研究者総覧

小原 幸弘 (コハラ ユキヒロ)

  • 大学院医学系研究科 医学専攻 助教
Last Updated :2020/08/04

研究者情報

学位

  • 博士(獣医学)(日本獣医生命科学大学)

論文上での記載著者名

  • Yukihiro Kohara
  • Y Kohara

科研費研究者番号

  • 50792214

J-Global ID

研究キーワード

  • 骨粗鬆症   関節リウマチ   骨折治癒   マクロファージ   脂肪細胞   骨細胞   骨芽細胞   破骨細胞   骨代謝   

研究分野

  • ライフサイエンス / 実験病理学 / 骨代謝
  • ライフサイエンス / 解剖学 / 獣医解剖・組織・発生学

経歴

  • 2019年02月 - 現在  愛媛大学大学院医学系研究科分子病理学講座助教
  • 2016年02月 - 2019年01月  国立長寿医療研究センター運動器疾患研究部ポスドク

学歴

  • 2012年04月 - 2016年03月   日本獣医生命科学大学大学院   獣医学専攻   博士課程
  • 2006年04月 - 2012年03月   日本獣医生命科学大学   獣医学部   獣医学科

所属学協会

  • アメリカ骨代謝学会   日本組織細胞化学会   日本病理学会   日本骨代謝学会   

研究活動情報

論文

  • New Insights into the Pathogenesis of Diabetic Nephropathy: Proximal Renal Tubules Are Primary Target of Oxidative Stress in Diabetic Kidney
    #Ryuma Haraguchi, Yukihiro Kohara, Kanako Matsubayashi, Riko Kitazawa, Sohei Kitazawa, #corresponding author
    Acta Histochem. Cytochem. 53 2 2020年04月 [有り][無し]
     研究論文(学術雑誌)
  • Yukihiro Kohara, Ryuma Haraguchi, Riko Kitazawa, Yuuki Imai, Sohei Kitazawa
    International journal of molecular sciences 21 8 2020年04月 [有り][無し]
     研究論文(学術雑誌) 
    The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0-48 h (early stage of osteoclast differentiation) or 48-96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.
  • Yukihiro Kohara, Ryuma Haraguchi, Riko Kitazawa, Sohei Kitazawa
    Biochemical and biophysical research communications 523 4 961 - 965 2020年03月 [有り][無し]
     
    Low density lipoprotein receptor-related protein 1 (LRP1), a multifunctional cell surface protein, is expressed in bone marrow-derived macrophages. While LRP1 is thought to be a suppressor of osteoclast differentiation at late stages, its function at early stages remains unclear. Here we demonstrate that Lrp1 stable knockdown by lentiviral short hairpin RNA in macrophage cell line RAW264 cells inhibited RANKL-induced osteoclast formation and osteoclastic master transcription factor Nfatc1 mRNA expression as assessed by quantitative RT-PCR. Furthermore, knockdown of the Lrp1 gene suppressed not only differentiation, but also proliferation, and inhibitory effects on osteoblastic ALP activity by osteoclast-derived humoral factors. Thus, we propose that LRP1 in macrophages is required for both differentiation into osteoclasts and osteoclast-osteoblast interactions.
  • Localization of DLL1- and NICD-positive osteoblasts in cortical bone during postnatal growth in rats.
    Yukihiro Kohara, Soehi Kitazawa, Riko Kitazawa, Ryuma Haraguchi, Kiyotaka Arai, Hajime Amasaki, Satoshi Soeta
    Biochemical and Biophysical Research Communications in press 2020年 [有り][無し]
  • Ryuma Haraguchi, Riko Kitazawa, Yukihiro Kohara, Aoi Ikedo, Yuuki Imai, Sohei Kitazawa
    International journal of molecular sciences 20 23 2019年11月 [有り][無し]
     
