Language：English   Publishing type：Research paper (scientific journal)  

Development of the mammalian telencephalon, which is the most complex region of the central nervous system, is precisely orchestrated by many signaling molecules. Wnt signaling derived from the cortical hem, a signaling center, is crucial for telencephalic development including cortical patterning and the induction of hippocampal development. Secreted protein R-spondin (Rspo) 1-4 and their receptors, leucine-rich repeat-containing G-protein-coupled receptor (Lgr) 4-6, act as activators of Wnt signaling. Although Rspo expression in the hem during the early stages of cortical development has been reported, comparative expression analysis of Rspos and Lgr4-6 has not been performed. In this study, we examined the detailed spatiotemporal expression patterns of Rspo1-4 and Lgr4-6 in the embryonic and postnatal telencephalon to elucidate their functions. In the embryonic day (E) 10.5-14.5 telencephalon, Rspo1-3 were prominently expressed in the cortical hem. Among their receptors, Lgr4 was observed in the ventral telencephalon, and Lgr6 was highly expressed throughout the telencephalon at the same stages. This suggests that Rspo1-3 and Lgr4 initially regulate telencephalic development in restricted regions, whereas Lgr6 functions broadly. From the late embryonic stage, the expression areas of Rspo1-3 and Lgr4-6 dramatically expanded; their expression was found in the neocortex and limbic system, such as the hippocampus, amygdala, and striatum. Increased Rspo and Lgr expression from the late embryonic stages suggests broad roles of Rspo signaling in telencephalic development. Furthermore, the Lgr+ regions were located far from the Rspo+ regions, especially in the E10.5-14.5 ventral telencephalon, suggesting that Lgrs act via a Rspo-independent pathway.

DOI： 10.1016/j.gep.2023.119333

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s10266-022-00775-9

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Other Link： https://link.springer.com/article/10.1007/s10266-022-00775-9/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)  

Aberrant osteoclast formation and activation are the hallmarks of osteolytic metastasis. Extracellular vesicles (EVs), released from bone metastatic tumor cells, play a pivotal role in the progression of osteolytic lesions. However, the mechanisms through which tumor cell-derived EVs regulate osteoclast differentiation and function have not been fully elucidated. In this study, we found that 4T1 bone metastatic mouse mammary tumor cell-derived EVs (4T1-EVs) are taken up by mouse bone marrow macrophages to facilitate osteoclastogenesis. Furthermore, treatment of mature osteoclasts with 4T1-EVs promoted bone resorption, which was accompanied by enhanced survival of mature osteoclasts through the negative regulation of caspase-3. By comparing the miRNA content in 4T1-EVs with that in 67NR nonmetastatic mouse mammary tumor cell-derived EVs (67NR-EVs), miR-92a-3p was identified as one of the most enriched miRNAs in 4T1-EVs, and its transfer into mature osteoclasts significantly reduced apoptosis. Bioinformatic and Western blot analyses revealed that miR-92a-3p directly targeted phosphatase and tensin homolog (PTEN) in mature osteoclasts, resulting in increased levels of phospho-Akt. Our findings provide novel insights into the EV-mediated regulation of osteoclast survival through the transfer of miR-92a-3p, which enhances mature osteoclast survival via the Akt survival signaling pathway, thus promoting bone resorption.

DOI： 10.1111/cas.15557

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

OBJECTIVE: Rice peptide has antibacterial properties that have been tested in planktonic bacterial culture. However, bacteria form biofilm at disease sites and are resistant to antibacterial agents. The aim of this study was to clarify the mechanisms of action of rice peptide and its amino acid substitution against periodontopathic bacteria and their antibiofilm effects. DESIGN: Porphyromonas gingivalis and Fusobacterium nucleatum were treated with AmyI-1-18 rice peptide or its arginine-substituted analog, G12R, under anaerobic conditions. The amount of biofilm was evaluated by crystal violet staining. The integrity of the bacteria cytoplasmic membrane was studied in a propidium iodide (PI) stain assay and transmission electron microscopy (TEM). RESULTS: Both AmyI-1-18 and G12R inhibited biofilm formation of P. gingivalis and F. nucleatum; in particular, G12R inhibited F. nucleatum at lower concentrations. However, neither peptide eradicated established biofilms significantly. According to the minimum inhibitory concentration and minimum bactericidal concentration against P. gingivalis, AmyI-1-18 has bacteriostatic properties and G12R has bactericidal activity, and both peptides showed bactericidal activity against F. nucleatum. PI staining and TEM analysis indicated that membrane disruption by G12R was enhanced, which suggests that the replacement amino acid reinforced the electostatic interaction between the peptide and bacteria by increase of cationic charge and α-helix content. CONCLUSIONS: Rice peptide inhibited biofilm formation of P. gingivalis and F. nucleatum, and bactericidal activity via membrane destruction was enhanced by amino acid substitution.

