Osteoclast induction in periodontal tissue during experimental movement of incisors in osteoprotegerin-deficient mice.код для вставкиСкачать
THE ANATOMICAL RECORD 266:218 –225 (2002) DOI 10.1002/ar.10061 Osteoclast Induction in Periodontal Tissue During Experimental Movement of Incisors in Osteoprotegerin-Deficient Mice TAKAHIRO OSHIRO,1,2 AYA SHIOTANI,1,2 YOSHINOBU SHIBASAKI,1 2 AND TAKAHISA SASAKI * 1 Department of Orthodontics, School of Dentistry, Showa University, Tokyo, Japan 2 Department of Oral Histology, School of Dentistry, Showa University, Tokyo, Japan ABSTRACT Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor (TNF) receptor superfamily that negatively regulates osteoclastogenesis. The receptor activator of the NFKB ligand (RANKL) is one of the key regulatory molecules in osteoclast formation and binds to OPG. In this study, it was suggested that OPG and RANKL are involved in alveolar bone remodeling during orthodontic tooth movement. We examined RANKL localization and osteoclast induction in periodontal tissues during experimental movement of incisors in OPG-deficient mice. To produce orthodontic force, an elastic band was inserted between the upper right and left incisors for 2 or 5 days, and the dissected maxillae were examined for cytochemical and immunocytochemical localization of tartrate-resistant acid phosphatase (TRAP), vacuolar-type H⫹-ATPase, and RANKL. Compared to wild-type OPG (⫹/⫹) littermates, TRAP-positive multinucleated cells were markedly induced in the periodontal ligament (PDL) on the compressed side and in the adjacent alveolar bone of OPG-deficient mice. These multinucleated cells exhibited intense vacuolar-type H⫹-ATPase along the ruffled border membranes. Because of accelerated osteoclastic resorption in OPG-deficient mice, alveolar bone was severely destroyed and partially perforated at 2 and 5 days after force application. In both wild-type and OPG-deficient mice, RANKL expression became stronger at 2 and 5 days after force application than before force application. There was no apparent difference in intensity of RANKL expression between OPG (⫹/⫹) littermates and OPGdeficient mice. In both wild-type and OPG-deficient mice, expression of RANKL protein was detected in osteoblasts, fibroblasts, and osteoclasts mostly located in resorption lacunae. These results suggest that during orthodontic tooth movement, RANKL and OPG in the periodontal tissues are important determinants regulating balanced alveolar bone resorption. Anat Rec 266:218 –225, 2002. © 2002 Wiley-Liss, Inc. Key words: osteoprotegerin-deficient mice; osteoprotegerin; receptor activator of NFKB ligand; osteoclast; periodontal tissue; tooth movement Orthodontic tooth movement is mediated by the coupling of bone resorption on the compressed side of the periodontal ligament (PDL) and bone formation on the stretched side of the PDL (Yokoya et al., 1997; Chung et al., 1999; Sato et al., 2000a, b). Differentiation and cellular activities of both osteoblasts and osteoclasts are highly regulated by a wide variety of osteotropic hormones, inflammatory mediators, and growth factors (Suda et al., 1992). An imbalance of the cell functions of osteoblasts and osteoclasts, therefore, results in skeletal abnormalities such as osteopetrosis, osteoporosis, and Paget’s disease (Osier and Marks, 1992; Roodman, 1992). © 2002 WILEY-LISS, INC. During bone remodeling, the differentiation, maturation, and function of osteoclasts are regulated by osteoblast-derived factors. One of these, osteoprotegerin (OPG), is a novel secreted member of the tumor necrosis factor *Correspondence to: Takahisa Sasaki, Department of Oral Histology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Fax: ⫹81-3-3781-0255. E-mail: email@example.com Received 7 August 2001; Accepted 28 December 2001 Published online 00 Month 2002 219 PERIODONTAL TISSUE IN OPG (–/–) MICE (TNF) receptor superfamily that negatively regulates osteoclastogenesis (Simonet et al., 1997; Tsuda et al., 1997; Yasuda et al., 1998a, b). OPG appears to locally and/or systemically inhibit the differentiation of osteoclast precursors into mature osteoclasts, by interrupting the osteoblast– osteoclast precursor interaction (Simonet et al., 