA AAG AAG CCC TGG AAA TGA ATC AGA GAG T 3'. The oligo-DNA was labeled at their 3' end with Dig-11 dUTP (digoxigenin-labeled deoxyuridine triphosphate) as recommended by Boehringer Mannheim (Germany). The labeling mixture containing 1 M potassium cacodylate, 0.125 M Tris-HCl, 1.25 mg/mL bovine serum albumin (BSA), pH 6.6, 25 mM CoCl2, 1 mM DIG-dUTP, 10 mM dATP, 50 U TdT, and 1 µg of oligo DNA was incubated at 37°C for 15 min. The labeled DNA was extracted by ethanol precipitation with glycogen and 4 M lithium chloride, suspended in sterile water, and stored at -20°C.
In situ hybridization was carried out as described previously (Jayasurya et al., 2003). Formalin-fixed sections (4 µm) were de-waxed in xylene twice and rehydrated in graded series of alcohol and then in DEPC water. Sections were treated with 0.2 N hydrochloric acid (HCl) for 20 min and washed in 0.01% PBS-T (PBS and 0.1% Tween 20). They were rendered permeable with proteinase K (1 µg/mL) at 37°C for 10 min and post-fixed with 4% paraformaldehyde for 5 min. The sections were further treated with glycine (2 mg/mL), washed in PBS, and kept in 40% de-ionized formamide in 4x Sodium Chloride Sodium Citrate (SSC) until used for hybridization. The sections were incubated in prehybridization medium (DAKO, Carpinteria, CA, USA) for 2 hrs at 45°C. Hybridization was carried out at 45°C overnight with 1.5 µg/mL of pre-warmed Dig-oligo-DNA probe dissolved in the hybridization medium. The following day, the sections were washed with high stringency 5x at 45°C in 2x SSC with 0.1% Tween 20 (1 hr each time) and were incubated in a 5% blocking solution (Boehringer Mannheim) prepared in maleic acid buffer (150 mM maleic acid and 0.1 M NaCl in DEPC water) for 1 hr at RT. Polyclonal sheep anti-digoxigenin-alkaline phosphatase antiserum (Boehringer Mannheim) was later applied to the sections at a dilution of 1:1500 in 5% blocking buffer and incubated overnight at 4°C. Sections were washed in maleic acid buffer, followed by alkaline phosphatase buffer (1 M Tris HCl, pH 9.5, 1 M MgCl2, 5 M NaCl). Color was developed with a 1:200 dilution of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) (Boehringer Mannheim) in alkaline phosphatase solution, and sections were visualized without being counterstained. Control sections were incubated with the respective sense oligo DNA probes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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–1E). RANKL immunoreactivity was localized to cells scattered within the connective tissue stroma of the bone-resorbing tumors. The fibro-epithelial polyp which was used as a negative control also demonstrated distinct stained cells to RANKL (Fig. 1F). These stained cells were localized within the epithelial layer and were identified as dendritic cells. In addition to scattered cells within the connective tissue stroma, RANKL immunoreactivity present in the ameloblastoma, dentigerous cyst, odontogenic keratocyst, and radicular cyst specimens was also localized on endothelial cells (Fig. 1G).
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–2E). Immunoreactivity to TRAP, however, was negative in the fibro-epithelial polyp specimens (Fig. 2F). To identify the cells exhibiting positive immunoreactivity to TRAP, we carried out in situ hybridization using oligonucleotides specific for human calcitonin receptor (CTR). These cells stained positively for CTR, confirming their identity as osteoclasts (Figs. 2I, 2J).
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). Immunofluorescence of FITC-conjugated secondary antibody bound to TRAP showed fusion of pre-osteoclasts to form multi-nucleated osteoclasts toward the bone surface of the lesion (Fig. 2H). CTR mRNA was detected in TRAP-positive cells (Fig. 2I), with the negative control being shown in Fig. 2J. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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RANKL is expressed on the surfaces of pre-osteoblastic/stromal cells. Binding to its receptor, RANK on the surfaces of osteoclastic precursor cells results in the activation of a series of second-messenger systems (Hofbauer and Heufelder, 2001), resulting in differentiation, formation of multinucleated cells, and activation and survival of osteoclastic cells. OPG limits this cascade by blocking the effects of RANKL. Pro-resorptive cytokines such as TNF-
and interleukin-1 (IL-1) appear to modulate this system by stimulating M-CSF production, which increases the pool of osteoclastic cells, and also by directly increasing RANKL expression (Wong et al., 1997; Lacey et al., 1998; Hofbauer et al., 1999a). Other cytokines and hormones—such as TGF-ß (which increases OPG production) (Takai et al., 1998), parathyroid hormone (which increases RANKL/decreases OPG production) (Lee and Lorenz, 1999), 1,25 dihydroxyvitamin D3 (which increases RANKL production) (Kitazawa et al., 1999), glucocorticoids (which increases RANKL/decreases OPG production) (Hofbauer et al., 1999b), and estrogen (which increases OPG production) (Hofbauer et al., 1999b)—exert their effects on osteoclastogenesis by regulating osteoblastic/stromal cell production of OPG and RANKL. In addition, calcitonin acts directly on osteoclastic cells, and estrogen has been shown to induce apoptosis of osteoclasts as well as to inhibit osteoclast differentiation by interfering with RANK signaling (Hughes et al., 1996; Shevde et al., 2000; Srivastava et al., 2001).
Inflammatory cytokines, interleukins, prostaglandins (PGs), and TNF- are known to stimulate bone resorption through the up-regulation of RANKL. PGs are known to be synthesized by dental cysts (Kawamoto et al., 2002). Arachidonic acid metabolites and lipoxygenase products have been found in radicular and dentigerous cysts. IL-1, TNF-, and IL-6 are produced by the stellate reticulum cells in ameloblastoma (Horowitz et al., 2001), while both the odontogenic keratocyst and the radicular cyst are known to produce IL-1 and IL-6 (Miyamoto et al., 2001).
It is postulated that inflammatory cytokines produced by the above bone-resorbing lesions cause up-regulation of RANKL expression in osteoblasts and bone stromal cells. RANKL, which is also expressed by endothelial cells of blood vessels, induces recruitment of TRAP-positive precursor cells. Osteoclast progenitors recruited via blood vessels bind to RANKL on stromal cells and differentiate into TRAP-positive pre-osteoclasts (Fuller et al., 1998). These mononuclear precursor cells migrate from the blood to the connective tissue stroma and multinucleate toward the bone surface. The mature osteoclasts then carry out excavation activity on the bone, resulting in bone resorption.
In conclusion, the identification of RANKL as a mediator of osteoclastogenesis in ameloblastomas, dentigerous cysts, odontogenic keratocysts, and radicular cysts opens up a new area for investigations into the biology of these diseases. The fact that RANKL is required for osteoclast development suggests that agents which inhibit its activity may be therapeutic. It is possible that antibodies to RANKL which presently exist for the mouse could be developed for human use. Alternatively, soluble RANK and OPG, which inhibit osteoclast formation by blocking RANKL-RANK interaction, also have the potential to be developed for future therapeutic use.
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ACKNOWLEDGMENTS |
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Received January 21, 2003; Last revision January 16, 2004; Accepted January 29, 2004
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REFERENCES |
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