DOI： 10.1016/j.archoralbio.2020.104956

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Publishing type：Research paper (scientific journal)   Publisher：Nihon University School of Dentistry  

DOI： 10.2334/josnusd.20-0411

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

In bone tissues, gap junctions form direct links between the cytoplasm of an osteocyte and another adjacent osteocyte or osteoblast, which underlie both bone formation and bone resorption. We have previously demonstrated that alkaline phosphatase (ALP) and bone sialoprotein (BSP), which are osteoblast markers, were induced in mesenchymal stem cells (MSCs) co-cultured with osteoblast-like cell line. However, the molecular mechanism of this process has not been fully addressed. Furthermore, few advances have been made toward elucidating the communication networks that link the status of committed cells such as (pre-) adipocytes that differentiated from MSCs as well as osteoblasts. Therefore, the objective of the present study was to investigate the mechanism underlying the communication network between pre-adipocytes and osteoblasts. We evaluated the effect of co-culture with osteoblast on the cell status of pre-adipocytes using murine osteoblast-like cell line, MLO-A5, and pre-adipocyte-like cell line, 3T3-L1, respectively. The results presented here demonstrated that osteoblasts and pre-adipocytes communicate via gap junctions, and the ensuing drastic increase in ALP and BSP transcription in co-cultured pre-adipocytes was induced, at least partly, via heat shock protein family B member 1 (Hspb1). In addition, terminal differentiation into adipocytes was suppressed in pre-adipocytes during co-culture with osteoblast without loss of adipogenic differentiation ability. Interestingly, after co-culture with osteoblasts, isolated co-cultured pre-adipocytes were able to differentiate to adipocytes as well as original pre-adipocytes. These results suggest that gap junctional communication with osteoblasts suppressed adipogenic differentiation of pre-adipocytes without loss of adipogenic differentiation ability.

DOI： 10.1016/j.bone.2020.115596

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

To elucidate the underlying mechanism of secretory leukocyte protease inhibitor (SLPI)-induced cell migration, we compared SLPI-deleted human gingival carcinoma Ca9-22 (ΔSLPI) cells and original (wild-type: wt) Ca9-22 cells using several microscopic imaging methods and gene expression analysis. Our results indicated reduced migration of ΔSLPI cells compared to wtCa9-22 cells. The lamellipodia/dorsal ruffles were smaller and moved slower in ΔSLPI cells compared to wtCa9-22 cells. Furthermore, well-developed intermediate filament bundles were observed at the desmosome junction of ΔSLPI cells. In addition, Galectin4 was strongly expressed in ΔSLPI cells, and its forced expression suppressed migration of wtCa9-22 cells. Taken together, SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.

DOI： 10.1080/19336918.2020.1829264

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Inorganic polyphosphate (polyP), a linear polymer of orthophosphate, is found at high concentrations in osteoblasts. We demonstrated the effects of various polyP concentrations on the mineralization of rat osteoblast ROS17/2.8 cells. Mineralization of ROS17/2.8 was induced by a high polyP concentration (1 mg/mL), which was accompanied by an upregulation of the bone sialoprotein and osteocalcin. In contrast, a low polyP concentration (1 × 10-2 mg/mL) reduced mineralization without affecting the osteogenic gene expression. Furthermore, gene expression profiling and forced expression analysis indicated that phosphodiesterase 11a could be a candidate involved in the dose-dependent effect of polyP on osteoblast mineralization.

DOI： 10.1016/j.jphs.2018.10.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Several studies have demonstrated the remarkable properties of microbiota and their metabolites in the pathogenesis of several inflammatory diseases. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a bioactive metabolite generated by probiotic microorganisms during the process of fatty acid metabolism, has been studied for its protective effects against epithelial barrier impairment in the intestines. Herein, we examined the effect of HYA on gingival epithelial barrier function and its possible application for the prevention and treatment of periodontal disease. We found that GPR40, a fatty acid receptor, was expressed on gingival epithelial cells; activation of GPR40 by HYA significantly inhibited barrier impairment induced by Porphyromonas gingivalis, a representative periodontopathic bacterium. The degradation of E-cadherin and beta-catenin, basic components of the epithelial barrier, was prevented in a GPR40-dependent manner in vitro. Oral inoculation of HYA in a mouse experimental periodontitis model suppressed the bacteria-induced degradation of E-cadherin and subsequent inflammatory cytokine production in the gingival tissue. Collectively, these results suggest that HYA exerts a protective function, through GPR40 signaling, against periodontopathic bacteria-induced gingival epithelial barrier impairment and contributes to the suppression of inflammatory responses in periodontal diseases.

DOI： 10.1038/s41598-018-27408-y

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/jre.12418

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nihon University School of Dentistry  

<p>SIRT1 is a NAD-dependent histone deacetylase that is important in a wide variety of physiological and pathophysiological processes. Although many studies have examined the relationship between SIRT1 and cancer, the role of SIRT1 in tumor malignancy is controversial. Here, we examined the effects of the SIRT1 activator CAY10591 in gingival epithelial carcinoma Ca9-22 cells. CAY10591 treatment dose- and time-dependently increased SIRT1 level and activity. The treatment decreased cell growth and induced cell-cycle repressor p21 levels. In addition, dimethyl sulfoxide significantly reduced cellular invasion and migration, and CAY10591 enhanced this decrease. Quantitative PCR analysis showed that CAY10591 decreased expression of several invasion/migration promoter genes and induced repressor genes. Our findings suggest that CAY10591 suppresses cell growth and invasion/migration activity in gingival squamous cell carcinoma Ca9-22 cells.</p>

DOI： 10.2334/josnusd.16-0696

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Nihon University School of Dentistry  

DOI： 10.2334/josnusd.16-0534

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Polyphosphate [Poly(P)] has positive effects on osteoblast mineralization; however, the underlying mechanism remains unclear. In addition, it is unknown whether Poly(P) promotes mineralization in soft tissues. We investigated this by using various cells. Poly(P) concentrations of 1 and 0.5 mg/mL yielded high levels of mineralization in ROS17/2.8 osteoblast cells. Similarly, Poly(P) induced mineralization in cell types expressing alkaline phosphatase (ALP), namely, ATDC5 and MC3T3-E1, but not in CHO, C3H10T1/2, C2C12, and 3T3-L1 cells. Furthermore, forced expression of ALP caused Poly(P)-induced mineralization in CHO cells. These results suggest that ALP determines Poly(P)-induced mineralization in a cell-type independent manner.

