研究者データベース

田村 正人(タムラ マサト)
歯学研究院 口腔医学部門 口腔健康科学分野
教授

基本情報

所属

  • 歯学研究院 口腔医学部門 口腔健康科学分野

職名

  • 教授

学位

  • 歯学博士(1991年 東京医科歯科大学)

J-Global ID

研究キーワード

  • 骨芽細胞   骨形成   遺伝子発現   転写因子   Wntシグナル   tRNaseZ   Wnt   Cell Biology   

研究分野

  • ライフサイエンス / 常態系口腔科学
  • ライフサイエンス / 医化学
  • ライフサイエンス / 細胞生物学

職歴

  • 2001年09月 - 現在 北海道大学 大学院歯学研究院 教授
  • 1999年04月 - 2001年09月 鹿児島大学 歯学部 助教授
  • 1997年09月 - 1999年03月 鹿児島大学 歯学部 助手
  • 1992年04月 - 1997年08月 東京医科歯科大学 難治疾患研究所 助手
  • 1991年05月 - 1992年03月 鹿児島大学 歯学部 助手

研究活動情報

論文

  • Tatsuya Shimizu, Naomasa Fujita, Kiyomi Tsuji-Tamura, Yoshimasa Kitagawa, Toshiaki Fujisawa, Masato Tamura, Mari Sato
    Scientific reports 11 1 10298 - 10298 2021年05月13日 
    Ultrasound stimulation is a type of mechanical stress, and low-intensity pulsed ultrasound (LIPUS) devices have been used clinically to promote fracture healing. However, it remains unclear which skeletal cells, in particular osteocytes or osteoblasts, primarily respond to LIPUS stimulation and how they contribute to fracture healing. To examine this, we utilized medaka, whose bone lacks osteocytes, and zebrafish, whose bone has osteocytes, as in vivo models. Fracture healing was accelerated by ultrasound stimulation in zebrafish, but not in medaka. To examine the molecular events induced by LIPUS stimulation in osteocytes, we performed RNA sequencing of a murine osteocytic cell line exposed to LIPUS. 179 genes reacted to LIPUS stimulation, and functional cluster analysis identified among them several molecular signatures related to immunity, secretion, and transcription. Notably, most of the isolated transcription-related genes were also modulated by LIPUS in vivo in zebrafish. However, expression levels of early growth response protein 1 and 2 (Egr1, 2), JunB, forkhead box Q1 (FoxQ1), and nuclear factor of activated T cells c1 (NFATc1) were not altered by LIPUS in medaka, suggesting that these genes are key transcriptional regulators of LIPUS-dependent fracture healing via osteocytes. We therefore show that bone-embedded osteocytes are necessary for LIPUS-induced promotion of fracture healing via transcriptional control of target genes, which presumably activates neighboring cells involved in fracture healing processes.
  • Hidekazu Yamamoto, Kazuhiro Matsushita, Shogo Ito, Kei Kazama, Masato Tamura
    Journal of Maxillofacial and Oral Surgery 2021年04月24日
  • Kiyomi Tsuji-Tamura, Mari Sato, Misato Fujita, Masato Tamura
    Biochemical and biophysical research communications 529 3 596 - 602 2020年08月27日 [査読有り][通常論文]
     
    Glycine, a non-essential amino acid, exerts concentration-dependent biphasic effects on angiogenesis. Low-doses of glycine promote angiogenesis, whereas high-doses cause anti-angiogenesis. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling participates in angiogenesis of both physiological development, and pathological events including tumor and inflammation. We assessed the role of PI3K/Akt/mTOR signaling in vascular development, and the interaction with glycine, using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos expressing fluorescent proteins in vascular endothelial cells. Treatment with inhibitors of mTORC1 (rapamycin and everolimus), mTORC1/mTORC2 (KU0063794), PI3K (LY29400), and Akt (Akt inhibitor) decreased the development of intersegmental vessels (ISVs). These inhibitors cancelled the angiogenic effects of a low-dose of glycine, while acted synergistically with a high-dose of glycine in anti-angiogenesis. mTOR signaling regulates the gene expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and nitric oxide (NO) synthase (NOS), an enzyme for the synthesis of an angiogenic mediator NO. Expressions of VEGF and NOS were consistent with the vascular features induced by glycine and an mTOR inhibitor. Our results suggest that PI3K/Akt/mTOR signaling may interact with dose-dependent biphasic effects of exogenous glycine on in vivo angiogenesis. mTOR signaling is a key target for cancer therapy, thus, the combining mTOR inhibitors with glycine may be a potential approach for controlling angiogenesis.
  • Kiyomi Tsuji-Tamura, Mari Sato, Misato Fujita, Masato Tamura
    Biochemical and biophysical research communications 527 2 539 - 544 2020年06月25日 [査読有り][通常論文]
     
    Glycine, a non-essential amino acid, is involved in both angiogenesis and anti-angiogenesis. We hypothesized that glycine would exert dose-dependent different effects on angiogenesis. In this study, we investigated the effects of a broad range of concentrations of glycine on vascular development using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos. Effects of glycine transporter (GlyT) inhibitors (sarcosine and bitopertin) and a glycine receptor (GlyR) inhibitor (strychnine) were also examined in embryos in the absence or presence of glycine. After exposure to glycine and inhibitors, intersegmental vessels (ISVs) were observed by fluorescent microscopy. Low concentrations of glycine promoted the development of ISVs, whereas high concentrations reduced it. These effects of glycine could generally be reversed by treatment with GlyT and GlyR inhibitors. Furthermore, expressions of vascular endothelial growth factor (VEGF) (an angiogenic factor) and nitric oxide synthase (NOS) (an enzyme for nitric oxide synthesis) were associated with the dose-dependent effects of glycine. Our results suggest that glycine exerts dose-dependent biphasic effects on vascular development, which rely on GlyTs and GlyRs, and correlate with the expression of VEGF and NOS genes. At low concentrations, glycine acted as an angiogenic factor. In contrast, at high concentrations, glycine induced anti-angiogenesis. This evidence provides a novel insight into glycine as a unique target in angiogenic and anti-angiogenic therapy.
  • Mayumi Ukita, Kenji Matsushita, Masato Tamura, Taihiko Yamaguchi
    International journal of molecular medicine 45 2 607 - 614 2020年02月 [査読有り][通常論文]
     
    The morbidity of temporomandibular joint osteoarthritis (TMJOA) increases with age. Condylar articular cartilage degradation, which causes TMJOA, is known to be involved in articular chondrocyte metabolic imbalances in the temporomandibular joint (TMJ) and in other joints of the body. Epigenetic regulation, such as the chemical modification of DNA and histones, is implicated in cartilage homeostasis. However, few studies have been conducted on the epigenetic regulation of condylar articular cartilage degradation. The present study investigated the regulation of histone H3 lysine 9 (H3K9) methylation and its effects on the pathogenesis of degenerative TMJ cartilage disorders. The histone H3K9 methylation level was decreased in degenerated condylar articular cartilage in aged mice. Treatment with chaetocin (a selective H3K9 methylation inhibitor) reduced cell viability and promoted caspase‑3/7 activity in ATDC5 mouse chondroprogenitor cells. The inhibition of H3K9 methylation increased matrix metalloproteinase (Mmp)1 and Mmp13 mRNA expression in these cells. Furthermore, the expression levels of Sox9 and collagen α1(II) (Col2a1) mRNA, which are anabolic factors for chondrogenic differentiation, were also decreased by treatment with chaetocin, which is an inhibitor of histone methyltransferases. These results indicated that histone H3K9 methylation regulates chondrocyte homeostasis in terms of cell growth, apoptosis and gene expression, and highlighted a possible future therapy option for TMJOA.
  • Hamaya E, Fujisawa T, Tamura M
    International journal of molecular medicine 44 6 2336 - 2344 2019年12月 [査読有り][通常論文]
  • Vascular formation: in vitro differentiation of vascular endothelial cells from pluripotent stem cells
    Tsuji-Tamura K, Tamura M
    Hokkaido J Dent Sci 38 Sp 80 - 85 2017年09月 [査読無し][招待有り]
  • Yahara M, Tei K, Tamura M
    Molecular medicine reports 16 3 2779 - 2784 2017年09月 [査読有り][通常論文]
     
    Neuropeptide Y (NPY) is a major neural signaling molecule. NPY is produced by peripheral tissues, such as osteoblasts, and binds to the corresponding Y1 receptor that belongs to the G-protein-coupled receptor family. Osteoblast-specific Y1 receptor knockout mice exhibit high bone mass, indicating a role of the NPY-Y1 receptor axis in the regulation of bone homeostasis. In the bone microenvironment, peripheral nerve fibers and osteoblasts produce NPY. However, the effects of the Y1 receptor on osteoblasts remain unexplored. In the present study, an RNA interference approach was employed to target the Y1 receptor, in order to determine whether it may function to regulate the growth, differentiation and viability of osteoblasts. Knockdown of the Y1 receptor by small interfering RNA (siRNA) lead to induction of alkaline phosphatase (ALP) activity and mineralization in mouse MC3T3-E1 osteoblast cells. In addition, the mRNA expression levels of ALP, osteocalcin, collagen (I) alpha 1, and bone sialoprotein were significantly increased following transfection of a Y1 receptor siRNA. Furthermore, the mRNA expression levels of Runx2 and osterix were significantly increased; however, no significant alterations in cell proliferation and caspase-3/7 activity were observed in Y1 receptor siRNA-transfected cells when compared with non-targeting controls. The results demonstrate that Y1 receptor inhibition may increase osteoblastic differentiation, which indicates a role of the Y1 receptor in the regulation of osteoblastic differentiation.
  • 胚性幹細胞を用いた血管形成メカニズムの解明
    田村-辻 潔美, 田村 正人
    北海道歯誌 37 2 173 - 176 2017年03月 [査読無し][招待有り]
  • Sato M, Suzuki T, Kawano M, Tamura M
    Biomedical reports 6 2 223 - 231 2017年02月 [査読有り][通常論文]
  • Masato Tamura, Eiji Nemoto
    Japanese Dental Science Review 52 4 75 - 83 2016年11月01日 [査読有り][通常論文]
     
    Wnt signaling plays a central role in many processes during embryonic development and adult homeostasis. At least 19 types of Wnt ligands, receptors, transducers, transcription factors, and antagonists have been identified in mammals. Two distinct Wnt signaling pathways, the canonical signaling pathway and the noncanonical signaling pathway, have been described. Some Wnt signaling pathway components are expressed in the dental epithelium and mesenchyme during tooth development in humans and mice. Functional studies and experimental analysis of relevant animal models confirm the effects of Wnt signaling pathway on the regulation of developing tooth formation and adult tooth homeostasis. Mutations in some Wnt signaling pathway components have been identified in syndromic and non-syndromic tooth agenesis. This review provides an overview of progress in elucidating the role of Wnt signaling pathway components in the tooth and the resulting possibilities for therapeutic development.
  • Sakisaka Y, Kanaya S, Nakamura T, Tamura M, Shimauchi H, Nemoto E
    Biochemical and biophysical research communications 478 2 527 - 532 2016年09月 [査読有り][通常論文]
     
    Wnt3a is a secreted glycoprotein that activates the glycogen synthase kinase-3 beta (GSK3 beta)/beta-catenin signaling pathway through low-density-lipoprotein receptor-related protein (LRP)5/6 co-receptors. Wnt3a has been implicated in periodontal development and homeostasis, as well as in cementum formation. Recently, we have reported that Wnt3a increases alkaline phosphatase expression through the induction of osterix (Osx) expression in dental follicle cells, a precursor of cementoblasts. However, the molecular mechanism by which Wnt3a induces Osx expression is still unknown. In this study, we show that Wnt3a-induced Osx expression was inhibited in the presence of p38 mitogen-activated protein kinase (MAPK) inhibitors (SB203580 and SB202190) at gene and protein levels, as assessed by real-time PCR and immunocytohistochemistry, respectively. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist binding to LRP5/6 co-receptors, did not influence Wnt3a-mediated p38 MAPK phosphorylation, suggesting that Wnt3a activates p38 MAPK through LRP5/6-independent signaling. On the other hand, pretreatment with p38 MAPK inhibitors had no effects on the phosphorylated status of GSK3 beta and beta-catenin as well as beta-catenin nuclear translocation, but inhibited Wnt3a-mediated beta-catenin transcriptional activity. These findings suggest that p38 MAPK modulates canonical Wnt signaling at the beta-catenin transcriptional level without any crosstalk with the Wnt3a-mediated LRP5/6-GSK3 beta signaling axis and subsequent beta-catenin nuclear translocation. These findings expand our knowledge of the mechanisms controlling periodontal development and regeneration. (C) 2016 Elsevier Inc. All rights reserved.
  • Mari Sato, Masato Tamura
    Biochemistry and Biophysics Reports 6 179 - 184 2016年07月01日 [査読有り][通常論文]
     
    The regulation of early B cell development and the interaction of hematopoietic precursors with stromal cells in the bone marrow (BM) are controlled by various secreted signaling molecules. Several recent studies showed Wnt signaling involved in B-lymphogenesis through stromal cells. However, the molecules modulated by Wnt signaling in stromal cells regulating B-lymphogenesis have not been identified yet. Interleukin (IL)-7 and CXC chemokine ligand (CXCL) 12 are known to be express in stromal cells, and both molecules are essential for B-lymphogenesis. In the present study, we examined the role of Wnt signaling in regulating IL-7 and CXCL12 expression and in affecting B-lymphogenesis. In mouse stromal ST2 cells, expression of IL-7 and CXCL12 mRNA was augmented by noncanonical Wnt5a. When mouse BM-derived cells were cultured on Wnt5a-overexpressing ST2 cells, an increased number of B220+/IgM- B-lymphoid precursor cells was observed. These results show that Wnt5a regulates IL-7 gene expression in stromal cells and suggest the possibility that noncanonical Wnt regulates B-lymphogenesis via IL-7 expression in stromal cells.
  • Mayumi Ukita, Taihiko Yamaguchi, Noboru Ohata, Masato Tamura
    JOURNAL OF CELLULAR BIOCHEMISTRY 117 6 1419 - 1428 2016年06月 [査読有り][通常論文]
     
    Sclerostin, a secreted protein encoded by the Sost gene, is produced by osteocytes and is inhibited by osteoblast differentiation and bone formation. Recently, a functional association between bone and fat tissue has been suggested, and a correlation between circulating sclerostin levels and lipid metabolism has been reported in humans. However, the effects of sclerostin on adipogenesis remain unexplored. In the present study, we examined the role of sclerostin in regulating adipocyte differentiation using 3T3-L1 preadipocytes. In these cells, sclerostin enhanced adipocyte-specific gene expression and the accumulation of lipid deposits. Sclerostin also upregulated CCAAT/enhancer binding protein expression but not cell proliferation and caspase-3/7 activities. Sclerostin also attenuated canonical Wnt3a-inhibited adipocyte differentiation. Recently, the transcriptional modulator TAZ has been involved in the canonical Wnt signaling pathway. Sclerostin reduced TAZ-responsive transcriptional activity and TAZ-responsive gene expression. Transfection of 3T3-L1 cells with TAZ siRNA increased the lipid deposits and adipogenic gene expression. These results show that sclerostin upregulates adipocyte differentiation in 3T3-L1 cells, suggesting a possible role for the osteocyte-derived sclerostin as a regulator of fat metabolism and as a reciprocal regulator of bone and adipose tissues metabolism. J. Cell. Biochem. 117: 1419-1428, 2016. (c) 2015 Wiley Periodicals, Inc.
  • E. Nemoto, Y. Sakisaka, M. Tsuchiya, M. Tamura, T. Nakamura, S. Kanaya, M. Shimonishi, H. Shimauchi
    JOURNAL OF PERIODONTAL RESEARCH 51 2 164 - 174 2016年04月 [査読有り][通常論文]
     
    Background and ObjectiveDental follicle cells, putative progenitor cells for cementoblasts, osteoblasts and periodontal ligament cells, interplay with Hertwig's epithelial root sheath (HERS) cells during tooth root formation, in which HERS is considered to have an inductive role in initiating cementogenesis by epithelial-mesenchymal interaction. However, the specific mechanisms controlling the cementoblast/osteoblast differentiation of dental follicle cells are not fully understood. Canonical Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions. This study examined the possible expression of canonical Wnt ligand in HERS and the role of Wnt signaling during the cementoblast/osteoblast differentiation of dental follicle cells. Material and MethodsThe expression of Wnt3a, a representative canonical Wnt ligand, in HERS was assessed by immunohistochemistry. The differentiation and function of immortalized murine dental follicle cells were evaluated by measuring alkaline phosphatase (ALP, Alpl) activity and osteogenic gene expression. ResultsWe identified the expression of Wnt3a in HERS during mouse tooth root development by immunohistochemistry as well as in cultured human epithelial rest cells of Malassez by real-time polymerase chain reaction, while no expression of Wnt3a was detected in cultured dental mesenchymal cells. Exposure of immortalized murine dental follicle cells to Wnt3a-induced ALP activity as well as expression of the Alpl gene. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist, markedly attenuated the effect of Wnt3a on ALP expression. Furthermore, Wnt3a induced transcriptional activity of runt-related transcription factor 2 (Runx2) and expression of osterix at gene and/or protein levels. Treatment with osterix-small interfering RNA significantly inhibited Wnt3a-induced ALP expression at gene and protein levels. ConclusionThese findings suggest that HERS has a potential role in stimulating cementoblast/osteoblast differentiation of dental follicle cells via the Wnt/-catenin signaling pathway.
  • Masato Tamura, Mitsuoki Kawano, Mari Sato, Masayuki Nashimoto
    MOLECULAR MEDICINE REPORTS 12 4 6365 - 6369 2015年10月 [査読有り][通常論文]
     
    tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology with therapeutic potential. Recently, our group demonstrated that a heptamer, mh1 (Bcl-2), targeting human Bc1-2 mRNA, can be taken up by cells without the use of any transfection reagents and can induce the apoptosis of leukemia cells. However, little is known regarding the mechanism of naked small guide (sg)RNA uptake by cultured cells. Therefore, in the present study the effects of various inhibitors on the induction of apoptosis by naked sgRNA treatment were investigated in order to identify the uptake pathway required for sgRNA function in cultured cells. Addition of the endocytosis inhibitors chlorpromazine, nystatin or methyl-P-cyclodextrin together with naked effective sgRNA was unable to diminish the apoptosis-inducing effects of naked sgRNA or the reduction in target mRNA, suggesting that functional uptake of sgRNA by cells is clathrin-, caveolae- and raft-independent. Next, chloroquine, an inhibitor of lysosome acidification, and brefeldin A, an inhibitor that blocks protein transport from the Golgi apparatus to the endoplasmic reticulum were administered. In the presence of these compounds, the apoptosis-inducing effects of naked sgRNA were reduced. These results suggest that a vesicular transport process is involved in sgRNA-mediated TRUE gene silencing. A greater understanding of how naked sgRNAs enter cells and how they reach their target RNAs may aid in the design of more specifically-targeted and potent sgRNA drugs.
  • Yukihiko Sakisaka, Masahiro Tsuchiya, Takashi Nakamura, Masato Tamura, Hidetoshi Shimauchi, Eiji Nemoto
    EXPERIMENTAL CELL RESEARCH 336 1 85 - 93 2015年08月 [査読有り][通常論文]
     
    Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through beta-catenin-dependent canonical and beta-catenin-independent noncanonical pathways. Canonical Wnt/beta-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of beta-catenin as well as beta-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the beta-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. (C) 2015 Elsevier Inc. All rights reserved.
  • Sho Ninomiya, Mitsuoki Kawano, Takashi Abe, Tatsuya Ishikawa, Masayuki Takahashi, Masato Tamura, Yoshiaki Takahashi, Masayuki Nashimoto
    PLOS ONE 10 3 e0118631  2015年03月 [査読有り][通常論文]
     
    Several pieces of evidence suggest that small RNA degradation products together with tRNase Z(L) appear to form another layer of the whole gene regulatory network. The degraded RNAsuch as a 5'-half-tRNA and an rRNA fragment function as small guide RNA (sgRNA) to guide the enzyme to target RNA. We were curious whether there exist RNAs in plasma that can function as sgRNAs for tRNase Z(L), whether these RNAs are working as signaling molecules between cells to fulfill physiological roles, and whether there are any differences in plasma sgRNA species and levels between normal and pathological conditions. Here, we analyzed small plasma RNAs from three healthy persons and three multiple myeloma patients for potential sgRNAs by deep sequencing. We also examined small RNAs from peripheral blood mononuclear cells (PBMC) of three healthy persons and three myeloma patients and from various cultured human cell lines for sgRNAs. We found that read-number distribution patterns of plasma and PBMC RNAs differ between persons in the range of 5-40 nt and that there are many RNA species that exist significantly more or less abundantly in the plasma or PBMC of the myeloma patients than those of the healthy persons. Furthermore, we found that there are many potential sgRNAs in the 5-40-nt RNAs and that, among them, a 31-nt RNA fragment derived from 94-nt Y4-RNA, which can function as a 50-half-tRNA-type sgRNA, is overwhelmingly abundant in the plasma of 2/3 of the examinees. These observations suggest that the gene regulatory network via tRNase Z(L) and sgRNA may be extended intercellularly.
  • Satoshi Iizuka, Nobuhiko Oridate, Masayuki Nashimoto, Satoshi Fukuda, Masato Tamura
    PLOS ONE 9 12 e114121  2014年12月 [査読有り][通常論文]
     
    Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA). In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs.
  • Masayuki Takahashi, Reyad A. Elbarbary, Norihiro Watanabe, Atsushi Goto, Daichi Kamiya, Yoshihiro Watabe, Takayoshi Uchiyama, Miwako Narita, Masuhiro Takahashi, Yoshiaki Takahashi, Noriko Ishihara, Tatsuya Miyazawa, Tetsuo Yoshida, Mitsuoki Kawano, Masato Tamura, Masayuki Nashimoto
    LEUKEMIA RESEARCH 38 7 808 - 815 2014年07月 [査読有り][通常論文]
     
    tRNase-Z(L)-utilizing efficacious (TRUE) gene silencing is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the property of tRNase Z(L) that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA). To search for novel potential therapeutic sgRNAs for hematological malignancies, we screened a library composed of 156 sgRNAs, and found that 20 sgRNAs can efficiently induce apoptosis in leukemia and/or myeloma cells. Furthermore, we demonstrated that 4 of the 20 sgRNAs can reduce growth rates of HL60 cells in mouse xenograft models. (C) 2014 Elsevier Ltd. All rights reserved.
  • Masato Tamura, Maki Uyama, Yuri Sugiyama, Mari Sato
    MOLECULAR MEDICINE REPORTS 8 6 1807 - 1811 2013年12月 [査読有り][通常論文]
     
