Biochemical and Biophysical Research Communications 362 (2007) 460–466 www.elsevier.com/locate/ybbrc
Osteoclastic activity induces osteomodulin expression in osteoblasts Ken Ninomiya a,b,1, Takeshi Miyamoto a,b,c,*,1, Jun-ichi Imai e, Nobuyuki Fujita Toru Suzuki a,b, Ryotaro Iwasaki a,d, Mitsuru Yagi a,b, Shinya Watanabe e, Yoshiaki Toyama b, Toshio Suda a,* a
a,b
,
Department of Cell Differentiation, The Sakaguchi Laboratory, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan b Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan c Department of Musculoskeletal Reconstruction and Regeneration Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan d Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan e Department of Clinical Informatics, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan Received 27 July 2007 Available online 13 August 2007
Abstract Bone resorption by osteoclasts stimulates bone formation by osteoblasts. To isolate osteoblastic factors coupled with osteoclast activity, we performed microarray and cluster analysis of 8 tissues including bone, and found that among 10,490 genes, osteomodulin (OMD), an extracellular matrix keratan sulfate proteoglycan, was simultaneously induced with osteoclast-specific markers such as MMP9 and Acp5. OMD expression was detected in osteoblasts and upregulated during osteoblast maturation. OMD expression in osteoblasts was also detected immunohistochemically using a specific antibody against OMD. The immunoreactivity against OMD decreased in op/op mice, which lack functional macrophage colony stimulating factor (M-CSF) and are therefore defective in osteoclast formation, when compared to wild-type littermates. OMD expression in op/op mice was upregulated by M-CSF treatment. Since the M-CSF receptor c-Fms was not expressed in osteoblasts, it is likely that OMD is an osteoblast maturation marker that is induced by osteoclast activity. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Osteomodulin; Osteoclast; Osteoblast; Coupling
Bone is continuously remodeled via bone resorption by osteoclasts and bone formation by osteoblasts [1]. Osteoclasts are derived from hematopoietic stem cells, while osteoblasts are differentiated from mesenchymal stem cells [2,3]. Osteoclasts are multinuclear giant cells, and c-Fos and NFATc1 are both required for osteoclast differentiation [4,5]. M-CSF and RANKL are cytokines required
*
Corresponding authors. Address: Department of Cell Differentiation, The Sakaguchi Laboratory, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Fax: +81 3 5363 3475. E-mail addresses:
[email protected] (T. Miyamoto),
[email protected] (T. Suda). 1 These authors are contributed equally to this work. 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.193
for osteoclast differentiation, and that lack of these cytokines induces osteopetrosis in mice due to failure of osteoclast differentiation [6,7]. Thus, osteoclasts are required for appropriate bone development. In contrast, osteoblasts are bone-forming cells, and the transcription factors Runx2 and Osterix are both required for osteoblast differentiation [8,9]. TGFb and BMPs have also been reported to be involved in osteoblast differentiation [2]. Bone remodeling is a complex process requiring ‘coupling’ between osteoclastic and osteoblastic activities. Osteoclasts form resorption lacunae that are filled with new bone produced by osteoblasts. Disruption of the balance between osteoclast and osteoblast activity is observed in various disease states such as osteoporosis and bone destruction by metastatic tumors. Thus, understanding the ‘coupling’ process is
