ARTICLE |
Correspondence to: Hiroaki Nakamura, Dept. of Oral Morphology, Okayama U. Graduate School of Medicine and Dentistry, Shikata-cho 2-5-1, Okayama 700-8525, Japan. E-mail: nakam@md.okayama-u.ac.jp
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Summary |
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Osteoprotegerin (OPG), a soluble member of the tumor necrosis factor (TNF) receptor family, is an osteoclastogenesis inhibitory factor. We investigated the localization of OPG in rat tibia using a specific peptide antibody to clarify the role of OPG in bone remodeling. OPG reactivity was mainly seen on bone surfaces. In bone matrices, OPG was also localized on cartilage/bone interfaces and cement lines. However, labeling was scarcely detected in the region of contact between osteoclasts and stromal cells. Some osteoblasts and osteocytes showed weak labeling. Immunoreactivity was not seen in chondrocytes or osteoclasts. Immunoelectron microscopic observation revealed that OPG is localized on the bone surfaces under osteoclasts. These findings suggest that OPG derived from osteoblast lineage cells and/or serum may be concentrated on resorbed bone surfaces and subsequently on cement lines. OPG may play an important role in the prevention of excess bone resorption by inhibiting differentiation and activity of osteoclasts in bone remodeling. (J Histochem Cytochem 50:945953, 2002)
Key Words: osteoprotegerin (OPG), osteoblasts, osteoclasts, cement line, bone remodeling, immunohistochemistry
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Introduction |
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BONE VOLUME is maintained through the balance of bone formation by osteoblasts and bone resorption by osteoclasts. Regulation of the balance between osteoblastic and osteoclastic activity is a critical component of normal bone cell biology. Osteoblasts differentiate from mesenchymal stem cells and produce bone matrix. Osteoclasts are multinuclear giant cells that arise from the monocytemacrophage lineage of hematopoietic precursors (B (RANK), expressed in osteoclast lineage cells and RANK ligand (RANKL) produced by osteoblast lineage and stromal cells participates in this cellcell interaction (
Osteoprotegerin (OPG), a soluble member of the tumor necrosis factor (TNF) receptor family, works as a decoy by binding to RANKL and thus inhibits osteoclastogenesis by interrupting RANKL-mediated signaling (
We report here the immunolocalization by light and electron microscopy of OPG in rat tibia using a specific peptide antibody.
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Materials and Methods |
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All animal procedures were in accordance with the Guidelines for Animal Experiments, Okayama University Graduate School of Medicine and Dentistry.
Antibody Against OPG
A cysteine-conjugated peptide corresponding to residues 233247 (NAESVERIKRQHSSQ), which is a hydrophilic region in the death homologous domain of rat OPG (
Protein Extraction, SDS-PAGE, and Western Blotting
Tibias of 4-week-old rats were resected and bone segments of the proximal region were obtained. Enamel organs were also dissected out from lower incisors. Calvaria extracts were obtained from 2-day-old rats. Samples were dissolved in 100 µl of sample buffer containing 4% SDS, 20% glycerol, and 12% mercaptoethanol in 100 mM Tris-HCl (pH 6.8) and heated at 100C. We also used glutathione S-transferase (GST)OPG as a positive control.
SDS-PAGE was carried out with a 12% polyacrylamide gel. Samples were electrophoresed at 150 V for 60 min and were then transferred to a nitrocellulose membrane using 192 mM glycine and 20% methanol in 25 mM Tris-HCl (pH 8.3) at a constant amperage of 50 mA for 60 min. The membrane was immersed in 10% skim milk in 10 mM Tris-buffered saline (TBS) for 30 min to block nonspecific binding and washed with TBS containing 0.05% Tween-20. The membrane was incubated with anti-OPG antibody (1 µg/ml) for 12 hr at 4C, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit Ig G (Sigma; St Louis, MO) for 1 hr at room temperature (RT). Immunoreactivity was visualized using ECL Western blotting detection reagents (Amersham Pharmacia Biotech UK; Poole, UK) according to the manufacturer's instructions.
Preparation of Tissue for Immunohistochemistry
Five 4-week-old Wistar rats were used for IHC. The rats were anesthetized with sodium pentobarbital and perfused through the left ventricle with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.05 M phosphate buffer (pH 7.3). Tibias were resected, immersed in the perfusion fixative for 2 hr at 4C, and then decalcified in 5% EDTA (pH 7.3) for 1 week at 4C. Specimens from each rat were used for light and electron microscopic IHC.
Light Microscopic IHC
Specimens were dehydrated in graded ethanol and embedded in paraffin. One or two blocks obtained from each animal were cut with microtome. Seven-µm-thick sections were dewaxed with xylene and ethanol, immersed in PBS containing 10% bovine serum albumin (BSA) for 15 min, incubated in anti-OPG polyclonal antibody at a concentration of 5 µg/ml for 12 hr at 4C, and finally incubated in HRP-conjugated goat anti-rabbit Ig G diluted 1:100 for 1 hr at RT. After washing with PBS, immunoreactivity was visualized by immersion in a DABH2O2 solution (0.05% diaminobenzidine and 0.01% H2O2 in 0.05 M Tris-HCl buffer, pH 7.6) for 5 min at RT. For tartrate-resistant acid phosphatase (TRAP) detection, specimens were incubated in a mixture of 0.01% naphthol AS-BI phosphate (Sigma), 0.06% fast red violet LB salt (Sigma) and 50 mM L(+)-tartaric acid (Wako; Osaka, Japan) in 0.1 M acetate buffer (pH 5.0) for 30 min at 37C. They were then stained with 1% methyl green, dehydrated, cleared, and observed under a Leitz Vario Orthomat (Leitz Wetzlar; Wetzlar, Germany).
