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KAJI Hiroshi

    Department of Medicine　Professor/Senior Staff
Last Updated :2024/04/25

Researcher Information

URL

J-Global ID

Research Interests

  • 内分泌学 骨代謝学 再生医学 生理学   Endocrinology Bone and mineral metabolism   

Research Areas

  • Life sciences / Metabolism and endocrinology

Academic & Professional Experience

  • 2011 - Today  Kindai UniversityFaculty of Medicine主任教授
  • 2009 - 2011  Kobe UniversityGraduate School of Medicine特命准教授
  • 2002 - 2009  Kobe University大学院医学系研究科応用分子医学講座内分泌神経学分野助手(助教）

Education

  •        - 1989  Kobe University  School of Medicine  Faculty of Medicine

Association Memberships

  • JAPAN SOCIETY FOR MEDICAL EDUCATION   PHYSIOLOGICAL SOCIETY OF JAPAN   THE JAPANESE SOCIETY FOR REGENERTIVE MEDICINE(JSRM)   米国骨代謝学会   日本骨粗鬆症学会   日本糖尿病学会   日本骨代謝学会   日本内分泌学会   日本内科学会   

Published Papers

  • Plasminogen activator inhibitor-1 is involved in glucocorticoid-induced decreases in angiogenesis during bone repair in mice.
    Okada K; Niwa Y; Fukuhara K; Ohira T; Mizukami Y; Kawao N; Matsuo O; Kaji H
    J Bone Miner Metab in press 2024/04 [Refereed]
  • Mizukami Y; Kawao N; Ohira T; Hashimoto D; Okada K; Matsuo O; Kaji H
    Calcified Tissue Int in press 2024/03 [Refereed]
  • Kawao N; Matsumura D; Yamada A; Okumoto K; Ohira T; Mizukami Y; Hashimoto D; Kaji H
    Bone 181 (4) 117040  2024/02 [Refereed]
  • Ohira T; Ino Y; Kawao N; Mizukami Y; Okada K; Matsuo O; Hirano H; Kimura Y; Kaji H
    J Applied Physiol 136 (3) 643 - 658 2024/02 [Refereed]
  • Kaji H
    J Bone Miner Metab in press 2023/12 [Refereed][Invited]
  • Matsumura D; Kawao N; Okumoto K; Ohira T; Mizukami Y; Akagi M; Kaji H
    PLoS One 18 (6) e0287541  2023/06 [Refereed]
  • Mizukami Y; Kawao N; Takafuji Y; Ohira T; Okada K; Jo JI; Tabata Y; Kaji H
    PLoS One Public Library of Science (PLoS) 18 (4) e0284258 - e0284258 2023/04 [Refereed]
     
    Matrix vesicles (MtVs) are one of the extracellular vesicles (EVs) secreted by osteoblasts. Although MtVs have a classically-defined function as an initiator of ossification and recent findings suggest a role for MtVs in the regulation of bone cell biology, the effects of MtVs on bone repair remain unclear. In the present study, we employed collagenase-released EVs (CREVs) containing abundant MtVs from mouse osteoblasts. CREVs were administered locally in gelatin hydrogels to damaged sites after a femoral bone defect in mice. CREVs exhibited the characteristics of MtVs with a diameter <200 nm. The local administration of CREVs significantly promoted the formation of new bone with increases in the number of alkaline phosphatase (ALP)-positive cells and cartilage formation at the damaged site after the femoral bone defect. However, the addition of CREVs to the medium did not promote the osteogenic differentiation of ST2 cells or the ALP activity or mineralization of mouse osteoblasts in vitro. In conclusion, we herein showed for the first time that MtVs enhanced bone repair after a femoral bone defect partly through osteogenesis and chondrogenesis in mice. Therefore, MtVs have potential as a tool for bone regeneration.
  • Ohira T; Kawao N; Takafuji Y; Mizukami Y; Kaji H
    Exp Clin Endocrinol Diabetes 131 (4) 228 - 235 2022/12 [Refereed]
  • Takada Y; Takafuji Y; Mizukami Y; Ohira T; Kawao N; Okada K; Kaji H
    Calcified Tissue Int 112 (3) 377 - 388 2022/12 [Refereed]
     
    Extracellular vesicles (EVs) play crucial roles in physiological and pathophysiological processes. Although studies have described muscle-bone interactions via humoral factors, we reported that EVs from C2C12 muscle cells (Myo-EVs) suppress osteoclast formation. Current clinical evidence suggests that inflammation induces both sarcopenia and osteoporosis. Although tumor necrosis factor-α (TNF-α) is a critical proinflammatory factor, the influences of TNF-α on muscle-bone interactions and Myo-EVs are still unclear. In the present study, we investigated the effects of TNF-α stimulation of C2C12 cells on osteoclast formation and osteoblastic differentiation modulated by Myo-EVs in mouse cells. TNF-α significantly decreased the protein amount in Myo-EVs, but did not affect the Myo-EV size distribution. TNF-α treatment of C2C12 myoblasts significantly decreased the suppression of osteoclast formation induced by Myo-EVs from C2C12 myoblasts in mouse bone marrow cells. Moreover, TNF-α treatment of C2C12 myoblasts in mouse preosteoclastic Raw 264.7 cells significantly limited the Myo-EV-induced suppression of osteoclast formation and decreased the Myo-EV-induced increase in mRNA levels of osteoclast formation-related genes. On the other hand, TNF-α treatment of C2C12 muscle cells significantly decreased the degree of Myo-EV-promoted mRNA levels of Osterix and osteocalcin, as well as ALP activity in mouse mesenchymal ST-2 cells. TNF-α also significantly decreased miR196-5p level in Myo-EVs from C2C12 myoblasts in quantitative real-time PCR. In conclusion, TNF-α stimulation of C2C12 muscle cells blunts both the osteoclast formation suppression and the osteoblastic differentiation promotion that occurs due to Myo-EVs in mouse cells. Thus, TNF-α may disrupt the muscle-bone interactions by direct Myo-EV modulation.
  • Kawao N; Kawaguchi M; Ohira T; Ehara H; Mizukami Y; Takafuji Y; Kaji H
    J Cachexia Sarcopenia Muscle. 14 (1) 661 - 662 2022/11 [Invited]
  • Kinoshita Y; Takafuji Y; Okumoto K; Takada Y; Ehara H; Mizukami N; Kawao N; Jo J; Tabata Y; Kaji H
    J Bone Miner Metab 40 735 - 747 2022/08 [Refereed]
     
    INTRODUCTION: Irisin is a proteolytic product of fibronectin type II domain-containing 5, which is related to the improvement in glucose metabolism. Numerous studies have suggested that irisin is a crucial myokine linking muscle to bone in physiological and pathophysiological states. MATERIALS AND METHODS: We examined the effects of local irisin administration with gelatin hydrogel sheets and intraperitoneal injection of irisin on the delayed femoral bone repair caused by streptozotocin (STZ)-induced diabetes in female mice. We analyzed the femurs of mice using quantitative computed tomography and histological analyses and then measured the mRNA levels in the damaged mouse tissues. RESULTS: Local irisin administration significantly blunted the delayed bone repair induced by STZ 10 days after a femoral bone defect was generated. Local irisin administration significantly blunted the number of Osterix-positive cells that were suppressed by STZ at the damaged site 4 days after a femoral bone defect was generated, although it did not affect the mRNA levels of chondrogenic and adipogenic genes 4 days after bone injury in the presence or absence of diabetes. On the other hand, intraperitoneal injection of irisin did not affect delayed bone repair induced by STZ 10 days after bone injury. Irisin significantly blunted the decrease in Osterix mRNA levels induced by advanced glycation end products or high-glucose conditions in ST2 cells in the presence of bone morphogenetic protein-2. CONCLUSIONS: We first showed that local irisin administration with gelatin hydrogel sheets improves the delayed bone repair induced by diabetic state partially by enhancing osteoblastic differentiation.
  • Takafuji Y; Kawao N; Ohira T; Mizukami Y; Okada K; Jo J; Tabata Y; Kaji H
    Endocr J 70 (2) 161 - 171 2022/08 [Refereed]
     
    Humoral factors that are secreted from skeletal muscles can regulate bone metabolism and contribute to muscle-bone relationships. Although extracellular vesicles (EVs) play important roles in physiological and pathophysiological processes, the roles of EVs that are secreted from skeletal muscles in bone repair have remained unclear. In the present study, we investigated the effects of the local administration of muscle cell-derived EVs on bone repair in control and streptozotocin-treated diabetic female mice. Muscle cell-derived EVs (Myo-EVs) were isolated from the conditioned medium from mouse muscle C2C12 cells by ultracentrifugation, after which Myo-EVs and gelatin hydrogel sheets were transplanted on femoral bone defect sites. The local administration of Myo-EVs significantly improved delayed bone repair that was induced by the diabetic state in mice 9 days after surgery. Moreover, this administration significantly enhanced the ratio of bone volume to tissue volume at the damaged sites 9 days after surgery in the control mice. Moreover, the local administration of Myo-EVs significantly blunted the number of Osterix-positive cells that were suppressed by the diabetic state at the damage sites after bone injury in mice. Additionally, Myo-EVs significantly blunted the mRNA levels of Osterix and alkaline phosphatase (ALP), and ALP activity was suppressed by advanced glycation end product 3 in ST2 cells that were treated with bone morphogenetic protein-2. In conclusion, we have shown for the first time that the local administration of Myo-EVs improves delayed bone repair that is induced by the diabetic state through an enhancement of osteoblastic differentiation in female mice.
  • Ohira T; Kawano F; Goto K; Kaji H; Ohira Y
    Neurosci Biobehav Rev Elsevier BV 136 104617 - 104617 0149-7634 2022/05 [Refereed]
  • Kawaguchi M; Kawao N; Muratani M; Takafuji Y; Ishida M; Kinoshita Y; Takada Y; Mizukami Y; Ohira T; Kaji H
    J Cell Physiol 237 (5) 2492 - 2502 2022/05 [Refereed]
     
    Exercise is important for the prevention and treatment of sarcopenia and osteoporosis. Although the interactions between skeletal muscles and bone have recently been reported, the myokines linking muscle to bone during exercise remain unknown. We previously revealed that chronic exercise using treadmill running blunts ovariectomy-induced osteopenia in mice. We herein performed an RNA sequence analysis of the gastrocnemius and soleus muscles of male mice with or without chronic exercise to identify the myokines responsible for the effects of chronic exercise on the muscle/bone relationship. We extracted peripheral myelin protein 22 (PMP22) as a humoral factor that was putatively induced by chronic exercise in the soleus and gastrocnemius muscles of mice from the RNA sequence analysis. Chronic exercise significantly enhanced the expression of PMP22 in the gastrocnemius and soleus muscles of female mice. PMP22 suppressed macrophage-colony stimulating factor and receptor activator factor κB ligand-induced increases in the expression of osteoclast-related genes and osteoclast formation from mouse bone marrow cells. Moreover, PMP22 significantly inhibited osteoblast differentiation, alkaline phosphatase activity, and mineralization in mouse osteoblast cultures; however, the overexpression of PMP22 did not affect muscle phenotypes in mouse muscle C2C12 cells. A simple regression analysis revealed that PMP22 mRNA levels in the gastrocnemius and soleus muscles were positively related to cortical bone mineral density at the femurs of mice with or without chronic exercise. In conclusion, we identified PMP22 as a novel myokine induced by chronic exercise in mice. We first showed that PMP22 suppresses osteoclast formation and the osteoblast phenotype in vitro.
  • Okada K; Kawao N; Nakai D; Wakabayashi R; Horiuchi Y; Okumoto K; Kurashimo S; Takafuji Y; Matsuo O; Kaji H
    Int J Mol Sci MDPI AG 23 (1) 478 - 478 2022/01 [Refereed]
     
    Glucocorticoids delay fracture healing and induce osteoporosis. However, the mechanisms by which glucocorticoids delay bone repair have yet to be clarified. Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of plasminogen activators and an adipocytokine that regulates metabolism. We herein investigated the roles of macrophages in glucocorticoid-induced delays in bone repair after femoral bone injury using PAI-1-deficient female mice intraperitoneally administered with dexamethasone (Dex). Dex significantly decreased the number of F4/80-positive macrophages at the damaged site two days after femoral bone injury. It also attenuated bone injury-induced decreases in the number of hematopoietic stem cells in bone marrow in wild-type and PAI-1-deficient mice. PAI-1 deficiency significantly weakened Dex-induced decreases in macrophage number and macrophage colony-stimulating factor (M-CSF) mRNA levels at the damaged site two days after bone injury. It also significantly ameliorated the Dex-induced inhibition of macrophage phagocytosis at the damaged site. In conclusion, we herein demonstrated that Dex decreased the number of macrophages at the damaged site during early bone repair after femoral bone injury partly through PAI-1 and M-CSF in mice.
  • Renal failure suppresses muscle irisin expression and irisin blunts cortical bone loss in mice.
    Kawao N; Kawaguchi M; Ohira T; Ehara H; Mizukami Y; Takafuji Y; Kaji H
    J Cachexia Sarcopenia Muscle 13 (1) 758 - 771 2022/01 [Refereed]
  • Ehara H; Tatsumi K; Takafuji Y; Kawao N; Ishida M; Okada K; Mackman N; Kaji H
    PLoS One 16 (12) e0260754  2021/12 [Refereed]
     
    BACKGROUND: Tissue factor (TF) is the primary activator of the extrinsic coagulation protease cascade. Although TF plays roles in various pathological states, such as thrombosis, inflammatory diseases, cancer, and atherosclerosis, its involvement in bone metabolism remains unknown. MATERIALS AND METHODS: The present study examined the roles of TF in delayed bone repair induced by a diabetic state in mice using wild-type (WT) and low TF-expressing (LTF) male mice. A diabetic state was induced by intraperitoneal injections of streptozotocin (STZ). RESULTS: A prolonged diabetic state significantly reduced total and trabecular bone mineral densities (BMD) as well as cortical bone thickness in WT and LTF mice; these BMD parameters were similar between WT and LTF mice treated with or without STZ. The diabetic state induced in WT mice delayed the repair of the femur following injury. The diabetic state induced in LTF mice was associated with further delays in bone repair. In in vitro experiments, TF significantly decreased receptor activator of nuclear factor-κB ligand-induced osteoclast formation and osteoclastogenic gene expression in RAW264.7 cells. However, it did not affect the gene expression levels of runt-related transcription factor 2 and osterix as well as alkaline phosphatase activity in mouse primary osteoblasts. CONCLUSION: Low TF state was associated with enhanced bone repair delay induced by diabetic state in mice. The TF-induced suppression of bone remodeling may be a contributing factor to the protective effects of TF against delayed bone repair in a diabetic state.
  • Medical students who cannot advance to the next grade may have been aware of the possibility that they will not be able to advance: A case-controlled study at a medical school.
    Ikeda Y; Mitsui Y; Kaji H; Isogai N; Akagi M; Matsumura I
    J Med Educat 2021/07 [Refereed]
  • Ehara J; Takafuji Y; Tatsumi K; Okada K; Mizukami Y; Kawao N; Matsuo O; Kaji H
    Endocr J Japan Endocrine Society 68 (12) 1421 - 1428 0918-8959 2021/07 [Refereed]
  • Takafuji Y; Tatsumi K; Kawao N; Okada K; Muratani M; Kaji H
    PLoS One 16 (5) e0250741  2021/05 [Refereed]
     
    The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.
  • Serpinb1a suppresses osteoclast formation.
    Ishida M; Kawao N; Mizukami Y; Takafuji Y; Kaji H
    Biochem Biophys Rep 26 101004  2021/04 [Refereed]
  • Ishida M; Kawao N; Mizukami Y; Takafuji Y; Kaji H
    BMC Muscloskelt Disord 22 (1) 398  2021/04 [Refereed]
  • Kawao N; Iemura S; Kawaguchi M; Mizukami Y; Takafuji Y; Kaji H
    J Bone Miner Metab 39 (4) 547 - 557 2021/02 [Refereed]
  • Influences of microgravity on the crosstalk between muscle and bone.
    Kaji H
    Frontiers in Physiology Proceedings of the 2019 ISGP meeting 43 - 48 2020/12 [Invited]
  • Iemura S; Kawao N; Akagi M; Kaji H
    Exp Clin Endocrinol Diabet 2020 Dec 22. doi: 10.1055/a-1331-7021. Online ahead of print. 2020/12 [Refereed]
  • Okada K; Nishioka M; Kaji H
    Inflam Regener 40 (22) 22 - 22 2020/09 [Refereed][Invited]
     
    In bone tissues, metabolic turnover through bone resorption by osteoclasts and bone formation by osteoblasts, termed bone remodeling, is strictly controlled and maintains homeostasis. Fibrinolytic factors are expressed in osteoclasts and osteoblasts, and are involved in bone remodeling through bone resorption and formation. The repair/regeneration process after bone injury is divided into the acute inflammatory, repair, and remodeling stages. Osteoblasts, osteoclasts, chondrocytes, and macrophages involved in the bone repair process originate from hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stem cells (MSCs) in the bone marrow. Therefore, stem cells in the bone marrow may be strongly influenced by bone injury. The urokinase-type PA (u-PA)/plasminogen (Plg) system functions in macrophage accumulation/phagocytosis through chemokines in the acute inflammatory stage, and Plg increases blood vessel-related growth factor expression, being involved in vascularization in mice. Plasminogen activator inhivitor-1 (PAI-1) causes bone loss and delayed bone repair through the inhibition of osteoblast differentiation in a drug-induced diabetes model in mice. Plg is considered to induce transforming growth factor-β (TGF-β) production in macrophages in the bone repair process, TGF-β release from the extracellular matrix through the activation of matrix metalloproteinase-9 (MMP-9), and stromal cell-derived factor-1 (SDF-1) expression in endosteal preosteoblasts, leading to the induction of bone marrow HSPCs in mice. Based on the above, establishment of a fibrinolytic factor-targeting method efficiently promoting bone repair/regeneration and fracture healing, and development of a new osteoporosis treatment method and diagnostic marker are awaited.
  • Shimoide T; Kawao N; Morita H; Ishida M; Takafuji Y; Kaji H
    Calcif Tissue Int 107 180 - 190 2020/06 [Refereed]
  • Kawaguchi M; Kawao N; Takafuji Y; Ishida M; Kaji H
    Heliyon 6 (5) e03967  2020/05 [Refereed]
     
    Myonectin is a myokine, which is involved in the pathophysiology of diabetes and obesity, and various myokines are involved in the interactions between skeletal muscle and bone. However, roles of myonectin in bone have still remained unknown. We therefore examined the effects of myonectin on mouse osteoblast and osteoclast differentiation in vitro. Myonectin significantly suppressed the mRNA levels of osteogenic genes and alkaline phosphatase (ALP) activity in mouse osteoblasts. As for osteoclasts, myonectin significantly suppressed osteoclast formation as well as the mRNA levels of osteoclast-related genes enhanced by receptor activator nuclear factor κB ligand (RANKL) from mouse monocytic RAW264.7 cells. Moreover, myonectin significantly suppressed osteoclast formation from mouse bone marrow cells in the presence of macrophage-colony stimulating factor and RANKL. On the other hand, myonectin significantly suppressed RANKL-induced oxygen consumption rate and peroxisome proliferator-activated receptor γ coactivator-1β mRNA levels in RAW264.7 cells, although myonectin did not affect these mitochondrial biogenesis parameters in mouse osteoblasts. In conclusion, the present study demonstrated that myonectin suppresses the differentiation and ALP activity in mouse osteoblasts. Moreover, myonectin suppressed osteoclast differentiation from mouse bone marrow and RAW264.7 cells partly through an inhibition of mitochondrial biogenesis.
  • Naoyuki Kawao; Hironobu Morita; Shunki Iemura; Masayoshi Ishida; Hiroshi Kaji
    International journal of molecular sciences 21 (7) 2020/04 [Refereed]
     
    Mechanical unloading simultaneously induces muscle and bone loss, but its mechanisms are not fully understood. The interactions between skeletal muscle and bone have been recently noted. Although canonical wingless-related integration site (Wnt)/β-catenin signaling is crucial for bone metabolism, its roles in the muscle and bone interactions have remained unknown. Here, we performed comprehensive DNA microarray analyses to clarify humoral factors linking muscle to bone in response to mechanical unloading and hypergravity with 3 g in mice. We identified Dickkopf (Dkk) 2, a Wnt/β-catenin signaling inhibitor, as a gene whose expression was increased by hindlimb unloading (HU) and reduced by hypergravity in the soleus muscle of mice. HU significantly elevated serum Dkk2 levels and Dkk2 mRNA levels in the soleus muscle of mice whereas hypergravity significantly decreased those Dkk2 levels. In the simple regression analyses, serum Dkk2 levels were negatively and positively related to trabecular bone mineral density and mRNA levels of receptor activator of nuclear factor-kappa B ligand (RANKL) in the tibia of mice, respectively. Moreover, shear stress significantly suppressed Dkk2 mRNA levels in C2C12 cells, and cyclooxygenase inhibitors significantly antagonized the effects of shear stress on Dkk2 expression. On the other hand, Dkk2 suppressed the mRNA levels of osteogenic genes, alkaline phosphatase activity and mineralization, and it increased RANKL mRNA levels in mouse osteoblasts. In conclusion, we showed that muscle and serum Dkk2 levels are positively and negatively regulated during mechanical unloading and hypergravity in mice, respectively. An increase in Dkk2 expression in the skeletal muscle might contribute to disuse- and microgravity-induced bone and muscle loss.
  • Hironobu Morita; Hiroshi Kaji; Yoichi Ueta; Chikara Abe
    The journal of physiological sciences : JPS 70 (1) 17 - 17 2020/03 [Refereed]
     
    The peripheral vestibular organs are sensors for linear acceleration (gravity and head tilt) and rotation. Further, they regulate various body functions, including body stability, ocular movement, autonomic nerve activity, arterial pressure, body temperature, and muscle and bone metabolism. The gravitational environment influences these functions given the highly plastic responsiveness of the vestibular system. This review demonstrates that hypergravity or microgravity induces changes in vestibular-related physiological functions, including arterial pressure, muscle and bone metabolism, feeding behavior, and body temperature. Hopefully, this review contributes to understanding how human beings can adapt to a new gravitational environment, including the moon and Mars, in future.
  • Shunki Iemura; Naoyuki Kawao; Katsumi Okumoto; Masao Akagi; Hiroshi Kaji
    Journal of bone and mineral metabolism 38 (2) 161 - 171 2020/03 [Refereed]
     
    Androgen deficiency plays a crucial role in the pathogenesis of male osteoporosis and sarcopenia. Myokines have recently been identified as humoral factors that are involved in the interactions between muscle and bone; however, the influence of androgen deficiency on these interactions remains unclear. Therefore, we herein investigated the roles of humoral factors linking muscle to bone using orchidectomized mice with sarcopenia and osteopenia. Orchidectomy (ORX) significantly reduced muscle mass, grip strength, and trabecular bone mineral density (BMD) in mice. Among the myokines examined, ORX only significantly reduced fibronectin type III domain-containing 5 (Fndc5) mRNA levels in both the soleus and gastrocnemius muscles of mice. In simple regression analyses, Fndc5 mRNA levels in the soleus muscle positively correlated with trabecular BMD, but not cortical BMD. The administration of irisin, a product of Fndc5, significantly protected against the decrease induced in trabecular BMD, but not muscle mass, by androgen deficiency in mice. In conclusion, the present results demonstrated that androgen deficiency decreases the expression of irisin in the skeletal muscle of mice. Irisin may be involved in muscle/bone relationships negatively affected by androgen deficiency.
  • Okada K, Okamoto T, Okumoto K, Takafuji Y, Ishida M, Kawao N, Matsuo O, Kaji H
    Bone 2020/03 [Refereed]
  • Takafuji Y, Tatsumi K, Ishida M, Kawao N, Okada K, Kaji H
    Bone (2) 2020/02 [Refereed]
  • Ishida M, Tatsumi K, Okumoto K, Kaji H
    Stem Cells Dev 15 2020/02 [Refereed]
  • Micro RNA 196a-5p in extracellular vesicles secreted from myoblasts suppresses osteoclast-like cell formation in mouse cells.
    Takafuji Y; Tatsumi K; Kawao N; Okada K; Mutatani M; Kaji H
    Calcif Tissue Int 2020 [Refereed]
  • Naoyuki Kawao; Yoshimasa Takafuji; Masayoshi Ishida; Katsumi Okumoto; Hironobu Morita; Masafumi Muratani; Hiroshi Kaji
    PloS one 15 (2) e0228685  2020 [Refereed]
     
    The vestibular system controls balance, posture, blood pressure, and gaze. However, the roles of the vestibular system in energy and glucose metabolism remain unknown. We herein examined the roles of the vestibular system in obesity and impaired glucose metabolism using mice with vestibular lesions (VL) fed a high-sucrose/high-fat diet (HSHFD). VL was induced by surgery or arsenic. VL significantly suppressed body fat enhanced by HSHFD in mice. Glucose intolerance was improved by VL in mice fed HSHFD. VL blunted the levels of adipogenic factors and pro-inflammatory adipokines elevated by HSHFD in the epididymal white adipose tissue of mice. A β-blocker antagonized body fat and glucose intolerance enhanced by HSHFD in mice. The results of an RNA sequencing analysis showed that HSHFD induced alterations in genes, such as insulin-like growth factor-2 and glial fibrillary acidic protein, in the vestibular nuclei of mice through the vestibular system. In conclusion, we herein demonstrated that the dysregulation of the vestibular system influences an obese state and impaired glucose metabolism induced by HSHFD in mice. The vestibular system may contribute to the regulation of set points under excess energy conditions.
  • Akihiro Moritake; Naoyuki Kawao; Kiyotaka Okada; Masayoshi Ishida; Kohei Tatsumi; Osamu Matsuo; Masao Akagi; Hiroshi Kaji
    Modern rheumatology 29 (6) 959 - 963 2019/11 [Refereed]
     
    Objectives: Interleukin (IL)-1β and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis. On the other hand, plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, exerts functions in the pathogenesis of various diseases. However, the functional roles of PAI-1 in the chondrocytes have been still remained unknown.Methods: In the present study, we investigated the roles of PAI-1 in the effects of IL-1β on the chondrocytes using wild-type and PAI-1-deficient mice.Results: IL-1β significantly elevated PAI-1 mRNA levels in the chondrocytes from wild-type mice. PAI-1 deficiency significantly blunted the mRNA levels of TGF-β and IL-6 enhanced by IL-1β in murine chondrocytes. Moreover, PAI-1 deficiency significantly decreased the mRNA levels of MMP-13, -3 and -9 as well as MMP-13 activity enhanced by IL-1β in the chondrocytes. In addition, PAI-1 deficiency significantly reversed type II collagen mRNA levels suppressed by IL-1β in the chondrocytes. On the other hand, active PAI-1 treatment significantly enhanced the mRNA levels of MMP-13, -3 and -9 as well as decreased type II collagen mRNA levels in the chondrocytes from wild-type mice.Conclusion: We first demonstrated that PAI-1 is involved in MMP expression enhanced by IL-1β in murine chondrocytes. PAI-1 might be crucial for the cartilage matrix degradation and the impaired chondrogenesis by IL-1β in mice.
  • Takafuji Y; Tatsumi K; Ishida M; Kawao N; Okada K; Matsuo O; Kaji H
    J Cell Physiol 234 (6) 9687 - 9697 2019/06 [Refereed]
  • Terumasa Ikeda; Hiroshi Kaji; Yukinori Tamura; Masao Akagi
    Journal of Orthopaedic Science Elsevier BV 24 (3) 532 - 538 0949-2658 2019/05 [Refereed]
  • Naoyuki Kawao; Masayoshi Ishida; Hiroshi Kaji
    PloS one 14 (10) e0224403  2019 [Refereed]
     
    Muscle and bone masses are elevated by the increased mechanical stress associated with body weight gain in obesity. However, the mechanisms by which obesity affects muscle and bone remain unclear. We herein investigated the roles of obesity and humoral factors from adipose tissue in the recovery phase after reloading from disuse-induced muscle wasting and bone loss using normal diet (ND)- or high fat diet (HFD)-fed mice with hindlimb unloading (HU) and subsequent reloading. Obesity did not affect decreases in trabecular bone mineral density (BMD), muscle mass in the lower leg, or grip strength in HU mice. Obesity significantly increased trabecular BMD, muscle mass in the lower leg, and grip strength in reloading mice over those in reloading mice fed ND. Among the humoral factors in epididymal and subcutaneous adipose tissue, leptin mRNA levels were significantly higher in reloading mice fed HFD than in mice fed ND. Moreover, circulating leptin levels were significantly higher in reloading mice fed HFD than in mice fed ND. Leptin mRNA levels in epididymal adipose tissue or serum leptin levels positively correlated with the increases in trabecular BMD, total muscle mass, and grip strength in reloading mice fed ND and HFD. The present study is the first to demonstrate that obesity enhances the recovery of bone and muscle masses as well as strength decreased by disuse after reloading in mice. Leptin may contribute to the recovery of muscle and bone enhanced by obesity in mice.
  • Masayoshi Ishida; Naoyuki Kawao; Kiyotaka Okada; Kohei Tatsumi; Kazuko Sakai; Kazuto Nishio; Hiroshi Kaji
    Endocrinology 159 (11) 3775 - 3790 2018/11 [Refereed]
     
    It is well known that sex differences exist concerning the severity of osteoporosis and bone metabolism, suggesting that factors other than sex hormones might be responsible for sex differences of bone metabolism. We therefore examined sex differences of osteoblast phenotypes of mouse osteoblasts and then performed comparative gene expression analyses using a comprehensive DNA microarray between female and male osteoblasts. Alkaline phosphatase (ALP) activity, mineralization, and the expression of Osterix, ALP, and bone sialoprotein were significantly lower in mouse female osteoblasts compared with male osteoblasts. We identified Serpina3n, a novel serine protease inhibitor, as the gene whose expression has the highest ratio of females to males. A reduction in endogenous levels of Serpina3n by small interfering RNA significantly enhanced the mRNA levels of Runx2, ALP, osteocalcin, and type I collagen (Col1a1) in both male and female osteoblasts. Moreover, Serpina3n overexpression significantly suppressed the mRNA levels of Osterix, ALP, osteocalcin, and Col1a1 in MC3T3-E1 cells. Serpina3n overexpression did not affect Osterix, ALP, and osteocalcin mRNA levels enhanced by bone morphogenetic protein (BMP)-2 in ST2 cells, adipogenic differentiation in ST2 and 3T3-L1 cells, and receptor activator of nuclear factor κB ligand-induced osteoclast formation in RAW264.7 cells, although it significantly suppressed mineralization in ST2 cells differentiated into osteoblasts by BMP-2. In conclusion, we found Serpina3n as the most female osteoblast-dominant gene. Serpina3n exerts a suppression of the osteoblast phenotypes such as Col1a1 expression and ALP activity in differentiated osteoblasts, which might partly explain sex differences of the osteoblast phenotypes in mice.
  • Naoyuki Kawao; Hironobu Morita; Kazuaki Nishida; Koji Obata; Kohei Tatsumi; Hiroshi Kaji
    The journal of physiological sciences : JPS 68 (5) 609 - 616 2018/09 [Refereed]
     
    We recently reported that hypergravity with 3 g for 4 weeks affects muscle and bone through the vestibular system in mice. The purpose of this study was to investigate the effects of hypergravity with 2 g, which had no influence on circulating glucocorticoid level, on the gene levels in muscle and bone, as well as the roles of the vestibular system in those changes using vestibular lesioned (VL) mice. Hypergravity for 2 and 8 weeks or VL exerted little effects on the mRNA levels of muscle differentiation factors and myokines in the soleus muscle. Although hypergravity for 2 weeks significantly elevated alkaline phosphatase (ALP) and type I collagen mRNA levels in the tibia, VL significantly attenuated the levels of ALP mRNA enhanced by hypergravity. In conclusion, the present study suggests that a 2-g load for 2 weeks enhances osteoblast differentiation partly through the vestibular system in mice.
  • Naoyuki Kawao; Akihiro Moritake; Kohei Tatsumi; Hiroshi Kaji
    Calcified tissue international 103 (1) 24 - 34 2018/07 [Refereed]
     
    Mechanical unloading induces disuse muscle atrophy and bone loss, but the details in mechanism involved in those pathophysiological conditions are not fully understood. Interaction between muscle and bone has been recently noted. Here, we investigated the roles of humoral factors linking muscle to bone during mechanical unloading using mice with hindlimb unloading (HU) and sciatic neurectomy (SNX). HU and SNX reduced muscle volume surrounding the tibia, tissue weights of soleus and gastrocnemius muscle, and trabecular bone mineral density (BMD) in the tibia of mice. Among humoral factors linking muscle to bone, HU and SNX reduced fibronectin type III domain-containing 5 (FNDC5) mRNA levels in the soleus muscle of mice. Simple regression analysis revealed that FNDC5 mRNA levels in the soleus muscle were positively related to trabecular BMD in the tibia of control and HU mice as well as sham and SNX mice. Moreover, FNDC5 mRNA levels were negatively correlated with receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels in the tibia of control and HU mice. Irisin, a product of FNDC5, suppressed osteoclast formation from mouse bone marrow cells and RANKL mRNA levels in primary osteoblasts. FNDC5 mRNA levels elevated by fluid shear stress were antagonized by bone morphogenetic protein (BMP) and phosphatidylinositol 3-kinase (PI3K) signaling inhibitors in myoblastic C2C12 cells. In conclusion, the present study first showed that mechanical unloading reduces irisin expression in the skeletal muscle of mice presumably through BMP and PI3K pathways. Irisin might be involved in muscle/bone relationships regulated by mechanical stress in mice.
  • Kiyotaka Okada; Naoyuki Kawao; Kohei Tatsumi; Masayoshi Ishida; Yoshimasa Takafuji; Shinzi Kurashimo; Katsumi Okumoto; Kotaro Kojima; Osamu Matsuo; Hiroshi Kaji
    Bone reports 8 195 - 203 2018/06 [Refereed]
     
    We previously revealed that stromal cell-derived factor-1 (SDF-1) is involved in the changes in the number of bone marrow stem cells during the bone repair process in mice. Moreover, we reported that plasminogen (Plg) deficiency delays bone repair and the accumulation of macrophages at the site of bone damage in mice. We investigated the roles of Plg in the changes in bone marrow stem cells during bone repair. We analyzed the numbers of hematopoietic stem cells (HSC) and mesenchymal stem cells (MSCs) within bone marrow from Plg-deficient and wild-type mice after a femoral bone injury using flow cytometric analysis. Plg deficiency significantly blunted a decrease in the number of HSCs after bone injury in mice, although it did not affect an increase in the number of MSCs. Plg deficiency significantly blunted the number of SDF-1- and Osterix- or SDF-1- and alkaline phosphatase-double-positive cells in the endosteum around the lesion as well as matrix metalloprotainase-9 (MMP-9) activity and mRNA levels of SDF-1 and transforming growth factor-β (TGF-β) elevated by bone injury. TGF-β signaling inhibition significantly blunted a decrease in the number of HSCs after bone injury. The present study showed that Plg is critical for the changes in bone marrow HSCs through MMP-9, TGF-β, and SDF-1 at the damaged site during bone repair in mice.
  • Takeshi Shimoide; Naoyuki Kawao; Yukinori Tamura; Kiyotaka Okada; Yoshitaka Horiuchi; Katsumi Okumoto; Shinji Kurashimo; Masayoshi Ishida; Kohei Tatsumi; Osamu Matsuo; Hiroshi Kaji
    Endocrinology 159 (4) 1875 - 1885 0013-7227 2018/04 [Refereed]
     
    Copyright © 2018 Endocrine Society. Delayed fracture healing is a clinical problem in diabetic patients. However, the mechanisms of diabetic delayed bone repair remain unknown. Here, we investigate the role of macrophages in diabetic delayed bone repair after femoral bone injury in streptozotocin (STZ)-treated and plasminogen activator inhibitor-1 (PAI-1)–deficient female mice. STZ treatment significantly decreased the numbers of F4/80-positive cells (macrophages) but not granulocyte-differentiation antigen-1–positive cells (neutrophils) at the damaged site on day 2 after femoral bone injury in mice. It significantly decreased the messenger RNA (mRNA) levels of macrophage colony-stimulating factor, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and CD206 at the damaged site on day 2 after bone injury. Moreover, STZ treatment attenuated a decrease in the number of hematopoietic stem cells in bone marrow induced by bone injury. On the other hand, PAI-1 deficiency significantly attenuated a decrease in the number of F4/80-positive cells induced by STZ treatment at the damaged site on day 2 after bone injury in mice. PAI-1 deficiency did not affect the mRNA levels of iNOS and IL-6 in F4/80– and CD11b–double-positive cells from the bone marrow of the damaged femurs decreased by diabetes in mice. PAI-1 deficiency significantly attenuated the phagocytosis of macrophages at the damaged site suppressed by diabetes. In conclusion, we demonstrated that type 1 diabetes decreases accumulation and phagocytosis of macrophages at the damaged site during early bone repair after femoral bone injury through PAI-1 in female mice.
  • Yukinori Tamura; Naoyuki Kawao; Takeshi Shimoide; Kiyotaka Okada; Osamu Matsuo; Hiroshi Kaji
    Journal of bone and mineral metabolism 36 (2) 148 - 156 2018/03 [Refereed]
     
    We recently revealed that plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is involved in diabetes, osteoporosis and muscle wasting induced by glucocorticoid (GC) treatment in mice. In the present study, we investigated the detailed mechanisms by which GC induces muscle wasting through PAI-1 in vivo and in vitro. PAI-1 deficiency suppressed the mRNA levels of atrogin1 and muscle RING-Finger Protein 1 (MuRF1), ubiquitin ligases leading to muscle degradation, elevated by GC treatment in the gastrocnemius muscle of mice. In vitro study revealed that active PAI-1 treatment augmented the increase in atrogin1 mRNA levels enhanced by dexamethasone (Dex) in mouse myoblastic C2C12 cells. Moreover, a reduction in endogenous PAI-1 level by siRNA suppressed the mRNA levels of atrogin1 and MuRF1 enhanced by Dex in C2C12 cells. In contrast, a reduction in endogenous PAI-1 levels and active PAI-1 did not affect the phosphorylations of Akt and p70S6 kinase nor myogenic differentiation with or without Dex in C2C12 cells. In addition, PAI-1 deficiency blunted IGF-1 mRNA levels decreased by GC treatment in the gastrocnemius muscle of mice, although neither active PAI-1 nor a reduction in endogenous PAI-1 levels affected the levels of IGF-1 mRNA in C2C12 cells in the presence of Dex. In conclusion, our data suggest that paracrine PAI-1 is involved in GC-induced muscle wasting through the enhancement of muscle degradation in mice.
  • Naoyuki Kawao; Hironobu Morita; Koji Obata; Kohei Tatsumi; Hiroshi Kaji
    JOURNAL OF CELLULAR PHYSIOLOGY WILEY 233 (2) 1191 - 1201 0021-9541 2018/02 [Refereed]
     