    The longitudinal growth of long bone, regulated by an epiphyseal cartilaginous component known as the "growth plate", is generated by epiphyseal chondrocytes. The growth plate provides a continuous supply of chondrocytes for endochondral ossification, a sequential bone replacement of cartilaginous tissue, and any failure in this process causes a wide range of skeletal disorders. Therefore, the cellular and molecular characteristics of the growth plate are of interest to many researchers. Hedgehog (Hh), well known as a mitogen and morphogen during development, is one of the best known regulatory signals in the developmental regulation of the growth plate. Numerous animal studies have revealed that signaling through the Hh pathway plays multiple roles in regulating the proliferation, differentiation, and maintenance of growth plate chondrocytes throughout the skeletal growth period. Furthermore, over the past few years, a growing body of evidence has emerged demonstrating that a limited number of growth plate chondrocytes transdifferentiate directly into the full osteogenic and multiple mesenchymal lineages during postnatal bone development and reside in the bone marrow until late adulthood. Current studies with the genetic fate mapping approach have shown that the commitment of growth plate chondrocytes into the skeletal lineage occurs under the influence of epiphyseal chondrocyte-derived Hh signals during endochondral bone formation. Here, we discuss the valuable observations on the role of the Hh signaling pathway in the growth plate based on mouse genetic studies, with some emphasis on recent advances.
  • Kitazawa S, Haraguchi R, Kohara Y, Kitazawa R
    Acta histochemica et cytochemica 52 4 77 - 83 2019年08月 [有り][無し]
     
    The interleukin (IL)-4, 1,25(OH)2D3 and retinoic acid, increase surface expression of functional integrin αvβ3 on murine osteoclast precursors. All three agonists stimulate transcription of the β3 gene, leading to increased steady-state levels of mRNA this protein. By contrast, mRNA levels of αv remain unchanged. In each instance, the increase in the surface expression of the integrin results in increased migration of the cells onto an αvβ3 substrate. Because β3 subunit, except platelet where β3 subunit conform a dimer with αIIb, associates solely with αv subunit monogamously, while promiscuous αv subunit combines with various subunit, our present data support the idea that the β3 subunit governs the surface-expressed functional integrin complex.
  • Riko Kitazawa, Satomi Kinto-Shibahara, Ryuma Haraguchi, Yukihiro Kohara, Sohei Kitazawa
    Biochemical and biophysical research communications 515 2 268 - 274 2019年07月 [有り][無し]
     
    Receptor activator of NF-κB (RANK) expressed on osteoclasts and their precursors is a receptor for RANK ligand (RANKL). Signals transduced by RANKL-RANK interaction induce genes essential for the differentiation and function of osteoclasts. We have cloned a basic promoter region of the mouse RANK gene and have analyzed the transcription machinery by transcription factors such as PU.1 (-480), and MITF (-100). Here, we examined the regulatory mechanisms of RANK gene transcription through AP-1 binding site, agagctca (-240). RANK mRNA expression in pre-osteoclastic RAW264.7 cells was induced by Phorbol12-myristate13-acetate (PMA) and suppressed by protein kinase C (PKC) inhibitor calphostin C. In RAW264.7 cells, Fos knockdown by siRNA blocked the inducible effect of PMA on RANK expression. By EMSA, an oligonucleotide (-246/-238) showed DNA protein binding, the specificity of which was confirmed by block-shift assay with an anti-Fos antibody and by the addition of the excess of a cold consensus probe. Co-transfection with a Fos expression vector showed that Fos increased RANK promoter activity 6-fold in RAW264.7 cells, and the addition of PU.1 and MITF superinduced the activity more than twenty-fold by the addition of PU.1 and MITF. Mutagenesis of the putative AP-1 site (-240) blocked the inducible effect of Fos on promoter activity. Taken together, these results indicate that during the differentiation of bone marrow mono-nucleated cells into osteoclast precursors, RANK transcription is positively regulated by Fos/AP-1 through the binding element of its gene promoter, supporting the concept that Fos activation by continuous CSF-1 stimulation on macrophages triggers initial expression of RANK and, later, a positive feedback loop by RANKL-RANK interaction.
  • Macrophage-secreted Emilin2 Stimulates Chemotaxis and Differentiation in Stromal/Osteoblastic Cells
    Kohara Yukihiro, Watanabe Atsushi, Ogiso Noboru, Takeshita Sunao
    JOURNAL OF BONE AND MINERAL RESEARCH 33 92  2018年11月 [有り][無し]
  • Kazuhiko Matsuoka, Yukihiro Kohara, Yoshinori Naoe, Atsushi Watanabe, Masako Ito, Kyoji Ikeda, Sunao Takeshita
    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 33 8 1500 - 1512 2018年08月 [有り][無し]
     