K. Ninomiya et al. / Biochemical and Biophysical Research Communications 362 (2007) 460–466
crucial for the regulation of bone homeostasis. Recently, it was shown that the interaction between ephrinB2 expressed in osteoclasts and EphB4 expressed in osteoblasts was crucial for the regulation of bone homeostasis [1]. However, the molecular mechanisms of ‘coupling’ are still largely unknown, possibly due to their complicated nature. The microarray and its gene cluster analysis can subdivide genes into various groups based on the correlation of their expression across various tissues; this approach is a powerful tool to isolate a group of genes whose expression is synchronized in a tissue [10]. Osteoclasts express several osteoclastspecific markers such as Acp5, MMP9 and DC-STAMP [3,11–13], and these genes are therefore useful to detect groups of genes clustered with osteoclast-specific markers. Osteomodulin (OMD), also called osteoadherin, was originally isolated from bovine bone [14]. OMD is a cellbinding keratan sulfate proteoglycan that belongs to the family of leucine-rich repeat proteins (SLRPs) of the extracellular matrix, which are specifically expressed in osteoblasts [15]. It was reported that the expression of OMD was regulated by the transcription factor Runx2, Smads, or the cytokines TGFb1 and BMP2 [16–18]. SLRPs are known to have diverse functions such as regulation of matrix assembly, cell growth, and cell differentiation [19]. Asporin, a SLRP family protein, is involved in the regulation of chondrocyte activity mediated by TGFb [20], while biglycan, another member of the SLRPs, was reported to be involved in osteoblast differentiation [21]. Thus, SLRPs have crucial roles in regulating skeletal homeostasis and development. Here, we describe that OMD expression was detected in bone to be coupled with osteoclast activity, and that OMD expression was clustered with the osteoclastic molecules MMP9 and Acp5. OMD was expressed in osteoblasts and was upregulated during osteoblast differentiation. OMD expression was reduced in the bones of op/op mice lacking functional osteoclasts, although the M-CSF receptor c-Fms was not expressed in osteoblasts. Reduced OMD expression in op/op mice recovered after administration of M-CSF, indicating that OMD may be a maturation marker for osteoblasts activated by osteoclasts. Materials and methods Animals and injected reagent. All animals were purchased from Japan Crea (Tokyo, Japan) or born and kept under pathogen-free conditions, and cared for in accordance with the guidelines of Keio University School of Medicine. op/op mice were provided by Prof. S. Hayashi (Tottori University, Tottori, Japan). Five micrograms of recombinant human (rh) M-CSF (provided by Morinaga Milk Industry, Tokyo, Japan) in 100 ll PBS was injected peritoneally into 11-d-old op/op mice. Mice were sacrificed 5 days after injection. Microarray analysis. Total RNA was extracted and pooled from 8 different tissues including bone, bone marrow, peripheral blood, nucleus pulposus, skin, muscle, spinal cord, and brain of fifty 8-week-old male Wistar rats using TRIzol Reagent (Qiagen, GmbH, Hilden, Germany), and microarray and cluster analysis were undertaken as previously described [10]. In vitro culture of cells. For osteoblast differentiation in vitro, isolated primary osteoblasts from neonatal calvaria were cultured in aMEM medium containing 10% FCS at 37 °C in a humidified atmosphere of 5%
461
