研究者詳細 - 沢　禎彦

Effects of a chemically synthesized leucine-rich amelogenin peptide (csLRAP) on chondrogenic and osteogenic cells 査読

Yuko Matsuda, Yuji Hatakeyama, Kazuki Nakashima, Naoko Kamogashira, Junko Hatakeyama, Sachio Tamaoki, Yoshihiko Sawa, Hiroyuki Ishikawa

Journal of Hard Tissue Biology 26 ( 1 ) 51 - 60 2017年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Society of Hard Tissue Regenerative Biology

Amelogenin is one of the enamel matrices secreted by ameloblasts and is known to have at least 15 alternative mRNA splicing isoforms. The leucine rich amelogenin peptide (LRAP) is one of most abundant amelogenin isoforms and numerous studies have shown that amelogenin peptides including LRAP, are expressed in cartilage and bone. Lysosome-associated membrane protein-1 (LAMP-1) has been identified as amelogenin binding partner, however, the mechanism of action for LRAP in chondrogenesis or osteogenesis is still unclear. This study aimed to assay the effect of chemically synthesized LRAP (csLRAP) on chondrogenic or osteogenic differentiation with the involvement of LAMP-1. The csLRAP used in this study was generated by F-moc solid phase chemistry based on the mouse LRAP amino acid sequence and was added to the culture medium of the chondrogenic cells, ATDC5 and the osteoblast cells, MC3T3-E1. Both chondrogenic and osteoblast cells, in which an immunopositive reaction to LAMP-1 antibody was observed by immunocytochemistry, showed significant suppression in cell number in the presence of csLRAP at 10 μg/ml compared to that in control, but this effect was rescued in the presence of LAMP-1 antibody. On the other hand, the intensity of alcian blue staining in chondrogenic cells and alizarin red staining in osteoblast cells was significantly increased in the presence of csLRAP at a dose of 10 μg/ml after 4 weeks culture, and this effect was suppressed in the presence LAMP-1 antibody. Moreover, the chondrogenic or osteogenic differentiation marker genes including Sox9, Type II Collagen, Type X Collagen, Runx2, Alkaline Phosphatase, and Type I Collagen, were upregulated in the presence of csLRAP after one week of culture and were suppressed in the presence of LAMP-1 antibody. These results suggest that csLRAP could promote osteogenic and chondrogenic differentiation in vitro, and that LAMP-1 may be involved in the differentiation and proliferation of these cells.

DOI： 10.2485/jhtb.26.51

Scopus

レクチンを用いた歯根膜におけるオキシタラン線維の検出の検討

鴨頭奈央子, 藤田隆寛, 中島一記, 松田裕子, 畠山雄次, 沢禎彦, 石川博之

福岡歯科大学学会雑誌 42 ( 増補 ) 38 - 38 2016年12月

化学合成により作製されたLRAPの骨芽細胞分化に与える影響

松田裕子, 畠山雄次, 沢禎彦, 石川博之

福岡歯科大学学会雑誌 42 ( 増補 ) 35 - 35 2016年12月

Novel Monoclonal Antibody LpMab-17 Developed by CasMab Technology Distinguishes Human Podoplanin from Monkey Podoplanin. 査読国際誌

Yukinari Kato, Satoshi Ogasawara, Hiroharu Oki, Ryusuke Honma, Michiaki Takagi, Yuki Fujii, Takuro Nakamura, Noriko Saidoh, Hazuki Kanno, Mitsuo Umetsu, Satoshi Kamata, Hiroshi Kubo, Mitsuhiro Yamada, Yoshihiko Sawa, Kei-Ichi Morita, Hiroyuki Harada, Hiroyoshi Suzuki, Mika Kato Kaneko

Monoclonal antibodies in immunodiagnosis and immunotherapy 35 ( 2 ) 109 - 16 2016年4月

記述言語：英語掲載種別：研究論文（学術雑誌）

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3 is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN in flow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77-Asp82 of PDPN using enzyme-linked immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94% between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared with the other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for the PDPN-targeting diagnosis or therapy.