    Interactions between muscle and bone have been recently noted. We reported that the vestibular system plays crucial roles in the changes in muscle and bone induced by hypergravity in mice. However, the details of the mechanisms by which gravity change affects muscle and bone through the vestibular system still remain unknown. Here, we investigated the roles of humoral factors linking muscle to bone and myostatin-related factors in the hypergravity-induced changes in muscle and bone in mice with vestibular lesions (VL). Hypergravity elevated serum and mRNA levels of follistatin, an endogenous inhibitor of myostatin, in the soleus muscle of mice. VL blunted the hypergravity-enhanced levels of follistatin in the soleus muscle of mice. Simulated microgravity decreased follistatin mRNA level in mouse myoblastic C2C12 cells. Follistatin elevated the mRNA levels of myogenic genes as well as the phosphorylation of Akt and p70S6 kinase in C2C12 cells. As for bone metabolism, follistatin antagonized the mRNA levels of osteogenic genes suppressed by activin A during the differentiation of mesenchymal cells into osteoblastic cells. Moreover, follistatin attenuated osteoclast formation enhanced by myostatin in the presence of receptor activator of nuclear factor-B ligand in RAW 264.7 cells. Serum follistatin levels were positively related to bone mass in mouse tibia. In conclusion, the present study provides novel evidence that hypergravity affects follistatin levels in muscle through the vestibular system in mice. Follistatin may play some roles in the interactions between muscle and bone metabolism in response to gravity change.
  • Yuji Kanazawa; Keisuke Ikegami; Mitsugu Sujino; Satoshi Koinuma; Mamoru Nagano; Yuki Oi; Tomoya Onishi; Shinichi Sugiyo; Isao Takeda; Hiroshi Kaji; Yasufumi Shigeyoshi
    EXPERIMENTAL GERONTOLOGY PERGAMON-ELSEVIER SCIENCE LTD 98 153 - 161 0531-5565 2017/11 [Refereed]
     
    Aging is known to lead to the impaired recovery of muscle after disuse as well as the increased susceptibility of the muscle to damage. Here, we show that, in the older rats, reloading after disuse atrophy, causes the damage of the muscle fibers and the basement membrane (BM) that structurally support the muscle fibers. Male Wistar rats of 3-(young) and 20-(older) months of age were subjected to hindlimb-unloading for 2 weeks followed by reloading for a week. In the older rats, the soleus muscles showed necrosis and central nuclei fiber indicating the regeneration of muscle fibers. Furthermore, ectopic immunoreactivity of collagen IV, a major component of the BM, remained mostly associated with the necrotic appearance, suggesting that the older rats were impaired with the ability of repairing the damaged BM. Further, after unloading and reloading, the older rats did not show a significant alteration, although the young rats showed clear response of Col4a1 and Col4a2 genes, both coding for collagen IV. In addition, during the recovery phase, the young rats showed increase in the amount of Hsp47 and Sparc mRNA, which are protein folding-related factor genes, while the older rats did not show any significant variation. Taken together, our findings suggest that the atrophic muscle fibers of the older rats induced by unloading were vulnerable to the weight loading, and that attenuated reactivity of the BM-synthesizing fibroblast to gravity contributes to the fragility of muscle fibers in the older animals.
  • Akihiro Moritake; Naoyuki Kawao; Kiyotaka Okada; Kohei Tatsumi; Masayoshi Ishida; Katsumi Okumoto; Osamu Matsuo; Masao Akagi; Hiroshi Kaji
    BMC MUSCULOSKELETAL DISORDERS BIOMED CENTRAL LTD 18 392  1471-2474 2017/09 [Refereed]
     
    Background: Subchondral osteopenia is important for the pathophysiology of osteoarthritis (OA). Although previous studies suggest that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is related to bone metabolism, its role in OA remains unknown. We therefore investigated the roles of PAI-1 in the subchondral bone in OA model mice. Methods: Wild type (WT) and PAI-1-deficient (KO) mice were ovariectomized (OVX), and then destabilization of the medial meniscus (DMM) surgery was performed. Results: DMM and OVX significantly decreased the trabecular bone mineral density of the subchondral bone evaluated by quantitative computed tomography in PAI-1 KO mice. The effects of OVX and/or PAI-1 deficiency on the OARSI score for the evaluation of the progression of knee degeneration were not significant. PAI-1 deficiency significantly augmented receptor activator nuclear factor kappa B ligand mRNA levels enhanced by IL-1 beta in mouse primary osteoblasts, although it did not affect osteoblast differentiation. Moreover, PAI-1 deficiency significantly increased osteoclast formation from mouse bone marrow cells. Conclusion: We showed that PAI-1 deficiency accelerates the subchondral osteopenia after induction of OA in mice. PAI-1 might suppress an enhancement of bone resorption and subsequent subchondral osteopenia after induction of OA in mice.
  • Kiyotaka Okada; Kotarou Kojima; Katsumi Okumoto; Naoyuki Kawao; Osamu Matsuo; Hiroshi Kaji
    THROMBOSIS RESEARCH PERGAMON-ELSEVIER SCIENCE LTD 157 7 - 8 0049-3848 2017/09 [Refereed]
  • Yukinori Tamura; Haruko Fujito; Naoyuki Kawao; Hiroshi Kaji
    Diabetology International 8 (1) 52 - 58 2190-1678 2017/03 [Refereed]
     
    © 2016, The Japan Diabetes Society. We recently reported that vitamin D deficiency aggravates diabetic bone loss in mice. Although vitamin D affects both muscle and bone, the role of the vitamin D state in diabetic muscle loss and muscle-bone relationships remains unclear. In the present study, we examined the effects of vitamin D deficiency on muscle mass, muscle differentiation and muscle-derived humoral factors linking muscle to bone in diabetic female mice. Diabetes was induced in mice by streptozotocin (STZ) injection after feeding with a normal or vitamin D-deficient diet for 6 weeks. Quantitative computed tomography analysis showed that tibial muscle mass was significantly decreased in diabetic mice compared with control mice 4 weeks after induction of diabetes. Vitamin D deficiency accelerated muscle loss in diabetic mice. Vitamin D deficiency augmented the decreases in Pax7 mRNA levels and the increases in muscle RING-Finger Protein-1 and atrogin-1 mRNA levels induced by diabetes in the gastrocnemius muscle of mice. Moreover, vitamin D deficiency decreased the mRNA levels of insulin-like growth factor-1, fibroblast growth factor-2 and osteoglycin in muscle of diabetic mice. In conclusion, we demonstrated that vitamin D deficiency aggravates muscle loss induced by diabetes in female mice. Vitamin D may exert significant effects on the maintenance of the musculoskeletal system partly through the muscle-bone relationships in diabetic state.
  • Ikeda T; Kaji H; Akagi M
    Acta Med Kinki Univ Kindai University Medical Association 42 (1) 17 - 23 0386-6092 2017 [Refereed]
     
    [Abstract]Glucocorticoid(GC) therapy leads to an increase in fracture risk, and it is well recognized that bisphosphonates are effective for the treatment of GC-induced osteoporosis. We performed a non-randomized prospective study to clarify the effects of alendronate or alfacalcidol on the change in the trabecular bone score(TBS) at the lumbar spine and bone metabolic indices in 94 patients receiving high-dose GC therapy. The mean initial daily GC dose of predonisolone was 27.6±6.9 mg/day. Although the bone mineral density(BMD) at the lumbar spine continued to significantly decrease for 12 months in the alfacalcidol group, BMD increased above the baseline level at 3 and 6 months in the alendronate group. Alendronate significantly reduced urinary NTx levels from the baseline for 12 months, although alfacalcidol did not affect them. TBS was significantly decreased at 6 and 9 months in the alfacalcidol group. There were no significant differences in TBS between the alfacalcidol and alendronate groups for 12 months. TBS was significantly correlated with BMD at the lumbar spine in both groups for 12 months. In conclusion, alendronate treatment did not affect TBS at the lumbar spine in patients treated with GC, although TBS decreased with alfacalcidol.
  • Hiroshi Kaji
    Comprehensive Physiology JOHN WILEY & SONS INC 6 (4) 1873 - 1896 2040-4603 2016/10 [Refereed][Invited]
     
    Adipose tissue has recently been reevaluated as an endocrine organ, and adipose-tissue-derived endocrine factors are termed adipokines. Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of PAs, which convert plasminogen into plasmin, a critical protease involved in fibrinolysis. PAI-1 induces fibrinogenesis by suppressing intravascular and tissue fibrinolysis. Moreover, PAI-1 exerts various cellular effects independently of fibrinolysis. Although PAI-1 is expressed in various tissues, its expression is regulated by numerous growth factors, cytokines, and hormones in a paracrine and endocrine manner. Adipocyte-derived PAI-1, predominantly expressed in visceral fat, is released into the circulation in parallel with increased fat mass, and it functions as a crucial adipokine that negatively affects physiological metabolism and vascular biology. Elevated PAI-1 levels induce insulin resistance and metabolic abnormalities during proinflammatory processes involving several cytokines and chemokines in diabetes. Several studies have indicated that PAI-1 plays crucial roles in insulin actions on liver, muscle, and fat. Accumulated fat and enhanced adipose tissue-derived PAI-1 influence metabolism and vessels in relation to macrophage infiltration, chronic inflammation, and free fatty acid release in obese states. PAI-1-induced fibrinolysis abnormalities are associated with metabolic syndrome, leading to cardiovascular disease through dysregulated vascular coagulation, endothelial dysfunction, and metabolic abnormalities. Adipose tissue-derived PAI-1 is involved in insulin resistance, osteoporosis, and sarcopenia induced by glucocorticoid excess in mice. Moreover, PAI-1 is involved in the other pathological states, such as nonalcoholic fatty liver disease, and cancer. As such, PAI-1 may be exploited as a marker of disease activity as well being a target for clinical drug development. (C) 2016 American Physiological Society.
  • Takeshi Shimoide; Naoyuki Kawao; Yukinori Tamura; Hironobu Morita; Hiroshi Kaji
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 479 (3) 602 - 606 0006-291X 2016/10 [Refereed]
     
    Skeletal muscle hypertrophy and wasting are induced by hypergravity and microgravity, respectively. However, the mechanisms by which gravity change regulates muscle mass still remain unclear. We previously reported that hypergravity increases muscle mass via the vestibular system in mice. In this study, we performed comparative DNA microarray analysis of the soleus muscle from mice kept in 1 or 3 g environments with or without vestibular lesions. Mice were kept in 1 g or 3 g environment for 4 weeks by using a centrifuge 14 days after surgical bilateral vestibular lesions. FKBP5 was extracted as a gene whose expression was enhanced by hypergravity through the vestibular system. Stable FKBP5 overexpression increased the phosphorylations of Akt and p70 S6 kinase (muscle protein synthesis pathway) and myosin heavy chain, a myotube gene, mRNA level in mouse myoblastic C2C12 cells, although it reduced the mRNA levels of atrogin-1 and MuRF1, muscle protein degradation-related genes. In conclusion, we first showed that FKBP5 is induced by hypergravity through the vestibular system in anti-gravity muscle of mice. Our data suggest that FKBP5 might increase muscle mass through the enhancements of muscle protein synthesis and myotube differentiation as well as an inhibition of muscle protein degradation in mice. (C) 2016 Elsevier Inc. All rights reserved.
  • Naoyuki Kawao; Hironobu Morita; Koji Obata; Yukinori Tamura; Katsumi Okumoto; Hiroshi Kaji
    Physiological reports 4 (19) 2051-817X 2016/10 [Refereed]
     
    Gravity changes concurrently affect muscle and bone as well as induce alterations in vestibular signals. However, the role of vestibular signals in the changes in muscle and bone induced by gravity changes remains unknown. We therefore investigated the effects of vestibular lesions (VL) on the changes in muscle and bone induced by 3 g hypergravity for 4 weeks in C57BL/6J mice. Quantitative computed tomography analysis revealed that hypergravity increased muscle mass surrounding the tibia and trabecular bone mineral content, adjusting for body weight in mice. Hypergravity did not affect cortical bone and fat masses surrounding the tibia. Vestibular lesions blunted the increases in muscle and bone masses induced by hypergravity. Histological analysis showed that hypergravity elevated the cross-sectional area of myofiber in the soleus muscle. The mRNA levels of myogenic genes such as MyoD, Myf6, and myogenin in the soleus muscle were elevated in mice exposed to hypergravity. Vestibular lesions attenuated myofiber size and the mRNA levels of myogenic differentiation markers enhanced by hypergravity in the soleus muscle. Propranolol, a β-blocker, antagonized the changes in muscle induced by hypergravity. In conclusion, this study is the first to demonstrate that gravity changes affect muscle and bone through vestibular signals and subsequent sympathetic outflow in mice.
  • Naoyuki Kawao; Masato Yano; Yukinori Tamura; Katsumi Okumoto; Kiyotaka Okada; Hiroshi Kaji
    JOURNAL OF BONE AND MINERAL METABOLISM SPRINGER JAPAN KK 34 (5) 517 - 525 0914-8779 2016/09 [Refereed]
     
    Fibrodysplasia ossificans progressiva (FOP) is a disorder of skeletal malformations and progressive heterotopic ossification. The constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2), is responsible for the pathogenesis of FOP. Although transfection of the causal mutation of FOP into myoblasts enhances osteoclast formation by transforming growth factor-beta (TGF-beta), the role of osteoclasts in heterotopic ossification is unknown. We therefore examined the effects of alendronate, SB431542 and SB203580 on heterotopic ossification induced by the causal mutation of FOP. Total bone mineral content as well as numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated and alkaline phosphatase (ALP)-positive cells in heterotopic bone were significantly higher in muscle tissues implanted with ALK2 (R206H)-transfected mouse myoblastic C2C12 cells than in the tissues implanted with empty vector-transfected cells in nude mice. Alendronate, an aminobisphosphonate, did not affect total mineral content or numbers of TRAP-positive multinucleated and ALP-positive cells in heterotopic bone, which were enhanced by the implantation of ALK2 (R206H)-transfected C2C12 cells, although it significantly decreased serum levels of cross-linked C-telopeptide of type I collagen, a bone resorption index. Moreover, neither SB431542, an inhibitor of TGF-beta receptor type I kinase, nor SB203580, an inhibitor of p38 mitogen-activated protein kinase, affected the increase in heterotopic ossification due to the implantation of ALK2 (R206H)-transfected C2C12 cells. In conclusion, the present study indicates that osteoclast inhibition does not affect heterotopic ossification enhanced by FOP-related mutation.
  • Kaji H
    Bonekey Reports 5 826  2016 [Refereed][Invited]
  • Tanaka K; Kanazawa I; Kaji H; Sugimoto T
    Bone 95 5 - 10 2016 [Refereed]
  • Kiyotaka Okada; Naoyuki Kawao; Masato Yano; Yukinori Tamura; Shinzi Kurashimo; Katsumi Okumoto; Kotarou Kojima; Hiroshi Kaji
    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM AMER PHYSIOLOGICAL SOC 310 (1) E15 - E23 0193-1849 2016/01 [Refereed]
     
    Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in 75 mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The number of HSCs in the bone marrow of mice with damaged femurs was significantly lower than the number of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Meanwhile, the number of MSCs in the bone marrow of mice with damaged femurs was significantly higher than that of the contralateral femurs. Both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease in HSC and increase in MSC populations within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury.
  • Akihito Shiomi; Naoyuki Kawao; Masato Yano; Kiyotaka Okada; Yukinori Tamura; Katsumi Okumoto; Osamu Matsuo; Masao Akagi; Hiroshi Kaji
    BONE ELSEVIER SCIENCE INC 79 233 - 241 8756-3282 2015/10 [Refereed]
     
    The mechanism of postmenopausal osteoporosis is not fully understood. alpha(2)-Antiplasmin (alpha(2)-AP) is the primary inhibitor of plasmin in the fibrinolytic system, but is known to have activities beyond fibrinolysis. However, its role in bone metabolism and the pathogenesis of osteoporosis remains unknown. In the current study, we therefore examined the effects of alpha(2)-AP deficiency on ovariectomy (OVX)-induced bone loss by using wildtype and alpha(2)-AP-deficient mice. Quantitative computed tomography analysis revealed that alpha(2)-AP deficiency blunted OVX-induced trabecular bone loss in mice. Moreover, alpha(2)-AP deficiency significantly blunted serum levels of bone-specific alkaline phosphatase, cross-linked C-telopeptide of type I collagen, and interleukin (IL)-1 beta elevated by OVX. alpha(2)-AP treatment elevated the levels of IL-1 beta and tumor necrosis factor (TNF)-alpha mRNA in RAW 264.7 cells, although it suppressed osteoclast formation induced by receptor activator of nuclear factor-kappa B ligand. alpha(2)-AP treatment activated ERK1/2 and p38 MAP kinase pathways in RAW 264.7 cells, and these MAP kinase inhibitors antagonized the levels of IL-1 beta mRNA elevated by alpha(2)-AP. The data demonstrate that alpha(2)-AP is linked to bone loss due to OVX, through a mechanism that depends in part on the production of IL-1 beta and TNF-alpha in monocytes. (C) 2015 Elsevier Inc. All rights reserved.
  • Yukinori Tamura; Naoyuki Kawao; Masato Yano; Kiyotaka Okada; Katsumi Okumoto; Yasutaka Chiba; Osamu Matsuo; Hiroshi Kaji
    DIABETES AMER DIABETES ASSOC 64 (6) 2194 - 2206 0012-1797 2015/06 [Refereed]
     
    Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic mechanism, however, is not completely understood. In this study, we used plasminogen activator inhibitor-1 (PAI-1)-deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting. Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1 suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover, the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the current study indicated that PAI-1 is involved in GC-induced glucose metabolism abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic target to mitigate the adverse effects of GC.
  • Naoyuki Kawao; Hiroshi Kaji
    JOURNAL OF CELLULAR BIOCHEMISTRY WILEY-BLACKWELL 116 (5) 687 - 695 0730-2312 2015/05 [Refereed][Invited]
     
    Sarcopenia and osteoporosis have recently been noted for their relationship with locomotive syndrome and increased number of older people. Sarcopenia is defined by decreased muscle mass and impaired muscle function, which may be associated with frailty. Several clinical data have indicated that increased muscle mass is related to increased bone mass and reduced fracture risk. Genetic, endocrine and mechanical factors as well as inflammatory and nutritional states concurrently affect muscle tissues and bone metabolism. Several genes, including myostatin and a-actinin 3, have been shown in a genome-wide association study (GWAS) to be associated with both sarcopenia and osteoporosis. Vitamin D, growth hormone and testosterone as well as pathological disorders, such as an excess in glucocorticoid and diabetes, affect both muscle and bone. Basic and clinical research of bone metabolism and muscle biology suggests that bone interacts with skeletal muscle via signaling from local and humoral factors in addition to their musculoskeletal function. However, the physiological and pathological mechanisms related to muscle and bone interactions remain unclear. We found that Tmem119 may play a critical role in the commitment of myoprogenitor cells to the osteoblast lineage. We also reported that osteoglycin and FAM5C might be muscle-derived humoral osteogenic factors. Other factors, including myostatin, osteonectin, insulin-like growth factor I, irisin and osteocalcin, may be associated with the interactions between muscle tissues and bone metabolism. J. Cell. Biochem. 116: 687-695, 2015. (C) 2014 Wiley Periodicals, Inc.
  • Naoyuki Kawao; Yukinori Tamura; Yoshitaka Horiuchi; Katsumi Okumoto; Masato Yano; Kiyotaka Okada; Osamu Matsuo; Hiroshi Kaji
    PLOS ONE PUBLIC LIBRARY SCIENCE 10 (4) e0123982  1932-6203 2015/04 [Refereed]
     
    Macrophages play crucial roles in repair process of various tissues. However, the details in the role of macrophages during bone repair still remains unknown. Herein, we examined the contribution of the tissue fibrinolytic system to the macrophage functions in bone repair after femoral bone defect by using male mice deficient in plasminogen (Plg(-/-)), urokinase-type plasminogen activator (uPA(-/-)) or tissue-type plasminogen activator (tPA(-/-)) genes and their wild-type littermates. Bone repair of the femur was delayed in uPA(-/-) mice until day 6, compared with wild-type (uPA(+/+)) mice. Number of Osterix-positive cells and vessel formation were decreased in uPA(-/-) mice at the bone injury site on day 4, compared with those in uPA(+/+) mice. Number of macrophages and their phagocytosis at the bone injury site were reduced in uPA(-/-) and Plg(-/-), but not in tPA(-/-) mice on day 4. Although uPA or plasminogen deficiency did not affect the levels of cytokines, including TNF-alpha, IL-1 beta, IL-6, IL-4 and IFN-gamma mRNA in the damaged femur, the elevation in CCL3 mRNA levels was suppressed in uPA(-/-) and Plg(-/-), but not in tPA(-/-) mice. Neutralization of CCL3 antagonized macrophage recruitment to the site of bone injury and delayed bone repair in uPA(+/+), but not in uPA(-/-) mice. Our results provide novel evidence that the tissue fibrinolytic system contributes to the induction of macrophage recruitment and CCL3 at the bone injury site, thereby, leading to the enhancement of the repair process.
  • Naoyuki Kawao; Yukinori Tamura; Katsumi Okumoto; Masato Yano; Kiyotaka Okada; Osamu Matsuo; Hiroshi Kaji
    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM AMER PHYSIOLOGICAL SOC 307 (3) E278 - E288 0193-1849 2014/08 [Refereed]
     
    Further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Recently, we reported that plasminogen is crucial for bone repair through enhancement of vessel formation. However, the details of the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in the bone repair process still remain unknown. Herein, we examined the effects of plasminogen activators on bone repair after a femoral bone defect using tPA-deficient (tPA(-/-)) and uPA-deficient (uPA(-/-)) mice. Bone repair of the femur was delayed in tPA-/- mice, unlike that in wild-type (tPA(-/-)) mice. Conversely, the bone repair was comparable between wild-type (uPA(+/+)) and uPA(-/-) mice. The number of proliferative osteoblasts was decreased at the site of bone damage in tPA(-/-) mice. Moreover, the proliferation of primary calvarial osteoblasts was reduced in tPA(-/-) mice. Recombinant tPA facilitated the proliferation of mouse osteoblastic MC3T3-E1 cells. The proliferation enhanced by tPA was antagonized by the inhibition of endogenous annexin 2 by siRNA and by the inhibition of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in MC3T3-E1 cells. Vessel formation as well as the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 alpha(HIF-1 alpha) were decreased at the damaged site in tPA(-/-) mice. Our results provide novel evidence that tPA is crucial for bone repair through the facilitation of osteoblast proliferation related to annexin 2 and ERK1/2 as well as enhancement of vessel formation related to VEGF and HIF-1 alpha at the site of bone damage.
  • Ken-ichiro Tanaka; Ippei Kanazawa; Toru Yamaguchi; Shozo Yano; Hiroshi Kaji; Toshitsugu Sugimoto
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 450 (1) 482 - 487 0006-291X 2014/07 [Refereed]
     
    Vitamin D deficiency and advanced glycation end products (AGEs) are suggested to be involved in the pathogenesis of osteoporosis and sarcopenia. However, the effects of vitamin D and AGEs on myogenesis and the interaction between muscle and bone remains still unclear. We previously showed that osteoglycin (OGN) is secreted from myoblasts and stimulates osteoblastic differentiation, suggesting that it plays important roles in the interaction between muscle and bone. The aim of this study is thus to examine the effects of vitamin D and AGEs on myoblastic differentiation of C2C12 cells and osteoblastic differentiation of osteoblastic MC3T3-E1 cells through OGN expression. 1 alpha,25-dihydroxyvitamin D-3 (1,25D) and eldecalcitol, an active vitamin D analog, induced the expression of MyoD, myogenin and OGN, and these effects were abolished by vitamin D receptor (VDR) suppression by siRNA in C2C12 cells. Moreover, conditioned medium from 1,25D-pretreated C2C12 cells stimulated the expression of type I collagen and alkaline phosphatase in MC3T3-E1 cells, compared to control medium from 1,25D-untreated C2C12 cells. In contrast, conditioned medium from VDR-suppressed and 1,25D-pretreated C2C12 cells showed no effects. AGE2 and AGE3 suppressed the expression of MyoD, myogenin and OGN in C2C12 cells. Moreover, 1,25D blunted the AGEs' effects. In conclusion, these findings showed for the first time that active vitamin D plays important roles in myogenesis and muscle-induced osteoblastogenesis through OGN expression. Active vitamin D treatment may rescue the AGEs-induced sarcopenia as well as - suppressed osteoblastic differentiation via OGN expression in myoblasts. (C) 2014 Elsevier Inc. All rights reserved.
  • Terumasa Ikeda; Kouichi Maruyama; Hiroshi Kaji; Masao Akagi
    MODERN RHEUMATOLOGY SPRINGER 24 (4) 671 - 676 1439-7595 2014/07 [Refereed]
     
    Objectives. Glucocorticoid (GC) is usually used for the treatment of systemic inflammatory diseases. We performed the prospective study to clarify the effects of alendronate or alfacalcidol on bone metabolic indices and bone mineral density (BMD) in 90 patients treated with GC for ophthalmologic diseases without systemic disorders for 12 months. Methods. BMD was measured with dual-energy X-ray absorptiometry. Serum bone-specific alkaline phosphatase (BAP) and urinary Type I collagen cross-linked N-telopeptide (NTx) were measured as bone metabolic indices. Results. BMD values in the alendronate group were significantly higher than those in the alfacalcidol group during 12 months. Alendronate significantly reduced urinary NTX levels from the baseline during 12 months, although alfacalcidol did not affect them. Serum BAP levels in the alendronate group were significantly lower than those in the alfacalcidol group during 9 months. The effects of alendronate on BMD and NTx in male patients seemed to be somewhat potent compared with those in female patients. Conclusions. Alendronate is effective to prevent BMD loss and bone resorption induced by GC treatment in patients with ophthalmic diseases without systemic disorders. There might be sex differences in the potency of alendronate effects.
  • Masato Yano; Naoyuki Kawao; Katsumi Okumoto; Yukinori Tamura; Kiyotaka Okada; Hiroshi Kaji
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 289 (24) 16966 - 16977 0021-9258 2014/06 [Refereed]
     
    Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-beta, whereas the inhibition of TGF-beta signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-beta in muscle tissues.
  • Yukinori Tamura; Shigeshi Mori; Shigeki Asada; Naoyuki Kawao; Shigeru Ueshima; Hiroshi Kaji; Junichiro Yamamoto; Masao Akagi; Osamu Matsuo
    Thrombosis Journal 12 (1) 11  2014/05 [Refereed]
     
    Background: Deep venous thrombosis (DVT), which is often associated with pulmonary embolism (PE), is a serious complication after total knee arthroplasty (TKA). In the present study, we examined the overall thrombotic and thrombolytic status using Global Thrombosis Test (GTT) in non-anticoagulated blood of patients undergoing TKA to develop the predictable marker for the incidence of DVT.Methods: DVT was diagnosed using doppler ultrasonography a day after the surgery in 31 patients with osteoarthritis (n = 24), rheumatoid arthritis (n = 6) and ankylosing spondylitis (n = 1) by the well-trained operator. We measured overall thrombotic and thrombolytic status using GTT and other biomarkers, which is associated with blood coagulation and fibrinolysis, before and immediately after the surgery.Results: Newly-generated DVT during the operation was detected in 11 of 31 patients (35.4%) 1 day after TKA. There were no differences in markers of coagulation (PT and APTT), platelet activity (platelet aggregation-induced by ADP and collagen) and fibrinolysis (FDP and D-dimer) between non-DVT and DVT group both before and after the surgery. Both Pre- and Post-operative GTT-occlusion times (OT), an index of platelet reactivity, were tended to be shorter, but not significant, in DVT group compared with non-DVT group. Pre-operative GTT-lysis time (LT), an index of thrombolytic activity, was significantly shorter in DVT group compared with non-DVT group, while there were no differences in post-operative value of this index between DVT group and non-DVT group, suggesting overall thrombolytic activity was enhanced in DVT group before surgery.Conclusions: Our data suggest that enhancement of pre-operative thrombolytic activity assessed by GTT may be a predictable marker for the incidence of DVT after TKA. © 2014 Tamura et al.; licensee BioMed Central Ltd.
  • Yukinori Tamura; Naoyuki Kawao; Masato Yano; Kiyotaka Okada; Osamu Matsuo; Hiroshi Kaji
    ENDOCRINOLOGY ENDOCRINE SOC 155 (5) 1708 - 1717 0013-7227 2014/05 [Refereed]
     
    We previously demonstrated that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is involved in type 1 diabetic bone loss in female mice. PAI-1 is well known as an adipogenic factor induced by obesity. We therefore examined the effects of PAI-1 deficiency on bone and glucose and lipid metabolism in high-fat and high-sucrose diet (HF/HSD)-induced obese female mice. Female wild-type (WT) and PAI-1-deficient mice were fed with HF/HSD or normal diet for 20 weeks from 10 weeks of age. HF/HSD increased the levels of plasma PAI-1 in WT mice. PAI-1 deficiency suppressed the levels of blood glucose, plasma insulin, and total cholesterol elevated by obesity. Moreover, PAI-1 deficiency improved glucose intolerance and insulin resistance induced by obesity. Bone mineral density (BMD) at trabecular bone as well as the levels of osterix, alkaline phosphatase, and receptor activator of nuclear factor kappa B ligand mRNA in tibia were decreased by HF/HSD in WT mice, and those changes by HF/HSD were not affected by PAI-1 deficiency. HF/HSD increased the levels of plasma TNF-alpha in both WT and PAI-1-deficient mice, and the levels of plasma TNF-alpha were negatively correlated with trabecular BMD in tibia of female mice. In conclusion, we revealed that PAI-1 deficiency does not affect the trabecular bone loss induced by obesity despite the amelioration of insulin resistance and hyperlipidemia in female mice. Our data suggest that the changes of BMD and bone metabolism by obesity might be independent of PAI-1 as well as glucose and lipid metabolism.
  • Li Mao; Yukinori Tamura; Naoyuki Kawao; Kiyotaka Okada; Masato Yano; Katsumi Okumoto; Hiroshi Kaji
    BONE ELSEVIER SCIENCE INC 61 102 - 108 8756-3282 2014/04 [Refereed]
     
    Type 1 diabetes is associated with an increased fracture risk, an impaired fracture healing, and an increased vitamin D insufficiency. However, the role of vitamin D in diabetic bone repair process remains unclear. We therefore examined the effects of vitamin D deficiency on the impaired bone repair in streptozotocin (STZ)-induced diabetes using female mice. Diabetes was induced by STZ injection into female mice after feeding with normal or vitamin D-deficient diet for 6 weeks from the age of 4 weeks. A femoral bone defect was induced in mice 4 weeks after induction of diabetes. The repair of damaged site on the femur was significantly delayed at days 7 and 10 after bone defect by diabetic state in mice, as assessed by quantitative computed tomography, while vitamin D deficiency did not affect the bone repair both in mice with normal and diabetic state. The decreases in bone mineral density (BMD) at cortical and trabecular bone by diabetic state were significantly augmented by vitamin D deficiency in tibia at the undamaged side in mice. Diabetic state blunted the levels of osteogenic and chondrogenic genes enhanced by vitamin D deficiency. Moreover, vitamin D deficiency significantly aggravated the decreases in osteocalcin and IGF-1 mRNA by diabetic state. In conclusion, our study showed that vitamin D deficiency aggravates the decrease in BMD by diabetic state in female mice, although vitamin D deficiency did not affect bone repair delayed by diabetic state. (C) 2013 Elsevier Inc. All rights reserved.
  • Ken-ichiro Tanaka; Hiroshi Kaji; Toru Yamaguchi; Ippei Kanazawa; Lucie Canaff; Geoffrey N. Hendy; Toshitsugu Sugimoto
    CALCIFIED TISSUE INTERNATIONAL SPRINGER 94 (4) 454 - 464 0171-967X 2014/04 [Refereed]
     
    The osteoinductive factors BMP-2 and Tmem119 that promote the differentiation of myoblasts into osteoblasts, each increase the levels of the other. However, the relative contributions of BMP-2 and Tmem119 to the osteogenic differentiation and the mechanisms involved are incompletely understood. In the present study, we examined the relationship among BMP-2, Tmem119, and the PERK-eIF2 alpha-ATF4 endoplasmic reticulum (ER) stress response pathway in the differentiation of C2C12 myoblasts into osteoblastic cells. Both BMP-2 and Tmem119 induced levels of the osteoblast markers Runx2, Osterix, Col1a1, ALP, and osteocalcin, as well as mineralization. BMP-2 activation of the ER stress sensor PERK stimulated phosphorylation of eIF2 alpha and led to increased biosynthesis of the osteoblast differentiation factor ATF4. When dephosphorylation of eIF2 alpha was blocked by the selective inhibitor salubrinal, the osteogenic effects of BMP-2 and Tmem119 were enhanced further. Although BMP-2 stimulated both P-eIF2 alpha and ATF4 levels, Tmem119 had no effect on P-eIF2 alpha but stimulated ATF4 only. Reduction in endogenous Tmem119 levels by siRNA reduced both basal and BMP-2-stimulated levels of the ATF4 protein. In conclusion, BMP-2 stimulates differentiation of myoblasts into osteoblasts via the PERK-eIF2 alpha-ATF4 pathway but in addition stimulates Tmem119, which itself increases ATF4. Hence, BMP-2 stimulates ATF4 both dependently and independently of the PERK-eIF2 alpha ER stress response pathway.
  • Li Mao; Naoyuki Kawao; Yukinori Tamura; Katsumi Okumoto; Kiyotaka Okada; Masato Yano; Osamu Matsuo; Hiroshi Kaji
    PLOS ONE PUBLIC LIBRARY SCIENCE 9 (3) e92686  1932-6203 2014/03 [Refereed]
     
    Previous studies suggest that fracture healing is impaired in diabetes; however, the underlying mechanism remains unclear. Here, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in the impaired bone repair process by using streptozotocin (STZ)-induced diabetic female wild-type (PAI-1(+/+)) and PAI-1-deficient (PAI-1(-/-)) mice. Bone repair and the number of alkaline phosphatase (ALP)-positive cells at the site of a femoral bone damage were comparable in PAI-1(+/+) and PAI-1(-/-) mice without STZ treatment. Although the bone repair process was delayed by STZ treatment in PAI-1(+/+) mice, this delayed bone repair was blunted in PAI-1(-/-) mice. The reduction in the number of ALP-positive cells at the site of bone damage induced by STZ treatment was attenuated in PAI-1(-/-) mice compared to PAI-1(+/+) mice. On the other hand, PAI-1 deficiency increased the levels of ALP and type I collagen mRNA in female mice with or without STZ treatment, and the levels of Osterix and osteocalcin mRNA, suppressed by diabetic state in PAI-1(+/+) mice, were partially protected in PAI-1(-/-) mice. PAI-1 deficiency did not affect formation of the cartilage matrix and the levels of types II and X collagen and aggrecan mRNA suppressed by STZ treatment, although PAI-1 deficiency increased the expression of chondrogenic markers in mice without STZ treatment. The present study indicates that PAI-1 is involved in the impaired bone repair process induced by the diabetic state in part through a decrease in the number of ALP-positive cells.
  • Kaji H
    J Bone Metab 21 29 - 40 2014 [Refereed][Invited]
  • M. Yano; N. Kawao; Y. Tamura; K. Okada; H. Kaji
    EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES JOHANN AMBROSIUS BARTH VERLAG MEDIZINVERLAGE HEIDELBERG GMBH 122 (1) 7 - 14 0947-7349 2014/01 [Refereed]
     
    Previous studies have suggested some interactions between muscle tissues and bone metabolism. The constitutively activating mutation (R206H) of the BMP type I receptor, activin-like-kinase 2 (ALK2), causes fibrodysplasia ossificans progressiva (FOP), which is characterized by extensive ossifications within muscle tissues. In the present study, we revealed that Tmem176b mRNA levels were upregulated by stable transfection of ALK2 (R206H) in mouse myoblastic C2C12 cells. Transient Tmem176b overexpression elevated levels of osteoblast differentiation markers, such as Osterix and alkaline phosphatase, as well as mineralization in C2C12 cells. In addition, Tmem176b overexpression elevated the levels of these markers in mouse osteoblastic MC3T3-E1 cells. On the other hand, Tmem176b overexpression suppressed the levels of myogenic markers, such as MyoD and myogenin in C2C12 cells, although it did not affect the levels of chondrogenic markers, such as type II and X collagens. In conclusion, the present study is the first to demonstrate that Tmem176b induces the differentiation of myoblasts into an osteoblast lineage.
  • Li Mao; Masato Yano; Naoyuki Kawao; Yukinori Tamura; Kiyotaka Okada; Hiroshi Kaji
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOC 60 (12) 1309 - 1319 0918-8959 2013/12 [Refereed]
     
    Fibrodysplasia ossificans progressiva (FOP) is a skeletal disorder with progressive heterotopic ossification in skeletal muscle. A mutation causing constitutive activation in a bone morphogenetic protein (BMP) type 1 receptor [ALK2(R206H)] is found in most patients with FOP. However, the details in the heterotopic ossification of muscle in FOP and the role of matrix metalloproteinase-10 (MMP-10) in bone remain to be fully elucidated. In the present study, we investigated the role of MMP-10 in the differentiation of mouse myoblastic C2C12 cells into osteoblasts. MMP-10 was extracted as a factor, whose expression was most extensively enhanced by ALK2 (R206H) transfection in C2C12 cells. MMP-10 significantly augmented the levels of Osterix, type 1 collagen, alkaline phosphatase (ALP) and osteocalcin mRNA as well as ALP activity enhanced by BMP-2 in C2C12 cells. Moreover, a reduction in endogenous MMP-10 levels by siRNA significantly decreased the levels of Runx2, Osterix, type 1 collagen, ALP and osteocalcin mRNA enhanced by BMP-2 in these cells. In addition, MMP-10 increased the phosphorylation of Smad1/5/8 as well as enhanced the levels of Smad6 and Smad7 mRNA induced by BMP-2. In conclusion, the present study first demonstrated that MMP-10 promotes the differentiation of myoblasts into osteoblasts by interacting with the BMP signaling pathway. MMP-10 may play some important role in the heterotopic ossification of muscle in FOP.
  • Koji Sugioka; Aya Kodama; Kiyotaka Okada; Mihoko Iwata; Koji Yoshida; Shunji Kusaka; Chota Matsumoto; Hiroshi Kaji; Yoshikazu Shimomura
    EXPERIMENTAL EYE RESEARCH ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD 115 13 - 21 0014-4835 2013/10 [Refereed]
     