CO2 in air for three days, and cells were treated with 10 mM b-glycerophosphate (Calbiochem, La Jolla, CA, USA) and 50 ng/ml ascorbic acid (Sigma-Aldrich, St. Louis, MO) for the indicated periods. C2C12 myoblasts were maintained or treated with BMP2 (R&D, McKinley Place, MN, USA) as described [22]. To generate osteoclasts in vitro, bone marrow mononuclear cells were cultured with M-CSF and RANKL, and osteoclastogenesis was evaluated by tartrate resistance acid phosphatase (TRAP) staining as described [23,24]. Osteoblast–osteoclast coculture experiments were performed as described [11]. RT-PCR and real-time PCR analysis. Total RNA was extracted and RT-PCR was performed using the RNeasy mini kit (Qiagen). First-strand cDNA was prepared with a reverse transcriptase-polymerase chain reaction (RT-PCR) kit (Clontech Laboratories, Palo Alto, CA, USA) and PCR analysis was undertaken as previously described [23]. Real-time RTPCR was performed using the Applied Biosystems 7500 Fast Real-Time Polymerase Chain Reaction System (Applied Biosystems, Foster City, CA, USA). b-Actin was used as an internal control and primer sets used for RT-PCR were as follows: b-actin-sense, 5 0 -TGAGAGGGAAATCGT GCGTGAC-3 0 and b-actin-antisense 5 0 -AAGAAGGAAGGCTGGAAA AGAG-3 0 ; OMD-sense, 5 0 -GAAGCAAGCATTCTACATTCCAAGG-3 0 and OMD-antisense 5 0 -GCTTATTTTGGTCCACACGAAGGT-3 0 ; Col1a1-sense, 5 0 -TTCACCTACAGCACGCTTGTG-3 0 and Col1a1-antisense 5 0 -GATGACTGTCTTGCCCCAAGTT-3 0 ; Catk-sense, 5 0 -GCAGA GGTGTGTACTATGA-3 0 and Catk-antisense 5 0 -GCAGGCGTTGTTC TTATT-3 0 ; c-fms-sense, 5 0 -CTGTGAATGGCTCTGATGTCCTGTTC TG-3 0 and c-fms-antisense 5 0 -CTCCCACTTCTCATTGTAGGGCAAC TGA-3 0 . Primer sets of runx2 and osteocalcin for real-time PCR analysis were purchased from Takara (Otsu, Shiga, Japan). Establishment of polyclonal anti-OMD antibody. A polyclonal antiOMD antibody was provided by Dr. Bleicher [25] or established by immunizing rabbits with a synthetic peptide corresponding to the sequence CTLEGQEVSDEHYNS. Immunofluorescence staining. For OMD, ALP, Osteopontin (OPN) and Cathepsin K (CatK) staining, femurs and tibias were dissected from 6- to 8-week-old mice, fixed with 10% formalin, decalcified in a 10% EDTA solution in PBS (pH 7.4) for 2 weeks at 4 °C, embedded in paraffin and cut into 4 lm-thick sections. For Osteocalcin staining, frozen sections were prepared as described [10,26]. Frozen sections were fixed in 4% PFA for 10 minutes at 4 °C, and paraffin sections were deparaffinized before staining. For antigen retrieval, sections were incubated with 1mg/ml pepsin (Dako, Carpinteria, CA) in 0.2 N HCl for 30 min at 37 °C. Then, sections were pretreated with blocking buffer (DAKO) for 30 min at room temperature and stained as follows: rabbit anti-OMD antibody, mouse anti-ALP antibody (R&D), goat anti-OPN antibody (Sigma), goat anti-OCN antibody (Sigma), and mouse anti-CatK (Fuji Chemical Co., Takaoka, Japan) overnight at 4 °C followed by Alexa Fluor488-conjugated anti-mouse IgG (Molecular Probes), Alexa Fluor546-conjugated anti-mouse IgG (Molecular Probes) and TOTO3 (Invitrogen) for nuclear staining for 1 h at room temperature. To confirm the specificity of the anti-OMD antibody, the synthetic peptide used for antibody establishment was incubated with the anti-mouse OMD antibody for 30 min at room temperature. Immunoreactivity was detected by confocal microscope (Olympus, Tokyo, Japan). Flow cytometric analysis. Primary osteoblasts derived from mouse neonatal calvaria, the osteoblastic cell line MC3T3E1 cells, and the preosteoclast cell line RAW264.7 cells were stained with rat anti-mouse c-Fms (clone AFS98) followed by PE-conjugated anti-rat Ig and analyzed using FACS Calibur (Becton–Dickinson Immunocytometry Systems, San Jose, CA).