1997; Akatsu et al., 1998; Yasuda et al., 1998a, b). OPG also inhibits in vitro osteoclastogenesis elicited through distinct signaling pathways stimulated by 1,25(OH)2D3, PTH, or IL-11 (Simonet et al., 1997; Tsuda et al., 1997; Matsuzaki et al., 1998; Yasuda et al., 1998a). Therefore, OPG inhibits ovariectomy-induced bone loss in rats (Bateman et al., 2000). In contrast, OPG deficiency results in severe osteoporosis in both humans and experimental animals (Bucay et al., 1998; Mizuno et al., 1998; Yano et al., 1999). Another osteoblast-derived factor, the receptor activator of the NFkB ligand (RANKL), has been identified as a member of the membrane-associated TNF ligand family and an important regulatory molecule of osteoclastogenesis (Lacey et al., 1998; Yasuda et al., 1998b; Matsuzaki et al., 1998; Tsukii et al., 1998; Fuller et al., 1998; Jimi et al., 1999; Takami et al., 1999; Udagawa et al., 1999). RANKL was found to induce osteoclast differentiation from hemopoietic precursors and stimulate their bone resorptive activity (Lacey et al., 1998; Matsuzaki et al., 1998; Yasuda et al., 1998b; Tsukii et al., 1998; Fuller et al., 1998; Jimi et al., 1999; Takami et al., 1999; Udagawa et al., 1999). RANKL is a ligand of OPG and is expressed on the plasma membrane of osteoblasts/stromal cells (Lacey et al., 1998; Tsukii et al., 1998; Yasuda et al., 1998a, b). OPG is a soluble decoy receptor for RANKL, and its inhibition of osteoclast differentiation is due to direct binding to a ligand for OPG expressed on osteoblasts/stromal cells (Matsuzaki et al., 1998; Yasuda et al., 1998a, b; Udagawa et al., 1999, 2000). The resorptive activity of osteoclasts induced by soluble RANKL or osteoblasts is completely inhibited by the simultaneous addition of soluble OPG (Udagawa et al., 1999). Therefore, osteoclast differentiation and function are thought to be regulated by the counterbalancing influences of RANKL and OPG. We recently localized RANKL expression in PDL tissue during orthodontic tooth movement (Shiotani et al., 2001). However, the involvement of RANKL and OPG in PDL tissue during orthodontic tooth movement has not yet been examined. In that regard, OPG-deficient mice provide a useful animal model for osteoporosis without other abnormalities (Bucay et al., 1998; Mizuno et al., 1998). From this study of OPG-deficient mice, we report immunocytochemical evidence suggesting that OPG is an important negative regulator of osteoclast induction in periodontal tissues during experimental movement of incisors. MATERIALS AND METHODS Animal Use Protocol and Experiments Throughout the experiments, the animals were maintained following the principles of laboratory animal care established by the NIH. The animal use protocol was reviewed, and all experiments were conducted according to the animal experimental guide approved by the Animal Experiment Committee, Showa University. Eight-week-old OCIF/JcL OPG (–/–) mice and their wild-type OPG (⫹/⫹) littermates (Saitama Experimental Animals Supply Co. Ltd., Saitama, Japan) (Mizuno et al., 1998) were used in the experiment. An elastic band (a stretched piece of rubber) was set between the right and left incisors in the maxilla of OPG-deficient and wild-type mice for 2 or 5 days. To maintain the elastic band between the incisors, both of its ends were fixed with resin. As baseline controls, both wild-type and OPG-deficient mice before elastic band insertion were used as day 0 specimens. After tooth movement, and after the mice were killed with an overdose with ethyl ether anesthesia, the mice were fixed by intracardiac perfusion with either a mixture of 4% formaldehyde and 0.1% glutaraldehyde in 0.M sodium cacodylate buffer (pH 7.3) for immunohistochemistry, or 2.5% glutaraldehyde in the same buffer for ultrastructural observation. The dissected maxillae, including incisors, were decalcified in 10% EDTA solution for 4 weeks. Histological, Cytochemical, and Immunohistochemical Examinations Decalcified bone tissues were routinely embedded in paraffin. Sections were stained with hematoxylin and eosin. The other sections were stained for tartrate-resistant acid phosphatase (TRAP) as described previously (Udagawa et al., 1999, 2000) and examined with