    Transforming growth factor-beta (TGF-beta) is one of the main epithelial mesenchymal transition (EMT)-inducing factors. In general, TGF-beta-induced EMT promotes cell migration and invasion. TGF-beta also acts as a potent regulator of pericellular proteolysis by regulating the expression and secretion of plasminogen activators. Urokinase-type plasminogen activator (uPA) is a serine protease that binds to its cell surface receptor (uPAR) with high affinity. uPA binding to uPAR stimulates uPAR's interaction with transmembrane proteins, such as integrins, to regulate cytoskeletal reorganization and cell migration, differentiation and proliferation. However, the influence of TGF-beta and the uPA/uPAR system on EMT in retinal pigment epithelial (RPE) cells is still unclear. The purpose of this study was to determine the effect of TGF-beta 2, which is the predominant isoform in the retina, and the uPA/uPAR system on RPE cells. In this study, we first examined the effect of TGF-beta 2 and/or the inhibitor of uPA (u-PA-STOP (R)) on the proliferation of a human retinal pigment epithelial cell line (ARPE-19 cells). Treatment with TGF-beta 2 or u-PA-STOP (R) suppressed cell proliferation. Combination treatment of TGF-beta 2 and u-PA-STOP (R) enhanced cell growth suppression. Furthermore, western blot analysis, fibrin zymography and real-time reverse transcription PCR showed that that TGF-beta 2 induced EMT in ARPE-19 cells and that the expression of uPA and uPAR expression was up-regulated during EMT. The TGF-beta inhibitor SB431542 suppressed TGF-beta 2-stimulated uPA expression and secretion but did not suppress uPAR expression. Furthermore, we seeded ARPE-19 cells onto Transwell chambers and allowed them to invade the collagen matrix in the presence of TGF-beta 2 alone or with TGF-beta 2 and u-PA-STOP (R). TGF-beta 2 treatment induced ARPE-19 cell invasion into the collagen gel. Treatment with a combination of TGF-beta 2 and the uPA inhibitor strongly inhibited ARPE-19 cell invasion compared with treatment with TGF-beta 2 alone. Furthermore, the interaction between uPA and ARPE-19 cells was analyzed using a surface plasmon biosensor system. The binding of uPA to ARPE-19 cells was observed. In addition, TGF-beta 2 significantly promoted the binding activity of uPA to ARPE-19 cells in a time-dependent or cell-number-dependent fashion. These results indicate that TGF-beta-induced EMT-associated phenotype changes in ARPE-19 cells and the invasiveness of ARPE-19 cells into a collagen gel matrix are mediated, at least in part, by uPA. (C) 2013 Elsevier Ltd. All rights reserved.
  • Yukinori Tamura; Naoyuki Kawao; Kiyotaka Okada; Masato Yano; Katsumi Okumoto; Osamu Matsuo; Hiroshi Kaji
    Diabetes 62 (9) 3170 - 3179 0012-1797 2013/09 [Refereed]
     
    In diabetic patients, the risk of fracture is high because of impaired bone formation. However, the details of the mechanisms in the development of diabetic osteoporosis remain unclear. In the current study, we investigated the role of plasminogen activator inhibitor (PAI)-1 in the pathogenesis of type 1 diabetic osteoporosis by using PAI-1deficient mice. Quantitative computed tomography analysis showed that PAI-1 deficiency protected against streptozotocin-induced bone loss in female mice but not in male mice. PAI-1 deficiency blunted the changes in the levels of Runx2, osterix, and alkaline phosphatase in tibia as well as serum osteocalcin levels suppressed by the diabetic state in female mice only. Furthermore, the osteoclast levels in tibia, suppressed in diabetes, were also blunted by PAI-1 deficiency in female mice. Streptozotocin markedly elevated the levels of PAI-1 mRNA in liver in female mice only. In vitro study demonstrated that treatment with active PAI-1 suppressed the levels of osteogenic genes and mineralization in primary osteoblasts from female mouse calvaria. In conclusion, the current study indicates that PAI-1 is involved in the pathogenesis of type 1 diabetic osteoporosis in females. The expression of PAI-1 in the liver and the sensitivity of bone cells to PAI-1 may be an underlying mechanism. © 2013 by the American Diabetes Association.
  • Ken-ichiro Tanaka; Toru Yamaguchi; Hiroshi Kaji; Ippei Kanazawa; Toshitsugu Sugimoto
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 438 (3) 463 - 467 0006-291X 2013/08 [Refereed]
     
    Advanced glycation end products (AGES) are involved in bone quality deterioration in diabetes mellitus. We previously showed that AGE2 or AGE3 inhibited osteoblastic differentiation and mineralization of mouse stromal ST2 cells, and also induced apoptosis and decreased cell growth. Although quality management for synthesized proteins in endoplasmic reticulum (ER) is crucial for the maturation of osteoblasts, the effects of AGEs on ER stress in osteoblast lineage are unknown. We thus examined roles of ER stress in AGE2- or AGE3-induced suppression of osteoblastogenesis of ST2 cells. An ER stress inducer, thapsigargin (TG), induced osteoblastic differentiation of ST2 cells by increasing the levels of Osterix, type 1 collagen (Coil), alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA. AGE2 or AGE3 suppressed the levels of ER stress sensors such as IRE1 alpha, ATF6 and OASIS, while they increased the levels of PERK and its downstream molecules, ATF4. A reduction in PERK level by siRNA did not affect the AGEs-induced suppression of the levels of Osterix, Coll and OCN mRNA. In conclusion, AGEs inhibited the osteoblastic differentiation of stromal cells by suppressing ER stress sensors and accumulating abnormal proteins in the cells. This process might accelerate AGEs-induced suppression of bone formation found in diabetes mellitus. (C) 2013 Elsevier Inc. All rights reserved.
  • Naoyuki Kawao; Yukinori Tamura; Katsumi Okumoto; Masato Yano; Kiyotaka Okada; Osamu Matsuo; Hiroshi Kaji
    JOURNAL OF BONE AND MINERAL RESEARCH WILEY-BLACKWELL 28 (7) 1561 - 1574 0884-0431 2013/07 [Refereed]
     
    The further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Plasminogen is a critical factor of the tissue fibrinolytic system, which mediates tissue repair in the skin and liver. However, the role of the fibrinolytic system in bone regeneration remains unknown. Herein, we investigated bone repair and ectopic bone formation using plasminogen-deficient (Plg-/-) mice. Bone repair of the femur is delayed in Plg-/- mice, unlike that in the wild-type (Plg+/+) mice. The deposition of cartilage matrix and osteoblast formation were both decreased in Plg-/- mice. Vessel formation, macrophage accumulation, and the levels of vascular endothelial growth factor (VEGF) and transforming growth factor- (TGF-) were decreased at the site of bone damage in Plg-/- mice. Conversely, heterotopic ossification was not significantly different between Plg+/+ and Plg-/- mice. Moreover, angiogenesis, macrophage accumulation, and the levels of VEGF and TGF- were comparable between Plg+/+ and Plg-/- mice in heterotopic ossification. Our data provide novel evidence that plasminogen is essential for bone repair. The present study indicates that plasminogen contributes to angiogenesis related to macrophage accumulation, TGF-, and VEGF, thereby leading to the enhancement of bone repair.
  • Hiroshi Kaji
    CURRENT OPINION IN CLINICAL NUTRITION AND METABOLIC CARE LIPPINCOTT WILLIAMS & WILKINS 16 (3) 272 - 277 1363-1950 2013/05 [Refereed][Invited]
     
    Purpose of review This review summarizes the recent articles and perspectives about the linkage between muscle and bone. Moreover, it focuses on common, clinically important signals affecting both muscle and bone. Recent findings The clinical significance of sarcopenia has recently been highlighted, and muscle mass and muscle strength affect osteoporosis differently. The link between muscle and bone is also important from the viewpoint of exercise therapy. The clinical evaluation of vitamin D insufficiency has been developed, and vitamin D action is important for both muscle and bone. Although several studies have suggested that there are some interactions between muscle tissues and bone, we found a novel local regulator that might induce osteoblast differentiation of myoblasts. Moreover, several factors were proposed as muscle-derived soluble factors that induce bone anabolic action. There have been identified linkages from bone to muscle, such as osteocyte-producing or bone marrow mesenchymal cell-producing factors affecting muscle. Summary The links between muscle and bone are not fully understood at the present time. However, the development of research on the interactions between muscle and bone will be crucial for the development of novel drugs for sarcopenia and osteoporosis, as well as for the understanding of the physiological and pathological relationships of muscle and bone.
  • Yukinori Tamura; Masato Yano; Naoyuki Kawao; Katsumi Okumoto; Shigeru Ueshima; Hiroshi Kaji; Osamu Matsuo
    Journal of Nutritional Science 2 1 - 10 2048-6790 2013 [Refereed]
     
    The effects of Enzamin on obesity-related metabolic disorders in obese db/db mice were examined to explore a novel agent for the prevention of insulin resistance. Db/db mice were treated with water containing Enzamin (0·1 and 1·0 %) for 8 weeks from 6 weeks of age. Enzamin treatment at 1·0 %, but not at 0·1 %, significantly decreased the fasting plasma glucose, serum total cholesterol and TAG levels in db/db mice, without affecting body weight gain and body fat composition. Furthermore, insulin sensitivity and glucose tolerance were improved by the treatment of db/db mice with 1·0 % Enzamin. Immunohistochemical studies and gene expression analysis showed that 1·0 % Enzamin treatment suppressed macrophage accumulation and inflammation in the adipose tissue. In addition, 1·0 % Enzamin treatment increased serum adiponectin in db/db mice. Treatment with 1·0 % Enzamin also significantly suppressed the expression of NADPH oxidase subunits, suggesting an antioxidative effect for Enzamin in the adipose tissue. Furthermore, in vitro experiments demonstrated that the lipopolysaccharide-induced inflammatory reaction was significantly suppressed by Enzamin treatment in macrophages. Enzamin treatment increased the expression of GLUT4 mRNA in muscle, but not GLUT2 mRNA in the liver of db/db mice. Enzamin also increased the mRNA expression of carnitine palmitoyltransferase 1a (CPT1a, muscle isoform) in db/db mice, whereas Enzamin treatment did not affect the mRNA expression of CPT1b (liver isoform) in db/db mice. In conclusion, our data indicate that Enzamin can improve insulin resistance by ameliorating impaired adipocytokine expression, presumably through its anti-inflammatory action, and that Enzamin possesses a potential for preventing the metabolic syndrome. © The Author(s) 2013.
  • Masanori Yamazaki; Shin-ichi Suzuki; Shinji Kosugi; Takahiro Okamoto; Shinya Uchino; Akihiro Miya; Tsuneo Imai; Hiroshi Kaji; Izumi Komoto; Daishu Miura; Masanobu Yamada; Takashi Uruno; Kiyomi Horiuchi; Ai Sato; Akira Miyauchi; Masayuki Imamura; Akihiro Sakurai
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOC 59 (9) 797 - 807 0918-8959 2012/09 [Refereed]
     
    The morbidity and mortality of individuals with multiple endocrine neoplasia type 1 (MEN I) can be reduced by early diagnosis of MENI and related endocrine tumors. To find factors contributing to early diagnosis, we collected clinical information on MENI patients through a MEN study group, "MEN Consortium of Japan" and analyzed the time of initial symptom-dependent detection of parathyroid tumors, gastro-entero-pancreatic neuroendocrine tumors (GEPNETs) and pituitary tumors, and that of tumor detection-dependent MENI diagnosis in 560 patients. Main tumors were identified up to 7.0 years after symptoms appeared and there was no difference in age at the diagnosis of GEPNETs alone between probands and family members. In patients with typical symptoms (peptic ulcers, urolithiasis, fasting hypoglycemia, bone fracture/loss and amenorrhea), the mean interval between symptom manifestation and tumor detection was extended up to 9.6 years. In particular, 21.7% (5/23) of patients with amenorrhea were diagnosed with pituitary tumors in under one year. In patients with peptic ulcers (from parathyroid tumors or GEPNETs) and urolithiasis (from parathyroid tumors), the interval was positively correlated with age at tumor detection. The interval between tumor detection and MENI diagnosis was also prolonged to approximately four years in patients with fasting hypoglycemia (from GEPNETs) and amenorrhea. A substantial delay in the diagnosis of symptom-related tumors and subsequent MENI and inadequate screening of GEPNETs in family members were indicated. A greater understanding of MEN! may assist medical practitioners to make earlier diagnoses, to share patients' medical information and to give family members sufficient disease information.
  • Masato Yano; Yoshifumi Inoue; Takako Tobimatsu; Geoffrey N. Hendy; Lucie Canaff; Toshitsugu Sugimoto; Susumu Seino; Hiroshi Kaji
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOC 59 (8) 653 - 662 0918-8959 2012/08 [Refereed]
     
    The transforming growth factor (TGF)-beta family members, bone morphogenetic protein (BMP)-2 and TGF-beta that signal via the receptor-regulated Smads (R-Smads) induce bone formation in vivo. The inhibitory Smads (I-Smads), Smad6 and Smad7, negatively regulate TGF-beta family ligand signaling by competing with R-Smads for binding to activated type I receptors, and preventing R-Smad activation, Hence, the I-Smads potentially act as suppressors of bone formation although their effects on phenotypic changes in mature osteoblasts are unclear. While Smad7 inhibits both BMP and TGF-beta signaling, Smad6 is less effective in inhibiting TGF-beta signaling. The present study was performed to examine the role of Smad7 on the phenotype of mouse osteoblastic MC3T3-E1 cells. We employed stable Smad7-transfected MC3T3-E1 cells to examine the role of Smad7 in osteoblast proliferation, differentiation and mineralization. Stable Smad7 overexpression significantly inhibited the absorbance in the MTT-dye assay and inhibited the levels of PCNA compared with those in empty vector-transfected cells. Smad7 overexpression suppressed the type 1 collagen mRNA and protein levels. Moreover, Smad7 inhibited ALP activity and mineralization of osteoblastic cells. The effects of stable overexpression of Smad6 were similar to those of Smad7 suggesting the changes mediated by either I-Smad occurred by inhibition of BMP rather than TGF-beta signaling. In addition, PTH-(1-34) elevated the levels of Smad7 in parental MC3T3-E1 cells. In conclusion, the present study demonstrated that Smad7, as well as Smad6, inhibits proliferation, differentiation and mineralization of mouse osteoblastic cells. Therefore, I-Smads are important molecular targets for the negative control of bone formation.
  • Hiroshi Kaji
    JOURNAL OF BONE AND MINERAL METABOLISM SPRINGER JAPAN KK 30 (4) 381 - 387 0914-8779 2012/07 [Refereed][Invited]
     
    Menin, a product of the MEN1 gene, is related to the ontogeny of several cancers such as MEN1 and sporadic endocrine tumors, although it is considered to be a tumor suppressor. Many proteins interact with menin, and it is involved in various biological functions in several tissues. Menin plays some physiological and pathological roles related to transforming growth factor-beta (TGF-beta) signaling pathway in the parathyroid, and it is implicated in the tumorigenesis of parathyroid tumors. In bone, the bone phenotype was observed in some menin-deleted mice. Menin is considered to support BMP-2- and Runx2-induced differentiation of mesenchymal cells into osteoblasts by interacting with Smad1/5, Runx2, beta-catenin and LEF-1, although it has different effects on osteoblasts at later differentiation stages through TGF-beta-Smad3 and AP-1 pathways. Further research is expected to shed more light on the role of menin in bone.
  • Ken-ichiro Tanaka; Yoshifumi Inoue; Geoffrey N. Hendy; Lucie Canaff; Takenobu Katagiri; Riko Kitazawa; Toshihisa Komori; Toshitsugu Sugimoto; Susumu Seino; Hiroshi Kaji
    BONE ELSEVIER SCIENCE INC 51 (1) 158 - 167 8756-3282 2012/07 [Refereed]
     
    Bone morphogenetic proteins (BMPs) are critical for bone regeneration and induce ectopic bone formation in vivo. The constitutively activating mutation (R206H) of the BMP type 1 receptor, activin A type 1 receptor/activin-like kinase 2 (ACVR1/ALK2), underlies the molecular pathogenesis of fibrodysplasia ossificans progressiva (FOP) in which heterotopic ossification occurs in muscle tissue. In the present study, we performed a comparative DNA microarray analysis between stable empty vector- and ALK2(R206H)-transfected mouse myoblastic C2C12 cells. Forty genes were identified whose expression was increased >3.5 times in the experimental group versus the control. The bone formation-related factor, Tmem119, was included in this group. Osteoblast differentiation markers and mineralization were enhanced in C2C12 cells stably expressing Tmem119. Differentiation of myoblastic cells into myotubes was suppressed but differentiation into chondrocytes was little affected. Transcriptional activity of the BMP-2 signaling molecules, Smad1/5, was increased even in the absence of exogenous BMP-2. Endogenous BMP-2 levels positively correlated with Tmem119 levels. A BMP-2/4 neutralizing antibody and dorsomorphin, an ALK2 inhibitor, antagonized Tmem119-enhanced alkaline phosphatase (ALP) levels. Tmem119 siRNA antagonized the BMP-2-induced ALP and osteocalcin, but not Runx2 and Osterix, mRNAs, in C2C12 cells. In conclusion, Tmem119 levels were increased by the FOP-associated constitutively activating ALK2 mutation in myoblasts. The data show that Tmem119 promotes the differentiation of myoblasts into osteoblasts and the interaction with the BMP signaling pathway likely occurs downstream of Runx2 and Osterix in myoblasts. Tmem119 may play a critical role in the commitment of myoprogenitor cells to the osteoblast lineage. (C) 2012 Elsevier Inc. All rights reserved.
  • Ken-ichiro Tanaka; Erika Matsumoto; Yoshiko Higashimaki; Takenobu Katagiri; Toshitsugu Sugimoto; Susumu Seino; Hiroshi Kaji
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 287 (15) 11616 - 11628 0021-9258 2012/04 [Refereed]
     
    The interaction between muscle tissues and bone metabolism is incompletely understood. We hypothesized that there might be some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We, therefore, performed comparative DNA microarray analysis between mouse myoblastic C2C12 cells transfected with either stable empty vector or ALK2 (R206H), the mutation that constitutively activates the bone morphogenetic protein (BMP) receptor, to search for muscle-derived bone anabolic factors. Twenty-five genes whose expression was decreased to <1/4, were identified; these included osteoglycin (OGN). Stable overexpression of OGN significantly decreased the levels of Runx2 and Osterix mRNA compared with those in cells transfected with vector alone in MC3T3-E1 cells. On the other hand, it significantly enhanced the levels of alkaline phosphatase (ALP), type I collagen (Col1), and osteocalcin (OCN) mRNA as well as beta-catenin and mineralization. A reduction in endogenous OGN level showed the opposite effects to those of OGN overexpression in MC3T3-E1 and mouse calvarial osteoblastic cells. Transient OGN overexpression significantly suppressed the levels of Runx2, Osterix, ALP, Col1, and OCN mRNA induced by BMP-2 in C2C12 cells. The conditioned medium from OGN-overexpressed and OGN-suppressed myoblastic cells enhanced and decreased, respectively, the levels of ALP, Col1, and beta-catenin in MC3T3-E1 cells. Moreover, OGNincreased Smad3/4-responsive transcriptional activity as well as Col1 mRNA levels independently of endogenous TGF-beta in these cells. In conclusion, this study suggests that OGN may be a crucial humoral bone anabolic factor that is produced by muscle tissues.
  • Naoyuki Kawao; Nobuo Nagai; Yukinori Tamura; Yoshitaka Horiuchi; Katsumi Okumoto; Kiyotaka Okada; Yasuhiro Suzuki; Kazuo Umemura; Masato Yano; Shigeru Ueshima; Hiroshi Kaji; Osamu Matsuo
    THROMBOSIS AND HAEMOSTASIS SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN 107 (4) 749 - 759 0340-6245 2012/04 [Refereed]
     
    Urokinase-type plasminogen activator (u-PA) and plasminogen play a primary role in liver repair through the accumulation of macrophages and alteration of their phenotype. However, it is still unclear whether u-PA and plasminogen mediate the activation of macrophage phagocytosis during liver repair. Herein, we investigated the morphological changes in macrophages that accumulated at the edge of damaged tissue induced by a photochemical reaction or hepatic ischaemia-reperfusion in mice with u-PA (u-PA(-/-)) or plasminogen (Plg(-/-).) gene deficiency by using transmission electron and fluorescence microscopy. In wildtype mice, the macrophages aligned at the edge of the damaged tissue and extended a large number of long pseudopodia. These macrophages clearly engulfed cellular debris and showed well-developed organelles, including lysosome-like vacuoles, nuclei, and Golgi complexes. In wildtype mice, the distribution of the Golgi complex in these macrophages was biased towards the direction of the damaged tissue, indicating the extension of their pseudopodia in this direction. Conversely, in u-PA(-/-) and plg(-/-) mice, the macrophages located at the edge of the damaged tissue had few pseudopodia and less developed organelles. The Golgi complex was randomly distributed in these macrophages in u-PA(-/-) mice. Furthermore, interferon gamma and IL-4 were expressed at a low level at the border region of the damaged tissue in u-PA(-/-) mice. Our data provide novel evidence that u-PA and plasminogen are essential for the phagocytosis of cellular debris by macrophages during liver repair. Furthermore, u-PA plays a critical role in the induction of macrophage polarity by affecting the microenvironment at the edge of damaged tissue.
  • Akihiro Sakurai; Shinichi Suzuki; Shinji Kosugi; Takahiro Okamoto; Shinya Uchino; Akihiro Miya; Tsuneo Imai; Hiroshi Kaji; Izumi Komoto; Daishu Miura; Masanobu Yamada; Takashi Uruno; Kiyomi Horiuchi; Akira Miyauchi; Masayuki Imamura
    CLINICAL ENDOCRINOLOGY WILEY 76 (4) 533 - 539 0300-0664 2012/04 [Refereed]
     
    Objective Multiple endocrine neoplasia type 1 (MEN1) is less well recognized in Asian countries, including Japan, than in the West. The clinical features and optimal management of MEN1 have yet to be clarified in Japan. The aim of this study was to clarify the clinical features of Japanese patients with MEN1. Design/Patients We established a MEN study group designated the ` MEN Consortium of Japan' in 2008, and asked physicians and surgeons to provide clinical and genetic information on patients they had treated. Of 680 registered patients, 560 were analysed. Measurements Clinical and genetic features of Japanese patients with MEN1 were examined. Results Primary hyperparathyroidism, gastroenteropancreatic neuroendocrine tumours (GEPNET), and pituitary tumours were seen in 94 4%, 58 6% and 49 6% of patients, respectively. The prevalence of insulinoma was higher in the Japanese than in the West (22% vs 10%). In addition, 37% of patients with thymic carcinoids were women, while most were men in western countries. The MEN1 mutation positive rate was 91 7% in familial cases and only 49 3% in sporadic cases. Eight novel mutations were identified. Despite the availability of genetic testing for MEN1, the application of genetic testing, especially presymptomatic diagnosis for at-risk family members appeared to be insufficient. Conclusions We established the first extensive database for Asian patients with MEN1. Although the clinical features of Japanese patients were similar to those in western countries, there were several characteristic differences between them.
  • Lucie Canaff; Jean-Francois Vanbellinghen; Hiroshi Kaji; David Goltzman; Geoffrey N. Hendy
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 287 (11) 8584 - 8597 0021-9258 2012/03 [Refereed]
     
    Multiple endocrine neoplasia type 1 (MEN1) is characterized by tumors of the parathyroid, enteropancreas, and anterior pituitary. The MEN1 gene encodes the tumor suppressor menin of 610 amino acids that has multiple protein partners and activities. The particular pathways that, when lost, lead to tumorigenesis are not known. We demonstrated that members of a three-generation MEN1 kindred are heterozygous for a donor splice site mutation at the beginning of intron 3 (IVS3 + 1G -> A). Lymphoblastoid cells of a mutant gene carrier had, in addition to the wild-type menin transcript, an aberrant transcript resulting from use of a cryptic splice site within exon III that splices to the start of exon IV. The predicted menin Delta(184-218) mutant has an in-frame deletion of 35 amino acids but is otherwise of wild-type sequence. The transfected menin Delta(184-218) mutant was well expressed and fully able to mediate the normal inhibition of the activity of the transcriptional regulators JunD and NF-kappa B. However, it was defective in mediating TGF-beta-stimulated Smad3 action in promoter-reporter assays in insulinoma cells. Importantly, lymphoblastoid cells from an individual heterozygous for the mutation had reduced TGF-beta-induced (Smad3) transcriptional activity but normal JunD and NF-kappa B function. In addition, the mutant gene carrier lymphoblastoid cells proliferated faster and were less responsive to the cytostatic effects of TGF-beta than cells from an unaffected family member. In conclusion, the menin mutant exhibits selective loss of the TGF-beta signaling pathway and loss of cell proliferation control contributing to the development of MEN1.
  • Ken-ichiro Tanaka; Erika Matsumoto; Yoshiko Higashimaki; Toshitsugu Sugimoto; Susumu Seino; Hiroshi Kaji
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 418 (1) 134 - 139 0006-291X 2012/02 [Refereed]
     
    Muscle mass is related to higher bone mass and a reduction in fracture risk. However, the interactions between muscle tissues and bone metabolism are incompletely understood and there might be some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We therefore investigated the role of FAM5C in osteoblast differentiation and the interactions between muscle and bone. A reduction of endogenous FAM5C by siRNA reduced the levels of osterix, alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA as well as the levels of type 1 collagen and B-catenin in mouse osteoblastic MC3T3-E1 cells and mouse calvarial osteoblasts, although FAM5C overexpression significantly antagonized the levels of osterix. ALP and OCN mRNA induced by bone morphogenetic protein-2 in C2C12 cells. The conditioned medium from FAM5C-overexpressed and beta-suppressed C2C12 cells increased and decreased the levels of osterix, ALP and OCN mRNA in MC3T3-E1 cells, respectively. In conclusion, the present study is the first to show that FAM5C enhances osteoblast differentiation in differentiated osteoblasts, and that the effects of the conditioned medium from FAM5C-modulated myoblastic cells were positively correlated with the effects of FAM5C on osteoblast phenotype in osteoblasts. FAM5C might be an important humoral bone anabolic factor produced from muscle cells. (C) 2012 Elsevier Inc. All rights reserved.
  • Bone anabolic action of parathyroid hormone.
    Kaji H
    Acta Med Kinki Univ 37 57 - 62 2012 [Invited]
  • Susumu Takano; Hiroshi Kaji; Fujio Hayashi; Kanae Higashiguchi; Sachie Joukei; Yoshiaki Kido; Juro Takahashi; Kayo Osawa
    Analytical chemistry insights 7 23 - 30 2012 [Refereed]
     
    Measurement of ionized calcium is more important than measurement of total calcium in serum samples. In the present study, equations were derived from complexation and acid dissociation equilibrium equations, and were used to determine the concentration of ionized calcium from the observed serum concentrations of total calcium, albumin, total protein, and inorganic phosphate. The ionized calcium concentration was calculated in 67 serum samples from healthy subjects and 34 outpatients previously identified as having abnormal serum calcium levels. The correlation coefficient between our method (y) and the calcium-ion-selective electrode method (x) was 0.953 and the linear regression equation was y = 0.97x at pH 7.4 with a factor of α = 0.21, which was based on the differences between the concentrations of calcium phosphorus compounds obtained by the electrode method and by calculation. The developed calculation is as useful and accurate as the electrode method, and therefore extremely useful for clinical diagnoses.
  • Effects of serum from a fibrodysplasia ossificans profressiva patients on osteoblastic cells.
    Hisa I; Kawara A; Katagiri T; Sugimoto T; Kaji H
    Open J Endocr Metab Dis 2 1 - 6 2012 [Refereed]
  • Yasuo Imanishi; Jun Hashimoto; Wataru Ando; Keisuke Kobayashi; Takafumi Ueda; Yuki Nagata; Akimitsu Miyauchi; Hajime M. Koyano; Hiroshi Kaji; Takatoshi Saito; Koichi Oba; Yasato Komatsu; Tomoaki Morioka; Katsuhito Mori; Takami Miki; Masaaki Inaba
    JOURNAL OF BONE AND MINERAL METABOLISM SPRINGER TOKYO 30 (1) 93 - 99 0914-8779 2012/01 [Refereed]
     
    Oncogenic osteomalacia (OOM), or tumor-induced osteomalacia, is a rare disease characterized by renal phosphate wasting and osteomalacia. It arises due to the secretion of fibroblast growth factor 23 (FGF-23) from causative tumors. Matrix extracellular phosphoglycoprotein (MEPE) is predominantly expressed in odontoblasts, osteoblasts, and osteocytes. Although the presence of MEPE mRNA has been reported in some OOM tumors, little is known about the prevalence of MEPE expression in OOM tumors. In this study, the expression of MEPE and FGF-23 in OOM tumors was investigated at the transcriptional and translational levels. Eleven causative OOM tumors were analyzed by quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry for MEPE and FGF-23 expression. Hemangiopericytomas and giant cell tumors, pathological diagnoses that are common in cases of OOM, were obtained from non-osteomalacic patients and analyzed as controls. The gene expression level of FGF23 and MEPE in OOM tumors was 10(4)- and 10(5)-times higher, respectively, than in non-OOM tumors. Immunohistochemical staining revealed that FGF-23 protein was expressed in all OOM tumors, and MEPE was expressed in 10 out of 11 OOM tumors. Thus, MEPE expression was common in OOM tumors, similar to FGF-23. These results indicate that, in addition to the hypophosphatemic effects of FGF-23, MEPE or the MEPE-derived acidic serine aspartate-rich MEPE-associated motif peptide may contribute to decreased bone mineralization in OOM patients.
  • H. Kaji; Y. Imanishi; T. Sugimoto; S. Seino
    EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES JOHANN AMBROSIUS BARTH VERLAG MEDIZINVERLAGE HEIDELBERG GMBH 119 (7) 440 - 444 0947-7349 2011/07 [Refereed]
     
    Wnt-beta-catenin signaling is important for bone formation. Sclerostin inhibits bone formation mainly by suppressing this signal, and several studies suggest that the suppression of sclerostin expression contributes to the bone anabolic action of parathyroid hormone (PTH). We therefore examined serum sclerostin levels using enzyme-linked immunosolvent assay in 18 patients with postmenopausal osteoporosis, 9 postmenopausal women with primary hyperparathyroidism (pHPT) and 7 patients with osteomalacia. Serum levels of sclerostin were significantly lower in the group with pHPT, compared with those with postmenopausal osteoporosis. Moreover, serum sclerostin levels were significantly lower in the group with tumor-induced osteomalacia, but not in the group with osteomalacia without tumor, compared with those with postmenopausal osteoporosis. In patients with pHPT, serum sclerostin levels were significantly and negatively correlated to serum calcium and PTH levels. In patients with postmenopausal osteoporosis, serum levels of sclerostin levels were significantly and positively related to serum calcium and creatinine levels. In conclusion, we showed that serum sclerostin levels are decreased presumably through endogenous PTH elevation in postmenopausal women with pHPT, compared with the patients with postmenopausal osteoporosis.
  • Mika Yamauchi; Hiroshi Kaji; Kiyoko Nawata; Shin Takaoka; Toru Yamaguchi; Toshitsugu Sugimoto
    CALCIFIED TISSUE INTERNATIONAL SPRINGER 88 (5) 362 - 369 0171-967X 2011/05 [Refereed]
     
    Vitamin D insufficiency is related to an increase in PTH, which might be critical for an increase in bone fragility. However, the role of endogenous PTH in vitamin D insufficiency-induced fracture risk remains unclear. The present study was performed to examine the relationships among vitamin D insufficiency, bone fragility, and PTH in 202 Japanese postmenopausal women. Serum 25-hydroxyvitamin D (25[OH]D) levels were measured. The percentages of subjects with 25(OH)D levels below 10, 15, and 20 ng/ml were 5.0, 41.0, and 80.7%, respectively. Serum 25(OH)D levels were negatively related to age and serum levels of Cr and PTH; they were positively related to bone mineral density (BMD). In multiple regression analysis, BMD was significantly related to 25(OH)D levels when adjusted for age, body mass index (BMI), and serum levels of Cr and PTH. Multiple logistic regression analysis showed that lower 25(OH)D levels were significantly related to prevalent fracture risk when adjusted for age, BMI, serum levels of Cr and PTH, as well as femoral neck BMD. The proportion of subjects with prevalent fractures was significantly higher in the group with lower PTH and lower 25(OH)D than in the group with lower PTH and higher 25(OH)D or higher PTH and higher 25(OH)D. In conclusion, vitamin D insufficiency was found to be related to prevalent fracture risk independently of PTH. Functional hypoparathyroidism, rather than functional hyperparathyroidism, might be a risk factor for bone fragility in vitamin D insufficiency.
  • Itoko Hisa; Yoshifumi Inoue; Geoffrey N. Hendy; Lucie Canaff; Riko Kitazawa; Sohei Kitazawa; Toshihisa Komori; Toshitsugu Sugimoto; Susumu Seino; Hiroshi Kaji
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 286 (11) 9787 - 9796 0021-9258 2011/03 [Refereed]
     
    The mechanisms whereby the parathyroid hormone (PTH) exerts its anabolic action on bone are incompletely understood. We previously showed that inhibition of ERK1/2 enhanced Smad3-induced bone anabolic action in osteoblasts. These findings suggested the hypothesis that changes in gene expression associated with the altered Smad3-induced signaling brought about by an ERK1/2 inhibitor would identify novel bone anabolic factors in osteoblasts. We therefore performed a comparative DNA microarray analysis between empty vector-transfected mouse osteoblastic MC3T3-E1 cells and PD98059-treated stable Smad3-overexpressing MC3T3-E1 cells. Among the novel factors, Tmem119 was selected on the basis of its rapid induction by PTH independent of later increases in endogenous TGF-beta. The levels of Tmem119 increased with time in cultures of MC3T3-E1 cells and mouse mesenchymal ST-2 cells committed to the osteoblast lineage by BMP-2. PTH stimulated Tmem119 levels within 1 h as determined by Western blot analysis and immunocytochemistry in MC3T3-E1 cells. MC3T3-E1 cells stably overexpressing Tmem119 exhibited elevated levels of Runx2, osteocalcin, alkaline phosphatase, and beta-catenin, whereas Tmem119 augmented BMP-2-induced Runx2 levels in mesenchymal cells. Tmem119 interacted with Runx2, Smad1, and Smad5 in C2C12 cells. In conclusion, we identified a Smad3-related factor, Tmem119, that is induced by PTH and promotes differentiation in mouse osteoblastic cells. Tmem119 is an important molecule in the pathway downstream of PTH and Smad3 signaling in osteoblasts.
  • Y. Inoue; G. N. Hendy; L. Canaff; S. Seino; H. Kaji
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG KG 43 (3) 183 - 187 0018-5043 2011/03 [Refereed]
     
    Menin promotes the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage by interacting with the BMP-2 signaling molecules Smad1/5, and Runx2. However, the relationship between menin and the Wnt-beta-catenin pathway in bone is unclear. Reduction of menin expression by transfection of a menin antisense construct did not alter the levels of beta-catenin in mouse mesenchymal C2C12 and osteoblastic MC3T3-E1 cells. However, menin co-immunoprecipitated with beta-catenin as well as LEF-1 in C2C12 and MC3T3-E1 cells. Reduction of menin expression by antisense menin transfection antagonized beta-catenin-induced transcriptional activity of the pGL3-OT luciferase reporter construct in C2C12 and MC3T3-E1 cells. Antisense menin transfection antagonized the BMP-2 and beta-catenin-stimulated increases in Runx2 and alkaline phosphatase levels in C2C12 cells. The data show that menin interacts with beta-catenin in mouse mesenchymal and osteoblastic cells, and suggest that the interaction is important for osteoblast differentiation.
  • Ryo Okazaki; Toshitsugu Sugimoto; Hiroshi Kaji; Yoshio Fujii; Masataka Shiraki; Daisuke Inoue; Itsuro Endo; Toshio Okano; Takako Hirota; Issei Kurahashi; Toshio Matsumoto
    JOURNAL OF BONE AND MINERAL METABOLISM SPRINGER TOKYO 29 (1) 103 - 110 0914-8779 2011/01 [Refereed]
     
    Vitamin D insufficiency is a risk for both skeletal and nonskeletal health. However, some ambiguity remains about threshold serum 25(OH)D for vitamin D insufficiency. To determine the threshold serum 25(OH)D to maintain normal calcium availability without elevation in serum parathyroid hormone (PTH) among Japanese subjects with various calcium intakes, we conducted a multicenter prospective open-labeled study. We recruited 107 ambulatory subjects without disorders affecting vitamin D metabolism to whom oral vitamin D-3 800 IU/day for 4 weeks or 1,200 IU/day for 8 weeks was given. Serum 25(OH)D, PTH, calcium, phosphate, and magnesium were measured before and after vitamin D-3 supplementation. Calcium intake was assessed by questionnaires. When all the data were combined, serum 25(OH)D was negatively correlated with PTH. The cubic spline curve between serum 25(OH)D and PTH indicated PTH reached its plateau between 35 and 40 pg/ml at 25(OH)D between 25 and 30 ng/ml. Vitamin D-3 supplementation increased serum 25(OH)D and decreased PTH. Change in PTH correlated positively with baseline serum 25(OH)D. From the regression analyses, baseline serum 25(OH)D above 28 ng/ml corresponded to the threshold level without reduction in PTH after vitamin D-3 supplementation. In multivariate regression analyses, age but not calcium intake was a significant determinant of PTH. We concluded that a serum 25(OH)D level of 28 ng/ml was identified as a threshold for vitamin D insufficiency necessary to stabilize PTH to optimal levels.
  • Y. Inoue; I. Hisa; S. Seino; H. Kaji
    EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES JOHANN AMBROSIUS BARTH VERLAG MEDIZINVERLAGE HEIDELBERG GMBH 118 (10) 719 - 723 0947-7349 2010/11 [Refereed]
     