Results Osteomodulin expression is linked with the osteoclast markers MMP and Acp5 We performed microarray and cluster analysis in 8 tissues including bone, bone marrow, peripheral blood,
462
K. Ninomiya et al. / Biochemical and Biophysical Research Communications 362 (2007) 460–466
nucleus pulposus, skin, muscle, spinal cord, and brain for 11,464 rat transcripts derived from 10,490 independent genes. We focused on the gene cluster containing the osteoclastic markers MMP9 and Acp5 since osteoclastic activity is considered to be required for the proper activation of osteoblasts. OMD was found in this cluster, and the specific expression of OMD occurred in bone as detected by microarray analysis (Fig. 1A). The expression of OMD was analyzed in osteoblasts and osteoclasts to determine which cells express OMD in bone (Fig. 1B). Only weak OMD expression was detected in osteoclasts, while abundant expression was observed in osteoblasts. Thus, we designated OMD as an osteoblastic molecule coupled with osteoclastic activity.
cin, a late osteoblast differentiation marker, was induced in a manner similar to OMD, suggesting that OMD is a late osteoblastic molecule. The expression of OMD was also induced in C2C12 cells treated with BMP2 (Fig. 2B and C). OMD expression was induced in the mesenchymal cell line C2C12 cells by BMP2 in a dose-dependent as well as a time-dependent manner. The expression of OMD was induced in C2C12 cells by BMP2 even later than the increase in osteocalcin expression, suggesting that OMD expression was detected at the terminal stage of osteoblast differentiation. These results indicate that OMD is expressed in osteoblasts and upregulated upon osteoblast activation. OMD protein is expressed in osteoblasts
OMD expression is induced in osteoblastic cells during osteoblastogenesis In order to analyze the regulation of OMD expression in osteoblasts, primary osteoblasts were isolated from mouse neonatal calvaria and cultured in the presence of ascorbic acid and b-glycerolphosphate to induce osteoblastic maturation. Expression of OMD was analyzed during in vitro osteoblastogenesis (Fig. 2A). The expression of OMD was upregulated in osteoblasts during differentiation in a time dependent manner together with an increase of the transcription factor runx2, which is essential for osteoblast differentiation. In this culture, osteocal-
To determine whether protein expression of OMD could be detected in bone, we established a polyclonal antibody against OMD (Fig. 3A). OMD expression was detected in cells overexpressing OMD using the antiOMD antibody, and the immunoreactivity was abolished by addition of the synthetic OMD peptide that was used for immunization. Next, we performed immunohistochemical analysis of OMD in bone (Fig. 3B). Immunoreactivity was evident in osteoblasts and was abrogated by treatment with the synthetic OMD peptide, thereby demonstrating the specificity of the anti-OMD antibody (Fig. 3B). The expression pattern of OMD in osteoblasts was examined by double staining of OMD with other osteoblastic markers such as alkaline-phosphatase (ALP), Osteopontin (OPN) and Osteocalcin (OCN) (Fig. 3C) in trabecular and cortical bone. Broad expression of OMD relative to ALP was observed in trabecular bone, while merged OMD expression with ALP was detected in the trabecular bone surface of the metaphysis region under a growth plate (Fig. 3C). Interestingly, OMD expression was also detected in osteocytes located in cortical bone. Partially merged expression of OPN with OMD was detected in trabecular and cortical bone (Fig. 3C). Furthermore, OMD co-localized with OCN in osteoblasts, particularly those located on the surface of the cortical bone (Fig. 3C). Thus, OMD expression was detected in osteoblasts and osteocytes, and some osteoblasts co-expressed OMD with ALP, OPN or OCN. OMD expression is reduced in op/op mice
Fig. 1. Osteomodulin expression is correlated with MMP and Acp5. (A) Total RNA was extracted from indicated eight tissues, and microarray and cluster analysis was undertaken. (B) Total RNA was extracted from primary osteoblasts and cultured osteoclasts, and RT-PCR analysis was undertaken to detect expression of OMD and the internal control, b-actin. Col1a1 and CatK expression were analyzed as positive controls for osteobaslts and osteoclasts, respectively.