an Olympus VANOX light microscope (Tokyo, Japan). Other decalcified bone tissues were embedded in LR white resin (London Resin, Basingstoke, UK), which was polymerized at –20°C under ultraviolet rays. Ultrathin sections mounted on Formvar-coated nickel grids were first treated with 10% bovine serum albumin (BSA) in 0.01 M phosphate-buffered saline (PBS) for 1 hr to block nonspecific binding of antibody. The sections were then incubated with rabbit antiserum raised against either RANKL (donated by Dr. M. Gillespie, St. Vincent’s Institute of Medical Research, Victoria, Australia) (Kartsogiannis et al., 1999) diluted 1:100 with 1% BSA/PBS or vacuolar-type H⫹-ATPase (donated by Dr. Moriyama, Hiroshima University, Hiroshima, Japan) diluted 1:500 with 1% BSA/ PBS, overnight at 4°C. After incubation, the sections were rinsed with PBS and incubated with goat anti-rabbit IgG, conjugated with 10 nm colloidal gold particles (BioCell Research Laboratories, Cardiff, UK) diluted 1:100 with PBS for 1 hr at room temperature. After rinsing with PBS and distilled water, the sections were stained with 2% uranyl acetate. For light microscopy, decalcified bone tissues were embedded in paraffin, and sections were processed for RANKL localization by the biotin-streptavidinhorseradish peroxidase method, using a Histofine SAB-PO kit (Nichirei Co. Ltd., Tokyo, Japan). The sections were incubated for 2 hr with the primary antibody at room temperature. RESULTS In our previous experiment (Yokoya et al., 1997) on movement of rat molars induced by elastic band insertion, we showed that the number of osteoclasts steadily increased on the compressed side of the PDL from 1 to 7 days after force application. At 4 days after elastic band insertion, many osteoclasts, which were immunostained for vacuolar-type H⫹-ATPase, appeared along the alveolar bone surfaces on the compressed side of the PDL (Yokoya et al., 1997). We therefore examined PDL tissues on the compressed side on days 0, 2, and 5 after elastic band insertion in both wild-type and OPG-deficient mice. In wild-type mice, the alveolar bone surfaces facing the compressed side of the PDL on both days 0 and 2 were very 220 OSHIRO ET AL. smooth and osteoclasts were seldom observed (Fig. 1a and b). The mean number of osteoclasts per unit length (1 mm) of alveolar bone surfaces at the compressed side was only 1.15 ⫾ 1.62 (SD) on day 0, and 3.55 ⫾ 1.95 (SD) on day 2. On day 5, osteoclasts were increased in number (8.62 ⫾ 3.24) along the alveolar bone surfaces (Fig. 1c). On the other hand, in OPG-deficient mice, even on day 0, many osteoclasts (5.76 ⫾ 1.49) were already observed not only along the alveolar bone surfaces but also within wide vascular canals of bone (Fig. 2a). On day 2, the osteoclast number increased to 8.20 ⫾ 1.95 (SD), and due to accelerated osteoclastic bone resorption alveolar bone structures were markedly destroyed, exhibiting irregular bone surfaces and enlarged vascular canals and medullary cavities (Fig. 2b). On day 5, the osteoclast number further increased to 10.66 ⫾ 5.70 (SD). Although osteoclastic resorption was less prominent than on day 2, alveolar bone had become very thin, and in some cases completely perforated by PDL tissue (Fig. 2c). Thus, osteoclasts in OPGdeficient mice on days 0, 2, and 5 appeared to be markedly increased compared to those in wild-type mice. We then examined the cytochemical characteristics of these osteoclasts. In both wild-type and OPG-deficient mice, osteoclasts located along the alveolar bone surfaces were strongly stained for TRAP (Fig. 3). Ultrastructurally, these osteoclasts exhibited well-developed ruffled borders, consisting of deep and regular membrane infoldings toward the cytoplasm, and accumulation of many pale vacuoles in the cytoplasm proximal to the ruffled borders (Fig. 4). Immunoelectron microscopic localization of vacuolartype H⫹-ATPase demonstrated deposition of many immunogold particles, mainly along the limiting membranes of pale vacuoles and along the ruffled border membranes of these osteoclasts (Fig. 4). We further examined RANKL expression in the PDL on the compressed side in both wild-type and OPG-deficient mice. RANKL expression was hardly observed in the PDL on