    The osteoclast-derived collagen triple helix repeat containing 1 (CTHRC1) protein stimulates osteoblast differentiation, but the underlying mechanism remains unclear. Here, we identified Wnt-activated inhibitory factor 1 (WAIF1)/5T4 as a cell-surface protein binding CTHRC1. The WAIF1-encoding Trophoblast glycoprotein (Tpbg) gene, which is abundantly expressed in the brain and bone but not in other tissues, showed the same expression pattern as Cthrc1. Tpbg downregulation in marrow stromal cells reduced CTHRC1 binding and CTHRC1-stimulated alkaline phosphatase activity through PKCδ activation of MEK/ERK, suggesting a novel WAIF1/PKCδ/ERK pathway triggered by CTHRC1. Unexpectedly, osteoblast lineage-specific deletion of Tpbg downregulated Rankl expression in mouse bones and reduced both bone formation and resorption; importantly, it impaired bone mass recovery following RANKL-induced resorption, reproducing the phenotype of osteoclast-specific Cthrc1 deficiency. Thus, the binding of osteoclast-derived CTHRC1 to WAIF1 in stromal cells activates PKCδ-ERK osteoblastogenic signaling and serves as a key molecular link between bone resorption and formation during bone remodeling. © 2018 American Society for Bone and Mineral Research.
  • Osteoclast-secreted Cthrc1 Regulates Bone Remodeling through Waif1, a Receptor on Stromal/Osteoblastic Cells.
    Yukihiro Kohara, Kazuhiko Matsuoka, Masako Ito, Kyoji Ikeda, Sunao Takeshita
    JOURNAL OF BONE AND MINERAL RESEARCH 32 S32 - S32 2017年12月 [有り][無し]
  • Yukihiro Kohara, Satoshi Soeta, Yayoi Izu, Kiyotaka Arai, Hajime Amasaki
    Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft 208 58 - 68 2016年11月 [有り][無し]
     
    In the groove of Ranvier (GOR), osteoblast lineages form bone bark, which develops into endosteal cortical bone. This ossification process is thought to be regulated by the microenvironment in the GOR. Type VI collagen (Col VI), an extracellular matrix (ECM) protein found in the periosteum/perichondrium, mediates osteoblast differentiation via the cell-surface receptor neural/glial antigen 2 (NG2) chondroitin sulfate proteoglycan. In order to clarify the function of Col VI during osteoblast differentiation in the GOR, in the present study, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the rat tibia proximal end during postnatal growing periods by immunohistochemistry. Our data revealed that Col VI accumulated in the ECM of the GOR middle layer and that Col VI accumulation was reduced and disappeared in the inner and middle lower regions. Runt-related transcription factor 2-immunoreactive pre-osteoblasts expressed NG2 in Col VI-immunopositive areas. However, Osterix-immunoreactive mature osteoblasts were only found in the Col VI-immunonegative area. These findings indicate that Col VI provided a characteristic microenvironment in the GOR and that NG2-Col VI interactions may regulate the differentiation of osteoblast lineages prior to terminal maturation.
  • K. Arai, K. Takahashi, A. Yasuda, N. Kanno, Y. Kohara, M. Michishita, Y. Harada, Y. Hara
    JOURNAL OF COMPARATIVE PATHOLOGY 155 2-3 199 - 206 2016年08月 [有り][無し]
     研究論文(学術雑誌) 
    Muscle lesions and decreased numbers of peripheral nerve branches have been reported in the soft palates of dogs presenting with brachycephalic airway obstruction syndrome (BAOS). Myosin adenosine triphosphatase staining was employed to investigate whether muscle lesions in the elongated soft palate (ESP) of dogs with BAOS reflect the presence of denervation. Soft palates were collected from nine brachycephalic dogs during surgical intervention for BAOS and from five healthy beagle dogs as controls. In the control soft palates, myofibres with relatively uniform diameters and a random mosaic pattern of type I and II myofibres were observed in the palatinus muscle (PM), while almost all of the myofibres in the levator veli palatini muscle (LVPM) were of type II. In the ESPs, small group atrophy, large group atrophy and angular-shaped atrophy were observed in myofibres of the PM and rarely in the LVPM. Fibre type grouping and an increase in type IIC myofibres were found only in the PM. Morphometric analysis of ESPs revealed a significant increase in the number of type I and II myofibres in the PM showing atrophy or hypertrophy compared with controls. A significant increase in atrophic type II myofibres was found in the LVPM of affected dogs. Myopathy consistent with denervation was observed in the PM, but rarely in the LVPM, of ESP specimens. The results suggest that the myopathy seen in dogs with ESP may partly reflect atrophy of myofibres resulting from damage to peripheral nerve branches, with subsequent reinnervation of myofibres. (C) 2016 Elsevier Ltd. All rights reserved.
  • Yukihiro Kohara, Satoshi Soeta, Yayoi Izu, Hajime Amasaki
    Journal of anatomy 226 5 478 - 88 2015年05月 [有り][無し]
     