    The canonical Wnt signaling pathway is crucial for the regulation of bone mass in humans and for the development of osteoblasts. MicroRNAs (miRs) represent a class of non-coding RNAs, similar to 22 nucleotides in length, that regulate gene expression by targeting mRNAs for cleavage or translational repression. Several previous studies have demonstrated the involvement of miRNAs in modulating gene expression in osteoblasts and regulating osteoblast differentiation. In the present study, microRNA profiling was conducted using Wnt3a-C2C12 cells; C2C12 cells were transfected with a Wnt3a expression plasmid to activate canonical Wnt signaling. miR-34b-5p and miR-34c were identified to be upregulated by the activation of canonical Wnt signaling in C2C12 cells. Expression of mature miR-34b/c increased from low levels at day 0 to maximum levels at day 28 of MC3T3-E1 cell differentiation. To analyze the effects of these miRNAs on osteoblast differentiation, an antisense inhibitor was transfected into MC3T3-E1 cells and osteoblast-related gene expression was investigated. Knockdown of miR34b/c enhanced osteocalcin mRNA expression; however, alkaline phosphatase mRNA expression and activity were decreased by miR34b/c inhibition. These results indicated that miR-34b/c regulates gene expression by targeting regulators of the osteogenic pathways and thereby contributes to osteoblast differentiation.
  • Naoko Kurebayashi, Mari Sato, Toshiaki Fujisawa, Kazuaki Fukushima, Masato Tamura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 439 4 506 - 510 2013年10月 [査読有り][通常論文]
     
    The neuropeptide Y (NPY) system is known as one of the major neural signaling pathways. NPY, produced by peripheral tissues including osteoblasts, is known to bind to the Y1 receptor. Recently, osteoblast-specific Y1 receptor knockout mice were developed and were found to have a high bone mass phenotype, indicating a role for the NPY-Y1 receptor axis as a regulator of bone homeostasis. However, regulation of Y1 receptor expression during osteoblastic differentiation remains unexplored. In the present study, we examined the role of bone morphogenetic protein (BMP) 2 signaling in regulating Y1 receptor expression. In C2C12 cells, expression of Y1 receptor mRNA was induced by BMP2. This induction was also observed after co-transfection with Smad1 and Smad4, the intracellular signaling molecules of the BMP2 signaling pathway. In a transfection assay, Smad1/4 up-regulated transcriptional activity through interaction with the Y1 receptor gene promoter. Following transfection of MC3T3-E1 cells with siRNA for the Y1 receptor, the expression of alkaline phosphatase, osteocalcin, Runx2 and osterix were increased. These results show that BMP2 signaling regulates Y1 receptor gene expression, and raises the possibility that NPY acts in osteoblasts via an autocrine mechanism. (C) 2013 Elsevier Inc. All rights reserved.
  • Maki Uyama, Masamitsu Kawanami, Masato Tamura
    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 31 5 1243 - 1247 2013年05月 [査読有り][通常論文]
     
    Systemic intermittent administration of parathyroid hormone (PTH) stimulates bone formation in animals and humans, and recombinant human PTH1-34 (teriparatide) is used clinically for the treatment of osteoporosis. In this study, we investigated the regulation of gene expression by intermittent PTH administration in MC3T3-E1 osteoblastic cells. We found that intermittent PTH1-34 administration downregulated Wiskott-Aldrich syndrome protein family member (Wasf) 2 mRNA expression. Wnt inhibitor, IWP-2, and protein kinase C inhibitor, Go6976, inhibited this downregulation. However, continuous PTH did not affect Wasf2 expression. Transfection of Wasf2 siRNA reduced bone sialoprotein (BSP) mRNA expression in a similar manner following intermittent PTH administration in MC3T3-E1 cells. These results identify Wasf2 as a novel target of intermittent PTH administration via the Wnt and phosphoinositide-dependent protein kinase signaling pathways, and the resulting regulation of BSP expression may contribute to the anabolic effects of PTH.
  • Masayuki Takahashi, Reyad A. Elbarbary, Aiko Nakashima, Mayumi Abe, Norihiro Watanabe, Miwako Narita, Masuhiro Takahashi, Masato Tamura, Tetsuo Yoshida, Masayuki Nashimoto
    Cancer Letters 328 2 362 - 368 2013年01月28日 [査読有り][通常論文]
     
    tRNase ZL-utilizing efficacious gene silencing is a gene control technology, which is based on the property that tRNase ZL can cleave any target RNA under the direction of an appropriate small guide RNA (sgRNA). To find therapeutic sgRNAs to cure hematological malignancies, we investigated behavior of heptamer-type sgRNA. We demonstrated that a heptamer, mh1(Bcl-2), which targets the human Bcl-2 mRNA, can be taken up by cells without any transfection reagents and that it can induce apoptosis of the leukemia cells. Mouse xenograft experiments showed that a median survival of the mh1(Bcl-2)-treated mice was longer than that of the control mice. © 2012 Elsevier Ireland Ltd.
  • Maki Uyama, Mari M. Sato, Masamitsu Kawanami, Masato Tamura
    GENES TO CELLS 17 7 548 - 558 2012年07月 [査読有り][通常論文]
     
    In eukaryotic cells, degradation of most intracellular proteins is carried out by the ubiquitin-proteasome pathway. Recent investigations suggest that bone metabolism is also regulated by this pathway. The clinical efficacy of bortezomib, a 26S proteasome inhibitor used as an anticancer drug, has been linked to an increase in bone formation. In this study, we show that proteasome inhibitors induce expression of osteoblastic differentiation-related genes such as osteocalcin and alkaline phosphatase in C2C12 cells. In contrast, myogenic differentiation is inhibited. Among the proteasome inhibitors tested, bortezomib induced the greatest increase in osteocalcin expression. Although these effects were similar to that of bone morphogenetic protein (BMP) 2, proteasome inhibitors did not induce transcriptional activity of Smad1/4-dependent reporter or BMP2 signaling target gene expression. Transient transfection of osteocalcin promoter-luciferase constructs with bortezomib resulted in an increase in luciferase activity. Mutation of OSE2, but not OSE1, sites of the osteocalcin promoter diminished the bortezomib-induced activity. Also, Runx2 binding activity and protein levels were induced by bortezomib treatment. These results suggest that the bortezomib induces osteoblastic differentiation by modifying the activity of Runx2 and that the function of the proteasome in controlling degradation of differentiation-related transcription factors plays an important role in osteoblast differentiation.
  • Yoshiyuki Wada, Morimichi Mizuno, Yoshinobu Nodasaka, Masato Tamura
    INTERNATIONAL JOURNAL OF ORAL & MAXILLOFACIAL IMPLANTS 27 4 849 - 858 2012年07月 [査読有り][通常論文]
     
    Purpose: This study investigated the effect of enamel matrix derivative (EMD) on spreading, proliferation, and differentiation of osteoblasts cultured on zirconia disks with smooth and rough surfaces. Materials and Methods: EMD was added to the culture medium or coated on zirconia disks that had machined (smooth) or sandblasted (rough) surfaces. The effects of EMD on cell proliferation of MC3T3-E1 osteoblastic cells were examined using a hemocytometer. Osteoblastic differentiation was examined by histologic analysis of alkaline phosphatase (ALP) activity and the degree of mineralization. ALP activity was also measured quantitatively. Scanning electron microscopic analysis was performed to observe cell morphology. Enzyme-linked immunosorbent assay of osteocalcin and reverse-transcriptase polymerase chain reaction of osteocalcin, osteopontin, and type 1 collagen were performed to investigate the expression of osteoblast-related genes. Results: The addition of EMD to the medium enhanced the spreading, proliferation, and differentiation of osteoblasts cultured on zirconia. However, when it was coated on zirconia, EMD reduced osteoblastic spreading and adhesion in the early stage of culture, although it enhanced proliferation and differentiation of osteoblasts in later stages. A promotive effect of EMD on osteocalcin mRNA expression, mineralization, and ALP activity of osteoblasts cultured on the rough surface was observed. Conclusions: EMD may contribute to treatment with zirconia implants via its promotion of osteoblastic proliferation and activity. However, the procedure for application of EMD may be a crucial factor for the outcome of implants. INT J ORAL MAXILLOFAC IMPLANTS 2012;27:849-858.
  • Eiji Nemoto, Yukari Ebe, Sousuke Kanaya, Masahiro Tsuchiya, Takashi Nakamura, Masato Tamura, Hidetoshi Shimauchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 422 4 627 - 632 2012年06月 [査読有り][通常論文]
     
    Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through beta-catenin-dependent canonical and beta-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway. (C) 2012 Elsevier Inc. All rights reserved.
  • Masayuki Takahashi, Reyad A. Elbarbary, Mayumi Abe, Mari Sato, Tetsuo Yoshida, Yoji Yamada, Masato Tamura, Masayuki Nashimoto
    PLOS ONE 7 6 e38496  2012年06月 [査読有り][通常論文]
     
    tRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase Z(L), which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5'-half-tRNA, RNA heptamer, hook RNA, and similar to 14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase Z(L) cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by similar to 50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.
  • Ryuichi Fujisawa, Masato Tamura
    FRONTIERS IN BIOSCIENCE-LANDMARK 17 1891 - 1903 2012年01月 [査読有り][通常論文]
     
    Mammalian bones are composed of calcium phosphate crystals in a protein matrix. The major form of the calcium phosphate is hydroxyapatite. The most abundant matrix protein in bone is type I collagen. Collagen contributes to the mechanical properties of bone and is necessary for calcification of the tissue. In addition to collagen, several acidic proteins are present as minor components. Osteocalcin is a gamma-carboxyglutamic acid-containing protein of bone, which has an affinity to hydroxyapatite and can prevent crystal growth. Bone sialoprotein (BSP) and osteopontin are acidic glycophosphoproteins of bone. These proteins have RGD cell-attachment sequences and consecutive sequences of acidic amino acids. The poly glutamic acid sequences of BSP act as possible nucleation sites for hydroxyapatite crystals. Dentin phosphoprotein is the major non-collagenous protein of dentin. This protein has (Asp-Ser-Ser) repeat sequences, in which most of the Ser residues are phosphorylated. Some of these acidic matrix proteins are immobilized on the collagen fibrils and induce nucleation of hydroxyapatite crystals. They can also modulate crystal shape by adsorption on a specific face of the crystals.
  • Maki Uyama, Mari Sato, Masamitsu Kawanami, Masato Tamura
    Interface Oral Health Science 2011 164 - 166 2012年01月01日 [査読有り][通常論文]
     
    In eukaryotic cells, degradation of most intracellular proteins is carried out by the ubiquitin-proteasome pathway. This pathway is the therapeutic target in several diseases. Recent observations suggest that bone metabolism is regulated by the ubiquitin-proteasome pathway, and that proteasome activity is critical for activation of osteogenic transcription factors. The clinical efficacy of bortezomib, a 26S proteasome inhibitor used as an anticancer drug in the treatment of multiple myeloma, has been linked to an increase in bone markers. Also, bortezomib has been reported to induce differentiation of osteoblasts both in vitro and in vivo. From our studies, bortezomib not only induces osteoblastic differentiation but also inhib its myogenic differentiation in pluripotent C2C12 cells in culture.
  • Takako Sano, Masayuki Takahashi, Tadasuke Nozaki, Yoshiaki Takahashi, Masato Tamura, Masayuki Nashimoto
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 416 3-4 427 - 432 2011年12月 [査読有り][通常論文]
     
    tRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a novel technology for suppressing gene expression. TRUE gene silencing is based on a unique enzymatic property of mammalian tRNase Z(L), which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like or micro-pre-tRNA-like complex formed between the target RNA and artificial small guide RNA (sgRNA). sgRNA is divided into four groups, 5'-half-tRNA, RNA heptamer, hook RNA, and similar to 14-nt linear RNA. One of the final destinations of TRUE gene silencing is to generate cancer therapeutic sgRNAs, and from a pharmacological point of view, heptamer-type sgRNA appears to be the most appropriate for this purpose. In this paper, we present two strategies to expand the utility of heptamer-type sgRNA: one is about locked nucleic acid (LNA) modifications of heptamers and the other is about usage of double heptamers. We showed that RNA heptamers with LNA modifications can work as sgRNA in vitro and in vivo. We also demonstrated that two consecutively aligned heptamers can guide target RNA cleavage by human tRNase Z(L) as efficiently as a corresponding 14-nt sgRNA in vitro and that a double heptamer can work much better than a 14-nt sgRNA in vivo. (C) 2011 Elsevier Inc. All rights reserved.
  • Masato Tamura, Mari M. Sato, Masayuki Nashimoto
    INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY 43 5 760 - 767 2011年05月 [査読有り][通常論文]
     
    CXCL12 (stromal cell-derived factor-1, SDF-1), produced by stromal and endothelial cells including cells of the bone marrow, binds to its receptor CXCR4 and this axis regulates hematopoietic cell trafficking. Recently, osteoclast precursor cells were found to express CXCR4 and a potential role for the CXCL12-CXCR4 axis during osteoclast precursor cell recruitment/retention and development was proposed as a regulator of bone resorption. We examined the role of canonical Wnt signaling in regulating the expression of CXCL12 in bone marrow stromal cells. In mouse stromal ST2 cells, CXCL12 mRNA was expressed, while its expression was reduced in Wnt3a over-expressing ST2 (Wnt3a-ST2) cells or by treatment with lithium chloride (LiCl). Wnt3a decreased CXCL12 levels in culture supernatants from mouse bone marrow stromal cells. The culture supernatant from Wnt3a-ST2 cells also reduced migratory activity of bone marrow-derived cells in a Transwell migration assay. Silencing of glycogen synthase kinase-3 beta decreased CXCL12 expression, suggesting that the canonical Wnt signaling pathway regulates CXCL12 expression. In a transfection assay, LiCl down-regulated the activity of a reporter gene, a 1.8 kb fragment of the 5'-flanking region of the CXCL12 gene. These results show that canonical Wnt signaling regulates CXCL12 gene expression at the transcriptional level, and this is the first study linking chemokine expression to canonical Wnt signaling. (C) 2011 Elsevier Ltd. All rights reserved.
  • Minqi Li, Yukie Seki, Paulo H. L. Freitas, Masaki Nagata, Taku Kojima, Sara Sultana, Sobhan Ubaidus, Takeyasu Maeda, Junko Shimomura, Janet E. Henderson, Masato Tamura, Kimimitsu Oda, Zhusheng Liu, Ying Guo, Reiko Suzuki, Tsuneyuki Yamamoto, Ritsuo Takagi, Norio Amizuka
    JOURNAL OF ELECTRON MICROSCOPY 59 3 227 - 236 2010年06月 [査読有り][通常論文]
     
    The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but not with an inhibitor of MAPK (SB203580) or JAK2 (AG490). Altogether, these findings suggest that FGFR3 down-regulates PTH/PTHrP receptor gene expression via the JAK/STAT signaling in chondrocytic cells.
  • Minqi Li, Yukie Seki, Paulo H.L. Freitas, Masaki Nagata, Taku Kojima, Sara Sultana, Sobhan Ubaidus, Takeyasu Maeda, Junko Shimomura, Janet E. Henderson, Masato Tamura, Kimimitsu Oda, Zhusheng Liu, Ying Guo, Reiko Suzuki, Tsuneyuki Yamamoto, Ritsuo Takagi, Norio Amizuka
    Journal of Electron Microscopy 59 3 227 - 236 2010年06月 [査読無し][通常論文]
     
    The signaling axis comprising the parathyroid hormone (PTH)-related peptide (PTHrP), the PTH/PTHrP receptor and the fibroblast growth factor receptor 3 (FGFR3) plays a central role in chondrocyte proliferation. The Indian hedgehog (IHH) gene is normally expressed in early hypertrophic chondrocytes, and its negative feedback loop was shown to regulate PTH/PTHrP receptor signaling. In this study, we examined the regulation of PTH/PTHrP receptor gene expression in a FGFR3-transfected chondrocytic cell line, CFK2. Expression of IHH could not be verified on these cells, with consequent absence of hypertrophic differentiation. Also, expression of the PTH/PTHrP receptor (75% reduction of total mRNA) and the PTHrP (50% reduction) genes was reduced in CFK2 cells transfected with FGFR3 cDNA. Interestingly, we verified significant reduction in cell growth and increased apoptosis in the transfected cells. STAT1 was detected in the nuclei of the CFK2 cells transfected with FGFR3 cDNA, indicating predominance of the JAK/STAT signaling pathway. The reduction in PTH/PTHrP receptor gene in CFK2 cells overexpressing FGFR3 was partially blocked by treatment with an inhibitor of JAK3 (WHI-P131), but not with an inhibitor of MAPK (SB203580) or JAK2 (AG490). Altogether, these findings suggest that FGFR3 down-regulates PTH/PTHrP receptor gene expression via the JAK/STAT signaling in chondrocytic cells. The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved.
  • Masato Tamura, Eiji Nemoto, Mari M. Sato, Aiko Nakashima, Hidetoshi Shimauchi
    Frontiers in Bioscience - Elite 2 4 1405 - 1413 2010年01月06日 [査読有り][通常論文]
     
    Signaling by the Wnt plays a central role in many processes during embryonic development and adult homeostasis. At least 19 types of Wnts, several families of secreted antagonists and multiple receptors have been identified. Two distinct Wnt signaling pathways, the canonical pathway and the noncanonical pathway have been described. Functional studies and experimental analysis of relevant animal models confirmed the effects of Wnt on regulation of developing mineralized tissue formation and adult homeostasis. In osteoblasts, the canonical Wnt pathway modulates differentiation, proliferation and mineralization, while it blocks apoptosis and osteoclastogenesis by increasing osteoprotegerin. Functional crosstalk between Wnt and bone morphogenetic protein signaling during osteoblastic differentiation has been reported. Recently, non-canonical Wnt signaling was shown to play a role in bone formation. The Wnt signaling pathway also plays an important role not only in tooth formation but also in differentiation and proliferation of cementoblasts and odontoblasts in the tooth. This present review provides an overview of progress in elucidating the role of Wnt signaling pathways in bone and tooth and the resulting possibilities for therapeutic potential.
  • Naoto Okubo, Akira Ishisaki, Tadashi Iizuka, Masato Tamura, Yoshimasa Kitagawa
    JOURNAL OF VASCULAR RESEARCH 47 5 369 - 383 2010年 [査読有り][通常論文]
     
    Objective: To evaluate whether fibroblasts derived from periodontal ligament retain the ability to differentiate into putative vascular cells and construct vascular cell-specific marker-positive blood vessel structures. We also evaluated the morphological features of the structure and investigated the intracellular molecular mechanism underlying the angiogenic activity of these cells. Methods: Single cell-derived cultures (SCDCs) were established from primary rat ligament fibroblast cultures, and their expression of ligament cell-, mesenchymal stem cell-and vascular cell-specific markers was evaluated by RT-PCR and immunocytochemistry. The ability of the cells to construct a blood vessel structure was evaluated in a three-dimensional type I collagen scaffold. The morphological and immunohistological characteristics of the structure were then evaluated. Results: Each SCDC expressed endothelial cell (EC)-specific and smooth muscle cell-specific markers, in addition to mesenchymal stem cell- and ligament cell-specific markers. SCDC2 cells, which abundantly expressed the EC markers Flk-1 and Tie-2, vigorously constructed a blood vessel structure in a phosphoinositide 3-kinase activation-dependent manner. Conclusion: Periodontal ligament fibroblasts have the potential to construct an EC marker-positive blood vessel-like structure. Consequently, the fibroblastic lineage in ligament tissue could be a candidate precursor for construction of a vascular system around damaged ligament tissue to facilitate its regeneration. Copyright (C) 2010 S. Karger AG, Basel
  • Mari M. Sato, Yasutaka Yawaka, Masato Tamura
    INTERFACE ORAL HEALTH SCIENCE 2009 158 - 160 2010年 [査読有り][通常論文]
     
    MicroRNAs (miRNAs) are small noncoding RNAs that are emerging as important posttranscriptional gene regulators. Many miRNAs are expressed in a tissue-specific manner, which suggests that they have specific biological roles in the specification of tissues. In this chapter, we summarize the currently available data on the regulation of miRNA expression by bone morphogenetic protein (BMP)-2. These studies open new avenues for the study of BMP signaling and miRNA biogenesis.
  • Amizuka N, Li M, Tamura M, Oda K
    Clinical calcium 19 935 - 943 7 2009年07月 [査読有り][通常論文]
  • Eiji Nemoto, Yohei Koshikawa, Sousuke Kanaya, Masahiro Tsuchiya, Masato Tamura, Martha J. Somerman, Hidetoshi Shimauchi
    BONE 44 5 805 - 812 2009年05月 [査読有り][通常論文]
     
    Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3 beta, increased beta-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) activity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast differentiation and promotes cell proliferation. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies. (C) 2009 Elsevier Inc. All rights reserved.
  • Mari M. Sato, Masayuki Nashimoto, Takenobu Katagiri, Yasutaka Yawaka, Masato Tamura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 383 1 125 - 129 2009年05月 [査読有り][通常論文]
     
    MicroRNAs (miRNAs) are small non-coding RNAs that are emerging as important post-transcriptional gene regulators. miR-206 is unique in that it is expressed only in skeletal muscle, including the myoblastic C2C12 cell line. In C2C12 cells, miR-206 expression was reduced dramatically after bone morphogenetic protein (BMP)-2 treatment. The down-regulation of miR-206 expression was also observed after co-transfection with constitutively-active Smad1 and Smad4, which are the intracellular signaling molecules of the BMP pathway. BMP-2 also reduced miR-206 expression in the presence of alpha-amanitin in a similar manner to that in the absence of alpha-amanitin. Moreover, the expression of pri-miR-206 was increased upon BMP-2 treatment for 6 h compared to that in the absence of BMP-2. These results suggested that BMP-2 down-regulates miR-206 expression at the post-transcriptional level, by inhibiting the processing of pri-miR-206 into mature miR-206, and that BMP-2 Could regulate miRNA biogenesis by a novel mechanism. Crown Copyright (C) 2009 Published by Elsevier Inc. All rights reserved.
  • K. Shirai, A. Ishisaki, T. Kaku, M. Tamura, Y. Furuichi
    JOURNAL OF PERIODONTAL RESEARCH 44 2 238 - 247 2009年04月 [査読有り][通常論文]
     
    A blood supply is indispensable for the regeneration of damaged or lost periodontal ligament (PDL) tissue. Mesenchymal stem cell-like activity of cells derived from the PDL has been identified by their capacity to form fibrous and osseous tissue and cementum. However, it remains to be clarified whether the cells have an ability to build the capillary network of blood vessels. This study evaluated the potential of cells derived from the PDL to construct a blood vessel-like structure and examined how growth factors controlled the multipotency of the cells. The ability of a swine PDL fibroblast cell line, TesPDL3, to construct a blood vessel-like structure was evaluated on and in the self-assembling peptide scaffold, PuraMatrix(TM). In addition, the ability of the cells to form mineralized nodules was evaluated on type I collagen-coated plastic plates. In some cases, fibroblast growth factor (FGF)-2 and bone morphogenetic protein (BMP)-2 were added to these cultures. The status of the expression of vascular and osteoblastic cell-specific markers in the cells was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence analyses. The TesPDL3 cells not only formed mineralized nodules in response to BMP-2 stimulation but also constructed tube-like structures in response to FGF-2 stimulation. Intriguingly, FGF-2 inhibited the BMP-2-induced formation of mineralized nodules. Conversely, BMP-2 inhibited the FGF-2-induced formation of tube-like structures. Periodontal ligament fibroblasts have the potential to differentiate not only into osteoblastic but also into vascular cell lineages. The destiny of the cells was reciprocally regulated by BMP-2 and FGF-2.
  • Yoshiyuki Wada, Morimichi Mizuno, Masato Tamura
    ARCHIVES OF ORAL BIOLOGY 54 4 306 - 312 2009年04月 [査読有り][通常論文]
     