op/op mice show strong osteopetrosis due to osteoclast differentiation failure especially in the juvenile stage [6], and the ‘coupling’ process activated by osteoclastic activity is considered to be inhibited in their bones. Thus, the expression of OMD was analyzed in bones of op/op mice (Fig. 4A). The expression of Cathepsin K, a marker of activated osteoclasts, was reduced in op/op mice when compared to wild-type littermates, and the reduced expression was rescued by M-CSF treatment (Fig. 4A). Abundant OMD expression was detected in osteoblasts
K. Ninomiya et al. / Biochemical and Biophysical Research Communications 362 (2007) 460–466
463
Fig. 2. OMD is upregulated in osteoblasts during differentiation. (A) Primary osteoblasts were cultured in the presence or absence of b-glycerol phosphate and ascorbic acid for the indicated periods, and real-time PCR analysis was undertaken to detect OMD, runx2 and osteocalcin. Data are shown as the relative expression of OMD, runx2 and osteocalcin to b-actin, respectively. (B and C) C2C12 cells were cultured in various concentrations of BMP2 for 48 h, and expression of OMD, runx2 and osteocalcin relative to b-actin were examined by real-time PCR (B). C2C12 cells were cultured in the presence of 300ng/ml BMP2 for the indicated periods, and OMD, runx2 and osteocalcin expression relative to b-actin were examined by real-time PCR.
surrounding Cathepsin K-positive osteoclasts in wild-type mice, however, such strong OMD expression was not detected in op/op mice. Interestingly, M-CSF treatment of op/op mice induced Cathepsin K-positive osteoclast formation accompanied by induction of strong OMD expression around osteoclasts. Since the expression of the M-CSF receptor c-Fms was not detected in osteoblasts (Fig. 4B), osteoclastic activity is likely to play an indirect role in upregulating OMD expression in osteoblasts. Taken together, our results indicate that OMD is a late osteoblastic molecule whose in vivo expression is stimulated by osteoclastic activity in a ‘coupling’ manner.
Discussion A well-controlled balance between the activities of boneresorbing osteoclasts and bone-forming osteoblasts is required for the maintenance of bone homeostasis [1]. Osteoblasts express cytokines M-CSF and RANKL, which induce bone-resorbing osteoclasts [2,3]. Osteoblasts also express osteoprotegerin (OPG), a decoy soluble receptor of RANKL that prevents osteoclast formation [2,3]. Thus osteoclastogenesis is tightly controlled by osteoblastderived factors. In contrast, osteoclastic activity is required for appropriate bone formation following bone resorption. However, the molecular mechanisms underlying osteoclas-
464
K. Ninomiya et al. / Biochemical and Biophysical Research Communications 362 (2007) 460–466
Fig. 3. OMD protein is expressed in osteoblasts in vivo. (A) Total lysate was extracted from parental C2C12 cells, or OMD or Mock-transduced C2C12 cells, and western blot analysis was performed to detect OMD expression by using a rabbit anti-OMD polyclonal antibody with or without synthetic OMD peptide followed by HRP-conjugated goat anti-rabbit Ig’ antibody. (B) Decalcified paraffin sections of 8-week-old mouse tibia were stained with rabbit anti-OMD antibody in the presence or absence of synthetic OMD peptide followed by Alexa488-conjugated goat anti-rabbit antibody. TOTO3 was used for nuclear staining. (C) Paraffin or frozen sections of 8-week-old mouse tibia were stained with rabbit anti-OMD antibody together with mouse anti-ALP, OPN or OCN followed by Alexa488-conjugated goat anti-rabbit antibody and Alexa546-conjugated goat anti-mouse antibody.