DOI： 10.1007/s00774-015-0719-6

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Other Link： http://link.springer.com/article/10.1007/s00774-015-0719-6/fulltext.html

Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.canlet.2016.05.028

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nihon University School of Dentistry  

Porphyromonas gingivalis is important in the development of marginal periodontitis. However, the precise role and localization of P. gingivalis in chronic periapical periodontitis remain unclear. Thus, methods that can detect P gingivalis in formalin-fixed and paraffin-embedded (FFPE) tissue samples are needed. We assessed a technique combining loop mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for detection of P gingivalis, using 110 FFPE tissue samples of chronic apical periodontitis. PCR-LAMP specifically detected P gingivalis with high sensitivity in FFPE tissue samples, and the sensitivity of the technique was higher than that of PCR or LAMP alone. The results of immunohistochemistry (IHC) confirmed the specificity of PCR-LAMP. IHC showed that P gingivalis was localized hi a granular layer of chronic apical periodontitis, a region that correlated with the localization of macrophages. This is the first study to describe the localization of P gingivalis in human periapical periodontitis. In conclusion, PCR-LAMP was an effective tool for detecting P gingivalis in periapical periodontitis. In addition, IHC results improve our understanding of the role of P gingivalis in the progression of periapical periodontitis.

DOI： 10.2334/josnusd.15-0665

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Polymeric immunoglobulin receptor (pIgR) plays an important role in mucosal immune systems. Secretory immunoglobulin A, composed of secretory component of pIgR and a dimeric form of immunoglobulin A, is secreted on mucosal surfaces and serves as a biological defense factor. pIgR gene expression is reportedly induced by activation of the transcription factor nuclear factor (NF)-kappa B. On the other hand, secretory leukocyte protease inhibitor (SLPI) is a glycoprotein that functions as a serine protease inhibitor. In alveolar epithelial cells, SLPI increases the level of I kappa B beta, which indicates that it is an inhibitor of NF-kappa B at the protein level. Taken together, SLPI may regulate plgR expression; however, the specific mechanism by which this occurs is unclear. Therefore, the aim of this study was to elucidatethe influence of SLPI on pIgR expression. SLPI and pIgR localized in goblet cells and ciliated epithelial cells of the gastrointestinal tract, respectively. No cells were detected in which SLPI and pIgR were co-expressed. In addition, recombinant human SLPI stimulation of an epithelial cell line (HT-29) decreased the pIgR expression. The pIgR expression was also higher in SLPI-deficient Ca9-22 cells than in wild-type Ca9-22 cells. Furthermore, a luciferase assay using a NF-kappa B reporter plasmid and real-time RT-PCR analysis indicated that when SLPI was present, the transcriptional activity of NF-kappa B protein was suppressed, which was accompanied by anincrease in the protein, but not the mRNA,expression of I kappa B beta. These results demonstrate that SLPI down-regulates pIgR expression through the NF-kappa B signaling pathway by inhibiting degradation of I kappa B beta protein. (C) 2015 The Authors. Published by Elsevier Ltd.

DOI： 10.1016/j.molimm.2015.07.021

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Recently, earthworms have become a useful model for research into the immune system, and it is expected that results obtained using this model will shed light on the sophisticated vertebrate immune system and the evolution of the immune response, and additionally help identify new biomolecules with therapeutic applications. However, for earthworms to be used as a genetic model of the invertebrate immune system, basic molecular and genetic resources, such as an expressed sequence tag (EST) database, must be developed for this organism. Next-generation sequencing technologies have generated EST libraries by RNA-seq in many model species. In this study, we used Illumina RNA-sequence technology to perform a comprehensive transcriptome analysis using an RNA sample pooled from sterile-cultured Eisenia andrei. All clean reads were assembled de novo into 41,423 unigenes using the Trinity program. Using this transcriptome data, we performed BLAST analysis against the GenBank non-redundant (NR) database and obtained a total of 12,285 significant BLAST hits. Furthermore, gene ontology (GO) analysis assigned 78 unigenes to 24 immune class GO terms. In addition, we detected a unigene with high similarity to beta-1,3-glucuronyltransferase 1 (GlcAT-P), which mediates a glucuronyl transfer reaction during the biosynthesis of the carbohydrate epitope HNK-1 (human natural killer-1, also known as CD57), a marker of NK cells. The identified transcripts will be used to facilitate future research into the immune system using E. andrei.

DOI： 10.1371/journal.pone.0118587

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We examined cell-to-cell interaction between pre-osteoblasts and osteocytes using MC3T3-E1 and MLO-Y4, respectively. First, GFP expressing MC3T3-E1 (E1-GFP) cells were generated to isolate the cells from co-culture with MLO-Y4. No changes were observed in the expression of osteogenic transcription factors Runx2, Osterix, Dlx5 and Msx2, but expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP) in E1-GFP co-cultured with MLO-Y4 was 300-400-fold greater than that in mono-cultured E1-GFP. In addition, mineralized nodule formation was drastically increased in co-cultured E1-GFP cells compared to mono-cultured cells. Patch clamp assay showed the presence of gap junctions between E1-GFP and MLO-Y4. Furthermore, when the gap junction inhibitor carbenoxolone (CBX) was added to the culture, increased expression of ALP and BSP in E1-GFP co-cultured with MLO-Y4 was suppressed. These results suggest that gap junction detected between pre-osteoblasts and osteocytes plays an important role on the terminal differentiation of pre-osteoblasts. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2014.10.128