    Objective: In this study, we investigated the effects of enamel matrix derivative (EMD) on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) expression of osteoblasts in the presence of lipopolysaccharide (LPS). Study design: OPG and RANKL gene expression and protein synthesis of MC3T3-E1 osteoblastic cells were examined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Results: LPS inhibited OPG gene expression and protein synthesis, and stimulated RANKL gene expression and soluble RANKL synthesis. EMD enhanced OPG gene expression and protein synthesis, and inhibited RANKL gene expression and soluble RANKL synthesis. Furthermore, EMD neutralized the effects of LPS on OPG and RANKL expression in osteoblasts. Conclusions: EMD might regulate the function of osteoblasts; by elevating the ratio of OPG/RANKL gene expression, which is downregulated by LPS, and suppress the induction of osteoclastogenesis. Thereby, EMD might contribute to periodontal tissue regeneration. (C) 2009 Elsevier Ltd. All rights reserved.
  • Reyad A. Elbarbary, Hiroaki Takaku, Masato Tamura, Masayuki Nashimoto
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 379 4 924 - 927 2009年02月 [査読有り][通常論文]
     
    Pathogenic angiogenesis in various diseases including cancer, autoimmune diseases, and age-related macular degeneration is thought to be regressed with anti-angiogenic drugs. TRUE gene silencing is a new technology to eliminate a specific mRNA using synthetic sgRNA and cellular tRNase Z(L). To discover anti-angiogenic sgRNAs, we applied TRUE silencing to the VEGF gene. We examined eight sgRNAs for efficacy in targeting exogenous human VEGF mRNA. Many of them worked efficiently in 293 and HeLa cells. Two of them downregulated the endogeneous VEGF gene expression in HeLa cells very efficiently, and the efficacy of these two sgRNAs surpassed that of siRNA extremely. (C) 2009 Elsevier Inc. All rights reserved.
  • Mari M. Sato, Aiko Nakashima, Masayuki Nashimoto, Yasutaka Yawaka, Masato Tamura
    GENES TO CELLS 14 2 141 - 153 2009年02月 [査読有り][通常論文]
     
    Wnt/beta-catenin signaling plays an important role in the developing skeletal system. Our previous studies demonstrated that Wnt/beta-catenin signaling inhibits the ability of bone morphogenetic protein (BMP)-2 to suppress myotube formation in the multipotent mesenchymal cell line C2C12 and that this inhibition is mediated by Id1. In this study, we examined the role of intracellular signaling by Wnt/beta-catenin and BMP-2 in regulating the expression of osteoprotegerin (OPG) and of the receptor activator of NF kappa B ligand (RANKL). OPG expression was induced by Wnt/beta-catenin signaling in C2C12 cells and osteoblastic MC3T3-E1 cells. Silencing of glycogen synthase kinase-3 beta also increased OPG expression. In contrast, R expression was suppressed by Wnt/beta-catenin signaling. In a transfection assay, beta-catenin induced the activity of a reporter gene, a 1.5 kb fragment of the 5'-flanking region of the OPG gene. Deletion and mutation analysis revealed that Wnt/beta-catenin signaling regulates transcription of OPG via a promoter region containing two Wnt/beta-catenin responsive sites. BMP-2 enhanced Wnt/beta-catenin-dependent transcriptional activation of the OPG promoter. In response to BMP-2 stimulation, Smad 1 and 4 interacted with Wnt/beta-catenin responsive sites. These results show that the regulation of OPG expression is mediated through two transcription pathways that involve the OPG promoter.
  • Ryuichi Fujisawa, Morimichi Mizuno, Masato Tamura
    CELLS TISSUES ORGANS 189 1-4 60 - 64 2009年 [査読有り][通常論文]
     
    Dentin phosphoprotein, the major noncollagenous protein in dentin, has effects on differentiation of odontoblast-like cells. This study was designed to investigate the effect of the protein on apoptosis of the cells. The odontoblast-like cells were prepared from the pulp cells of rat incisors. Apoptosis was detected by measuring caspase-3 activity by using DEVD-AMC as a fluorescent substrate. The cells formed calcification nodules in the presence of 2-glycerophosphate and expressed dentin sialophosphoprotein. Apoptosis was not observed in the cells through the differentiation stages. Then, apoptosis was induced by raising inorganic phosphate concentration in the medium. Elevation of phosphate concentration to 5 m M reduced the number of viable cells and increased caspase-3 activity, indicating the induction of apoptosis. Addition of bovine dentin phosphoprotein in the medium suppressed phosphate-induced apoptosis. Phosvitin and poly( Asp) had similar antiapoptotic effects. Copyright (C) 2008 S. Karger AG, Basel
  • Yoshiyuki Wada, Hidekazu Yamamoto, Satoshi Nanbu, Morimichi Mizuno, Masato Tamura
    JOURNAL OF PERIODONTOLOGY 79 2 341 - 347 2008年02月 [査読有り][通常論文]
     
    Background: Enamel matrix derivative (EMD) plays a crucial role in periodontal tissue regeneration. However, the precise mechanism of tissue regeneration by EMD remains obscure. The purpose of this study was to clarify what factors present in EMD show bioactivity. Methods: We first examined the effect of EMD on MC3T3-E1 osteoblastic cells. To evaluate the differentiation, the expression of osteoblast-related genes was measured by reverse transcription-polymerase chain reaction, and the osteocalcin (OCN) content was measured by enzyme-linked immunosorbent assay. Alkaline phosphatase activity and the mineralization were examined histologically. EMD (intact EMD) was filtrated to separate the soluble fraction (soluble EMD), and the effects of soluble and intact EMD were examined. Neutralization of the bioactivity of EMD was performed using a polyclonal antibody against porcine transforming growth factor-beta TGF-beta). Results: EMD inhibited the expression of osteoblastic phenotypes, and we used the inhibitory effect of EMD on osteoblastic differentiation as a benchmark of activity of EMD. The soluble fraction separated from EMD inhibited osteoblast-related gene expression and OCN synthesis. Soluble EMD suppressed the OCN gene level within 24 hours, and the effect of soluble EMD mimicked that of TGF-beta (10 ng/ml). The antibody against TGF-beta diminished the inhibitory effect of soluble EMD on OCN gene expression. Conclusions: The inhibitory effect of EMD on OCN gene expression of osteoblastic cells is neutralized by the antibody against TGF-beta in it. This result might indicate that EMD contains TGF-beta and that it participates in the bioactivity of EMD.
  • Miho Ibi, Akira Ishisaki, Matsuo Yamamoto, Satoshi Wada, Takaharu Kozakai, Aiko Nakashima, Jynichiro Iida, Sonshin Takao, Yuichi Izumi, Atsuro Yokoyama, Masato Tamura
    ARCHIVES OF ORAL BIOLOGY 52 10 1002 - 1008 2007年10月 [査読有り][通常論文]
     
    Objective: The periodontal ligament (PDL) is a fibrous connective tissue composed of heterogeneous cell types, including PDL fibroblasts. It is not clear whether cells within the PDL fibroblast population retain the potency to differentiate into other cell types. Design: In the present study, clonal cell lines, derived from Clawn miniature swine PDLs, were established by gene transfection for a human telomerase reverse transcriptase, and characterized. Results: These cell lines, denoted TesPDL1-4, had PDL fibroblasts that showed fibroblastic morphology and expressed procollagen alpha 1(I), osteopontin, periostin and alkaline phosphatase mRNA. Under the specific culture conditions, TesPDL3 cells also have the ability to express CD31, vascular endothelial cadherin, von Willebrand factor, osteocalcin, and to form extracellular mineralized nodules. Conclusions: Our data indicate that TesPDL3 cells have unique properties of expressing several phenotype of fibroblasts, vascular endothelial cells and osteoblasts in cultures. (C) 2007 Elsevier Ltd. All rights reserved.
  • A. Nakashima, H. Takaku, H. S. Shibata, Y. Negishi, M. Takagi, M. Tamura, M. Nashimoto
    GENE THERAPY 14 1 78 - 85 2007年01月 [査読有り][通常論文]
     
    We have been developing a unique system for the down-regulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3'-processing endoribonuclease (tRNase Z(L)) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase Z(L) in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase Z(L) method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase Z(L) directed by sgRNA not owing to a simple antisense effect. We also show that tRNase Z(L) together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3 beta by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.
  • A Nakashima, M Tamura
    FRONTIERS IN BIOSCIENCE 11 1667 - 1678 2006年05月 [査読有り][通常論文]
     
    During bone remodeling, degradation of skeletal connective tissue is regulated, at least in part, by the balance between matrix metalloproteinases ( MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs), their natural inhibitors. Recently, the Wnt signaling pathway has been demonstrated to play a crucial role in the regulation of bone formation. Here, we investigated a potential role for Wnt signaling and functional cross-talk with bone morphogenetic protein (BMP)-2 in mRNA expression of MMPs, TIMPs and bone matrix proteins in pluripotent C2C12 cells. To assess the functional contribution of Wnt signaling, we have generated C2C12 cell lines stably over-expressing Wnt3a or Wnt5a, and then treated these cells with BMP-2 for 24 h. In these cultures, MMP-13 mRNA expression was induced by BMP-2 in Wnt3a over-expressing C2C12 (Wnt3a-C2C12) cells but not in either Wnt5a over-expressing C2C12 (Wnt5aC2C12) cells or vehicle-transfected C2C12 cells. MMP-13 mRNA was induced in these cells by addition of BMP-2 for 12 h and the enhancement lasted up to 48 h. These effects were observed in a dose-dependent manner. Enzymatic activity of MMP-13 also induced in Wnt3a- C2C12 cells by addition of BMP-2. However, membrane type-1 matrix metalloproteinase (MT1- MMP) and MMP-2 mRNA expression was not affected by either Wnt3a or BMP-2. In contrast, TIMP-1 mRNA expression was suppressed by BMP-2 in Wnt3a-C2C12 cells but not in Wnt5a-C2C12 cells. Our results show that expression of MMP-13 and TIMP-1 is regulated by Wnt signaling combined with BMP-2 in osteoblastic differentiation, and this signaling may in part mediate MMP-13 and TIMP-1 production during bone formation and/or remodeling.
  • A Nakashima, T Katagiri, M Tamura
    JOURNAL OF BIOLOGICAL CHEMISTRY 280 45 37660 - 37668 2005年11月 [査読有り][通常論文]
     
    Loss of function of the Wnt co-receptor, lipoprotein receptor-related protein 5, decreases bone formation, and a point mutation in this gene results in high bone mass, indicating the importance of this signaling pathway in bone formation. However, the exact mechanism is currently unknown. We examined a potential role for Wnt signaling and functional cross-talk of bone morphogenetic protein 2 (BMP-2) in osteoblast differentiation. To assess the contribution of Wnt, we generated C2C12 cells over-expressing Wnt3a or Wnt5a and treated these with BMP-2. We showed that expression of matrix extracellular phosphoglycoprotein was induced by BMP-2 in Wnt3a over-expressing C2C12 cells but not in Wnt5a over-expressing C2C12 cells. Over-expression of Wnt3a blocked BMP-2-induced inhibition of myotube formation in C2C12 cells when switched to low mitogen medium. In these cultures, expression of inhibitor of DNA binding/differentiation ( Id) 1, a helix-loop-helix protein induced by BMP-2, decreased in stable Wnt3a- but not in Wnt5a-expressing cells. This suppression is mediated by a GC-rich region of the BMP-2-responsive element of the Id1 gene promoter, and interaction between Smad1/4 and beta-catenin is crucial for Wnt-mediated suppression of the BMP-2 response in C2C12 cells. Overexpression of the inhibitor of canonical Wnt signaling, Dickkopf, inhibits this suppression. In contrast, BMP-2 or Smad1/4 up-regulated Wnt3a or activated beta-catenin-induced lymphoid-enhancing factor 1/T cell factor-dependent transcriptional activity. These findings identify functional cross-talk of Id1 expression between Wnt and BMP signaling and demonstrate a novel mechanism for Wnt regulation of the BMP-2 response, linking Id1 expression to Wnt/beta-catenin signaling.
  • K Bandow, T Ohnishi, M Tamura, Semba, I, Y Daikuhara
    JOURNAL OF CELLULAR PHYSIOLOGY 201 2 236 - 243 2004年11月 [査読有り][通常論文]
     
    Hepatocyte growth factor (HGF) stimulates the migration of myogenic cells during the development of skeletal muscles. The inactivation of HGF genes or that of its receptor, c-met, in mice causes hypoplasia of skeletal muscle organs, such as the tongue. Basic fibroblast growth factor (FGF-2) also induces migration of skeletal myoblasts. A comparison of the functions of HGF and FGF-2 in myogenesis revealed the crucial effect of HGF in the development of skeletal muscles. Unlike FGF-2, HGF induced migration of myoblasts from the developing mouse tongue. The differences between the activities of HGF and FGF-2 were determined by comparing their effects on the expression of matrix metalloproteinase-9 (MMP-9) in myoblasts, C2C12 cells, cultured in collagen-coated dishes. The results showed that HGF, but not FGF-2, stimulated MMP-9 expression, and that the stimulation was mediated through the activation of phosphoinositide 3-kinase (PI3K) which was not associated with FGF-2 signal transduction. Nevertheless, both growth factors exerted almost the same effect on the reduction of myogenin expression in, and on the proliferation of, C2C12 cells, suggesting that HGF, rather than FGF-2, plays a crucial role in the generation of skeletal muscles, including the tongue. Moreover,the specific role of HGF through the PI3K signal pathway is the induction of MMP-9 expression in, and the migration of, myoblasts. (C) 2004 Wiley-Liss, Inc.
  • H Takita, JWM Vehof, JA Jansen, M Yamamoto, Y Tabata, M Tamura, Y Kuboki
    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A 71A 1 181 - 189 2004年10月 [査読有り][通常論文]
     
    To evaluate the osteoinductive effects of recombinant human bone morphogenetic protein (rhBMP)-2 during the early stages of rat ectopic bone formation, we prepared two distinct carriers. Two carriers, insoluble bone matrix (IBM) and fibrous glass membrane (FGM) were combined with rhBMP-2 and implanted into the backs of rats to evaluate the osteoinductive effects of the two rhBMP-2 carrier systems. Insoluble bone matrix particle size was 320 to 620 mum. Fibrous glass membrane was constructed from unwoven glass fibers 1 mum in diameter. Alkaline phosphatase (ALP) activity and type II collagen were detected in IBM/ rhBMP-2 at 5 days postimplantation. Calcium (Ca) was also detected in IBM/rhBMP-2 at 7 and 9 days postimplantation. In contrast, ALP and type II collagen were detected in FGM/ rhBMP-2 at 7 days. Calcium was undetected, indicating that the bone formation in IBM/rhBMP-2 proceeded faster than in FGM/rhBMP-2 during the early stage of BMP-induced osteogenesis. In addition, mRNA expression level of KDR, a receptor for vascular endothelial growth factor, was also increased in IBM/rhBMP-2. To investigate the in vivo release profile of rhBMP-2, iodine 125 (1211)-labeled BMP-2-incorporating IBM and FGM implants were inserted into the back subcutis of mice. More than 60% of the rhBMP-2 was released from the IBM/rhBMP-2 carrier within 1 day after implantation, whereas 50% of the rhBMP-2 was released from the FGM/rhBMP-2 10 days postimplantation. These results indicated that osteo- and chondrogenesis depends highly upon the geometry of the carrier and the in situ retention of rhBMP-2 during the early stage of rhBMP-2 induced bone formation. (C) 2004 Wiley Periodicals, Inc.
  • Xia Zhang Gui, Morimichi Mizuno, Kiyomi Tsuji, Masato Tamura
    Endocrine 24 1 15 - 24 2004年06月 [査読有り][通常論文]
     
    Matrix extracellular phosphoglycoprotein (MEPE)/ osteoblast/osteocyte factor 45 (OF45) is a recently isolated RGD-containing matrix protein that acts as the tumor-derived phosphaturic factor in oncogenic hypophosphatemic osteomalacia. It is also highly expressed by osteoblasts and osteocytes. We examined the regulation of MEPE/OF45 mRNA expression in osteoblastic cells derived from high-density cultures of primary rat bone marrow stromal cells incubated with dexamethasone, β-glycerophosphate, and ascorbic acid. The level of MEPE/OF45 mRNA in these cells was down-regulated by the addition of fibroblast growth factor 2 (FGF2) for 48 h. These effects were observed in a dose-dependent manner between 2 and 10 ng/mL. FGF2 also reduced the expression of osteocalcin mRNA in these cells. In contrast, bone sialoprotein mRNA expression was increased by FGF2, while α1(I) procollagen mRNA expression was not altered. Additionally, neither Runx2 and osterix mRNA expression nor cell proliferation were affected by the addition of FGF2 in these high-density cultures, indicating that regulation by FGF2 may not be dependent on these transcription factors or on the proliferation of cells. Experiments using actinomycin D indicated that FGF2 decreased the stability of the MEPE/OF45 mRNA. Moreover, inhibition of a specific mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK) by PD98059 blocked FGF2-regulated MEPE/OF45 expressions, indicating that this regulation requires the MAPK pathway. These results suggest that MEPE/OF45 gene is one of the targets of FGF2 and may play an important role during bone formation and calcification. © 2004 by Humana Press Inc. All rights of any nature whatsoever reserved.
  • GX Zhang, M Mizuno, K Tsuji, M Tamura
    ENDOCRINE 24 1 15 - 24 2004年06月 [査読有り][通常論文]
     
    Matrix extracellular phosphoglycoprotein (MEPE)/ osteoblast/osteocyte factor 45 (OF45) is a recently isolated RGD-containing matrix protein that acts as the tumor-derived phosphaturic factor in oncogenic hypophosphatemic osteomalacia. it is also highly expressed by osteoblasts and osteocytes. We examined the regulation of MEPE/OF45 mRNA expression in osteoblastic cells derived from high-density cultures of primary rat bone marrow stromal cells incubated with dexamethasone, beta-glycerophosphate, and ascorbic acid. The level of MEPE/OF45 mRNA in these cells was down-regulated by the addition of fibroblast growth factor 2 (FGF2) for 48 h. These effects were observed in a dose-dependent manner between 2 and 10 ng/mL. FGF2 also reduced the expression of osteocalcin mRNA in these cells. In contrast, bone sialoprotein mRNA expression was increased by FGF2, while all (1) procollagen mRNA expression was not altered. Additionally, neither Runx2 and osterix mRNA expression nor cell proliferation were affected by the addition of FGF2 in these high-density cultures, indicating that regulation by FGF2 may not be dependent on these transcription factors or on the proliferation of cells. Experiments using actinomycin D indicated that FGF2 decreased the stability of the MEPE/OF45 mRNA. Moreover, inhibition of a specific mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK) by PD98059 blocked FGF2-regulated MEPE/OF45 expressions, indicating that this regulation requires the MAPK pathway. These results suggest that MEPE/OF45 gene is one of the targets of FGF2 and may play an important role during bone formation and calcification.
  • K Tsuji, K Uno, GX Zhang, M Tamura
    JOURNAL OF BONE AND MINERAL METABOLISM 22 2 94 - 103 2004年03月 [査読有り][通常論文]
     
    We studied the mRNA expression of osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), tissue inhibitor of matrix metalloprotease (TIMP)-1 and -2, and matrix metalloprotease (MMP)-1 and -2 by human periodontal ligament (PDL) cells under intermittent tensile stress using a Flexercell Strain Unit. Analysis by reverse transcriptase-polymerase chain reaction showed that mechanical force upregulated OPG mRNA. We also demonstrated that the protein concentration of OPG in conditioned medium increased upon loading with tensile stress, as determined by enzyme-linked immunosorbent assay. TIMP-1 and -2 mRNA levels also increased, whereas levels of RANKL, MMP-1, and MMP-2 mRNA were barely affected. We further examined the effect of loading with tensile stress and addition of Salmonella abortus equi lipopolysaccharide (LPS) on the mRNA expression of PDL cells. The amount of OPG mRNA induced by mechanical strain was found to decrease with the addition of LPS to cultures. The induction of OPG mRNA expression by stretching was inhibited in the presence of indomethacin or genistein, whereas TIMP-1 mRNA expression induced by stretching was inhibited by the addition of cycloheximide, suggesting that tensile stress regulates cyclooxygenase activities, tyrosine phosphorylation, and de novo protein synthesis in PDL cells through the induction of OPG and TIMP-1 mRNA expression. These results provide evidence that the mechanical stimulus of stretching is responsible for the observed regulation of bone resorption and tissue degradation in PDL tissue.
  • Y Sato, M Kikuchi, N Ohata, M Tamura, Y Kuboki
    JOURNAL OF PERIODONTOLOGY 75 2 243 - 248 2004年02月 [査読有り][通常論文]
     
    Background: Therapies using biologically active, soluble factors such as growth factors or cytokines have been investigated for potential clinical use in regenerating lost periodontal tissue due to periodontitis. Basic fibroblast growth factor (bFGF, FGF-2) is a multifunctional growth factor that has a variety of effects including induction of proliferation and morphogenesis in a wide range of cells and tissues including periodontal ligament tissue. Methods: In this study, we examined the effects of bFGF on the regeneration of cementum and periodontal ligament in experimentally induced partial defects in a beagle dog model. bFGF in a Collagen gel was applied to the defects and root surfaces, and the teeth were replanted. Results: Eight weeks post-surgery, formation of cementum on denuded dentin was enhanced by application of 0.1, 1, or 5 mug of bFGF in a Collagen gel compared to Collagen gel containing vehicle. Histological analyses revealed that at 4 weeks post-surgery, random periodontal ligament fibers had bound to dentin, but were attached only to denuded dentin to which 0.1, 1, or 5 mug of bFGF in Collagen gel had been applied. At 8 weeks post-surgery, we observed the formation of dense fibers bound to alveolar bone and newly synthesized cementum in teeth treated with 1 mug of bFGF. Conclusion: These results suggest that basic fibroblast growth factor in a collagen gel is a suitable therapy for damaged periodontal ligament and could lead to readily achievable methods of treatment for periodontal disease.
  • K Wada, M Mizuno, T Komori, M Tamura
    JOURNAL OF CELLULAR PHYSIOLOGY 198 1 40 - 47 2004年01月 [査読有り][通常論文]
     
