記述言語：日本語   出版者・発行元：(一社)日本小児外科学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Paired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated. METHODS: PRRX1 expression in human Malignant peripheral nerve sheath tumours (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA-seq and structural prediction were used to identify proteins interacting with PRRX1. RESULTS: High expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2 A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumour malignancy-related gene sets including mTORC1, KRAS and SRC signalling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1-TOP2A interaction. CONCLUSION: Targeting the PRRX1-TOP2A interaction in malignant tumours with high PRRX1 expression might provide a novel tumour-selective therapeutic strategy.

DOI： 10.1038/s41416-024-02632-8

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：日本レックリングハウゼン病学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

T-cell acute leukemia and lymphoma have a poor prognosis. Although new therapeutic agents have been developed, their therapeutic effects are suboptimal. α-Pinene, a monoterpene compound, has an antitumor effect on solid tumors; however, few comprehensive investigations have been conducted on its impact on hematologic malignancies. This report provides a comprehensive analysis of the potential benefits of using α-pinene as an antitumor agent for the treatment of T-cell tumors. We found that α-pinene inhibited the proliferation of hematologic malignancies, especially in T-cell tumor cell lines EL-4 and Molt-4, induced mitochondrial dysfunction and reactive oxygen species accumulation, and inhibited NF-κB p65 translocation into the nucleus, leading to robust apoptosis in EL-4 cells. Collectively, these findings suggest that α-pinene has potential as a therapeutic agent for T-cell malignancies, and further investigation is warranted.

DOI： 10.1111/cas.16086

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記述言語：日本語   出版者・発行元：(一社)日本移植学会  

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society of Hematology  

T-cell acute leukemia and lymphoma have poor prognoses, necessitating the development of novel therapeutic agents. In this study, we investigated the antitumor activity of α-pinene, a monoterpene compound, against T-cell tumors. We evaluated the effects of α-pinene on cell growth inhibition in vitro using the murine T-cell tumor cell line EL-4 and the human-derived T-cell tumor cell line Molt-4. In addition, we assessed the effects of limonene, another monoterpene with reported tumor-suppressive effects, as a comparative agent.

Both α-pinene and limonene demonstrated concentration- and time-dependent inhibition of cell growth in both the EL-4 and Molt-4 cell lines (Figure 1). Moreover, α-pinene exhibited greater potency than limonene in inhibiting the growth of various hematologic malignancies. Importantly, α-pinene and limonene had minimal effects on the growth of normal murine spleen T cells. Further investigation of the mechanisms of action of α-pinene revealed its ability to induce apoptosis and cell cycle arrest in EL-4 and Molt-4 cells. Transcriptomic profiling of α-pinene-treated EL-4 cells using RNA-seq identified differentially expressed genes associated with cellular damage and mitochondrial dysfunction. Increased intracellular ROS levels and mitochondrial impairment were observed in the T-cell tumors treated with α-pinene. The activation of intrinsic apoptotic pathways involving EGR1, p53, BCL-2, and BAX was found to contribute to α-pinene-induced apoptosis in T-cell tumors. Moreover, α-pinene inhibited the NF-κB signaling pathway, resulting in the reduced nuclear translocation of NF-κB p65 and decreased total intracellular NF-κB p65 levels in EL-4 cells. Additionally, we discovered that α-pinene induced ferroptosis, a newly identified programmed cell death process, in EL-4 cells through lipid peroxidation and iron accumulation. Activation of the system x c−/GSH/GPX4 axis and upregulation of iron transporters, such as Slc39a8 and TfR1, were observed in α-pinene-induced ferroptosis. Treatment with the ferroptosis inhibitor Fer-1 partially reversed the tumor-inhibiting effect of α-pinene in EL-4 cells. Finally, we administrated α-pinene to mice subcutaneously injected with luciferase-expressing EL-4 to evaluate the efficacy of α-pinene in vivo. The tumor growth was significantly inhibited by α-pinene treatment (vehicle: 1055 ± 101 mm 3; α-pinene: 701 ± 82 mm 3, p < 0.01, Figure 2) without adverse effects on body weight or behavior. Immunohistochemistry analyses revealed a significant increase of CD8 + T-cells (vehicle: 8.1 ± 2.7 cells / field of view; α-pinene: 43.1 ± 12.2 cells / field of view , p < 0.05) and NK1.1 + cells (vehicle: 5.1 ± 1.0 cells / field of view ; α-pinene: 41.1 ± 9.8 cells / field of view , p < 0.01) in the tumors, suggesting that α-pinene may have an indirect antitumor effect by recruiting immune cells into tumors.

Overall, our findings demonstrate the antitumor activity of α-pinene against T-cell tumors through the induction of apoptosis, cell cycle arrest, and ferroptosis. α-Pinene shows promise as a potential therapeutic agent for T-cell tumors and warrants further investigation for its clinical application.

DOI： 10.1182/blood-2023-181447

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Production of cartilaginous particles for regenerative medicine requires a large supply of chondrocytes and development of suitable production techniques. Previously, we successfully produced human induced pluripotent stem cell (hiPSC)-derived limb bud mesenchymal cells (ExpLBM cells) with a high chondrogenic differentiation potential that stably proliferate. It may be possible to use these cells in combination with a stirred bioreactor to develop a tissue-engineered cell culture technology with potential for scale-up to facilitate production of large amounts of cartilaginous particles. ExpLBM cells derived from 414C2 and Ff-I 14s04 (human leukocyte antigen homozygous) hiPSCs were seeded into a stirred bioreactor containing cartilage induction medium. To characterize the cartilaginous particles produced, we performed real-time quantitative reverse transcription-polymerase chain reaction and histological analyses. Additionally, we transplanted the cartilage tissue into osteochondral defects of immunocompromised rats to assess its functionality, and evaluated engraftment of the grafted tissue. We successfully produced large amounts of cartilaginous particles via cartilage induction culture in a stirred bioreactor. This tissue exhibited significantly increased expression levels of type II collagen (COL2), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), as well as positive Safranin O and Toluidine blue staining, indicating that it possesses characteristics of hyaline cartilage. Furthermore, engrafted tissues in osteochondral knee defects of immunodeficient rats were positively stained for human vimentin, COL2, and ACAN as well as with Safranin O. In this study, we successfully generated large amounts of hiPSC-derived cartilaginous particles using a combination of tissue engineering techniques. This method is promising as a cartilage regeneration technology with potential for scale-up.

DOI： 10.1016/j.bbrc.2023.149146

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

With the advancement of tissue engineering technologies, implantable materials have been developed for use in facial plastic surgery, including auriculoplasty and rhinoplasty. Tissue-engineered cartilage comprising only cells and cell-produced extracellular matrix is considered valuable as there is no need to consider problems associated with the absorption of scaffold materials and the generation of immune responses. However, it is exceedingly difficult to produce large-sized complex shapes of cartilage without the use of scaffolds. In this study, we describe the production of shape-designable cartilage using a novel cell self-aggregation technique (CAT) and chondroprogenitor cells derived from human induced pluripotent stem cells as the source. The method described does not require special equipment such as bio-3D printers, and the produced tissue can be induced into well-matured cartilage with abundant cartilage matrix in vitro. Using CAT, we were able to generate cartilage in the form of rings or tubes with adjustable inner diameter and curvature, over a range of several centimeters, without the use of scaffolds. The in vitro fabrication of shape-designable cartilage using CAT will undoubtedly contribute to developments in the field of facial plastic surgery.&#xD.

DOI： 10.1088/1748-605X/ad02d1

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(一社)日本移植学会  

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記述言語：日本語   出版者・発行元：(一社)日本移植学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Cell sheet fabrication for articular cartilage regenerative medicine necessitates a large number of chondrocytes of consistent quality as a cell source. Previously, we have developed human-induced pluripotent stem cell (iPSC)-derived expandable PRRX1+ limb-bud mesenchymal cells (ExpLBM) with stable expansion and high chondrogenic capacity, while in this study; our ExpLBM technology was combined with cell sheet engineering to assess its potential as a stable cell source for articular cartilage regeneration. METHODS: ExpLBM cells derived from human-induced pluripotent stem cells (hiPSCs), including 414C2 and Ff-KVs09 (HLA homozygous), were seeded onto a culture plate and two-dimensional chondrogenic induction (2-DCI) was initiated. After 2-DCI, ExpLBM-derived chondrocytes were stripped and transferred to temperature-responsive culture inserts and the chondrocyte sheets were histologically examined or transplanted into osteochondral knee defects of immunodeficient rats. RESULTS: Immunohistochemistry revealed that ExpLBM-derived cell sheets were positive for Safranin O, COL2, and ACAN but that they were negative for COL1 and RUNX2. Furthermore, the engrafted tissues in osteochondral knee defects in immunodeficient rats were stained with SafO, human VIMENTIN, ACAN, and COL2. CONCLUSIONS: The present study is the first to report the chondrocyte sheet fabrication with hiPSC-derived cell source. hiPSC-derived ExpLBM would be a promising cell source for cell sheet technology in articular cartilage regenerative medicine.

DOI： 10.1186/s13287-023-03252-4

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記述言語：日本語   出版者・発行元：日本レックリングハウゼン病学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Here, we present a protocol for the selective differentiation of human pluripotent stem cells mimicking human developmental processes into expandable PRRX1+ limb-bud mesenchymal (ExpLBM) cells. This approach enables expansion through serial passage while maintaining capacity for chondrogenic differentiation. For complete details on the use and execution of this protocol, please refer to Yamada et al. (2021, 2022).

DOI： 10.1016/j.xpro.2022.101786

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Tendon regeneration is difficult because detailed knowledge about tendon progenitor cells (TPCs), which produce tenocytes to repair tendon tissue, has not been revealed. Mohawk homeobox (MKX) is a marker of TPCs or tenocytes, but a human pluripotent stem cell (hPSC)-based reporter system that visualizes MKX+ cells has not been developed. Here, we established an hPSC-derived MKX-tdTomato reporter cell line and tested the induction ratio of MKX-tdTomato+ cells using our stepwise/xeno-free differentiation protocol. MKX-tdTomato+ cells were generated with high efficiency and expressed tendon-specific markers, including MKX, SCX, TNMD, and COL1A1. Our MKX-tdTomato hPSC line would be a useful tool for studying the development or regeneration of tendon tissue.

DOI： 10.1186/s13287-022-03203-5

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Three-dimensional cell constructs comprising only tissue-specific cells and extracellular matrix secreted by them would be ideal transplants, but their fabrication in a cell aggregation manner without cell scaffolds relies on random cell self-aggregation, making the control of their size and shape difficult. In this study, we propose a method to fabricate band-shaped tissues by inducing the self-aggregation of cell sheets using the developed cell self-aggregation technique (CAT). Acting as cell aggregation stoppers, silicone semicircular pillars were attached to two positions equidistant from both short ends of the rounded rectangular culture groove and coated with a specifically charged biomimetic polymer as a CAT-inducing surface. Mesenchymal stem cells, chondrocytes, and skeletal myoblast cells seeded on the surface of the culture grooves formed band-shaped aggregates between the two aggregation stoppers following spontaneous detachment with aggregation of the cell sheet from the outer edge of the grooves during day one of culture. The aggregated chondrocyte band matured into a cartilage-like plate with an abundant cartilage matrix while retaining its band shape after two weeks of chondrogenic cultivation. Additionally, the aggregates of mesenchymal stem cells and myoblast cell bands could patch the induced collagen membrane derived from rat subcutaneous tissue like a bandage immediately after their formation and successfully mature into fat and muscle tissues, respectively. These results indicate that, depending on the cell type, scaffold-free band-shaped cell aggregates produced by CAT have the potential to achieve tissue regeneration that follows the shape of the defect viain vitromaturation culture orin vivoorganization.