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

An analytical study of cell-cell communications between murine osteoblast-like MLO-A5 cells and bone marrow mesenchymal stem cell (BMSC)-like C3H10T1/2 cells was performed. C3H10T1/2 cells expressing green fluorescent protein (10T-GFP cells) were generated to enable the isolation of the BMSC-like cells from co-cultures with MLO-A5 cells. The mRNA expression levels of several osteogenic transcription factors (Runx2, Osterix, Dlx5, and Msx2) did not differ between the co-cultured and mono-cultured 10T-GFP cells, but those of alkaline phosphatase (ALP) and bone sialoprotein (BSP) were 300- to 400-fold higher in the co-cultured cells. Patch clamp and biocytin transfer assays revealed gap junction-mediated communication between co-cultured 10T-GFP and MLO-A5 cells. The addition of a gap junction inhibitor suppressed the increases in the expression levels of the ALP and BSP mRNAs in co-cultured 10T-GFP cells. Furthermore, the histone acetylation levels were higher in co-cultured 10T-GFP cells than in mono-cultured 10T-GFP cells. These results suggest that osteoblasts and BMSCs associate via gap junctions, and that gap junction-mediated signaling induces histone acetylation that leads to elevated transcription of the genes encoding ALP and BSP in BMSCs.

DOI： 10.1089/scd.2014.0060

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The 75 kDa transmembrane protein, p75(NTR), is a marker of mesenchymal stem cells (MSCs). Isolated MSCs are capable of differentiating into osteoblasts, but the molecular function of p75(NTR) in MSCs and osteoblasts is poorly understood. The aim of this study was to examine the function of p75(NTR) in the human MG63 osteoblast cell line compared to the murine MC3T3E-1 pre-osteoblast cell line.
MG63 cells and MC3T3-E1 cells expressing exogenous p75(NTR) protein (denoted as p75-MG63 and p75GFP-E1, respectively) were generated to compare osteogenic differentiation and cell proliferation abilities. Overexpression of p75(NTR) induced alkaline phosphatase activity and the mRNA expression of osteoblast-related genes such as osterix and bone sialoprotein in both p75-MG63 and p75GFP-E1. Interestingly, exogenous p75(NTR) stimulated cell proliferation and cell cycle progression in p75GFP-E1, but not in p75-MG63.
To elucidate any different effects of p75(NTR) expression on osteogenic differentiation and cell proliferation, we examined the mRNA expression of tropomyosin receptor kinase (trk) genes (trkA, trkB, trkC) and Nogo receptor (NgR), which are binding partners of p75(NTR). Although trkA, trkB, and trkC were detected in both p75-MG63 and p75GFP-E1, only NgR was detected in p75-MG63. We then used the K252a inhibitor of the trks to identify the signaling pathway for osteogenic differentiation and cell pmliferation. Inhibition of trks by K252a suppressed p75(NTR)-mediated osteogenic differentiation of p75GFP-E1, whereas deletion of the GDI domain in p75(NTR) from the p75-MG63 produced enhanced cell proliferation compared to p75-MG63. These results suggest that p75(NTR) signaling associated with Lrk receptors promotes both cell proliferation arid osteoblast differentiation, but that p75(NTR)-mediated proliferation may be suppressed by signaling from the p75(NTR)/NgR complex. (C) 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.diff.2014.01.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Dexamethasone (Dex) regulates osteoblastic and adipocytic differentiation in mesenchymal progenitor cells through regulation of Wnt/beta-catenin signaling. To elucidate the regulatory mechanisms underlying the effects of Dex, we examine the expression of Axin2, which is an intracellular inhibitor of Wnt/beta-catenin signaling, in ROB-C26 clonal mesenchymal progenitor cells (C26). We observed the induction of Axin2 mRNA in C26 cells in response to Dex treatment. Treatment with a glucocorticoid receptor (GR) antagonist, mifepristone, showed that Dex-induced up-regulation of Axin2 is mediated by the GR. In the absence of Dex, gene silencing by using Axin2-targeted short hairpin RNA increased the number of alkaline phosphatase (ALP)-positive and nuclear beta-catenin-positive cells and ALP activity. In the presence of Dex, Axin2 knockdown resulted in an increased number of ALP-positive and nuclear beta-catenin-positive cells. Furthermore, Axin2 knockdown in Dex-treated cells suppressed adipocyte differentiation (as determined by reduced Oil Red O staining), reduced the number of PPAR gamma-positive and aP2-positive cells and decreased the mRNA expression of PPAR gamma 2 and aP2. These results suggest that Axin2 plays a key role in adipocyte and osteoblastic differentiation by controlling beta-catenin expression.

DOI： 10.1007/s00441-013-1696-5

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s12565-013-0208-8

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Other Link： http://link.springer.com/article/10.1007/s12565-013-0208-8/fulltext.html

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Labridae is a family of shallow-water coastal fish that belong to the Labroidei suborder in the Perciformes order. There has however been little comparative dissection or phylogenetic research on this family, which resulted in confusion regarding species classification. Therefore morphological characters of 12 species of Labridae containing three subfamilies and seven genera were compared for the purpose of determining the characteristics of the subfamilies and genera of Labridae and deducing their phylogenic relationship. We compiled a phylogenetic tree by using the computer program PAUP 4.0b10 and a data set composed of 17 meristic and osteological characters. In our phylogenic tree, the two species of Bodianinae, three species of Cheilininae and seven species of Corinae each formed monophyletic groups, supporting the conventional classification by lateral line shape. Furthermore, the group of three species of Cheilininae and the group of two species of Bodianinae were shown to be more closely related, because they have several common morphological characteristics, such as neuroarticular processes and anterior teeth on premaxilla. Between seven species of Corinae, not only was the group of two species of Halichoeres more closely related to Coris dorsomacula, but also, this group of three species may be closely related to the three species of Thalassoma. Suezichthys gracilis was also found to have separated from the other Corinae species at the earliest stage. (C) 2013 Wiley Periodicals, Inc.