DOI： 10.1089/mab.2015.0077

Differentiation of Apical Bud Cells in a Newly Developed Apical Bud Transplantation Model Using GFP Transgenic Mice as Donor. 査読国際誌

Naoki Maruo, Ryuji Sakagami, Yasunori Yoshinaga, Kazuhiko Okamura, Yoshihiko Sawa

PLoS one 11 ( 3 ) e0150766 2016年

記述言語：英語掲載種別：研究論文（学術雑誌）

Rodent mandibular incisors have a unique anatomical structure that allows teeth to grow throughout the lifetime of the rodent. This report presents a novel transplantation technique for studying the apical bud differentiation of rodent mandibular incisors. Incisal apical end tissue with green fluorescent protein from transgenic mouse was transplanted to wild type mice, and the development of the transplanted cells were immunohistologically observed for 12 weeks after the transplantation. Results indicate that the green fluorescent apical end tissue replaced the original tissue, and cells from the apical bud differentiated and extended toward the incisal edge direction. The immunostaining with podoplanin also showed that the characteristics of the green fluorescent tissue were identical to those of the original. The green fluorescent cells were only found in the labial side of the incisor up to 4 weeks. After 12 weeks, however, they were also found in the lingual side. Here the green fluorescent cementocyte-like cells were only present in the cementum close to the dentin surface. This study suggests that some of the cells that form the cellular cementum come from the apical tissue including the apical bud in rodent incisors.

DOI： 10.1371/journal.pone.0150766

Intracellular Signaling Pathway Activation via TGF-β Differs in the Anterior and Posterior Axis During Palatal Development

Higa Arisa, Oka Kyoko, Kira-Tatsuoka Michiko, Tamura Shougo, Itaya Satoshi, Toda Masako, Ozaki Masao, Sawa Yoshihiko

Journal of Hard Tissue Biology 25 ( 2 ) 195 - 204 2016年

記述言語：英語出版者・発行元：硬組織再生生物学会

It is important to understand the different mechanisms involved in anterior hard and posterior soft palate development to prevent and treat patients with cleft palate. Genetic analyses of humans and gene-mutated mice with cleft palate have shown that TGF-β signaling has a critical role in palatogenesis. However, the intracellular signaling pathway of TGF-β during palatogenesis in the anterior-posterior axis has not yet been fully understood. In the present study, the expression patterns of intracellular molecules Smad2/3 and phospho-p38 (Pp38) were examined at embryonic days 13.5, 14.0, and 14.5 (E13.5, E14.0, and E14.5) in mice. It was found that Smad3 was activated in anterior palatal mesenchyme and in the medial edge epithelium (MEE) region, with TGF-β3 expressed at E13.5. On the other hand, Pp38 was more expressed in posterior palatal mesenchyme and strongly expressed in the entire palatal epithelium at E13.5. These opposing expression patterns between Smad3 and Pp38 in palatal mesenchyme were also observed at E14.0. Interestingly, Pp38 expression was inhibited in MEE from E14.0. Generally, from E14.5, the tissue specificities of hard and soft palate started showing their characteristics following the activation of cell differentiation in palatal mesenchyme, and the medial edge seam (MES) of the palatal epithelium started to disappear for fusion to occur. At this stage, Smad3 was also more expressed in anterior palatal mesenchyme, while expression of Pp38 was activated in posterior palatal mesenchyme. Pp38 expression was inhibited, but Smad3 was activated in the MES. These results suggest that TGF-β signaling plays various roles, such as in cell proliferation and differentiation of palatal mesenchyme and in the disappearance of the MES, through different intracellular signaling pathways in anterior-posterior palatal mesenchyme and epithelium.