day 0 in wild-type mice (Fig. 5a). However, on day 2, RANKL immunostaining was clearly observed in osteoblasts facing the alveolar bone surfaces and in some PDL fibroblasts (Fig. 5b). On day 5, in addition to osteoblasts and PDL fibroblasts, RANKL immunostaining was observed in multinucleated osteoclasts located in the resorption lacunae of alveolar bone (Fig. 5c). On the other hand, in OPG-deficient mice, even on day 0, RANKL expression was detected in osteoblasts facing the alveolar bone surfaces and in some PDL fibroblasts (Fig. 6a). On both days 2 and 5, RANKL immunostaining was clearly observed in osteoblasts facing the alveolar bone surfaces, PDL fibroblasts, and multinucleated osteoclasts (Fig. 6b and c). There was no apparent difference in the intensity of RANKL immunostaining between wild-type and OPG-deficient mice on days 2 and 5 (Figs. 5b and c, and 6b and c). In immunoelectron microscopic examination of both wild-type and OPG-deficient mice, deposition of immunogold particles for RANKL localization was mainly observed in the cytoplasm and cisterns of the rough-surfaced endoplasmic reticulum (RER) of osteoblasts (Fig. 7). Some immunogold particles were observed along the plasma membrane of these osteoblasts/stromal cells. Similar subcellular localization of RANKL was also observed in PDL fibroblasts (data not shown). RANKL localization in osteoclasts was detected as deposition of immunogold particles in the cytoplasm and along the ruffled border membranes (Fig. 8). In these immunocytochemical examinations, neg- ative control sections showed that replacement of the primary antibody with nonimmune normal rabbit serum resulted in sparse immunoreaction throughout the tissue sections (data not shown). DISCUSSION This study showed that, in OPG-deficient mice, OPG and RANKL in the periodontal tissue are involved in alveolar bone remodeling during experimental tooth movement. OPG, RANK, and their ligand, RANKL, coordinate in regulating bone density and structure by the well-balanced modulation of osteoclast differentiation from hematopoietic precursors. The soluble form of OPG is reported to capture and bind their cognate ligand, RANKL, and prevent these ligands from activating their target cells, osteoclasts. O’Brien et al. (2001) reported that recombinant human OPG caused osteoclasts to detach from the bone surfaces and attach to the adjacent periosteum. OPG transgenic mice show a marked decrease in mature osteoclasts, but not in the number of osteoclast precursors (Simonet et al., 1997). These results suggest that OPG affects the later unknown stage of osteoclast differentiation. In this regards, Akatsu et al. (1998) reported that OPG inhibited the survival of osteoclasts formed in mouse marrow cultures in a dose- and timedependent manner, and suggested that OPG affected the osteoclast number by promoting apoptosis. Murakami et al. (1998) also reported that apoptosis of osteoclasts was mediated by up-regulation of OPG, and that this phenomenon was induced by TGF-␤1. Our results indicate that OPG is a key negative regulator of osteoclastogenesis in PDL tissue during tooth movement. We previously localized RANKL expression in PDL fibroblasts and osteoblasts on the compressed side of the PDL (Shiotani et al., 2001). Because cell-to-cell interaction between osteoblasts/stromal cells and osteoclast precursors is essential for osteoclast formation (Lacey et al., 1998; Matsuzaki et al., 1998; Tsukii et al., 1998), our observation suggests that these osteoblasts/stromal cells and fibroblasts in PDL are involved in supporting osteoclast differentiation during tooth movement. Therefore, bone remodeling during orthodontic tooth movement is thought to be regulated by OPG, RANK, and RANKL to maintain well-balanced alveolar bone structure and bone mass. In our results, RANKL expression was increased at 2 and 5 days after force application compared to that on day 0 in both wild-type and OPG-deficient mice. This suggests that osteoclast differentiation is critically regulated by RANKL, which is produced as a local factor by osteoblasts/ stromal cells in response to mechanical stress such as tooth movement, as well as osteotropic factors. Interestingly, except for that on