DOI： 10.1088/1748-605X/ac9c7f

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Our previous gene profiling analysis showed that the transcription cofactor vestigial-like 3 (VGLL3) gene expression was upregulated by mechanical tension in the mouse cranial suture, coinciding with accelerated osteoblast differentiation. Therefore, we hypothesized that VGLL3 plays a significant role in osteogenic differentiation. To clarify the function of VGLL3 in osteoblasts, we examined its expression characteristics in mouse bone tissue and the osteoblastic cell line MC3T3-E1. We further examined the effects of Vgll3 knockdown on osteoblast differentiation and bone morphogenetic protein (BMP) signaling. In the mouse cranial suture, where membranous ossification occurs, VGLL3 was immunohistochemically detected mostly in the nucleus of osteoblasts, preosteoblasts, and fibroblastic cells. VGLL3 expression in MC3T3-E1 cells was transient and peaked at a relatively early stage of differentiation. RNA sequencing revealed that downregulated genes in Vgll3-knockdown cells were enriched in gene ontology terms associated with osteoblast differentiation. Interestingly, most of the upregulated genes were related to cell division. Targeted Vgll3 knockdown markedly suppressed the expression of major osteogenic transcription factors (Runx2, Sp7/osterix, and Dlx5) and osteoblast differentiation. It also attenuated BMP signaling; moreover, exogenous BMP2 partially restore osteogenic transcription factors' expression in Vgll3-knockdown cells. Furthermore, overexpression of Vgll3 increased the expression of osteogenic transcription factors. These results suggest that VGLL3 plays a critical role in promoting osteoblast differentiation and that part of the process is mediated by BMP signaling. Further elucidation of VGLL3 function will increase our understanding of osteogenesis and skeletal disease etiology.

DOI： 10.1007/s00223-022-00997-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Stem cell-based therapies and experimental methods rely on efficient induction of human pluripotent stem cells (hPSCs). During limb development, the lateral plate mesoderm (LPM) produces limb-bud mesenchymal (LBM) cells that differentiate into osteochondroprogenitor cells and form cartilage tissues in the appendicular skeleton. Previously, we generated PRRX1-tdTomato reporter hPSCs to establish the protocol for inducing the hPSC-derived PRRX1+ LBM-like cells. However, surface antigens that assess the induction efficiency of hPSC-derived PRRX1+ LBM-like cells from LPM have not been identified. Here, we used PRRX1-tdTomato reporter hPSCs and found that high pluripotent cell density suppressed the expression of PRRX1 mRNA and tdTomato after LBM-like induction. RNA sequencing and flow cytometry suggested that PRRX1-tdTomato+ LBM-like cells are defined as CD44high CD140Bhigh CD49f-. Importantly, other hPSC lines, including four human induced pluripotent stem cell lines (414C2, 1383D2, HPS1042, HPS1043) and two human embryonic stem cell lines (SEES4, SEES7), showed the same results. Thus, an appropriate cell density of hPSCs before differentiation is a prerequisite for inducing the CD44high CD140Bhigh CD49f- PRRX1+ LBM-like cells.

DOI： 10.3390/ijms23052661

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Optogenetics, a technology to manipulate biological phenomena thorough light, has attracted much attention in neuroscience. Recently, the Magnet System, a photo-inducible protein dimerization system which can control the intracellular behavior of various biomolecules with high accuracy using light was developed. Furthermore, photoactivation systems for controlling biological phenomena are being developed by combining this technique with genome-editing technology (CRISPR/Cas9 System) or DNA recombination technology (Cre-loxP system). Herein, we review the history of optogenetics and the latest Magnet System technology and introduce our recently developed photoactivatable Cre knock-in mice with temporal-, spatial-, and cell-specific accuracy.

DOI： 10.18926/AMO/63202

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記述言語：日本語   出版者・発行元：公益社団法人 日本薬理学会  

[Background] To achieve the disease modeling of skeletal dysplasia, human induced pluripotent stem cells (iPSCs) will be useful but the induction method for limb bud mesenchymal (LBM) cells, which can give rise to most of the future limb elements - bone, cartilage and tendon/ligament, has not been established. In this study, we developed the induction and expansion protocol of LBM cells from human iPSCs, and a high-throughput drug screening system using human LBM cells differentiated from iPSC which is derived from patients with skeletal dysplasia.

[Method] Paired-related homeobox 1 (PRRX1) serves as a marker for the LBM. To assess the induction efficiency of PRRX1 positive cells during each differentiation step, we established PRRX1-tdTomato reporter human iPSC line. Lateral plate mesoderm cells (LPM) derived from PRRX1-tdTomato reporter were used to establish the LBM-inducing method that recapitulates human developmental process. The chondrogenic capacity of expandable LBM (ExpLBM) cells was assessed using our two- or three- dimensional chondrogenic induction method (2DCI or 3DCI). In addition, ExpLBM cells differentiated from iPSCs derived from patients with type II collagenopathy (COL2pathy), one of the skeletal dysplasia arising from mutations in COL2A1, were used to develop high-throughput screening system.

[Results] By activating WNT signaling and inhibiting BMP/TGFb/headge hog signaling pathways, almost all LPM cells could be induced to PRRX1 positive LBM cells. Interestingly, we found the defined culture method that can not only stably expand LBM cells (ExpLBM) but also maintain their PRRX1 expression. ExpLBM formed Alcian Blue positive nodules under 2DCI condition and Safranin O positive cartilaginous particles under 3DCI condition. 2DCI-based high-throughput screening system found that several chemicals improved the chondrogenic capacity of ExpLBM derived from COL2pathy patient.

[Conclusion] ExpLBM cells will be a potential tool to study human bone development, cartilage regeneration and drug discovery using disease-specific human iPSCs.

DOI： 10.1254/jpssuppl.95.0_2-o-087

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/biof.1774

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/biof.1774

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1-tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells' chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.

DOI： 10.1038/s41551-021-00778-x

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lung adenocarcinoma (LUAD) is the most common types among lung cancers generally arising from terminal airway and understanding of multistep carcinogenesis is crucial to develop novel therapeutic strategy for LUAD. Here we used human induced pluripotent stem cells (hiPSCs) to establish iHER2-hiPSCs in which doxycycline induced the expression of the oncoprotein human epidermal growth factor receptor 2 (HER2)/ERBB2. Lung progenitors that differentiated from iHER2-hiPSCs, which expressed NKX2-1/TTF-1 known as a lung lineage maker, were cocultured with human fetal fibroblast and formed human lung organoids (HLOs) comprising alveolar type 2-like cells. HLOs that overexpressed HER2 transformed to tumor-like structures similar to atypical adenomatous hyperplasia, which is known for lung precancerous lesion and upregulated the activities of oncogenic signaling cascades such as RAS/RAF/MAPK and PI3K/AKT/mTOR. The degree of morphological irregularity and proliferation capacity were significantly higher in HLOs from iHER2-hiPSCs. Moreover, the transcriptome profile of the HLOs shifted from a normal lung tissue-like state to one characteristic of clinical LUAD with HER2 amplification. Our results suggest that hiPSC-derived HLOs may serve as a model to recapitulate the early tumorigenesis of LUAD and would provide new insights into the molecular basis of tumor initiation and progression.

DOI： 10.1002/ijc.33713

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFβ. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.

DOI： 10.1172/JCI141566

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Paired related homeobox 1 (PRRX1) is a marker of limb bud mesenchymal cells, and deficiency of p53 or Rb in Prrx1-positive cells induces osteosarcoma in several mouse models. However, the regulatory roles of PRRX1 in human osteosarcoma have not been defined. In this study, we performed PRRX1 immunostaining on 35 human osteosarcoma specimens to assess the correlation between PRRX1 level and overall survival. In patients with osteosarcoma, the expression level of PRRX1 positively correlated with poor prognosis or the ratio of lung metastasis. Additionally, we found PRRX1 expression on in 143B cells, a human osteosarcoma line with a high metastatic capacity. Downregulation of PRRX1 not only suppressed proliferation and invasion but also increased the sensitivity to cisplatin and doxorubicin. When 143B cells were subcutaneously transplanted into nude mice, PRRX1 knockdown decreased tumor sizes and rates of lung metastasis. Interestingly, forskolin, a chemical compound identified by Connectivity Map analysis using RNA expression signatures during PRRX1 knockdown, decreased tumor proliferation and cell migration to the same degree as PRRX1 knockdown. These results demonstrate that PRRX1 promotes tumor malignancy in human osteosarcoma.

DOI： 10.1016/j.tranon.2020.100960

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Medication-related osteonecrosis of the jaw (MRONJ) is a severe pathological condition associated mainly with the long-term administration of bone resorption inhibitors, which are known to induce suppression of osteoclast activity and bone remodeling. Bone Morphogenetic Protein (BMP)-2 is known to be a strong inducer of bone remodeling, by directly regulating osteoblast differentiation and osteoclast activity. This study aimed to evaluate the effects of BMP-2 adsorbed onto beta-tricalcium phosphate (β-TCP), which is an osteoinductive bioceramic material and allows space retention, on the prevention and treatment of MRONJ in mice. Tooth extraction was performed after 3 weeks of zoledronate (ZA) and cyclophosphamide (CY) administration. For prevention studies, BMP-2/β-TCP was transplanted immediately after tooth extraction, and the mice were administered ZA and CY for an additional 4 weeks. The results showed that while the tooth extraction socket was mainly filled with a sparse tissue in the control group, bone formation was observed at the apex of the tooth extraction socket and was filled with a dense connective tissue rich in cellular components in the BMP-2/β-TCP transplanted group. For treatment studies, BMP-2/β-TCP was transplanted 2 weeks after tooth extraction, and bone formation was followed up for the subsequent 4 weeks under ZA and CY suspension. The results showed that although the tooth extraction socket was mainly filled with soft tissue in the control group, transplantation of BMP-2/β-TCP could significantly accelerate bone formation, as shown by immunohistochemical analysis for osteopontin, and reduce the bone necrosis in tooth extraction sockets. These data suggest that the combination of BMP-2/β-TCP could become a suitable therapy for the management of MRONJ.

DOI： 10.3390/ijms21197028

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Core Binding Factors (CBFs) are a small group of heterodimeric transcription factor complexes composed of DNA binding proteins, RUNXs, and a non-DNA binding protein, CBFB. The LH surge increases the expression of Runx1 and Runx2 in ovulatory follicles, while Cbfb is constitutively expressed. To investigate the physiological significance of CBFs, we generated a conditional mutant mouse model in which granulosa cell expression of Runx2 and Cbfb was deleted by the Esr2Cre. Female Cbfbflox/flox;Esr2cre/+;Runx2flox/flox mice were infertile; follicles developed to the preovulatory follicle stage but failed to ovulate. RNA-seq analysis of mutant mouse ovaries collected at 11 h post-hCG unveiled numerous CBFs-downstream genes that are associated with inflammation, matrix remodeling, wnt signaling, and steroid metabolism. Mutant mice also failed to develop corpora lutea, as evident by the lack of luteal marker gene expression, marked reduction of vascularization, and excessive apoptotic staining in unruptured poorly luteinized follicles, consistent with dramatic reduction of progesterone by 24 h after hCG administration. The present study provides in vivo evidence that CBFs act as essential transcriptional regulators of both ovulation and luteinization by regulating the expression of key genes that are involved in inflammation, matrix remodeling, cell differentiation, vascularization, and steroid metabolisms in mice.