DOI： 10.2485/jhtb.25.195

CiNii Article

CiNii Books

Immunohistochemical expression of fibrillin-1 and fibrillin-2 during tooth development 査読

その他リンク： https://jlc.jst.go.jp/DN/JLC/20022363242?from=CiNii

M. Kira-Tatsuoka, K. Oka, E. Tsuruga, M. Ozaki, Y. Sawa

Journal of Periodontal Research 50 ( 6 ) 714 - 720 2015年12月

Intermittent Administration of Parathyroid Hormone Ameliorates Periapical Lesions in Mice 査読国際誌

Masato Otawa, Ryuichiro Tanoue, Hirofumi Kido, Yoshihiko Sawa, Junro Yamashita

JOURNAL OF ENDODONTICS 41 ( 5 ) 646 - 651 2015年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

Introduction: Intermittent administration of parathy raid hormone (PTH) promotes oral osseous wound healing and protects against ligature-induced alveolar bone loss. However, its therapeutic value on periapical periodontitis is unknown. The goal of this study was to determine the effect of intermittent PTH administration on the progression of periapical periodontitis. Methods: Seven lymphotoxin alpha deficient mice received pulp exposures of mandibular first and second molars. Exposed pulp in the right mandible was covered with plaque-contaminated fibrin, whereas exposed pulp in the left mandible was left open. After 4 weeks, the perk apical tissues were examined to determine the effect of plaque-contaminated fibrin to induce periapical lesions. Fourteen mice received pulp exposure covered with plaque-contaminated fibrin. PTH (40 Ag/kg/d) was administered intermittently to half of the mice for 3 weeks beginning 1 week after pulp exposure. The remaining half received saline injections as the vehicle control. At sacrifice, mandibles and tibiae were harvested and processed for histologic examination. Evaluation of neutrophils and blood vessels was performed after staining with immunofluorescence, and periradicular bone was histomorphometrically analyzed. Results: The exposed pulp covered with plaque-contaminated fibrin resulted in significantly larger periapical lesions compared with the control. Intermittent PTH administration reduced the size of periapical lesions significantly. Significantly less neutrophil infiltration around the root apex was found in PTH-treated animals compared with the control. Conclusions: PTH treatment suppressed periapical inflammation by reducing neutrophil infiltration and protected against tissue destruction by periapical periodontitis.

DOI： 10.1016/j.joen.2014.12.008

歯周病原菌由来LPSによるTLRを介した糖尿病性腎症の促進について

高田俊輔, 梶原弘一郎, 石川博之, 沢禎彦

福岡歯科大学学会雑誌 40 ( 増補 ) 31 - 31 2014年12月

ヒト無色素毛様体上皮細胞およびヒト歯根膜線維芽細胞におけるオキシタラン線維の分解過程

川越慈, 敦賀英知, 石川博之, 沢禎彦

福岡歯科大学学会雑誌 40 ( 増補 ) 36 - 36 2014年12月

マウス下顎切歯歯胚の幹細胞におけるSox2およびOct4の発現査読

丸尾直樹, 岡村和彦, 沢禎彦, 坂上竜資

Journal of Oral Biosciences Supplement 2014 160 - 160 2014年9月

Immunohistochemical Study of Amelogenin and Lysosome-Associate Membrane Proteins (LAMPs) in Cartilage 査読

Yuji Hatakeyama, Junko Hatakeyama, Kyoko Oka, Eichi Tsuruga, Tetsuichiro Inai, Hisashi Anan, Yoshihiko Sawa

INTERNATIONAL JOURNAL OF MORPHOLOGY 32 ( 2 ) 618 - 626 2014年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC CHILENA ANATOMIA