    In rodents, the long bone diaphysis is expanded by forming primary osteons at the periosteal surface of the cortical bone. This ossification process is thought to be regulated by the microenvironment in the periosteum. Type VI collagen (Col VI), a component of the extracellular matrix (ECM) in the periosteum, is involved in osteoblast differentiation at early stages. In several cell types, Col VI interacts with NG2 on the cytoplasmic membrane to promote cell proliferation, spreading and motility. However, the detailed functions of Col VI and NG2 in the ossification process in the periosteum are still under investigation. In this study, to clarify the relationship between localization of Col VI and formation of the primary osteon, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the periosteum of rat femoral diaphysis during postnatal growing periods by immunohistochemistry. Primary osteons enclosing the osteonal cavity were clearly identified in the cortical bone from 2 weeks old. The size of the osteonal cavities decreased from the outer to the inner region of the cortical bone. In addition, the osteonal cavities of newly formed primary osteons at the outermost region started to decrease in size after rats reached the age of 4 weeks. Immunohistochemistry revealed concentrated localization of Col VI in the ECM in the osteonal cavity. Col VI-immunoreactive areas were reduced and they disappeared as the osteonal cavities became smaller from the outer to the inner region. In the osteonal cavities of the outer cortical regions, Runx2-immunoreactive spindle-shaped cells and mature osteoblasts were detected in Col VI-immunoreactive areas. The numbers of Runx2-immunoreactive cells were significantly higher in the osteonal cavities than in the osteogenic layers from 2 to 4 weeks. Most of these Runx2-immunoreactive cells showed NG2-immunoreactivity. Furthermore, PCNA-immunoreactivity was detected in the Runx2-immunoreactive spindle cells in the osteonal cavities. These results indicate that Col VI provides a characteristic microenvironment in the osteonal cavity of the primary osteon, and that differentiation and proliferation of the osteoblast lineage occur in the Col VI-immunoreactive area. Interaction of Col VI and NG2 may be involved in the structural organization of the primary osteon by regulating osteoblast lineages.
  • Kunihiko Terakado, Takuya Yogo, Yukihiro Kohara, Satoshi Soeta, Yoshinori Nezu, Yasuji Harada, Yasushi Hara, Hajime Amasaki, Masahiro Tagawa
    Veterinary journal (London, England : 1997) 202 1 48 - 52 2014年10月 [有り][無し]
     
    Conjunctival epithelial and goblet cell P2Y2 nucleotide receptors regulate ion transport and secretory function. Diquafosol is a P2Y2 purinergic receptor agonist that stimulates secretion of aqueous tear components from conjunctival epithelial cells and secretion of mucin from conjunctival goblet cells. In humans suffering from keratoconjunctivitis sicca (dry eye), topical administration of diquafosol improves corneal epithelial integrity and stabilises the tear film. The aim of the present study was to investigate P2Y2 receptor expression and to determine the effect of topical administration of diquafosol on mucin and aqueous tear production in dogs. Canine conjunctival P2Y2 receptor expression was evaluated by Western blotting and immunohistochemical analysis. The effect of diquafosol on mucin secretion was evaluated by examining mucin-5 subtype AC (MUC5AC) concentration in tears. The effect of diquafosol on aqueous secretions was evaluated by performing the Schirmer tear test (STT) and phenol red thread test. Expression of the P2Y2 receptor was confirmed in canine bulbar and palpebral conjunctivae and receptors were identified at the conjunctival epithelial and goblet cell surface. Tear MUC5AC concentration significantly increased after administration of 3% diquafosol ophthalmic solution, although neither STT nor phenol red thread test values showed any significant change after diquafosol instillation. Topical ocular administration of 3% diquafosol might improve corneal epithelial disorders in dogs through stabilisation of the tear film, by virtue of an increase in MUC5AC secretion.
  • K. Terakado, T. Yogo, Y. Kohara, S. Soeta, Y. Nezu, Y. Harada, Y. Hara, H. Amasaki, M. Tagawa
    VETERINARY PATHOLOGY 50 4 664 - 667 2013年07月 [有り][無し]
     研究論文(学術雑誌) 
    The objectives of this study were to investigate the normal histological localization of aquaporin (AQP) 5 protein in the lacrimal and nictitating membrane glands and to compare this localization in healthy and keratoconjunctivitis sicca (KCS) dogs. Lacrimal and nictitating membrane glands of 5 healthy Beagles and nictitating membrane glands of 5 KCS dogs (3 Beagles and 2 mongrel dogs: 0-13 years) were used for the present study. The owners of the KCS dogs did not consent to perform biopsies of the lacrimal glands. The localization and distribution of AQP5 protein were investigated by an immunohistochemical technique. In immunohistochemical staining, AQP5 was localized in the apical site of acinar epithelial and ductal epithelial cells from both the lacrimal and nictitating membrane glands in healthy dogs. However, AQP5 was not detected in the 5 KCS dogs. These results for immunohistochemical AQP5 localization might correlate with the deficiency in tear secretion found in KCS dogs.