DOI： 10.1038/s41598-020-64257-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Cre-loxP recombination system is widely used to generate genetically modified mice for biomedical research. Recently, a highly efficient photoactivatable Cre (PA-Cre) based on reassembly of split Cre fragments has been established. This technology enables efficient DNA recombination that is activated upon blue light illumination with spatiotemporal precision. In this study, we generated a tTA-dependent photoactivatable Cre-loxP recombinase knock-in mouse model (TRE-PA-Cre mice) using a CRISPR/Cas9 system. These mice were crossed with ROSA26-tdTomato mice (Cre reporter mouse) to visualize DNA recombination as marked by tdTomato expression. We demonstrated that external noninvasive LED blue light illumination allows efficient DNA recombination in the liver of TRE-PA-Cre:ROSA26-tdTomato mice transfected with tTA expression vectors using hydrodynamic tail vein injection. The TRE-PA-Cre mouse established here promises to be useful for optogenetic genome engineering in a noninvasive, spatiotemporal, and cell-type specific manner in vivo.

DOI： 10.1016/j.bbrc.2020.03.015

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.

DOI： 10.1038/s41467-020-16038-6

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記述言語：日本語   出版者・発行元：(一社)中部日本整形外科災害外科学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In drug addiction, environmental stimuli previously associated with cocaine use readily elicit cocaine-associated memories, which persist long after abstinence and trigger cocaine craving and consumption. Although previous studies suggest that the medial prefrontal cortex (mPFC) is involved in the expression of cocaine-addictive behaviors, it remains unclear whether excitatory and inhibitory neurons in the mPFC are causally related to the formation and retrieval of cocaine-associated memories. To address this issue, we used the designer receptors exclusively activated by designer drugs (DREADD) technology combined with a cocaine-induced conditioned place preference (CPP) paradigm. We suppressed mPFC neuronal activity in a cell-type- and timing-dependent manner. C57BL/6J wild-type mice received bilateral intra-mPFC infusion of an adeno-associated virus (AAV) expressing inhibitory DREADD (hM4Di) under the control of CaMKII promotor to selectively suppress mPFC pyramidal neurons. GAD67-Cre mice received bilateral intra-mPFC infusion of a Cre-dependent AAV expressing hM4Di to specifically silence GABAergic neurons. Chemogenetic suppression of mPFC pyramidal neurons significantly attenuated both the acquisition and expression of cocaine CPP, while suppression of mPFC GABAergic neurons affected neither the acquisition nor expression of cocaine CPP. Moreover, chemogenetic inhibition of mPFC glutamatergic neurons did not affect the acquisition and expression of lithium chloride-induced conditioned place aversion. These results suggest that the activation of glutamatergic, but not GABAergic, neurons in the mPFC mediates both the formation and retrieval of cocaine-associated memories.

DOI： 10.1111/adb.12723

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記述言語：日本語   出版者・発行元：(株)羊土社  

ペリサイト(周皮細胞)は、血管を構成する血管内皮細胞の周囲に接着し、血管の安定化や機能維持において重要な役割を担う。特に、中枢神経系においては、血液脳関門の形成・維持においてペリサイトの存在が不可欠であることが知られている。正常な神経機能を維持するうえで、血液脳関門は必須な保護機構であり、加齢などに伴う血液脳関門の機能低下はさまざまな神経変性疾患を引き起こす原因の1つであると考えられている。これらの理由から、近年、ペリサイトによる血液脳関門の機能維持メカニズムは大きな注目を集めている。(著者抄録)

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Global gene deletion studies have established that Runt-related transcription factor-2 (Runx2) is essential during skeletogenesis for osteoblastic differentiation in both intramembranous and endochondral ossification processes. However, the postnatal significance of Runx2 in vivo is poorly understood because a global Runx2 deletion causes perinatal lethality. In this study, we generated tamoxifen-induced Runx2 global deficient mice by crossing Runx2flox mice with ROSA26-CreERT2 mice (Rosa26-CreERT2; Runx2flox/flox). Four-week-old mice were intraperitoneally treated with tamoxifen for five consecutive days, sacrificed, and analyzed six weeks after tamoxifen administration. Deletion of Runx2 led to low bone mass, which is associated with decreased bone formation and bone resorption as well as excessive bone marrow adiposity. Collectively, postnatal Runx2 absolutely plays an important role in maintaining the homeostasis of bone tissues not only in bone mass, but also in the bone marrow environment.

DOI： 10.1016/j.bbrc.2019.07.014

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記述言語：日本語   出版者・発行元：(一社)日本骨代謝学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Runx2 plays an essential role in embryonic disc tissue development in mice. However, the role of runt-related transcription factor 2 (Runx2) in postnatal disc tissue growth and development has not been defined. In the present studies, we generated Runx2 conditional knockout (KO) mice (Runx2Agc1ER ), in which Runx2 was deleted in Aggrecan-expressing cells in disc tissue at postnatal 2-weeks of age. We then analyzed changes in disc tissue growth and development using histology and immunohistochemical methods in 3-month-old mice. We found that large vacuolated notochordal cells were accumulated in the nucleus pulposus (NP) in Runx2 KO mice. The growth plate cartilage tissue in the disc was thicker in Runx2 KO mice. We also found a significant upregulation of Indian hedgehog (Ihh) expression in the cells in NP cells and in annulus fibrosus cells of Runx2 KO mice. These results demonstrated that Runx2 may play an important role in postnatal disc tissue development through interacting with Ihh signaling.

DOI： 10.1002/jcp.27410

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記述言語：日本語   出版者・発行元：(公社)日本補綴歯科学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, an increasing number of studies have demonstrated side effects of BMP-2 therapy. A deeper understanding of the effect of BMP-2 on cells other than those involved directly in bone remodeling is of fundamental importance to promote a more effective delivery of BMP-2 to patients. In this study, we aimed to investigate the effect of BMP-2 in the marrow environment. First, BMP-2 adsorbed onto titanium implants was delivered at the tooth extraction socket (marrow-absent site) or in the mandible marrow of beagle dogs. BMP-2 could induce marked bone formation around the implant at the tooth extraction socket. Surprisingly, however, no bone formation was observed in the BMP-2-coated titanium implants inserted in the mandible marrow. In C57BL/6 mice, BMP-2 adsorbed in freeze-dried collagen pellets could induce bone formation in marrow-absent calvarial bone. However, similar to the canine model, BMP-2 could not induce bone formation in the femur marrow. Analysis of osteoblast differentiation using Col1a1(2.3)-GFP transgenic mice revealed a scarce number of osteoblasts in BMP-2-treated femurs, whereas in the control group, osteoblasts were abundant. Ablation of femur marrow recovered the BMP-2 ability to induce bone formation. In vitro experiments analyzing luciferase activity of C2C12 cells with the BMP-responsive element and alkaline phosphatase activity of MC3T3-E1 osteoblasts further revealed that bone marrow cells inhibit the BMP-2 effect on osteoblasts by direct cell-cell contact. Collectively, these results showed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application. © 2018 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.3598

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In drug addiction, environmental stimuli previously associated with cocaine use readily elicit cocaine-associated memories, which persist long after abstinence and trigger cocaine craving and consumption. Although previous studies suggest that the medial prefrontal cortex (mPFC) is involved in the expression of cocaine-addictive behaviors, it remains unclear whether excitatory and inhibitory neurons in the mPFC are causally related to the formation and retrieval of cocaine-associated memories. To address this issue, we used the designer receptors exclusively activated by designer drugs (DREADD) technology combined with a cocaine-induced conditioned place preference (CPP) paradigm. We suppressed mPFC neuronal activity in a cell-type- and timing-dependent manner. C57BL/6J wild-type mice received bilateral intra-mPFC infusion of an adeno-associated virus (AAV) expressing inhibitory DREADD (hM4Di) under the control of CaMKII promotor to selectively suppress mPFC pyramidal neurons. GAD67-Cre mice received bilateral intra-mPFC infusion of a Cre-dependent AAV expressing hM4Di to specifically silence GABAergic neurons. Chemogenetic suppression of mPFC pyramidal neurons significantly attenu

DOI： 10.1111/adb.12723

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bmc.2018.12.016

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1248/yakushi.18-00173-F

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1254/fpj.153.67

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

The mesenchymal stem cell (MSC) is a type of tissue stem cell. In clinical studies, cultured MSCs have shown important therapeutic effects on diseases via both the reduction of neurological defects and the regulation of immune responses. However, in vivo MSC localization, function, and properties are poorly understood; therefore, the molecular understanding of MSC hierarchy is less advanced compared to hematopoietic stem cell hierarchy. Runt-related transcription factor 2 (Runx2) is an essential transcriptional regulator of osteoblast differentiation from MSCs. Runx2 deficiency in Paired-related homeobox 1 (Prrx1)-derived cells (Runx2Prrx1-/- mice) results in defective intramembranous ossification. Double-positive cells for Prrx1-GFP, and stem cell antigen-1 (Sca1) (Prrx1+Sca1+ cells) in the calvaria, express Runx2 at lower levels, and are more homogeneous and primitive compared with Prrx1+Sca1- cells. Our results suggest that osteoblast differentiation in vivo may begin at the Prrx1+Sca1+ MSC stage, with sequential progression to Prrx1+Sca1- cells, followed by Osterix+Prrx1-Sca1- osteoblast precursors, which eventually form mature α1(I)-collagen+ osteoblasts. This research will enable us to better understand the in vivo molecular biology features of MSCs, leading to their therapeutic applications for tissue repair and regeneration.

DOI： 10.1248/yakushi.18-00173-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The pathophysiological role of mammalian target of rapamycin complex 1 (mTORC1) in neurodegenerative diseases is established, but possible therapeutic targets responsible for its activation in neurons must be explored. Here we identified solute carrier family 38a member 1 (SNAT1, Slc38a1) as a positive regulator of mTORC1 in neurons. Slc38a1 flox/flox and Synapsin I-Cre mice were crossed to generate mutant mice in which Slc38a1 was selectively deleted in neurons. Measurement of 2,3,5-triphenyltetrazolium chloride (TTC) or the MAP2-negative area in a mouse model of middle cerebral artery occlusion (MCAO) revealed that Slc38a1 deficiency decreased infarct size. We found a transient increase in the phosphorylation of p70S6k1 (pp70S6k1) and a suppressive effect of rapamycin on infarct size in MCAO mice. Autophagy inhibitors completely mitigated the suppressive effect of SNAT1 deficiency on neuronal cell death under in vitro stroke culture conditions. These results demonstrate that SNAT1 promoted ischemic brain damage via mTOR-autophagy system.