Amelogenin is one of the enamel matrix proteins secreted by ameloblasts during enamel formation in tooth development. Recent studies showed that the amelogenin is expressed in chondrocyte. Lysosome-associated membrane proteins (LAMPs) have been identified as binding partner proteins to amelogenin and it has been suggested they act as signaling receptors of amelogenin. The purpose of this study is to clarify the localization of amelogenin and LAMPs in growth plate cartilage and cartilaginous nodules in micromass culture. Mouse knee joints including tibia growth plate at 4 weeks old and micromass cultures of limb bud mesenchymal cells after 2 weeks were fixed in paraformaldehyde, routinely processed, sections were cut and immunostained with amelogenin, collagen type II and type X, LAMP-1 and -3. The positive immunoreaction of amelogenin was observed both in proliferation and hypertrophic zone cartilage of growth plate after enzymatic pretreatment in immunostaining. Furthermore, cartilaginous nodules in micromass culture were immunopositive to amelogenin. The chondrocytes in the proliferation zone of the growth plate were immunopositive to LAMP-1 but weakly stained in the chondrocytes of hypertrophic zone. These observations indicate that amelogenin may be present in cartilage matrix produced in vivo and in vitro and amelogenin may involve cartilage formation through the LAMP-1 signaling pathway.

Expression of Toll-Like Receptor 2 in Glomerular Endothelial Cells and Promotion of Diabetic Nephropathy by Porphyromonas gingivalis Lipopolysaccharide 査読国際誌

Yoshihiko Sawa, Shunsuke Takata, Yuji Hatakeyama, Hiroyuki Ishikawa, Eichi Tsuruga

PLOS ONE 9 ( 5 ) e97165 2014年5月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

The toll-like receptor (TLR) has been suggested as a candidate cause for diabetic nephropathy. Recently, we have reported the TLR4 expression in diabetic mouse glomerular endothelium. The study here investigates the effects of the periodontal pathogen Porphyromonas gingivalis lipopolysaccharide (LPS) which is a ligand for TLR2 and TLR4 in diabetic nephropathy. In laser-scanning microscopy of glomeruli of streptozotocin-and a high fat diet feed-induced type I and type II diabetic mice, TLR2 localized on the glomerular endothelium and proximal tubule epithelium. The TLR2 mRNA was detected in diabetic mouse glomeruli by in situ hybridization and in real-time PCR of the renal cortex, the TLR2 mRNA amounts were larger in diabetic mice than in non-diabetic mice. All diabetic mice subjected to repeated LPS administrations died within the survival period of all of the diabetic mice not administered LPS and of all of the non-diabetic LPS-administered mice. The LPS administration promoted the production of urinary protein, the accumulation of type I collagen in the glomeruli, and the increases in IL-6, TNF-alpha, and TGF-beta in the renal cortex of the glomeruli of the diabetic mice. It is thought that blood TLR ligands like Porphyromonas gingivalis LPS induce the glomerular endothelium to produce cytokines which aid glomerulosclerosis. Periodontitis may promote diabetic nephropathy.

DOI： 10.1371/journal.pone.0097165

Microfibril-associated glycoprotein-1 controls human ciliary zonule development in vitro. 査読

Takahiro Fujita, Eichi Tsuruga, Kaori Yamanouchi, Yoshihiko Sawa, Hiroyuki Ishikawa

Acta histochemica et cytochemica 47 ( 1 ) 11 - 7 2014年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

The ciliary zonule in the eye, also known as Zinn's zonule, is composed of oxytalan fibers, which are bundles of microfibrils consisting mainly of fibrillin-1. However, it is still unclear which of the microfibril-associated molecules present in the ciliary zonule controls oxytalan fibers. Microfibril-associated glycoprotein-1 (MAGP-1) is the only microfibril-associated molecule identified in the human ciliary zonule. In the present study, we used siRNA against MAGP-1 in cultures of human non-pigmented ciliary epithelial cells to examine the extracellular deposition and appearance of fibrillin-1 employing Western blotting and immunofluorescence. MAGP-1 suppression led to a reduction of fibrillin-1 deposition. Immunofluorescence also confirmed that RNAi-mediated down-regulation of MAGP-1 led to suppression of fiber development. These results suggest that MAGP-1 plays a crucial role in the extracellular deposition of fibrillin-1 during formation of the human ciliary zonule.