    In mammalian cells, several observations indicate not only that phosphate transport probably regulates local inorganic phosphate (Pi) concentration, but also that Pi affects normal cellular metabolism, which in turn regulates apoptosis and the process of mineralization. To elucidate how extracellular Pi regulates cellular functions of pre-osteoblastic cells, we investigated the expression of type III sodium (Na)-dependent Pi transporters in rat bone marrow stromal cells and ROB-C26 pre-osteoblastic cells. The mRNA expression level of gibbon ape leukemia virus receptor (Glvr)-2 was increased by the addition of Pi in rat bone marrow stromal cells, but not in ROB-C26 or normal rat kidney (NRK) cells. In contrast, the level of Glvr-1 mRNA was not altered by the addition of extracellular Pi in these cells. The induction of Glvr-2 mRNA by Pi was inhibited in the presence of cycloheximide (CHX). Moreover, mitogen-activated protein kinase (MEK) /extracellular-signal-regulated kinase (ERK) pathway inhibitors; U0126 (1.4-diamino-2, 3-dicyano-1, 4-bis [2-amino-phenylthio] butadiene) and PD98059 (2'-Amino-3'-methoxyflavone) inhibited inducible Glvr-2 mRNA expression, but p38 MEK inhibitor SB203580 [4-(4'-fluorophenyl)-2-(4'-methyl-sulfinylphenyl)-5-(4'pyridyl) imidazole] did not inhibit the induction of Glvr-2 mRNA expression, suggesting that extracellular Pi regulates de novo protein synthesis and MEK/ERK activity in rat bone marrow stromal cells, and through these, induction of Glvr-2 mRNA. Although Pi also induced osteopontin mRNA expression in rat bone marrow stromal cells but not in ROB-C26 and NRK cells, changes in cell viability with the addition of Pi were similar in both cell types. These data indicate that extracellular Pi regulates Glvr-2 mRNA expression, provide insights into possible mechanisms whereby Pi may regulate protein phosphorylation, and suggest a potential role for the Pi transporter in rat bone marrow stromal cells. (C) 2003 Wiley-Liss, Inc.
  • T Nakamura, M Yamamoto, M Tamura, Y Izumi
    JOURNAL OF PERIODONTAL RESEARCH 38 6 597 - 605 2003年12月 [査読有り][通常論文]
     
    Objectives: Growth/differentiation factor-5 (GDF-5), a member of the transforming growth factor-beta superfamily, shows a close structural relationship to bone morphogenetic proteins and plays crucial roles in skeletal, tendon, and ligament morphogenesis. The mRNA encoding GDF-5 is also expressed during odonto-genesis, especially in dental follicle tissue. While this suggests that GDF-5 participates in the formation of alveolar bone and the periodontal ligament, cementum, and dental root, the physiologic role of GDF-5 in these tissues in adulthood remains unclear. We therefore investigated GDF-5 effects upon cultures of human periodontal ligament (HPDL) cells. Material and methods: HPDL cells were obtained from healthy periodontal ligaments of individuals. Tetrazolium reduction assay was carried out for cell proliferation assay. Alkaline phosphatase (ALP) activity was estimated by measuring light absorbance at 405 nm. Reverse transcription-polymerase chain reaction (RT-PCR) and northern analysis were performed for gene expression in cultured HPDL cells. Sulfated glycosaminoglycan (sGAG) synthesis was evaluated by histochemical staining and a quantitative dye-binding method. Results: Expression of GDF-5 and its receptor was demonstrated in HPDL cells by RT-PCR. ALP activity in HPDL cells was significantly decreased by addition of rhGDF-5 at 10-1000 ng/ml (p < 0.05). Although northern analysis showed little change in gene expression for collagen alpha2(I) in rhGDF-5-stimulated HPDL cells, rhGDF-5 dose-dependently enhanced cell proliferation. This proliferative effect persisted for 16 d. Alcian blue staining and dye-binding assays indicated that sGAG synthesis was enhanced by rhGDF-5. Conclusion: rhGDF-5 may provide an environment fostering periodontal healing or regeneration by affecting extracellular matrix metabolism.
  • Masato Tamura, Chikako Nashimoto, Noriko Miyake, Yasushi Daikuhara, Kozo Ochi, Masayuki Nashimoto
    Nucleic Acids Research 31 15 4354 - 4360 2003年08月01日 [査読有り][通常論文]
     
    Mammalian tRNA 3′ processing endoribonuclease (3′-tRNase) can cleave any RNA at any site under the direction of small guide RNA (sgRNA) in vitro. sgRNAs can be as short as heptamers, which are much smaller than small interfering RNAs of ∼21 nt. Together with such flexibility in substrate recognition, the ubiquity and the constitutive expression of 3′-tRNase have suggested that this enzyme can be utilized for specific cleavage of cellular RNAs by introducing appropriate sgRNAs into living cells. Here we demonstrated that the expression of chloramphenicol acetyltransferase can be downregulated by an appropriate sgRNA which is introduced into Madin-Darby canine kidney epithelial cells as an expression plasmid or a synthetic 2′-O-methyl RNA. We also showed that 2′-O-methyl RNA heptamers can attack luciferase mRNAs with a high specificity and induce 3′-tRNase-mediated knock-down of the mRNAs in 293 cells. Furthermore, the MTT cell viability assay suggested that an RNA heptamer can downregulate the endogenous Bcl-2 mRNA in Sarcoma 180 cells. This novel sgRNA/3′-tRNase strategy for destroying specific cellular RNAs may be utilized for therapeutic applications.
  • K Kakimoto, M Machigashira, T Ohnishi, T Kajihara, Semba, I, T Setoguchi, M Tamura, Y Izumi, Y Daikuhara
    ARCHIVES OF ORAL BIOLOGY 47 9 655 - 663 2002年09月 [査読有り][通常論文]
     
    Hepatocyte growth factor (HGF), also known as scatter factor, is a broad-spectrum and multifunctional cytokine required for the development, growth and regeneration of various organs and tissues. The expression of HGF in human gingival fibroblasts is induced by inflammatory cytokines such as interleukin 1. Thus, although it is possible that content of HGF in gingival crevicular fluid (GCF) in periodontitis is increased, this has not so far been reported because the volume of GCF is too small to determine HGF by the available enzyme-linked immunosorbent assay (ELISA). A recently developed, highly sensitive ELISA for HGF, with a detection limit of I pg/ml sample, has now enabled HGF to be measured in GCF. The mean HGF content in GCF from sites with clinically healthy gingiva, defined by the absence of overt signs of gingival inflammation and a probing depth (PD) <3 mm, was 1.7 ng/ml, and that of periodontitis, defined by obvious alveolar bone loss detected by radiographic examination and a PD greater than or equal to3 mm, was 3.23 ng/ml. Although treating the periodontitis did not significantly decrease the HGF concentration despite significantly improved clinical scores such as PD and Gingival Index, the total amount of HGF in GCF did decrease significantly after treatment. HGF was expressed by gingival fibroblasts and inflammatory cells as determined by in situ hybridization. HGF-activator (HGFA), which converts inactive pro-HGF to active mature HGF, was detected in gingival epithelial cells by immunostaining. The expression of HGFA was also confirmed in gingival tissue by reverse transcription-polymerase chain reaction (RT-PCR). These findings indicate that HGF is synthesized and activated in gingiva that is clinically healthy or associated with periodontitis. (C) 2002 Elsevier Science Ltd. All rights reserved.
  • Y Sato, H Takita, N Ohata, M Tamura, Y Kuboki
    JOURNAL OF BIOCHEMISTRY 131 6 877 - 886 2002年06月 [査読有り][通常論文]
     
    We previously isolated pleiotrophin (PTN) from bovine bone as a protein and showed that it stimulated osteoblastic growth and differentiation. Further details of its function, however, have not been fully clarified. The aim of this paper was to elucidate the effects of PTN on bone morphogenetic protein (BMP)-induced ectopic osteogenesis. Recombinant human BMP (rhBMP)-2 (1.2 mug) was combined with a fibrous glass membrane, which had been established as an effective carrier. Various amounts of the purified bovine PTN (5, 10, 50, and 100 mug) or rhPTN (5 and 10 mug) were added to the rhBMP-2/carrier composites and implanted into rats subcutaneously as reported. It was found that the amount of bone induced in the system increased with the addition of 10 mug of either purified PTN or rhPTN. However, the amount of bone decreased with the addition of 50 or 100 mug of purified PTN dose-dependently, as judged by both alkaline phosphatase activity and calcium content in the retrieved implants. It was concluded that purified PTN or rhPTN, at ratios of concentration of 10-100 mug of PTN to 1.2 mug of rhBMP-2 in the carrier, regulated the ectopic bone-inducing activity of rhBMP-2.
  • M Nashimoto, C Nashimoto, M Tamura, RL Kaspar, K Ochi
    JOURNAL OF MOLECULAR BIOLOGY 312 5 975 - 984 2001年10月 [査読有り][通常論文]
     
    Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various precursor (pre)-tRNAs. We investigated what effect the autoantigen La has on 3' processing, since the La protein is known to bind to a 3'-terminal uridine tract of pre-tRNAs. We tested sixteen different pre-tRNA(Arg) substrates containing various 3' trailers with or without a 5 leader sequence for in vitro processing by pig 3 tRNase, and for gel-retardation in the presence or absence of human La protein. The R-TUUU series consists of four pre-tRNAs containing 6, 8, 11 and 15 nt 3' trailers ending with UUU and no 5' leader, while the R-TAGC series consists of the same four pre-tRNAs as R-TUUU except that the terminal sequence is AGC. The R-6LTUUU and R-6LTAGC series are derived from R-TUUU and R-TAGC, respectively, by adding a 6 nt 5' leader. La differentially inhibited their processing and bound to the pre-tRNAs; the 50 % inhibitory concentrations for the R-TUUU, R-TAGC, R-6LTUUU, and R-6LTAGC series were 82 to > 850, > 850, 2 to 292 and 573 to 785 nM, respectively, and the dissociation constants were 10 to 840, > 850, 3 to 203 and 155 to 520 nM, respectively. These results indicate that both the terminal sequence UUU and the 5' leader contribute to more severe inhibition of 3' processing via tighter interaction with La. With respect to the R-TUUU and R-6LTUUU series, on the whole, the La inhibition was enhanced as the 3' trailer lengths decreased. Taken together, our results suggest that the La protein sterically hinders 3' tRNase from binding a pre-tRNA molecule probably near the cleavage site. (C) 2001 Academic Press.
  • Tamura M, Daikuhara Y
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 45 1152 - 1157 6 Suppl 2000年04月 [査読有り][通常論文]
  • M Nashimoto, M Tamura, RL Kaspar
    BIOCHEMISTRY 38 37 12089 - 12096 1999年09月 [査読有り][通常論文]
     
    Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trailer after the discriminator nucleotide from precursor tRNA (pre-tRNA), To elucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase, A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleotide, and a 3' trailer was cleaved more efficiently than the full-length wild type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher than that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor stem, a discriminator nucleotide, and a 3' trailer, while the enzyme never cleaved a "microhelix" that is composed of a T loop, an acceptor stem, a discriminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have one to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently than R-ATM5, A helix ("minihelix Delta 1") one base pair smaller than minihelices was a good substrate, while small helices containing a continuous 10-base pair stem were poor substrates. The cleavage of these three small substrates occurred after the discriminator and one to three nucleotides downstream of the discriminator. From these results, we conclude that minimum substrates for efficient cleavage by mammalian 3' tRNase are minihelices or minihelices Delta 1, in which there seem to be no essential bases.
  • M Nashimoto, DR Wesemann, S Geary, M Tamura, RL Kaspar
    NUCLEIC ACIDS RESEARCH 27 13 2770 - 2776 1999年07月 [査読有り][通常論文]
     
    Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various pre-tRNAs without 5' leader nucleotides. To examine how 5' leader sequences affect 3' processing efficiency, we performed in vitro 3' processing reactions with purified pig 3' tRNase and pre-tRN(Arg)s containing a 13-nt 3' trailer and a 5' leader of various lengths. The 3' processing was slightly stimulated by 5' leaders containing up to 7 nt, whereas leaders of 9 nt or longer severely inhibited the reaction. Structure probing indicated that the 5' leader sequences had little effect on pre-tRNA folding, Similar results were obtained using pre-tRNA(Val)s containing a 5' leader of various lengths, We also investigated whether 3' tRNase can remove 3' trailers that are stably base-paired with 5' leaders to form an extended acceptor stem, Even such small 5' leaders as 3 and 6 nt, when base-paired with a 3' trailer, severely hindered removal of the 3' trailer by 3' tRNase.
  • M Nashimoto, M Tamura, RL Kaspar
    JOURNAL OF MOLECULAR BIOLOGY 287 4 727 - 740 1999年04月 [査読有り][通常論文]
     
    Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNA(Arg) variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12 - N and 13 - N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase; (C) 1999 Academic Press.
  • M Tamura, M Noda
    JOURNAL OF CELLULAR BIOCHEMISTRY 72 2 167 - 176 1999年02月 [査読有り][通常論文]
     
    Several members of the basic helix-loop-helix (bHLH) type of transcription factors have now been reported, and almost every member of this class has been implicated in transcriptional regulation in cell type determination and differentiation. Previously, we reported that dominant negative HLH proteins are involved in osteoblastic phenotype expression, such as osteocalcin, and hence differentiation (Tamura and Noda [1994] J. Cell Biol. 126:773-782). In this work, we used degenerate PCR cloning in order to identify cDNA clones encoding bHLH proteins expressed in osteoblastic osteosarcoma ROS17/2.8 cells. Sequence analyses of the 47 clones revealed that 11 clones encoded products with a characteristic motif of the bHLH transcription factor family. Of these clones, sequences in the amplified region of seven clones were homologous to the mouse twist, and three clones were homologous to the mouse twist-related HLH protein, Dermo-l. To confirm Dermo-l mRNA expression in osteoblastic cells, we performed reverse transcription polymerase chain reaction (RT-PCR) analysis using mRNA from ROS17/2.8 cells and MC3T3-E1 cells by Dermo-l specific primers and Northern blot analysis. These analyses demonstrated that Dermo-l mRNA was expressed in these osteoblast-like cell lines. Nucleotide sequence analysis of the partial rat Dermo-l cDNA cloned from ROS17/2.8 library revealed that it has the highest degree of homology with the mouse Dermo-l cDNA, and the partial amino acid sequence deduced from the obtained rat Dermo-l was identical with the corresponding region of the mouse Dermo-l amino acid sequence. To further examine the role of Dermo-l in the regulation of osteoblastic differentiation, we examined mRNA levels of Dermo-l and twist in C3H10T1/2 cells treated with recombinant human bone morphogenetic protein (rhBMP)-2. Using the RT-PCR method, the mRNA levels of Dermo-l and twist were found to be decreased by the treatment with rhBMP-2 in C3H10T1/2 cells. We also observed that the mRNA level of Dermo-l was decreased about fourfold by the treatment with rhBMP-2 in C3H10T1/2 cells by Northern blot analysis. Moreover, Dermo-1 mRNA was detected at lower levels in 21-day-old differentiated MC3T3-E1 cells compared with 3-day-old undifferentiated MC3T3-E1 cells. These results suggested that Dermo-l could be involved in the osteoblastic differentiation in a negative manner. J. Cell. Biochem. 72:167-176, 1999. (C) 1999 Wiley-Liss, Inc.
  • T Sakuta, M Tokuda, M Tamura, E Jimi, T Ikebe, T Koga, S Nagaoka, H Takada
    JOURNAL OF DENTAL RESEARCH 77 8 1597 - 1605 1998年08月 [査読有り][通常論文]
     
    In a previous study, we demonstrated that the amount of interleukin (IL)-8 mRNA expressed by human gingival fibroblasts stimulated with lipopolysaccharide (LPS) from Prevotella intermedia ATCC 25611 is increased by pre-treatment with beta or gamma interferon (IFN-beta or -gamma). Ln the present study, we identified the regulatory effects of these IFNs on IL-8 mRNA expression and IL-8 production by human gingival fibroblasts. Priming with IFN-alpha (alpha), -beta, or -gamma upregulated the IL-8 mRNA expression in response to P. intermedia LPS, whereas co-stimulation with these IFNs reduced the amount of mRNA expressed by the cells. The regulation of IL-8 mRNA expression induced by recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) or rHuIL-1 alpha was similar to that induced by LPS. The IL-8 mRNA expression in response to P, intermedia LPS was enhanced by IFN-gamma independently of ne novo protein synthesis, and was regulated, at least in part, at the transcriptional level. The IL-8 mRNA accumulation in response to P, intermedia LPS was inhibited by tosylphenyl-alanyl chloromethyl ketone, an inhibitor of NF-kappa B activation, although the NF-kappa B activation itself was not altered by IFN-gamma. These findings suggest that IFNs might be capable of both enhancing end inhibiting inflammatory responses in periodontal tissues through the dual regulation of IL-8 production by gingival fibroblasts in response to bacterial components and cytokines.
  • M Nashimoto, S Geary, M Tamura, R Kaspar
    NUCLEIC ACIDS RESEARCH 26 11 2565 - 2571 1998年06月 [査読有り][通常論文]
     
    Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize and cleave any target RNA that forms a precursor tRNA-like complex with another RNA. Various sets of RNA molecules were tested to identify the smallest RNA that can direct target RNA cleavage by 3' tRNase. A 3' half tRNA(Arg) was cleaved efficiently by 3' tRNase in the presence of small 5' half tRNA(Arg) variants, the D stem-loop region of which was partially deleted, Remarkably, 3' tRNase also cleaved the 3' half tRNA(Arg) in the presence of a 7 nt 5' tRNA(Arg) composed only of the acceptor stem region with a catalytic efficiency comparable with that of cleavage directed by an intact 5' half tRNA(Arg). The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNA(Arg) variants decreased. No heptamer-directed cleavage of a 3' half tRNA(Arg) without T stem base pairs was detected. A heptamer also directed cleavage of an HIV-1 RNA containing a stable hairpin structure. These findings suggest that in the presence of an RNA heptamer, 3' tRNase can discriminate and eliminate target RNAs that possess a stable hairpin adjacent to the heptamer binding sequence from a large complex RNA pool.
  • Y Liu, A Nifuji, M Tamura, JM Wozney, EN Olson, M Noda
    JOURNAL OF CELLULAR BIOCHEMISTRY 67 1 66 - 74 1997年10月 [査読有り][通常論文]
     
    We examined the mRNA expression of scleraxis, a non-myogenic helix-loop-helix type transcription factor in C2C12 myogenic cells. Scleraxis mRNA has been shown to be expressed in sclerotome and perichondrium of the embryos. We found that C2C12 cells express 1.2 kb scleraxis mRNA constitutively. Since BMP was reported to induce ectopic bone formation when implanted in muscle, we examined the effects of BMP on scleraxis expression. Scleraxis mRNA expression in C2C12 cells was suppressed by the treatment with BMP2. This suppression was observed at 200 ng/ml but not at the lower concentrations. BMP2 treatment suppressed scleraxis mRNA level within 24 h and lasted at least up to 48 h. Electrophoresis mobility shift assay showed that the proteins in the crude nuclear extracts prepared from C2C12 cells bound to an Scx-E-box sequence, CATGTG, which is preferentially recognized by scleraxis. This binding was competed out by 100-fold molar excess of cold Scx-E-box sequence but not by the one with mutations in the E-box. This band was supershifted by the addition of antiserum raised against scleraxis. BMP2 treatment suppressed the Scx-E binding activity in C2C12 cells. This suppression of the Sex E-box binding activity was in parallel to the BMP2 suppression of the transcriptional activity of the Scx-E-CAT reporter gene transfected into C2C12 cells. These data indicated that although the default pathway for C2C12 cells is to differentiate into muscle cells, these cells do express non-myogenic transcription factor, scleraxis, whose expression is suppressed by BMP2. (C) 1997 Wiley-Liss, Inc.
  • N Mataga, M Tamura, N Yanai, T Shinomura, K Kimata, M Obinata, M Noda
    JOURNAL OF BONE AND MINERAL RESEARCH 11 11 1646 - 1654 1996年11月 [査読有り][通常論文]
     
    We established a clonal chondrocyte-like cell line (TC6, TC stands for large (T) under bar immortalized chondrocyte-like cell line) derived from articular cartilage of transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen gene. TC6 cells exhibited spindle-like or polygonal morphology and grew well at 33 degrees C in alpha-minimal essential medium supplemented with 0.5% fetal bovine serum. After confluence, these cells formed nodules that were positive for staining with alcian blue, Northern blot analysis demonstrated that these cells expressed messenger RNAs (mRNA) of the genes encoding cartilage-specific proteins such as type II procollagen, link protein, and aggrecan, Furthermore, the expression of type II procollagen and link protein genes in TC6 cells was regulated by parathyroid hormone and basic fibroblast growth factor, suggesting the presence of the receptors for the hormone and cytokine. The expression of link protein mRNA in TC6 cells was regulated in a time-dependent manner and was enhanced in culture within a week and increased continuously up to 10-fold by the end of 4 weeks. Expression of mRNAs encoding type II procollagen and versican/PG-M also increased moderately during the culture period. TC6 cells expressed type I procollagen mRNA, however, its level declined along with time in culture in contrast to the enhancement of the genes encoding cartilage-specific molecules in these cells, Interestingly, alkaline phosphatase mRNA expression was barely detectable in the TC6 cells in their growing phase while it was enhanced dramatically more than 7-fold by day 14 in culture. These results indicate that the TC6 cells could serve as an excellent model for the studies on chondrocyte physiology.
  • M Tamura, S Nagaoka, M Kawagoe
    JOURNAL OF ENDODONTICS 22 5 240 - 243 1996年05月 [査読有り][通常論文]
     
    Interstitial collagenase (matrix metalloprotease-1) is a member of a family of matrix metalloproteases and is thought to play a role in degradation of the extracellular components, such as collagens in normal extracellular matrix remodeling and in many disease processes, We examined interstitial collagenase mRNA expression in human dental pulp fibroblast cultures by Northern blot analysis, These cells did not express interstitial collagenase mRNA in an unstimulated condition, Inflammatory cytokines, such as interleukin-1 alpha, induced interstitial collagenase mRNA expression in these cells, The interstitial collagenase mRNA levels began to increase after 2-h exposure, reaching a maximum after 8 h, then dropping to the unstimulated level at 48 h, These effects were observed in a dose-dependent manner in a dose range of 0.1 to 10 ng/ml, Transforming growth factor-beta reduced the levels of interstitial collagenase mRNA expression that were induced by interleukin-1 alpha. These observations suggest that interstitial collagenase mRNA expression in human dental pulp fibroblasts is regulated by the inflammatory cytokines and that interstitial collagenase may play a role in tissue degradation in inflamed dental pulp.
  • S Nagaoka, M Tokuda, T Sakuta, Y Taketoshi, M Tamura, H Takada, M Kawagoe
    JOURNAL OF ENDODONTICS 22 1 9 - 12 1996年01月 [査読有り][通常論文]
     