DOI： 10.1038/s42003-019-0582-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. However, therapeutic strategies against malignant gliomas have not been completely established. Runt-related transcription factor 2 (Runx2) is an essential gene for skeletal development but its regulatory role in the malignant progression of glioma remains unclear. Here we investigated expression levels of RUNX2 in glioma tissues and its regulatory effects on aberrant growth of glioma cells. RUNX2 mRNA levels were higher in GBM tissues than that of normal brains or low-grade gliomas. RUNX2 protein was detected in five out of seven human GBM cell lines and its level was positively correlated with proliferative capacity. Stable transduction of dominant-negative Runx2 in rat-derived C6 glioma cells not only inhibited the promoter activity containing Runx2 response element, but also decreased mRNA expression levels of Runx2 target genes, such as Mmp13 and Spp1, as well as the proliferative capacity. Furthermore, transient introduction of Runx2-targeted siRNAs into C6 glioma cells significantly decreased mRNA expression levels of Mmp13 and Spp1 and the proliferative capacity. Furthermore, Runx2 knockdown suppressed both Ccnd1 mRNA expression and activation of the Ccnd1 promoter by forskolin, a PKA-activating reagent, in C6 glioma cells. Our results demonstrate that cross-talk between cAMP/PKA signaling and RUNX2 promotes a malignant phenotype of glioma cells.

DOI： 10.1007/s11064-018-2626-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Erk5 belongs to the mitogen-activated protein kinase (MAPK) family. Following its phosphorylation by Mek5, Erk5 modulates several signaling pathways in a number of cell types. In this study, we demonstrated that Erk5 inactivation in mesenchymal cells causes abnormalities in skeletal development by inducing Sox9, an important transcription factor of skeletogenesis. We further demonstrate that Erk5 directly phosphorylates and activates Smurf2 (a ubiquitin E3 ligase) at Thr249, which promotes the proteasomal degradation of Smad proteins and phosphorylates Smad1 at Ser206 in the linker region known to trigger its proteasomal degradation by Smurf1. Smads transcriptionally activated the expression of Sox9 in mesenchymal cells. Accordingly, removal of one Sox9 allele in mesenchymal cells from Erk5-deficient mice rescued some abnormalities of skeletogenesis. These findings highlight the importance of the Mek5-Erk5-Smurf-Smad-Sox9 axis in mammalian skeletogenesis.

DOI： 10.1242/dev.164004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-018-28529-0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates.

DOI： 10.1002/jcp.26285

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記述言語：日本語   出版者・発行元：日本結合組織学会  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1210/en.2018-00011

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-018-21000-0

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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 NEUROSCIENCE  

Circadian rhythm disturbances are well established in neurological diseases. However, how these disruptions cause homeostatic imbalances remains poorly understood. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) is a major circadian clock transcriptional activator, and Bmal1 deficiency in male Bmal1nestin-/- mice induced marked astroglial activation without affecting the number of astrocytes in the brain and spinal cord. Bmal1 deletion caused blood-brain barrier (BBB) hyperpermeability with an age-dependent loss of pericyte coverage of blood vessels in the brain. Using Nestin-green fluorescent protein (GFP) transgenic mice, we determined that pericytes are Nestin-GFP+ in the adult brain. Bmal1 deletion caused Nestin-GFP+ pericyte dysfunction, including the downregulation of platelet-derived growth factor receptor β (PDGFRβ), a protein necessary for maintaining BBB integrity. Knockdown of Bmal1 downregulated PDGFRβ transcription in the brain pericyte cell line. Thus, the circadian clock component Bmal1 maintains BBB integrity via regulating pericytes.SIGNIFICANCE STATEMENT Circadian rhythm disturbances may play a role in neurodegenerative disorders, such as Alzheimer's disease. Our results revealed that one of the circadian clock components maintains the integrity of the blood-brain barrier (BBB) by regulating vascular-embedded pericytes. These cells were recently identified as a vital component for the control of BBB permeability and cerebral blood flow. Our present study demonstrates the involvement of circadian clock component Bmal1 in BBB homeostasis and highlights the role of Bmal1 dysfunction in multiple neurological diseases.

DOI： 10.1523/JNEUROSCI.3639-16.2017

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bmc.2017.08.025

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Publishing Group  

Runx2 may play an important role in development of osteoarthritis (OA). However, the specific role of Runx2 in articular chondrocyte function and in OA development in adult mice has not been fully defined. In this study, we performed the destabilization of the medial meniscus (DMM) surgery at 12-week-old mice to induce OA in adult Runx2 Agc1CreER mice, in which Runx2 was specifically deleted in Aggrecan-expressing chondrocytes by administering tamoxifen at 8-weeks of age. Knee joint samples were collected 8- and 12-weeks post-surgery and analyzed through histology, histomorphometry and micro-computed tomography (μCT). Our results showed that severe OA-like defects were observed after DMM surgery in Cre-negative control mice, including articular cartilage degradation and subchondral sclerosis, while the defects were significantly ameliorated in Runx2 Agc1CreER KO mice. Immunohistochemical (IHC) results showed significantly reduced expression of MMP13 in Runx2 Agc1CreER KO mice compared to that in Cre-negative control mice. Results of quantitative reverse-transcription PCR (qRT-PCR) demonstrated that expression of the genes encoding for matrix degradation enzymes was significantly decreased in Runx2 Agc1CreER KO mice. Thus, our findings suggest that inhibition of Runx2 in chondrocytes could at least partially rescue DMM-induced OA-like defects in adult mice.

DOI： 10.1038/s41598-017-02490-w

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：John Wiley and Sons Inc.  

We have previously shown that endochondral ossification is finely regulated by the Clock system expressed in chondrocytes during postnatal skeletogenesis. Here we show a sophisticated modulation of bone resorption and bone mass by the Clock system through its expression in bone-forming osteoblasts. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Period1 (Per1) were expressed with oscillatory rhythmicity in the bone in vivo, and circadian rhythm was also observed in cultured osteoblasts of Per1::luciferase transgenic mice. Global deletion of murine Bmal1, a core component of the Clock system, led to a low bone mass, associated with increased bone resorption. This phenotype was recapitulated by the deletion of Bmal1 in osteoblasts alone. Co-culture experiments revealed that Bmal1-deficient osteoblasts have a higher ability to support osteoclastogenesis. Moreover, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced receptor activator of nuclear factor κB ligand (Rankl) expression was more strongly enhanced in both Bmal1-deficient bone and cultured osteoblasts, whereas overexpression of Bmal1/Clock conversely inhibited it in osteoblasts. These results suggest that bone resorption and bone mass are regulated at a sophisticated level by osteoblastic Clock system through a mechanism relevant to the modulation of 1,25(OH)2D3-induced Rankl expression in osteoblasts. © 2017 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.3053

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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  

We have previously shown that endochondral ossification is finely regulated by the Clock system expressed in chondrocytes during postnatal skeletogenesis. Here we show a sophisticated modulation of bone resorption and bone mass by the Clock system through its expression in bone-forming osteoblasts. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Period1 (Per1) were expressed with oscillatory rhythmicity in the bone in vivo, and circadian rhythm was also observed in cultured osteoblasts of Per1::luciferase transgenic mice. Global deletion of murine Bmal1, a core component of the Clock system, led to a low bone mass, associated with increased bone resorption. This phenotype was recapitulated by the deletion of Bmal1 in osteoblasts alone. Co-culture experiments revealed that Bmal1-deficient osteoblasts have a higher ability to support osteoclastogenesis. Moreover, 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ]-induced receptor activator of nuclear factor κB ligand (Rankl) expression was more strongly enhanced in both Bmal1-deficient bone and cultured osteoblasts, whereas overexpression of Bmal1/Clock conversely inhibited it in osteoblasts. These results suggest that bone resorption and bone mass are regulated at a sophisticated level by osteoblastic Clock system through a mechanism relevant to the modulation of 1,25(OH)2 D3 -induced Rankl expression in osteoblasts. © 2017 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.3053

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/febs.14019

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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-BLACKWELL  

Similar to neurons, microglia have an intrinsic molecular clock. The master clock oscillator Bmal1 modulates interleukin-6 upregulation in microglial cells exposed to lipopolysaccharide. Bmal1 can play a role in microglial inflammatory responses. We previously demonstrated that gliotransmitter ATP induces transient expression of the clock gene Period1 via P2X7 purinergic receptors in cultured microglia. In this study, we further investigated mechanisms underlying the regulation of pro-inflammatory cytokine production by clock molecules in microglial cells. Several clock gene transcripts exhibited oscillatory diurnal rhythmicity in microglial BV-2 cells. Real-time luciferase monitoring also showed diurnal oscillatory luciferase activity in cultured microglia from Per1::Luciferase transgenic mice. Lipopolysaccharide (LPS) strongly induced the expression of pro-inflammatory cytokines in BV-2 cells, whereas an siRNA targeting Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), a core positive component of the microglial molecular clock, selectively inhibited LPS-induced interleukin-6 (IL-6) expression. In addition, LPS-induced IL-6 expression was attenuated in microglia from Bmal1-deficient mice. This phenotype was recapitulated by pharmacological disruption of oscillatory diurnal rhythmicity using the synthetic Rev-Erb agonist SR9011. Promoter analysis of the Il6 gene revealed that Bmal1 is required for LPS-induced IL-6 expression in microglia. Mice conditionally Bmal1 deficient in cells expressing CD11b, including microglia, exhibited less potent upregulation of Il6 expression following middle cerebral artery occlusion compared with that in control mice, with a significant attenuation of neuronal damage. These results suggest that the intrinsic microglial clock modulates the inflammatory response, including the positive regulation of IL-6 expression in a particular pathological situation in the brain, GLIA 2016. GLIA 2017;65:198-208.

DOI： 10.1002/glia.23087

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/MCB.01075-15

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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-BLACKWELL  

Activating transcription factor 3 (Atf3) has been implicated in the pathogenesis of various diseases, including cancer and inflammation, as well as in the regulation of cell proliferation and differentiation. However, the involvement of Atf3 in developmental skeletogenesis and joint disease has not been well studied to date. Here, we show that Atf3 is a critical mediator of osteoarthritis (OA) development through its expression in chondrocytes. ATF3 expression was markedly up-regulated in the OA cartilage of both mice and humans. Conditional deletion of Atf3 in chondrocytes did not result in skeletal abnormalities or affect the chondrogenesis, but alleviated the development of OA generated by surgically inducing knee joint instability in mice. Inflammatory cytokines significantly up-regulated Atf3 expression through the nuclear factor-kB (NF-kB) pathway, while cytokine-induced interleukin-6 (Il6) expression was repressed, in ATF3-deleted murine and human chondrocytes. Mechanistically, Atf3 deficiency decreased cytokine-induced Il6 transcription in chondrocytes through repressing NF-kB signalling by the attenuation of the phosphorylation status of IkB and p65. These findings suggest that Atf3 is implicated in the pathogenesis of OA through modulation of inflammatory cytokine expression in chondrocytes, and the feed-forward loop of inflammatory cytokines/NF-kB/Atf3 in chondrocytes may be a novel therapeutic target for the treatment for OA. Copyright (C) 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DOI： 10.1002/path.4739

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Bone homeostasis is maintained by the sophisticated coupled actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here we identify activating transcription factor 3 (ATF3) as a pivotal transcription factor for the regulation of bone resorption and bone remodeling under a pathological condition through modulating the proliferation of osteoclast precursors. The osteoclast precursor-specific deletion of ATF3 in mice led to the prevention of receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL)-induced bone resorption and bone loss, although neither bone volume nor osteoclastic parameter were markedly altered in these knockout mice under the physiological condition. RANKL-dependent osteoclastogenesis was impaired in vitro in ATF3-deleted bone marrow macrophages (BMM). Mechanistically, the deficiency of ATF3 impaired the RANKL-induced transient increase in cell proliferation of osteoclast precursors in bone marrow in vivo as well as of BMM in vitro. Moreover, ATF3 regulated cyclin D1 mRNA expression though modulating activator protein-1-dependent transcription in the osteoclast precursor, and the introduction of cyclin D1 significantly rescued the impairment of osteoclastogenesis in ATF3-deleted BMM. Therefore, these findings suggest that ATF3 could have a pivotal role in osteoclastogenesis and bone homeostasis though modulating cell proliferation under pathological conditions, thereby providing a target for bone diseases.

