研究者詳細 - 山本　直史

Inhibition of SMAD2 expression prevents murine palatal fusion 査読

N Shiomi, XM Cui, T Yamamoto, T Saito, CF Shuler

DEVELOPMENTAL DYNAMICS 235 ( 7 ) 1785 - 1793 2006年7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：WILEY-LISS

Transforming growth factor (TGF)-beta 3 is known to regulate the disappearance of murine medial edge epithelium (MEE) during palatal fusion. Our previous studies showed that SMAD2, a TGF-beta signaling mediator, was expressed and phosphorylated primarily in the MEE and that SMAD2 phosphorylation in the MEE was temporospatially regulated by TGF-beta 3. The goal of this study was to examine the requirement for SMAD2 to complete the developmental events necessary for palatal fusion. SMAD2 expression was inhibited with Smad2 siRNA transfection into palatal tissues in vitro. The results showed that Smad2 siRNA transfection resulted in the maintenance of MEE cells in the palatal midline. Western blot and immunofluorescence analyses confirmed that the endogenous SAUD2 and phospho-SMAD2 levels were reduced following siRNA transfection. The SMAD3 level was not altered by the Smad2 siRNA transfection. The persistence of the MEE and the decreased SMAD2/phospho-SMAD2 levels were coincident with increased MEE cell proliferation. Addition of exogenous TGF-beta 3 increased p-SMAD2 level but not the total SMAD2 level. Therefore, exogenous TGF-beta 3 was not able to induce p-SMAD2 enough to rescue the palatal phenotype in the Smad2 siRNA group. The results indicated that the endogenous SMAD2 level is crucial in the regulation of disappearance of MEE during palatal fusion.

DOI： 10.1002/dvdy.20819

培養ヒト歯根膜線維芽細胞における高浸透圧刺激によるsgk発現に関する研究査読

明貝文夫, 妹尾京子, 山城圭介, 山本直史, 小柳津功介, 新井英雄, 西村英紀, 高柴正悟

日本歯科保存学雑誌 48 ( 6 ) 939 - 945 2005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

浸透圧の上昇に応答して発現上昇するとされるsgk遺伝子が培養ヒト歯根膜線維芽細胞(HPLF)においてどのような発現動態を示すか調べるとともに,sgk発現におけるアクチン細胞骨格の関与について検討した.HPLFにおいても浸透圧の上昇に伴ってsgk発現は上昇することが明らかになり,この発現変化はアクチン細胞骨格に関係する刺激伝達系によって制御されていることが示唆された

CiNii Article

Effect of N '-nitrosonornicotine (NNN) on murine palatal fusion in vitro 査読

T Saito, XM Cui, T Yamamoto, N Shiomi, P Bringas, CF Shuler

TOXICOLOGY 207 ( 3 ) 475 - 485 2005年2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 SCI IRELAND LTD

Maternal smoking has been linked to an increased risk for orofacial clefts. N'-nitrosonornicotine (NNN) is one of the tobacco-specific nitrosamines that has been shown to be Linked to the deleterious effects of tobacco and could be linked to the formation of cleft palate birth defects. The effect of NNN on palatal fusion was examined using an in vitro organ culture model of palatal development. The organ cultures were exposed to NNN (0.0 1, 0. 1, 1, 10 and 100 mM) and the effects on palatal development characterized at defined points. Palatal fusion was evaluated at embryonic day 13 (E 13) + 72 h by characterizing the remaining medial edge epithelium (MEE) and determining the extent of fusion compared to controls. The NNN-treated group (I MM) had more MEE remaining in the palatal midline than the untreated group at E 13 + 72 h (P < 0.05). Changes in cell proliferation in the MEE resulting from NNN exposure were examined by BrdU incorporation in replicating DNA. Changes in the pattern of MEE cell death were examined by TUNEL. BrdU incorporation and TUNEL staining showed that the NNN (1 mM)-treated palates had more MEE cell proliferation and less apoptosis than the untreated-palates at E 13 + 24 h (P < 0.05). The mechanism altered by NNN was further evaluated by characterizations of extracellular signal-regulated kinase (ERK) 1/2, p38 and c-jun amino-terminal kinase (INK). NNN at I mM induced ERK1/2 phosphorylation, but reduced p38 phosphorylation (P&LT;0.05, P&LT; 0.01, respectively) in the MEE. The results suggest that NNN inhibited palatal fusion by effects on cell proliferation and MEE cell death. &COPY; 2004 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.tox.2004.10.015

Overexpression of Smad2 in Tgf-beta 3-null mutant mice rescues cleft palate 査読

XM Cui, N Shiomi, JC Chen, T Saito, T Yamamoto, Y Ito, P Bringas, Y Chat, CF Shuler

DEVELOPMENTAL BIOLOGY 278 ( 1 ) 193 - 202 2005年2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：ACADEMIC PRESS INC ELSEVIER SCIENCE

Transforming growth factor (TGF)-beta3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-beta3. No phospho-SMAD2 was identified in the MEE in Tgf-beta3-null mutant mice (Tgf-beta(-1-)), which was correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in Tgf-beta3(-/-) mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-beta3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue could be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences. The rescued palatal fusion in Tgf-beta3(-/-)/K14-Smad2 mice, however, never proceeded to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate. The K14-Smad2 transgene was unable to restore all the functional outcomes of TGF-beta3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF-beta3 signaling and the mechanism of secondary palatal fusion. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2004.10.023