DOI： 10.1002/ar.22687

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Publishing type：Research paper (scientific journal)   Publisher：Quintessence Publishing  

DOI： 10.11607/jomi.te25

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.

DOI： 10.1016/j.diff.2012.07.001

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Dexamethasone (Dex) stimulates the differentiation of mesenchymal progenitor cells into adipocytes and osteoblasts. However, the mechanisms underlying Dex-induced differentiation have not been clearly elucidated. We examined the effect of Dex on the expression and activity of Wnt/β-catenin signal-related molecules in a clonal mesenchymal progenitor cell line, ROB-C26 (C26). Dex induced the mRNA expression of Wnt antagonists, dickkopf-1 (Dkk-1), and Wnt inhibitory factor (WIF)-1. Immunocytochemical analysis showed that the downregulation of β-catenin protein expression by Dex occured concomitantly with the increased expression of the PPARγ protein. Dex decreased phosphorylation of Ser9-GSK3β and expression of active β-catenin protein. To examine the effects of Dex on Wnt/β-catenin activity, we used immunocytochemistry to analyze TCF/LEF-mediated transcription during Dex-induced adipogenesis in Wnt indicator (TOPEGFP) C26 cells. Our results demonstrated that Dex repressed TCF/LEF-mediated transcription, but induced adipocyte differentiation. Treatment with a GSK3β inhibitor attenuated Dex-induced inhibition of TCF/LEF-mediated transcriptional activity, but suppressed Dex-induced adipocyte differentiation, indicating that adipocyte differentiation and inhibition of Wnt/β-catenin activity by Dex are mediated by GSK3β activity. Furthermore, β-catenin knockdown not only suppressed Dex-induced ALP-positive osteoblasts differentiation but also promoted Dex-induced adipocytes differentiation. These results suggest that inhibition of β-catenin expression by Dex promotes the differentiation of mesenchymal progenitor cells into adipocytes.

DOI： 10.1007/s00418-012-1007-3.

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/j.1439-0264.2011.01103.x

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Language：English   Publishing type：Research paper (scientific journal)  

Lamin A/C gene encodes a nuclear membrane protein, and mutations in this gene are associated with diverse degenerative diseases that are linked to premature aging. While lamin A/C is involved in the regulation of tissue homeostasis, the distinct expression patterns are poorly understood in the mesenchymal cells differentiating into adipocytes. Here, we examined the expression of lamin A/C in a rat mesenchymal progenitor cell-line, ROB-C26 (C26). Immunocytochemical analysis showed that lamin A/C was transiently down-regulated in immature adipocytes, but its expression increased with terminal differentiation. To elucidate the role of lamin A/C expression on mesenchymal cell differentiation, lamin A/C expression was suppressed using short hairpin RNA (shRNA) molecules in C26 cells. In the absence of adipogenic stimuli, lamin A/C shRNA decreased alkaline phosphatase (ALP) activity, but induced preadipocyte factor -1 (Pref-1) mRNA expression. In the presence of adipogenic stimuli, lamin A/C knockdown promotes adipocytes differentiation, as assessed by the detection of an increase in Oil Red O staining. RT-PCR analysis showed that lamin A/C shRNA resulted in increased mRNA expression of PPARγ2 and aP2 during adipocyte differentiation. These results suggest that decreased lamin A/C expression levels not only suppress osteoblast phenotypes but also promote adipocyte differentiation in C26 cells.

DOI： 10.1007/s00418-011-0890-3

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Authorship：Lead author,　Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：OMICS Publishing Group  

DOI： 10.4172/2161-1122.1000e105

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Language：English   Publishing type：Part of collection (book)   Publisher：Nova Science Publishers, Inc.  

Periodontal diseases are featured by chronic infection, inflammation and degeneration of the periodontal tissue that is induced by anaerobic gram-negative bacteria (e.g., Porphyromonas gingivalis). We have recently reported that butyrate, a metabolite produced by periodontopathic bacteria, induced death of the gingival epithelial cells mainly via autophagy. Another study showed that the release of high-mobility group box-1 (HMGB1), a molecule that acts as a group of proinflammatory cytokine when released extracellularly, was detected in the culture medium of gingival epithelial cells after butyrate-induced cell death. These results suggest a possible connection between autophagy and periodontal diseases. P. gingivalis, a causative bacterium of periodontal diseases and butyrate producer, can persist and proliferate in host epithelial cells. The bacterium is internalized in phagosomes of the host cells and then transferred to autophagosomes, where it persists and proliferates. Inhibition of autophagy by 3-methyladenine or wortmannin caused lysosomal fusion with phagosomes containing P. gingivalis, and the lysosomal enzymes digest the bacterium. This suggests that P. gingivalis can utilize a part of the host autophagy mechanism for their own protection from being damaged by host anti-bacterial system. In this chapter, we provide an overview of the relationship between autophagy and the processes of periodontal disease and propose a hypothesis for the mechanism of periodontal disease via autophagy. © 2012 by Nova Science Publishers, Inc. All rights reserved.