    Alendronate, an aminobisphosphonate, is an effective reagent to reduce fracture risk in osteoporotic patients. Although several studies suggest that bisphosphonates affect osteoblast differentiation, how they affect the genes relating to the mineralization step remains unknown. The present study was performed to clarify the effects of alendronate on mineralization and its related genes in mouse osteoblastic MC3T3-E1 cells. Alendronate at 10(-8) and 10(-7) M induced mineralization in MC3T3-E1 cells. As for the genes that suppress mineralization, alendronate enhanced the level of PC-1 mRNA in a dose-dependent manner in 7-day cultures in semi-quantitative RT-PCR, although it reduced the levels of PC-1 mRNA in 21-day cultures. On the other hand, alendronate did not affect the levels of ANK, osteopontin and matrix Gla protein mRNA in both 7- and 21-day cultures. Moreover, alendronate reduced the level of osteocalcin mRNA at 10(-7) and 10(-6) M in 14-day cultures of these cells. As for the expression of alkaline phosphatase (ALP), an important positive regulator of mineralization in osteoblasts, alendronate enhanced the levels of ALP mRNA and protein at 10(-7)-10(-5) M. In conclusion, low-dose alendronate induced mineralization ill mouse osteoblastic cells. The regulation of PC-1, osteocalcin and ALP by alendronate might play some role in these effects.
  • Hiroshi Kaji; Mika Yamauchi; Toru Yamaguchi; Takashi Shigematsu; Toshitsugu Sugimoto
    JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM ENDOCRINE SOC 95 (10) 4635 - 4642 0021-972X 2010/10 [Refereed]
     
    Context: The effect of mild renal dysfunction on bone mineral density and fracture risk is uncertain. Objective: We evaluated whether mild renal dysfunction would affect bone mineral density (BMD) and the risk of vertebral fractures (VFs) in 659 postmenopausal women. Main Outcome Measures: Creatinine clearance (CCr) and the estimated glomerular filtration rate (eGFR) were calculated using the Cockcroft-Gault and the Modification of Diet in Renal Disease formulas, respectively. BMD was measured by dual-energy x-ray absorptiometry. Renal function was categorized by the criteria of the Kidney Disease Outcomes Quality Initiative Committee. Results: Comparison of fracture prevalence by chronic kidney disease stages revealed that the group of stage 3 or greater by eGFR had a significantly higher rate of VFs (45.3%) than stages 1 (23.8%) and 2 (25.3%) groups. In the stage 2 group, there were significant positive correlations between eGFR and BMD values at the femoral neck and radius as well as between CCr and BMD values at all sites. Moreover, postmenopausal women with VFs had lower eGFR and CCr than those without VFs in stage 2. When multivariable logistic regression analysis was performed with the presence of VFs as a dependent variable and CCr levels adjusted for years after menopause, smoking habit, alcohol intake, and lumbar spine BMD as an independent variable, CCr levels were identified as a factor associated with the presence of VFs in postmenopausal women with chronic kidney disease stage 2. Conclusions: The present study indicates that postmenopausal women with mild renal dysfunction are at increased risk for BMD decrease and VFs. (J Clin Endocrinol Metab 95: 4635-4642, 2010)
  • Dong Yu; Yuko Nagamura; Satoko Shimazu; Junko Naito; Hiroshi Kaji; Seiki Wada; Munehiro Honda; Lian Xue; Toshihiko Tsukada
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOC 57 (9) 825 - 832 0918-8959 2010/09 [Refereed]
     
    Menin is lost by the sequential inactivation of both MEN1 alleles in subsets of non-hereditary endocrine tumors as well asthose associated with multiple endocrine neoplasia type 1 (MEN I), an autosomal dominant hereditary cancer syndrome characterized by multiple tumors including parathyroid, pituitary and enteropancreatic endocrine tumors. Loss of menin has been reported to be associated with lowered caspase 8 expression and resistance to apoptosis in murine fibroblasts and in pancreatic islet tumors arising in heterozygous MEN I gene knockout mice, the animal model of the human MEN I syndrome. We confirmed by menin-knockdown experiments with specific siRNA that menin is crucial for caspase 8 expression in human culture cells while overexpression of menin did not increase caspase 8 protein over basal levels. We then examined expression of menin, caspase 8 and cyclin-dependent kinase inhibitors p27(Kip1) and p15(Ink4b) by Western blotting in human parathyroid tumors surgically resected from patients with MEN I and those with non-hereditary primary hyperparathyroidism. The menin and p27(Kip1) expression levels were correlated with MEN1 mutation status that was confirmed by DNA analysis. The caspase 8 and p15(Ink4b) protein levels were variable among tumors, and were not correlated with menin protein levels. These findings suggest that human endocrine tumors lacking menin may not always exhibit lowered caspase 8 expression and hence may not be resistant to apoptosis-inducing therapy.
  • H. Kaji; M. Yamauchi; T. Yamaguchi; T. Sugimoto
    OSTEOPOROSIS INTERNATIONAL SPRINGER LONDON LTD 21 (9) 1585 - 1590 0937-941X 2010/09 [Refereed]
     
    Urinary deoxypyridinoline (DPD) level was associated with prevalent vertebral fractures in glucocorticoid (GC)-treated postmenopausal women independently of lumbar spine bone mineral density (BMD). Bone metabolic indices are the potential predictors of bone fragility. However, their diagnostic efficiency for identifying the risk of GC-induced vertebral fractures is still unclear. We therefore evaluated whether bone metabolic indices would assess the risk of vertebral fractures in GC-treated women. One hundred seventy-five women treated with GC for more than 6 months were enrolled in this study. Both premenopausal and postmenopausal women with vertebral fractures had significantly higher urinary DPD levels than those without vertebral fractures. When multivariable logistic regression analysis was performed with the presence of vertebral fractures as a dependent variable and each of DPD or osteocalcin level adjusted for age, weight, height, current and maximum doses of GC, duration of GC treatment, as well as lumbar spine BMD as an independent variable, DPD level was identified as a factor associated with the presence of vertebral fractures in postmenopausal women but not in premenopausal women. Urinary DPD level was significantly associated with prevalent vertebral fractures in GC-treated postmenopausal women independently of lumbar spine BMD.
  • H. Kaji; Y. Kuroki; Y. Murakawa; I. Funakawa; Y. Funasaka; F. Kanda; T. Sugimoto
    OSTEOPOROSIS INTERNATIONAL SPRINGER LONDON LTD 21 (9) 1565 - 1571 0937-941X 2010/09 [Refereed]
     
    This prospective study, in the very early phase after initiation of glucocorticoid (GC) treatment, showed that alendronate was effective in suppressing accelerated bone resorption and subsequent decrease in bone mineral density (BMD) at the lumbar spine of patients with high-dose GC treatment. How bisphosphonates affect bone metabolism and BMD of patients with high-dose GC in the early phase, especially within 1 month is unclear. We examined the prospective effects of daily 5 mg alendronate on bone metabolism and BMD in 20 patients with high-dose GC (at least 40 mg prednisolone/day) and compared them to 34 high-dose GC-treated patients without alendronate. Serum levels of calcium decreased at day 28 in the alendronate group. Urinary calcium excretion significantly increased after day 7 in both groups. The increase in serum parathyroid hormone (PTH) level at day 7 in the control group was not observed in the alendronate group, but PTH levels increased at day 28 and month 3 in the alendronate group. As for the bone turnover markers, the serum osteocalcin level decreased in both alendronate and control groups, but serum bone-type alkaline phosphatase levels did not show significant changes. Although the urinary type I collagen cross-linked N-telopeptide (NTX) level showed significant increases on days 7 and 28 in the control group; such early increases in urinary NTX were not observed in the alendronate group. Thereafter, the urinary NTX levels fell slowly in the alendronate group significantly. BMD at the lumbar spine significantly decreased from month 1 in the control group, whereas in the alendronate group, BMD at the lumbar spine maintained almost the same level at all time points observed. Alendronate was effective in suppressing bone resorption and subsequent BMD decrease at the lumbar spine in patients with high-dose GC treatment.
  • Kazuhiro Uenishi; Takuo Fujita; Hiromi Ishida; Yoshio Fujii; Mutsumi Ohue; Hiroshi Kaji; Midori Hirai; Mikio Kakumoto; Steven A. Abrams
    NUTRIENTS MDPI AG 2 (7) 752 - 761 2072-6643 2010/07 [Refereed]
     
    With the use of stable isotopes, this study aimed to compare the bioavailability of active absorbable algal calcium (AAACa), obtained from oyster shell powder heated to a high temperature, with an additional heated seaweed component (Heated Algal Ingredient, HAI), with that of calcium carbonate. In 10 postmenopausal women volunteers aged 59 to 77 years (mean +/- S. D., 67 +/- 5.3), the fractional calcium absorption of AAACa and CaCO(3) was measured by a dual stable isotope method. (44)Ca-enriched CaCO(3) and AAACa were administered in all subjects one month apart. After a fixed-menu breakfast and pre-test urine collection (Urine 0), (42)Ca-enriched CaCl(2) was intravenously injected, followed by oral administration of 44Ca-enriched CaCO(3) without carrier 15 minutes later, and complete urine collection for the next 24 hours (Urine 24). The fractional calcium absorption was calculated as the ratio of Augmentation of (44)Ca from Urine 0 to Urine 24/ augmentation of (42)Ca from Urine 0 to Urine 24. Differences and changes of (44)Ca and (42)Ca were corrected by comparing each with (43)Ca. Fractional absorption of AAACa (mean +/- S. D., 23.1 +/- 6.4), was distinctly and significantly higher than that of CaCO(3) (14.7 +/- 6.4; p = 0.0060 by paired t-test). The mean fractional absorption was approximately 1.57-times higher for AAACa than for CaCO(3). The serum 25(OH) vitamin D level was low (mean +/- S. D., 14.2 +/- 4.95 ng/ml), as is common in this age group in Japan. Among the parameters of the bone and mineral metabolism measured, none displayed a significant correlation with the fractional absorption of CaCO(3) and AAACa. Higher fractional absorption of AAACa compared with CaCO(3) supports previous reports on the more beneficial effect of AAACa than CaCO(3) for osteoporosis.
  • Akira Ishii; Yasuo Imanishi; Keisuke Kobayashi; Jun Hashimoto; Takafumi Ueda; Akimitsu Miyauchi; Hajime M. Koyano; Hiroshi Kaji; Takatoshi Saito; Koichi Oba; Yasato Komatsu; Masafumi Kurajoh; Yuki Nagata; Hitoshi Goto; Kenichi Wakasa; Toshitsugu Sugimoto; Takami Miki; Masaaki Inaba; Yoshiki Nishizawa
    CALCIFIED TISSUE INTERNATIONAL SPRINGER 86 (6) 455 - 462 0171-967X 2010/06 [Refereed]
     
    Oncogenic osteomalacia (OOM) is a rare disease characterized by renal phosphate wasting and osteomalacia and is caused by the secretion of fibroblast growth factor 23 (FGF-23) from causative tumors. Scintigraphy with octreotide, which binds to somatostatin receptors (SSTRs), is a useful way to locate causative tumors in OOM patients. However, the therapeutic effects of octreotide acetate are still controversial. Two OOM patients were administered octreotide acetate intramuscularly. Ten causative OOM tumors, including two resected from the patients participating in the octreotide administration study, were examined for expression of genes encoding SSTRs by quantitative real-time RT-PCR and immunohistochemistry. Octreotide therapy did not improve hypophosphatemia in either case, despite temporal decreases in FGF-23 levels in one patient. The mean expression levels of SSTR1, SSTR3, and SSTR5 were similar in the OOM and non-OOM tumors. Expression of SSTR2 was significantly higher in the OOM tumors than in the non-OOM tumors. Immunohistochemical examinations revealed the presence of SSTR2A, SSTR2B, and SSTR5 in both the OOM and non-OOM tumors. The expression of SSTR genes in OOM tumors contributes to positive imaging using octreotide scintigraphy. However, the levels of SSTRs seem to be insufficient for the octreotide therapy to improve hypophosphatemia. Further studies are needed to clarify the mechanisms by which FGF-23 secretion from OOM tumors is suppressed by octreotide acetate.
  • H. Kaji; I. Hisa; Y. Inoue; T. Sugimoto
    EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES JOHANN AMBROSIUS BARTH VERLAG MEDIZINVERLAGE HEIDELBERG GMBH 118 (6) 371 - 376 0947-7349 2010/06 [Refereed]
  • Yasuo Kuroki; Hiroshi Kaji; Seiji Kawano; Fumio Kanda; Yutaka Takai; Michiko Kajikawa; Toshitsugu Sugimoto
    INTERNAL MEDICINE JAPAN SOC INTERNAL MEDICINE 49 (10) 897 - 902 0918-2918 2010 [Refereed]
     
    Objective Glucocorticoid (GC) causes various metabolic abnormalities; however, few prospective studies have examined the changes in glucose and lipid metabolism in newly GC-treated patients. Methods and Patients The present study was therefore performed to analyze markers of glucose and lipid metabolism on days 0, 3, 7, 14, 28 and at month 3 of treatment in patients starting GC therapy. Then, we analyzed the relationships between the changes in these parameters and the initial dose of prednisolone (PSL), separating groups into different regimens by the GC dose. Results The fasting plasma glucose (FPG) level transiently increased on day 3 of PSL administration but was restored by day 7. The immunoreactive insulin (IRI) level and HOMA-R transiently increased on day 3 and then fell, although remaining significantly higher than each basal level by day 7. A transient elevation in FPG level on day 3 was observed only in groups with a PSL dose >= 40 mg. On the other hand, total cholesterol and low-density lipoprotein cholesterol levels increased on day 3 of PSL administration and similar levels were maintained after day 7. High density-lipoprotein cholesterol levels were significantly increased on day 3; subsequently then gradually increased from days 3 to day 28. Triglyceride levels did not change during treatment. No relationship was apparent between the GC dose and the changes in each lipid parameter. Conclusion GC treatment induced changes in FPG, IRI, LDL-CHOL and HDL-CHOL levels from day 3 after start of GC. The dose of GC seemed to influence glucose metabolism, but not lipid metabolism.
  • K. Hayashi; M. Yamamoto; Y. Murakawa; M. Yamauchi; H. Kaji; T. Yamaguchi; T. Sugimoto
    OSTEOPOROSIS INTERNATIONAL SPRINGER LONDON LTD 20 (11) 1889 - 1894 0937-941X 2009/11 [Refereed]
     
    Eighty-seven male Japanese subjects taking prednisolone a parts per thousand yen5 mg for more than 6 months and 132 age- and body mass index (BMI)-matched control subjects were examined. Multiple regression analysis adjusted for age and BMI showed that spinal bone mineral density (BMD) in the prednisolone group was not associated with prevalent vertebral fractures (VFs). Glucocorticoid (GC) treatment is known to increase the risk for bone fractures. However, the association between VFs and BMD in GC-treated male patients remains unclear. Eighty-seven male subjects taking prednisolone a parts per thousand yen5 mg for more than 6 months and 132 age- and BMI-matched control subjects were examined using lateral thoracic and lumbar spine radiographs and spine dual energy X-ray absorptiometry. The presence of GC use was an independent risk factor for VFs adjusted for age and BMI (odds ratio 10.93, P < 0.001). By receiver operating characteristic analysis, the absolute BMD values for detecting VFs were higher and the sensitivity and specificity were lower in the GC group than in the control group (0.936 vs 0.825 g/cm(2) and 53.5% vs 74.0%, respectively). Multiple regression analysis adjusted for age and BMI showed that spinal BMD in the GC group was not associated with prevalent VFs, even after adding current and past maximum GC doses as independent variables. These results show that lumbar BMD values are not associated with prevalent VFs in GC-treated male patients, suggesting that bone fragility in male GC users is affected by bone quality rather than by BMD.
  • H. Kaji; M. Yamauchi; R. Nomura; T. Sugimoto
    EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES JOHANN AMBROSIUS BARTH VERLAG MEDIZINVERLAGE HEIDELBERG GMBH 117 (10) 633 - 636 0947-7349 2009/11 [Refereed]
     
    Several studies suggest that mild PTH excess does not have any deteriorative effects on bone mineral density (BMD) in several-year-longitudinal studies of patients with mild primary hyperparathyroidism (pHPT) without parathyroidectomy (PTX). However, it remains unknown about the change in bone geometry in pHPT patients without PTX. We examined the longitudinal effects of mild PTH excess on cortical bone geometry in postmenopausal patients with mild pHPT without PTX by using peripheral quantitative computed tomography (pQCT), and we compared them with normal and hypoparathyroidism women. Nine postmenopausal female patients who were diagnosed as pHPT, six postmenopausal female patients with hypoparathyroidism (3 idiopathic and 3 postoperative), and thirty postmenopausal control subjects participated in this study. Radial volumetric (v) BMD and several bone geometry parameters were measured by pQCT at basal line and after 2 years. Cortical vBMD was significantly lower in pHPT group. Moreover, total area and periosteal circumferences were significantly higher in pHPT group. Total and cortical vBMD were significantly decreased after 2 years in control group. However, they were stable in pHPT group after 2-year follow-up. As for bone geometry, cortical thickness and area were also stable in pHPT group during 2-year follow-up, although they were significantly reduced in control group and hypoparathyroidism group. In conclusion, the present longitudinal study revealed that there were no significant changes in radial vBMD and cortical bone geometry in postmenopausal women with mild pHPT, whereas age-related thinning of cortical bone as well as decrease of vBMD were observed in the control and patients with hypoparathyroidism.
  • Clemens Bergwitz; Santanu Banerjee; Hilal Abu-Zahra; Hiroshi Kaji; Akimitsu Miyauchi; Toshitsugu Sugimoto; Harald Jueppner
    JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM OXFORD UNIV PRESS INC 94 (11) 4267 - 4274 0021-972X 2009/11 [Refereed]
     
    Background: Homozygous mutations in fibroblast growth factor (FGF23) have recently been described as the genetic cause of one form of hyperphosphatemic tumoral calcinosis (HFTC). However, it remained unclear to date how these mutations lead to loss of biologically active FGF23 in the circulation. Methods: We here report a novel homozygous mutation, c. 385T > C in FGF23 exon 2, which changes codon 129 from serine to proline (S129P) in a previously described individual affected by HFTC. The S129P mutation as well as two known FGF23 mutations, S71G and S129F, were introduced into an expression vector encoding wild-type (wt) human (h) FGF23 to yield [P129]hFGF23, [F129]hFGF23, and [G71] hFGF23; whole lysates, glycoprotein fractions, and conditioned media from HEK293 and COS-7 cells expressing these constructs were subjected to Western blot analysis using affinity-purified goat anti-hFGF23(51-69) and anti-hFGF23(206-222) antibodies. Results: We detected 25-and 32-kDa protein species in total lysates of HEK293 cells expressing wt-hFGF23. The 32-kDa band, representing O-glycosylated hFGF23, was not detectable in the glycoprotein fraction of lysates from HEK293 cells expressing [P129] hFGF23, and in comparison with wt-FGF23 only small amounts of [P129] hFGF23 were secreted into the medium. Similar results were obtained for cells expressing [G71] hFGF23 and [F129] hFGF23. Conclusion: Our data for the first time directly show that FGF23 mutations associated with HFTC impair O-glycosylation in vitro resulting in poor secretion of the mutant hormone thereby explaining the characteristic hyperphosphatemic phenotype of homozygous carriers in vivo. (J Clin Endocrinol Metab 94: 4267-4274, 2009)
  • Yoshifumi Inoue; Lucie Canaff; Geoffrey N. Hendy; Itoko Hisa; Toshitsugu Sugimoto; Kazuo Chihara; Hiroshi Kaji
    JOURNAL OF CELLULAR BIOCHEMISTRY WILEY-LISS 108 (1) 285 - 294 0730-2312 2009/09 [Refereed]
     
    Parathyroid hormone (PTH) exerts an anabolic action on bone but the mechanisms are incompletely understood. We showed previously that PTH interacts with the canonical Wnt-beta-catenin signaling pathway via the transforming growth factor (TGF)-beta signaling molecule, Smad3, to modulate osteoblast differentiation and apoptosis. Here, we examined which actions of Smad3 are TGF-beta-independent in stimulating the osteoblast phenotype and PTH-induced Wnt-beta-catenin signaling. For this, the TGF-beta receptor type 1 [activin receptor-like kinase (ALK5)] inhibitor (SB431542), and a Smad3 mutant in which the site normally phosphorylated by ALK5 is mutated from SSVS to AAVA, was used. PTH induced total beta-catenin and reduced phosphorylated beta-catenin levels at 1, 6, and 24 h in mouse osteoblastic MC3T3-E1 cells. Transient transfection of Smad3AAVA inhibited the PTH induction of total beta-catenin and reduction of phosphorylated beta-catenin levels at 6 and 24 h, but not at 1 h, indicating that the early effects occur independently of TGF-beta receptor signaling. On the other hand, MC3T3-E1 cell clones in which Smad3AAVA was stably expressed demonstrated elevated beta-catenin levels, although alkaline phosphatase (ALP) activity and mineralization were unaltered. In contrast, MC3T3-E1 cell clones in which wild-type Smad3 was stably expressed exhibited increased ALP activity and mineralization that were decreased by the ALK5 inhibitor, SB431542, although the beta-catenin levels induced in these cells were not modulated. In conclusion, the present study indicates that PTH induces osteoblast beta-catenin levels via Smad3 independently of, and dependently on, TGF-beta in the early and later induction phases, respectively. J. Cell. Biochem. 108: 285-294, 2009. (C) 2009 Wiley-Liss, Inc.
  • Masahiro Yamamoto; Toru Yamaguchi; Mika Yamauchi; Hiroshi Kaji; Toshitsugu Sugimoto
    JOURNAL OF BONE AND MINERAL RESEARCH AMER SOC BONE & MINERAL RES 24 (4) 702 - 709 0884-0431 2009/04 [Refereed]
     
    Although patients with type 2 diabetes (T2DM) have an increased risk of hip fracture, risk of vertebral fracture (VF) and its association with BMD are still unclear. We examined Japanese T2DM patients (161 men >50 yr and 137 postmenopausal women) and non-DM controls (76 and 622, respectively) by lateral spine radiography and DXA at the lumbar spine (L), femoral neck (FN), and radius (R). Logistic regression analysis adjusted for age, body mass index, and L-BMD showed that the presence of T2DM was an independent risk factor for prevalent VFs in women (OR=1.86, p=0.019) and men (OR=4.73, p<0.001). BMD at any site, however, was not significantly associated with the presence of prevalent VFs in T2DM patients, in contrast to the significant association in controls (at least p=0.010). Comparison of T2DM patients with and without VFs showed no significant differences in BMD values, bone markers, or diabetes status. Receiver operating characteristic analysis showed that the absolute L-, FN-, and R-BMD values for detecting prevalent VFs were higher in T2DM patients than controls, whereas their sensitivity and specificity were lower. T2DM patients may have an increased risk of VFs independent of BMD or diabetic complication status, suggesting that bone quality may define bone fragility in T2DM.
  • Hironori Bando; Naoko Hashimoto; Yushi Hirota; Kazuhiko Sakaguchi; Itoko Hisa; Yoshifumi Inoue; Yasuo Imanishi; Susumu Seino; Hiroshi Kaji
    INTERNAL MEDICINE JAPAN SOC INTERNAL MEDICINE 48 (5) 353 - 358 0918-2918 2009 [Refereed]
     
    A 49-year-old woman was admitted to our hospital for back pain with marked thoracic and extremity deformities leading to bed-rest for three years. She was diagnosed with hypophosphatemic osteomalacia based on her symptoms, X-ray and bone scintigram, high serum alkaline phosphatase level, and low serum levels of both phosphorus and 1,25 dihydroxyvitamin D(3) with inhibition of phosphorus reabsorption. Fanconi syndrome with renal tubular acidosis, vitamin D deficiency and primary biliary cirrhosis were related to the pathogenesis of osteomalacia in this case. Several causal diseases may be concomitantly responsible for acceleration of the severity of osteomalacia in this patient.
  • Hiroshi Kaji; Itoko Hisa; Yoshifumi Inoue; Junko Naito; Toshitsugu Sugimoto; Masato Kasuga
    JOURNAL OF BONE AND MINERAL METABOLISM SPRINGER JAPAN KK 27 (1) 76 - 82 0914-8779 2009/01 [Refereed]
     
    Bisphosphonate is an effective drug to reduce fracture risk in osteoporotic patients; however, factors affecting the efficacy of bisphosphonate treatment are not fully known, especially in Japanese patients. In the present study, we examined the relationships between an increase in lumbar spine bone mineral density (BMD) by bisphosphonates and several pretreatment parameters, including biochemical, bone/mineral, and body composition indices, in 85 postmenopausal osteoporotic patients treated with alendronate or risedronate. BMD increase was measured by dual-energy X-ray absorptiometry at the lumbar spine before and 2 years after treatment. BMD increase at the lumbar spine was observed as independent of age, height, weight, body mass index, and fat mass, although lean body mass seemed slightly related. On the other hand, fasting plasma glucose (FPG) levels were significantly and positively related to BMD increase at the lumbar spine. In multiple regression analysis, FPG levels were not significantly related to BMD increase at the lumbar spine when lean body mass was considered. As for bone/mineral parameters, BMD increase at the lumbar spine was not significantly related to serum levels of calcium, parathyroid hormone (PTH), and alkaline phosphatase or urinary levels of deoxypiridinoline and calcium excretion. As for BMD parameters, Z-scores of BMD at any site and bone geometry parameters obtained by forearm peripheral quantitative computed tomography were not significantly related to BMD increase at the lumbar spine. BMD increases at the lumbar spine were similar between groups with or without vertebral fractures. In conclusion, BMD increase at the lumbar spine by bisphosphonate treatment was not related to any pretreatment parameters, including body size, body composition, and bone/mineral metabolism in postmenopausal Japanese women with primary osteoporosis, although FPG correlated partly to BMD through lean body mass.
  • H. Kaji; J. Naito; Y. Inoue; H. Sowa; T. Sugimoto; K. Chihara
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG KG 40 (11) 746 - 751 0018-5043 2008/11 [Refereed]
     
    Statins possess pleiotropic effects in several tissues. Among them, their bone anabolic actions have been recently noted. We have proposed that Smad3, a TGF-beta-signaling molecule, is a promoter of bone formation. However, whether statins would affect TGF-beta-Smad3 pathway in osteoblasts is still unknown. The present study was performed to examine the effects of statin on Smad3 expression and cell apoptosis by employing Mouse osteoblastic MC3T3-E1 and rat osteoblastic UMR-106 cells. Statins (pitavastatin, mevastatin, and simvastatin) as well as alendronate increased the levels of Smad3 in MC3T3-E1 cells. The effects of pitavastatin on Smad3 levels were observed from 3 hours and later. Pitavastatin induced the expression of TGF-beta, and cycloheximide, a protein synthesis inhibitor, antagonized the increased levels of pitavastatin on Smad3. On the other hand, pitavastatin antagonized dexamethasone- or etoposide-induced apoptosis in a close-dependent manner, and Smad3 inactivation by dominant negative Smad3 or an inhibition of endogenous TGF-beta action by SB431542 antagonized anti-apoptotic effects of pitavastatin, indicating that pitavastatin suppressed osteoblast apoptosis partly through TGF-beta-Smad3 pathway. In conclusion, the present study has demonstrated for the first time that statin Suppressed cell apoptosis partly through TGF-beta-Smad3 pathway in osteoblastic cells.
  • Hiroshi Kaji; Mika Yamauchi; Rikako Nomura; Toshitsugu Sugimoto
    JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM ENDOCRINE SOC 93 (8) 3045 - 3050 0021-972X 2008/08 [Refereed]
     
    Context: Cortical bone geometry is one of the most important components of bone strength. Excess endogenous PTH or intermittent PTH administration affects cortical bone geometry; however, the changes in cortical bone geometry in patients with primary hyperparathyroidism (pHPT) after parathyroidectomy (PTX) remain unknown. Objective: The present study was performed to examine the longitudinal effects of treating endogenous PTH excess on cortical bone geometry in postmenopausal patients with pHPT by using peripheral quantitative computed tomography. Patients: Twenty postmenopausal pHPT patients and 30 postmenopausal control subjects matched for age participated in this study. Main Outcome Measures: Volumetric bone mineral density (vBMD), cortical bone geometric parameters, polar strength strain index, and polar cross-sectional moment of inertia were measured using peripheral quantitative computed tomography at the radius during the year after PTX. Results: After 1 yr, total and cortical vBMD significantly increased after PTX in the pHPT group (2.9 and 1.6%, respectively), whereas they significantly decreased in the control group (-2.1 and -1.3%, respectively). Significant decreases in cortical thickness and area were observed in the control group (-3.0 and -2.5%, respectively). In contrast, the pHPT group showed increases in cortical thickness and area (8.5 and 7.6%, respectively) as well as polar strength strain index 1 year after PTX. Conclusion: The present longitudinal study showed significant beneficial changes in volumetric BMD, cortical bone geometry, and bone strength index after PTX in postmenopausal women with pHPT.
  • Yasuo Kuroki; Hiroshi Kaji; Seiji Kawano; Fumio Kanda; Yutaka Takai; Michiko Kajikawa; Toshitsugu Sugimoto
    JOURNAL OF BONE AND MINERAL METABOLISM SPRINGER JAPAN KK 26 (3) 271 - 278 0914-8779 2008/05 [Refereed]
     
    Glucocorticoid (GC) therapy induces rapid bone loss, but the early changes in calcium and bone metabolism in patients treated with GC have not been clarified. To investigate the changes in calcium and bone metabolism during the early stage of GC therapy, we analyzed various biochemical markers of bone metabolism. The serum levels of calcium (Ca), phosphorus, parathyroid hormone (PTH), osteocalcin (OC), bone alkaline phosphatase (BAP), and type I collagen cross-linked N-telopeptide (NTx), as well as the urinary levels of Ca, creatinine, and NTx, were measured on days 0, 3, 7, and 28 of GC therapy. The subjects were divided into the following four groups: 9 patients receiving pulse therapy (P), 18 patients receiving prednisolone (PSL) at doses >= 40 mg/day (H), 9 patients receiving PSL at doses >= 20 mg/day (M), and 11 patients receiving PSL at doses <= 10 mg/day (S). The serum OC level showed a marked decrease on day 3 of GC therapy (-41.2% +/- 6.6%, P < 0.01), while the BAP level decreased gradually. Both serum and urinary NTx levels significantly increased on day 7 of GC therapy (9.9% +/- 4.5%, P < 0.05, and 42.2% +/- 10.6%, P < 0.01, respectively). Urinary Ca excretion was increased on day 3 of GC therapy and continued to increase until 4 weeks, while intact PTH showed an increase on day 3 and then remained constant until 4 weeks. In groups P and H, there were significant early changes in OC, BAP, NTx, and intact PTH levels, as well as urinary Ca excretion. Even a PSL dose of < 10 mg/day caused a decrease in the serum OC level. In conclusion, the biochemical markers of Ca and bone metabolism showed different kinetics depending on the dose of GC, and it is important for patients on high-dose GC therapy to receive prophylaxis for bone loss from the start of GC treatment.
  • Hiroshi Kaji; Mika Yamauchi; Kazuo Chihara; Toshitsugu Sugimoto
    CALCIFIED TISSUE INTERNATIONAL SPRINGER 82 (3) 182 - 190 0171-967X 2008/03 [Refereed]
     
    Glucocorticoid (GC) excess causes a great increase in fracture risk, but the effects of GC excess on cortical bone geometry are unknown. The present study was performed to examine the effects of GC excess on cortical bone geometry in both premenopausal and postmenopausal women. Ninety-six women receiving oral GC treatments and 10 women with Cushing syndrome (CS) were each compared to age-matched control subjects using peripheral quantitative computed tomography. Total area, periosteal circumference, and polar strength strain index (SSIp) were significantly lower in GC-treated patients compared with control subjects in premenopausal women but not in postmenopausal women. Moreover, cortical area and thickness as well as periosteal circumference and SSIp were significantly lower in patients with CS compared to controls in premenopausal women but not in postmenopausal women. Total area, cortical area, cortical thickness, periosteal circumference, as well as SSIp were significantly lower in GC-treated patients with vertebral fractures compared to those without vertebral fractures in premenopausal women but not in postmenopausal women. In conclusion, endogenous or exogenous GC excess affects bone geometry of forearms of premenopausal, but not postmenopausal, women. These effects of GC excess on bone geometry may provide a strength loss mechanism beneath increased vertebral fracture risk.
  • Yoshifumi Inoue; Hiroshi Kaji; Itoko Hisa; Takako Tobimatsu; Junko Naito; Mei-Fway Iu; Toshitsugu Sugimoto; Kazuo Chihara
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOC 55 (1) 57 - 65 0918-8959 2008/02 [Refereed]
     
    Controversy still exists about whether vitamin D status is related to the severity of primary hyperparathyroidism (pHPT), although vitamin D insufficiency is frequent in pHPT. The present study was therefore performed to examine the relationships between vitamin D status and various parameters in 30 postmenopausal pHPT patients. BMD values were measured by dual-energy x-ray absorptiometry at the lumbar spine (L2-4), femoral neck (FN) and distal one third of the radius (Rad 1/3). Serum levels of 25 hydroxy-vitamn D-3 [25(OH)D] and 1,25-dihydroxy vitamin D3 [1,25(OH),D3] were 15.8 +/- 3.5 mu g/l and 69.3 +/- 33.3 ng/l in pHPT patients, respectively. Serum levels of calcium and PTH seemed to be negatively correlated to serum 25(OH)D levels, although the differences were not significant. However, when subjects with the highest serum PTH levels (PTH> 1000 pg/ml) were excluded from the analysis, the correlation was significant between serum 25(OH)D levels and PTH, indicating that vitamin D status affects the severity of pHPT when severe cases were excluded. In addition, serum levels of 1,25(OH)(2)D-3 were significantly and negatively correlated to serum 25(OH)D levels. On the other hand, serum levels of 25(OH)D were significantly and positively correlated to BMD (Z-score) at the lumbar spine, but not at the radius and femoral neck; however, serum 25(OH)D levels were not correlated to the levels of any bone metabolic indices measured. Moreover, serum levels of 25(OH)D were not related to urinary calcium and the tubular reabsorption rate of phosphorus, and they were similar in groups with and without renal stones. In conclusion, vitamin D status seemed to be related to the severity of disease in postmenopausal patients with pHPT. In particular, the relationship between serum 25(OH)D level and BMD at the lumbar spine was predominant.
  • H. Kaji; M. Yamauchi; K. Chihara; T. Sugimoto
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG KG 40 (1) 60 - 65 0018-5043 2008/01 [Refereed]
     
    Although the role of PITH (parathyroid hormone) has been debated in glucocorticoid (CC)-induced osteoporosis (GIO), clinical data about the relation of endogenous PTH to bone metabolism in patients treated with GC are still lacking. The present study was performed to examine the relationship of PTH to bone metabolic indices, bone mineral density (BMD), and bone geometry in 174 female patients treated with oral GC for more than 6 months. Dual-energy X-ray absorptiometry and peripheral quantitative computed tomography (pQCF) were employed for the assessment of BMD and bone geometry. No elevation of serum PTH levels was observed in patients treated with GC. Although serum levels of osteocalcin were not related to serum PTH levels, urinary levels of deoxypiridinoline were positively correlated. Serum PTH levels were negatively related to BMD at any site. In pQCT, serum PTH levels were negatively correlated to both trabecular and cortical volumetric BMD. As for bone morphometric indices, serum PTH levels were significantly related to endocortical circumferences, cortical thickness, and cortical area. Moreover, serum PTH levels were significantly higher in patients with vertebral fractures, compared with those without vertebral fractures in GC-treated patients. In the present study, serum PTH levels were related to the elevation of bone resorption marker, decreased BMD, cortical thinning, and an increase of vertebral fracture risk. The elevation of sensitivity to PTH in bone might play some role in the pathogenesis of GIO.
  • Itoko Hisa; Hiroshi Kaji; Yoshifumi Inoue; Toshitsugu Sugimoto; Kazuo Chihara
    INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE E-CENTURY PUBLISHING CORP 1 (4) 319 - 326 1940-5901 2008 [Refereed]
     
    How glucose levels affect bone in patients with primary hyperparathyroidism is unknown, although the prevalence of impaired glucose metabolism is higher in patients with primary hyperparathyroidism. The present study was performed to examine the relationships between fasting plasma glucose (FPG) and various indices in 93 postmenopausal women with primary hyperparathyroidism. Bone mineral density (BMD) and body composition were measured by dual-energy Xray absorptiometry. Body weight, body mass index (BMI), fat mass and % fat were positively related to FPG. Serum levels of calcium and parathyroid hormone (PTH) as well as bone metabolic indices were not related to FPG and immunoreactive insulin levels. As for BMD, FPG was positively related to the Z scores of BMD at the lumbar spine and femoral neck, although it was not significantly related to the Z-score of BMD at the radius. On the other hand, immunoreactive insulin levels were not significantly related to BMD parameters at any sites. In multiple regression analysis, FPG was significantly related to BMD (Z score) at the lumbar spine and femoral neck, when body weight, BMI, immunoreactive insulin, PTH, and bone resorption indices were considered; however, these relationships at the lumbar spine were not significant when fat mass was considered. In conclusion, the present study indicated that FPG levels were positively related to BMD at the lumbar spine and femoral neck in postmenopausal women with primary hyperparathyroidism.
  • M. Oka; T. Kamo; E. Sasaki; H. Kaji; H. Nishizawa; Y. Imanishi; C. Nishigori
    BRITISH JOURNAL OF DERMATOLOGY BLACKWELL PUBLISHING 157 (1) 198 - 200 0007-0963 2007/07 [Refereed]
  • M. Yamamoto; T. Yamaguchi; M. Yamauchi; H. Kaji; T. Sugimoto
    CALCIFIED TISSUE INTERNATIONAL SPRINGER 80 (6) 353 - 358 0171-967X 2007/06 [Refereed]
     