DOI： 10.1038/srep30918

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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-BLACKWELL  

The circadian clock controls many behavioral and physiological processes beyond daily rhythms. Circadian dysfunction increases the risk of cancer, obesity, and cardiovascular and metabolic diseases. Although clinical studies have shown that bone resorption is controlled by circadian rhythm, as indicated by diurnal variations in bone resorption, the molecular mechanism of circadian clock-dependent bone resorption remains unknown. To clarify the role of circadian rhythm in bone resorption, aryl hydrocarbon receptor nuclear translocator-like (Bmal1), a prototype circadian gene, was knocked out specifically in osteoclasts. Osteoclast-specific Bmal1-knockout mice showed a high bone mass phenotype due to reduced osteoclast differentiation. A cell-based assay revealed that BMAL1 upregulated nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (Nfatc1) transcription through its binding to an E-box element located on the Nfatc1 promoter in cooperation with circadian locomotor output cycles kaput (CLOCK), a heterodimer partner of BMAL1. Moreover, steroid receptor coactivator (SRC) family members were shown to interact with and upregulate BMAL1: CLOCK transcriptional activity. Collectively, these data suggest that bone resorption is controlled by osteoclastic BMAL1 through interactions with the SRC family and binding to the Nfatc1 promoter. (C) 2016 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.2803

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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-BLACKWELL  

This study evaluates the pathological role of the stress sensor activating transcription factor-3 (ATF3) in ischemic neurotoxicity. Upregulation of the transcript and protein for ATF3 was seen 2-10 hr after reperfusion in the ipsilateral cerebral hemisphere of mice with transient middle cerebral artery occlusion for 2 hr. Immunohistochemical analysis confirmed the expression of ATF3 by cells immunoreactive for a neuronal marker in neocortex, hippocampus, and striatum within 2 hr after reperfusion. In murine neocortical neurons previously cultured under ischemic conditions for 2 hr, transient upregulation of both Atf3 and ATF3 expression was similarly found during subsequent culture for 2-24 hr under normoxia. Lentiviral overexpression of ATF3 ameliorated the neurotoxicity of glutamate (Glu) in cultured murine neurons along with a slight but statistically significant inhibition of both Fluo-3 and rhodamine-2 fluorescence increases by N-methyl-D-aspartate. Similarly, transient upregulation was seen in Atf3 and ATF3 expression during the culture for 48 hr in neuronal Neuro2A cells previously cultured under ischemic conditions for 2 hr. Luciferase reporter analysis with ATF3 promoter together with immunoblotting revealed the possible involvement of several transcription factors responsive to extracellular and intracellular stressors in the transactivation of the Atf3 gene in Neuro2A cells. ATF3 could be upregulated to play a role in mechanisms underlying mitigation of the neurotoxicity mediated by the endogenous neurotoxin Glu at an early stage after ischemic signal inputs. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/jnr.23723

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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  

We have shown marked promotion of both proliferation and neuronal differentiation in pluripotent P19 cells exposed to the green tea amino acid theanine, which is a good substrate for SLC38A1 responsible for glutamine transport. In this study, we evaluated the activity of the mammalian target of rapamycin (mTOR) kinase pathway, which participates in protein translation, cell growth and autophagy in a manner relevant to intracellular glutamine levels, in murine neural progenitor cells exposed to theanine. Exposure to theanine promoted the phosphorylation of mTOR and downstream proteins in neurospheres from embryonic mouse neocortex. Although stable overexpression of SLC38A1 similarly facilitated phosphorylation of mTOR-relevant proteins in undifferentiated P19 cells, theanine failed to additionally accelerate the increased phosphorylation in these stable transfectants. Theanine accelerated the formation of neurospheres from murine embryonic neocortex and adult hippocampus, along with facilitation of both 5-bromo-2'-deoxyuridine incorporation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction in embryonic neurospheres. In embryonic neurospheres previously exposed to theanine, a significant increase was seen in the number of cells immunoreactive for a neuronal marker protein after spontaneous differentiation. These results suggest that theanine activates the mTOR signaling pathway for proliferation together with accelerated neurogenesis in murine undifferentiated neural progenitor cells.

DOI： 10.1016/j.bbrep.2015.09.021

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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-BLACKWELL  

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Although interferon-related developmental regulator 1 (Ifrd1) has been identified as a transcriptional coactivator/repressor in various cells, little attention has been paid to its role in osteoblastogenesis and bone homeostasis thus far. Here, we show that Ifrd1 is a critical mediator of both the cell-autonomous regulation of osteoblastogenesis and osteoblast-dependent regulation of osteoclastogenesis. Osteoblast-specific deletion of murine Ifrd1 increased bone formation and decreased bone resorption, causing high bone mass. Ifrd1 deficiency enhanced osteoblast differentiation and maturation along with increased expression of Runx2 and osterix (Osx). Mechanistically, Ifrd1 deficiency increased the acetylation status of p65, a component of NF-B, at residues K122 and K123 via the attenuation of the interaction between p65 and histone deacetylase (HDAC). This led to the nuclear export of p65 and a decrease in NF-B-dependent Smad7 expression and the subsequent enhancement of Smad1/Smad5/Smad8-dependent transcription. Moreover, a high bone mass phenotype in the osteoblast-specific deletion of Ifrd1 was markedly rescued by the introduction of one Osx-floxed allele but not of Runx2-floxed allele. Coculture experiments revealed that Ifrd1-deficient osteoblasts have a higher osteoprotegerin (OPG) expression and a lower ability to support osteoclastogenesis. Ifrd1 deficiency attenuated the interaction between -catenin and HDAC, subsequently increasing the acetylation of -catenin at K49, leading to its nuclear accumulation and the activation of the -catenin-dependent transcription of OPG. Collectively, the expression of Ifrd1 in osteoblasts repressed osteoblastogenesis and activated osteoclastogenesis through modulating the NF-B/Smad/Osx and -catenin/OPG pathways, respectively. These findings suggest that Ifrd1 has a pivotal role in bone homeostasis through its expression in osteoblasts in vivo and represents a therapeutic target for bone diseases. (c) 2015 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.2720

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER/PLENUM PUBLISHERS  

We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100 mu M in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.

DOI： 10.1007/s11064-015-1591-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COMPANY OF BIOLOGISTS LTD  

Runt-related transcription factor 2 (Runx2) is an essential transcriptional regulator of osteoblast differentiation and its haploinsufficiency leads to cleidocranial dysplasia because of a defect in osteoblast differentiation during bone formation through intramembranous ossification. The cellular origin and essential period for Runx2 function during osteoblast differentiation in intramembranous ossification remain poorly understood. Paired related homeobox 1 (Prx1) is expressed in craniofacial mesenchyme, and Runx2 deficiency in cells of the Prx1 lineage (in mice referred to here as Runx2(prx1)(-/-)) resulted in defective intramembranous ossification. Runx2 was heterogeneously expressed in Prx1-GFP(+) cells located at the intrasutural mesenchyme in the calvaria of transgenic mice expressing GFP under the control of the Prx1 promoter. Double-positive cells for Prx1-GFP and stem cell antigen-1 (Sca1) (Prx1(+)Sca1(+) cells) in the calvaria expressed Runx2 at lower levels and were more homogeneous and primitive than Prx1(+)Sca1(-) cells. Osterix (Osx) is another transcriptional determinant of osteoblast lineages expressed by osteoblast precursors; Osx is highly expressed by Prx1(-)Runx2(+) cells at the osteogenic front and on the surface of mineralized bone in the calvaria. Runx2 deficiency in cells of the Osx lineage (in mice referred to here as Runx2(osx)(-/-)) resulted in severe defects in intramembranous ossification. These findings indicate that the essential period of Runx2 function in intramembranous ossification begins at the Prx1(+)Sca1(+) mesenchymal stem cell stage and ends at the Osx(+)Prx1(-)Sca1(-) osteoblast precursor stage.

DOI： 10.1242/dev.128793

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/jcp.24988

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

The synthesis of type I collagen, the main component of bone matrix, precedes the expression of Runx2, the earliest determinant of osteoblast differentiation. We hypothesized that the energetic needs of osteoblasts might explain this apparent paradox. We show here that glucose, the main nutrient of osteoblasts, is transported in these cells through Glut1, whose expression precedes that of Runx2. Glucose uptake favors osteoblast differentiation by suppressing the AMPK-dependent proteasomal degradation of Runx2 and promotes bone formation by inhibiting another function of AMPK. While RUNX2 cannot induce osteoblast differentiation when glucose uptake is compromised, raising blood glucose levels restores collagen synthesis in Runx2-null osteoblasts and initiates bone formation in Runx2-deficient embryos. Moreover, RUNX2 favors Glut1 expression, and this feedforward regulation between RUNX2 and Glut1 determines the onset of osteoblast differentiation during development and the extent of bone formation throughout life. These results reveal an unexpected intricacy between bone and glucose metabolism.

DOI： 10.1016/j.cell.2015.05.029

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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  

Background
Several chemicals have been widely used to evaluate the involvement of free Ca2+ in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca2+-sensitive reagents in diverse cultured cells.
Methodology/Principal Findings
In rat astrocytic C6 glioma cells loaded with the fluorescent Ca2+ dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca2+ chelator EGTA irrespective of added CaCl2. The addition of the Ca2+ ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn2+ ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn2+ chelator N, N, N', N'-tetrakis(2-pyridylmethyl) ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters.
Conclusions/Significance
Taken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses mediated by Ca2+ in diverse cells enriched of Zn2+.