tic-activity induced osteoblastogenesis or the molecular cascade induced in osteoblasts following bone resorption are largely unknown. In the present study, we isolated OMD, a member of the SLRPs, as a late osteoblastic molecule whose expression was coupled with osteoclastic activity in vivo. Deficiency of osteoclastogenesis is observed in several mouse models due to a lack of osteoclast-inducing factors, intracellular molecules or transcription factors essential for osteoclast formation [4–7]. In osteoclast-deficient mouse models, osteopetrosis was observed, indicating that osteoclastic activity was required for proper bone homeostasis. We have found that multinucleation of osteoclasts upregulated the bone resorption efficiency of osteoclasts, and that cell–cell fusion of osteoclasts plays a role in regulating physiological bone mass in vivo [11]. Recently, ephrinB2, a transmembrane ligand expressed in osteoclasts, was demonstrated to stimulate bone formation via its receptor EphB4 in osteoblasts [1]. Microarray analysis is a powerful tool for isolating tissue-specific molecules among various tissues [10]. In the current study, we employed microarray and cluster analysis to isolate a group of genes expressed in a tissue-specific and synchronized manner with osteoclastic
molecules. OMD was isolated as a coupling gene with the osteoclastic genes Acp5 and MMP9, and its expression was reduced in osteoclast-deficient op/op mice in vivo. Since it was difficult to precisely duplicate the coupling process of osteoclasts and osteoblasts in vitro, microarray analysis among various tissues is a useful tool to isolate molecules involved in the ‘coupling’ process in vivo. To date, several ECM proteins have been reported to play a crucial role in bone homeostasis. Osteonecin knockout mice display osteopenia and decreased bone formation [27], while Biglycan-deficient mice have been reported to show a phenotype characterized by growth failure, reduced bone formation and age-related severe osteopenia [21]. Thus, control of ECM proteins is crucial for the maintenance of bone homeostasis. Possible interaction of OMD with integrin-avb3 has been reported [14,28]. Integrin-avb3 is highly expressed in osteoclasts, and OMD may interact with osteoclasts through integrin-avb3. On the other hand, TGFb, one of the most important osteogenic growth factors that stimulates early osteoblast differentiation, was shown to interact with Asporin, a family protein of the SLRPs [20]. Since OMD is a member of the SLRPs, OMD may regulate TGFb signals for osteoblastognesis.
K. Ninomiya et al. / Biochemical and Biophysical Research Communications 362 (2007) 460–466
465
Fig. 4. OMD expression is reduced in op/op mice. (A) Decalcified tibial paraffin sections of 8-week-old op/op, wild-type littermates or op/op mice that have been treated with 5 lg M-CSF intraperitoneally were stained with rabbit anti-OMD antibody and mouse anti-Cathepsin K (CatK) antibody followed by Alexa488-conjugated goat anti-rabbit Ig antibody and Alexa546-conjugated goat anti-mouse Ig antibody. TOTO3 was used for nuclear staining. Arrows represent CatK-positive osteoclasts. (B) (Left): c-fms expression was analyzed in primary osteoblasts or cultured osteoclasts by RT-PCR. (Right) primary osteoblasts, osteoblastic MC3T3E1 cells and preosteoclastic RAW264.7 cells were stained with rat anti-c-Fms antibody followed by PE-conjugated antirat Ig antibody and examined by FACS Calibur.
Further studies are needed to determine the function of OMD in bone homeostasis.
[2]
Acknowledgments
[3]
We thank Y. Sato and A. Kumakubo for technical support. T. Miyamoto was supported by a Grant-in-Aid for Young Scientists (B), Japan. T. Suda was supported by a Grant-in-Aid from Specially Promoted Research of the Ministry of Education, Science, Sports and Culture, Japan. K. Ninomiya was supported by a Grant-in-Aid from the 21st century COE Program of the Ministry of Education, Culture, Sports, Science and Technology, Japan, to Keio University. The authors have no conflicting financial interests.