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BENTHAM SCIENCE PUBL LTD  

SSH-BM-I is a new synthetic compound that is synthesized from Bromomelatonin by using a newly developed synthetic method. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to SSH-BM-I, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of SSH-BM-I on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In the present study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of SSH-BM-I on apoptosis by analyzing Caspase3/7 activity, translocation of Phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. We found that SSH-BM-I suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of Caspase activation and translocation of PS. Furthermore, SSH-BM-I up-regulated Bcl-2 expression and down-regulated Bax expression. These results suggest that SSH-BM-I has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

DOI： 10.2174/157018011797655287

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

Streptococcus mutans is a bacterial cause of dental caries that is resistant to bacitracin. The aim of this study was to elucidate the mbrABCD-related bacitracin resistance mechanism of S. mutans. Transcriptome data demonstrated that the expression levels of 33 genes were induced more than twofold by bacitracin. Fourteen genes were selected from the upregulated genes, and defective mutants of these genes were constructed for measurement of their sensitivity to bacitracin. Among the mutants, only the mbrA- or mbrB-deficient mutants exhibited 100- to 121-fold greater sensitivity to bacitracin when compared with the wild-type strain. Moreover, knockout of the mbrC and mbrD genes abolished the bacitracin-induced mbrAB upregulation. These results suggest that both mbrC and mbrD are required for mbrAB upregulation that confers the bacitracin-resistant phenotype on S. mutans. [Supplementary Table: available only at http://dx.doi.org/10.1254/jphs.11052SC]

DOI： 10.1254/jphs.11052SC

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5 h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.pep.2011.02.005

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC  

We describe a novel role for CD271 in the differentiation of mesenchymal stem cells (MSCs), including deciduous dental pulp stem cells (DDPSCs) and murine multipotent MSCs (C3H10T1/2 cells). The CD271(+) sub-population of deciduous dental pulp cells (CD271(+)/DDPSCs) and the forced expression of CD271 in C3H10T1/2 (10T271) were analyzed by fluorescence-activated cell sorting. CD271 expression was detected in DDPSCs that expressed both CD44 and CD90, simultaneously, and the clonogenic capacity of the CD271(+)/DDPSCs was higher than that of the CD271(-)/DDPSCs that expressed both CD44 and CD90. Further, the differentiation of CD271(+)/DDPSCs into osteoblasts and adipocytes was inhibited although CD271(-)/DDPSCs were capable of differentiating into osteoblasts and adipocytes. CD271 was overexpressed in C3H10T1/2 cells, which have the potential to differentiate into osteoblasts, adipocytes, chondrocytes, and myocytes. CD271 inhibited the differentiation of C3H10T1/2 cells into any of these lineages. These results indicate a role for CD271 in inhibiting the differentiation of MSCs.

DOI： 10.1089/scd.2010.0299

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

SSH-BM-I was synthesized from tryptamine by using a newly developed synthetic method, and it has structural similarity to bromomelatonin. Recently, it had been reported that SSH-BM-I increases osteoblasts in scales of gold fish. However, the effect of SSH-BM-I on osteoblast differentiation in mammalian cells has not yet been examined. Therefore, this study examined the effect of SSH-BM-I on osteoblast differentiation in mesenchymal progenitor-like cells and mature osteoblast-like cells. SSH-BM-I enhanced terminal osteoblast differentiation, as indicated by mineralization, which was accompanied by upregulation of the osteogenic marker genes bone sialoprotein (BSP) and osteocalcin (OC). However, in mesenchymal progenitor ROB-C26 cultures, no mineralized nodules were observed regardless of SSH-BM-I treatment, although BMP-2 was able to induce nodule formation in these cells. Furthermore, BMP-2 induced nodule formation was suppressed by SSH-BM-I treatment in ROB-C26 cultures. We further investigated the impact of the timing and duration of SSH-BM-I treatment on osteoblast differentiation. The effect of SSH-BM-I treatment on osteoblast differentiation of ROB-C26 in the presence of BMP-2 switches from negative to positive sometime between day 6 and 9, because SSH-BM-I treatment enhanced the formation of mineralized nodules when it was started on day 9, but suppressed nodule formation when it was started at day 6 or earlier. These results suggest that the stimulatory effects of SSH-BM-I on the formation of mineralized nodules depend on the degree of cell differentiation.

DOI： 10.1254/jphs.10329FP

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Osteoblasts and adipocytes originate from common mesenchymal progenitor cells and although a number of compounds can induce osteoblastic and adipogenic differentiation from progenitor cells, the underlying mechanisms have not been elucidated. The present study examined the synergistic effects of dexamethasone (Dex) and bone morphogenetic protein (BMP)-2 on the differentiation of clonal mesenchymal progenitor cells isolated from rat calvaria into osteoblasts and adipocytes, as well as the effects of the timing of treatment. Cells were cultured for various periods of time in the presence of Dex and/or BMP-2. When cells were treated with Dex+BMP-2 during the early phase of differentiation, they differentiated into adipocytes. However, when cells were treated with Dex+BMP-2 during the late phase of differentiation, a synergistic effect on in vitro matrix mineralization was observed. To examine differences between the early and late phases of differentiation, ALP activity was measured in the presence of BMP-2. ALP activity increased markedly on Day 9, corresponding to the onset of the synergistic effect of Dex. Dex treatment inhibited osterix (OSX) expression in cells committed to adipogenic differentiation, but not in cells committed to osteogenic differentiation following BMP-2 treatment. The isoform2 OSX promoter region was found to be involved in the effects of Dex on cells during the early phase of differentiation. Furthermore, cells stably expressing OSX (isoform2) formed mineralized nodules even though they had been treated with Dex+BMP-2 during the early phase of differentiation. It appears that Dex modulates osteogenesis and adipogenesis in mesenchymal stem cells by regulating OSX expression. J. Cell. Physiol. 226: 739-748, 2011. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jcp.22392

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE PHARMACOLOGICAL SOC  

MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA I on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA I had no effect on cell proliferation or cell cycle progression. However, MS-IPA I suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1 treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1 treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway.