DOI： 10.1371/journal.pone.0127421

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

Posttraumatic stress disorder is a long-lasting psychiatric disease with the consequence of hippocampal atrophy in humans exposed to severe fatal stress. We demonstrated a positive correlation between the transient decline of 5-bromo-2 '-deoxyuridine (BrdU) incorporation in the hippocampal dentate gyrus (DG) and long-lasting behavioral abnormalities in mice with traumatic stress. Here, we investigated pharmacological properties of theanine on the declined BrdU incorporation and abnormal behaviors in mice with traumatic stress. Prior daily oral administration of theanine at 50-500 mg/kg for 5 days significantly prevented the decline of BrdU incorporation, while theanine significantly prevented the decline in the DG even when administered for 5 days after stress. Consecutive daily administration of theanine significantly inhibited the prolonged immobility in mice with stress in forced swimming test seen 14 days later. Although traumatic stress significantly increased spontaneous locomotor activity over 30 min even when determined 14 days later, the increased total locomotion was significantly ameliorated following the administration of theanine at 50 mg/kg for 14 days after stress. These results suggest that theanine alleviates behavioral abnormalities together with prevention of the transient decline of BrdU incorporation in the hippocampal DG in adult mice with severe traumatic stress. (C) 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

DOI： 10.1016/j.jphs.2014.12.018

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1254/fpj.144.178

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掲載種別：研究論文（学術雑誌）   出版者・発行元：2  

DOI： 10.1254/fpj.144.98

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

Runt-related transcription factor-2 (Runx2) is the master regulator of osteoblastogenesis with an ability to promote differentiation of mesenchymal stem cells into the osteoblastic lineage. We have previously shown constitutive and functional expression of Runx2 by astroglial cells. In this study, we investigated the possible expression of Runx2 by both murine microglia and microglial cell line BV-2 cells. Runx2 expression was seen in cultured microglia and BV-2 cells, while sustained exposure to 1 mM ATP led to a significant but transient increase in mRNA and corresponding protein expression of Runx2 within 24 h. The increase in Runx2 expression was invariably prevented by several chemicals with antagonistic properties for P2X7 purinergic receptor, calmodulin and calcineurin in BV-2 cells, with a P2X7 receptor agonist more than quadrupling Runx2 expression. A significant increase in Runx2 expression was seen in osteoclastic cells, but not in osteoblastic or chondrocytic cells, when exposed to a high concentration of ATP. In BV2-cells with control siRNA, a significant decrease was found in the number of cells with at least one process within 3 h after the exposure to 1 mM ATP, followed by an increase up to 24 h. However, Runx2 siRNA significantly deteriorated the property to induce delayed process extension during 6-24 h after exposure to ATP along with drastically decreased Runx2 protein levels. These results suggest that Runx2 is constitutively and functionally expressed by microglial cells with responsiveness to ATP for upregulation in the murine brain. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2014.04.010

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記述言語：英語   出版者・発行元：公益社団法人 日本薬理学会  

DOI： 10.1254/fpj.144.305

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その他リンク： http://search.jamas.or.jp/link/ui/2015075200

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-BLACKWELL  

Global gene deletion studies in mice and humans have established the pivotal role of runt related transcription factor-2 (Runx2) in both intramembranous and endochondral ossification processes during skeletogenesis. In this study, we for the first time generated mice carrying a conditional Runx2 allele with exon 4, which encodes the Runt domain, flanked by loxP sites. These mice were crossed with 1(I)-collagen-Cre or 1(II)-collagen-Cre transgenic mice to obtain osteoblast-specific or chondrocyte-specific Runx2 deficient mice, respectively. As seen in Runx2(-/-) mice, perinatal lethality was observed in 1(II)-Cre;Runx2(flox/flox) mice, but this was not the case in animals in which 1(I)-collagen-Cre was used to delete Runx2. When using double-staining with Alizarin red for mineralized matrix and Alcian blue for cartilaginous matrix, we observed previously that mineralization was totally absent at embryonic day 15.5 (E15.5) throughout the body in Runx2(-/-) mice, but was found in areas undergoing intramembranous ossification such as skull and clavicles in 1(II)-Cre;Runx2(flox/flox) mice. In newborn 1(II)-Cre;Runx2(flox/flox) mice, mineralization impairment was restricted to skeletal areas undergoing endochondral ossification including long bones and vertebrae. In contrast, no apparent skeletal abnormalities were seen in mutant embryo, newborn, and 3-week-old to 6-week old-mice in which Runx2 had been deleted with the 1(I)-collagen-Cre driver. These results suggest that Runx2 is absolutely required for endochondral ossification during embryonic and postnatal skeletogenesis, but that disrupting its expression in already committed osteoblasts as achieved here with the 1(I)-collagen-Cre driver does not affect overtly intramembranous and endochondral ossification. The Runx2 floxed allele established here is undoubtedly useful for investigating the role of Runx2 in particular cells. (c) 2013 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.1945

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記述言語：日本語   出版者・発行元：PHARMACEUTICAL SOC JAPAN  

In this review, we would outline the possible signaling system for three types of amino acids including glutamate (Glu), gamma-aminobutyric acid (GABA) and D-serine (D-Ser) to play a role as an extracellular signal mediator in mechanisms underlying maintenance of the cellular homeostasis in skeletal tissues. Although Glu and GABA has been thought to be an excitatory/inhibitory amino acid neurotransmitter in the mammalian central nervous system, our molecular biological analyses give rise to a novel function for Glu and GABA as an autocrine and/or paracrine factor in three types of distinct cell types including osteoblasts, osteoclasts and chondrocytes in bone tissues. Moreover, D-Ser plays a pivotal role in osteoclastogenesis through a mechanism related to the incorporation of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts. Accordingly, bone formation and maintenance seems to be under control by amino acid signaling in skeletal tissues as seen with neurotransmission in the brain.

DOI： 10.1248/yakushi.13-00062

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記述言語：日本語   出版者・発行元：1  

脳梗塞発症時には，梗塞部位における細胞外グルタミン酸（Glu）濃度の異常な上昇に伴って，N-methyl-D-aspartate（NMDA）受容体の過度の活性化を介する遅発性神経細胞死が引き起こされる．動物実験では脳虚血負荷後に海馬CA1領域および扁桃体に限局する遅発性の神経細胞脱落が観察されるが，この脳内部位選択的な脆弱性の出現機序は未だ明らかにはなっていない．我々はこの選択的脆弱性を引き起こす"障害因子"の探索を行ってきたが，最近ミトコンドリアでのエネルギー代謝調節に重要な役割を果たすアンカップリングプロテイン（uncoupling protein：UCP）の一つであり，かつミトコンドリアにCa²⁺を輸送するユニポーターの候補分子であるUCP2が，選択的に海馬由来神経細胞で高発現する事実を見出した．さらに，培養神経細胞にレンチウイルスベクターを用いてUCP2を過剰発現させると，Glu神経毒性が著明に増強されることが明らかとなった．また，NMDA受容体を人工的に発現させたHEK293細胞を使用した解析では，UCP2がNMDA曝露に伴うミトコンドリア内Ca²⁺流入を選択的に促進させるだけでなく，この際にはNMDA受容体とUCP2が相互作用する可能性を確認した．一方，エタノールの薬理作用出現に，NMDA受容体活性化後のUCP2を介するミトコンドリア内Ca²⁺流入が関与する可能性を見出した．本稿では，我々の上記研究成果を踏まえて，Glu神経毒性におけるミトコンドリア因子の関与に関する新知見を紹介したい．

DOI： 10.1254/fpj.142.13

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その他リンク： https://jlc.jst.go.jp/DN/JALC/10021335814?from=CiNii

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  

Background: We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca2+ levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca2+ levels in cultured murine neocortical neurons.
Methodology/Principal Findings: In neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca2+ levels determined by Rhod-2 at concentrations of 0.1 to 100 mu M. Further addition of 250 mM ethanol significantly inhibited the increase by glutamate and NMDA in Rhod-2 fluorescence, while similarly potent inhibition of the NMDA-induced increase was seen after exposure to ethanol at 50 to 250 mM in cultured neurons. Lentiviral overexpression of UCP2 significantly accelerated the increase by NMDA in Rhod-2 fluorescence in neurons, without affecting Fluo-3 fluorescence for intracellular Ca2+ levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca2+ levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca2+ levels irrespective of UCP2 overexpression. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by ethanol in neurons exposed to glutamate. In HEK293 cells with NMDAR containing GluN2B subunit, more efficient inhibition was similarly induced by ethanol at 50 and 250 mM on the NMDA-induced increase in mitochondrial Ca2+ levels than in those with GluN2A subunit. Decreased protein levels of GluN2B, but not GluN2A, subunit were seen in immunoprecipitates with UCP2 from neurons with brief exposure to ethanol at concentrations over 50 mM.
Conclusions/Significance: Ethanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca2+ incorporation and cell death after NMDAR activation in neurons.

DOI： 10.1371/journal.pone.0069718

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：5  

Background:We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2′-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques.Methodology/Principal Findings:Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca2+ ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter.Conclusions/Significance:These results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells. © 2013 Takarada et al.

DOI： 10.1371/journal.pone.0063947

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Pre-chondrocytes undergo cellular differentiation stages during chondrogenesis under the influence by different transcription factors such as sry-type high mobility group box-9 (Sox9) and runt-related transcription factor-2 (Runx2). We have shown upregulation by parathyroid hormone (PTH) of the clock gene Period-1 (Per1) through the cAMP/protein kinase A signaling pathway in pre-chondrocytic ATDC5 cells. Here, we investigated the role of Per1 in the suppression of chondrogenic differentiation by PTH. In ATDC5 cells exposed to 10 nM PTH, a drastic but transient increase in Per1 expression was seen only 1 h after addition together with a prolonged decrease in Sox6 levels. However, no significant changes were induced in Sox5 and Runx2 levels in cells exposed to PTH. In stable Per1 transfectants, a significant decrease in Sox6 levels was seen, with no significant changes in Sox5 and Sox9 levels, in addition to the inhibition of gene transactivation by Sox9 allies. Knockdown of Per1 by siRNA significantly increased the Sox6 and type II collagen levels in cells cultured for 24-60 h. These results suggest that Per1 plays a role in the suppressed chondrocytic differentiation by PTH through a mechanism relevant to negative regulation of transactivation of the Sox6 gene during chondrogenesis. © The Japanese Pharmacological Society.

DOI： 10.1254/jphs.13091FP

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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-BLACKWELL  

The underlying mechanisms are still unclear for the neuroprotective properties of nicotine to date, whereas we have shown functional expression of nicotinic acetylcholine receptors (nAChRs) responsible for the influx of extracellular Ca2+ in cultured rat cortical astrocytes. In this study, we investigated the possible involvement of astrocytic nAChRs in the neuroprotection by this agonist. Exposure to nicotine predominantly induced mRNA expression of glial cell line-derived neurotrophic factor (GDNF) among the different neurotrophic factors examined in cultured astrocytes, in a manner sensitive to nAChR antagonists, nifedipine, and aCa2+ chelator. Nicotine significantly increased GDNF in a concentration-dependent manner in cultured astrocytes but not in neurons or neural progenitors even at the highest concentration used. In cultured astrocytes, a transient increase was seen in the expression of mRNA and corresponding protein for GDNF during sustained exposure to nicotine for 24 hr. Cytotoxicity mediated by oxidative, calcium, mitochondrial, or endoplasmic reticulum stress was invariably protected against in cortical neurons cultured with conditioned medium from astrocytes previously exposed to nicotine, and preincubation with the anti-GDNF antibody reduced the neuroprotection by conditioned medium from astrocytes exposed to nicotine. Intraperitoneal administration of nicotine transiently increased the number of cells immunoreactive for both GDNF and glial fibrillary acidic protein in rat cerebral cortex. These results suggest that astrocytic nAChRs play a role in the neuroprotection against different cytotoxins after predominant upregulation of GDNF expression through a mechanism relevant to the acceleration of extracellular Ca2+ influx in rat brain in a particular situation. (c) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/jnr.23101

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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  

Background: Neural progenitor is a generic term used for undifferentiated cell populations of neural stem, neuronal progenitor and glial progenitor cells with abilities for proliferation and differentiation. We have shown functional expression of ionotropic N-methyl-D-aspartate (NMDA) and gamma-aminobutyrate type-A receptors endowed to positively and negatively regulate subsequent neuronal differentiation in undifferentiated neural progenitors, respectively. In this study, we attempted to evaluate the possible functional expression of nicotinic acetylcholine receptor (nAChR) by undifferentiated neural progenitors prepared from neocortex of embryonic rodent brains.
Methodology/Principal Findings: Reverse transcription polymerase chain reaction analysis revealed mRNA expression of particular nAChR subunits in undifferentiated rat and mouse progenitors prepared before and after the culture with epidermal growth factor under floating conditions. Sustained exposure to nicotine significantly inhibited the formation of neurospheres composed of clustered proliferating cells and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction activity at a concentration range of 1 mu M to 1 mM without affecting cell survival. In these rodent progenitors previously exposed to nicotine, marked promotion was invariably seen for subsequent differentiation into cells immunoreactive for a neuronal marker protein following the culture of dispersed cells under adherent conditions. Both effects of nicotine were significantly prevented by the heteromeric alpha 4 beta 2 nAChR subtype antagonists dihydro-beta-erythroidine and 4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine, but not by the homomeric alpha 7 nAChR subtype antagonist methyllycaconitine, in murine progenitors. Sustained exposure to nicotine preferentially increased the expression of Math1 among different basic helix-loop-helix proneural genes examined. In undifferentiated progenitors from embryonic mice defective of NMDA receptor subunit-1, nicotine was still effective in significantly inhibiting the proliferation.
Conclusions/Significance: Functional alpha 4 beta 2 nAChR subtype would be constitutively expressed to play a role in the mechanism underlying the determination of proliferation and subsequent differentiation fate into a neuronal lineage in association with preferential promotion of Math1 expression in undifferentiated neural progenitors of developing rodent neocortex independently of NMDA receptor activation.