[4]
[5]
[6]
[7]
References [1] C. Zhao, N. Irie, Y. Takada, K. Shimoda, T. Miyamoto, T. Nishiwaki, T. Suda, K. Matsuo, Bidirectional ephrinB2-EphB4
signaling controls bone homeostasis, Cell. Metab. 4 (2006) 111–121. G. Karsenty, E.F. Wagner, Reaching a genetic and molecular understanding of skeletal development, Dev. Cell. 2 (2002) 389–406. S.L. Teitelbaum, F.P. Ross, Genetic regulation of osteoclast development and function, Nat. Rev. Genet. 4 (2003) 638–649. A.E. Grigoriadis, Z.Q. Wang, M.G. Cecchini, W. Hofstetter, R. Felix, H.A. Fleisch, E.F. Wagner, c-Fos: a key regulator of osteoclastmacrophage lineage determination and bone remodeling, Science 266 (1994) 443–448. M. Asagiri, K. Sato, T. Usami, S. Ochi, H. Nishina, H. Yoshida, I. Morita, E.F. Wagner, T.W. Mak, E. Serfling, H. Takayanagi, Autoamplification of NFATc1 expression determines its essential role in bone homeostasis, J. Exp. Med. 202 (2005) 1261–1269. H. Yoshida, S. Hayashi, T. Kunisada, M. Ogawa, S. Nishikawa, H. Okamura, T. Sudo, L.D. Shultz, S. Nishikawa, The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene, Nature 345 (1990) 442–444. Y.Y. Kong, H. Yoshida, I. Sarosi, H.L. Tan, E. Timms, C. Capparelli, S. Morony, A.J. Oliveira-dos-Santos, G. Van, A. Itie, W. Khoo, A. Wakeham, C.R. Dunstan, D.L. Lacey, T.W. Mak, W.J. Boyle, J.M. Penninger, OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis, Nature. 397 (1999) 315–323.
466
K. Ninomiya et al. / Biochemical and Biophysical Research Communications 362 (2007) 460–466
[8] T. Komori, H. Yagi, S. Nomura, A. Yamaguchi, K. Sasaki, K. Deguchi, Y. Shimizu, R.T. Bronson, Y.H. Gao, M. Inada, M. Sato, R. Okamoto, Y. Kitamura, S. Yoshiki, T. Kishimoto, Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts, Cell 89 (1997) 755–764. [9] K. Nakashima, X. Zhou, G. Kunkel, Z. Zhang, J.M. Deng, R.R. Behringer, B.D. Crombrugghe, The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation, Cell 108 (2002) 17–29. [10] N. Fujita, T. Miyamoto, J. Imai, N. Hosogane, T. Suzuki, M. Yagi, K. Morita, K. Ninomiya, K. Miyamoto, H. Takaishi, M. Matsumoto, H. Morioka, H. Yabe, K. Chiba, S. Watanabe, Y. Toyama, T. Suda, CD24 is expressed specifically in the nucleus pulposus of intervertebral discs, Biochem. Biophys. Res. Commun. 338 (2005) 1890–1896. [11] M. Yagi, T. Miyamoto, Y. Sawatani, K. Iwamoto, N. Hosogane, N. Fujita, K. Morita, K. Ninomiya, T. Suzuki, K. Miyamoto, Y. Oike, M. Takeya, Y. Toyama, T. Suda, DC-STAMP is essential for cell–cell fusion in osteoclasts and foreign body giant cells, J. Exp. Med. 202 (2005) 345–351. [12] T. Miyamoto, The dendritic cell-specific transmembrane protein DCSTAMP is essential for osteoclast fusion and osteoclast boneresorbing activity, Mod. Rheumatol. 16 (2006) 341–342. [13] M. Yagi, K. Ninomiya, N. Fujita, T. Suzuki, R. Iwasaki, K. Morita, N. Hosogane, K. Matsuo, Y. Toyama, T. Suda, T. Miyamoto, Induction of DC-STAMP by Alternative Activation and Downstream Signaling Mechanisms, J. Bone. Miner. Res. 22 (2007) 992–1001. [14] M. Wendel, Y. Sommarin, D. Heinegard, Bone matrix proteins: isolation and characterization of a novel cell-binding keratan sulfate proteoglycan (osteoadherin) from bovine bone, J. Cell. Biol. 141 (1998) 839–847. [15] Y. Sommarin, M. Wendel, Z. Shen, U. Hellman, D. Heinegard, Osteoadherin, a cell-binding keratan sulfate proteoglycan in bone, belongs to the family of leucine-rich repeat proteins of the extracellular matrix, J. Biol. Chem. 273 (1998) 16723–16729. [16] E.S. Tasheva, B. Klocke, G.W. Conrad, Analysis of transcriptional regulation of the small leucine rich proteoglycans, Mol. Vis. 7 (2004) 758–772. [17] E. Balint, D. Lapointe, H. Drissi, C. Meijden, D.W. Young, A.J. Winjen, J. L Stein, G.S. Stein, J.B. Lian, Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP2-mediated osteoblast differentiation, J. Cell. Biochem. 89 (2003) 401–426.