day 0, there was no difference in the intensity of RANKL expression between wild-type and OPG-deficient mice. Consistent with our results, Udagawa et al. (2000) reported that OPG deficiency did not affect RANKL mRNA levels expressed by osteoblasts from wild-type, heterozygous, and OPG-deficient mice that showed an equivalent level. In addition, RANKL mRNA levels in osteoblasts were similarly up-regulated by treatment with 1,25(OH)2D3 in each of the three OPG genetic backgrounds (Udagawa et al., 2000). RANKL expression on day 0 in OPG-deficient mice may be closely related to osteoclast induction. PERIODONTAL TISSUE IN OPG (–/–) MICE Fig. 1. a– c: Light micrographs of PDL tissue on the compressed side in wild-type mice on days (a) 0, (b) 2, and (c) 5 after force application. A few osteoclasts (arrowheads) are seen in the PDL proper on day 5. AB: alveolar bone, PDL: periodontal ligament, De: dentine. ⫻330. Fig. 2. a– c: Light micrographs of PDL tissue on the compressed side 221 in OPG-deficient mice on days (a) 0, (b) 2, and (c) 5 after force application. Osteoclasts (arrowheads) are seen in the PDL proper and within alveolar bone on days 0, 2, and 5. On days 2 and 5, alveolar bone is prominently perforated. AB: alveolar bone, PDL: periodontal ligament, De: dentine. ⫻330. 222 OSHIRO ET AL. Fig. 3. Intense TRAP staining of osteoclasts (arrowheads) in an OPG-deficient mouse on day 2. AB: alveolar bone. ⫻330. Fig. 4. Immunoelectron micrograph of vacuolar-type H⫹-ATPase in an osteoclast in an OPG-deficient mouse on day 2. Immunogold particles are deposited along the ruffled border membranes. ⫻37,500. OPG was found in the conditioned medium of human embryonic lung fibroblasts, 1MR-90 (Tsuda et al., 1997). In addition, mRNA of the TNF receptor, TR1 (identical to OPG), was abundantly expressed in primary osteoclasts, osteogenic sarcoma cell lines, and primary fibroblasts (Kwon et al., 1998). Transforming growth factor (TGF)-␤1 increased OPG mRNA level and the secretion of OPG protein in primary osteoblasts and osteoblastic MC3T3-1 and ST2 cell lines (Murakami et al., 1998; Takai et al., 1998). TGF-␤1 markedly increased the steady-state OPG mRNA level in a dose-dependent manner, but suppressed RANKL mRNA expression and inhibited the formation of TRAP-positive osteoclast-like cells in the presence of 1,25(OH)2D3 (Takai et al., 1998). In this regards, expression of TGF-␤1 mRNA and protein was increased in osteoblasts on the stretched side of the PDL during experimental tooth movement (Nagai et al., 1999). They also localized type-II receptors for TGF-␤1 in osteoclasts on the compressed side of the PDL. These results suggest that: 1) TGF-␤1 negatively regulates osteoclastogenesis through OPG induction by osteoblasts/stromal cells, and 2) OPG and RANKL in the local microenvironment are important determinants regulating balanced and site-specific osteoclastic bone resorption during orthodontic tooth movement. In other words, during tooth movement, stimulation of osteoclastic bone resorption on the compressed side of the PDL and inhibition of resorption on the stretched side are thought to be regulated by RANKL and OPG, respectively. Osteoblasts/stromal cells in the PDL tissue are thought to play important regulatory roles to cause sitespecific bone resorption in orthodontic tooth movement. It is also interesting that RANKL is expressed in osteoclasts, particularly in the ruffled border membranes. Because RANKL mRNA was detected in osteoclasts (Kartsogiannis et al., 1999), RANKL is thought to be produced in osteoclasts rather than by osteoclastic incorporation of soluble RANKL proteins, which are secreted by osteoblasts/stromal cells in a paracrine manner. Our ultrastructural localization of RANKL in osteoclasts suggests that RANKL has regulatory function(s) associated with resorptive function at the ruffled border membranes. In association with these observations, RANKL was reported to induce increased pseudopodial motility, associated with increased cell spreading in osteoclasts, and to stimulate resorption lacuna formation on cocultured bone slices (Fuller et al., 1998). In addition, soluble RANKL was reported to prolong the survival of cultured osteoclasts in a dose-dependent manner and enhance their resorptive activity in cocultured dentine slices (Fuller et al., 1998; Udagawa et al., 1999; Jimi et al., 1999). It can be concluded from these results that, in addition to the regulation of osteoclastogenesis by osteoblastic RANKL, RANKL PERIODONTAL TISSUE IN OPG (–/–) MICE Fig. 5. a– c: RANKL immunostaining in PDL tissue on the compressed side in wild-type mice on days (a) 0, (b) 2, and (c) 5 after force application. RANKL is expressed in osteoblasts, fibroblasts, and osteoclasts. AB: alveolar bone, PDL: periodontal ligament, De: dentine. ⫻330. 