    Although the association between diabetes and osteoporosis has been studied, it remains unclear if the pathogenesis of vertebral fractures in patients with type 2 diabetes would be similar to those without diabetes. One hundred and fifty female diabetic patients without apparent proteinuria as well as 716 women without diabetes (control group) were examined by lateral thoracic and lumbar spine radiographs as well as dual-energy X-ray absorptiometry. Vertebral fractures were found in 26 (17.3%) and 158 (22.1%) subjects in the diabetic and control groups, respectively. Diabetic patients had higher absolute and age-matched (Z score) values of lumbar bone mineral density (L-BMD) than controls despite their significantly higher mean age. By receiver operating characteristic (ROC) analysis, the absolute L-BMD values for detecting vertebral fractures were higher and sensitivity and specificity were lower in diabetic patients than controls (0.816 g/cm(2) vs. 0.716 g/cm(2) and 66.0% vs. 74.8%, respectively). Logistic regression analysis adjusted for age, body weight, and height also showed that L-BMD was not significantly associated with the presence of vertebral fractures in diabetic patients (odds ratio [OR] = 0.61, 95% confidence interval [CI] 0.34-1.09 per standard deviation increase, P = 0.0954), in contrast to the significant association in controls (OR = 0.23, 95% CI 0.16-0.33, P < 0.0001). These results show that L-BMD is not sensitive enough to assess the risk of vertebral fractures in female diabetic patients and suggest that bone fragility not defined by BMD might be related to the risk of vertebral fractures in them.
  • Mari Imanaka; Keiji Iida; Hitoshi Nishizawa; Hidenori Fukuoka; Ryoko Takeno; Kentaro Takahashi; Hiroshi Kaji; Yutaka Takahashi; Yasuhiko Okimura; Hidesuke Kaji; Yasuo Imanishi; Kazuo Chihara
    INTERNAL MEDICINE JAPAN SOC INTERNAL MEDICINE 46 (18) 1577 - 1583 0918-2918 2007 [Refereed]
     
    A 16-year-old girl presented with McCune-Albright syndrome associated with acromegaly and fibrous dysplasia. Brain MRI demonstrated a pituitary tumor. X-ray films showed bone deformities, and (TmO4)-Tm-99 bone scintigraphy revealed increased uptake of radioactivity in the affected bones. Although the serum FGF23 level was increased, the serum calcium, phosphate, and active vitamin D levels were all within normal limits. GNAS gene mutation was detected at neither codon 201 nor 227 by conventional PCR-based direct sequencing analysis. We performed a selective PCR with peptide nucleic acid (PNA) clamping to increase the sensitivity for gene mutation detection and identified the R201C GNAS mutation.
  • H. Kaji; J. Naito; H. Sowa; T. Sugimoto; K. Chihara
    EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES JOHANN AMBROSIUS BARTH VERLAG MEDIZINVERLAGE HEIDELBERG GMBH 114 (10) 599 - 604 0947-7349 2006/11 [Refereed]
     
    Hepatitis C-associated osteosclerosis (HCAO) is a rare syndrome characterized by severe, acquired, generalized osteosclerosis and hyperostosis in adults who are infected with the hepatitis C virus. However, the detail of the pathogenesis of HCAO is still unknown. We examined the effects of serum of the HCAO patient on the proliferation, alkaline phosphatase (ALP) activity and transforming growth factor (TGF)-beta-Smad signaling in mouse osteoblastic cells. The patient was compatible with HCAO, characterized by high bone mass, bone thickening and bone pain with normal lamelar bone. The serum from the HCAO patient increased the levels of TGF-beta and Smad3 expression in osteoblastic MC3T3-E1 cells, compared with the control subject. Moreover, the serum from the HCAO patient significantly augmented TGF-beta-induced transcriptional activity with luciferase assay using 3TP-Lux with a Smad3-specific responsive element. In addition, the serum from the HCAO patient significantly stimulated the MTT intensity, the level of proliferating cell nuclear antigen expression, a proliferation marker, and ALP activity in MC3T3-E1 cells, compared with that from the control subject. In conclusion, the present study indicated that the serum from the HCAO patient stimulated TGF-beta-Smad signaling, as well as the proliferation and ALP activity in osteoblastic cells. Some soluble factors other than parathyroid hormone might be related to the pathogenesis of HCAO.
  • H. Kaji; J. Naito; H. Sowa; T. Sugimoto; K. Chihara
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG KG 38 (11) 740 - 745 0018-5043 2006/11 [Refereed]
     
    Smad3, a critical component of the TGF-beta signaling pathways, plays an important role in the regulation of bone formation. However, how Smad3 affects osteoblast at the different differentiation stage remains still unknown. In the present study, we examined the effects of Smad3 on osteoblast phenotype by employing mouse bone marrow ST-2 cells and mouse osteoblastic MC3T3-E1 cells at the different differentiation stage. Smad3 overexpression significantly inhibited bone morphogenetic protein-2 (BMP-2)-induced ALP activity in ST-2 cells, indicating that Smad3 suppresses the commitment of pluripotent mesenchymal cells into osteoblastic cells. Smad3 increased the levels of COLI and ALP mRNA at 7 day cultures in MC3T3-E1 cells, and its effects on COLI were decreased as the culture periods progress, although its effects on ALP were sustained during 21 day cultures. Smad3 overexpression enhanced the level of Runx2 and OCN mRNA at 14 day and 21 day cultures. Smad3 increased the levels of MGP and NPP-1 mRNA, although the extent of increase in MGP and NPP-1 was reduced and enhanced during the progression of culture period, respectively. Smad3 did not affect the level of ANK mRNA. On the other hand, Smad3 enhanced the level of MEPE mRNA at 14 and 21 day cultures, although Smad3 decreased it at 7 day cultures. In conclusion, Smad3 inhibits the osteoblastic commitment of ST-2 cells, while promotes the early stage of differentiation and maturation of osteoblastic committed MC3T3-E1 cells. Also, Smad3 enhanced the expression of mineralization-related genes at the maturation phase of MC3T3-E1 cells.
  • H Kaji; MF Iu; J Naito; T Sugimoto; K Chihara
    JOURNAL OF BONE AND MINERAL METABOLISM SPRINGER TOKYO 24 (4) 349 - 352 0914-8779 2006/07 [Refereed]
  • Junko Naito; Hiroshi Kaji; Hideaki Sowa; Riko Kitazawa; Sohei Kitazawa; Toshihiko Tsukada; Geoffrey N. Hendy; Toshitsugu Sugimoto; Kazuo Chihara
    Endocrine HUMANA PRESS INC 29 (3) 485 - 490 1355-008X 2006/06 [Refereed]
     
    In some patients with multiple endocrine neoplasia type 1 (MEN1) it is not possible to identify a germline mutation in the MEN1 gene. We sought to document the loss of expression and function of the MEN1 gene product, menin, in the tumors of such a patient. The proband is an elderly female patient with primary hyperparathyroidism, pancreatic islet tumor, and breast cancer. Her son has primary hyperparathyroidism. No germline MEN1 mutation was identified in the proband or her son. However, loss of heterozygosity at the MEN1 locus and complete lack of menin expression were demonstrated in the proband's tumor tissue. The proband's cultured parathyroid cells lacked the normal reduction in proliferation and parathyroid hormone secretion in response to transforming growth factor-β. This assessment provided insight into the molecular pathogenesis of the patient and provides evidence for a critical requirement for menin in the antiproliferative action of transforming growth factor-β. © 2006 by Humana Press Inc. All rights of any nature whatsoever reserved.
  • H. Kaji; R. Nomura; M. Yamauchi; K. Chihara; T. Sugimoto
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG KG 38 (6) 411 - 416 0018-5043 2006/06 [Refereed]
     
    Patients with primary hyperparathyroidism (pHPT) have reduced bone mineral density (BMD). Although pHPT causes high bone turnover, the exact metabolic bone markers useful for predicting changes in BMD after parathyroidectomy (PTX) remain elusive. The present study was performed to examine the relationship between bone metabolic indices and BMD changes after PTX in 29 pHPT Japanese patients, which received PTX successfully. BMD values were measured by dual-energy X-ray absorptiometry in the lumbar spine and distal one third of radius. As for bone metabolic indices, serum bone-type alkaline phosphates (BAP), serum osteocalcin (OCN), urinary deoxypiridinoline (Dpd), and urinary type I collagen cross-linked N-telopeptides (NTX) were measured. The study included 10 male and 19 female patients (17 postmenopausal). Urinary Dpd, but not NTX was significantly correlated with serum BAP and OCN. Either bone formation or bone resorption indices were significantly and highly correlated with Z-score of BMD in the radius, but not at lumbar spine. Urinary Dpd was significantly correlated with BMD changes at both lumbar spine and radius and at all time points over the two years after PTX. These correlations were most potent among bone metabolic indices in this study. The measurement of urinary Dpd would be useful for predicting long-term changes in BMD at radial and lumbar spine after PTX than other bone metabolic indices.
  • T Tobimatsu; H Kaji; H Sowa; J Naito; L Canaff; GN Hendy; T Sugimoto; K Chihara
    ENDOCRINOLOGY ENDOCRINE SOC 147 (5) 2583 - 2590 0013-7227 2006/05 [Refereed]
     
    PTH, via the PTH/PTH-related protein receptor type 1 that couples to both protein kinase A (PKA) and protein kinase C (PKC) pathways, and the canonical Wnt-beta-catenin signaling pathway play important roles in bone formation. In the present study we have examined the interaction between the PTH and Wnt signaling pathways in mouse osteoblastic MC3T3-E1 cells. PTH dose- and time-dependently increased the concentrations of beta-catenin. The PKA activator, forskolin, and the PKC activator, phorbol 12-myristate-13-acetate, as well as the PTH analog, [NIe(8,18),Tyr(34)] human PTH-(3-34) amide, all increased beta-catenin levels. Both H-89, a specific PKA inhibitor, and PKC inhibitors, staurosporine and calphostin C, antagonized PTH stimulation of beta-catenin levels. TGF-beta as well as transfection of the TGF-beta-signaling molecule, Smad3, enhanced beta-catenin levels, and this was antagonized by transfection of a dominant-negative Smad3. The transcriptional activity of transfected dominant-active beta-catenin was enhanced by PTH, an effect that was antagonized by cotransfection of a dominant-negative Smad3. PTH as well as LiCl2, which mimics the effects of the Wnt-beta-catenin pathway, rescued the dexamethasone- and etoposide-induced apoptosis of osteoblastic cells. In conclusion, the data demonstrate that PTH stimulates osteoblast beta-catenin levels via Smad3, and that both PKA and PKC pathways are involved. The canonical Wnt-beta-catenin pathway is likely to be involved in the antiapoptotic actions of PTH by acting through Smad3 in osteoblasts.
  • H Kaji; T Tobimatsu; J Naito; MF Iu; M Yamauchi; T Sugimoto; K Chihara
    OSTEOPOROSIS INTERNATIONAL SPRINGER LONDON LTD 17 (4) 627 - 633 0937-941X 2006/04 [Refereed]
     
    Glucocorticoid (GC) causes bone loss and an increase in bone fragility. However, fracture risk was found to be only partly explained by bone mineral density in GC-treated patients (GC patients). Although GC causes a change in the distribution of fat in the body, the relationship between body composition and fracture risk in GC patients remains unknown. The present study examined the relationship between the presence or absence of vertebral fractures and various indices, including body composition, in 92 premenopausal GC patients, 122 postmenopausal GC patients and 122 postmenopausal age-matched control subjects. Dual-energy X-ray absorptiometry was employed to analyze body composition. Percentage lean body mass (LBM), % fat and % trunk fat were not significantly different between postmenopausal GC patients and the control women. When groups with and without vertebral fractures were compared, % LBM and % fat were significantly higher and lower in groups with vertebral fractures, respectively, in postmenopausal GC patients, but not in the postmenopausal control women, although % trunk fat was not significantly different between groups with and without vertebral fractures. Femoral neck BMD was negatively correlated with % LBM and positively correlated with % fat. In premenopausal GC patients, % trunk fat was significantly higher in the fracture group, although % LBM and % fat were not significantly different between groups with and without vertebral fractures. The present study revealed that body composition is related to vertebral fracture risk in GC-treated patients. Lower % fat can be included in the determination of vertebral fractures in postmenopausal GC-treated patients. The influence of body composition on vertebral fracture risk may be different between the pre- and postmenopausal state in GC patients.
  • H Kaji; M Yamauchi; K Chihara; T Sugimoto
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOCIETY 53 (1) 27 - 34 0918-8959 2006/02 [Refereed]
     
    Glucocorticoid (GC)-induced osteoporosis (GIO) is a serious problem for patients taking GC therapy. GC increases risk for fracture. However, there are controversies regarding the threshold of bone mineral density (BMD) in patients with GIO. The present study aimed to examine the relationship between the presence or absence of vertebral fracture and various indices including BMD in 136 female Japanese patients treated with oral GC (102 patients with autoimmune diseases). Moreover, we analyzed the cut-off values of BMD for incidence of vertebral fracture in patients with oral GC use and compared these values with those in control subjects. BMD was measured by dual-energy X-ray absorptiometry of the lumbar spine, femoral neck, and distal one third of radius. We compared various indices between patients taking oral GC with and without vertebral fracture. Age, body height, and body weight were significantly greater, shorter, and lower in the group with vertebral fracture, respectively. As for BMD, age-matched BMD seemed lower in the fracture group, although the differences were significant between both groups only at the femoral neck. Duration of GC treatment was Ionizer in the fracture group. Cut-off values of BMD at lumbar spine, femoral neck, and distal radius were higher in patients with GC treatment compared with those of control group [GC vs control (g/cm(2)): 0.807 vs 0.716 at lumbar spine; 0.611 vs 0.581 at femoral; 0.592 vs 0.477 at radius). The sensitivity and specificity were lower ill patients with GC treatment compared with those of control group. The present Study demonstrated that the thresholds of BMD for vertebral fracture were higher in Japanese female patients with oral GC treatment at any site compared with postmenopausal Subjects. The factors other than BMD were considered to affect bone strength and vertebral fracture risk.
  • T Yamaguchi; M Kanatani; M Yamauchi; H Kaji; T Sugishita; DJ Baylink; S Mohan; K Chihara; T Sugimoto
    CALCIFIED TISSUE INTERNATIONAL SPRINGER 78 (1) 18 - 24 0171-967X 2006/01 [Refereed]
     
    We previously found that serum levels of insulin-like growth factor I (IGF-I) and IGF-binding protein (IGFBP)-3, but not IFGBP-2, were associated with bone mineral density (BMD) and the risk of vertebral fractures. The aim of the present study was to investigate the roles of IGFBP-4 and -5 in age-dependent bone loss and vertebral fracture risk in postmenopausal Japanese women and to compare them with those of IGF-I and IGFBP-3. One hundred and ninety-three Japanese women aged 46-88 years (mean 62.5) were enrolled in the cross-sectional study. BMD was measured at the lumbar spine, femoral neck, ultradistal radius (UDR), and total body by dual-energy X-ray absorptiometry. Serum levels of IGFBP-4 and -5 as well as IGF-I and IGFBP-3 were measured by radioimmunoassay. Serum levels of IGF-I, IGFBP-3, and IGFBP-5 declined with age, while serum IGFBP-4 increased with age. Multiple regression analysis was performed between BMD at each skeletal site and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, and serum creatinine. BMD at the UDR was significantly and positively correlated with all serum levels of IGF-I and IGFBPs measured (P < 0.01), while BMD at the femoral neck was correlated with none of them. Serum IGF-I level was significantly and positively correlated with BMD at all sites except the femoral neck (P < 0.01), while serum IGFBP-3 and -4 levels were significantly and positively correlated with only radial BMD (P < 0.01). Serum IGFBP-5 level was positively correlated with UDR BMD (P < 0.001) and negatively correlated with total BMD (P < 0.05). Serum IGF-I, IGFBP-3, and IFGBP-5 levels were significantly lower in women with vertebral fractures than in those without fractures (mean +/- SD: 97.1 +/- 32.1 vs. 143.9 +/- 40.9 ng/dl, P < 0.0001; 2.18 +/- 1.02 vs. 3.23 +/- 1.07 mu g/ml, P < 0.0001; 223.6 +/- 63.3 vs. 246.5 +/- 71.5 ng/ml, P = 0.0330, respectively). When multivariate logistic regression analysis was performed with the presence of vertebral fractures as a dependent variable and serum levels of IGF-I and IGFBPs adjusted for age, body weight, height, serum creatinine, and serum alubumin as independent variables, IGF-I and IGFBP-3 were selected as indices affecting the presence of vertebral fractures [odds ratio (OR) = 0.29, 95% confidential interval (CI) 0.15-0.57 per SD increase, P = 0.0003 and OR = 0.31, 95% CI 0.16-0.61 per SD increase, P = 0.0007, respectively]. To compare the significance values, IGF-I, IGFBP-3, and age were simultaneously added as independent variables in the analysis. IGFBP-3 was more strongly associated with the presence of vertebral fractures than IGF-I and age (P = 0.0006, P = 0.0148, and P = 0.0013, respectively). Thus, after comprehensive measurements of serum levels of IGF-I and IGFBPs, it seems that serum IGF-I level is most efficiently associated with bone mass and that serum IGFBP-3 level is most strongly associated with the presence of vertebral fractures in postmenopausal women among the IGF system components examined.
  • H Kaji; R Kosaka; M Yamauchi; K Kuno; K Chihara; T Sugimoto
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOCIETY 52 (6) 659 - 666 0918-8959 2005/12 [Refereed]
     
    Peripheral quantitative computed tomography (pQCT) is useful to measure volumetric bone mineral density (vBMD) distinguishing trabecular from cortical bones as well as quantity of bone geometry. In the present study, we examined the effects of age, grip strength and smoking on vBMD, bone geometry and bone strength index (polar strength strain index (SSIp)), and then compared with the differences between female and male by employing pQCT in Japanese 252 female and 230 male subjects. Age was negatively correlated with vBMD, cortical area (Ct.Ar) and cortical thickness (Ct.Th) as well as SSIp in both sexes, and the correlation coefficients were higher in female, compared with those in male. Although age was correlated with endocortical circumferences (En.Le) in both sexes, periosteal circumferences (Ex.Le) were correlated with age only in male. Volumetric BMD, Ct.Ar, Ct.Th and SSIp were significantly lower in the group with vertebral fractures, although En.Le and Ex.Le were similar between subjects with and without vertebral fractures. Grip strength was positively correlated with vBMD, Ct.Ar, Ct.Th as well as SSIp. The extent of correlation was much higher in female, compared with that in male. Ct.vBMD, Ct.Ar, Ct.Th and SSIp, but not trabecular vBMD, were significantly lower in the group with high Brinkman index (number of cigarettes smoked per day) x (duration of smoking (years)) in female. These parameters were not significantly different between groups with high and low Brinkman index in male. In conclusion, the present study demonstrated that age, grip strength and smoking affected forearm vBMD, bone Geometry and bone strength index by pQCT. These effects were greater in female, compared with those in male.
  • MF Iu; H Kaji; J Naito; H Sowa; T Sugimoto; K Chihara
    JOURNAL OF BONE AND MINERAL METABOLISM SPRINGER TOKYO 23 (6) 450 - 455 0914-8779 2005/11 [Refereed]
     
    Glucocorticoid (GC)-induced osteoporosis (GIO) is frequently seen in patients with excessive GC. Numerous questions remain to be clarified about the pathogenesis and treatment of GIO, and the mechanism of GC-inhibited bone formation is not well known. Several studies suggest that parathyroid hormone (PTH) and hormone replacement therapy are effective for GIO. We therefore investigated whether PTH and estrogen would affect cell proliferation and alkaline phosphatase ( ALP) activity inhibited by dexamethasone (Dex) in mouse osteoblastic cell-line MC3T3-E1 cells. Low-dose (10(-11) M) PTH as well as 10(-8) M 17-beta-estradiol (17 beta-E-2) significantly attenuated Dex-inhibited ALP activity, although 10(-8) M PTH did not affect it. ICI 182780 (10(-8) M) antagonized the effects of 17 beta-E-2 on Dex-suppressed ALP activity. Neutralizing anti-IGF-I antibody (3 mu g/ml) blocked the reverse effects of 17 beta-E-2 on ALP activity suppressed by Dex. PTH (10(-11) M), but not 17 beta- E-2, significantly attenuated [H-3] thymidine incorporation inhibited by Dex. On the other hand, PTH and estrogen did not affect the level of 11-beta-hydrosteroid dehydrogenase type I mRNA increased by Dex. In conclusion, the present study demonstrated that low-dose PTH and estrogen reversed Dex-inhibited ALP activity in the mouse osteoblastic cell-line.
  • H Kaji; M Kanatani; T Sugimoto; K Chihara
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG KG 37 (10) 589 - 592 0018-5043 2005/10 [Refereed]
     
    Statins stimulate bone formation partly by inducing osteoblast differentiation, although there is controversy about the effects of statins on bone mineral density and fracture risk. Several studies have revealed that statins suppress bone resorption. However, the mechanism by which statins inhibit bone resorption is still unclear. The present study was performed to clarify the effects of statins on osteoclast formation as well as the levels of osteoprotegerin (OPG) and receptor activator of NFKB ligand (RANKL) mRNA in mouse bone-cell cultures by semiquantitative RT-PCR. 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D-3] significantly stimulated osteoclast formation and 10(-6) M statins (mevastatin and simvastatin) significantly antagonized osteoclast formation stimulated by 1,25(OH)(2)D-3 in mouse bone-cell cultures, including both osteoblasts and osteoclasts. 10(-6) M mevastatin and simvastatin increased the level of OPG mRNA in mouse bone-cell cultures. On the other hand, 10(-6) M mevastatin and simvastatin inhibited the level of RANKL mRNA in these cultures. In conclusion, the present study demonstrates that statins inhibit osteoclast formation in mouse bone-cell cultures. Moreover, statins also increased and decreased the levels of OPG and RANKL mRNA expression in these cultures, respectively. The modulation of OPG/RANKL may be involved in the inhibition of osteoclast formation by statins.
  • H Kaji; M Yamauchi; K Chihara; T Sugimoto
    EUROPEAN JOURNAL OF ENDOCRINOLOGY BIO SCIENTIFICA LTD 153 (3) 373 - 378 0804-4643 2005/09 [Refereed]
     
    Background and objective: Primary hyperparathyroidism (pHPT) is one of the causal diseases that induce secondary osteoporosis. Although patients with pHPT have reduced bone mineral density (MM) especially at the cortical bone, there have been controversies about risk of fracture. Moreover, no reports have been available about the threshold of BMD for fractures in pHPT patients. Methods: BMD values were measured by dual-energy x-ray absorptiometry at lumbar spine, femoral neck and distal one third of radius. Various indices were compared in 116 female pHPT patients and 716 control subjects. Moreover, we analyzed relationship between the cut-off values of BMD and the prevalence of vertebral fractures in pHPT and control subjects. Results: The prevalence of subjects with vertebral fractures was lower in pHPT patients, compared with that of control subjects. Age and body height were significantly higher and lower in pHPT women with vertebral fractures, respectively. Lumbar spine BMD was significantly lower in pHPT women with vertebral fractures, presumably due to their increased age. There were no differences in femoral neck and radius BMD or in bone metabolic indices between pHPT women with and without vertebral fractures. On the other hand, age-matched BMD was not significantly different between both groups at any measured site. Cut-off values of BMD at lumbar spine and femoral neck were lower in postmenopausal pHPT patients, compared with those of the postmenopausal control group. Moreover, cut-off values of BMD at radius was much lower in postmenopausal pHPT patients, compared with those of the postmenopausal control group (pHPT vs control (g/cm(2)): 0.670 vs 0.706 at lumbar spine; 0.549 vs 0.570 at femoral; 0.394 vs 0.474 at radius). Sensitivity and specificity of vertebral fractures was lower in pHPT patients, compared with those in control group. Conclusions: The present cross-sectional study demonstrated that thresholds of BMD for vertebral fractures were lower especially at radial bone in female patients with pHPT, compared with those in the control group.
  • GN Hendy; H Kaji; H Sowa; JJ Lebrun; L Canaff
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG KG 37 (6) 375 - 379 0018-5043 2005/06 [Refereed][Invited]
     
    Pituitary: Menin is a Smad3-interacting protein; inactivation of menin blocks transforming growth factor (TGF)-beta and activin signaling, antagonizing their growth-inhibitory properties in anterior pituitary cells. Menin is also required for the activin-induced inhibition of prolactin expression mediated by the Smads and the transcription factor, Pit-1. The interaction between menin and Smad3 is direct. Parathyroid: In cultured parathyroid cells from uremic hemodialysis patients, in which the menin signaling pathways are probably still intact, menin inactivation achieved by menin antisense oligonucleotides leads to loss of TGF-beta inhibition of parathyroid cell proliferation and parathyroid hormone (PTH) secretion. Moreover, TGF-beta does not affect the proliferation and PTH production of parathyroid cells from multiple endocrine neoplasia type 1 (MEN1) patients. Osteoblast: Men1-null mouse fetuses that die at day 12 or earlier have cranial/facial hypoplasias implicating menin in bone development. Menin is required for the commitment of multipotential mesenchymal stem cells into the osteoblast lineage. This is achieved by menin interacting physically and functionally with bone morphogenetic protein (BMP)-2 regulated Smads, such as Smad1 and Smad5, and the key osteoblast regulator, Runx2. These interactions are lost as the committed osteoblasts differentiate further at which time menin interacts with Smad3, mediating the negative regulation of Runx2 by TGF-beta. Menin also suppresses osteoblast maturation, partly by inhibiting the differentiation actions of JunD.
  • MF Iu; H Kaji; H Sowa; J Naito; T Sugimoto; K Chihara
    JOURNAL OF ENDOCRINOLOGY SOC ENDOCRINOLOGY 185 (1) 131 - 138 0022-0795 2005/04 [Refereed]
     
    Central in the pathogenesis of glucocorticoid (GC)induced osteoporosis is the effects of GC on bone formation. However, the mechanism of GC-inhibited bone formation is not well known. Transforming growth factor (TGF)-beta is most abundant in bone matrix compared with other tissues, and we have recently proposed that Smad3, a TGF-beta signaling molecule, is important for promoting bone formation. However, no reports have been available about the effects of GC on Smad3 in osteoblasts. In the present study, we investigated whether dexamethasone (Dex), an active GC analog, would affect the expression and activity of Smad3 in mouse osteoblastic MC3T3-E1 and rat osteoblastic UMR-106 cells. Dex significantly suppressed Smad3-stimulated alkaline phosphatase (ALP) activity, although it did not affect TGF-beta-inhibited ALP activity in MC3T3-E1 cells. Moreover, pretreatment with Dex suppressed TGF-beta-enhanced expression of type I collagen in MC3T3-E1 and UMR-106 cells. In the luciferase assay using p3TP-Lux with a Smad3-specific response element, Dex significantly suppressed the transcriptional activity induced by TGF-beta as well as Smad3. However, Dex did not affect the expression of Smad3 in these cells at both mRNA and protein levels. In conclusion, the present study indicates that Dex inhibits ALP activity and type I collagen expression, presumably by suppressing Smad3-induced transcriptional activity but not by modulating Smad3 expression in osteoblastic cells.
  • M Yamauchi; T Yamaguchi; H Kaji; T Sugimoto; K Chihara
    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM AMER PHYSIOLOGICAL SOC 288 (3) E608 - E616 0193-1849 2005/03 [Refereed]
     
    We have previously shown that the extracellular calcium-sensing receptor (CaR) is expressed in various bone marrow-derived cell lines and plays an important role in stimulating their proliferation and chemotaxis. It has also been reported that the CaR modulates matrix production and mineralization in chondrogenic cells. However, it remains unclear whether the CaR plays any role in regulating osteoblast differentiation. In this study, we found that mineralization of the mouse osteoblastic MC3T3-E1 cells was increased when the cells were exposed to high calcium (2.8 and 3.8 mM) or a specific CaR activator, NPS-R467 (1 and 3 muM). Next, we stably transfected MC3T3-E1 cells with either a CaR antisense vector (AS clone) or a vector containing the inactivating R185Q variant of the CaR (DN clone) that has previously been shown to exert a dominant negative action. Alkaline phosphatase activities were decreased compared with controls in both the AS and DN clones. However, the levels of type I procollagen and osteopontin mRNA in the AS clone, as detected by Northern blotting, were almost the same as in the controls. On the other hand, the expression of osteocalcin, which is expressed at a later stage of osteoblastic differentiation, was significantly reduced in both the AS and DN clones. Mineralization was also decreased in both clones. In conclusion, this study showed that the abolition of CaR function results in diminishing alkaline phosphatase activity, osteocalcin expression, and mineralization in mouse osteoblastic cells. This suggests that the CaR may be involved in osteoblastic differentiation.
  • J Naito; H Kaji; H Sowa; GN Hendy; T Sugimoto; K Chihara
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 280 (6) 4785 - 4791 0021-9258 2005/02 [Refereed]
     
    Mice null for menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, exhibit cranial and facial hypoplasia suggesting a role for menin in bone formation. We have shown previously that menin is required for the commitment of multipotential mesenchymal stem cells into the osteoblast lineage in part by interacting with the bone morphogenetic protein (BMP)-2 signaling molecules Smad1/5, and the key osteoblast transcriptional regulator, Runx2 (Sowa H., Kaji, H., Hendy, G. N., Canaff, L., Komori, T., Sugimoto, T., and Chihara, K. (2004) J. Biol. Chem. 279,40267-40275). However, menin inhibits the later differentiation of committed osteoblasts. The activator protein-1 (AP-1) transcription factor, JunD, is expressed in osteoblasts and has been shown to interact with menin in other cell types. Here, we examined the consequences of menin-JunD interaction on osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells. JunD expression, assessed by immunoblot, gradually increased during osteoblast differentiation. Stable expression of JunD enhanced expression of the differentiation markers, Runx2, type 1 collagen (COL1), and osteocalcin (OCN) and alkaline phosphatase (ALP) activity and mineralization. Hence, JunD promotes osteoblast differentiation. In MC3T3-E1 cells in which menin expression was reduced by stable menin antisense DNA transfection, JunD levels were increased. When JunD and menin were co-transfected in MC3T3-E1 cells, they co-immunoprecipitated. JunD overexpression increased the transcriptional activity of an AP-1 luciferase reporter construct, and this activity was reduced by co-transfection of menin. Therefore, JunD and menin interact both physically and functionally in osteoblasts. Furthermore, menin overexpression inhibited the ALP activity induced by JunD. In conclusion, the data suggest that menin suppresses osteoblast maturation, in part, by inhibiting the differentiation actions of JunD.
  • Q Chen; H Kaji; M Kanatani; T Sugimoto; K Chihara
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG KG 36 (10) 674 - 678 0018-5043 2004/10 [Refereed]
     
    The role that androgens play in the regulation of bone metabolism has been substantiated in animals and humans. We previously demonstrated that testosterone inhibits osteoclast differentiation stimulated by parathyroid hormone through the androgen receptor in mouse bone-cell cultures. However, the details of this mechanism are still unknown. The present study was aimed at examining whether testosterone would affect the mRNA levels of osteoprotegerin (OPG) and receptor activator of NfkappaB ligand (RANKL) in mouse bone-cell cultures as well as mouse osteoblastic cell-line, MC3T3-E1 cells by employing semi-quantitative RT-PCR. Testosterone increased OPG mRNA expression in both mouse bone-cell cultures and MC3T3-E1 cells. 10-8 M PTH-(1-34) as well as 10-8M 1,25-dihydroxyvita-min D3 [1,25(OH)2D3] inhibited OPG mRNA expression in mouse bone cells. 10-8 M testosterone antagonized OPG mRNA expression inhibited by 10-8 M PTH-(1-34), but failed to affect OPG mRNA expression inhibited by 10-8 M 1,25(OH)2D3. 10-8 M alpha-dehydrotestosterone, a non-aromatizable androgen, increased OPG mRNA expression. On the other hand, testosterone did not affect RANKL mRNA expression in MC3T3-E1 or mouse bone cells. In conclusion, the present study demonstrated that testosterone increased OPG mRNA expression in mouse bone-cell cultures and the osteoblastic cell line. These effects are likely to take place through the androgen receptor.
  • H Sowa; H Kaji; GN Hendy; L Canaff; T Komori; T Sugimoto; K Chihara
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 279 (39) 40267 - 40275 0021-9258 2004/09 [Refereed]
     
    Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, however, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G. N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. ( 2003) J. Biol. Chem. 278, 21058 - 21069). Here, we have examined the mechanism of action of menin in regulating osteoblast differentiation using the mouse bone marrow stromal ST2 and osteoblast MC3T3-E1 cell lines. In ST2 cells, reduced menin expression achieved by transfection of menin antisense DNA ( AS) antagonized bone morphogenetic protein (BMP)-2-induced alkaline phosphatase activity and osteocalcin and Runx2 mRNA expression. Menin was co-immunoprecipitated with Smad1/5 in ST2 and MC3T3-E1 cells, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5 in ST2 cells, but not MC3T3-E1 cells. Menin was co-immunoprecipitated with the key osteoblast regulator, Runx2, and AS antagonized Runx2 transcriptional activity and the ability of Runx2 to stimulate alkaline phosphatase activity only in ST2 cells but not in MC3T3-E1 cells. In the osteoblast MC3T3-E1 cells, transforming growth factor-beta and its signaling molecule, Smad3, negatively regulated Runx2 transcriptional activity. Menin and Smad3 were co-immunoprecipitated, and combined menin and Smad3 overexpression antagonized, whereas menin and the dominant-negative Smad3DeltaC together enhanced BMP-2-induced transcriptional activity of Smad1/5 and Runx2. Smad3 alone had no effect. Therefore, menin interacts physically and functionally with Runx2 in uncommitted mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts the interaction of menin and the transforming growth factor-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late-tage differentiation.
  • H Sowa; H Kaji; R Kitazawa; S Kitazawa; T Tsukamoto; S Yano; T Tsukada; L Canaff; GN Hendy; T Sugimoto; K Chihara
    CANCER RESEARCH AMER ASSOC CANCER RESEARCH 64 (6) 2222 - 2228 0008-5472 2004/03 [Refereed]
     
    Primary hyperparathyroidism is a common endocrine disorder caused by parathyroid gland enlargement and excessive parathyroid hormone (PTH) secretion. However, the precise mechanisms of tumorigenesis of the parathyroids are unknown. Here we have investigated the roles of transforming growth factor (TGF)-beta and menin, the product of the multiple endocrine neoplasia type 1 (Men1) gene, in the proliferation and PTH production of parathyroid cells from either patients with secondary hyperparathyroidism or Men1. TGF-beta was expressed in the parathyroid endocrine cells. Addition of TGF-beta to parathyroid cells from patients with secondary hyperparathyroidism inhibited their proliferation and PTH secretion. These responses to TGF-beta were lost when menin was specifically inactivated by antisense oligonucleotides. Moreover, TGF-beta did not affect the proliferation and PTH production of parathyroid cells from a Men1 patient. These results indicate that menin is required for TGF-beta action in the parathyroid. We conclude that TGF-beta is an important autocrine/paracrine negative regulator of parathyroid cell proliferation and PTH secretion and that loss of TGF-beta signaling due to menin inactivation contributes to parathyroid tumorigenesis.
  • H Sowa; H Kaji; MF Iu; T Tsukamoto; T Sugimoto; K Chihara
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 278 (52) 52240 - 52252 0021-9258 2003/12 [Refereed]
     
    Although several studies indicated that parathyroid hormone (PTH) exerted anabolic action on bone, its precise mechanisms have been unknown. On the other hand, transforming growth factor beta (TGF-beta), abundantly stored in bone matrix, stimulates bone formation with a local injection in rodents. Although our previous study suggested that Smad3 is an important molecule for the stimulation of bone formation, no reports have been available about the effects of PTH on Smad3. In this present study, we examined the effects of PTH on Smad3 and the physiological significance in mouse osteoblastic cells. PTH promoted the expression of Smad3 mRNA within 10 min and the protein level in a dose-dependent manner in MC3T3-E1 and rat osteoblastic UMR-106 cells. Protein kinase A (PKA) activator as well as protein kinase C (PKC) activators increased Smad3 protein level, and both PKA and PKC inhibitors antagonized PTH-induced Smad3, indicating that PTH promotes the production of Smad3 through both PKA and PKC pathways. Next, we examined anti-apoptotic effects of PTH and Smad3 in these cells, employing trypan blue, transferase-mediated nick end labeling, and Hoechst staining. Pretreatment with PTH or overexpression of Smad3 decreased the number of apoptotic cells induced by dexamethasone and etoposide. Moreover, a dominant negative mutant, Smad3DeltaC, abrogated PTH-induced anti-apoptotic effects. On the other hand, PTH augmented TGF-beta-induced transcriptional activity. Furthermore, PTH enhanced TGF-beta-induced production of type I collagen, whereas it did not affect TGF-beta-reduced proliferation in MC3T3-E1 cells. These observations indicated that PTH amplified the anabolic effects of TGF-beta by accelerating the transcriptional activity of Smad3. In conclusion, we first demonstrated that PTH-Smad3 axis exerts anti-apoptotic effects in osteoblasts and reinforces the anabolic action by TGF-beta in osteoblasts. Hence, PTH-Smad3 axis might be involved in the bone anabolic action of PTH.
  • QX Chen; H Kaji; MF Iu; R Nomura; H Sowa; M Yamauchi; T Tsukamoto; T Sugimoto; K Chihara
    JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM ENDOCRINE SOC 88 (10) 4655 - 4658 0021-972X 2003/10 [Refereed]
     
    Peripheral quantitative computed tomography (pQCT) is useful for evaluating volumetric bone mineral density (vBMD) as well as bone mineral density (BMD) of cortical and trabecular bones separately. Although PTH affects cortical and trabecular bones differently, the effects of endogenous PTH on vBMD and bone geometry have not previously been examined with pQCT. We, therefore, investigated the effects of an excess and a deficiency of endogenous PTH on bone by employing dual-energy x-ray absorptiometry and pQCT in 36 female patients with primary hyperparathyroidism (hyper), nine female patients with idiopathic or postoperative hypoparathyroidism (hypo), and 100 normal controls matched to age, gender, and body size (cont). Lumbar BMD by dual-energy x-ray absorptiometry was higher in the order: hypo > cont = hyper, and radius-1/3 BMD was significantly higher in the order: hypo > cont > hyper. The area of radius-1/3 was significantly higher in hyper than in cont. As for pQCT, trabecular vBMD was significantly higher in the order: hypo > cont > hyper at the 4% site ( hypo, 157.5 +/- 36.7 mg/cm(3); cont, 123.4 +/- 47.5 mg/cm(3); hyper, 98.4 +/- 41.7 mg/cm(3)). Cortical vBMD was higher in the order: hypo > cont > hyper at the 20% site (hypo, 1141.1 +/- 53.1 mg/cm(3); cont, 1090.2 +/- 72.9 mg/cm(3); hyper, 1038.6 +/- 89.1 mg/cm(3)). Total bone area and endosteal and periosteal circumferences were significantly higher in hyper than in cont and hypo. Cortical area and thickness were higher in the order: hypo > cont > hyper. Bone strength indices were not significantly different among the three groups. In conclusion, vBMD evaluation revealed that an excess of endogenous PTH was catabolic for both cortical and trabecular bones, and that bone mass (especially trabecular bone mass) was preserved under a condition of deficient endogenous PTH. An excess of endogenous PTH stimulated periosteal bone formation, which might partly compensate for a decrease in bone strength induced by low BMD.
  • QX Chen; H Kaji; R Nomura; H Sowa; M Yamauchi; T Tsukamoto; T Yamaguchi; A Kobayashi; T Sugimoto; K Chihara
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOCIETY 50 (5) 527 - 534 0918-8959 2003/10 [Refereed]
     