Identification of genes differentially regulated in rat alveolar bone wound healing by subtractive hybridization 査読

T Ohira, F Myokai, N Shiomi, K Yamashiro, T Yamamoto, Y Murayama, H Arai, F Nishimura, S Takashiba

JOURNAL OF DENTAL RESEARCH 83 ( 7 ) 546 - 551 2004年7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：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R

Periodontal healing requires the participation of regulatory molecules, cells, and scaffold or matrix. Here, we hypothesized that a certain set of genes is expressed in alveolar bone wound healing. Reciprocal subtraction gave 400 clones from the injured alveolar bone of Wistar rats. Identification of 34 genes and analysis of their expression in injured tissue revealed several clusters of unique gene regulation patterns, including the up-regulation at 1 wk of cytochrome c oxidase regulating electron transfer and energy metabolism, presumably occurring at the site of inflammation; up-regulation at 2.5 wks of pro-alpha-2 type I collagen involving the formation of a connective tissue structure; and up-regulation at 1 and 2 wks and down-regulation at 2.5 and 4 wks of ubiquitin carboxyl-terminal hydrolase 13 involving cell cycle, DNA repair, and stress response. The differential expression of genes may be associated with the processes of inflammation, wound contraction, and formation of a connective tissue structure.

Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion 査読

T Yamamoto, XM Cui, CF Shuler

DEVELOPMENTAL BIOLOGY 260 ( 2 ) 512 - 521 2003年8月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. We hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates failed to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation were localized in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibited EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stopped proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S0012-1606(03)00275-6

Gene profiling in human periodontal ligament fibroblasts by subtractive hybridization 査読

T Yamamoto, F Myokai, F Nishimura, T Ohira, N Shiomi, K Yamashiro, H Arai, Y Murayama, S Takashiba

JOURNAL OF DENTAL RESEARCH 82 ( 8 ) 641 - 645 2003年8月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INT AMER ASSOC DENTAL RESEARCHI A D R/A A D R

Genes expressed by human periodontal ligament fibroblasts (HPFs)are likely to be associated with specific functions of the ligament. The aim of this study is to profile genes expressed highly by HPFs. A library (6 x 103 pfu)was constructed, followed by subtraction of HPF cDNAs with human gingival fibroblast (HGF) cDNAs. Reverse-dot hybridization revealed that 33 clones expressed higher levels of specific mRNAs in HPFs than in HGFs. These were mRNAs for known genes including several associated with maturation and differentiation of cells. None had been reported in PFs. One clone, PDL-29 identified as a COX assembly factor, showed much stronger mRNA expression in HPFs than in HGFs in culture. In rat periodontium, however, PDL-29 mRNA expression was similar in PFs and GFs. These results suggest that HPFs express many previously unreported genes associated with maturation and differentiation, but expression can differ in vitro and in vivo.

TGF-beta3-dependent SMAD2 phosphorylation and inhibition of MEE proliferation during palatal fusion 査読

Cui XM, Chai Y, Chen J, Yamamoto T, Ito Y, Bringas P, Shuler CF

Dev Dyn 227 ( 3 ) 387 - 394 2003年7月

Unique genes induced by mechanical stress in periodontal ligament cells 査読

F Myokai, M Oyama, F Nishimura, T Ohira, T Yamamoto, H Arai, S Takashiba, Y Murayama

JOURNAL OF PERIODONTAL RESEARCH 38 ( 3 ) 255 - 261 2003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：BLACKWELL MUNKSGAARD

Objectives: The aim of this study is to isolate mechanical stress-induced genes (MSGens) from human periodontal ligament (PDL) cells and to analyze profiles of the mRNA expression of these genes.
Background: Differential expression of genes in PDL cells under physiological stress such as occlusal force is thought to be orchestrated not only for the remodeling of PDL itself but also for the repair and regeneration of periodontal tissues. However, little is known about the genes expressed in PDL cells under mechanical stress.
Methods: The cDNA from mechanical stress-applied human PDL cells was subtracted against the cDNA from static control cells. The subtracted cDNA was amplified by polymerase chain reaction (PCR) and cloned for further analysis.
Results: Among 68 independent clones isolated, 15 contained DNA fragments greater than 250 bp. Reverse Northern analysis revealed a marked induction of MSGen-15 and MSGen-28 mRNA expression in the mechanical stress-applied cells. However, little difference in the magnitude of expression for the other MSGens was detected between the stress-applied cells and the control cells. After nucleotide sequencing and the analysis of homology with known genes, five clones were identified; ribosomal protein S27 (MSGen-9), MRG 15 (MSGen-15), androgen-binding protein (MSGen-18), cathepsin H (MSGen-28), and cytochrome c (MSGen-47). Interestingly, it has been reported that MRG 15 is a novel transcription factor involved in the regulation of cell growth and senescence. The remaining 10 clones, classified into six sequence types, had no significant homology with any known genes.
Conclusions: These results suggest that many known and unknown genes are expressed in response to mechanical stress in PDL cells, and that a transcription factor, MRG 15, may be responsible for molecular events in PDL cells under mechanical stress.

DOI： 10.1034/j.1600-0765.2003.00602.x