[18] A.P. Rehn, A.M. Chalk, M . Wendel, Differential regulation of osteoadherin (OSAD) by TGF-beta1 and BMP-2, Biochem. Biophys. Res. Commun. 349 (2006) 1057–1064. [19] R.J. Waddington, H.C. Roberts, R.V. Sugars, E. Schonherr, Differential roles for small leucine-rich proteoglycans in bone formation, Eur. Cell. Mater. 6 (2003) 12–21. [20] H. Kizawa, I. Kou, A. Iida, A. Sudo, Y. Miyamoto, A. Fukuda, A. Mabuchi, A. Kotani, A. Kawakami, S. Yamamoto, A. Uchida, K. Nakamura, K. Notoya, Y. Nakamura, S. Ikegawa, An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to osteoarthritis, Nat. Genet. 37 (2005) 138–144. [21] T. Xu, P. Bianco, L.W. Fisher, G. Longenecker, E. Smith, S. Goldstein, J. Bonadio, A. Boskey, A.M. Heegaard, B. Sommer, K. Satomura, P. Dominguez, C. Zhao, A.B. Kulkarni, P.G. Robey, M.F. Young, Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice, Nat. Genet. 20 (1998) 78–82. [22] T. Katagiri, A. Yamaguchi, M. Komaki, E. Abe, N. Takahashi, T. Ikeda, V. Rosen, J.M. Wozney, A. Fujisawa-Sehara, T. Suda, Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage, J. Cell. Biol. 127 (1994) 1755–1766. [23] T. Miyamoto, F. Arai, O. Ohneda, K. Takagi, D.M. Anderson, T. Suda, An adherent condition is required for formation of multinuclear osteoclasts in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B ligand, Blood 15 (2000) 4335–4343. [24] T. Miyamoto, O. Ohneda, F. Arai, K. Iwamoto, S. Okada, K. Takagi, D.M. Anderson, T. Suda, Bifurcation of osteoclasts and dendritic cells from common progenitors, Blood 15 (2001) 2544–2554. [25] R. Buchaille, M.L. Couble, H. Magloire, F. Bleicher, Expression of the small leucine-rich proteoglycan osteoadherin/osteomodulin in human dental pulp and developing rat teeth, Bone 27 (2000) 265–270. [26] K. Morita, T. Miyamoto, N. Fujita, Y. Kubota, K. Ito, K. Takubo, K. Miyamoto, K. Ninomiya, T. Suzuki, R. Iwasaki, M. Yagi, H. Takaishi, Y. Toyama, T. Suda, Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification, J. Exp. Med. 204 (2007) 1613–1623. [27] A.M. Delany, M. Amling, M. Priemel, C. Howe, R. Baron, E. Canalis, Osteopenia and decreased bone formation in osteonectindeficient mice, J. Clin. Invest. 105 (2000) 915–923. [28] M. Lucchini, M.L. Couble, A. Romeas, M.J. Staquet, F. Bleicher, H. Magloire, J.C. Farges, Alpha v beta 3 integrin expression in human odontoblasts and co-localization with osteoadherin, J. Dent. Res. 83 (2004) 552–556.