    Interleukin (IL)-8 mRNA expression was investigated in human dental pulp fibroblast cultures after stimulation with lipopolysaccharide (LPS) prepared from Prevotella intermedia and inflammatory cytokines. The expression of IL-8 mRNA and the release of IL-8 induced by P. intermedia LPS in pulpal fibroblast cultures were detected by Northern blot analysis and ELISA, respectively. The sufficient concentration of P. intermedia LPS on the IL-8 mRNA expression was 0.1 mu g/ml in pulpal fibroblast cultures. IL-8 mRNA levels began to increase after 2 h of exposure, reached a maximum at 4 to 8 h, and declined after 48 h, reaching the unstimulated level by 60 h. IL-8 production by the pulpal fibroblasts began to increase after 8 h of exposure upon stimulation with 10 mu g/ml of P. intermedia LPS. By contrast Salmonella LPS and synthetic lipid A did not increase IL-8 mRNA concentrations in pulpal fibroblast cultures. Recombinant human IL-1 alpha, beta, and tumor necrosis factor-alpha were capable of stimulating these cells to express IL-8 mRNA, but natural human interferon-beta, gamma, and recombinant human IL-6 were incapable in our assay. These results suggest that pulpal fibroblasts are immunoresponsive cells and can elaborate IL-8 upon stimulation with P. intermedia LPS.
  • HN MATSUMOTO, M TAMURA, DT DENHARDT, M OBINATA, M NODA
    ENDOCRINOLOGY 136 9 4084 - 4091 1995年09月 [査読有り][通常論文]
     
    We established bone marrow stromal cell lines that support tartrate-resistant acid phosphatase-positive multinucleated cell [TRAcP(+)MNC] formation by using transgenic mice harboring simian virus 40 large T antigen gene. The morphology of these TM cell lines (large T-immortalized marrow cells) was spindle-like at sparse cell density, whereas it became smaller and cuboidal at confluence. The TM cell lines showed diverse ranges of activity in supporting TRAcP(+)MNC formation when they were examined in the cocultures with spleen cells in the presence of 1 alpha,25-dihydroxyvitamin D-3. Among these cell lines, TM8 supported the TRAcP(+)MNC formation most efficiently (from 400-1500 cells/well) when cocultured with spleen cells. Another bone marrow-derived cell line, TM5, supported TRAcP(+)MNC formation in the coculture assay, whereas the efficiency was approximately one fifth that of TM8. Interestingly, TM8 cells also supported TRAcP(+)MNC formation even in the cocultures at low serum concentration (0.5% fetal bovine serum) with an efficiency yielding over 200 TRAcP(+)MNCs/well. TM8 cells expressed certain levels of macrophage colony-stimulating factor and stem cell factor messenger RNAs (mRNAs), but low levels of c-fms mRNA. Expression of c-Kit mRNA in TM5 and TM8 cells was undetectable. 1 alpha,25-Dihydroxyvitamin D-3 treatment enhanced the expression of osteopontin mRNA more than 10-fold in these cells, indicating the presence of the receptor for this steroid. These TRAcP(+)MNCs, which developed in the cocultures of the TM8 and spleen cells, formed pits when cultured on bone slices, indicating that they were capable of resorbing bone. The various levels of expression of these genes and the difference in the supporting activities for the TRAcP(+)MNC development in the diverse TM cell lines suggest the heterogeneity in the marrow cell populations in vivo regarding their activity in supporting osteoclastogenesis.
  • Tamura M, Ichikawa F, Guillerman RP, Deuel TF, Nodal M
    Endocrine 3 21 - 24 1 1995年01月 [査読有り][通常論文]
  • M TAMURA, M NODA
    JOURNAL OF CELL BIOLOGY 126 3 773 - 782 1994年08月 [査読有り][通常論文]
     
    To elucidate regulatory mechanism(s) underlying differentiation of osteoblasts, we examined involvement of helix-loop-helix (HLH)-type transcription factors in osteoblast-specific expression of a phenotypic marker gene which encodes osteocalcin, a major noncollagenous bone matrix protein, exclusively expressed in osteoblasts. Overexpression of a dominant negative HLH protein, Id-1, decreased the activity of the 1.1-kb osteocalcin gene promoter cotransfected into rat osteoblastic osteosarcoma ROS17/2.8 cells. Analysis of deletion mutants revealed that a 264-bp fragment of osteocalcin promoter (-198 to +66) was sufficient for the Id-1-dependent suppression. Furthermore, the activity of the same promoter fragment (-198 to +66) was enhanced when antisense Id-1 expression vector was cotransfected. This osteocalcin gene promoter region contains two sites of an E-box motif, a consensus binding site for HLH proteins, which we refer to as OCE1 (CACATG, at -102) and OCE2 (CAGCTG, at -149), respectively. Mutagenesis in OCE1 but not OCE2 led to greater than 50% reduction in transcriptional activity of the osteocalcin gene promoter. Electrophoresis mobility shift assay indicated that factors in nuclear extracts prepared from ROS17/2.8 cells bound to the 30-bp oligonucleotide probe containing the E-box motif of OCE1. This binding was competed out by OCE1 oligonucleotide but neither by OCmE1 oligonucleotide in which E-box motif was mutated nor by OCE2. The OCE1-binding activity in the nuclear extracts of ROS17/2.8 cells was reduced by 70% when bacterially expressed Id-1 protein was added to the reaction mixture, suggesting the involvement of HLH proteins in the DNA/protein complex formation. In contrast to the osteoblast-like cells, OCE1-binding activity in the nuclear extracts of C3H10T1/2 fibroblasts was very low. However, when these fibroblasts were treated with recombinant human bone morphogenetic protein-2 which induced expression of osteocalcin as well as other phenotypic markers of osteoblasts, OCE1-binding activity was increased similar to 40-fold, indicating that OCE1 would be involved in the tissue-specific expression of the osteocalcin gene. These findings indicated for the first time that osteoblast-specific gene transcription is regulated via the interaction between certain E-box binding transcription factor(s) in osteoblasts and the OCE1 sequence in the promoter region of the osteocalcin gene.
  • M TAMURA, H SHIMOKAWA, S SASAKI
    DNA SEQUENCE 5 1 63 - 66 1994年 [査読有り][通常論文]
     
    Interstitial collagenase (EC 3.4.24.7, MMP-1) is a member of a family of metalloproteinases and is thought to play a role in extracellular matrix remodeling. We have isolated and sequenced a cDNA for bovine interstitial collagenase from a periodontium fibroblast cDNA library. An insert of the cDNA we isolated was 2,025 bp containing an open reading frame which encodes a sequence of 469 amino acids. The identity at the amino acid level between bovine and human interstitial collagenase was 88%, between bovine and rabbit 85% and between bovine and porcine 87%. However, sequence similarity of mouse and rat interstitial collagenases to that of bovine was revealed 55%.
  • H TAKADA, Y KAWABATA, M TAMURA, K MATSUSHITA, H IGARASHI, H OHKUNI, Y TODOME, T UCHIYAMA, S KOTANI
    INFECTION AND IMMUNITY 61 12 5252 - 5260 1993年12月 [査読有り][通常論文]
     
    During an etiological study of Kawasaki disease (mucocutaneous lymph node syndrome [MCLS]), we found that dominant viridans streptococcal strains on tooth surfaces and in the throat of both MCLS patients and non-MCLS control children formed erythrogenic and biologically active, extracellular products. In this study, we demonstrated that erythrogenic culture supernatant concentrates of representative strains (two Streptococcus mitis and two Streptococcus oralis), when injected intravenously, induced serum tumor necrosis factor alpha, interleukin-6 (IL-6), and gamma interferon in muramyldipeptide- or Propionibacterium acnes-primed C3H/HeN mice. The concentrates also induced tumor necrosis factor alpha, IL-6, and thymocyte-activating factor (essentially IL-1) in murine peritoneal macrophage, human monocyte, and human whole-blood cultures. An erythrogenic, heat-labile extracellular protein fraction (F-1) that was concentrated from the culture supernatants of a representative S. mitis strain exhibited the above-mentioned cytokine-inducing activity. This partially purified F-1 fraction also induced thymocyte-activating factor and IL-6 in human umbilical vascular endothelial cell and gingival fibroblast cultures.
  • M TAMURA, N ARAKAKI, H TSUBOUCHI, H TAKADA, Y DAIKUHARA
    JOURNAL OF BIOLOGICAL CHEMISTRY 268 11 8140 - 8145 1993年04月 [査読有り][通常論文]
     
    Human hepatocyte growth factor (hHGF) was first purified from plasma of patients with fulminant hepatic failure (Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Miyazaki, H., Hashimoto, S., and Daikuhara, Y. (1988) J. Clin. Invest. 81, 414-419) and is now identified to be the same protein as the scatter factor (Weidner, K. M., Arakaki, N., Hartmann, G., Vandekerckhove, J., Weingart, S., Rieder, H., Fonatsch, C., Tsubouchi, H., Hishida, T., Daikuhara, Y., and Birchmeier, W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7001-7005) and tumor cytotoxic factor (Shima, N., Nakao, M., Ogaki, F., Tsuda, E., Murakami, A., and Higashio, K. (1991) Biochem. Biophys. Res. Commun. 180, 1151-1158), and it is known to be produced by fibroblasts in culture. Here we report that inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) stimulate production of hHGF from human embryonic lung fibroblasts, MRC-5, and human gingival fibroblasts, GF-5. Recombinant human IL-1alpha (rhIL-1alpha) and recombinant human TNF-alpha (rhTNF-alpha) increased hHGF levels in culture supernatants of MRC-5 and GF-5 cells dose-dependently as determined by an enzyme-linked immunosorbent assay for hHGF. The half-maximal stimulatory concentrations of rhIL-1alpha and rhTNF-alpha were about 1 ng/ml and 10 units/ml, respectively. rhIL-1beta showed almost the same effect as IL-1alpha on stimulation of production of immunoreactive hHGF from the two cell lines. However, rhIL-6 failed to show the stimulatory effect on hHGF production by the cells in the range of 2-200 units/ml. Human interferon-beta and -gamma also did not show the stimulatory activity. Stimulation of hHGF production was observed 6-12 h after addition of rhIL-1alpha or rhTNF-alpha and lasted at least 48 h, and the observed stimulation of hHGF production by cytokines was suppressed by addition of corresponding antiserum. hHGF mRNA levels of MRC-5 cells increased by addition of rhIL-1alpha and rhTNF-alpha in a dose-dependent manner as determined by Northern blot analysis using cDNA for hHGF as a probe. In addition, results from nuclear run-off transcription experiments showed that the two cytokines regulated increasing hHGF gene expression at transcriptional levels rather than a change in mRNA stability. These observations indicate that the inflammatory cytokines modulate the production and secretion of hHGF by fibroblasts and may play an important role for tissue repair and regeneration.
  • M TAMURA, M TOKUDA, S NAGAOKA, H TAKADA
    INFECTION AND IMMUNITY 60 11 4932 - 4937 1992年11月 [査読有り][通常論文]
     
    LiPopolysaccharides (LPS) prepared from Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis by hot phenol-water extraction induced interleukin-8 (IL-8) mRNA in normal human gingival fibroblast cultures, as demonstrated by Northern (RNA) blot analysis. IL-8 mRNA levels began to increase after a 2-h expoSUre, reached a maximum after 12 h, and then dropped to the unstimulated level at 48 h. IL-8 mRNA levels were also enhanced in a dose-dependent manner. By contrast, LPS specimens from various Salmonella species with S and R chemotypes and from bacterial and synthetic lipid A preparations did not increase IL-8 mRNA levels in fibroblasts. Although recombinant human IL-1alpha induced IL-8 mRNA expression in fibroblast cultures, an antiserum to recombinant human IL-lalpha did not decrease the IL-8 mRNA accumulation induced by B. intermedius LPS. Fibroblasts primed with natural human gamma interferon (IFN-gamma) expressed higher IL-8 mRNA levels upon stimulation with B. intermedius LPS, but not with Salmonella LPS, compared with nontreated cells. Natural human IFN-beta exhibited a similar priming effect on the fibroblasts, and antiserum to IFN-beta added to the cultures together with B. intermedius LPS decreased the IL-8 mRNA levels. Therefore, endogenous IFN-beta enhanced IL-8 mRNA production in response to B. intermedius LPS in fibroblasts.
  • Tamura M
    Kokubyo Gakkai zasshi. The Journal of the Stomatological Society, Japan 58 1 284 - 299 1 1991年03月 [査読有り][通常論文]
  • 田村 正人
    口腔病學會雜誌 58(1), 284-299, 1991 58 1 284 - 299 1991年 [査読有り][通常論文]

書籍

  • 口腔生化学 第6版
    田村 正人 (担当:分担執筆範囲:4章 結合組織と上皮組織の生化学)
    医歯薬出版 2018年09月
  • Interface Oral Health Science 2007
    Springer 2008年 (ISBN: 9784431766896)
  • Possible etiological role in Kawasaki disease of viridans group streptococci producting erythrogenitic toxin : Report on the project study of the research committee on Kawasaki disease, Japan Heart Foundation. (jointly worked)
    Proceeding of the Fourth International Symposium on Kawasaki Disease, American Heart Association. Futura Publising Co. , Inc. 1993年
  • Human Amelogenin Gene. Tooth Enamel V (共著)
    Florence Publishers 1989年
  • Human Amelogenin Gene. (jointly worked)
    Tooth Enamel V Florence Publishers, 1989年

その他活動・業績

  • 血管内皮細胞におけるmTORC1/mTORC2阻害剤の効果
    田村 潔美, 田村 正人, 小川 峰太郎 Journal of Oral Biosciences Supplement 2018 160 -160 2018年09月 [査読無し][通常論文]
  • 血管内皮細胞の伸張機能に着目した、血管形成の新たな指標の探索
    田村 潔美, 田村 正人, 小川 峰太郎 Journal of Oral Biosciences Supplement 2017 206 -206 2017年09月 [査読無し][通常論文]
  • PI3K-AktとmTORC1シグナルは血管内皮細胞の伸長機能を制御する
    田村 潔美, 田村 正人, 小川 峰太郎 Journal of Oral Biosciences Supplement 2016 228 -228 2016年09月 [査読無し][通常論文]
  • 向阪幸彦, 金谷聡介, 須藤瑞樹, 田村正人, 島内英俊, 根本英二 日本歯周病学会会誌(Web) 57 126 2015年08月17日 [査読無し][通常論文]
  • ハイドロキシアパタイトを使用した上顎洞底挙上術 コーンビームCTによる形態変化
    山本 英一, 和田 義行, 上林 毅, 吉谷 正純, 柴多 浩一, 林 理, 吉川 修平, 松崎 紘一, 藤沢 隆一, 田村 正人 日本口腔検査学会雑誌 7 (1) 47 -55 2015年03月 [査読無し][通常論文]
     
    目的:ラテラルウインドウテクニックならびにオステオトームテクニックで骨増生を行った上顎洞底部の経時的な形態変化をコーンビームCTで比較検討した。方法:カルシタイト、自家骨およびPRPを移植材として用い、画像の特徴的経時変化と、術直後と5ヵ月後の断面積および高さを比較した。結果:上顎洞底挙上後、直ちに上顎洞粘膜の肥厚がみられ、続いて移植材が外側に移動しがら体積を増し、移植材の中心部分に低吸収域がみられた。その後、体積を減じ、さらに外側方向に移動し安定した。この傾向は、ラテラルウインドウテクニック法に比べ、オステオトームテクニック法が顕著であった。結論:上顎洞底挙上術後、創部は静的に経過するのでは無く、経時的に変化しうるものであることが判明した。よって、骨増生を行う際には、増生部位、増生量、移植材等を、それぞれの特徴を踏まえた上で選択することが肝要であると思われた。(著者抄録)
  • 山本 英一, 南部 聡, 松﨑 紘一, 上林 毅, 和田 義行, 藤沢 隆一, 田村 正人 北海道歯学雑誌 35 (2) 164 -172 2015年03月 [査読有り][通常論文]
     
    上顎洞底挙上術にはオステオトームテクニックとラテラルウインドウテクニックがある.前者は短時間に低侵襲で施行できる長所を有するものの,盲目的に行う手技であるため熟練を要し,さらに十分な初期固定を得ることが困難で適応症例が限られる.一方で,後者は広範に支持骨を増生することが可能で幅広く適応できるものの,上顎洞側壁を開窓する際,非常に薄い上顎洞粘膜を損傷せず必要かつ十分な範囲で剥離するのは困難で手術侵襲も高い.特に,伸展性が乏しい上顎洞粘膜の剥離や,上顎洞底部に複雑な骨隔壁が存在している場合,剥離は著しく難易度を増す.一度上顎洞粘膜を破損すると修復が困難となり,予定の増生量を確保できず,結果インプラントの埋入そのものが不可能になることもある. 今回,上顎洞底部の骨が薄く従来ならオステオトームテクニックは適応とならずラテラルウインドウテクニックを選択する症例に対し,手術侵襲の軽減,移植材の節約,そして確実性・安全性向上のため,オステオトームテクニックそのものが可能となる最低限度の骨増生をラテラルウインドウテクニックで行い,その後,二期的にオステオトームテクニックで上顎洞底部を挙上し,咬合圧負担に耐えられるまでに骨を増生した. また,増生部の経時的な形態変化をコーンビームCTで観察した.同一FOVにて,近傍の皮質骨と,増生部とのROI値の比を比較した.移植後2か月に中心部に透過領域が発現したが,それ以降,骨化度は増加していった. 本手法は,ラテラルウインドウテクニック単独の際の欠点とされる広範囲にわたる上顎洞粘膜剥離,侵襲の大きさを軽減できる手技である.当然,偶発症の発症も少なくできる.また,移植材の必要量も節約できる.よって,今回われわれは,上顎洞底部の骨が薄く,かつ上顎洞粘膜の剥離が困難な場合の対処法の一つとして極めて有用な手技と考えたため報告する.For maxillary sinus elevation, there are two major procedures, the Osteotome Technique and Lateral Window Technique. Each technique has its own advantages and disadvantages. The Osteotome Technique can be performed in a shorter time span, but should be done blindly, and initial implant fixation to the bone is limited. It is not possible in every case. On the other hand, the Lateral Window Technique can be applied is broad cases, but it inevitably requires a wide sinus membrane elevation with limited laceration. Particular to this case, the ability to stretch the membrane and with the small wall located at the floor of the sinus, thin membrane elevation is extremely difficult. Once the membrane is lacerated, the implantation itself is difficult. In the present case, we employed a two-stage method for the bone augmentation that should be conventionally performed in a lateral window technique alone because of the thin sinus floor height. Our method consisted of the procedure of the Lateral Window technique which aims the bone augmentation for preparing a minimum condition at which the sequential Osteotome Technique itself could be done safely with a certain height of bone, and that of a secondary osteotome technique which attempts a firm fixation of the implant body for functional occlusion. A morphological evaluation of the graft by a serial cone-beam CT revealed that the ratio of the pixel value at the interest point to that of the control point in the same FOV had decreased two months after surgery, but became higher afterwards. We think this method is superior to the conventional one in terms of lower invasiveness, smaller volume requirement of the filling bone, and higher certainty. Adverse effects are also decreased. We regard this two-stage method as one of the options when handling cases with a low sinus floor in which ordinal implant placement can't be carried out.
  • 向阪幸彦, 根本英二, 須藤瑞樹, 金谷聡介, 田村正人, 島内英俊 日本歯科保存学会学術大会プログラムおよび講演抄録集(Web) 140th C5 (WEB ONLY) 2014年06月06日 [査読無し][通常論文]
  • TRUE gene silencing法による頭頸部癌細胞の増殖抑制(Growth inhibition of head and neck squamous carcinoma cells by sgRNA targeting cyclin D1 mRNA on TRUE gene silencing)
    飯塚 さとし, 折舘 伸彦, 梨本 正之, 福田 諭, 田村 正人 日本癌学会総会記事 72回 209 -209 2013年10月 [査読無し][通常論文]
  • 佐藤真理, 田村正人 北海道歯学雑誌 31 (2) 124-126 -126 2010年12月15日 [査読無し][通常論文]
  • Eiji Nemoto, Yohei Koshikawa, Sousuke Kanaya, Masahiro Tsuchiya, Masato Tamura, Martha J. Somerman, Hidetoshi Shimauchi INTERFACE ORAL HEALTH SCIENCE 2009 113 -+ 2010年 [査読有り][通常論文]
     
    Writ signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. Exposure to Wnt3a inhibited the expression of the osteocalcin (OCN) gene. This effect was accompanied by decreased gene expression of Runx2. Pretreatment with Dickkopf-1 attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of Runx2. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.
  • 大久保直登, 田村正人, 飯塚正, 加茂政晴, 客本斉子, 帖佐直幸, 高橋典子, 北川善政, 石崎明 J Oral Biosci 51 (Supplement) 103 2009年08月 [査読無し][通常論文]
  • 【ホルモンと骨粗鬆症UPDATE】カルシウム・リン調節系ホルモンと骨 骨・軟骨に対するPTH/PTHrP作用における形態学的知見
    網塚 憲生, 李 敏啓, 田村 正人, 織田 公光 Clinical Calcium 19 (7) 935 -943 2009年06月 [査読無し][通常論文]
     
    副甲状腺ホルモン(parathyroid hormone:PTH)および副甲状腺ホルモン関連ペプチド(parathyroid hormone-related peptide:PTHrP)は同一の受容体であるPTH/PTHrP受容体に作用することが知られており、PTHは生後の骨代謝に、PTHrPは胎生期の軟骨に対して主に影響を及ぼす。PTHは骨芽細胞前駆細胞の増殖促進に作用し、その後の細胞分化・機能には破骨細胞とのカップリングが必要になることが示唆されている。また、軟骨形成におけるPTHrPのC末端領域の作用も明らかにされつつある。ここでは、近年報告されたPTHおよびPTHrPの骨・軟骨における作用について総説する。(著者抄録)
  • 南部 聡, 山本 英一, 和田 義行, 磯村 治男, 神田 昌巳, 松崎 紘一, 田村 正人, 小川 優, 松沢 耕介 日本口腔検査学会雑誌 1 (1) 28 -30 2009年 [査読有り][通常論文]
  • 田村 正人 Dental medicine research 28 (3) 2008年11月30日 [査読無し][通常論文]
  • 佐藤 真理, 田村 正人, 八若 保孝 小児歯科学雑誌 46 (2) 317 -317 2008年 [査読無し][通常論文]
  • M. Sato, A. Nakashima, M. Nashimoto, A. Ishisaki, Y. Yawaka, M. Tamura JOURNAL OF BONE AND MINERAL RESEARCH 22 S92 -S92 2007年09月 [査読無し][通常論文]
  • 南部 聡, 藤沢 隆一, 田村 正人 北海道歯学雑誌 28 (1) 2 -9 2007年06月 [査読有り][通常論文]
  • 白井要, 石崎明, 賀来亨, 田村正人, 古市保志 日本歯周病学会学術大会プログラムおよび講演抄録集 50th 157 2007年04月30日 [査読無し][通常論文]
  • Naoto Okubo, Akira Ishisaki, Matsuo Yamamoto, Miho Ibi, Satoshi Wada, Yoshimasa Kitagawa, Masato Tamura JOURNAL OF PHARMACOLOGICAL SCIENCES 103 165P -165P 2007年 [査読無し][通常論文]
  • Aiko Nakashima, Masayuki Nashimoto, Masato Tamura Journal of Oral Biosciences 49 (1) 54 -64 2007年 [査読無し][通常論文]
     