DOI： 10.1254/jphs.10208FP

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Alkaline phosphatase (ALP) is generally believed to be a faithful marker of osteoblast differentiation, and its expression is induced by bone morphogenetic protein-2 (BMP-2) and dexamethasone (Dex). However, the effects of combined administration of BMP-2 and Dex on ALP transcription have not been extensively examined. In this study, we found that BMP-2 and Dex synergistically increase ALP levels in mouse C3H10T1/2 pluripotent stem cells. However, switching from one inducer to the other, by adding BMP-2 or Dex to cell cultures at different times, was no more effective than continuous treatment with either inducer alone. A significant induction of ALP mRNA expression was observed only in cells continuously treated with both inducers. This result suggests that both BMP-2 and Dex may act in the same pathway or at the same stage of differentiation. A luciferase assay using ALP promoter deletion constructs showed that a region of the promoter containing a putative signal transducer and activator of transcription 3 (STAT3) response element (SRE) responds to treatment with a combination of BMP-2 and Dex. Furthermore, a ChIP assay indicated that STAT3 bound to the SRE. In addition, a STAT3 siRNA suppressed the synergistic effect of BMP-2 and Dex on ALP levels. These results indicate that STAT3 may play an important role in regulating ALP expression. To our knowledge, this is the first time that STAT3 has been implicated in the regulation of ALP expression by BMP-2 and Dex. These findings raise the possibility of developing new strategies for the enhancement of bone formation using a combination of BMPs and Dex. J. Cell. Physiol. 223: 123-133, 2010. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jcp.22017

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.tripleo.2009.08.028

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN ENDOCRINE SOC  

SST-VEDI-1(VEDI-1) is a new synthetic compound that is synthesized from tryptamine, and has structural similarity to the SSH-BM family of compounds. However, the biological effects of VEDI-1 have yet to be well characterized. A recent report has demonstrated that SSH-BM-type compounds can stimulate osteoblast activity in cultured scales of goldfish. In this study, we examined the effects of VEDI-1 on osteoblastic differentiation as well as its effects on apoptosis, which is known to be closely related to osteoblastic differentiation. We found that VEDI-1 enhanced the formation of mineralized nodules in rat osteoblast cell lines, including ROS17/2.8 cells, and in mouse pre-osteoblast cell lines, including MC3T3-E1 cells, in a dose dependent manner, which was accompanied by increased expression of late osteoblast markers, bone sialoprotein (BSP) and osteocalcin (OC). Furthermore, VEDI-1 inhibited apoptotic cell death and regulated the expression of proteins in the Bcl-2 family. These results suggest that VEDI-1 may facilitate late differentiation of osteoblasts and may have an inhibitory effect on apoptosis.

DOI： 10.1507/endocrj.K08E-340

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Intercellular adhesion molecul-1 (ICAM-1) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of adhesion molecules and plays perdominant roles in recruitment and trafficking of leucocytes to sites of inflammation. ICAM-1 expression in intestinal epithelial cells (IECs) is enhanced by several stimuli, such as proinflammatory cytokines, bacterial infections or pathogen-associated molecular patterns. One of these stimuli, double-stranded RNA (dsRNA), is a by-product of viral replication and can be recognized by its cognate receptor Toll-like receptor 3 (TLR-3). In spite of expression of both TLR-3 and ICAM-1 in IECs, correlation between TLR-3-signalling and ICAM-1 expression has never been examined in IECs. In the present study, we investigated whether poly I:C, an analogue of dsRNA, can stimulate the expression of ICAM-1 in IEC line, HT-29. Poly I:C-stimulation up-regulated the expression of ICAM-1 mRNA by real-time polymerase chain reaction. Enhanced expression of ICAM-1 was confirmed in protein level by immunofluoresense cell staining and enzyme-linked immunosorbent assay by measuring the released soluble ICAM-1 in culture supernatant. As the stimulation effect was reduced by pre-treatment of the cells with anti-TLR-3 antibody, poly I:C-binding signal was thought to be sensed by TLR-3 on the surface of HT-29. The results of luciferase assay and nuclear factor kappa-b (NF-kB) inhibitor treatment experiments indicated that the downstream signal was mainly transduced by transcription factor, NF-kB. All these results demonstrated the connection between TLR-3 signalling and ICAM-1 expression in HT-29 cells and indicated the importance of coordinated function of both innate and adaptive immunity against viral infections.

DOI： 10.1111/j.1365-2249.2009.03892.x

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

SST-VEDI-1(VEDI-1) is a new synthetic compound which is synthesized from tryptamine. However, the effect of VEDI-1 on various bio-phenomena in cells has not yet been examined. Tryptamine is one of the known trace amines. Trace amines are present in the central nervous system at very low concentrations and they are generally considered to have potent sympathomimetic actions. On the other hand, SSH-BM-I and SSH-BM-II-type compounds have been demonstrated to stimulate osteoblast activity in the cultured scales of goldfish. These compounds are also synthesized from tryptamine. VEDI-1 has a similar chemical structure to that of SSH-BM-I and SSH-BM-II-type compounds. Therefore, this study examined the effect of VEDI-1 on osteoblastic differentiation. VEDI-1 inhibited the osteoblast differentiation identified by mineralization, which was accompanied by the down-regulation of the expression of an osteogenic transcription factor, Osterix (OSX). Furthermore, as well as VEDI-1-treatment, the suppression of the OSX expression by stable-transfection with OSX/shRNA decreased the formation of mineralized nodules. These results suggest a possibility that VEDI-1 inhibits the osteoblast differentiation by suppressing the OSX expression.