    Parathyroid cancer is rare but relatively frequent in Japan compared to Western countries. Surgical parathyroidectomy is the primary choice for radical treatment of primary hyperparathyroidism (pHPT), hence it is important to distinguish malignant from benign tumor in the determination of surgical indication as well as method of operation. However, it is not easy to diagnose parathyroid cancer prior to operation. In the present study, we analyzed the background data, biochemical data and bone mineral density (BMD) of 131 patients with pHPT (111 benign and 20 malignant). BMD of the lumbar spine and mid-radius was measured by dual-energy X-ray absorptiometry. Serum levels of calcium, alkaline phosphatase (ALP), and parathyroid hormone (PTH) were significantly higher in malignant group compared to benign one. The extent of elevation of mid PTH seemed to be higher than that of intact PTH in malignant group. Age-, gender-, and race-adjusted BMD of distal one-third of radius was significantly decreased in malignant group compared to benign one, although that of lumbar spine was not significantly different between the two groups, indicating that osteopenia was marked in the region which was rich in cortical bone in malignant group. On the other hand, serum levels of calcium, ALP, and mid PTH as well as age were selected as predictors of malignancy in univariate logistic regression analysis, while serum level of intact PTH was not selected. In conclusion, radial BMD was lower in malignant group compared to benign one in pHPT. Serum levels of calcium, ALP and mid PTH were useful to predict malignancy of affected parathyroid glands in pHPT patients.
  • H Sowa; H Kaji; L Canaff; GN Hendy; T Tsukamoto; T Yamaguchi; K Miyazono; T Sugimoto; K Chihara
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 278 (23) 21058 - 21069 0021-9258 2003/06 [Refereed]
     
    The physiological roles of menin, the product of the multiple endocrine neoplasia type 1 gene, are not known. Homozygous menin knockout mice exhibit cranial and facial hypoplasia. We, therefore, investigated the role of menin in the regulation of osteoblastic differentiation. Menin antisense oligonucleotides (AS-oligo) reduced endogenous menin expression in the C3H10T1/2 (10T1/2) mouse mesenchymal stem cells and antagonized alkaline phosphatase ( ALP) activity and the expression of type I collagen, Runx2/cbfa1 (Runx2), and osteocalcin (OCN) induced by bone morphogenetic protein 2 (BMP-2). AS-oligo did not affect adipogenic markers ( Oil red staining and PPARgamma expression) and chondrogenic markers ( Alcian blue staining and type IX collagen) induced by BMP-2 in 10T1/2 cells. Menin co-immunoprecipitated with Smad1 and Smad5, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5. In osteoblastic MC3T3-E1 cells, AS-oligo affected neither BMP-2-stimulated ALP activity nor the expression of Runx2 and OCN. Stable inactivation of menin in MC3T3-E1 cells increased ALP activity, mineralization, and the expression of type I collagen and OCN. In 21-day cultures of MC3T3-E1 cells and BMP-2-treated 10T1/2 cells, endogenous menin expression increased up to day 14 and declined thereafter. These data indicate that menin inactivation specifically inhibits the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage, mediated by menin and Smad1/5 interactions. Menin is important for both early differentiation of osteoblasts and inhibition of their later differentiation, and it might be crucial for intramembranous ossification.
  • T Makino; T Sugimoto; H Kaji; T Yamaguchi; R Kitazawa; M Yamauchi; H Sowa; QX Chen; R Nomura; T Tsukamoto; K Chihara
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOC 50 (2) 215 - 219 0918-8959 2003/04 [Refereed]
     
    A 63-year-old man was admitted to our hospital for the evaluation of hypercalcemia and anterior neck mass. Laboratory findings revealed hypercalcemia, hypophosphatemia, and hypercalciuria, as well as elevated serum levels of parathyroid hormone (PTH) and alkaline phosphatase. Computerized tomography and magnetic resonance images showed that the mass contained a cystic area. Parathyroid scintigraphy using either Tc-99m-sestamibi alone or Tl-201-chloride in conjunction with Tc-99m-pertechnetate for thyroid image subtraction showed uptake of the radioactivity into the cyst wall, suggesting that the mass originated from the parathyroid. Fine needle aspiration biopsy revealed that the cyst fluid was serous and bloody with extremely high concentrations of both PTH and CA19-9. The patient was diagnosed as primary hyperparathyroidism caused by parathyroid cyst and cervical exploration was performed. The cyst was dissected away along with the right lobe of the thyroid gland. After tumor removal, serum calcium and PTH levels were normalized. Histological study showed that the tumor possessed malignant potential with capsular invasion as well as moderate cellular atypia with trabecular pattern in arrangement. Parathyroid cells in the wall of the cystic tumor were immunostained positively for CA 19-9, suggesting that CA 19-9 in the cyst fluid was produced from the cells.
  • S Yano; T Sugimoto; T Tsukamoto; T Yamaguchi; T Hattori; KI Sekita; H Kaji; S Hattori; A Kobayashi; K Chihara
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG KG 35 (4) 259 - 264 0018-5043 2003/04 [Refereed]
     
    Purpose: To examine longitudinal changes of bone mineral density (BMD) after parathyroidectomy (PTx) in patients undergoing maintenance hemodialysis (HD) with severe secondary hyperparathyroidism (HPT) to determine which factor contributes most to bone changes. Methods: Fifteen Japanese HD patients who had been refractory to medical therapy were subject to PTx with autotransplantation. We measured BMD by dual energy Xray absorptiometry (DXA) at the lumbar spine (L2 - 4 BMD) and the distal 1/3 region of the radius (1/3R BMD) at 1, 3, 6,12, 24, and 36 months after PTx. Results: Baseline Z-score of BMD was markedly low at 1/3R (-3.07) and slightly low at L2 - 4 (-0.59) in this group. A significant increase in L2 - 4 BMD was observed as early as one month after PTx, which was sustained afterwards. Annual percent changes in L2 - 4 and 1/3R BMD were +15.6% and +6.4%, respectively. The annual percent changes in BMD at both sites were positively associated with preoperative intact PTH levels (L2 - 4; r = 0.642, p = 0.010, 1/3R; r = 0.884, p < 0.001) and total alkaline phosphatase (ALP) levels (L2 - 4; r = 0.663, p = 0.007, 1/3R; r=0.858, p<0.001). Stepwise multiple regression. analysis revealed that serum levels of intact PTH and ALP were the best predictors of both percentage and net changes in radial BMD with high determination coefficients (r(2) > 0.8). Conclusion: Successful PTx following appropriate supplementation with vitamin D and calcium provides a marked increase in lumbar BMD and a modest increase in radial BMD in HD patients with secondary HPT. Preoperative levels of PTH and ALP are useful for predicting postoperative changes in bone mass.
  • H Kaji; S Hattori; K Sekita; T Sugimoto; K Chihara
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOCIETY 50 (2) 127 - 133 0918-8959 2003/04 [Refereed]
     
    Background: It remains undetermined whether bone mineral density (BMD) of hemodialysis (HD) patients with diabetes mellitus (DM) is reduced or not. The present study was performed to compare BMD between HD patients with DM and chronic glomerulonephritis (CGN). We analyzed the factors which affected BMD of 139 patients with chronic HD enrolled in this cross-sectional study. Methods: BMD of forearm [1/3-radius (R) and ultradistal (UD)-R] were measured by dual-energy x-ray absorptiometry. Results: 1/3R-BMD and UDR-BMD of DM patients were comparable to those of CGN patients. In DM patients, body mass index (BMI) was positively correlated with UDR-BMD, serum levels of PTH and creatinine were negatively correlated with 1/3R-BMD, and serum level of beta2-microglobulin (betaMG) was positively correlated with UDR-BMD. In CGN patients, duration of HD and height were negatively correlated with 1/3R- and UDR-BMD, serum levels of uric acid and triglyceride were positively correlated with 1/3R- and UDR-BMD, and serum level of total cholesterol was positively correlated with UDR-BMD. In stepwise regression analysis, BMI and serum levels of creatinine and betaMG were selected for UDR-BMD in DM patients, although serum PTH level was selected for 1/3R-BMD. Conclusion: BMD at forearm was comparable between DM and CGN in HD patients. The factors affecting BMD seemed to be different between DM and CGN.
  • H Sowa; H Kaji; T Yamaguchi; T Sugimoto; K Chihara
    JOURNAL OF BIOLOGICAL CHEMISTRY AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC 277 (39) 36024 - 36031 0021-9258 2002/09 [Refereed]
     
    Transforming growth factor (TGF) beta inhibits alkaline phosphatase (ALP) activity and mineralization in mouse osteoblastic MC3T3-E1 cells, whereas local administration of TGF-beta stimulates bone formation in vivo. We recently demonstrated that Smad3, a TGF-beta signaling molecule, promotes ALP activity and mineralization in MC3T3-E1 cells. Moreover, the target disruption of Smad3 in mouse is reported to cause a decrease in bone mineral density. These findings indicate that Smad3 plays an important role in the regulation of bone formation. However, why the effects of TGF-beta and Smad3 on ALP activity and mineralization are different remains unknown. The purpose of the present study is to clarify the role of mitogen-activated protein kinase (MAPK) in TGF-beta and Smad3 pathways in osteoblast. TGF-beta activated extracellular signal-regulated kinases/p42/p44 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in mouse osteoblastic MC3T3-E1 cells. The expression of dominant negative type Smad3, Smad3DeltaC, affected neither TGF-beta-activated ALAPKs nor TGF-beta-inhibited ALP activity. Specific inhibitors of ERK1/2 activation (PD98059 and U0126), as well as JNK inhibitors (curcumin and dicumarol) antagonized the inhibitory effects of TGF-beta on ALP activity and mineralization, whereas the specific inhibitor of p38 MAPK (SB203580) did not affect them. PD98059 and curcumin enhanced Smad3-induced ALP activity and mineralization, whereas SB203580 inhibited them. In the luciferase reporter assay using 3TP-lux with the specific Smad3-responsive element, PD98059, and curcumin enhanced TGF-beta- and Smad3-induced transcriptional activity in MC3T3-E1 cells. On the other hand, TGF-beta-induced production of type I collagen was antagonized by curcumin but not by PD98059. The present study indicated that TGF-beta-responsive ERK1/2 and JNK cascades negatively regulate Smad3-induced transcriptional activity as well as ALP activity and mineralization in osteoblasts.
  • T Sugimoto; H Kaji; D Nakaoka; M Yamauchi; S Yano; T Sugishita; DJ Baylink; S Mohan; K Chihara
    EUROPEAN JOURNAL OF ENDOCRINOLOGY BIO SCIENTIFICA LTD 147 (3) 339 - 348 0804-4643 2002/09 [Refereed]
     
    Background: There has been increasing evidence that the growth hormone (GH)-IGF-I axis plays an important part in the maintenance of bone mass. However, controversy still exists as to the effect of GH treatment on bone mineral density (BMD) in elderly patients with osteoporosis. Objective: To investigate the effect of low-dose GH treatment on markers of body composition and bone turnover, serum concentrations of IGF-I and IGF-binding proteins (IGFBPs), and BMD at the radius and lumbar spine in eight elderly Japanese women with osteoporosis. Methods: Participants were treated with GH as a single daily subcutaneous injection (0.125 IU/kg per week; 0.00595 mg/kg per day) for 48 weeks. Results: Markers of bone formation and bone resorption were both increased up to 24 weeks of GH treatment. The bone formation markers remained increased during GH treatment, whereas the bone resorption markers returned to baseline values after 24 weeks of GH treatment. GH treatment caused a rapid (within 2 weeks) and sustained increase in serum IGF-I concentration. As for IGFBPs, serum concentrations of IGFBPs-2, -3 and -4 did not change significantly during GH treatment. In contrast, GH treatment caused a gradual increase in serum IGFBP-5 concentration, with a significant increase seen 48 weeks after the start of GH treatment. Radial BMD seemed to be increased during the late period of GH treatment, although the change was not significant. Lumbar BMD did not change during GH treatment. GH treatment caused a significant increase in hand grip strength. None of the GH-treated participants had new fractures and side effects such as edema and joint pain. Radial BMD was significantly increased after discontinuation of GH treatment for another 48 weeks and a similar tendency was observed at the lumbar spine (7.1 +/- 2.3% above pretreatment values for the radius and 3.6 +/- 2.0% for the lumbar spine). Conclusions: Low-dose GH treatment attenuated the decrease in muscle strength and bone mass in elderly women without side effects, although changes in nutrition and exercise might affect BMD. The present findings provide useful information regarding the use of low-dose GH treatment in elderly women with osteoporosis.
  • H Sowa; H Kaji; T Yamaguchi; T Sugimoto; K Chihara
    JOURNAL OF BONE AND MINERAL RESEARCH AMER SOC BONE & MINERAL RES 17 (7) 1190 - 1199 0884-0431 2002/07 [Refereed]
     
    Transforming growth factor (TGF) beta is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-beta signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [H-3]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-beta on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-beta inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L-azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.
  • H Kaji; M Suzuki; S Yano; T Sugimoto; K Chihara; S Hattori; K Sekita
    AMERICAN JOURNAL OF NEPHROLOGY KARGER 22 (4) 325 - 331 0250-8095 2002/07 [Refereed]
     
    Background. The incidence and mortality of hip fractures were several times greater in the patients with hemodialysis (HD) than in the general population. Although patients with end-stage renal disease develop renal osteodystrophy, few published data examined the risk factor of hip fractures in the dialysis population. Methods: The present study was performed to compare various indices and bone mineral density (BMD) of HD patients with or without the history of hip fractures. Moreover, we analyzed the factors which predicted hip fractures in 183 patients with chronic HD enrolled in the cross-sectional study. Results: Serum level of alkaline phosphatase was significantly higher in HD patients with hip fractures, compared to those without hip fractures. Oral calcium carbonate dose was significantly lower in HD patients with hip fractures. 1/3 radius (R)-BMD and ultradistal (UD)R-BMD were significantly lower in HD patients with hip fractures. However, lumbar spine (LS)-BMD was comparable in HD patients with or without hip fractures. Although there was no significant differences of BMD between with and without hip fractures in diabetes mellitus, UDR-BMD of patients with hip fractures was significantly lower than that of patients without hip fractures in chronic glomerulonephritis. Radial BMD was lower in female patients with hip fractures, compared to without hip fractures, although there were no significant differences in male patients. In multiple logistic regression analysis, oral calcitriol dose and 1/3R-BMD were selected as a risk factor of hip fractures in HD patients. Conclusion: Radial BMD was lower in HD patients with hip fractures. However, its contribution is different, depending on gender and the original disease leading to HD. Radial BMD and oral calcitriol dose seemed to be important to predict the risk of hip fractures. Copyright (C) 2002 S. Karger AG, Basel.
  • H Kaji; T Sugimoto; D Nakaoka; Y Okimura; H Kaji; H Abe; K Chihara
    CLINICAL ENDOCRINOLOGY BLACKWELL SCIENCE LTD 55 (2) 175 - 181 0300-0664 2001/08 [Refereed]
     
    OBJECTIVE Skeletal Involvement is a common clinical feature in acromegalic patients. Although several recent reports are available concerning bone mineral density (BMD) in acromegaly, the controversy still exists as to whether BMD of acromegalic patients is increased or not. The present study was performed to examine biochemical bone metabolic indices and BMD as well as body composition in 26 Japanese patients with active acromegaly and 26 control subjects matched for age, sex, race and height in a cross-sectional study. MEASUREMENTS BMD of the lumbar spine and femoral neck, as well as body composition, was measured by dual-energy X-ray absorptiometry. Midradial BMD was measured by single-photon absorptiometry. We also determined serum levels of IGF-I, IGFBP-3 and osteocalcin (OC) as well as urinary levels of deoxy-pyridinoline (D-Pyr) and CrossLaps. RESULTS Percent lean body mass was increased and percent fat mass was decreased in the acromegalic patients compared to control subjects. Serum levels of OC, as well as urinary levels of D-Pyr and CrossLaps, were significantly higher in acromegalic patients compared to control subjects (9.8 +/- 1.2 vs. 5.7 +/- 0.77 for OC; 11.8 +/- 1.66 vs. 5.0 +/- 0.49 for D-Pyr; 437.6 +/- 68.4 vs. 156.5 +/- 39.6 for CrossLaps). Z scores of BMD at mid-radius as well as lumbar spine and femoral neck were significantly higher in acromegalic patients compared to control subjects (1.086 +/- 0.311 vs. -0.060 +/- 0.274 for mid-radius; 1.022 +/- 0.280 vs. 0.319 +/- 0.165 for lumbar spine; 1.292 +/- 0.347 vs. 0.232 +/- 0.264 for femoral neck). CONCLUSIONS The present study revealed that a decrease in percent fat mass and an increase in percent lean body mass were observed in Japanese patients with active acromegaly. Bone mineral density at all sites and bone metabolic markers were also increased in acromegaly. The present findings provide additional evidence that the GH/IGF-I axis might play an important role in the maintenance of bone mass as well as the regulation of body composition in Japanese adults.
  • Y Tokukoda; S Takata; H Kaji; R Kitazawa; T Sugimoto; K Chihara
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOC 48 (4) 443 - 452 0918-8959 2001/08 [Refereed]
     
    There is accumulating evidence that interleukin-1 (IL-1) levels are increased locally at the site of active bone resorption in a variety of diseases including osteoporosis, periodontal disease and rheumatoid arthritis. However, the pathogenic role of IL-1 in bone loss remains to be fully elucidated. We present here additional evidence that IL-1 beta enhances endothelial activation and thereby stimulates mobilization of peripheral blood mononuclear cells (PBMCs) from luminal to abluminal spaces across the endothelium. Furthermore, IL-1 beta stimulates the differentiation of PBMCs into osteoclast-like cells with bone-resorbing activity in the presence of human osteoblastic SaOS-2 cells without systemic hormones. These findings provide circumstantial evidence for the hypothesis that IL-1 beta generated in the bone microenviroment plays a stimulatory role in PBMC mobilization from the peripheral circulation and their subsequent differentiation into osteoclast-like cells in the bone tissue. In addition, the present study supports the notion that osteoclast progenitor cells might be derived from the peripheral circulating blood mononuclear cells in human.
  • H Kaji; L Canaff; JJ Lebrun; D Goltzman; GN Hendy
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 98 (7) 3837 - 3842 0027-8424 2001/03 [Refereed]
     
    Multiple endocrine neoplasia type I (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary, The MEN1 gene encodes a nuclear protein called menin, In MEN1 carriers inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. Here, we show that menin inactivation by antisense RNA antagonizes transforming growth factor type beta -mediated cell growth inhibition. Menin interacts with Smad3, and antisense menin suppresses transforming growth factor type beta -induced and Smad3-induced transcriptional activity by inhibiting Smad 3/4-DNA binding at specific transcriptional regulatory sites. These results implicate a mechanism of tumorigenesis by menin inactivation.
  • M Kanatani; T Sugimoto; H Kaji; K Ikeda; K Chihara
    EUROPEAN JOURNAL OF ENDOCRINOLOGY BIO SCIENTIFICA LTD 144 (3) 263 - 269 0804-4643 2001/03 [Refereed]
     
    Background: Although there have been some case reports suggesting that bone in patients with pseudohypoparathyroidism (PHP) might respond to parathyroid hormone (PTH), no information is available as to whether serum PTH concentration is related to bone metabolic markers or to bone mineral density (BMD) in PHP, Objective: To address these relationships, by comparing intact serum PTH, bone metabolic markers and BMD in patients with PHP with those in patients with idiopathic hypoparathyroidism (IHP) and postoperative hypoparathyroidism (OHP), Methods: Intact serum PTH, bone metabolic markers (osteocalcin, tartrate-resistant acid phosphatase, pyridinoline, deoxypyridinoline) and BMD by dual-energy X-ray absorptiometry or single-photon absorptiometry were measured in patients with PHP Ia (n = 2) and PHP Ib (n = 8). The results were compared with those in patients with IHP (n = 5) and OHP (n = 14). Results: All bone metabolic markers measured were present in significantly greater amounts in patients with PHP Ib than in those with IHP+OHP. The Z score (standard deviation of average BMD at each age) of the BMD of femoral neck was significantly lower in patients with PHP Ib than in those with IHP+OHP, The Z scores of BMD of lumbar spine and radius were also lower in patients with PHP Ib than in those with IHP+OHP, but the difference was not significant. Moreover, the intact serum PTH concentrations were significantly and positively related to bone metabolic marker levels in all patients, and the intact serum PTH concentrations were significantly and negatively related to BMD of lumbar spine in PHP patients. Conclusions: These results suggest that PTH stimulates bone turnover in PHP Ib patients, resulting in a relatively lower BMD in PHP Ib patients than in IHP+OHP patients. The present study indicates that bones of most cases of PHP could respond to PTH.
  • QX Chen; H Kaji; T Sugimoto; K Chihara
    FEBS LETTERS ELSEVIER SCIENCE BV 491 (1-2) 91 - 93 0014-5793 2001/02 [Refereed]
     
    Androgens play an important role in the regulation of bone metabolism in animals and humans. The present study was performed to investigate whether androgens would affect osteoclast formation stimulated by parathyroid hormone (PTH) in mouse bone cell cultures and its mechanism. Testosterone as well as alpha -dihydrotestosterone (DHT) concentration-dependently inhibited osteoclast formation induced by PTH-(1-34). 10(-8) M ICI 182780, an estrogen receptor inhibitor, did not affect PTH-induced osteoclast formation antagonized by 10-8 M testosterone, although it completely antagonized the effects of 10-8 M 17 beta -estradiol, Moreover, 3 muM 4-androsten-4-ol-3, 17-dione, an aromatase inhibitor, did not affect PTH-induced osteoclast formation antagonized by testosterone, Hydroxyflutamide, an androgen receptor antagonist, concentration-dependently antagonized the inhibitory effects of testosterone as well as DHT on PTH-stimulated osteoclast formation. In conclusion, the present study first demonstrated that testosterone inhibited osteoclast formation stimulated by PTH through the androgen receptor, but not through the production of intrinsic estrogen in mouse bone cell cultures, (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B,V, All rights reserved.
  • D Nakaoka; T Sugimoto; H Kaji; M Kanzawa; S Yano; M Yamauchi; T Sugishita; K Chihara
    OSTEOPOROSIS INTERNATIONAL SPRINGER-VERLAG LONDON LTD 12 (7) 548 - 554 0937-941X 2001 [Refereed]
     
    The present study analyzed the factors that determine bone mineral density (BMD) and predict spinal fracture risk in postmenopausal Japanese women. Two hundred and five postmenopausal Japanese women aged 48-84 years (mean age 64 years) were enrolled in the cross-sectional study. BMD of the lumbar spine, femoral neck and total body as well as body composition were measured by dual-energy X-ray absorptiometry (DXA). Mid-radial BMD was measured by single-photon absorptiometry. We also determined serum levels of insulin-like growth factor (IGF)-I, IGF binding protein-2, -3 and osteocalcin as well as urinary levels of pyridinoline (Pyr), deoxy-Pyr (D-Pyr) and growth hormone. Multiple regression analysis revealed that lean body mass (LBM) was positively correlated with BMD at all sites. In contrast, femoral neck BMD was highly related to fat mass as well as LBM, although fat mass was not an independent correlate of total body and mid-radial BMD. LBM and urinary D-Pyr were crucial determinants at all sites except the mid-radius in stepwise regression analysis. Fat mass and serum IGF-I were determinants of femoral neck and mid-radial BMD, respectively. In terms of reproductive history, parity affected lumbar BMD. Factors affecting BMD differed according to the site. On the other hand, lumbar BMD as well as serum levels of IGF-I and albumin were selected as predictors of spinal fracture risk in multiple logistic regression analysis. Lumbar BMD, serum IGF-I and LBM were selected in women with lumbar BMD above 0.727 g/cm(2). In conclusion, the present study indicates that LBM is a more important determinant of BMD than fat mass at any site except the femoral neck. Age, serum IGF-I and urinary D-Pyr were also determinants of BMD, dependent on the regions measured. Lumbar BMD and LBM as well as serum levels of IGF-I and albumin were useful markers which predicted the risk of osteoporotic spinal fractures in postmenopausal Japanese women.
  • M Nasu; T Sugimoto; H Kaji; K Chihara
    JOURNAL OF ENDOCRINOLOGY SOC ENDOCRINOLOGY 167 (2) 305 - 313 0022-0795 2000/11 [Refereed]
     
    Although there is clinical evidence showing that combined therapy with parathyroid hormone (PTH and estrogen is additively effective in increasing the bone mass of patients with osteoporosis, the mechanism of the interaction between these hormones remains unclear. The present study was performed to determine whether estrogen would affect osteoblast proliferation and function modulated by PTH in human osteoblastic SaOS-2 cells. Human PTH-(1-34) significantly inhibited [H-3]thymidine (TdR) incorporation, which was attenuated by 24 h pretreatment with 10(-10) to 10(-7) M 17 beta -estradiol (17 beta -E-2) in a concentration-dependent manner. PTH significantly stimulated alkaline phosphatase (ALP) activity, collagen synthesis and type-1 procollagen mRNA expression after pretreatment with 17 beta -E-2 in these cells. Tamoxifen, an anti-estrogen, antagonized these 17 beta -E-2-induced effects. Pretreatment with insulin-like growth factor-I (IGF-I) mimicked estrogen action, and coincubation of 3 mug/ml anti-IGF-I antibody antagonized the effects of 17 beta -E-2 as well as those of IGF-I. In the presence of 17 beta -E-2 pretreatment, PTH strongly stimulated IGF-binding protein (IGFBP)-5 mRNA expression in these cells, and recombinant IGFBP-5 increased type-1 procollagen mRNA expression and ALP activity. In conclusion, estrogen attenuates PTH-induced inhibition of osteoblast proliferation and PTH stimulates osteoblast function in the presence of estrogen pretreatment. IGF-I and/or IGFBP-5 seemed to be involved in the estrogen-induced modulation of PTH action on osteoblast proliferation and function.
  • H Kaji; L Canaff; D Goltzman; GN Hendy
    CANCER RESEARCH AMER ASSOC CANCER RESEARCH 59 (20) 5097 - 5101 0008-5472 1999/10 [Refereed]
     
    The multiple endocrine neoplasia type 1 gene product, menin, interacts with Jun D. The physiological role of menin in cell cycle control and the manner in which its inactivation contributes to tumorigenesis remain unknown. In the present study, the expression of menin was examined at various cell cycle stages in GH4C1 cells, a rat pituitary cell line. Cells synchronized at the G(1)-S-phase boundary expressed menin at a lower level than G(0)-G(1)-synchronized cells. The expression of menin increased as the cells entered S phase, at which time Jun D expression also increased. In contrast, cells synchronized at the G(2)-M phase expressed lower levels of menin. At G(0)-G(1), G(1)-S, and G(2)-M phases of the cell cycle, menin was found predominantly in the nucleus. In summary, we show that in pituitary cells, menin is a nuclear protein whose expression is cell-cycle regulated. The data suggest that menin has an important role in cell growth regulation.
  • M Kanatani; T Sugimoto; Y Takahashi; H Kaji; R Kitazawa; K Chihara
    JOURNAL OF BONE AND MINERAL RESEARCH BLACKWELL SCIENCE INC 13 (5) 854 - 862 0884-0431 1998/05 [Refereed]
     
    Several lines of evidence indicate that estrogen inhibits parathyroid hormone (PTH)-induced bone resorption in vivo and in vitro, However, its precise mechanism remains unknown, The present study was performed to investigate whether osteoclast precursor cells possess the receptors for PTH/PTH-related protein (PTHrP) and/or estrogen and to clarify the mechanism by which estrogen affects PTH-induced osteoclast-like cell (Ocl) formation. The polymerase chain reaction (PCR) product corresponding in size to the mouse PTH/PTHrP receptor cDNA was detected in mouse hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as in osteoblastic MC3T3-E1 cells. The nucleotide sequence of the PTH/PTHrP receptor PCR product of hemopoietic blast cells was found to be 95.4% identical to that of PTH/PTHrP receptor cDNA of rat osteoblastic ROS cells. The PCR product corresponding in size to the mouse estrogen receptor cDNA was detected in mouse hemopoietic blast cells supported by GM-CSF as web as in MC3T3-E1 cells, The nucleotide sequence of the estrogen receptor PCR product of hemopoietic blast cells was completely identical to that of mouse estrogen receptor cDNA. 17 beta-estradiol (17 beta-E-2) but not 17 alpha-E-2, dose dependently antagonized Ocl formation stimulated by human (h) PTR(1-34) at a minimal effective concentration of 10(-10) M in the hemopoietic blast cell culture. 17 beta-E-2 also significantly inhibited Ocl formation stimulated by 10(-8) M hPTHrP(1-34), while it did not affect 1,25-dihydroxyvitamin D-3-induced Ocl formation. EIolvever, 10(-8) M 17 beta-E-2 significantly inhibited Ocl formation stimulated by dibutyryladenosine cAMP (10(-4) M) and Sp-cAMPS (10(-4) M), an activator of cAMP-dependent protein kinase (PKA) as well as forskolin (10(-5) M). In contrast, 17 beta-E-2 did not affect Ocl formation by either phorbol myristate acetate (10(-7) hi), an activator of protein kinase C (PKC), or A23187 (10(-7) hi), a calcium ionophore, The pretreatment with 17 beta-E-2 significantly inhibited Ocl formation induced by the combined treatment with PTH and PKC inhibitors (H7 or staurosporine), while it did not affect Ocl formation stimulated by the combined treatment with RID and Rp-cAMPS, a PKA inhibitor. The present data indicate that estrogen inhibits RID-stimulated Ocl formation by directly acting on hemopoietic blast cells, possibly through blocking a PRA pathway but not a calcium/PKC pathway.
  • M Nasu; T Sugimoto; H Kaji; J Kano; K Chihara
    ENDOCRINE JOURNAL JAPAN ENDOCRINE SOCIETY 45 (2) 229 - 234 0918-8959 1998/04 [Refereed]
     
    The effects of carboxyl-terminal (C-) PTH fragments, (35-84), (53-84) and (69-84), on the proliferation and function of osteoblastic UMR-106 cells were compared with those of amino-terminal (N-) (1-34) and intact (I-) (1-84) PTH. I-PTH as well as N-PTH at 10(-8)M significantly inhibited [H-3] thymidine incorporation and stimulated alkaline phosphatase activity in UMR-106 cells. No C-PTH fragments affected them. In contrast, the expression of type-1 procollagen mRNA in these cells was stimulated by all C-PTH fragments, inhibited by N-PTH and not affected by I-PTH. All C-PTH fragments except (69-84) as well as N-PTH and I-PTH stimulated IGFBP-5 mRNA expression. The present study suggests that the C-portion of the PTH molecule exercises biological activities in mRNA expression of type-1 procollagen as well as IGFBP-5 in osteoblasts, and that it might be involved in the anabolic action of PTH on bone in vivo.
  • H Kaji; T Sugimoto; M Kanatani; K Nishiyama; M Nasu; K Chihara
    JOURNAL OF CELLULAR PHYSIOLOGY WILEY-LISS 172 (1) 55 - 62 0021-9541 1997/07 [Refereed]
     
    There have been several lines of evidence that parathyroid hormone (PTH) stimulates production of insulinlike growth factor I (IGF-I) in bone and that IGF-I stimulates osteoclast formation. Thus, the present study was performed to clarify the possible role of IGF-I in PTH-stimulated osteoclastlike cell formation and the role of PTH-responsive dual signal transduction systems (cyclic [c] AMP-dependent protein kinase [PKA] and calcium/protein kinase C [PKC]) in its mechanism. Treatment with anti-IGF-I antibody (1-10 mu g/ml) partially but significantly blocked hPTH-(1-34)-stimulated osteoclastlike cell formation in unfractionated mouse bone cell cultures, although it did not affect osteoclastlike cell formation stimulated by 1,25-dihydroxyvitamin D-3. Rp-cAMPS (10(-4) M), a direct PKA inhibitor, as well as two types of PKC inhibitors, H-7 (10 mu M) and staurosporine (3 nM), and dantrolene (10(-5) M), an inhibitor of calcium mobilization from intracellular calcium stores, all significantly blocked PTH-stimulated osteoclastlike cell formation. Anti-IGF-I antibody (3 mu g/ml) significantly blocked osteoclastlike cell formation stimulated by 10(-4) M dbcAMP, 10(-4) M Sp-cAMPS, a direct PKA activator, and 10(-5) M forskolin in mouse bone cell cultures. Dibutyryl cAMP, forskolin, and hPTH-(1-34) significantly stimulated mRNA expression of both IGF-I and IGF-binding protein 5 (IGFBP-5) in these cultures, but neither 10(-7) M PMA, a PKC activator, nor 10(-7) M A23187 did. Moreover, anti-IGF-I antibody significantly blocked osteoclastlike cell formation stimulated by the conditioned medium from MC3T3-E1 cells pretreated with 10(-8) PTH-(1-34), which induced IGF-I and IGFBP-5 mRNA expression in these cells. In conclusion, the present study indicates that IGF-I mediates osteoclastlike cell formation stimulated by PTH and that the PKA pathway is involved in its mechanism. However, IGF-I does not seem to be the sole effector molecule to be active in this system. (C) 1997 Wiley-Liss, Inc.
  • H Kaji; T Sugimoto; M Kanatani; K Nishiyama; K Chihara
    JOURNAL OF BONE AND MINERAL RESEARCH BLACKWELL SCIENCE INC 12 (5) 734 - 741 0884-0431 1997/05 [Refereed]
     
    Although an excess of glucocorticoid induces secondary osteoporosis, the mechanism still remains unclear, particularly in regard to glucocorticoid-stimulated bone resorption. We examined the effects of dexamethasone (Dex) on osteoclast-like cell formation and bone-resorbing activity by employing mouse bone and spleen cell cultures and further investigated whether Dex would modulate osteoclast-like cell formation stimulated by several bone-resorbing factors. Dex stimulated osteoclast-like cell formation in stromal cell-containing mouse bone cell cultures in a concentration-dependent manner. Also, Dex significantly stimulated osteoclast-like cell formation from hemopoietic blast cells in spleen cell cultures derived from 5-fluorouracil-pretreated mice. In contrast, Dex (10(-8) M) did not affect the bone-resorbing activity of mature osteoclasts. Pretreatment with 10(-8) M Dex significantly enhanced osteoclast-like cell formation in unfractionated mouse bone cell cultures stimulated by 10(-8) M human (h) parathyroid hormone (PTH) (1-34), 10(-8) M hPTH-related protein (1-34) and 10(-6) M prostaglandin E-2, but not by 10(-8) M 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3). Moreover, pretreatment with 10(-8) M Dex significantly enhanced osteoclast-like cell formation stimulated by both forskolin and dbcAMP. In contrast, pretreatment with 10(-8) M Dex significantly inhibited osteoclast-like cell formation in mouse spleen cell cultures stimulated by both 10(-8) M hPTH (1-34) and 10(-8) M 1,25(OH)D-2(3). These findings suggest that Dex stimulates osteoclast-like cell formation, at least in part by directly acting on hemopoietic blast cells. They further suggest that Dex enhances osteoclast-like cell formation stimulated by PTH and prostaglandin E-2 through an indirect pathway via cells other than hemopoietic blast cells.
  • H Kaji; T Sugimoto; M Kanatani; K Chihara
    JOURNAL OF BONE AND MINERAL RESEARCH BLACKWELL SCIENCE INC 11 (7) 912 - 920 0884-0431 1996/07 [Refereed]
     
    The present study aas performed to examine the effect of the high concentration of extracellular calcium ([Ca2+](e)) on osteoclast-like cell formation and bone-resorbing activity in the presence or absence of osteoblasts. High [Ca2+](e) (3 and 5 mM) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although high [Ca2+](e) did not affect the formation of osteoclast-like cells from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in mouse spleen cell cultures. The osteoclast-like cells, newly formed by high [Ca2+](e) in the presence of osteoblasts, possessed the ability to form pits on the dentine slices, The conditioned medium from osteoblastic MC3T3-E1 cells treated with high [Ca2+](e) (5 mM) significantly increased the formation of osteoclast-like cells from hemopoietic blast cells, compared with the control medium, Dantrolene, an inhibitor of calcium mobilization from the intracellular calcium pool, and indomethacin significantly blocked high [Ca2+](e)-stimulated osteoclast-like cell formation in the presence of osteoblasts, although voltage-dependent calcium channel blockers and anti-insulin-like growth factor I antibody did not affect it. High [Ca2+](e), however, significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures, although high [Ca2+](e) inhibited bone-resorbing activity in isolated rabbit osteoclasts. An increase in the extracellular magnesium concentration (5 mM) affected neither osteoclast-like cell formation nor bone-resorbing activity. In conclusion, high [Ca2+](e) stimulated osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts, presumably via osteoblasts.
  • H Kaji; T Sugimoto; M Kanatani; M Nasu; K Chihara
    ENDOCRINOLOGY ENDOCRINE SOC 137 (6) 2217 - 2224 0013-7227 1996/06 [Refereed]
     
    Several lines of evidence have previously indicated that estrogen inhibits PTH-induced bone resorption in vivo and in vitro. However, its mechanism remains unknown. Therefore, the present study was performed to investigate the effect of estrogen on PTH-stimulated osteoclast-like cell formation and clarify its mechanism. 17 beta-estradiol (17 beta-E(2)) significantly antagonized osteoclast-like cell formation stimulated by 10(-8) M human (h) PTH-(1-34) as well as 10(-8) M hPTH-related peptide (PTHrP)-(1-34) in osteoblast-containing mouse bone cell cultures. The conditioned medium derived from osteoblastic SaOS-2 cells or MC3T3-E1 cells pretreated with both PTH-(1-34) (10(-8) M) and 17 beta-E(2)(10(-8) M) stimulated osteoclast-like cell formation from hemopoietic blast cells more weakly than conditioned medium from cells pretreated with PTH-(1-34) alone. Moreover, 10(-8) M 17 beta-E(2) significantly blocked the formation of osteoclast-like cells stimulated by 10(-8) M hPTH-(1-34) in spleen cell cultures derived from 5-fluorouracil-pretreated mice. On the other hand, 10(-8) M 17 beta-E(2), significantly inhibited osteoclast-like cell formation stimulated by dbcAMP (10(-4) M) and Sp-cAMPS (10(-4) M), as well as forskolin (10(-5) M) in mouse bone cell cultures. In contrast, 10(-8) M 17 beta-E(2), did not affect PMA (10(-7) M)-, A23187 (10(-7) M)-, or BAYK-8644 (5 x 10(-6) M)-stimulated osteoclast-like cell formation. In conclusion, the present study demonstrated that estrogen inhibits PTH-stimulated osteoclast-like cell formation by directly acting on hemopoietic blast cells as well as by indirectly acting on them via osteoblasts. The inhibitory effects of estrogen on PTH-stimulated osteoclast-like cell formation seemed to be mediated through blocking the cAMP-dependent protein kinase pathway but not by blocking calcium/protein kinase C.
  • K Nishiyama; T Sugimoto; H Kaji; M Kanatani; T Kobayashi; K Chihara
    ENDOCRINOLOGY ENDOCRINE SOC 137 (1) 35 - 41 0013-7227 1996/01 [Refereed]
     