    Recently, signaling by Wnt and its coreceptor, lipoprotein receptor-related protein (LRP) 5, has been suggested to be involved in the regulation of bone formation and bone mass. However, the mechanism by which Wnt signaling achieves this regulation is currently unknown. To assess the functional contribution of Wnt signaling to osteoblastic differentiation, we generated C2C12 cell lines that overexpress either Wnt3a or Wnt5a and treated these cell lines with bone morphogenetic protein (BMP)-2. Expression of bone matrix protein, matrix extracellular phosphoglycoprotein (MEPE), and matrix metalloproteinase (MMP)-13 was induced by BMP-2 in C2C12 cells overexpressing Wnt3a but not Wnt5a. Functional crosstalk between Wnt and BMP signaling during osteoblastic differentiation was also identified. We have now developed a unique system for downregulating gene expression that involves cutting a specific mRNA using the long form of tRNA 3' processing endoribonuclease (tRNase ZL) under the direction of a small guide RNA (sgRNA). Any potential off-target effects of sgRNA would be expected to be much less serious than those induced by siRNA because the sgRNA targets complementary RNA of only 7–14 nt, a much smaller length of RNA than that targeted by siRNA. We compared the relative efficiency of the sgRNA/tRNase ZL and RNAi methods using an exogenous luciferase gene target. We found that our new method may be more effective at suppressing gene expression. Here, we also show that tRNaseZL, together with sgRNA, can downregulate expression of the endogenous human genes Bcl-2 and GSK-3β by degradation of their mRNAs. These data suggest that the TRUE (tRNase ZL-utilizing efficacious) gene silencing technology has the potential to be used as a therapeutic agent in the treatment of several diseases. © 2007, Japanese Association for Oral Biology. All rights reserved.
  • Aiko Nakashima, Masayuki Nashimoto, Masato Tamura Journal of Oral Biosciences 49 (1) 54 -64 2007年 [査読無し][通常論文]
     
    Recently, signaling by Wnt and its coreceptor, lipoprotein receptor-related protein (LRP) 5, has been suggested to be involved in the regulation of bone formation and bone mass. However, the mechanism by which Wnt signaling achieves this regulation is currently unknown. To assess the functional contribution of Wnt signaling to osteoblastic differentiation, we generated C2C12 cell lines that overexpress either Wnt3a or Wnt5a and treated these cell lines with bone morphogenetic protein (BMP)-2. Expression of bone matrix protein, matrix extracellular phosphoglycoprotein (MEPE), and matrix metalloproteinase (MMP)-13 was induced by BMP-2 in C2C12 cells overexpressing Wnt3a but not Wnt5a. Functional crosstalk between Wnt and BMP signaling during osteoblastic differentiation was also identified. We have now developed a unique system for downregulating gene expression that involves cutting a specific mRNA using the long form of tRNA 3' processing endoribonuclease (tRNase ZL) under the direction of a small guide RNA (sgRNA). Any potential off-target effects of sgRNA would be expected to be much less serious than those induced by siRNA because the sgRNA targets complementary RNA of only 7–14 nt, a much smaller length of RNA than that targeted by siRNA. We compared the relative efficiency of the sgRNA/tRNase ZL and RNAi methods using an exogenous luciferase gene target. We found that our new method may be more effective at suppressing gene expression. Here, we also show that tRNaseZL, together with sgRNA, can downregulate expression of the endogenous human genes Bcl-2 and GSK-3β by degradation of their mRNAs. These data suggest that the TRUE (tRNase ZL-utilizing efficacious) gene silencing technology has the potential to be used as a therapeutic agent in the treatment of several diseases. © 2007, Japanese Association for Oral Biology. All rights reserved.
  • 山本英一, 藤沢隆一, 和田義行, 田村正人 北海道歯学雑誌 27 (2) 78 -85 2006年12月15日 [査読有り][通常論文]
  • M. Sato, A. Nakashima, T. Katagiri, A. Ishisaki, Y. Yawaka, M. Tamura JOURNAL OF BONE AND MINERAL RESEARCH 21 S253 -S253 2006年09月 [査読無し][通常論文]
  • 中島 愛子, 田村 正人 北海道歯学雑誌 27 (1) 2 -12 2006年06月15日 [査読無し][通常論文]
  • 浦本 洋一, 滝田 裕子, 久保木 芳徳, 田村 正人 北海道歯学雑誌 27 (1) 13 -23 2006年06月15日 [査読無し][通常論文]
  • M Ibi, A Ishisaki, M Yamamoto, S Wada, A Yokoyama, M Tamura JOURNAL OF PHARMACOLOGICAL SCIENCES 100 81P -81P 2006年 [査読無し][通常論文]
  • 鈴木 敏雄, 滝田 裕子, 田村 正人, 横山 敦郎 北海道歯学雑誌 26 (2) 134 -145 2005年12月15日 [査読無し][通常論文]
  • 百合草 健圭志, 石崎 明, 田村 正人, 北川 善政 北海道歯学雑誌 26 (2) 172 -181 2005年12月15日 [査読無し][通常論文]
  • 和田 悟史, 山本 松男, 石崎 明, 田村 正人, 飯田 順一郎 北海道歯学雑誌 26 (2) 246 -254 2005年12月 [査読無し][通常論文]
     
    クラウン系医用ミニブタの歯根膜由来細胞株の樹立を試みた.ミニブタの下顎より前臼歯を抜去し,歯根膜組織を採取し培養した.この初代培養細胞にヒトtelomere reverse transcriptase(TERT)遺伝子発現プラスミドを導入し,テロメラーゼ活性を高めて不死化させ,27種類の細胞株を得た.得られた樹立細胞株の中から,線維芽細胞様の形態を示し比較的増殖傾向の高い3種の細胞株を選択し,I型コラーゲンα1鎖,オステオポンチン,オステオカルシンならびにRunx2のmRNA発現をRT-PCRにより調べたところ,細胞株間でこれらの発現に違いが認められた.遺伝子発現が最も顕著であった細胞株は,MC3T3-E1細胞と同様に高いアルカリフォスファターゼ活性を示したが,細胞間基質石灰化能力は有していなかった
  • A Nakashima, T Katagiri, H Takaku, A Minagawa, M Nashimoto, M Tamura JOURNAL OF BONE AND MINERAL RESEARCH 20 (9) S138 -S138 2005年09月 [査読無し][通常論文]
  • 鈴木 豊典, 田村 正人, 戸塚 靖則 北海道歯学雑誌 25:179-190. (2) 179 -190 2004年12月15日 [査読無し][通常論文]
  • T Suzuki, H Takita, A Yokoyama, M Tamura JOURNAL OF BONE AND MINERAL RESEARCH 19 S111 -S111 2004年10月 [査読無し][通常論文]
  • GX Zhang, M Mizuno, K Tsuji, M Tamura JOURNAL OF BONE AND MINERAL RESEARCH 19 S250 -S251 2004年10月 [査読無し][通常論文]
  • A Nakashima, T Katagiri, M Tamura JOURNAL OF BONE AND MINERAL RESEARCH 19 S74 -S75 2004年10月 [査読無し][通常論文]
  • Tamura, M, Zhang, X A, Nakashima, A Connective Tissue 36 (3) 139 -147 2004年09月25日 [査読無し][通常論文]
     
    Vascularization represents a key mechanism involved in bone growth, cartilage production and bone formation, and is crucial during fracture repair in adults. Changes in this process might induce pathologic conditions such as osteoarthritis or ectopic hone formation. Recent studies identified several endogeneous positive or negative regulators involved in angiogenic processes such as matrix remodeling,cell migration and vessel maturation and these factors were identified as growth factors, matrix metalloproteases, cytokines and integrins. suggesting a molecular mechanisms that could explain how endothelial cells invade tissues and form new blood vessels. Most molecules that have angiogenic activity also enhance osteogenesis in animal experimental models. In this review, we briefly summarize the functions of angiogenic factors and their roles in bone formation and discuss possible future therapeutic uses.
  • 藤沢 隆一, 水野 守道, 田村 正人 Journal of oral biosciences 46 (5) 2004年09月01日 [査読無し][通常論文]
  • 中島愛子, 田村正人 北海道歯学雑誌 25 (2) 134 -137 2004年06月15日 [査読無し][通常論文]
  • 滝田 裕子, 田村 正人, 久保木 芳徳 日本歯周病学会会誌 46 2004年04月25日 [査読無し][通常論文]
  • K Wada, M Mizuno, T Komori, M Tamura JOURNAL OF BONE AND MINERAL RESEARCH 18 S142 -S142 2003年09月 [査読無し][通常論文]
  • 坂東 健二郎, 大西 智和, 田村 正人, 大工原 恭 歯科基礎医学会雑誌 45 (5) 2003年09月01日 [査読無し][通常論文]
  • 藤沢 隆一, 水野 守道, 田村 正人 歯科基礎医学会雑誌 45 (5) 2003年09月01日 [査読無し][通常論文]
  • R. Fujisawa, M. Mizuno, M. Tamura JOURNAL OF DENTAL RESEARCH 82 B76 -B76 2003年06月 [査読無し][通常論文]
  • 宮本 哲朗, 水野 守道, 田村 正人, 川浪 雅光 歯科基礎医学会雑誌 44 (6) 530 -540 2002年12月20日 [査読無し][通常論文]
  • 宮本 哲朗, 水野 守道, 田村 正人, 川浪 雅光 歯科基礎医学会雑誌 44 (5) 2002年09月20日 [査読無し][通常論文]
  • 柿元 協子, 町頭 三保, 大西 智和, 梶原 武弘, 仙波 伊知郎, 田村 正人, 大工原 恭 歯科基礎医学会雑誌 44 (5) 2002年09月20日 [査読無し][通常論文]
  • 滝田 裕子, 佐藤 靖子, 久保木 芳徳, 田村 正人 歯科基礎医学会雑誌 44 (5) 2002年09月20日 [査読無し][通常論文]
  • 藤沢 隆一, 水野 守道, 田村 正人 歯科基礎医学会雑誌 44 (5) 2002年09月20日 [査読無し][通常論文]
  • 辻 潔美, 張 桂霞, 田村 正人 歯科基礎医学会雑誌 44 (5) 2002年09月20日 [査読無し][通常論文]
  • 張 桂霞, 水野 守道, 辻 潔美, 滝田 裕子, 田村 正人 歯科基礎医学会雑誌 44 (5) 2002年09月20日 [査読無し][通常論文]
  • Y Sato, H Takita, N Ohata, M Tamura, Y Kuboki JOURNAL OF BONE AND MINERAL RESEARCH 17 S326 -S326 2002年09月 [査読無し][通常論文]
  • 宮本 哲朗, 水野 守道, 田村 正人, 川浪 雅光 歯科基礎医学会雑誌 44 (6) 530 -540 2002年 [査読無し][通常論文]
     
    高等動物の骨髄間質細胞のなかには, 多分化能を有する間葉系幹細胞が存在し, 培養下においても, 種々の条件によりさまざまな細胞に分化しうると考えられている. われわれは, これまでにラット骨髄間質細胞をI型コラーゲンとともに培養することにより, 骨芽細胞への分化が誘導されることを報告してきた. 本研究では, 細胞の三次元的配置と細胞分化との関連を明らかにする目的で, ラット骨髄間質細胞を通常の培養用ディシュにて単層培養した群 (2D群), I型コラーゲンをコートしたディシュにて培養した群 (COAT群) ならびにI型コラーゲンゲル内にて三次元培養を行った群 (3D群) にてそれぞれ培養し, 遺伝子発現を比較検討した. 3D群において, ほかの2群と比較して, アルカリホスファターゼ活性が高く, 骨シアロプロテインおよびオステオカルシンの遺伝子発現が誘導された. 本研究により, ラット骨髄間質細胞をI型コラーゲンゲル内で三次元的に培養することにより, 骨芽細胞の分化が誘導されることを明らかにした.
  • M Tamura, M Nashimoto, C Nashimoto, N Miyake, K Ochi, Y Daikuhara MOLECULAR BIOLOGY OF THE CELL 12 361A -361A 2001年11月 [査読無し][通常論文]
  • 柿元協子, 町頭三保, 大西智和, 梶原武弘, 仙波伊知郎, 瀬戸口尚志, 田村正人, 和泉雄一, 大工原恭 生化学 73 (8) 1011 2001年08月25日 [査読無し][通常論文]
  • 坂東 健二郎, 田村 正人, 大西 智和, 仙波 伊知郎, 大工原 恭 歯科基礎医学会雑誌 43 (5) 2001年08月20日 [査読無し][通常論文]
  • 田村 正人 鹿児島大学歯学部紀要 21 15 -25 2001年03月25日 [査読無し][通常論文]
     
    Elucidation of molecular mechanisms controlling differentiation and function of osteoblasts is one of the major subjects in bone biology. Differentiation of cells is controlled at the level of transcription by various classes of transcription factors that have been identified through biochemical and genetic means. I showed an expression of rat osteocalcin gene in osteoblasts was mediated by basic helix-loop-helix (bHLH) type of transcription factors. Our study presented evidence that the E-box sequence, OCE1,and transcription factors interacting with this motif, are involved in osteoblast-specific osteocalcin gene transcription. Furthermore, I performed degenerate PCR cloning in order to identify cDNA clones encoding bHLH proteins expressed in osteoblasts. We got clones which sequences in the amplified region were homologous to the mouse twist-related HLH protein, Dermo-1. Dermo-1 mRNA was expressed in osteoblast-1ike cell lines, and could be involved in the osteoblastic differentiation in a negative manner. Current many studies using gene deficient mice revealed how osteoblast differentiation and bone remodeling are controlled through many other type of transcription factors, Cbfal and homeobox protein family. Karsenty et al. showed that Cbfal is necessary for differentiation of mesenchymal progenitor cells to osteoblasts, and Cbfal controls bone formation by differentiated osteoblasts. Extracellular signals including hormones, growth factors, or cytokines as well as their intracellular mediators regulate osteoblast function. In this paper, I reviewed new understandings of the molecular control of osteoblast differentiation and function.
  • 坂東健二郎, 田村正人, 大西智和, 仙波伊知郎, 大工原恭 生化学 72 (8) 904 -904 2000年08月25日 [査読無し][通常論文]
  • 田村 正人, 大工原 恭 蛋白質核酸酵素 45 (6) 1152 -1157 2000年04月 [査読無し][通常論文]
  • 硬組織の形成機構とそれらを応用した再生医療への展開
    鹿児島県歯科医師会会報 559 8 -9 2000年 [査読無し][通常論文]
  • Structure and function of hepatocyte growth factor/scatter factor (HGF/SF) (共著)
    Current Topics in Biochemical Research 2, 149-159 2000年 [査読無し][通常論文]
  • DR Wesemann, S Geary, M Tamura, RL Kaspar, M Nashimoto FASEB JOURNAL 13 (7) A1412 -A1412 1999年04月 [査読無し][通常論文]
  • 骨の細胞におけるbasic Helix-loop-Helix型転写因子(共著)
    腎と骨代謝 12 273 -278 1999年 [査読無し][通常論文]
  • オステオネクチンの構造と生理的機能(共著)
    日骨代謝誌 6 34 -40 1998年 [査読無し][通常論文]
  • M Tamura, M Noda JOURNAL OF BONE AND MINERAL RESEARCH 12 F216 -F216 1997年08月 [査読無し][通常論文]
  • 二藤 彰, 山下 照仁, 田村 正人, 黒尾 誠, 鍋島 陽一, 野田 政樹 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 15 (2) 1997年06月20日 [査読無し][通常論文]
  • 力学的な刺激環境下における培養骨芽細胞の増殖能に関する研究(共著)
    5 87 -89 1997年 [査読無し][通常論文]
  • ノックアウトマウスを利用した硬組織研究
    アニテックス 9 194 -199 1997年 [査読無し][通常論文]
  • 歯をつくる遺伝子たち(共著)
    アニテックス 9 313 -318 1997年 [査読無し][通常論文]
  • 長岡 成孝, 川越 昌宜, 田村 正人, 徳田 雅行 日本臨床歯内療法学会雑誌 = The journal of Japan Endodontic Association 17 (2) 162 -166 1996年12月25日 [査読無し][通常論文]
  • 大井田 新一郎, 田村 正人, 飯村 忠浩, 鈴木 ミチ子, 下川 仁弥太 日本分子生物学会年会プログラム・講演要旨集 19 1996年08月01日 [査読無し][通常論文]
  • 俣賀 宣子, 田村 正人, 帯刀 益夫, 野田 政樹 日本骨代謝学会雑誌 = Japanese journal of bone metabolism 14 (2) 1996年06月01日 [査読無し][通常論文]
  • β-Adrnergic receptor kinase 1 mRNA expression in human dental pulp. (共著)
    J. Jpn Endodontics Assoc. 17 162 -166 1996年 [査読無し][通常論文]
  • Relationship between transferin receptor expression and proliferating activity of the tumor cells. (共著)
    Asian J. Oral Maxilofacial surgery 8,73-78 1996年 [査読無し][通常論文]
  • IL-8 mRNA expression in human pulpal fibroblasts. (共著) Proceedings of the Internatinal Conference on Dentin/Pulp Complex 1995 and the International Meeting on Clinical Topics of Dentin/Pulp Complex.
    Quintessence Publishing Co., Ltd. 308-309 1996年 [査読無し][通常論文]
  • S Nagaoka, M Tokuda, T Sakuta, H Takada, M Kawagoe, M Tamura DENTIN/PULP COMPLEX 308 -309 1996年 [査読無し][通常論文]
     
    We investigated the interleukin (IL)-8 messenger RNA (mRNA) expression in human dental pulp fibroblast cultures after stimulation with lipopolysaccharide (LPS) prepared from Prevotella intermedia, Salmonella abortus-equi, and synthetic Escherichia coli-type lipid A. The expression of IL-8 mRNA and the release of IL-g induced by P. intermedia LPS in pulpal fibroblast cultures were detected by Northern blot analysis and enzyme-linked immunosorbent assay, respectively. The sufficient concentration of P. intermedia LPS on the IL-8 mRNA expression was 0.1 mu g/mL in pulpal fibroblast cultures. IL-8 mRNA levels began to increase after 2 hr of exposure, reached a maximum at 4 to 8 hr, and declined after 48 hr. IL-g production by the pulpal fibroblasts began to increase after 8 hr of exposure upon stimulation with 10 mu g/mL, of P. intermedia LPS. By contrast Salmonella LPS, and synthetic lipid A, did not increase IL-8 mRNA concentrations in pulpal fibroblast cultures. These results suggest that pulpal fibroblasts are immunoresponsive cells and can elaborate IL-8 upon stimulation with P. intermedia LPS.
  • Relationship between transferin receptor expression and proliferating activity of the tumor cells. (jointly worked)
    Asian J. Oral Maxilofacial surgery 8 73 -78 1996年 [査読無し][通常論文]
  • β-Adrnergic receptor kinase 1 mRNA expression in human dental pulp. (jointly worked)
    J. Jpn Endodontics Assoc. 17 162 -166 1996年 [査読無し][通常論文]
  • Identification of a DNA sequence involved in osteoblast-specific gene expression via interaction with helix-loop-helix(HLH)-type transcriptional factors.
    The Thirs International forum on Calcified Tissue and Bone Metabolism(International Bone Forum 1994). Published by Chugai Pharmaceutical Co. , Ltd. 32 -35 1995年 [査読無し][通常論文]
  • 口腔ビリダンスレンサ球菌の菌体外産物によるサイトカインの誘導(共著)
    第三次川崎病原因究明対策委員会報告書 75 -83 1994年 [査読無し][通常論文]
  • M TAMURA, J WOZNEY, M NODA JOURNAL OF BONE AND MINERAL RESEARCH 8 S179 -S179 1993年08月 [査読無し][通常論文]
  • H NAKAYAMA, M TAMURA, M OBINATA, M NODA JOURNAL OF BONE AND MINERAL RESEARCH 8 S393 -S393 1993年08月 [査読無し][通常論文]
  • 二藤 彰, 大井田 新一郎, 田村 正人, 山口 聰, 武田 弘資, 岩城 博, 下川 仁弥太, 佐々木 哲, 天笠 光雄 日本口腔外科学会誌 39 (2) 103 -109 1993年 [査読無し][通常論文]
     
    A cDNA clone of murine bone morphogenetic protein (BMP) was obtained by polymerase chain reaction, using oligonucleotides as primers derived from human BMP2 and BMP4. Messenger RNAs were isolated from neonatal murine tissue and used for the synthesis of cDNA with reverse transcriptase.<BR>Specifically amplified cDNAs were cloned into bluescript plasmid and subjected to sequencing. One clone which partially encoded murine BMP2 was obtained. The nucleotide sequence, 554 bp in length, shared 88.6% identity with that of human BMP2 and the deduced amino acids including the mature, biologically active protein exhibited 96.2% homology. Within the C-terminal 114 amino acids of the protein, seven cysteine residues were completely conserved which is characteristic of TGF-β superfamily. Substitution of only three amino acids was found when compared with the corresponding human sequence. Isolation of the full length cDNA is now in progress.
  • Possible etiological role in Kawasaki disease of viridans group streptococci producting erythrogenitic toxin : Report on the project study of the research committee on Kawasaki disease, Japan Heart Foundation. (共著)
    Proceeding of the Fourth International Symposium on Kawasaki Disease, American Heart Association. Futura Publising Co. 60-76 1993年 [査読無し][通常論文]
  • 丸岡 豊, 大井田 新一郎, 飯村 忠浩, 二藤 彰, 田村 正人, 武田 弘資, 佐々木 哲, 榎本 昭二 歯科基礎医学会雑誌 35 (1) 64 -74 1993年 [査読無し][通常論文]
     
    骨形成タンパク質 (BMP) は骨誘導を起こさせる強力なサイトカインであり, 骨誘導能を持つ骨補填材料への利用が期待されている。その臨床応用へ向けての遺伝子工学的アプローチの第一歩として, 今回, 著者らはヒト骨肉腫由来細胞株MG63より作製したcDNA-mRNA混合物から, polymerase chain reaction (PCR) 法により, 2種のプライマーを用いてヒトBMP -2およびヒトBMP-4のcDNAの増幅, 単離を試みた。その結果ヒトBMP-2の成熟活性領域をコードすると思われる塩基配列をもつ4つのクローンの増幅に成功した。このうちの1つのcDNAについて, PCR法により3'末端に終止コドンを付加し, またプロ鎖をコードする領域に存在した塩基欠失部分の削除を行った。このようにしてつくられたcDNAには, なおいくつかの塩基の置換もあったが, 成熟活性領域の塩基をアミノ酸に翻訳すると天然のものと全く同じであり, hBMP-2の成熟活性部位の全領域をコードするcDNAを得ることができた。
  • M TAMURA, F ICHIKAWA, T DEUEL, M NODA JOURNAL OF BONE AND MINERAL RESEARCH 7 S164 -S164 1992年08月 [査読無し][通常論文]
  • Jpn. J. Oral Biol. 34, 612-615 1992年 [査読無し][通常論文]
  • アメロジェニン(エナメル蛋白)の構造と石灰化における役割(共著)
    日骨代謝誌 9 165 -172 1991年 [査読無し][通常論文]
  • オステオネクチンの構造と発現に関する研究(共著)
    日骨代謝誌 8 20 -24 1990年 [査読無し][通常論文]
  • オステオネクチンの発現に関する研究(共著)
    日骨代謝誌 7 21 -25 1989年 [査読無し][通常論文]