DOI： 10.1267/ahc.13038

Distribution of podoplanin-expressing cells in the mouse nervous systems. 査読

Miwa Tomooka, Chiaki Kaji, Hiroshi Kojima, Yoshihiko Sawa

Acta histochemica et cytochemica 46 ( 6 ) 171 - 7 2013年12月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Podoplanin is a mucin-type glycoprotein which was first identified in podocytes. Recently, podoplanin has been successively reported as a marker for brain and peripheral nerve tumors, however, the distribution of podoplanin-expressing cells in normal nerves has not been fully investigated. This study aims to examine the podoplanin-expressing cell distribution in the mouse head and nervous systems. An immunohistochemical study showed that the podoplanin-positive areas in the mouse peripheral nerve and spinal cord are perineurial fibroblasts, satellite cells in the dorsal root ganglion, glia cells in the ventral and dorsal horns, and schwann cells in the ventral and dorsal roots; in the cranial meninges the dura mater, arachnoid, and pia mater; in the eye the optic nerve, retinal pigment epithelium, chorioidea, sclera, iris, lens epithelium, corneal epithelium, and conjunctival epithelium. In the mouse brain choroid plexus and ependyma were podoplanin-positive, and there were podoplanin-expressing brain parenchymal cells in the nuclei and cortex. The podoplanin-expressing cells were astrocyte marker GFAP-positive and there were no differences in the double positive cell distribution of several portions in the brain parenchyma except for the fornix. The results suggest that podoplanin may play a common role in nervous system support cells and eye constituents.

DOI： 10.1267/ahc.13035

Leucine Rich Amelogenin Peptideによる軟骨形成の誘導

畠山純子, 畠山雄次, 松本典祥, 春名千英子, 諸冨孝彦, 泉利雄, 沢禎彦, 笹野泰之, 阿南壽

日本歯科保存学雑誌 56 ( 6 ) 560 - 569 2013年12月

記述言語：日本語出版者・発行元：(NPO)日本歯科保存学会

目的:アメロジェニンスプライシングバリアントの一つであるleucine rich amelogenin peptide(LRAP)は,破骨細胞と骨芽細胞の成熟,歯根膜細胞の遊走および増殖を促進するシグナルペプチドとして示唆されている.近年,軟骨にアメロジェニンの発現が報告されている.しかし,LRAPが軟骨形成に関与するかについての詳細は不明である.本研究の目的は,軟骨形成におけるLRAPの機能を明らかにすることである.材料と方法:C57BL6/CrSlcマウスの胎生10日齢の肢芽を摘出後,トリプシン/コラゲナーゼ処理により細胞を分離し,マイクロマスカルチャーを行った.ブタリコンビナントLRAP(マウスLRAPと〜85%の相同性,Dr.Gibson(ペンシルバニア大学,USA)より供与)と陽性コントロールとしてマウスリコンビナントGrowth Differentiation Factor-5(GDF-5,R & D Systems,USA)を,1,10,100ng/mlずつ培地に添加した.培養開始2,4,6日後にアルシアンブルー染色を行った.24,48時間後にBrdU assayを行い,細胞の増殖を確認した.12,24,48時間後にRNAを抽出し,軟骨のマーカーであるSox9,II型コラーゲン(Col2a1)およびAggrecanの半定量的RT-PCRを行った.有意差検定にはANOVAとBonferroni's post hoc testを用いた.結果:添加24・48時間後で,LRAP添加0.1,1,10ng/mlのいずれにおいても,濃度依存的にBrdU陽性細胞がコントロール群と比較して有意に増加していた.コントロール群では添加2日後にアルシアンブルー染色陽性結節の出現がみられ,培養期間が長くなると陽性結節の染色性が増加したが,LRAPおよびGDF-5を添加した群では,濃度依存的・培養期間依存的にアルシアンブルーの染色性が強くなった.LRAP添加群では,24・48時間後にSox9の遺伝子発現が,また48時間後にCol2a1およびAggrecanの遺伝子発現上昇がコントロール群と比較して有意に上昇した.結論:これらの知見は,アメロジェニンスプライシングバリアントの一つであるLRAPが,軟骨形成を促進していることを示唆している.(著者抄録)