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Muneaki Ishijima Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10, Kanda-Surugadai 2-Chome, Chiyoda-Ku, Tokyo 101-0062, Japan
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA
Department of Orthopaedics, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

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Kunikazu Tsuji Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10, Kanda-Surugadai 2-Chome, Chiyoda-Ku, Tokyo 101-0062, Japan
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA
Department of Orthopaedics, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

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Susan R Rittling Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10, Kanda-Surugadai 2-Chome, Chiyoda-Ku, Tokyo 101-0062, Japan
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA
Department of Orthopaedics, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

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Teruhito Yamashita Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10, Kanda-Surugadai 2-Chome, Chiyoda-Ku, Tokyo 101-0062, Japan
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA
Department of Orthopaedics, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

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Hisashi Kurosawa Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10, Kanda-Surugadai 2-Chome, Chiyoda-Ku, Tokyo 101-0062, Japan
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA
Department of Orthopaedics, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

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David T Denhardt Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10, Kanda-Surugadai 2-Chome, Chiyoda-Ku, Tokyo 101-0062, Japan
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA
Department of Orthopaedics, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

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Akira Nifuji Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10, Kanda-Surugadai 2-Chome, Chiyoda-Ku, Tokyo 101-0062, Japan
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA
Department of Orthopaedics, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

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Yoichi Ezura Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10, Kanda-Surugadai 2-Chome, Chiyoda-Ku, Tokyo 101-0062, Japan
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA
Department of Orthopaedics, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

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Masaki Noda Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 3-10, Kanda-Surugadai 2-Chome, Chiyoda-Ku, Tokyo 101-0062, Japan
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082, USA
Department of Orthopaedics, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

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Mechanical stress to bone plays a crucial role in the maintenance of bone homeostasis. It causes the deformation of bone matrix and generates strain force, which could initiate the mechano-transduction pathway. The presence of osteopontin (OPN), which is one of the abundant proteins in bone matrix, is required for the effects of mechanical stress on bone, as we have reported that OPN-null (OPN−/−) mice showed resistance to unloading-induced bone loss. However, cellular mechanisms underlying the phenomenon have not been completely elucidated. To obtain further insight into the role of OPN in mediating mechanical stress effect on bone, we examined in vitro mineralization and osteoclast-like cell formation in bone marrow cells obtained from hind limb bones of OPN−/− mice after tail suspension. The levels of mineralized nodule formation of bone marrow cells derived from the femora subjected to unloading were decreased compared with that from loaded control in wild-type mice. However, these were not decreased in cells from OPN−/− mice after tail suspension compared with that from loaded OPN−/− mice. Moreover, while spreading of osteoclast-like cells derived from bone marrow cells of the femora subjected to unloading was enhanced compared with that from loaded control in wild-type mice, this enhancement of spreading of these cells derived from the femora subjected to unloading was not recognized compared with those from loaded control in OPN−/− mice. These data provided cellular bases for the effect of the OPN deficiency on in vitro reduced mineralized nodule formation by osteoblasts and on enhancement of osteoclast spreading in vitro induced by the absence of mechanical stress. These in vitro results correlate well with the resistance to unloading-induced bone loss in OPN−/− mice in vivo, suggesting that OPN has an important role in the effects of unloading-induced alterations of differentiation of bone marrow into osteoblasts and osteoclasts.

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