共同研究・競争的資金等の研究課題

  • 骨芽細胞が産生する新規SLRPを介した脂肪細胞分化・脂質代謝の制御機構の解明
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2024年03月 
    代表者 : 田村 正人, 田村 潔美
  • ”舌の老い”とは?エピジェネティクスの視点からオーラルフレイルに迫る
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2024年03月 
    代表者 : 浮田 万由美, 山口 泰彦, 田村 正人, 松下 健二
  • 骨細胞が産生・分泌する分子による脂肪組織・脂質代謝制御の分子機構
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 田村 正人, 田村 潔美
     
    本研究では,骨から脂肪組織へのシグナル分子としてのsclerostinが脂肪組織や肝臓などの脂質代謝系にどのような機構を介し影響を及ぼすのか,その詳細な分子機構を調べ,neuropeptide Yの脂質代謝に及ぼす効果と骨細胞が産生し分泌されるsclerostinの関係を明らかにする.また,骨組織と脂肪組織の異種組織間の恒常性維持のための機能的なクロストークに関して,骨細胞におけるsclerostinの産生調節と脂質代謝の相関について明らかにすることを目的としている.本研究により,個体レベルにおいてこれまで明らかではなかった骨と脂質代謝との関連性の解明を目指すものである.今年度は,脂質代謝の主要臓器である肝臓に着目する研究を行った.肝臓組織からmRNAを抽出し,acyl-CoA carboxylaseなどの脂肪酸合成系やHMG-CoA reductaseなどのコレステロール代謝酵素の酵素のmRNAをqRT-PCRを用いて測定したところ,発現量の変化が観察された.すなわち骨細胞の産生するsclerostinが脂肪組織の脂肪細胞のみならず,肝臓における脂肪酸合成やコレステロール代謝といった生体にとって重要な脂質代謝系に対しても影響を及ぼしえることが明らかとなった.また,SLRP (small leucine rich proteoglycan) class IIに分類されるオステオアドへリンの発現プラスミドを作成し,MLO-Y4細胞にトランスフェクトしオステオアドへリンを過剰発現させたところ,sclerostinの産生が亢進されるという結果が得られた.本年度得られた結果から,骨細胞において脂質代謝に影響を及ぼすsclerostinの産生がプロテオグリカンによって調節され,脂肪組織における脂質代謝に影響を及ぼしていることが示唆されるという新たな知見を得ることができた.
  • 骨と脂肪組織の臓器間クロストークに関わる分子とその調節機構の解明
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 田村 正人
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2012年04月 -2015年03月 
    代表者 : 折舘 伸彦, 梨本 正之, 田村 正人
     
    頭頸部癌は世界では年間50万人以上が罹患する.頭頸部癌の治療は手術,放射線治療,化学治療を複合的に組み合わせているが,予後に関しては、この20年で著しい改善は認められていない.その原因のひとつとして頭頸部癌の化学治療抵抗性が挙げられる,本研究の成果は治療成績向上が頭打ちの頭頸部癌治療において新治療法の開発へとつながる可能性を示している,化学療法併用放射線治療が頭頸部癌治療の重要なモダリティーとして確立した今日にあって,その治療効果を高める補助療法の整備は医療者サイドからも患者サイドからも強く望まれるものであり,低侵襲・低コスト・高効率の補助療法を供する可能性をも示している
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2010年04月 -2014年03月 
    代表者 : 田村 正人, 梨本 正之, 根本 英二, 佐藤 真理
     
    骨芽細胞において古典的Wntシグナルは,造血幹細胞の遊走を促進するCXCL12の産生を負に調節していた.この作用により破骨細胞分化も抑制された.骨細胞から造血幹細胞へのシグナル伝達分子として,古典的Wntシグナルを抑制する分子であるsclerostinを同定した.骨芽細胞においてNeuropeptide Yの受容体の発現がBMPによって調節されるとともに,オートクラインに制御されていた.本研究から,骨細胞・骨芽細胞とWntシグナルを介して,造血系と全身の異種組織間とに機能的なクロストークの存在が明らかになった.
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2011年 -2013年 
    代表者 : 田村 正人, 川野 光興
     
    ユビキチン―プロテアソーム系は骨形成や骨吸収の過程で種々の細胞内シグナル伝達分子の制御に関与している.本研究では,プロテオソーム阻害剤の一つであるBortezomibが筋分化を抑制し,Runx2のユビキチン―プロテアソームシステムによる分解を抑制することにより,骨芽細胞分化を誘導することを明らかにした.また,Bortezomibは骨芽細胞分化に関与するマイクロRNAの発現も誘導した.さらに,ユビキチンリガーゼ(E3)阻害剤やそのノックダウンによって骨芽細胞分化を促進する効果があることを見出した.
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2009年 -2011年 
    代表者 : 根本 英二, 島内 英俊, 田村 正人, 多田 浩之
     
    Wnt蛋白は、個体の発生や生体組織の機能維持に至る様々な局面で機能する分泌型の糖タンパク分子である。Wnt5aを代表とする非古典的経路のシグナルはどのような機能を有するかどうか未だ不明な点が多く残されている。本研究において、実験動物および培養細胞を用いて解析を行なったところ、未分化な細胞が骨を作る細胞に分化するにつれてWnt5aが強く発現してくることを明らかにした。さらに、その遺伝子の発現を消失させたところ、骨の成長が抑制されることを証明した。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2008年 -2010年 
    代表者 : 田村 正人
     
    昨年度までに同定した骨芽細胞分化に機能するmiRNAは,古典的WntシグナルもしくはBMP-2によって発現量が増加するmiRNAである.骨芽細胞の分化段階にけるこれらのmiRNAの発現量の変化を調べるために,MC3T3-E1細胞を4週間培養し,1週ごとに全RNAを回収した.このRNAを用いてStep-OneによるリアルタイムPCR法によって発現量の変動を定量化した.骨芽細胞の分化が進むにつれて,これらのmiRNAの発現量は増大した.またこれらのmiRNAをantagomiRもしくはsgRNAを用いてノックダウンした.リアルタイムPCR法でこれらのmiRNAがほぼ検出限界以下までノックダウンさせることに成功した.これらのノックダウンによって骨芽細胞分化に関連するmRNAの発現を調べたところ,オステオカルシンのmRNAは有意に減少しなかったが,アルカリフォスファターゼmRNAは著しく減少していた.以上の結果から,これらのmiRNAは,骨芽細胞分化に関連するmiRNAである可能性が高いと考えられた.そこで,これらのmiRNAの標的となるmRNAの候補を,データベースより検索した. さらに,TRUEジーンサイレンシング法を用いたmiRNA抑制性sgRNA発現プラスミドのライブラリーを未分化間葉系細胞培養系に導入し,アルカリフォスファターゼ活性染色やリアルタイムRT-PCR法を用いて,Runx2,osterix,I型コラーゲン,オステオカルシン,ALP,BSP,MEPE,MMP-13などのmRNAの発現量を測定した.これらより骨芽細胞分化と関連するmRNAが誘導されるsgRNAが見出された.これらのsgRNAの骨・軟骨形成に及ぼす影響及び内軟骨性骨形成に対する影響について検討を行った.sgRNA発現プラスミドもしくは2'-O-methyl化sgRNAの導入によって骨形成マーカーの増加が観察され,これらのsgRNAは骨形成活性を有している可能性が考えられた.
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2007年 -2009年 
    代表者 : 田村 正人, 梨本 正之, 根本 英二, 石崎 明
     
    骨形成と骨吸収の調節のしくみに関して,骨芽細胞と破骨細胞の分化と機能にWntシグナルと骨誘導因子(BMP)シグナルの2つのシグナル伝達経路のクロストークが重要な役割を果たしていることを明らかにした.骨吸収阻害活性を有するオステオプロテグリンがこれらの2つのシグナルの標的分子であり,その調節の機構として,それぞれのシグナル伝達経路のシグナル分子間の相互作用を介していることを見出した.また,ある種のmiRNAが骨芽細胞分化に関与しBMPはそのプロセシングを調節した.
  • 日本学術振興会:科学研究費助成事業 萌芽研究
    研究期間 : 2006年 -2007年 
    代表者 : 田村 正人
     
    標的GSK-3β mRNAについて,切断予定部位の塩基配列の解析から5'-half-tRNA型(sgGSKH)及び数種のlinear型(sgGSKL)のsgRNAを設計した.293細胞及びC2C12細胞のGSK-3βは2'-O-メチル修飾したsgGSKHもしくはsgGSKLの導入によって著しく減少した.これらの結果は,sgRNAとtRNaseZLによって標的であるGSK-3β mRNAを有効に切断しノックダウンしたと考えられた.また,培養系においてfluorescein標識したsgGSKLは,リポフェクタミンを使用せず単独で添加しても細胞内に有効に取り込まれることが明らかになった.これらのsgGSKLをマウス頭蓋骨に単独で注入したところ,2週後の骨は有意に増加した.また,このsgGSKLの有効性について,マウス大腿骨骨折モデルを用いて内軟骨性骨形成に対する影響を検討した.マウス大腿骨を骨折させ,仮骨形成する時期に2'-O-メチル修飾したsgGSKLをelectrophorationにより導入した.3日後,1週間後,2週間後における骨折治癒の経過を単純エックス線装置で観察したが,骨折治癒に対する影響は観察されなかった.hydrodynamics gene transfer法を用いて,この2'-O-メチルsgGSKL RNAを全身投与し全身の骨量の検討を行ったが,この投与法では有意な変化が認められなかった.sgRNAの投与方法,投与量等の検討が必要ではあるが,新たな骨・軟骨組織の形成誘導法としての可能性を示唆する成果が得られた.
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2006年 -2007年 
    代表者 : 石崎 明, 田村 正人
     
    歯根膜は顎骨と歯根との間に存在する線維性結合組織であり、線維芽細胞を含む多種類の細胞群で構成されている。しかし、これらの細胞が歯根膜中の各細胞の共通の前駆細胞から分化していくのか、それともそれぞれ細胞に分化することを運命づけられた前駆細胞が存在して、それぞれの成熟した細胞に分化していくのかはいまだ明らかになっていない。そこで、本研究ではクラウン系医用ミニブタ歯根膜から採取した歯根膜由来細胞にヒトTERT遺伝子発現プラスミド(pCl-neohTERT)を導入し、樹立された細胞株を用いてこれらの細胞の多分化能力とその分子メカニズムを明らかにすることを目的として研究を進めた。 hTERTを導入しクローン化されたミニブタ歯根膜由来線維芽細胞様細胞4種(TesPDL1-4)のうち、TesPDL3細胞が示す多分化能力の詳細について検討した。TesPDL3細胞は石灰化誘導培地にて培養すると、骨芽細胞あるいはセメント芽細胞様の細胞間基質石灰化能力をもつことが明らかとなった。また、TesPDL3細胞は血管内皮細胞マーカーであるCD31、VE-cadherin、及びvWF因子を発現すること、同時に血管平滑筋細胞マーカーであるh1-CalponinやSmoothelinを発現することが判明した。加えて、この細胞を3次元培養すると、線維芽細胞増殖因子(FGF)依存的に血管様チューブ構造物を形成することが判明した。 以上の結果から、歯根膜中には多分化能力をもつ細胞が存在すること、また、これらの細胞が骨芽細胞やセメント芽細胞の他に、血管内皮細胞や血管平滑筋細胞の起源となる可能性が示唆された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2005年 -2006年 
    代表者 : 瀬戸 かん一, 丸山 一雄, 根岸 洋一, 田村 正人, 滝澤 知子, 関谷 秀樹
     
    非ウイルスベクターによるWnt3a遣伝子を、超音波を用いて局所注射により導入し、その後通常通り仮骨延長を行うことを発案した。 低出力超音波パルス照射群、FGFに対しても仮骨延長を行い、比較検討を行う予定で、マウス骨折モデルの仮骨部分に対して、遺伝子導入した後の骨形成効果について、比較検討した。 その結果、仮骨周囲の筋組織にFGF遺伝子が導入されることによって、持続的にFGFタンパクを仮骨へ向かって放出することが確認された。そのFGFタンパクによって骨化は促進した。 Wnt3a導入群では、軟骨仮骨のVolumeは増大したが、骨化は促進されなかった。 超音波刺激群ではFGFシグナル、COX2-PGE2生成系の双方を活性化することで、骨芽細胞や線維芽細胞の増殖を促し、同時に超音波によって活性化されたCOX2-PGE2生成系は血管成長因子やMMPsの活性化を行い、骨のリモデリングが起こることを明らかにした(投稿中)。 結果的には、仮骨形成時にWnt3a導入、仮骨成熟時にFGF導入、リモデリング促進補助に超音波刺激を用いれば効率よくシャフトの太い延長骨が得られることが予想された。 さらに、WNT関連遺伝子やFGF等の骨形成遺伝子の非ウイルスベクター合成を行い、培養骨系細胞にelectrophoration(電気的導入)遣伝子導入法により導入し、発現効率を検討したものと同じベクターを用いてマイクロバブル封入を試みた。 electrophorationで得られた結果を基にすると、線維性仮骨への遺伝子導入効率はきわめて悪く、周囲骨膜・筋組織への骨形成遺伝子導入により遺伝子発現させることで、仮骨部分への持続的なタンパク形成と放出による効果を得られたことを利用して、マイクロバブルを筋組織・筋細胞に導入する実験系を確立させた。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2004年 -2006年 
    代表者 : 田村 正人, 梨本 正之, 石崎 明
     
    本研究では,未分化な筋芽細胞株であるC2C12細胞にcanonicalなWntとして知られるWnt3aならびにnon-canonicalなWntであるWnt5aを過剰発現させた細胞株を作製した.C2C12細胞では,骨基質タンパク質の一つであるMEPEを産生していないが,Wnt3a-C2C12細胞ではBMP-2によってMEPEやMMP-13の発現が大幅に誘導された.これらの結果から,骨芽細胞の分化においてWntとBMP-2の2つのシグナルが協調して特異的に骨基質タンパク質やMMPの発現を調節していることが明らかになった.次に,骨芽細胞分化においてWntとBMP-2の2つのシグナルの間でどのように調節しているのか検討した.canonical WntシグナルがBMP-2の誘導するId1 mRNAの発現を調節することを見出した.また,BMP-2はC2C12細胞の筋管形成を抑制するが,Wnt3a-C2C12細胞ではこのBMP-2の筋分化抑制作用が低下していた.すなわち,WntとBMP-2の2つのシグナルの間にクロストークがあることがわかった.さらに,Wnt3aによりOPG濃度は著しく増大した.一方,RANKLの発現はWnt/β-catenin及びBMP-2いずれによっても抑制された.活性型β-catenin応答領域を特定するために-253までの領域の4つのLef/Tcf1認識候補部位の変異レポーターコンストラクトを作製した.転写活性の検討ならびにクロマチンIP法を用いた結合因子の同定を行った.以上の研究からWnt/β-cateninシグナルの標的遺伝子としてOPGを同定し,転写活性化機構の詳細を明らかにした.骨芽細胞においてWnt/β-cateninシグナルとBMPシグナルが協調してOPGを介した破骨細胞の分化と機能を調節している可能性が示された.
  • 日本学術振興会:科学研究費助成事業 特定領域研究
    研究期間 : 2004年 -2004年 
    代表者 : 田村 正人, 梨本 正之
     
    tRNA前駆体の3'トレーラー切断酵素(tRNaseZ)と,短いガイドRNA(sgRNA)を用いて,細胞内mRNAを塩基配列特異的に切断するRNA切断法を,研究代表者らは見出し報告してきた.本研究は,この新たな遺伝子発現制御法を個体レベルに応用し,抗腫瘍性RNAヘプタマー分子(sgRNAの一種)を開発して有効ながん治療法として実用化を目指するものである.まず,tRNase Zが,新しいタイプのsgRNAであるhook RNAの存在下で4-7 base RNA cutterとして機能しうるという発見がなされ,これと同等の構造をとりうる一部のmiRNAがhook RNAとして機能しうる可能性が考えられた.哺乳動物細胞において,実際にtRNase ZがmRNAを切断していることをより厳密に証明するために,テトラサイクリン存在下でtRNaseZを過剰発現する293細胞を樹立した.northern blotならびにwestern blotにより,tRNase Zの発現増加が確認された.この細胞において,標的RNAと塩基対合しうるsgRNAによって,ルシフェラーゼ活性の抑制効果が増大するか検討したところ,その効果は有意に増加した.この結果は細胞内においてtRNase ZによってmRNAの切断が引き起こされていることを証明するものである. また,Sarcoma180細胞においてアポトーシスを増加させたRNAヘプタマーBclHepを種々の濃度でDoxorubicinと併用し,MM6乳癌細胞をC3H/Heマウスに移植した固形癌担癌マウスの腫瘍局部に導入し,腫瘍径ならびに腫瘍重量を測定した.本研究から,tRNase Zならびにhook RNAを使用する遺伝子発現抑制法をがん治療に応用しうると考えられた.
  • 日本学術振興会:科学研究費助成事業 萌芽研究
    研究期間 : 2003年 -2004年 
    代表者 : 田村 正人
     
    マウス筋芽細胞であるC2C12細胞を培養し本研究に用いた.この細胞にBMP2を加えて培養するとアルカリフォスファターゼ(ALP)活性が増加するとともに,骨芽細胞に特異的なオステオカルシンの発現が誘導され,骨芽細胞へ分化しうる.ノーザンプロット法を用い細胞接着因子のmRNA発現を検討したところ,E-カドヘリンならびにα5 integrinの発現が見られた.カドヘリンの細胞内裏打ちタンパク質であるβ-cateninはLef/Tcf1と結合してDNAに結合し転写調節の機能も有している.そこで,Lef/Tcf1応答領域レポーター(pTOP-FLASH)をC2C12細胞にトランスフェクトした.GSK-3によるリン酸化部位を欠失させた活性型β-cateninもしくはWnt3aを導入すると,pTOP-FLASHの活性は増大した.他方,non-canonical WntであるWnt-5aによっては,その活性は増加しなかった.Wnt3aと同時にBMP2を添加したところ,その活性は低下した.これらの結果から,C2C12細胞においてもWntのcanonical経路が存在しBMP2はこれに抑制的に機能することがわかった.RNAiを用いたノックダウンを行うために,α5 integrin等のインテグリンやGSK3βの一部のmRNA配列相当の合成ヌクレオチドをU6プロモーターベクターに挿入したコンストラクトを作製した.このプラスミドをC2C12細胞にトランスフェクトさせノックダウンを行い,機能解析を行ったところ,ALP活性,オステオカルシンmRNA発現の減少ならびにId1遺伝子のBMP2応答領域に対する反応性が低下し,骨芽細胞への分化が抑制された,これらの結果からインテグリンからのシグナルはβ-cateninを介して骨芽細胞分化を制御してい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 基盤研究(C)
    研究期間 : 2003年 -2003年 
    代表者 : 川浪 雅光, 山田 了, 鈴木 邦明, 田村 正人, 山本 松男, 吉江 弘正
     
    歯周組織再生には、再生にかかわる細胞同士の情報伝達システム全体を解析し、そのシステムを律速するプロセス全体を活性化することが必要となる。最近の遺伝子やタンパクの解析技術の発展により、情報伝達蛋白質の解明が技術的にも容易になってきている。本研究は、現時点におけるわが国の内外の歯周組織再生にかかわる細胞の情報伝達システムに関しての研究の動向を調査し、研究計画申請のための基礎資料を収集することであった。そこで、研究分担者を以下の専門領域にわけて、それぞれが、歯周組織再生にかかわる細胞や細胞間伝達物質について以下の分担テーマ毎に現時点における最新の内外の研究報告について調査研究した。 1、歯周組織再生における歯肉・歯根膜・歯槽骨由来細胞の果たす役割(川浪) 2、歯肉粘膜結合組織由来細胞と細胞間伝達物質 (1)庄司:上皮と結合組織間の情報伝達 (2)田村:免疫担当細胞、血管内皮細胞の情報伝達 3、歯根膜由来細胞と細胞間伝達物質 (1)鈴木:イオン輸送ポンプによる情報伝達 (2)山田:骨形成蛋白による情報伝達 (3)吉江:PRP(血小板多血漿)による情報伝達 (4)池澤:線維芽細胞増殖因子による情報伝達 (5)野口:セメント芽細胞における情報伝達 (6)西村:走化性因子による情報伝達 4、歯槽骨由来細胞と細胞間伝達物質 (1)小林:成長因子による情報伝達 (2)向後:MIFによる情報伝達 (3)菅谷:骨髄幹細胞の情報伝達 (4)山本:機能性スキャホールドによる情報伝達 それぞれの調査研究結果の情報を基に、「細胞間情報伝達システムの解明による歯周組織再生」基盤研究(A)の研究計画の立案、および申請を行った。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2001年 -2002年 
    代表者 : 田村 正人
     
    骨芽細胞に存在するRNA結合タンパク質による翻訳制御が,骨芽細胞の分化に対してどのように機能するかについて明らかにする目的で本研究を行った.分化した骨芽細胞としてマウスMC3T3細胞、未分化間葉細胞としてマウスC3H10T1/2細胞を培養し,それらの細胞からtotal RNAを抽出した.また,種々のRNA binding domain (RBD)で保存されている塩基配列を利用して十数個のオリゴヌクレオチドプライマーを合成した.それらプライマーならびに骨芽細胞から抽出したRNAを用いてdegenerative RT-PCR法を用いDNAを増幅し,複数のcDNA断片が得られた.得られたcDNA断片をpGEM-Tベクターに挿入し,dideoxy法によって塩基配列を決定した.得られたcDNAは,既知のRNA結合タンパク質と同一であるもの(nucleolin)や構造が類似しているが未だ報告のない新規のRNA結合タンパク質(RNaseZ)と考えられるクローンがあった.得られたRNA結合タンパク質のcDNAをInteinタグを有する発現ベクターに組み込み,融合タンパクを大腸菌に発現させInteinのタンパク質スプライシング活性を利用して精製を行った.この組換えタンパクを抗原としてウサギを免疫し,ポリクローナル抗体を作製した.マウスオステオカルシン遺伝子のさまざまな長さの5'非翻訳領域ならびに3'非翻訳領域をプローブとしたゲルシフトアッセイを行ない,RNA/タンパク複合体に対する抗体の効果を検討した.抗nucleolin抗体によって,スーパーシフトが観察されRNAにこのタンパク質が相互作用していることが明らかになった.さらに,得られたRNA結合タンパク質cDNAを哺乳動物発現用ベクターに挿入させたプラスミドを,C3H10T1/2,W-20等の未分化間葉系細胞にトランスフェクトした.Stableに発現する細胞株を選択し,これらの細胞についてアルカリフォスファターゼ,オステオポンチン等の骨芽細胞の形質を調べたところ,オステオポンチンの発現上昇が見られた.以上の結果は,RNA結合タンパク質の骨芽細胞の分化との相関性に関する分子基盤を明確に見出したものであるといえる.
  • 文部科学省:科学研究費補助金(基盤研究(A))
    研究期間 : 2000年 -2002年 
    代表者 : 大工原 恭, 梶原 武弘, 町頭 三保, 田村 正人, 杉山 明子, 柿元 協子, 梶原 武弘
     