DOI： 10.1093/jb/mvn164

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

Dentin matrix protein 1 (DMP1) is an acidic phosphorylated extracellular protein and essential for mineralization of dentin and bone; however, the precise mechanism regulating DMP1 expression is not fully understood. A synthetic glucocorticoid (GC), dexamethasone (Dex), promotes an early osteoblast differentiation of a mesenchymal progenitor, ROB-C26 (C26), in parallel with inductive expression of an osteoblast-specific transcription factor, Runx2, and other extracellular matrix proteins such as osteocalcin and bone sialoprotein (BSP). We have examined the effect of Dex on DMP1 expression via induction of Runx2 in C26 cells. Real time RT-PCR showed that Dex increases DMP1 mRNA expression levels at time- and dose-dependent manners and a GC antagonist, RU486, drastically inhibited DMP1 mRNA expression levels. Furthermore, Dex increased the luciferase activity of six-repeated osteoblast-specific cis-acting element 2 (6 x OSE2), which is the binding sequence of Runx2, suggesting that Dex stimulates DMP1 expression via activation of Runx2. However, unexpected results showed that overexpression of exogenous Runx2 depressed DMP1 mRNA expression level, even after cells had been treated with Dex, while downregulated expression of endogenous Runx2 enhanced Dex-induced DMP1 mRNA expression level. These results imply that large amounts of exogenous Runx2 inhibit DMP1 expression, whereas small amounts are more effective for Dex-induced DMP1 expression in C26 cells. Therefore, Dex may activate some factors that inhibit negative action of Runx2 on DMP1 expression. Since mitogen-activating protein kinase (MAPK) phosphatase-1 (MKP-1) has been reported to affect the Dex-induced osteoblast differentiation via decrease of Runx2-phosphorylation, we focus on the relationship between MKP-1 and DMP1 expression. Dex increases MKP-1 expression, and overexpression of exogenous MKP-1 showed significant increase of luciferase activity of 6 x OSE up to the level detected in Dex-treated C26 cells. However, no inductive DMP1 mRNA expression level was found in C26 cells unlike BSP and OPN. These results suggest that although MKP-1 increases DNA-binding activity of Runx2, DMP1 expression may require the collaboration of MKP-1 and additive factors to stimulate Runx2-mediated DMP1 expression in the post-transcriptional event of Dex-treated C26 cells. (C) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.cellbi.2007.08.033

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We examined the effects of dexamethasone (Dex), a synthetic glucocorticoid, on the formation of mineralized bone nodules and the gene expressions of the late ostcoblastic markers, bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN) in mature osteoblast ROS17/2.8 cells. Treatment of ROS17/2.8 cells with Dex resulted in the induction of mineralization accompanied with increasing BSP and OC expressions. Previous reports have demonstrated that BSP and OC expressions are regulated by Runx2. Then, we hypothesized that Dex might promote osteoblastic differentiation and mineralization on ROS 17/2.8 by Runx2. In this study, no effect was observed in mRNA and protein expression of Runx2. However, the transcriptional activity of Runx2 was enhanced by Dex treatment. Furthermore, the Dex-induced BSP and OC expressions decreased after the transfection of Runx2 small-interfering RNAs (siRNAs). These results suggested that the enhancement of Runx2 transcriptional activity by Dex treatment may be followed by the activation of ostcoblast marker genes, such as BSP and OC to thereby produce a bone-specific matrix that subsequently becomes mineralized. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.07.192

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The Fox family of transcription factors is expressed in various organs and tissues during development, and is involved in a variety of developmental and cellular differentiation processes. Foxc2 mRNA is strongly expressed in mesoderm-derived tissues in the embryo, but the molecular mechanism of Foxc2-induced cellular differentiation and the physiological role of Foxc2 are unclear.
In mouse myoblast C2C12 cells, over-expression of Foxc2 increased the expression of desmin, the muscle-specific member of the intermediate filament family of proteins, and induced the synthesis of myotubes. Transient expression of Foxc2 increased MyoD mRNA and protein levels, as assessed by real-time PCR and Western blot, respectively. Chromatin immunoprecipitation (ChIP) analysis showed that Foxc2 does not bind to the promoter region of the MyoD gene, which indicated that Foxc2 does not directly activate MyoD. In contrast to reports that Foxc2 regulates the production of basement membrane components in endothelial cells, we found no evidence of Foxc2-mediated regulation of Collagen type IV alpha 1 (Col4a1) or Col4a2 in myoblast cells. Taken together, these results indicate that Foxc2 plays an important role in the development of the mesenchyme through the regulation of MyoD gene expression. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.05.009

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

CENP-H is a constitutive centromere component that localizes to the centromere throughout the cell cycle. Because CENP-H is colocalized with CENP-A and CENP-C, it is thought to be an inner centromere protein. We previously generated a conditional loss-of-function mutant of CENP-H and showed that CENP-H is required for targeting of CENP-C to the centromere in chicken DT40 cells. In the present study, we used this mutant to identify the functional region of chicken CENP-H necessary for centromere targeting and cell viability. This region was found by yeast two-hybrid analysis to interact with Hec1, which is a member of the Nuf2 complex that transiently localizes to the centromere during mitosis. Coimmunoprecipitation experiments revealed that CENP-H interacts with the Nuf2 complex in chicken DT40 cells. Photobleaching experiments showed that both Hec1 and CENP-H form stable associations with the centromeres during mitosis, suggesting that Hec1 acts as a structural component of centromeres during mitosis. On the basis of these results and previously published data, we propose that the Nuf2 complex functions as a connector between the inner and outer kinetochores.

DOI： 10.1128/MCB.25.5.1958-1970.2005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENT-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.

DOI： 10.1093/emboj/20.16.4603

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Authorship：Lead author   Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Venue：岐阜  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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