223 Fig. 6. a– c: RANKL immunostaining in PDL tissue on the compressed side in OPG-deficient mice on days (a) 0, (b) 2, and (c) 5 after force application. RANKL is expressed in osteoblasts, fibroblasts, and osteoclasts. AB: alveolar bone, PDL: periodontal ligament, De: dentine. ⫻330. 224 OSHIRO ET AL. Fig. 7. Immunoelectron micrograph showing RANKL localization in an osteoblast. Immunogold particle deposition is seen in RER cisterns and throughout the cytoplasm. ⫻37,500. Fig. 8. Immunoelectron micrographs showing RANKL localization in osteoclasts. Immunogold particles are deposited in the ruffled border. ⫻37,500. PERIODONTAL TISSUE IN OPG (–/–) MICE expressed in osteoclasts may have self-regulatory roles in their resorptive activity and survival. LITERATURE CITED Akatsu T, Murakami T, Nishikawa M, Ono K, Shinomiya N, Tsuda E, Mochizuki S, Yamaguchi K, Kinosaki M, Higashio K, Yamamoto M, Motoyoshi K, Nagata N. 1998. Osteoclastogenesis inhibitory factor suppresses osteoclast survival by interfering in the interaction of stromal cells with osteoclasts. Biochem Biophys Res Commun 250: 229 –234. Bateman TA, Dunstan CR, Ferguson VL, Lacey DL, Ayers RA, Simske SJ. 2000. Osteoprotegerin mitigates tail suspension-induced osteopenia. Bone 26:443– 449. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS. 1998. Osteoprotegerin-deficient mice develop early onset osteopororsis and arterial calcification. Genes Devel 12:1260 –1268. Chung HS, Sasaki T, Sato Y, Shibasaki Y. 1999. H⫹-ATPase inhibitor, bafilomycin A1, reduces bone resorption during experimental movement of rat molars. Orthodont Waves 58:183–192. Fuller K, Wong B, Fox S, Choi Y, Chambers TJ. 1998. TRANCE is necessary and sufficient for osteoclast-mediated activation of bone resorption in osteoclasts. J Exp Med 188:997–1001. Jimi E, Akiyama S, Tsurukai T, Okahashi N, Kobayashi K, Udagawa N, Nishimura T, Takahashi N, Suda T. 1999. Osteoclast differentiation factor act as a multifunctional regulator in murine osteoclast differentiation and function. J Immunol 163:434 – 442. Kartsogiannis V, Zhou H, Horwood NJ, Thomas RJ, Hards DK, Quinn JMW, Niforas P, Ng KW, Martin TJ, Gillespie MT. 1999. Localization of RANKL (receptor activator of NFKB ligand) mRNA and protein in skeletal and extraskeletal tissues. Bone 25:525–534. Kwon BS, Wang S, Udagawa N, Haridas V, Lee ZH, Kim KK, Oh K, Greene J, Li Y, Su J, Gentz R, Aggarwal BB, Ni J. 1998. TR1, a new member of the tumor necrosis factor receptor superfamily, induces fibroblast proliferation and inhibits osteoclastogenesis and bone resorption. FASEB J 12:845– 854. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Hsu H, Sullivan J, Hakins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senalddi G, Guo J, Delaney J, Boyle WJ. 1998. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176. Matsuzaki K, Udagawa N, Takahashi N, Yamaguchi K, Yasuda H, Shima N, Moronaga T, Toyama Y, Yabe Y, Higashio K, Suda T. 1998. Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures. Biochem Biophys Res Commun 246:199 –204. Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, Sato Y, Nakagawa N, Yasuda H, Mochizuki S, Gomibuchi T, Yano K, Shima N, Washida N, Tsuda E, Morinaga T, Higashio K, Ozawa H. 1998. Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem Biophys Res Commun 247:610–615. Murakami T, Yamamoto M, Yamamoto M, Ono K, Nishikawa M, Nagata N, Motoyoshi K, Akatsu T. 1998. Transforming growth factor-␤1 increases mRNA levels of osteoclastogenesis inhibitory factor in osteoblastic/stromal cells and inhibits the survival of murine osteoclast-like cells. Biochem Biophys Res Commun 252:747– 752. Nagai M, Yoshida A, Sato N, Wong DTW. 1999. Messenger RNA level and protein localization of transforming growth factor-␤1 in experimental tooth movement in rats. Eur J Oral Sci 107:475– 481. O’Brien EA, Williams JHH, Marshall MJ. 2001. Osteoprotegerin is produced when prostaglandin synthesis is inhibited causing osteoclasts to detach from the surface of mouse parietal bone and attach to the endocranial membrane. Bone 28:208 –214. Osier LK, Marks SC. 1992. Osteopetrosis. In: Rifkin BR, Gay CV, editors. Biology and physiology of the osteoclasts. Boca Raton: CRC Press. p 433– 453. Roodman GD. 1992. The osteoclast in Paget’s disease of bone. In: Rifkin BR, Gay CV, editors. Biology and physiology of the osteoclasts. Boca Raton: CRC Press. p 455– 473. 225 Sato Y, Shibasaki Y, Sasaki T. 2000a. Effects of bisphosphonate administration on root and bone resorption during experimental movement of rat molars. In: Davidovitch Z, Mah J, editors. Biological mechanisms of tooth movement and craniofacial adaptation. Birmingham: EBSCO Media. p 243–252. Sato Y, Sakai H, Kobayashi Y, Shibasaki Y, Sasaki T. 2000b. Bisphosphonate administration alters subcellular localization of vacuolartype H⫹-ATPase and cathepsin K in osteoclasts during experimental movement of rat molars. Anat Rec 260:72– 80. Shiotani A, Shibasaki Y, Sasaki T. 2001. Localization of receptor activator of NFKB ligand, RANKL, in periodontal tissues during experimental movement of rat molars. J Electron Microsc 50:365–369. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Amgen EST Program, Boyle WJ. 1997. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309 –319. Suda T, Takahashi N, Martin TJ. 1992. Modulation of osteoclast differentiation. Endocrinol Rev 13:66 –79. Takai H, Kanematsu M, Yano K, Tsuda E, Higashio K, Ikeda K, Watanabe K, Yamada Y. 1998. Transforming growth factor-␤stimulates the production of osteoprotegerin/osteoclastogenesis inhibitory factor by bone marrow stromal cells. J Biol Chem 273:27091– 27096. Takami M, Woo JT, Nagai K. 1999. Osteoblastic cells induce fusion and activation of osteoclasts through a mechanism independent of macrophage-colony-stimulating factor production. Cell Tissue Res 298:327–334. Tsuda E, Goto M, Mochizuki S, Yano K, Kobayashi F, Morinaga T, Higashio K. 1997. Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochem Biophys Res Commun 234:137–142. Tsukii K, Shima N, Mochizuki N, Yamaguchi K, Kionosaki M, Yano K, Shibata O, Udagawa N, Yasuda H, Suda T, Higashio K. 1998. Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1␣, 25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid hormone in the microenvironment of bone. Biochem Biophys Res Commun 246:337–341. Udagawa N, Takahashi N, Jimi E, Matsuzaki K, Tsurukai T, Itoh K, Nakagawa N, Yasuda H, Goto M, Tsuda E, Higashio K, Gillespie MT, Martin TJ, Suda T. 1999. Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation facor/RANKL but not macrophage colony-stimulating factor. Bone 25:517–523. Udagawa N, Takahashi N, Yasuda H, Mizuno A, Itoh K, Ueno Y, Shinki T, Gillespie MT, Martin TJ, Higashio K, Suda T. 2000. Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function. Endocrinology 141:3478 – 3484. Yano K, Tsuda E, Washida N, Kobayashi F, Goto M, Harada A, Ikeda K, Higashio K, Yamada Y. 1999. Immunological characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor: increased serum concentrations in postmenopausal women with osteoporosis. J Bone Miner Res 14:518 –527. Yasuda H, Shima N, Nakagawa N, Mochizuki S, Yano K, Fujise N, Sato Y, Goto M, Yamaguchi K, Kuriyama M, Kanno T, Murakami A, Tsuda E, Morinaga T, Higashio K. 1998a. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 139:1329 –1337. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda A, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T. 1998b. Osteoclast differentiation factor is a ligand for osteoprotegerin/ osteoclastogenesis-inhibitory factor and is identical to TRANCE/ RANKL. Proc Natl Acad Sci USA 95:3597–3602. Yokoya K, Sasaki T, Shibasaki Y. 1997. Distributional changes of osteoclasts and preosteoclastic cells in periodontal tissues during experimental tooth movement as revealed by quantitative immunohistochemistry of H⫹-ATPase. J Dent Res 76:580 –587.