    ペリオペーパーを用いて歯肉溝滲出液を採取し、このペリオペーパーからHGFを抽出して、研究代表者らが、平成11年度に新たに開発した高感度HGF ELISA(測定感度:2pg/ml)により測定した。その結果、健常部位歯肉からの歯肉溝滲出液中HGF濃度平均値は、1.70±0.73 (SD) ng/ml (n=9)、また、歯周炎部位歯肉からのそれは3.23±1.01 (SD) pg/ml (n=13)で、歯周炎では有意(P<0.001)に上昇していることが明らかとなった。これら歯肉溝滲出液中HGF濃度は、健常者血清中HGF濃度の約10倍である。従って、歯肉組織で実際にHGFが産生されていることが明らかとなった。さらに、歯周炎部位の歯肉溝滲出液中HGF濃度は、歯周炎の程度(ポケットの深さ、骨吸収レベル、歯周炎指数)と強い正の相関が認められ、歯周炎組織ではHGFの産生が促進されていることが示唆された。また、歯周炎歯肉を用いて免疫組織化学により産生細胞を検索したところ、歯肉組織の繊維芽細胞および炎症性の浸潤細胞がpositiveに染色され、さらに歯肉組織をRT-PCRで解析したところ、HGF mRNAが検出された。これにより、歯肉組織でHGFが合成・分泌されていることが確認された。次に、歯肉組織にHGFAが存在するか否かを免疫組織化学的に検討したところ、歯肉上皮がpositiveに染色され...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2000年 -2002年 
    代表者 : 長岡 成孝, 田村 正人, 徳田 雅行
     
    1.ノーザンブロットによる解析歯根膜由来線維芽細胞ならびに胎児歯小嚢由来細胞を10%ウシ胎児血清(FBS)存在下で培養し,コンフルエントに達したのち,1%FBSとし24時問培養を続けた.先に作製した種々のエナメルタンパクをこの培養系に添加し,さらに培養を続けた.コントロールとしては,「エムドゲイン」を添加したものを用いた.経時的に培養上清を採取し,一定時間後に細胞を集め,全RNAを抽出した.このRNAを用いて,ノーザンブロット解析を行った.オステオカルシンなどの基質タンパクならびにDermo-1, OSE-1等の転写因子およびアルカリフォスファターゼのcDNAをプローブとして用い,mRNA発現の変動を調べた.mRNA発現誘導を引き起こすタンパクについて,種々の欠失型タンパクを用い,その活性ドメインを同定した.今後これらの同定されたドメインについてPROSITE, Gen Bank等タンパク質のデータベースで,構造について検討を行う予定である.2.変異型高活性エナメルタンパク断片の作製これまでの実験で同定された生物活性を有するドメインについて,Kunel法を用いて塩基の置換,欠失,挿入等を行い発現コンストラクトを作製した.これらのプラスミドを用い,上記3の方法により変異型タンパクを発現させ,活性については,上記4の方法で検討し,高活性のエナメルタンパクを得るとともに,タンパクの...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1999年 -2000年 
    代表者 : 田村 正人, 大西 智和, 小窪 明子
     
    骨芽細胞において特異的に産生されるタンパク質であるオステオカルシン遺伝子の発現調節機構を明らかにすることは、骨芽細胞分化の解明のために非常に重要である。さきに研究代表者は、オステオカルシン遺伝子発現には転写開始点上流域に、骨芽細胞特異的な転写調節因子が結合し転写を活性化することを明らかにしてきた。しかし、骨芽細胞分化には転写段階のみならず転写後の翻訳段階での制御についても考慮する必要がある。本研究では、骨芽細胞に存在するRNA結合タンパク質による翻訳段階における制御が、骨芽細胞の分化に対してどのように機能するかについて明らかにした。オステオカルシンmRNAの骨誘導タンパク質(BMP)による翻訳制御調節を検討するために、シュークロースグラジィエント密度勾配遠心法によるポリゾーム解析を行った。骨芽細胞分化に伴ってリボゾームの多く結合したオステオカルシンmRNAが増加していた。次に、マウスオステオカルシン遺伝子をクローニングし、翻訳制御領域を同定するためさまざまな長さの5'非翻訳領域ならびに3'非翻訳領域を挿入したレポータープラスミドの作製を行った。カチオン性リポソームを用いた遺伝子導入法によってこれらのプラスミドを未分化間葉系細胞に導入し、組み換えBMP-2の添加による骨芽細胞分化によってレポーターの活性上昇が認められる領域を同定した。同定した領域の合成RNAをプローブとしたゲル...
  • 文部科学省:科学研究費補助金(基盤研究(A))
    研究期間 : 1997年 -2000年 
    代表者 : 野田 政樹, 田村 正人, 川口 奈奈子, 二藤 彰, 山下 照仁
     
    本基盤研究においては、骨粗鬆症の病態生理学の基礎となる骨芽細胞・破骨細胞の研究を展開した.その結果、本研究による現在までの骨吸収に関する研究領域の成果としては,カルシウム向性制御因子である副甲状腺ホルモン、ビタミンDやTGFβを含むホルモンおよびサイトカインによって制御されるオステオポンチンのノックアウトマウスを作成し(Journal of Bone and Mineral Research,1998)、このマウスにおいては、閉経後骨粗鬆症モデルを作成すると、骨吸収が明らかに抑制されることから、オステオポンチンが骨粗鬆症の成立の上で重要な促進的役割を持つ分子であることを発見した(Proceedings of the National Academy of Sciences U.S.A.,1999).更に、骨形成領域の研究の成果としては,骨芽細胞機能の制御の分子機構を解析し、新しいBMP制御分子TOBが、特異的に成体の動物の加齢後期において骨の形成の制御に関わることをin vivoで明らかにし(Cell,2000)、Smadの下流におけるCbfaの発現制御の機構(Journal of Biological Chemistry(JBC)1998)、ビタミンDによる抑制性の転写制御因子Idの発現阻害のメカニズム(JBC,1997a)、helix-loop-helix型転写因子Scl...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1997年 -1997年 
    代表者 : 田村 正人
  • 文部科学省:科学研究費補助金(国際学術研究)
    研究期間 : 1996年 -1997年 
    代表者 : 野田 政樹, Denhardt Dav, 川口 奈奈子, 田村 正人, 二藤 彰
     
    本研究においては、マウスオステオポンチン遺伝子のノックアウトマウスの作成を行い、その機能の解析を以下の如く遂行した。マウスオステオポンチンについては、その主要なエクソンを含むDNA領域を対象としてホモロガスリコンビネーションを行った。そのホモロガスリコンビネーションによって得られたES細胞をマウスの胚に導入し、この胚から得られたマウスを更に交配することにより、オステオポンチン遺伝子の発現しないホモのマイナス/マイナスの動物を作成した。当初オステオポンチンの遺伝子を欠損した細胞は十分な生殖細胞への遺伝子のトランスミッションが得られなかったが、その後の実験の追加により、生殖細胞へ至るものが得られ、これにより、オステオポンチン遺伝子を2つの遺伝子座共に持たないマウスが作成された。このマウスの脛骨ならびに大腿骨など長管骨より骨髄細胞を採取し、これを頭蓋より採取した骨芽細胞と共に、ビタミンD3の存在下に共存培養させ、これによって7日の後に形成される破骨細胞を酒石酸耐性酸性フォスファターゼ(TRAP)を指標として、破骨細胞と同定して、この形成効率を検討した。また、脾臓より未分化細胞を得て、同じくノックアウト動物より得られた骨芽細胞と共に共存培養をし、TRAPを指標とした破骨細胞様細胞の形成能を検討した。これらの共存培養の結果、ノックアウト動物より得られた脾臓細胞や骨髄細胞を用いた場合には...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 1996年 -1997年 
    代表者 : 大井田 新一郎, 田村 正人
     
    骨形成タンパク質(BMP)はTGF-βスーパーファミリーに属するタンパク質で、骨芽細胞の分化に強く関与していることが示唆されている。本研究ではBMPレセプターをクローニングすると共に、それらのレセプターを持っている、骨芽細胞様株細胞を用い、細胞内シグナル伝達の機構解析を行った。実験に用いたBMPは主にBMP-7(OP-1)で細胞はMC3T3-E1およびST-2細胞で方法としはDifferential Display法を用いBMPによって発現する遺伝子の解析をおこなった。主な結果は以下のごとくである。1) BMP-7でアルカリホスファターゼ活性が増加した。2) アルカリホスファターゼの活性上昇に続いてオステオカルシン遺伝子の発現を確認した。3) Differential Display法でBMP-7で16時間後に誘導される77個の遺伝子を単離した。4) 単離した遺伝子の多くは既知のものであった。5) BMPにより誘導される遺伝子、発現が抑制される遺伝子についてさらに詳細に検討した。これらの他に、TGF-βスーパーファミリーの細胞内シグナル伝達に関与しているsmad遺伝子の発現についても検討した。
  • 文部科学省:科学研究費補助金(試験研究(B), 基盤研究(A))
    研究期間 : 1995年 -1997年 
    代表者 : 田村 正人, 二藤 彰, 二藤 彰, 野田 政樹, 田村 正人
     
    本研究においては、胎生期のマウスより歯胚を取り出し、上皮と間葉系を分離の後、更に共存させて複合培養を行った。実験には13日及び16日の初期胚のマウスの歯胚を用いた。培養再構成組織より、total RNAを抽出し、polyA・RNAを調整後、サブトラクション法によって、上皮から間葉ならびに間葉から上皮へのシグナルを伝達する分子の候補となるcDNAを選択し、陽性のクローンを得ることができた。これらの歯胚の組織においては、肝細胞増殖因子が存在していることがこの研究の過程で明らかになった。Differential Display法を用いたサブストラクション法によって得られたcDNAについては、配列を決定し、データベース検索を行った。また、歯胚のエナメル形成に重要な機能を果たすと考えられているアメロゲニンの遺伝子の全長ならびに5'上流領域の6Kbのクローニングとその全塩基配列の決定を行った。更にマウスの胎児生腫瘍細胞である多能性幹細胞様細胞株C1を用い、その遺伝子ライブラリーを作成し、このライブラリーよりスクリーニングを行って、noggin遺伝子のクローニングを行った。塩素配列の決定により、nogginの全長のうちC末端側約110アミノ酸の部分をコードする部分のフラグメントが取れたことを確認した。このcDNAのフラグメントを用いてin situ hybridizationを行い、nog...
  • 文部科学省:科学研究費補助金(試験研究(B), 基盤研究(A))
    研究期間 : 1995年 -1997年 
    代表者 : 野田 政樹, 二藤 彰, 田村 正人
     
    本研究においては、ラットのオステオカルシンの上流約1キロベースのプロモーター領域をクローニングし、これをクロラムフェニコールアセチルトランスセラーゼ(CAT)に繋いだコンストラクトを用い、このコンストラクトによる転写活性の制御の確認を行った。オステオカルシンと共に、骨芽細胞の主要な産生蛋白であり、骨基質の90%を担うI型コラーゲンのプロモーターをクローニングし、これにルシフェラーゼに結合したリポーターを作成し、更にこれをマウスに導入したトランスジェニックマウスについてその発現の検討を行った。このトランスジェニックマウスでは、I型コラーゲンの発現に従って生体内でベータガラクトシダーゼの発現が起こり、これをベータガラクトシダーゼ活性によるモニタリングによって解析し、in vivoでの発現活性を確認した。最も長いI型コラーゲンのプロモーターを持つ3200ベースのベータガラクトシダーゼコントラクトについて、このマウスにおいてリポーターの発現が靱帯、骨を中心として、観察されることを検討した。これらのマウスに対し、骨粗鬆症の治療上の有効と思われる薬剤として、TGFβやBMPなど、サイトカインを動物に投与し、その投与部分における3200ベースのコンストラクトの発現様式を確認した。この結果、ベータガラクトシダーゼがI型コラーゲンの発現部分において高い発現を示すことが推察できた。このような実験...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1996年 -1996年 
    代表者 : 田村 正人, 野田 政樹
     
    細胞の分化に伴う組織特異的な遺伝子発現は,その多くが組織特異的な転写調節因子によって制御されていると考えらている.骨形成を担う骨芽細胞においても,未分化間葉系細胞から骨芽細胞への分化に何らかの組織特異的な転写調節因子が関与しているものと予想される.そこで本研究代表者はオステオカルシン遺伝子ならびにベーシックヘリックス-ループ-ヘリックス(bHLH)型の転写因子に注目し,研究を進めてきた.本研究代表者らは,オステオカルシン遺伝子の骨芽細胞での特異的な遺伝子発現に,このbHLH型の転写調節因子が関与していることなどを明らかにし既に報告している.本研究では,骨芽細胞におけるbHLH型の転写調節因子についての解析を行なった.すなわち,既に報告されているbHLH型の転写調節因子のなかでも組織特異的な因子のあいだでアミノ酸配列の保存されている領域についてのdegenerative Primerを作製した.種々の培養骨芽細胞株から抽出したRNAから合成したcDNAを鋳型として,これらプライマーを用いたRT-PCRを行なった.得られたフラグメントをサブクローニングし,得られたクローンの塩基配列の決定を行った.いくつかのクローンは,bHLH型の転写調節因子と思われるcDNAの塩基配列をもっていた.得られたbHLH型の転写調節因子と思われるcDNAについては,5'RACE法等を用いて全長の塩基配...
  • 文部科学省:科学研究費補助金(一般研究(A), 基盤研究(A))
    研究期間 : 1993年 -1996年 
    代表者 : 野田 政樹, 田村 正人, 二藤 彰
     
    骨粗鬆症の分子的基盤を明らかにする目的で,骨芽細胞並びに破骨細胞についてその細胞分化を制御する液性因子並びに細胞内において細胞の機能を調節する転写因子を中心に解析を行った.骨芽細胞においてはその分化と機能を制御する転写因子としてHelix-Loop-Helix型転写因子であるIdが発現することを明らかにし,その遺伝子制御がBMPによって調節を受けること,またレチノイン酸によって修飾を受けることを明らかにした.骨芽細胞に特異的に発現するオステオカルシンの上流にもHelix-Loop-Helix型転写因子が結合し,制御する機構が存在することを発見した.骨芽細胞の機能を調節する転写因子として,Helix-Loop-Helix型のADD1並びにHES1の存在とそれぞれのレチノイン酸並びにビタミンDによる制御機構が存在することを明らかにした.また骨芽細胞と共に軟骨細胞の研究を推進し,軟骨細胞株としてTC6細胞を樹立し,軟骨細胞を含む骨格系細胞において軟骨特異的な遺伝子の制御を制御するScleraxisの存在を明らかにした.更にこのScleraxisがTGFβ並びにFGFによって制御を受けることを示し,また軟骨の分化に重要な働きをするhedgehogが骨芽細胞でもTGFβによって制御されることを見出した.また骨芽細胞において,骨芽細胞の産生する主要な骨形成性のサイトカインであるTGFβの...
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1995年 -1995年 
    代表者 : 田村 正人
     
    ヘリックス‐ループ‐ヘリックス(HLH)型転写因子は,核内で転写制御を介して細胞の分化方向の決定を行う例が知られている.骨芽細胞の分化においても,これら因子が関与していることをすでに,本研究代表者は明らかにしてきた.本研究では,^<32>Pでラベルしたオステオカルシン遺伝子のE-box(OCE1)の二本鎖オリゴヌケレオチドをプローブにしてサウスウエスタン法によるスクリーニングを行った.まずROS17/2.8細胞やMC3T3E1細胞等の骨芽細胞様細胞のλgt11cDNAライブラリーを作製した.これらのcDNAライブラリーを用い、IPTGで融合タンパクを発現させプローブと結合させた.この方法によりいくつかのクローンが得られた.これらのクローンはプラスミドベクターにサブクローニング後,ジデオキシ法により塩基配列を決定した.また塩基配列より,アミノ酸配列に翻訳しタンパクの一次構造を決定し,コンピューターを用いタンパク質データベースで既知の転写因子との構造上の類似性を検討するとともに,種々のソフトウエアを用い構造に関する解析を行った.さらに,OCE1へ結合するタンパクについて,サウスウエスタン法によりその結合を検討した.すなわち,ROS17/2.8細胞,BMP‐2処理したC3H10T1/2細胞の核抽出液を未変性ポリアクリルアミド電気泳動し,ニトロセルロースメンブレンに転写した.^<32...
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1994年 -1994年 
    代表者 : 田村 正人
     
    ヘリックス-ループ-ヘリックス(HLH)型因子は,核内で転写制御を介して細胞の分化方向の決定を行う例が知られている.骨芽細胞分化においても,これら因子の関与が推測されたが,その本体は明らかにされていなかった.本研究では,骨芽細胞で特異的に産生されるオステオカルシン(OC)遺伝子のプロモーターに着目し,以下の結果を得た.(1)ld-1の発現ベクターをROS17/2.8細胞にトランスフェクトすると,OCのプロモーター活性が低下することを見いだし,種々の変異を用いた解析よりldに応答するE-boxを含む領域OCE1を同定した.(2)ゲルシフトアッセイよりROS17/2.8細胞の核抽出物にはOCE1に特異的に結合するタンパクが存在し,GST-ld-1タンパクの添加によりこの結合が減少した.μE5+μE2のオリゴヌクレオチドを加えても競合は見られず,E12/E47とは異なる因子の結合が示唆された.(3)OCE1への結合はC3H10T1/2細胞の核抽出物では見られないが,rhBMP-2処理で骨芽細胞への分化に伴い,OCE1への結合が観察された.(4)OCE2,3ならびに4のオリゴヌクレオチドを過剰に加えても競合は見られなかった.(5)DNaselフットプリント法から,OCE1部位におけるDNA-タンパク相互作用の存在が考えられた.(6)OCE1配列に結合するタンパクが,遺伝子産物として存...
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1993年 -1994年 
    代表者 : 田村 正人
     
    プライオトロフィン(PTN)は,神経突起の伸長活性を有するヘパリン結合性タンパクとして見いだされ,発生ならびに細胞分化において何らかの重要な機能を果たしているらしい。このタンパクは骨基質中にも存在する。本研究は,骨芽細胞様細胞の培養系を用いプライオトロフィンの産生調節に関してnRNAレベルで検討した。すなわち,種々の骨芽細胞様細胞を培養し骨芽細胞の機能に影響を及ぼすカルシウム代謝ホルモン,増殖因子やサイトカインを添加した。それらの細胞からRNAを抽出し,ラットPTN cDNAをプローブとして用いたノーザン解析でプライオトロフィンのmRNA量を調べた。ROS17/2.8,ROS25/1,UMR106ならびにMCT3T3-E1細胞において約1.8kbの単一バンドとして,培養時の血清濃度にかかわらずプライオトロフィンmRNAの発現が認められた。ROS17/2.8やMC3T3-E1細胞においては,活性型ビタミンDの添加濃度に依存して,プライオトロフィンmRNAの発現が減少した。一方,骨芽細胞の形質を有さないROS25/1細胞ではこの活性型ビタミンDの効果は認められなかった。またMC3T3-E1細胞のプライオトロフィンmRNAの発現は,副甲状腺ホルモンもしくはインターロイキン6によって増加することが認められた。これらの培養系を用いた検討の結果は,骨芽細胞のプライオトロフィンの産生が,活...
  • Structure and Function of tRNaseZ and regulation of gene expression using these enzyme
    Contract Research
  • Molecular Mechanisms for bone formation and resorption
    Cooperative Research

教育活動情報

主要な担当授業

  • 研究実習Ⅰ
    開講年度 : 2020年
    課程区分 : 学士課程
    開講学部 : 歯学部
  • 歯学研究概論
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学院
    キーワード : 歯学研究,基本的研究技法,基礎知識
  • 発表・論文執筆法演習Ⅰ
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学研究科
    キーワード : 研究計画,文献検索,研究のまとめ,口頭発表,論文作製
  • 発表・論文執筆法演習Ⅰ
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学院
    キーワード : 研究計画,文献検索,研究のまとめ,口頭発表,論文作製
  • 発表・論文執筆法演習Ⅱ
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学研究科
    キーワード : 研究計画,文献検索,研究のまとめ,口頭発表,論文作製
  • 発表・論文執筆法演習Ⅱ
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学院
    キーワード : 研究計画,文献検索,研究のまとめ,口頭発表,論文作製
  • 発表・論文執筆法演習Ⅲ
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学研究科
    キーワード : 研究計画,文献検索,研究のまとめ,口頭発表,論文作製
  • 発表・論文執筆法演習Ⅲ
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学院
    キーワード : 研究計画,文献検索,研究のまとめ,口頭発表,論文作製
  • 口腔分子生化学
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学研究科
    キーワード : 分子メカニズム,タンパク質,制御因子,細胞分化,細胞機能
  • 口腔分子生化学
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学院
    キーワード : 分子メカニズム,タンパク質,制御因子,細胞分化,細胞機能
  • 口腔分子生化学研究
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学研究科
    キーワード : タンパク質,核酸,分子メカニズム,制御因子,細胞分化,細胞機能
  • 口腔分子生化学研究
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学院
    キーワード : タンパク質,核酸,分子メカニズム,制御因子,細胞分化,細胞機能
  • 口腔生物学と医学
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学院
    キーワード : feeding behavior, vomiting, conditioned taste aversion (CTA), nausea, GLP-1, bacteriology, immunity, lipoprotein, salivary gland, oncogene, oral cancer, orofacial pain, tumor virus, dental materials, cementum, HIV, matrix cell biology
  • 歯学研究概論
    開講年度 : 2020年
    課程区分 : 博士後期課程
    開講学部 : 歯学研究科
    キーワード : 歯学研究,基本的研究技法,基礎知識
  • 関連臨床医学
    開講年度 : 2020年
    課程区分 : 学士課程
    開講学部 : 歯学部
    キーワード : 関連臨床医学、耳鼻咽喉科学、精神医学、臨床心理学、眼科、皮膚科、産婦人科
  • 研究実習Ⅱ
    開講年度 : 2020年
    課程区分 : 学士課程
    開講学部 : 歯学部
    キーワード : 研究,実験,コンピュータ,データ処理,文献検索,講演発表,論文執筆
  • 健康と社会
    開講年度 : 2020年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 口腔,血管,リンパ管,炎症,歯周組織,生体防御,常在細菌叢,う蝕(虫歯),歯周病,唾液, 口腔ガン,歯垢バイオフィルム
  • 生化学・口腔生化学実習
    開講年度 : 2020年
    課程区分 : 学士課程
    開講学部 : 歯学部
    キーワード : 生化学実習 口腔生化学実習
  • 生化学・口腔生化学
    開講年度 : 2020年
    課程区分 : 学士課程
    開講学部 : 歯学部
    キーワード : 生化学,口腔生化学,分子生物学
  • 歯科学概論Ⅱ
    開講年度 : 2020年
    課程区分 : 学士課程
    開講学部 : 歯学部
    キーワード : 歯科学、歯科医学
  • 歯科学概論Ⅰ
    開講年度 : 2020年
    課程区分 : 学士課程
    開講学部 : 歯学部
    キーワード : 歯科学、歯科医学


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