Electron Microscopic IHC
Specimens were dehydrated in graded N,N-dimethylformamide and then embedded in glycol methacrylate (GMA). Polymerization was performed under UV irradiation for 24 hr at 4C. At least one block from each rat was used for electron microscopic immunohistochemistry. Ultrathin GMA sections were cut with an Ultracut E ultramicrotome (ReichertJung; Vienna, Austria) and mounted on nickel grids. Specimens were immersed in PBS containing 10% BSA for 15 min, incubated in anti-OPG antibody at a concentration of 10 µg/ml for 12 hr at 4C, and finally incubated with 10-nm gold-conjugated goat anti-rabbit Ig G (British Biocell International; Cardiff, UK) diluted 1:40 for 1 hr at RT. They were then washed with PBS and distilled water and air-dried. Sections were stained with uranyl acetate and lead citrate or with 1% tannic acid and uranyl acetate. The samples were observed under a Hitachi H-800 transmission electron microscope (TEM) (Hitachi; Tokyo, Japan) at an accelerating voltage of 100 kV.
Controls
Control sections were incubated with preimmune serum or without any primary antibody. IHC procedures were performed in the same manner as for the non-control sections.
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Results |
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Western Blotting Analysis
Western blotting analysis revealed that the OPG peptide antibody reacts with a 90-kD band in tibia and calvaria extracts. On the other hand, two bands, at 55 kD and 90 kD, were seen in enamel organ extract from lower incisors. GSTOPG, used as a positive control, reacted with this antibody and formed a band at 70 kD. This band is consistent with the size expected from GSTOPG (Fig 1).
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Light Microscopic IHC
We performed double labeling of OPG IHC and TRAP enzyme histochemistry to examine the relationship between OPG localization and osteoclasts.
In this report, laminar structures between new bone matrices and old ones are referred to as cement lines. Bone surfaces that have a concave shape and sometimes lead to the bone surface under osteoclasts are referred to as resorbed bone surfaces. In longitudinal sections of the proximal region of tibias from 4-week-old rats, many chondroclasts and osteoclasts were attached to cartilage matrices in the erosion zone, where blood capillaries invade and cartilage is replaced by bone. Very little OPG-positive labeling was detected in this region. OPG immunoreactivity was mainly detected on surfaces of trabecular bone slightly distant from the growth plate in metaphysis (Fig 2A). Many OPG-positive lines represented resorbed bone surfaces (Fig 2B). Weak labeling was seen in some osteoblasts and osteocytes (Fig 2C). However, osteoblast lineage cells did not always show positive labeling. Osteoclasts did not show any positive immunoreactivity for OPG without TRAP staining (Fig 2C).
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In the trabecular bone of epiphyses, many OPG-positive lines were seen on the bone surface and in bone matrices. These lines in bone matrices corresponded to cement lines and cartilage/bone interfaces (Fig 2D).
Chondrocytes in epiphysial growth plate did not show positive immunoreactivity at any stage of differentiation. No reactivity was seen in cartilage matrix in the growth plate.
No specific immunoreactivity was detected in the control sections that were incubated without any primary antibody or with preimmune rabbit serum.
Electron Microscopic IHC
Under electron microscopy, many gold particles were detected under clear zones of osteoclasts (Fig 3) and resorbed bone surfaces. Gold particles were mainly distributed on the material that was slightly electron-dense. Sometimes a few gold particles were seen on the bone surface under ruffled borders. However, particles were scarcely seen in the regions of contact between osteoclasts and stromal cells.
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In bone trabeculae of epiphyses, cartilage/bone interfaces were located between cartilage matrices, which contain thin collagen fibrils and amorphous ground substances, and bone matrices, which were filled with thick collagen fibrils. Cement lines were seen as lines among collagen-rich bone matrices and were only slightly stained with uranyl acetate in GMA sections. Gold particles were concentrated at cartilage/bone interfaces and cement lines (Fig 4).
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Discussion |
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Our peptide antibody for OPG reacted with 90- and 55-kD bands in enamel organ extract by Western blotting. The 55-kD band corresponds to a monomer of OPG purified from human fibroblasts (
The immunoreactivity we observed in osteoblasts and osteocytes is consistent with previous findings concerning OPG mRNA expression and OPG localization in osteoblast lineage cells (2HS-glycoprotein, osteopontin, and albumin, are absorbed in bone matrices (
There is a question of why OPG is bound to resorbed bone surfaces. Because OPG is a heparin-binding protein (
OPG is found as an osteoclastogenesis inhibitory factor that acts as a soluble decoy receptor to neutralize the RANKRANKL interaction. OPG is believed to suppress bone resorption associated with osteoclast development. However, OPG has also been found to lower serum calcium levels in normal animals within 2 hr of administration (
Cement lines are believed to connect newly formed bone to older bone to maintain bone integrity and biomechanical strength (
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Acknowledgments |
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Supported in part by a grant (no. 13671903) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
We wish to thank Dr Noriyuki Nagaoka for technical support.
Received for publication October 16, 2001; accepted February 6, 2002.
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