DOI： 10.1371/journal.pone.0046177

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have previously shown transient promotion by parathyroid hormone of Period-1 (Per1) expression in cultured chondrocytes. Here we show the modulation by clock genes of chondrogenic differentiation through gene transactivation of the master regulator of chondrogenesis Indian hedgehog (IHH) in chondrocytes of the growth plate. Several clock genes were expressed with oscillatory rhythmicity in cultured chondrocytes and rib growth plate in mice, whereas chondrogenesis was markedly inhibited in stable transfectants of Per1 in chondrocytic ATDC5 cells and in rib growth plate chondrocytes from mice deficient of brain and muscle aryl hydrocarbon receptor nuclear translocator-like (BMAL1). Ihh promoter activity was regulated by different clock gene products, with clear circadian rhythmicity in expression profiles of Ihh in the growth plate. In BMAL1-null mice, a predominant decrease was seen in Ihh expression in the growth plate with a smaller body size than in wild-type mice. BMAL1 deficit led to disruption of the rhythmic expression profiles of both Per1 and Ihh in the growth plate. A clear rhythmicity was seen with Ihh expression in ATDC5 cells exposed to dexamethasone. In young mice defective of BMAL1 exclusively in chondrocytes, similar abnormalities were found in bone growth and Ihh expression. These results suggest that endochondral ossification is under the regulation of particular clock gene products expressed in chondrocytes during postnatal skeletogenesis through a mechanism relevant to the rhythmic Ihh expression.

DOI： 10.1074/jbc.M112.408963

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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  

Background: We previously demonstrated the functional expression in newborn rat neocortical astrocytes of glutamine transporter (GlnT = slc38a1) believed to predominate in neurons over astroglia in the brain. In order to evaluate the possible role of this transporter in neurogenesis, we attempted to establish stable transfectants of GlnT in mouse embryonal carcinoma P19 cells endowed to proliferate for self-renewal and differentiate into progeny cells such as neurons and astroglia, in addition to in vitro pharmacological profiling of the green tea ingredient theanine, which is shown to be a potent inhibitor of glutamine transport mediated by GlnT in cultured neurons and astroglia.
Methodology/Principal Findings: The full-length coding region of rat GlnT was inserted into a vector for gene transfection along with selection by G418, followed by culture with all-trans retinoic acid under floating conditions and subsequent dispersion for spontaneous differentiation under adherent conditions. Stable overexpression of GlnT led to marked increases in the size of round spheres formed during the culture for 4 days and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide reduction, with concomitant promotion of subsequent differentiation into cells immunoreactive for a neuronal marker protein. In these stable GlnT transfectants before differentiation, drastic upregulation was seen for mRNA expression of several proneural genes with a basic helix-loop-helix domain such as NeuroD1. Although a drastic increase was seen in NeuroD1 promoter activity in stable GlnT transfectants, theanine doubled NeuroD1 promoter activity in stable transfectants of empty vector (EV), without affecting the promoter activity already elevated in GlnT transfectants. Similarly, theanine promoted cellular proliferation and neuronal differentiation in stable EV transfectants, but failed to further stimulate the acceleration of both proliferation and neuronal differentiation found in stable GlnT transfectants.
Conclusions/Significance: GlnT would promote both proliferation and neuronal differentiation through a mechanism relevant to the upregulation of particular proneural genes in undifferentiated P19 cells.

DOI： 10.1371/journal.pone.0048270

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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-BLACKWELL  

We have shown the functional expression by chondrocytes of serine racemase (SR) which is responsible for the synthesis of D-serine (Ser) from L-Ser in cartilage. In this study, we evaluated the possible functional expression of SR by bone-forming osteoblasts and bone-resorbing osteoclasts. Expression of SR mRNA was seen in osteoblasts localized at the cancellous bone surface in neonatal rat tibial sections and in cultured rat calvarial osteoblasts endowed to release D-Ser into extracellular medium, but not in cultured osteoclasts differentiated from murine bone marrow progenitor cells. Sustained exposure to D-Ser failed to significantly affect alkaline phosphatase activity and Ca2+ accumulation in cultured osteoblasts, but significantly inhibited differentiation and maturation in a concentration-dependent manner at a concentration range of 0.11?mM without affecting cellular survival in cultured osteoclasts. By contrast, L-Ser promoted osteoclastic differentiation in a manner sensitive to the inhibition by D-Ser. Matured osteoclasts expressed mRNA for the amino acid transporter B0,+ (ATB0,+) and the system alanine, serine, and cysteine amino acid transporter-2 (ASCT2), which are individually capable of similarly incorporating extracellular L- and D-Ser. Knockdown of these transporters by siRNA prevented both the promotion by L-Ser and the inhibition by D-Ser of osteoclastic differentiation in pre-osteoclastic RAW264.7 cells. These results suggest that D-Ser may play a pivotal role in osteoclastogenesis through a mechanism related to the incorporation mediated by both ATB0,+ and ASCT2 of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts. J. Cell. Physiol. 227: 34773487, 2012. (C) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/jcp.24048

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記述言語：日本語   出版者・発行元：PHARMACEUTICAL SOC JAPAN  

L-Glutamate (Glu) and gamma-aminobutyric acid (GABA) has been thought to be an excitatory/inhibitory amino acid neurotransmitter in the mammalian central nervous system (CNS). Limited information is available in the literature with regard to an extracellular transmitter role of Glu and GABA in peripheral neuronal and non-neuronal tissues, whereas recent molecular biological analyses including ours give rise to a novel function for Glu and GABA as an autocrine and/or paracrine factor in a variety of cells derived from mesenchymal stem cells, in addition to other peripheral tissues including pancreas, adrenal, and pituitary glands. Emerging evidence suggests that Glu and GABA could play a dual role in mechanisms underlying maintenance of cellular homeostasis as a neurotransmitter in the CNS and as an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, therefore, we summarized the possible signaling by Glu and GABA as an extracellular signal mediator in mechanisms underlying maintenance of cellular homeostasis in mesenchymal stem cells, osteoblasts and chondrocytes.

DOI： 10.1248/yakushi.12-00191

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

We have previously shown the possible involvement of mitochondrial membrane potential disruption in the mechanisms underlying the neurotoxicity seen after activation of N-methyl-D-aspartate (NMDA) receptors (NMDAR) in primary cultured rat hippocampal neurons. In this study, we attempted to demonstrate a pivotal role of mitochondrial uncoupling protein-2 (UCP2) as a determinant of the NMDA neurotoxicity by using acquired NMDAR channels artificially orchestrated in HEK293 cells. In cells with overexpression of UCP2, immunoreactive UCP2 was exclusively detected at intracellular locations stained with the mitochondrial marker MitoTracker. In cells with acquired NMDAR channels, exposure to either NMDA or the calcium ionophore A23187 similarly led to a significant increase in cytosolic Ca2+ levels determined by Fluo-3 imaging irrespective of the overexpression of UCP2. By contrast, NMDA, but not A23187, was significantly more effective in increasing mitochondrial Ca2+ levels determined by Rhod-2 fluorescence imaging in cells transfected with NMDAR subunit and UCP2 expression vectors than in those without UCP2 overexpression. Overexpression of UCP2 significantly increased the number of cells stained with propidium iodide in cultures with acquired NMDAR channels, but failed to significantly affect that in cells exposed to A23187. Immunocytochemical and immunoprecipitation analyses similarly revealed the possible interaction between GluN1 subunit and UCP2 in HEK293 cells with acquired NMDAR channels and UCP2 overexpression. These results suggest that UCP2 could play a role as a determinant of the neurotoxicity mediated by NMDAR through a mechanism related to the unidentified interaction with the essential GluN1 subunit toward modulation of mitochondrial Ca2+ levels in neurons. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2012.03.019

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：40  

DOI： 10.1074/jbc.M112.344051

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

Necrotic damage leads to a massive leakage from injured cells of different intracellular constituents such as glutamate (Glu) and ATP, which are believed to play a role in the neuronal survival in the brain. In this study, we evaluated pharmacological properties of ATP, which is shown to be an endogenous inhibitor of N-methyl-D-aspartate (NMDA) receptors, on the neurotoxicity relevant to mitochondrial membrane potential disruption in cultured rat hippocampal neurons. Exposure to Glu or NMDA significantly inhibited cellular viability determined 24 and 48 h later, while simultaneous addition of 1 mM ATP significantly ameliorated the decreased viability in neurons exposed to Glu and NMDA, but not in those exposed to other cytotoxins. Both Glu and NMDA markedly increased intracellular free Ca2+ levels in a manner sensitive to blockade by the exposure to ATP, but not by that to adenosine. Exposure to ATP significantly delayed the rate of mitochondrial membrane potential disruption induced by Glu and NMDA. These results suggest that extracellular ATP would play a role as an endogenous antagonist endowed to protect rat hippocampal neurons from the excitotoxicity mediated by NMDA receptors in association with the delayed mitochondrial membrane potential disruption after the liberation from adjacent cells under necrotic death.

DOI： 10.1254/jphs.12034FP

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

In contrast to osteoblasts, little attention has been paid to expression profiles of different glutamatergic signaling machineries in osteocytes, which are the most abundant cells with a possible role as a mechanical sensor in bone. Here, we show that N-methyl-D-aspartate receptor (NMDAR) is expressed by osteocytic cells in five-weeks-old mouse tibiae in vivo as well as by osteocytic MLO-Y4 cells in vitro. Sustained exposure to the NMDAR antagonist dizocilpine significantly increased the number of cells with processes in cultured MLO-Y4 cells. These results suggest that NMDAR would be expressed by osteocytes with an unidentified role in the process extension.