MISC

受賞

  • 2018年09月 アメリカ骨代謝学会 ASBMR Young Investigator Travel Grant
     
    受賞者: 小原 幸弘
  • 2018年09月 プロテイン・アイランド・松山 PIM 2018 Poster Award
     
    受賞者: 小原 幸弘
  • 2017年09月 アメリカ骨代謝学会 ASBMR Young Investigator Award
     
    受賞者: 小原 幸弘
  • 2017年06月 日本骨代謝学会 ASBMR 2017 Travel Award
     
    受賞者: 小原 幸弘

共同研究・競争的資金等の研究課題

  • マクロファージが産生するEmilin2の骨代謝における機能解明と臨床応用の可能性
    日本学術振興会:科学研究費助成事業 若手研究
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 小原 幸弘
  • マクロファージが産生・分泌する骨芽細胞遊走因子の同定と機能解明
    日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 小原 幸弘
     
    マクロファージが骨芽細胞による骨形成を支持することが示唆されているが、その分子メカニズムについては不明である。本研究では、そのメカニズムを解明する過程でマクロファージの培養上清から、骨髄間質細胞株ST2の走化性を刺激する分子としてEmilin2を分離・同定することに成功した。さらにレトロウイルスベクターを用いて、マウス新生仔頭蓋冠由来骨芽細胞にEmilin2遺伝子を強発現させると、Runx2やOsteocalcinといった骨芽細胞分化マーカー遺伝子の発現上昇が認められ、石灰化が亢進したことから、Emilin2は骨芽細胞の分化を促進することが示唆された。骨髄間質細胞は骨芽細胞へと分化するだけでなく、脂肪細胞へと分化することも知られている。そこで、脂肪細胞前駆細胞株3T3-L1細胞にもEmilin2遺伝子を強発現させたところ、脂肪細胞分化マーカーであるAdipoq、Fabp4およびLpl遺伝子発現が低下し、脂肪滴形成も抑制された。したがって、Emilin2は骨髄間質細胞から脂肪細胞への分化を抑制しつつ、骨芽細胞への分化を促進していることが示唆された。マクロファージにIL-4を添加するとEmilin2 mRNA発現が上昇した。すなわち、組織修復型マクロファージでEmilin2発現が高いことが示唆された。抗Emilin2抗体を用いた骨組織の免疫組織染色では、Emilin2は骨芽細胞表面のキャノピー状の細胞で検出された。マクロファージマーカーであるF4/80との二重染色ではEmilin2を発現する細胞ではF4/80も検出されたことから、osteomacと呼ばれる骨組織常在型マクロファージがEmilin2を産生・分泌することで骨代謝を制御している可能性が示唆された。
  • 骨粗鬆症に対する創薬シーズの探索 -肝臓由来の破骨細胞形成阻害因子の分離と同定-
    愛媛大学:研究活性化事業(スタートアップ支援)
    研究期間 : 2019年 -2020年 
    代表者 : 小原 幸弘

その他

  • 査読経験のある雑誌 
    ・Cancer Management and Research ・Healthcare ・Cell Communication & Signaling ・Molecular and Cellular Biochemistry ・International Journal of Molecular Sciences


Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.