    Although the actions of GH on osteoblasts have been extensively investigated, its effects on osteoclasts remain unknown. In the present study, the effects of GH on bone resorption and osteoclast differentiation were examined in vitro. Bovine GH (bGH; 1-100 ng/ ml) significantly stimulated bone resorption by preexistent osteoclasts in stromal cell-containing mouse bone cell cultures, whereas it did not affect the bone-resorbing activity of isolated rabbit osteoclasts. When bGH was added to unfractionated bone cells after degeneration of preexistent osteoclasts, it concentration dependently stimulated osteoclast-like cell formation. GH also enhanced 1,25-dihydroxyvitamin D-3-induced osteoclast-like cell formation. Moreover, osteoclast-like cells newly formed from unfractionated bone cells in the presence of bGH possessed the ability to form pits on dentine slices. The conditioned medium from osteoblastic MC3T3-E1 cells or MC3T3-G2/PA-6 stromal cells pretreated with bGH stimulated osteoclast-like cell formation from mouse hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. On the other hand, the PCR products corresponding in size to the mouse GH receptor were detected in mouse hemopoietic blast cells as well as liver. GH concentration dependently stimulated osteoclast-like cell formation from these hemopoietic blast cells in the absence of stromal cells, and these osteoclast-like cells formed pits on dentine slices in the presence of MC3T3-G2/PA-6 stromal cells. The present study indicated for the first time that GH stimulates osteoclastic bone resorption through both its direct and indirect actions on osteoclast differentiation and through its indirect activation of mature osteoclasts, possibly via stromal cells, including osteoblasts.
  • H Kaji; T Sugimoto; M Kanatani; M Fukase; M Kumegawa; K Chihara
    JOURNAL OF BONE AND MINERAL RESEARCH BLACKWELL SCIENCE INC 11 (1) 62 - 71 0884-0431 1996/01 [Refereed]
     
    Prostaglandin E(2) (PGE(2)) is an important local regulator in bone, The present study was performed to investigate the effect of PGE(2) on osteoclast-like cell formation acid bone-resorbing activity of mature osteoclasts in the presence or absence of osteoblasts. PGE(2) (10(-8) to 10(-6) M) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although it did not affect osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in osteoblast-free mouse spleen cell cultures. The conditioned medium from osteoblastic UMR-106 cells pretreated with PGE(2) (10(-8) and 10(-6) hi) significantly stimulated osteoclast-like cell formation from hemopoietic blast cells, PGE(2) also significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures, In contrast, PGE(2) significantly inhibited the bone-resorbing activity and osteopontin mRNA expression in isolated rabbit osteoclasts, Rp-cAMPS, a direct protein kinase (PKA) antagonist, significantly inhibited PGE(2)-stimulated osteoclast-like cell formation and the bone-resorbing activity of mature osteoclasts, although protein kinase C inhibitors, dantrolene (an inhibitor of calcium release from the intracellular calcium pool) and voltage-dependent calcium channel blockers did not affect PGE(2)-stimulated osteoclast-like cell formation, In conclusion, PGE(2) stimulated osteoclast-like cell formation and bone-resorbing activity in mouse bone cell cultures presumably through osteoblasts. The activation of PKA is linked to PGE(2)-stimulated osteoclast-like cell formation and bone-resorbing activity.
  • M KANATANI; T SUGIMOTO; H KAJI; J KANO; K CHIHARA
    EUROPEAN JOURNAL OF ENDOCRINOLOGY SCANDINAVIAN UNIVERSITY PRESS 133 (5) 618 - 625 0804-4643 1995/11 [Refereed]
     
    22-Oxacalcitriol (OCT), a synthetic vitamin D-3 analog, can mimic the ability of calcitriol to differentiate leukemia and skin cells, to enhance the immune response and to suppress parathyroid hormone secretion, but has much less calcemic activity than that of calcitriol. The mechanism of this selective action remains not fully understood, and the actions of OCT on bone metabolism are little known. The present study was, therefore, designed to investigate the effects of OCT and calcitriol on: the proliferation and functions of osteoblastic MC3T3-E1 cells; osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells as well as from unfractionated bone cells in the presence of stromal cells; bone resorption; and the proliferation of MC3T3-E1 cells via monocytes. 22-Oxacalcitriol and calcitriol inhibited [H-3]thymidine (TdR) incorporation, alkaline phophatase activity and collagen synthesis of MC3T3-E1 cells to a similar degree. Both OCT (10(-10)-10(-8) mol/l) and calcitriol significantly and similarly stimulated osteoclast-like cell formation from both hemopoietic blast cells and unfractionated bone cells. 22-Oxacalcitriol (10(-10) and 10(-8) mol/l) significantly stimulated bone resorption, although to a slightly lesser degree than did calcitriol. Human monocyte-conditioned medium (CM) significantly stimulated TdR incorporation into MC3T3-E1 cells, On the other hand, CM obtained from monocytes treated with calcitriol (10(-10)-10(-8) mol/l) significantly inhibited TdR incorporation in a dose-related fashion, whereas CM obtained from monocytes treated with OCT (10(-10)-10(-8) mol/l) significantly stimulated TdR incorporation in a dose-related fashion. Thus, the actions of OCT on osteoblasts, osteoclast precursor cells and mature osteoclasts, possibly ria osteoblasts, are mostly similar to those of calcitriol in vitro. The low activity of OCT in mobilizing calcium from bone in vivo, therefore, appears to be due to some other factor. OCT and calcitriol differed only in their indirect effects on the proliferation of osteoblasts via monocytes, possibly through modulating the release from monocytes of local factors that affect bone remodelling.
  • M KANATANI; T SUGIMOTO; H KAJI; T KOBAYASHI; K NISHIYAMA; M FUKASE; M KUMEGAWA; K CHIHARA
    JOURNAL OF BONE AND MINERAL RESEARCH BLACKWELL SCIENCE PUBL INC CAMBRIDGE 10 (11) 1681 - 1690 0884-0431 1995/11 [Refereed]
     
    Although the action of bone morphogenetic protein (BMP) on osteoblast differentiation has been extensively investigated, its effect on osteoclast differentiation remains unknown. In the present study, in vitro effects of BMP-2 on osteoclast-like cell formation and bone resorption,were examined. BMP-2 (1-100 ng/ml) significantly stimulated bone resorption by preexistent osteoclast-like cells in mouse bone cell cultures containing stromal cells, whereas it did not affect the bone-resorbing activity of isolated rabbit osteoclast-like cells. When BMP-2 was added to unfractionated bone cells after degeneration of preexistent osteoclast-like cells, BMP-2, dose-dependently stimulated osteoclast-like cell formation at a minimal effective concentration of 10 pg/ml. BMP-2 also enhanced the osteoclast-like cell formation induced by 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3). Moreover, osteoclast-like cells newly formed by BMP-2 from unfractionated bone cells possessed the ability to form pits on dentine slices, Because these results indicated that BMP-2 directly or indirectly stimulated osteoclast differentiation and activity, we next examined the direct effect of BMP-2 on osteoclast precursors in the absence of stromal cells using hemopoietic blast cells derived from spleen cells. The mRNA for BMP-2/4 receptor was detected in hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as osteoblastic MC3T3-E1 cells and MC3T3-G2/PA6 stromal cells by RNase protection assay, BMP-2 dose-dependently stimulated osteoclast-like cell formation from hemopoietic blast cells supported by GM-CSF at a minimal effective concentration of 10 pg/ml. BMP-2 also enhanced 1,25(OH)(2)D-3-induced osteoclast-like cell formation from hemopoietic blast cells. The present data are the first to indicate that BMP-2 stimulates bone resorption through both direct stimulation of osteoclast formation and activation of mature osteoclasts, possibly via stromal cells, in vitro.
  • H KAJI; T SUGIMOTO; M KANATANI; M FUKASE; M KUMEGAWA; K CHIHARA
    LIFE SCIENCES PERGAMON-ELSEVIER SCIENCE LTD 56 (22) 1903 - 1913 0024-3205 1995/04 [Refereed]
     
    Although retinoic acid (RA) has been considered to be a bone-resorbing agent both in vivo and in vitro, its mechanism remains still unclear. The present study was performed to examine the effect of RA on osteoclast-like cell formation in the presence or absence of osteoblasts and to study whether RA would affect osteopontin mRNA expression in isolated rabbit osteoclasts. RA (10(-8) and 10(-6) M) significantly stimulated the formation of osteoclast-like cell in osteoblast-containing mouse bone cell cultures. Also, RA caused a stimulation of osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte macrophage-colony stimulating factor (GM-CSF) in mouse spleen cell cultures. However, RA did not affect blast cell number in these cultures and significantly inhibited GMCSF-stimulated proliferation of hemopoietic blast cells. On the other hand, RA stimulated the bone-resorbing activity of mature osteoclasts in mouse bone cell cultures. Moreover, RA caused a stimulation of osteopontin mRNA expression in isolated rabbit osteoclasts. The present study demonstrated for the first time that RA stimulated osteoclast-like cell formation, presumably through directly acting on the hemopoietic blast cells, and that RA stimulated osteopontin mRNA expression in isolated rabbit osteoclasts.
  • H KAJI; T SUGIMOTO; M KANATANI; M FUKASE; K CHIHARA
    ENDOCRINOLOGY ENDOCRINE SOC 136 (3) 842 - 848 0013-7227 1995/03 [Refereed]
     
    The role of the carboxyl (C)-terminal portion of PTH-related protein (PTHrP) in bone resorption continues to be controversial. The present study was performed to examine the effect of C-terminal PTHrP peptides on osteoclast-like cell formation as well as bone resorption in mice. C-Terminal PTHrP peptides [human (h) PTHrP-(107-139) and hPTHrP-(107-111); 10(-10)-10(-8) M] stimulated osteoclast-like cell formation in a concentration-dependent manner in osteoblast-containing mouse bone cell cultures. Moreover, osteoclast-like cells newly formed by these peptides possessed the ability to form pits on the dentine slices. The conditioned medium from UMR-106 cells and MC3T3-E1 cells pretreated with the C-terminal peptides did not affect osteoclast-like cell formation from mouse hemopoietic blast cells derived from spleen cells. The C-terminal peptides as well as hPTHrP-(1-34) stimulated osteoclast-like cell formation from mouse hemopoietic blast cells in the absence of osteoblasts, although both amino- and C-terminal peptides were unable to support hemopoietic blast cells. Protein kinase-C inhibitors (H-7 and staurosporine) almost completely inhibited the stimulation of osteoclast-like cell formation by the C-terminal peptides in both the presence and absence of osteoblasts. The C-terminal peptides did not affect bone resorption by mature osteoclasts in osteoblast-containing mouse bone cell cultures. The present study indicates that C-terminal PTHrP peptides possess the ability to stimulate osteoclast-like cell formation in both the presence and absence of osteoblasts, possibly through the pathway involving protein kinase-C activation.
  • H KAJI; T SUGIMOTO; M KANATANI; J KANO; M FUKASE; K CHIHARA
    EXPERIMENTAL AND CLINICAL ENDOCRINOLOGY & DIABETES JOHANN AMBROSIUS BARTH VERLAG 103 (5) 297 - 302 0947-7349 1995 [Refereed]
     
    There has been some evidence suggesting an important role of mononuclear cells at bone remodeling sites in the coupling of bone formation to bone resorption. Since cells of the monocyte-macrophage lineage produce important local regulators of bone remodeling, we examined effects of human monocytes-conditioned medium (CM) treated with retinoic acid on [H-3] thymidine incorporation (TdR) and alkaline phosphatase (ALP) activity of osteoblastic MC3T3-E1 cells. Treatment of MC3T3-E1 cells with retinoic acid (10(-8) to 10(-6) M) caused an inhibition of TdR in a dose-dependent manner and an inhibition of ALP activity at 10(-6) M. Conditioned medium from monocytes untreated with retinoic acid caused a stimulation of TdR and an inhibition of ALP activity in these cells. In contrast, treatment of monocytes with retinoic acid (10(-8) or 10(-6) M) abolished both stimulation of DNA synthesis and inhibition of ALP activity induced by CM. The present study suggested that retinoic acid modulated osteoblast proliferation and ALP activity not only directly but also indirectly, presumably through modulating the release of local regulators as to bone remodeling from monocytes.
  • H KAJI; T SUGIMOTO; A MIYAUCHI; M FUKASE; K TEZUKA; Y HAKEDA; M KUMEGAWA; K CHIHARA
    ENDOCRINOLOGY ENDOCRINE SOC 135 (1) 484 - 487 0013-7227 1994/07 [Refereed]
     
    Recent evidence indicates that osteopontin (Opn), one of the bone matrix proteins, plays an important role in the attachment of osteoclasts to bone matrix. Besides being elaborated by osteoblasts, this protein is also produced by osteoclasts. The present study was performed to examine the effect of calcitonin (CT) on Opn mRNA expression of isolated rabbit osteoclasis and to clarify the second messenger signaling of this effect. Eel CT inhibited Opn mRNA expression as well as bone-resorbing activity of isolated rabbit osteoclasts. Eel CT caused a transient increase in intracellular calcium followed by a sustained increase as well as an increase in cAMP production in these cells. Dibutyryl-cAMP (10(-4) M) and Sp-cAMPS (10(-4) M), an activator of cAMP-dependent protein kinase (PKA), as well as A23187 (10(-7) M), a calcium ionophore, and phorbol myristate acetate (10(-7) M), an activator of protein kinase C (PKC), caused a significant inhibition of Opn mRNA expression, and suppressed tone-resorbing activity of isolated osteoclasis. The present study is the first to demonstrate that CT inhibits Opn mRNA expression in isolated rabbit osteoclasis, presumably through the activation of PKA and calcium/PKC pathways, by which the bone-resorbing activity might be attenuated subsequently.
  • H KAJI; T SUGIMOTO; M KANATANI; A MIYAUCHI; T KIMURA; S SAKAKIBARA; M FUKASE; K CHIHARA
    ENDOCRINOLOGY ENDOCRINE SOC 134 (4) 1897 - 1904 0013-7227 1994/04 [Refereed]
     
    The controversy still exists about the biological activity of carboxyl (C)-terminal PTH fragments. The present study was performed to examine the effect of C-terminal PTH fragments on osteoclast-like cell formation and bone-resorbing activity. In contrast to human (h) PTH(1-34) or hPTH-(1-84), any C-terminal fragments examined [hPTH(3-84), hPTH-(53-84), and hPTH-(69-84)] did not affect cellular cAMP production and intracellular calcium in osteoblastic UMR-106 cells. Although hPTH-(1-84) caused an increase in cAMP production and intracellular calcium less effectively than hPTH-(1-34) in UMR-106 cells, the former caused a stimulation of osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures more effectively than the latter. All of the C-terminal fragments significantly stimulated osteoclast-like cell formation, and their effectiveness seemed to depend on the amino acid length of the fragments. The conditioned medium from UMR-106 cells pretreated with C-terminal PTH as well as amino-terminal PTH significantly stimulated osteoclast-like cell formation from mouse hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. Moreover, all of the C-terminal fragments stimulated osteoclast-like cell formation from hemopoietic blast cells even in the absence of osteoblasts, and their effectiveness seemed to depend on the length of fragments. As for bone-resorbing activity by mature osteoclasts, all of the C-terminal fragments stimulated bone resorption in osteoblast-containing mouse bone cell cultures, whereas these fragments did not affect the bone-resorbing activity of isolated rabbit osteoclasts. The present study first indicates that C-terminal PTH fragments stimulate osteoclast-like cell formation as weil as bone-resorbing activity by mature osteoclasts in the presence of osteoblasts and accelerate osteoclast-like cell formation from hemopoietic blast cells in the absence of osteoblasts.
  • Y KUROKI; S SHIOZAWA; T SUGIMOTO; M KANATANI; H KAJI; A MIYAUCHI; K CHIHARA
    CLINICAL AND EXPERIMENTAL IMMUNOLOGY BLACKWELL SCIENCE LTD 95 (3) 536 - 539 0009-9104 1994/03 [Refereed]
     
    The effect of culture supernatants of c-fos-transfected MC3T3-E1 osteoblastic cells on osteoclastic bone resorption was studied. Human c-fos cDNA was integrated in the expression vector pH8, and the cells were transfected using the calcium phosphate precipitation technique. Osteoclastic bone resorption was quantified by the pit formation assay, and the osteoclast maturation from precursor was assessed by the generation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC). The culture supernatants of MC3T3-E1 transfectants constitutively expressing c-fos gene enhanced osteoclast-like MNC formation from haematopoietic blast cells compared with those of control transfectants (P < 0.01). The culture supernatants also promoted osteoclastic bone resorption: the pit number, 118.7+/-38.5, was significantly higher than 19.0+/-10.1 of the control (P < 0.05). The absorption area, 12 394+/-3145 mm(2), was significantly larger than 1646+/-314 mm(2) of the control(P < 0.05). The culture supernatants also promoted bone resorption by purified chick osteoclasts (P < 0.05). The results show that constitutive expression of c-fos gene in osteoblastic MC3T3-E1 cells stimulates osteoclast maturation and osteoclastic bone resorption by releasing humoral mediator(s).
  • Junichi Kano; Toshitsugu Sugimoto; Masanori Kanatani; Hiroshi Kaji; Toru Yamaguchi; Masaaki Fukase; Kazuo Chihara
    Journal of Bone and Mineral Metabolism Springer-Verlag 12 (1) S39 - S43 0914-8779 1994/02 [Refereed]
     
    Although there is a recent evidence that PTH induces c-fos gene expression in osteoblasts, the physiological role of this expression remains unknown. We, therefore, employed c-fos antisense oligodeoxynucleotide (as-ODN) and sought to clarify the role of c-fos gene in the regulation of osteoblast proliferation and differentiation as well as osteoclast differentiation and bone-resorbing activity in the presence of osteoblasts by PTH and PTH-related protein (PTHrP). We employed osteoclastlike cell formation from mouse bone cells for the evaluation of osseoclast differentiation and the pit formation assay on the dentin slice in mouse bone cells for the evaluation of bone-resorbing activity by mature osteoclasts. Northern blot analysis revealed that both human (h)PTH-(1-34) and hPTHrP-(1-34) (10-8M) induced a transient c-fos gene expression to a similar degree in osteoblastic UMR-106 cells. Sp-cAMPS (10-4M), an activator of cAMP-dependent protein kinase (PKA), as well as dbcAMP induced a weak c-fos gene expression and Rp-cAMPS (10-4M), an inhibitor of PKA, almost completely antagonized these expressions. However, Rp-cAMPS only slightly blocked c-fos gene expression by PTH and PTHrP. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC) (10-7 to 10-6M), but not 4 alpha-phorbol 12, 13-didecanoate, incapable of activating PKC induced an intense expression of c-fos gene. Calcium ionophores (A23187 and ionomycin, 10-7 to 10-6M) did not induce the expression of c-fos gene. An inhibitor of PKC (H-7, 50 μM) almost completely blocked the c-fos gene expression by PTH and PTHrP as well as PMA. Pretreatment with 1 μM as-ODN significantly antagonized the inhibition of [3H] thymidine incorporation into UMR-106 cells and the stimulation of osteoclast-like cell formation by PTH and PTHrP, compared to pretreatment with the control oligodeoxynucleotide consisting of same nucleotides as as-ODN but with a random sequence. On the other hand, as-ODN did not affect an increase in alkaline phosphatase activity of UMR-106 cells and pit formation by PTH and PTHrP. In all experiments so far, the effects of PTHrP were virtually the same as those of PTH. The present study indicates, first, the direct involvement of PKC as well as PKA in PTH- and PTHrP-induced c-fos gene expression and, second, the participation of its expression in the regulation of osteoblast proliferation and osteoclast differentiation by PTH as well as PTHrP. © 1994 Japanese Society of Bone Metabolism Research.
  • A simple method for isolation of rabbit osteoclasts.
    Sato T; Kamioka H; Nemoto T; Tezuka K; Tanaka K; Sugimoto T; Kaji H; Miyauchi A; Yoshizawa K; Yamaguchi Y; Hakeda Y; Kumegawa M
    Dentistry in Japan 31 3 - 8 1994 [Refereed]
  • T SUGIMOTO; M KANATANI; J KANO; H KAJI; T TSUKAMOTO; T YAMAGUCHI; M FUKASE; K CHIHARA
    JOURNAL OF BONE AND MINERAL RESEARCH BLACKWELL SCIENCE INC 8 (12) 1445 - 1452 0884-0431 1993/12 [Refereed]
     
    The present study was performed to clarify the role of high calcium concentration and the appearance of mononuclear cells at the resorptive site in bone remodeling. Our recent study revealed that the high concentration of extracellular calcium ([Ca2+]e) stimulated DNA synthesis in osteoblastic MC3T3-E1 cells not only directly but also indirectly via monocytes. Human monocyte-conditioned medium (CM) significantly stimulated DNA synthesis and inhibited alkaline phosphatase (ALP) activity. In contrast, when monocytes were cultured at high [Ca2+]e concentrations (more than 3 mM), CM from these monocytes significantly stimulated ALP activity in MC3T3-E1 cells. Such stimulatory effect of CM was not observed at a high magnesium concentration (Mg2+, 5 mM). Treatment of monocytes with the calcium ionophore A23187 did not affect the CM-induced effect on DNA synthesis and ALP activity in these cells. To determine the migration potency of MC3T3-E1 cells and monocytes toward the high [Ca2+]e, chemotaxis assay was performed. The increasing [Ca2+]e (more than 3 mM) induced a chemotactic response of MC3T3-E1 cells as well as monocytes, but the high concentration of Mg2+ (5 mM) did not induce it. On the other hand, treatment with high [Ca2+]e (more than 3 mM) or CM significantly inhibited the 1,25-(OH)2D3-induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) from their precursors derived from mouse spleen cells. The present study indicated that an increase in [Ca2+]e stimulated DNA synthesis and ALP activity of osteoblasts via monocytes, induced chemotaxis of osteoblasts as well as monocytes, and inhibited the formation of TRAP-positive MNC, suggesting the importance of the high Ca2+ Concentration and mononuclear cells at the resorptive site in bone remodeling.
  • T SUGIMOTO; M KANATANI; H KAJI; T YAMAGUCHI; M FUKASE; K CHIHARA
    AMERICAN JOURNAL OF PHYSIOLOGY AMER PHYSIOLOGICAL SOC 265 (3) E367 - E373 0002-9513 1993/09 [Refereed]
     
    The second messenger signaling mechanisms of parathyroid hormone (PTH)- and PTH-related peptide (PTHRP)-stimulated osteoclast-like cell formation were investigated in mouse hemopoietic blast cells that possessed PTH binding sites. Human (h) PTH-(1-34) or hPTHRP-(1-34) resulted in a dose-dependent stimulation of tartrate-resistant acid phosphatase-positive multinucleated cells (MNC) formation. Pretreatment with [Nle8,18Tyr34]hPTH-(3-34) significantly blocked hPTH-(1-34)- and hPTHRP-(1-34)-stimulated MNC formation. Dibutyryladenosine 3',5'-cyclic monophosphate (10(-4) M) and forskolin (10(-5) M) as well as the stimulatory diastereoisomer of adenosine 3',5'-cyclic phosphorothioate (Sp-cAMPS), a direct activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) (10(-4) M), stimulated MNC formation, and Rp-cAMPS, an inhibitor of PKA activation (10(-4) M), almost completely inhibited MNC formation stimulated by the aforementioned agents but not by 1,25-dihydroxyvitamin D3. Moreover, Rp-cAMPS significantly blocked PTH- and PTHRP-stimulated MNC formation. Treatment with calcium ionophores (10(-8) and 10(-7) M) and phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator (10(-8) to 10(-6) M), but not 4alpha-phorbol 12,13-didecanoate, a phorbol incapable of activating PKC, stimulated MNC formation. Two PKC inhibitors [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride and staurosporine] equally blocked PTH- and PTHRP-stimulated MNC formation. The combined pretreatment with Rp-cAMPS and PKC inhibitors completely blocked PTH- and PTHRP-stimulated MNC formation. Present findings indicate that the activation of PKA and PKC is directly linked to PTH- and PTHRP-stimulated osteoclast-like cell formation from hemopoietic blast cells.
  • H KAJI; T SUGIMOTO; M FUKASE; K CHIHARA
    HORMONE AND METABOLIC RESEARCH GEORG THIEME VERLAG 25 (8) 421 - 424 0018-5043 1993/08 [Refereed]
     
    The present study was performed to compare the effect of parathyroid hormone-related peptide (PTHrP) on bone resorption with that of parathyroid hormone (PTH) and clarify the participation of PTHrP-responsive dual signal transduction systems involving cAMP-dependent protein kinase (PKA) and calcium/protein kinase C (Ca/PKC) in the stimulation of bone resorption by PTHrP. Bone resorbing activity was estimated as the number of pits formed on the dentine slice and total area of pits per slice in bone cells derived from 2 week-old mice. Human (h)PTHrP(1-34) (10(7)M) stimulated bone resorption as potent as hPTH-(1-34) (10(7)M) did. The stimulation of bone resorption by hPTHrP-(1-34) and hPTH-(1-34) was equally blocked by either simultaneous treatment with 10(-8)M eel calcitonin or pretreatment with 10-7 M [Nle8,18Tyr34]hPTH-(3-34)amide. Rp-cAMPS, an antagonist in the activation of PKA, equally attenuated bone resorption stimulated by PTHrP as well as by PTH. A23187 (10(-7) M) caused a significant stimulation of bone resorption. These findings indicate the direct involvement of PKA activation and a contributory role of an increase in cytosolic calcium in the stimulation of bone resorption by PTHrP and suggest that PTHrP stimulates bone resorption presumably through the same mechanism as PTH does.
  • H KAJI; T SUGIMOTO; M KANATANI; M FUKASE; K CHIHARA
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS 194 (1) 157 - 162 0006-291X 1993/07 [Refereed]
  • T. Sugimoto; M. Kanatani; H. Kaji; T. Yamaguchi; M. Fukase; K. Chihara
    American Journal of Physiology - Endocrinology and Metabolism 265 (3) E367 - E373 0002-9513 1993 [Refereed]
     
    The second messenger signaling mechanisms of parathyroid hormone (PTH)- and PTH-related peptide (PTHRP)-stimulated osteoclast-like cell formation were investigated in mouse hemopoietic blast cells that possessed PTH binding sites. Human (b) PTH · (1-34) or hPTHRP-(1-34) resulted in a dose-dependent stimulation of tartrate-resistant acid phosphatase-positive multinucleated cells (MNC) formation. Pretreatment with [Nle8,18Tyr34]hPTH-(3-34) significantly blocked hPTH-(1-34)- and hPTHRP-(1-34)-stimulated MNC formation. Dibutyryladenosine 3',5'-cyclic monophosphate (10-4 M) and forskolin (10-5 M) as well as the stimulatory diastereoisomer of adenosine 3',5'-cyclic phosphorothioate (Sp-cAMPS), a direct activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) (10-4 M), stimulated MNC formation, and Rp-cAMPS, an inhibitor of PKA activation (10-4 M), almost completely inhibited MNC formation stimulated by the aforementioned agents but not by 1,25-dihydroxyvitamin D3. Moreover, Rp- cAMPS significantly blocked PTH- and PTHRP-stimulated MNC formation. Treatment with calcium ionophores (10-8 and 10-7 M) and phorbol 12- myristate 13-acetate, a protein kinase C (PKC) activator (10-8 to 10-6 M), but not 4α-phorbol 12,13-didecanoate, a phorbol incapable of activating PKC, stimulated MNC formation. Two PKC inhibitors [1-(5- isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride and staurosporine] equally blocked PTH- and PTHRP-stimulated MNC formation. The combined pretreatment with Rp-cAMPS and PKC inhibitors completely blocked PTH- and PTHRP-stimulated MNC formation. Present findings indicate that the activation of PKA and PKC is directly linked to PTH- and PTHRP-stimulated osteoclast- like cell formation from hemopoietic blast cells.
  • Toshitsugu Sugimoto; Masaaki Fukase; Yasuo Imai; Junichi Kano; Tatsuya Kobayashi; Hiroshi Kaji; Riko Kitazawa; Toru Yamaguchi; Takuo Fujita
    Journal of Bone and Mineral Metabolism Springer-Verlag 11 (1) S22 - S25 0914-8779 1993/01 [Refereed]
     
    Although numerous studies have demonstrated that ipriflavone prevents bone loss, its effect on bone mass remains to be quantitatively assessed by dual-energy x-ray absorptiometry (DXA), a recently developed diagnostic technique which enables precise measurement of bone mineral density (BMD). In this study, we investigated the effect of ipriflavone on BMD in female outpatients with osteoporosis by means of DXA and single-photon absorptiometry (SPA). The study group consisted of 23 female patients with postmenopausal or senile osteoporosis (age: 50-80 years, mean 62.4 years body weight: 39-63 kg, mean 49.2 kg height: 145-163 cm, mean 150.8 cm). Nineteen untreated female outpatients (age: 61-65 years) served as a control group. BMD of L1-4 in both the study and control groups was measured by DXA before and after 1 year of treatment with and without ipriflavone (600 mg/day per os), but only 19 of the study group patients were evaluated for BMD by SPA at the radius. For DXA, the mean BMD in the study group was 0.651 g/cm2 before ipriflavone treatment and 0.647 g/cm2 after treatment, with a mean percent change in BMD of 99.5%. For SPA, the mean BMD was 0.495 g/cm2 before ipriflavone treatment and 0.504 g/cm2 after treatment, with a mean percent change in BMD of 101.6%. For patients in the study group aged 61-65 years (n=9), the mean percent change in BMD by DXA after 1 year of treatment was 101.6%. In contrast, the mean percent change in BMD for the control patients of the same age bracket (n=19) was 96.5%. The results by DXA confirmed that ipriflavone inhibits loss of bone mass, making it a useful agent for the treatment of postmenopausal and senile osteoporosis. © 1993 Japanese Society of Bone Metabolism Research.
  • H KAJI; T SUGIMOTO; M KANATANI; M FUKASE
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS 182 (3) 1356 - 1361 0006-291X 1992/02 [Refereed]

Books etc

  • QUICK 生理学・解剖学 人体の構造と機能・病態生理
    梶博史; 辰巳公平 (Contributor第１１章 運動器系の構造と機能)羊土社 2022/02 435 336-355
  • 副甲状腺・骨代謝疾患診療マニュアル改訂第２版
    KAJI HIROSHI (Contributor多発性内分泌腫瘍症に伴う副甲状腺機能亢進症)診断と治療社 2019
  • フレイルのみかた
    河尾直之; 梶博史 (Contributorフレイルと骨粗鬆症 p163-169)中外医学社 2018
  • Adaptation Biology and Medicine
    KAJI HIROSHI (ContributorGravity change and muscle/bone relationship.)Narosa Publishing House 2016
  • 骨・軟骨・関節治療のための新製品開発と臨床ニーズ
    梶 博史 (Contributor骨吸収のメカニズム(骨粗鬆症))技術情報協会 2015
  • 「骨」を知る５３の質問(太田博明編)
    梶 博史 (Contributor高齢女性が筋肉減少症に陥りやすいのは何故ですか)医薬ジャーナル社 2015
  • In: Balogh K, Patocs A (ed), SuperMEN1:Pituitary, parathyroid and pancreas
    Hendy GN; Kaji H; Canaff L (ContributorCellular function of menin.)Landes Bioscience and Springer Science+Business Media, USA 2009
  • 臨床検査データブック(高久史麿監修)
    梶 博史 (Contributor低カルボキシル化オステオカルシン(ucOC))医学書院 2009
  • Balogh K, Patocs A (ed), SuperMEN1:Pituitary, parathyroid and pancreas
    Kaji H; Canaff L (Joint workRole of menin in bone development.pp59-67)Landes Bioscience and Springer Science+Business Media 2009
  • 千原和夫、寺本明、藤枝憲二(編),成人成長ホルモン分泌不全症の臨床
    梶 博史 (Single work骨代謝, pp149-154)メディカルレビュー社 2006
  • 飯田喜俊(編),症候による内科診断ストラテジー
    梶博史; 馬場泰人 (Joint work肥満, pp123-133)メディカルサイエンスインターナショナル 1999
  • 飯田喜俊(編),症候による内科診断ストラテジー
    梶博史; 馬場泰人 (Joint workやせ, pp115-122)メディカルサイエンスインターナショナル 1999
  • 細井孝之(編),『本音で語る骨粗鬆症の診療』
    梶博史; 杉本利嗣 (Joint work鑑別診断のポイント：日常診療における臨床検査（pp30-38）)永井書店 1998

Conference Activities & Talks

  • シンポジウム 骨と他臓器の連環制御(オステオネットワーク): 骨と筋肉の連環因子とその制御機構  [Invited]
    梶博史
    第94回日本整形外科学会学術総会  2022/05
  • KSO-JOS-JSBMR Symposium. Sarcopenia Promising treatment and prevention  [Invited]
    Hiroshi Kaji
    International Congress of Osteoporosis 2021  2021/11
  • 筋・骨ネットワークとエクソソーム  [Invited]
    梶博史
    第45回長崎骨粗鬆症研究会  2021/09
  • シンポジウム3 骨・筋研究における最近の進歩: メカニカルストレスと筋-骨連関  [Invited]
    梶博史
    第94回日本内分泌学会学術総会  2021/04
  • 日本医学会連合加盟学会連携フォーラム「細胞外小胞の制御と機能」：筋骨格系と細胞外小胞  [Invited]
    梶博史
    第98回日本生理学会・第126回日本解剖学会合同大会  2021/03
  • 教育講演2 副甲状腺疾患の診断と治療の実際  [Invited]
    梶博史
    第21回日本内分泌学会近畿支部学術集会  2020/11
  • シンポジウム「サルコペニアを考える」 骨粗鬆症におけるサルコペニア  [Invited]
    梶博史
    第48回日本関節病学会  2020/10
  • 教育講演22:筋肉と骨ミネラル代謝の相互連関  [Invited]
    梶博史
    第93回日本内分泌学会学術総会  2020/07
  • 最近の骨粗鬆症治療の実際  [Invited]
    梶博史
    泉州地区臨床懇話会  2020/02
  • Gravity change and muscle/bone relationship  [Invited]
    KAJI HIROSHI
    第37回日本骨代謝学会学術集会Japn-Korea Joint Symposium 2 Topics in Basic Research  2019/10
  • 内科医による骨粗鬆症治療の実際  [Invited]
    梶 博史
    第3回骨粗鬆症ベーシックセミナー  2019/09
  • 筋・骨連関とマイオカイン  [Invited]
    梶 博史
    第5回日本筋学会学術集会  2019/08
  • 私の骨代謝・内分泌・糖尿病にわたる基礎・臨床融合研究の軌跡  [Invited]
    梶 博史
    2019 糖尿病・内分泌疾患ジャンプアップセミナー  2019/06
  • Influences of gravity change on the crosstalk between muscle and bone  [Invited]
    KAJI HIROSHI
    40th Annual Meeting of the International Society for Gravitational Physiology (ISGP) and Space Life Science and Medicine Meeting  2019/05
  • 骨と筋肉の連環因子とその制御機構  [Invited]
    梶 博史
    第92回日本整形外科学会学術総会  2019/05
  • ビタミンD欠乏症とサルコペニア  [Invited]
    梶 博史
    第92回日本内分泌学会学術総会  2019/05
  • 内科医による骨粗鬆症診療の実際  [Invited]
    梶 博史
    大阪府保険医協会 内科研究会  2019/04
  • 筋・骨ネットワークの展望  [Invited]
    梶 博史
    第20回Niigata Bone Research Conference  2019/02
  • 筋・骨ネットワークのアップデート  [Invited]
    梶 博史
    第6回臨床骨ネットワーク研究会  2019/02
  • 筋・骨連関  [Invited]
    梶 博史
    日本抗加齢協会 第3回学術フォーラム  2018/12
  • マイオカインと骨代謝  [Invited]
    KAJI HIROSHI
    第36回内分泌代謝学サマーセミナー  2018/08
  • PAI-1の骨代謝における意義  [Invited]
    KAJI HIROSHI
    第36回日本骨代謝学会学術集会  2018/07
  • サルコペニアと骨粗鬆症〜マイオカインの意義  [Invited]
    梶 博史
    第52回兵庫内分泌研究会  2018/06
  • 骨と臓器ネットワーク研究の展望  [Invited]
    KAJI HIROSHI
    第9１回日本内分泌学会学術総会  2018/04
  • 骨・筋連関の臨床的意義  [Invited]
    KAJI HIROSHI
    第26回臨床内分泌代謝UpDate  2016/11
  • Plasminogen activator inhibitor-1によるステロイド性糖尿病ならびに骨量減少制御  [Invited]
    KAJI HIROSHI
    第31回日本整形外科学会基礎学術集会  2016/10
  • Muscle-derived factor affecting bone metabolism.  [Invited]
    KAJI HIROSHI
    The 4th Seoul International Congress of Endocrinology and Metabolism (SICEM 2016)  2016/04
  • 骨粗鬆症とサルコペニア-筋と骨のネットワーク  [Invited]
    梶 博史
    第20回兵庫県骨・カルシウムを語る会  2015/10
  • 筋と骨のネットワーク  [Invited]
    梶 博史
    第５回整形外科連携フォーラム  2015/08
  • 糖尿病と骨粗鬆症  [Invited]
    梶 博史
    DM Conference in Tokyo Northeast  2015/07
  • 副甲状腺ホルモンの生理作用と薬理作用  [Invited]
    梶 博史
    第33回日本骨代謝学会学術集会  2015/07
  • Gravity change and muscle/bone relationship  [Invited]
    KAJI HIROSHI
    The 11th World Congress of the International Society for Adaptive Medicine  2015/05
  • 筋と骨代謝の相互関連  [Invited]
    梶 博史
    梶ヶ谷骨粗鬆症セミナー  2014/12
  • 骨代謝・再生における組織線溶系の新しい役割  [Invited]
    梶 博史
    島根大学大学院講義  2014/10
  • 筋と骨代謝の相互関連  [Invited]
    梶 博史
    第32回日本骨代謝学会学術集会  2014/07
  • Interaction between muscle and bone  [Invited]
    KAJI HIROSHI
    2nd Asia-Pacific Bone & Mineral Research Meeting  2014/05
  • 再生医療  [Invited]
    梶 博史
    金剛祭 医学展  2013/11
  • Linkage between muscle and bone.  [Invited]
    KAJI HIROSHI
    5th Hiroshima Conference on Education and Science in Dentistry  2013/10
  • 骨と筋・神経との連関  [Invited]
    梶 博史
    第３回神戸再生医療研究会  2013/07
  • 私の骨代謝研究における基礎・臨床融合の実践  [Invited]
    梶 博史
    生化学若い研究者の会 近畿支部 秋のセミナー  2013/03
  • CKD, 副甲状腺機能亢進症における骨粗鬆症について  [Invited]
    梶 博史
    第２回南河内骨の健康フォーラム  2013/03
  • 生活習慣病にともなう骨粗鬆症へのアプローチ  [Invited]
    梶 博史
    ベネット錠75mg新発売記念講演会  2013/02
  • 骨粗鬆症診療発展をめざした研究の展開  [Invited]
    梶 博史
    骨粗鬆症セミナー in 南大阪 2013  2013/01
  • 内科医が診る生活習慣病と骨粗鬆症  [Invited]
    梶 博史
    富田林Meet The Specialist  2012/09
  • 最近の骨粗鬆症診療と研究の展望  [Invited]
    梶 博史
    平成24年度大阪神緑会総会学術講演会  2012/06
  • 生活習慣病と骨粗鬆症について  [Invited]
    梶 博史
    第8回三田・北神戸症例検討会  2012/05
  • CKDと骨粗鬆症、筋骨連関へのアプローチ  [Invited]
    梶 博史
    第25回Kobe Parathyroid and Bone Forum (KPBF)  2012/04
  • 甲状腺機能異常と骨代謝  [Invited]
    梶 博史
    神戸甲状腺研究会  2012/02
  • 生活習慣病と骨粗鬆症の診療  [Invited]
    梶 博史
    兵庫県保険医協会 第472回診療内容向上研究会  2012/01
  • Mechanism of bone anabolic action by PTH: Role of Smad3, beta-catenin and Tmem119.  [Invited]
    KAJI HIROSHI
    3rd joint meeting of Japan Osteoporosis Society & Korean Society of Osteoporosis  2011/11
  • 骨粗鬆症治療の実際  [Invited]
    梶 博史
    関西医療薬学研究会  2011/09
  • 私の骨代謝とMENの研究  [Invited]
    梶 博史
    3緑会総会、記念講演会  2011/07
  • 骨代謝調節ホルモンの骨作用機構とMENの研究(学術賞受賞講演)  [Invited]
    梶 博史
    第29回日本骨代謝学会学術集会  2011/07
  • 甲状腺・副甲状腺疾患の臨床  [Invited]
    梶 博史
    兵庫県内科医会内科学セミナー  2011/03
  • 「年齢によって骨粗鬆症の治療を分けるべきか？」パネルディスカッション 高齢者  [Invited]
    梶 博史
    第17回近畿骨粗鬆症研究会  2011/02
  • 骨粗鬆症診療のための骨代謝の基礎  [Invited]
    梶 博史
    神戸市医師会学術講演会  2011/02
  • 食事とカルシウムで骨ケアしませんか  [Invited]
    梶 博史
    市民公開講座「あなたは大丈夫？骨粗しょう症」  2010/11
  • 骨粗鬆症の診断と治療  [Invited]
    梶 博史
    神戸市北区医師会学術講演会  2010/10
  • 糖尿病と骨粗鬆症について(ランチョンセミナー）  [Invited]
    梶 博史
    第52回日本老年医学会学術集会  2010/06
  • 糖尿病と骨粗鬆症について  [Invited]
    梶 博史
    芦屋市医師会学術講演会  2010/03
  • ビスフォスフォネート製剤がステロイド療法後の骨代謝マーカーの変動と骨密度におよぼす影響についての縦断的検討  [Invited]
    梶 博史
    第２７回日本骨代謝学会学術講演会  2009/07
  • 原発性及び続発性骨粗鬆症におけるビスホスホネート治療  [Invited]
    梶 博史
    第６回東北信内分泌談話会  2009/07
  • 最近の続発性骨粗鬆症の診断と治療  [Invited]
    梶 博史
    出雲医師会・薬剤師会 学術講演会  2009/02
  • 骨粗鬆症の診断と治療  [Invited]
    梶 博史
    灘区医師会生涯教育、学術講演会  2008/10
  • ステロイド性骨粗鬆症治療を中心としたアップデート  [Invited]
    梶 博史
    べネット17.5mg発売１周年記念講演会  2008/06
  • 原発性骨粗鬆症女性の治療（薬物療法）  [Invited]
    梶 博史
    第１４回近畿骨粗鬆症研究会  2008/02
  • 骨粗鬆症治療に関する最近の話題  [Invited]
    梶 博史
    明石市医師会学術講演会  2007/12
  • 原発性及び続発性骨粗鬆症の診療アップデート（ランチョンセミナー）  [Invited]
    梶 博史
    兵庫区医師会学術講演会  2007/11
  • ２型糖尿病と骨粗鬆症  [Invited]
    梶 博史
    第５８会兵庫県糖尿病懇話会  2007/11
  • ステロイド骨粗鬆症の診断と治療  [Invited]
    梶 博史
    あくとねる錠17.5mg錠新発売記念講演会  2007/09
  • 大規模臨床試験における骨粗鬆症治療薬１週間製剤の位置付け  [Invited]
    梶 博史
    骨粗鬆症治療の今後を考える（べねっと錠17.5mg錠新発売記念講演会）  2007/07
  • 原発性骨粗鬆症に関するアップデート  [Invited]
    梶 博史
    但馬内科医会学術集会  2007/07
  • 原発性及び続発性骨粗鬆症の診療アップデート（ランチョンセミナー）  [Invited]
    梶 博史
    第４４回日本リハビリテーション医学会学術集会  2007/06
  • 続発性骨粗鬆症診療と骨アナボリック治療への展望  [Invited]
    梶 博史
    第３回兵庫骨粗鬆症懇話会  2007/05
  • 骨粗鬆症診療に関する最近の話題  [Invited]
    梶 博史
    須磨区医師会学術講演会  2007/01
  • ステロイド骨粗鬆症における骨密度の骨折閾値について  [Invited]
    梶 博史
    第24回日本骨代謝学会学術集会  2006/07
  • MEN-1遺伝子の機能についての研究  [Invited]
    梶 博史
    第12回日本家族性腫瘍学会学術総会  2006/06
  • ステロイド骨粗鬆症の機序について  [Invited]
    梶 博史
    第５回骨粗鬆症セミナー  2005/07
  • 多発性内分泌腺腫症I型(MENーI)遺伝子の機能と骨形成機構に関する研究（研究奨励賞受賞講演）  [Invited]
    梶 博史
    第78回日本内分泌学会学術総会  2005/07
  • 最近の骨粗鬆症治療の話題  [Invited]
    梶 博史
    兵庫県病院薬剤師会東西神戸市部合同学術講演会  2004/12
  • 最近の骨粗鬆症の診断と治療  [Invited]
    梶 博史
    明石市医師会学術講演会  2004/09
  • MeninはSmad3と相互作用し、Menin不活化はTGF-b-Smadシグナルを阻害する  [Invited]
    梶 博史
    第12回骨細胞分子研究会  2001/11
  • MENーI遺伝子不活化による腫瘍発生の機序について  [Invited]
    梶 博史
    第２回Aging Science Forum  2001/04

MISC

  • 骨と筋肉の連関因子とその制御機構
    梶博史  日本整形外科学会雑誌  97-  (7)  482  -487  2023/07  [Invited]
  • メカニカルストレスからみた筋・骨連関
    高藤義正; 梶博史  糖尿病・内分泌代謝科  54-  (3)  313  -322  2022/03  [Invited]
  • 筋・骨連関のネットワークシステム
    河尾直之; 梶博史  老年内科  3-  (5)  615  -623  2021/07  [Invited]
  • 筋骨連関のニューサイエンス
    梶博史  White  8-  (1)  9  -11  2021/07  [Invited]
  • 骨と骨外臓器のネットワークシステム
    梶博史  日本臨床  78-  (12)  2010  -2015  2020/12  [Invited]
  • サルコペニアからみた骨粗鬆症
    梶博史  内分泌・糖尿病・代謝内科(骨粗鬆症のすべて)  51-  69  -75  2020/07  [Invited]
  • 骨分化初期過程におけるPAI-1の役割
    高藤義正; 梶博史  BIO Clinica  34-  799  -802  2019  [Invited]
  • 筋肉と骨
    梶 博史  腎と骨代謝  32-  181  -186  2019  [Invited]
  • 内科医による骨粗鬆症診療の実際
    梶 博史  大阪保険医雑誌  633-  51  -56  2019  [Invited]
  • 骨の老化
    梶 博史  内分泌・糖尿病・代謝内科  48-  308  -312  2019  [Invited]
  • 多発性内分泌腺腫症I型
    梶 博史  別冊日本臨床 領域別症候群シリーズ No.4 内分泌症候群（第３版）IV-その他の内分泌疾患を含めて-  343  -346  2019  [Invited]
  • オステオグリシンなどによるマイオカイン
    河尾直之; 梶博史  腎と骨代謝  32-  152  -157  2019  [Invited]
  • 骨修復初期におけるプラスミノーゲンのSDF-1とTGF-β発現調節による骨髄造血幹細胞誘導
    岡田 清孝; 河尾 直之; 辰巳 公平; 石田 昌義; 高藤 義正; 蔵下 伸治; 奥本 勝美; 松尾 理; 梶 博史  日本血栓止血学会誌  29-  (2)  206  -206  2018/05
  • 筋肉、マイオカインと骨・カルシウム代謝
    梶 博史  Clinical Calcium  28-  919  -925  2018  [Invited]
  • 骨・筋連関
    梶 博史  内分泌・糖尿病・代謝内科  46-  449  -454  2018  [Invited]
  • 筋・骨連関
    梶 博史  Medical Science Digest  43-  (9)  442  -445  2017/09  [Invited]
  • 森竹章公; 河尾直之; 岡田清孝; 辰巳公平; 石田昌義; 奥本勝美; 松尾理; 梶博史; 赤木將男  日本整形外科学会雑誌  91-  (8)  S1776  -S1776  2017/08
  • 重力変化が抗重力筋と骨の遺伝子発現におよぼす影響と前庭系の関与
    河尾 直之; 森田 啓之; 西田 一晃; 小畑 孝二; 石田 昌義; 辰巳 公平; 梶 博史  日本骨代謝学会学術集会プログラム抄録集  35回-  159  -159  2017/07
  • Plasminogen activator inhibitor-1(PAI-1)はマウス変形性膝関節症による軟骨下骨減少を抑制する
    森竹 章公; 河尾 直之; 岡田 清孝; 辰巳 公平; 石田 昌義; 奥本 勝美; 松尾 理; 赤木 將男; 梶 博史  日本骨代謝学会学術集会プログラム抄録集  35回-  195  -195  2017/07
  • サルコペニアと骨粗鬆症
    河尾直之; 梶博史  老年医学  55-  69  -73  2017  [Invited]
  • 河尾直之; 梶博史  Clinical Calcium  27-  73  -78  2017  [Invited]
  • 骨損傷後のSDF-1による骨髄造血幹細胞誘導におけるプラスミノーゲンの関与
    岡田 清孝; 河尾 直之; 児嶋 耕太郎; 蔵下 伸治; 奥本 勝美; 田村 行識; 下出 孟史; 松尾 理; 梶 博史  日本骨代謝学会学術集会プログラム抄録集  34回-  193  -193  2016/07
  • SDF-1/CXCR4系による骨修復再生過程での骨髄幹細胞の分化誘導機構
    岡田 清孝; 河尾 直之; 田村 行識; 蔵下 伸治; 奥本 勝美; 児嶋 耕太郎; 梶 博史  日本生理学雑誌  78-  (1)  18  -18  2016/01
  • SDF-1/CXCR4系による骨修復再生過程での骨髄幹細胞の分化誘導機構
    岡田 清孝; 河尾 直之; 田村 行識; 蔵下 伸治; 奥本 勝美; 児嶋 耕太郎; 梶 博史  日本生理学雑誌  78-  (1)  18  -18  2016/01
  • サルコペニア
    梶 博史  診断と治療  104-  1326  -1330  2016  [Invited]
  • 原発性副甲状腺機能亢進症の病態生理
    梶 博史  Clinical Calcium  26-  815  -820  2016  [Invited]
  • サルコペニアと骨代謝
    河尾直之; 梶博史  最新医学:別冊:診断と治療のABC  112-  47  -53  2016  [Invited]
  • 汐見光人; 汐見光人; 河尾直之; 矢野昌人; 岡田清孝; 田村行識; 奥本勝美; 松尾理; 梶博史; 赤木將男  近畿大学医学雑誌  40-  (3-4)  19A  -19A  2015/12
  • 田村行識; 松尾理; 梶博史  日本血栓止血学会雑誌  26-  619  -625  2015  [Invited]
  • フレイルと骨粗鬆症の関連
    梶 博史  PROGRESS IN MEDICINE  35-  1713  -1716  2015  [Invited]
  • Hepatic Osteodystrophyの治療
    梶 博史  Clinical Calcium  25-  1689  -1694  2015  [Invited]
  • 座談会「血管・骨の再生医学」
    倉林正彦; 梶博史; 星和人  O.Li.v.e.[骨代謝と生活習慣病の連関]  5-  76  -81  2015  [Invited]
  • サルコペニアと骨粗鬆症の関連・予防のための運動習慣.
    梶博史  O-Line  10-  2015  [Invited]
  • 田村行識; 梶博史  Clinical Calcium  381-  (386)  2015  [Invited]
  • 骨・筋肉連携
    梶博史  最新医学  70-  58  -63  2015  [Invited]
  • 骨修復過程におけるtPAの重要性
    河尾 直之; 田村 行識; 奥本 勝美; 矢野 昌人; 岡田 清孝; 松尾 理; 梶 博史  日本血栓止血学会誌  25-  (2)  260  -260  2014/04
  • ステロイドの骨代謝に及ぼす影響
    田村行識、梶博史  整形・災害外科  57-  (7)  847  -853  2014  [Invited]
  • サルコペニアと骨粗鬆症
    梶博史  THE BONE  28-  (3)  317  -321  2014  [Invited]
  • サルコペニア、骨粗鬆症に至る共通のメカニズムとは？
    梶博史  Geriatric Medicine 老年医学  52-  (4)  359  -362  2014  [Invited]
  • 糖尿病性骨粗鬆症の病態におけるPlasminogen Activator Inhibitor-1の役割
    梶 博史; 田村 行識; 河尾 直之; 岡田 清孝; 矢野 昌人; 奥本 勝美; 松尾 理  日本内分泌学会雑誌  89-  (1)  276  -276  2013/04
  • 筋・骨連関.
    梶博史  医学のあゆみ  247-  56  -60  2013  [Invited]
  • 副甲状腺機能低下症の病態生理.
    梶博史  腎と骨代謝  26-  273  -279  2013  [Invited]
  • 副甲状腺ホルモンとWntシグナル.
    田村行識; 梶博史  Clinical Calcium  23-  847  -852  2013  [Invited]
  • スクレロスチンと骨代謝.
    河尾直之; 梶博史  内科  111-  707  -710  2013  [Invited]
  • Linkage between muscle and bone.
    Kaji H  Proceedings of the 5th Hiroshima Conference on Education and Science in Dentistry  91  -93  2013  [Invited]
  • PTHによる骨形成促進作用のメカニズム.
    梶博史  Clinical Calcium  22-  321  -326  2012  [Invited]
  • 座談会「糖尿病と骨粗鬆症」.
    山岸昌一; 梶博史; 斉藤充; 吉澤達也  O.Li.v.e.[骨代謝と生活習慣病の連関]  2-  96  -106  2012  [Invited]
  • 筋芽細胞におけるTmem119の骨芽細胞分化促進機構について
    田中 賢一郎; 井上 喜文; 比佐 伊都子; 片桐 岳信; 北澤 理子; 北澤 荘平; 小守 壽文; 杉本 利嗣; 清野 進; 梶 博史  日本内分泌学会雑誌  87-  (1)  267  -267  2011/04
  • RAS(レニン・アンジオテンシン系)の異常と骨．
    梶博史  ホルモンと臨床  59-  391  -395  2011  [Invited]
  • 骨粗鬆症診療のための骨代謝の基礎.
    梶博史  神戸市医師会報  611-  53  -61  2011  [Invited]
  • GHと骨代謝.
    梶博史  内分泌・糖尿病・代謝内科  33-  260  -265  2011  [Invited]
  • PTHの骨アナボリック作用とサイトカイン.
    梶博史; 杉本利嗣  Clinical Calcium  20-  107  -113  2010  [Invited]
  • 内分泌学.
    千原和夫; 高橋裕; 飯田啓二; 梶博史; 加治秀介  日本医事新報  4371-  15  -21  2008  [Invited]
  • 内分泌疾患にともなう骨粗鬆症：成長ホルモン分泌不全症.
    梶博史  THE BONE  22-  149  -152  2008  [Invited]
  • ビタミンD依存症I型、II型.
    梶博史  日本内科学会雑誌  96-  85  -89  2007  [Invited]
  • 内分泌学.
    千原和夫; 高橋裕; 梶博史; 飯田啓二  日本医事新報  4319-  9  -12  2007  [Invited]
  • 活性型ビタミンDによる副甲状腺機能低下症の治療.
    梶博史  Clinical Calcium  17-  93  -98  2007  [Invited]
  • ピロリン酸と石灰化（TNSALP, PC-1, ANK）.
    梶博史  Clinical Calcium  17-  93  -98  2007  [Invited]
  • 副甲状腺ホルモンの骨吸収、骨形成への作用—投与法による差異.
    梶博史  Clinical Calcium  17-  34  -40  2007  [Invited]
  • 骨粗鬆症の治療：ビタミンDとカルシウム.
    梶博史; 杉本利嗣  内分泌・糖尿病科  22-  190  -195  2006  [Invited]
  • 骨代謝の基礎：成長ホルモン／IGF-Iとカルシウム骨代謝.
    梶博史; 千原和夫  内分泌・糖尿病科  23-  95  -99  2006  [Invited]
  • 内分泌症候群III: 多発性内分泌腺腫症I型.
    梶博史; 杉本利嗣  別冊 日本臨床 新領域別症候群シリーズ  3-  333  -336  2006  [Invited]
  • 責任遺伝子の特定された副甲状腺機能低下症: PTH遺伝子異常.
    梶博史  ホルモンと臨床  54-  9  -13  2006  [Invited]
  • 骨粗鬆症のアナボリック治療（PTHを中心に）.
    梶博史; 杉本利嗣  Clinical Calcium  16-  60  -65  2006  [Invited]
  • 高橋 裕; 梶 博史; 飯田 啓二; 千原 和夫  内科  96-  (6)  1052  -1057  2005/12  [Invited]
  • 副甲状腺ホルモンによる骨粗鬆症治療のエビデンスと展望.
    梶博史; 杉本利嗣  Clinical Calcium  15-  611  -615  2005  [Invited]
  • ビタミンDの筋骨格系作用における関連遺伝子群.
    梶博史; 杉本利嗣  Clinical Calcium  15-  834  -838  2005  [Invited]
  • 副甲状腺ホルモンの作用メカニズム.
    梶博史; 杉本利嗣  Clinical Calcium  15-  93  -97  2005  [Invited]
  • GHの骨代謝調節因子としての意義.
    梶博史; 千原和夫  日本臨床（臨床分子内分泌学３）  63-  551  -554  2005  [Invited]
  • 内分泌疾患と運動器障害.
    梶博史; 千原和夫  整形・災害外科  48-  1255  -1261  2005  [Invited]
  • ステロイド骨粗鬆症の成因.
    梶博史; 杉本利嗣  アレルギー・免疫  11-  266  -270  2004  [Invited]
  • 宗和秀明; 梶博史; 杉本利嗣; 千原和夫  最新医学  59-  (4)  965  -969  2004  [Invited]
  • 糖尿病性腎症と骨密度の関係は?
    梶博史; 杉本利嗣; 千原和夫  肥満と糖尿病  3-  408  -409  2004  [Invited]
  • ステロイド骨粗鬆症の臨床 診断・予防及び治療 その他の疾患.
    梶博史; 杉本利嗣  THE BONE  18-  375  -377  2004  [Invited]
  • 骨疾患の現状と骨代謝の基礎.
    梶博史; 杉本利嗣  Journal of Japanese Society of Hospital Pharmacists  40-  1379  -1381  2004  [Invited]
  • GnRH療法でのビスホスホネートの利用.
    梶博史; 杉本利嗣  Clinical Calcium  13-  192  -196  2003  [Invited]
  • 低カルシウム血症.
    梶博史; 杉本利嗣  救急・集中治療  15-  285  -290  2003  [Invited]
  • 推薦処方とその解説 胃切除の既往歴をもつ男性.
    梶博史; 杉本利嗣  今月の治療  11-  41  -42  2003  [Invited]
  • ビタミンD誘導体.
    梶博史; 杉本利嗣  骨粗鬆症治療  2-  209  -212  2003  [Invited]
  • ビタミンDとカルシウム製剤.
    梶博史; 杉本利嗣  Pharma Medica  21-  69  -72  2003  [Invited]
  • SERMとSARM.
    梶博史; 杉本利嗣  Clinical Calcium  13-  1463  -1466  2003  [Invited]
  • Menin変異による内分泌腫瘍発生のメカニズムと骨における生理機能についての研究.
    梶博史  上原記念生命科学財団研究報告集  17-  481  -483  2003  [Invited]
  • PTHの細胞内情報伝達系と骨吸収・骨形成への作用.
    梶博史; 杉本利嗣  Clinical Calcium  13-  39  -41  2002  [Invited]
  • MEN-1遺伝子不活化による腫瘍発生機構.
    梶博史; 杉本利嗣; 千原和夫  最新医学  57-  1718  -1722  2002  [Invited]
  • Menin, the multiple endocrine neoplasia type 1 (MEN1) gene product, menin, interacts with Smad3 and its inactivation blocks transforming growth factor type beta signaling.
    Hendy GN; Kaji H; Goltzman D; Lebrun JJ; Canaff L  Recent Res Devel Endocrinol  3-  41  -53  2002  [Invited]
  • 骨粗鬆症重症度を判断するマーカーとしての血中insulin-like growth factor-I(IGF-I)とIGF結合タンパク-3値の有用性.
    梶博史  体力研究  91-  9  -19  1996  [Invited]
  • Hiroshi Kaji; Toshitsugu Sugimoto; Masanori Kanatani; Akimitsu Miyauchi; Toru Yamaguchi; Masaaki Fukase; Kazuo Chihara  Journal of Bone and Mineral Metabolism  12-  (1)  S125  -S129  1994/02  [Invited]
  • Second messenger signaling of c-fos gene in the regulation of osteoblast proliferation and osteoclast differentiation by parathyroid hormone and parathyroid hormone-related protein.
    Kano J; Sugimoto T; Kanatani M; Kaji H; Yamaguchi T; Fukase M; Chihara K  J Bone Miner Metab  12-  S39  -S43  1994  [Invited]
  • 骨粗鬆症の治療 サイトカインと骨粗鬆症
    塚本達雄; 梶博史; 深瀬正晃  カレントテラピー  11-  1138  -1142  1993  [Invited]

Awards & Honors

  • 2011 日本骨代謝学会 学術賞
     
    受賞者: 梶 博史
  • 2006 三井生命厚生事業団医学研究助成
     JPN
  • 2005 第７８回日本内分泌学会研究奨励賞
     JPN
  • 2004 医学系研究奨励
     JPN

Research Grants & Projects

  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2023/04 -2026/03 
    Author : 梶 博史; 河尾 直之; 水上 優哉
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2021/04 -2023/03 
    Author : 高藤 義正; 梶 博史
     
    本研究は、筋と骨の連関機構(筋・骨連関)におけるエクソソーム(exo)の関与に着目し、筋由来exo(Myo-exo)の骨への作用を明らかにすることを目的としている。これまでに研究代表者は、Myo-exoが骨芽細胞の分化を促進し、破骨細胞の形成を抑制することをin vitroの実験系で明らかにしており、本研究ではMyo-exoの骨組織再生作用について、大腿骨欠損マウスモデルを用いて検討した。 研究に用いるMyo-exoはマウス筋細胞株であるC2C12細胞から回収した。コンフレントに培養したC2C12細胞を48時間培養した培養上清を回収し、遠心 (3,000×g)、フィルター濾過 (0.22 μm)によって細胞デブリなどの異物を除去した後、超遠心 (100,000×g)にて沈降したペレットをMyo-exoとして実験に用いた。得られたMyo-exoはBCAassay法によって蛋白量を定量した。また、Western blottingによるエクソソーム特異的な抗原(CD9, CD81)の発現を確認するとともに、Nanosightによる粒径解析によって、得られた粒子の大部分が200 nm以下の粒径であることを確認した。 健常マウスおよび骨修復が遅延する、ストレプトゾトシン誘発性糖尿病モデルマウスの大腿骨に0.8mm径の欠損を作製し、Myo-exoを局所移植した。移植担体として、1.5mm径に成型した生分解性のゼラチンハイドロゲルを用いた。移植9日後に欠損部近辺を動物用CT装置で撮影した。その結果、糖尿病マウスは健常マウスと比べて骨再生の遅延を認め、欠損部面積が大きかった。しかし糖尿病マウスにMyo-exoを移植することで、骨欠損部面積が有意に減少し、糖尿病によって引き起こされる骨修復の遅延がMyo-exoの局所移植によって改善することが明らかとなった。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2020/04 -2023/03 
    Author : 梶 博史; 河尾 直之
     
    高齢化社会の進展とともに健康寿命の延伸が喫緊の課題となり、骨粗鬆症とサルコペニアの予防・治療が重要となってきた。その中で、骨格筋と骨の相互関連（筋・骨連関）が注目されている。しかし、長期的な運動が筋・骨連関におよぼす影響とマイオカインの役割は不明である。そこで私共は、マウスにおいて、慢性的な運動負荷により、筋で産生され骨に影響をおよぼす新規マイオカインを探索し、その骨代謝におよぼす影響を検討した。8週間のトレッドミル運動を行ったマウス腓腹筋とヒラメ筋の網羅的RNAシークエンス解析により、慢性運動によって筋で発現が増加する因子としてperipheral myelin protein 22 (PMP22) を抽出した。さらに、慢性運動は卵巣摘出による骨量減少を回復し、腓腹筋PMP22発現を増加させた。In vitro解析において、PMP22は骨芽細胞の分化および石灰化を減少させた。一方、マウス骨髄細胞において、PMP22はRANKLによって誘導される破骨細胞形成を有意に抑制した。実験に用いたマウスでの単相関分析によって、腓腹筋とヒラメ筋のPMP22発現量は大腿骨の皮質骨骨密度と正の相関を示したが、海綿骨骨密度とは有意な相関はみられなかった。これらの実験結果より、マウスにおいて、慢性運動は骨格筋のPMP22発現を増加させることが明らかとなった。さらに、PMP22は、破骨細胞形成を抑制する作用により、慢性運動による骨量増加作用に寄与する新規のマイオカインであることが示唆された。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/06 -2020/03 
    Author : Morita Hironobu
     
    Under the hypothesis that plastic changes in the otolith vestibular system, which is a sensing organ of gravity, are involved in the causes of various medical problems associated with microgravity, we investigated the changes in otolith sensory cells and vestibular nuclei, which are primary neurons, caused by the microgravity environment, and obtained the following results. In addition to previously known functions, the vestibular system is involved in various body function regulation such as feeding, glucose metabolism, body temperature, blood pressure, regulation of muscle mass/bone mass. These functions change when exposed to different gravitational environments with strong plasticity. This plastic change involves changes in gene expression in the saccule and vestibular nuclei. It was suggested that the method of electrically stimulating the vestibular system might be useful as a countermeasure against plastic changes.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2017/04 -2019/03 
    Author : ISHIDA Masayoshi; KAJI Hiroshi
     
    It has been suggested that factors other than sex hormones may also be involved in sex differences in osteoporosis and bone metabolism. In primary mouse osteoblasts, the differentiation and calcification were significantly lower in females than males. We performed the comprehensive gene expression analyses and Serpina3n was identified as a gene whose expression level is predominantly higher in females than males. While the reduced endogenous Serpina3n significantly enhanced osteoblast differentiation, enhanced Serpina3n significantly suppressed osteoblast differentiation in a mouse osteoblast cell line. In addition, Serpina3n did not affect the differentiation of mouse mesenchymal stem cell line into osteoblasts, but significantly suppressed calcification. From the above results, Serpina3n, which is predominantly expressed in female osteoblasts, suppresses the phenotype in differentiated osteoblasts. It would be partially able to explain sex differences in osteoblast phenotypes.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2016/04 -2019/03 
    Author : KAWAO Naoyuki; MORITA hironobu; OKUMOTO katsumi
     
    We aimed to investigate the humoral factors linking muscle to bone induced by mechanical stress and effects of obesity on the relationships between muscle and bone. We found that hypergravity increased follistatin levels in skeletal muscle. Follistatin may have beneficial effects on bone metabolism as a humoral factor linking muscle to bone. Moreover, we reported that mechanical unloading reduced irisin expression in the skeletal muscle of mice and irisin may contribute to disuse-induced bone loss in mice. We also found that obesity enhances the recovery of bone and muscle masses as well as strength decreased by disuse after reloading in mice. Leptin may be involved in the recovery of muscle and bone enhanced by obesity in mice.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2016/08 -2018/03 
    Author : TATSUMI Kohei; KAJI Hiroshi; Mackman Nigel
     
    The objective of this study was to clarify the role of coagulation and fibrinolysis system, especially plasminogen activator inihibitor-1 (PAI-1) and tissue factor (TF), in the diathesis of oxaliplatin-induced liver sinusoidal obstruction syndrome (SOS). Repeated administration of oxaliplatin to the wild-type mice significantly increased PAI-1 mRNA levels in the liver, whereas TF mRNA levels were decreased. The increase rate of liver PAI-1 mRNA was significantly and negatively correlated with the rate of body weight loss of the mice.The increases of inflammatory cytokines and fibrosis markers in the liver of PAI-1 deficient mice were milder than WT mice. TF-deficient mice showed higher mortality and severe body weight loss compared with WT mice. Therefore, in the SOS pathogenesis, PAI-1 may play an important role in accelerating liver injury by forming multiple microthrombi in the liver microcirculation, resulting in enhanced inflammation and fibrosis.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2018/03 
    Author : KAJI Hiroshi
     
    The interactions between skeletal muscle and bone (muscle/bone relationships) have been recently noted. We investigated the influences of mechanical factors on muscle/bone relationships using the comprehensive gene analysis in mice. Our studies indicated the following conclusions: 1. Gravity change affects muscle and bone volume through the vestibular system. 2. Follistatin is involved in the effects of gravity change on muscle and bone as a humoral factor from muscle tissues. 3. Mechanical stress increases bone mineral density through the enhancement of irisin production from muscle.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2018/03 
    Author : OKADA Kiyotaka
     
    We previously revealed that stromal cell-derived factor-1 (SDF-1) is involved in the changes in the number of bone marrow stem cells during the bone repair process in mice. Moreover, we reported that plasminogen (Plg) deficiency delays bone repair. We investigated the roles of Plg in the changes in bone marrow stem cells during bone repair. Plg deficiency significantly blunted a decrease in the number of HSCs after bone injury in mice. Plg deficiency significantly blunted the number of SDF-1- and Osterix-double-positive cells in the endosteum around the lesion as well as mRNA levels of SDF-1 and transforming growth factor-b (TGF-b) elevated by bone injury. TGF-b signaling inhibition significantly blunted a decrease in the number of HSCs after bone injury. The present study showed that Plg is critical for the changes in bone marrow HSCs, TGF-b and SDF-1 at the damaged site during bone repair in mice.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2017/03 
    Author : ISHIDA Masayoshi; TATSUMI Kohei; KAJI Hiroshi
     
    Adipose-derived stem cells (ADSCs) were obtained from mouse subcutaneous adipose tissues digested by collagenase and centrifugation. Next, anti-inflammatory effects of ADSCs on the adipose tissues were examined in vitro culture system. In brief, the adipocytes were cultured in the addition of ADSC culture supernatant and the cells were analyzed the expressions of good and bad adipokines. We found out bad adipokines were decreased, on the other hand, good adipokines were not changed by the addition of ADSC supernatant. Further we examined the beneficial effect of ADSCs on the Type 2 diabetic mice. The mice were divided to two groups to be subjected to ADSC sheet transplantation and sham operation groups, respectively. After the operation,on Day 10, we performed the insulin tolerance test and found out the insulin resistance were recovered by ADSC transplantation compared with the sham operation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2013/04 -2016/03 
    Author : KAWAO Naoyuki; KAJI Hiroshi
     
    We aimed to investigate the role of the tissue fibrinolytic system in macrophage accumulation and their activation during bone repair and regeneration. We found that the tissue fibrinolytic system plays a crucial role in bone repair and regeneration. Moreover, the tissue fibrinolytic system contributes to macrophage accumulation at the damaged site of bone defect and their activation during bone repair. Since these findings suggest that the tissue fibrinolytic system is critical for the formation of a suitable microenvironment for bone repair and regeneration through macrophage accumulation and activation, the spatiotemporal regulation of the tissue fibrinolytic system is a promising strategy to meet the clinical needs for the regeneration of bone tissue for reconstruction. We would like to proceed the further study of the regulation of macrophage functions by the tissue fibrinolytic system for the development of the effective bone repair and regeneration.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : KAJI Hiroshi; KAWAO Naoyuki
     
    Previous evidence suggested that tissue-fibrinolytic system and subsequent proteolytic system and growth factor induction play important roles in tissue repair. We therefore investigated the roles of tissue-fibrinolytic system in bone repair and regeneration in this study. We established the model to evaluate the process of bone repair and regeneration by making a bone defect in mouse femorae. We demonstrated that plasminogen is crucial for bone repair process using plasminogen-deficient mice. Moreover, tissue-type plasminogen activator (tPA) is involved in bone repair using tPA-deficient mice. In that study, we showed that tPA enhances the proliferation of osteoblasts through Annexin 2 and MAP kinase.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2009 -2011 
    Author : KAJI Hiroshi
     
    The aim of our study is the identification of local ossification factors in muscle tissues and humoral bone anabolic factors produced from muscles in order to clarify the interactions between muscle and bone/mineral metabolism. We selected the factors with bone formation activity using the comprehensive gene analysis between muscle-derived cells and ossification signal-enhanced cells. Among them, Tmem119 is expected the factor, which locally induces muscle ossification. On the other hand, osteoglycin and FAM5C may be the candidates of novel humoral bone anabolic factors produced from muscles. We would like to proceed the further study of these factors as the putative targets for the development of the treatment of osteoporosis.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2007 -2010 
    Author : KITAZAWA Sohei; TAKENAKA Atsuhi; KAJI Hiroshi; KITAZAWA Riko
     
    Cytosine methylation in CpG-island, located usually regulatory region of many genes, is well recognized as an inhibitory epigenetic regulator. On the other hand, biological significance of the cytosine methylation other than CpG-island is unclear. By focusing morphology-oriented microscopic dissection methods, we analyzed cytosine methylation status of the BAMBI gene promoter in bladder cancer and of the P16 in renal tumor of diabetic rats, and found that some of the cytosine methylation outside of the CpG-island contributes to the tumor development and progression.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2005 -2006 
    Author : KAJI Hiroshi
     
    1 We examined the effects of parathyroid hormone (PTH), the putative potent bone forming reagent, on Smad3 and beta-catenin in osteoblastic cells. PTH and Smad3 induced the expression of beta-catenin. These actions of PTH were through both cAMP and calcium/protein kinase C systems. Moreover, PTH enhanced the transcriptional activity induced by beta-catenin. This PTH-Smad3-beta-catenin signal seemed to be associated with anti-apoptotic effects of PTH in osteoblasts. 2 We have clarified the significance of Smad3 in bone formation. Smad3 suppressed the differentiation into osteoblasts induced by bone morphogenetic protein-2 in mouse mesenchymal ST-2 cells. On the other hand, in mouse osteoblastic MC3T3-E1 cells, the induction of Smad3 on ALP activity and type I collagen expression decreased during differentiation, but the effects of Smad3 on the expressions of Runx2 and osteocalcin converted to induction from suppression during differentiation. Smad3 modulated the expression of other mineralization-related factors. 3 DNA microarray analysis with 45000 genes were performed between control and Smad3-overexpressed MC3T3-E1 cells treated with Erk1/2 inhibitor. The significance of several putative bone formation-related genes including known and unknown genes is being analyzed at the present. The expression vectors of unknown genes were made and their functions are investigated in osteoblasts. 4 MC3T3-E1 cells, which overexpressed Smad7, an inhibitory Smad, were developed. Smad7 suppressed proliferation, differentiation, the production of bone matrix proteins and mineralization in osteoblasts. Moreover, PTH induced the expression of Smad7, suggesting that Smad7 may be the target of the inhibition of bone formation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2003 -2004 
    Author : KAJI Hirohi; SUGIMOTO Toshitsugu
     
    We previously revealed that menin, a product of multiple endocrine neoplasia type I gene, is required for the early differentiation of osteoblast and inhibits it at later stage. Moreover, we proposed that Smad3, TGF-beta-specific signaling molecule, is important for bone formation. On the other hand, parathyroid hormone (PTH) is one of most potent bone formation stimulating agents. However, the mechanism, by which PTH stimulates bone formation, remains unknown. In the present study, we demonstrated that PTH-Smad3 axis exerts anti-apoptotic effects in osteoblasts and might be involved in the bone anabolic action of PTH. In addition, dexamethasone, a glucocorticoid analogue, inhibits alkaline phosphatase activity and type I collagen expression, presumably by suppressing Smad3-induced transcriptional activity but not by modulating Smad3 expression in osteoblasts. This evidence might be partially related to the inhibitory action of glucocorticoid on osteoblastic bone formation. On the other hand, we demonstrated that menin interacts physically and functionally with Runx2 as well as Smad 1/5 in uncommited mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts, the interaction of menin and TGF-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late stage differentiation. Moreover, menin suppresses osteoblast maturation, in part, by inhibiting the differentiation actions of JunD, AP-1 factor.

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