DOI： 10.1254/jphs.12068SC

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

Transferrin receptor-1 (TfR1) is a cell membrane-associated glycoprotein responsible for incorporation of the iron bound to transferrin through an endocytotic process from the circulating blood. Iron is believed to play a dual role as an active center of the electron transfer system in mitochondria and as an endogenous cytotoxin through promoted generation of reactive oxygen species in different eukaryotic cells. In this study, we evaluated expression profiles of different genes related to iron mobilization across plasma membranes in neuronal cells. Marked mRNA expression was seen for various iron-related genes such as TfR1 in cultured mouse neocortical neurons, while TfR1 mRNA levels were more than doubled during culture from 3 to 6 days. In mouse embryonal carcinoma P19 cells endowed to differentiate into neuronal and astroglial lineages, a transient increase was seen in both mRNA and corresponding protein for TfR1 in association with neuronal marker expression during culture with all-trans retinoic acid (ATRA). In neuronal Neuro2A cells cultured with ATRA, moreover, neurite was elongated together with increased expression of both mRNA and protein for TfR1. Overexpression of TfR1 significantly decreased the length of neurite elongated, however, while significant promotion was invariably seen in the neurite elongation in Neuro2A cells transfected with TfR1 siRNA as well as in Neuro2A cells cultured with an iron chelator. These results suggest that TfR1 would be highly expressed by neurons rather than astroglia to play a negative role in the neurite outgrowth after the incorporation of circulating transferrin in the brain. (C) 2011 Published by Elsevier B.V.

DOI： 10.1016/j.neuint.2011.08.018

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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-BLACKWELL  

Osteocytes are thought to play a role as a mechanical sensor through their communication network in bone. Although osteocytes are the most abundant cells in bone, little attention has been paid to their physiological and pathological functions in skeletogenesis. Here, we have attempted to delineate the pivotal functional role of osteocytes in regulation of bone remodeling under pathological conditions. We first found markedly increased osteoclastic differentiation by conditioned media (CM) from osteocytic MLO-Y4 cells previously exposed to hypoxia in vitro. Using microarray and real-time PCR analyses, we identified growth differentiation factor 15 (GDF15) as a key candidate factor secreted from osteocytes under hypoxia. Recombinant GDF15 significantly promoted osteoclastic differentiation in a concentration-dependent manner, with concomitant facilitation of phosphorylation of both p65 and inhibitory-kappa B in the presence of receptor activator of nuclear factor-kappa B ligand. To examine the possible functional significance of GDF15 in vivo, mice were subjected to ligation of the right femoral artery as a hypoxic model. A significant increase in GDF15 expression was specifically observed in tibias of the ligated limb but not in tibias of the normally perfused limb. Under these experimental conditions, in cancellous bone of proximal tibias in the ligated limb, a significant reduction was observed in bone volume, whereas a significant increase was seen in the extent of osteoclast surface/bone surface when determined by bone histomorphometric analysis. Finally, the anti-GDF15 antibody prevented bone loss through inhibiting osteoclastic activation in tibias from mice with femoral artery ligation in vivo, in addition to suppressing osteoclastic activity enhanced by CM from osteocytes exposed to hypoxia in vitro. These findings suggest that GDF15 could play a pivotal role in the pathogenesis of bone loss relevant to hypoxia through promotion of osteoclastogenesis after secretion from adjacent osteocytes during disuse and/or ischemia in bone. (c) 2012 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.1538

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記述言語：英語   出版者・発行元：2  

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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-BLACKWELL  

Objective AntiN-methyl-D-aspartate (anti-NMDA) receptor subunit NR2-reactive antibody may play a crucial role in neuronal manifestations of systemic lupus erythematosus (SLE). However, how NR2-reactive antibody acts as a critical modulator of the NMDA receptor is unknown. This study was undertaken to investigate the biologic function of NR2-reactive antibody in patients with SLE.
Methods. The study included 14 patients with SLE, 9 of whom had NR2-reactive antibody. We analyzed the effects of NR2-reactive antibody on cell viability and intracellular Ca2+ level. We also investigated the efficacy of zinc as a modulator of the intracellular Ca2+ level in the presence of NR2-reactive antibody.
Results. There was a significant inverse correlation between the NR2-reactive antibody titer and cell viability (R-2 = 0.67, P < 0.0001; n = 23), and there was a significant association between the NR2-reactive antibody titer and the intracellular Ca2+ level in NR1/NR2a-transfected HEK 293 cells (R-2 = 0.69, P < 0.0001). Intracellular Ca2+ levels were significantly higher in cells incubated with IgG derived from NR2reactive antibody-positive SLE patients than in those incubated with IgG derived from NR2-reactive antibody-negative SLE patients (P = 0.0002). The addition of zinc decreased the intracellular Ca2+ level in a dose-dependent manner. NR2-reactive antibodypositive SLE IgG weakened the efficacy of zinc as a negative modulator of the intracellular Ca2+ level.
Conclusion. Our findings indicate that NR2-reactive antibody decreases cell viability by Ca2+ influx in SLE through inhibition of the binding capacity of zinc.

DOI： 10.1002/art.30616

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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-BLACKWELL  

We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through amechanism associated with intracellularGSHdepletionmediated by the bidirectional cystine/Glu antiporter in osteoblasticMC3T3-E1 cells cultured in the absence of differentiation inducers. To further evaluate the possible role of the antiporter in osteoblastogenesis, in this study, we have established stable transfectants of the xCT subunit of the antiporter in MC3T3-E1 cells. Stable overexpression led to a significant facilitation of cellular proliferation determined by different indices with increased GSH levels and decreased ROS generation in addition to promoted [(14)C] cystine incorporation, while Glu failed to significantly inhibit cellular proliferation in stable xCT transfectants. In stable transfectants cultured under differentiation conditions, drastic decreases were invariably seen in Ca(2+) accumulation, alkaline phosphatase activity and several osteoblastic marker gene expressions, in addition to downregulation ofmRNA and corresponding protein for runt-related transcription factor-2 (Runx2). Runx2 promoter activitywas significantly promoted by the introduction ofRunx2 expression vector in amanner sensitive to the prevention by the co-introduction of xCT expression vector in MC3T3-E1 cells. In both MC3T3-E1 cells and murine calvarial osteoblasts cultured with differentiation inducers, transient transfection with xCT siRNA significantly increased Runx2 protein expression alongwith decreases in xCT mRNA expression and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction. These results suggest that the cystine/ Glu antiporter plays a pivotal role in cellular differentiation through a mechanismrelated to the regulation of transactivation of Runx2 essential for osteoblastogenesis toward maturation in osteoblastic cells. J. Cell. Physiol. 226: 2953-2964, 2011. (C) 2011 Wiley-Liss, Inc.

DOI： 10.1002/jcp.22642

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：38  

DOI： 10.1074/jbc.M111.253526

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

We have previously shown that mitochondrial membrane potential disruption is involved in mechanisms underlying differential vulnerabilities to the excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors between primary cultured neurons prepared from rat cortex and hippocampus. To further elucidate the role of mitochondria in the excitotoxicity after activation of NMDA receptors, neurons were loaded with the fluorescent dye calcein diffusible in the cytoplasm and organelles for determination of the activity of mitochondrial permeability transition pore (mPTP) responsible for the leakage of different mitochondrial molecules. The addition of CoCl2 similarly quenched the intracellular fluorescence except mitochondria in both cultured neurons, while further addition of NMDA led to a leakage of the dye into the cytoplasm in hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of viability in hippocampal neurons, while an activator of mPTP induced a similarly potent loss of viability in cortical and hippocampal neurons. Although NMDA was more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium indicator in hippocampal than cortical neurons, a mitochondrial calcium uniporter inhibitor significantly prevented the NMDA-induced loss of viability in hippocampal neurons. Expression of mRNA was significantly higher for the putative uniporter uncoupling protein-2 in hippocampal than cortical neurons. These results suggest that mitochondrial calcium uniporter would be at least in part responsible for the NMDA neurotoxicity through a mechanism relevant to promotion of mPTP orchestration in hippocampal neurons. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.neuint.2011.03.018

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

Clock genes are believed to play a pivotal role in the generation and oscillation of circadian rhythm as a central clock in the hypothalamic suprachiasmatic nucleus in the mammalian brain. In this study, mRNA expression was for the first time demonstrated with clock genes in both cultured murine microglia and microglial cell line BV-2 cells. Exposure to ATP transiently increased Period-1 (Per1) mRNA expression without affecting that of other clock genes in BV-2 cells, while a similarly transient increase was shown in Per1 mRNA expression in a manner sensitive to P2X7 purinergic receptor antagonists in cultured microglia exposed to ATP. In BV-2 cells transfected with a Per1 promoter luciferase reporter plasmid, ATP significantly increased luciferase activity in a manner sensitive to a P2X7-receptor antagonist. In both microglia and BV-2 cells, a significant increase by ATP was seen in the immunocytochemical fluorescence intensity of cells expressing Per1 protein, with mRNA expression of different P2 receptors including P2X7. Per1 siRNA significantly decreased the number of cells with processes in BV-2 cells exposed to ATP. These results suggest that ATP selectively promotes Per1 expression through gene transactivation after stimulation of P2X7 purinergic receptors in microglial cells.

DOI： 10.1254/jphs.11069FP

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：4  

Ischemic brain damage is believed to involve the drastic increase in extracellular glutamate levels after reperfusion and subsequent overactivation of both N-methyl-D-aspartate (NMDA) receptor (NMDAR) and non-NMDAR channels for delayed neuronal cell death mediated by Ca 2+ overload. In this study, we evaluated expression profiles of mRNA and corresponding proteins for different subunits of NMDAR and non-NMDAR in brains of rats with transient middle cerebral artery occlusion (MCAO). Cellular vitality was markedly reduced in proportion to increasing durations of MCAO for 1 to 8 h when determined 1 day after reperfusion. Within 7 days after reperfusion, MCAO for 2 h led to a gradual decrease in the neuronal marker microtubules-associated protein-2 (MAP2) level in the ipsilateral cerebral hemisphere, in addition to inducing a transient increase in the microglial marker CD11b expression without affecting the astroglial marker protein levels. MCAO for 2 h significantly decreased the expression of both mRNA and corresponding proteins for NR1, NR2A and NR2B subunits of NMDAR, but not for non-NMDAR subunits, in the ipsilateral hemisphere. These results suggest that NMDAR may be preferentially down-regulated in response to ischemic signal inputs amongst three different subtypes of ionotropic glutamate receptors in rats with MCAO.

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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  

Background: The view that gamma-aminobutyric acid (GABA) plays a functional role in non-neuronal tissues, in addition to an inhibitory neurotransmitter role in the mammalian central nervous system, is prevailing, while little attention has been paid to GABAergic signaling machineries expressed by adipocytes to date. In this study, we attempted to demonstrate the possible functional expression of GABAergic signaling machineries by adipocytes.
Methodology/Principal Findings: GABA(B) receptor 1 (GABA(B)R1) subunit was constitutively expressed by mouse embryonic fibroblasts differentiated into adipocytes and adipocytic 3T3-L1 cells in culture, as well as mouse white adipose tissue, with no responsiveness to GABA(B)R ligands. However, no prominent expression was seen with mRNA for GABA(B)R2 subunit required for heteromeric orchestration of the functional GABA(B)R by any adipocytic cells and tissues. Leptin mRNA expression was significantly and selectively decreased in adipose tissue and embryonic fibroblasts, along with drastically reduced plasma leptin levels, in GABA(B)R1-null mice than in wild-type mice. Knockdown by siRNA of GABA(B)R1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells.
Conclusions/Significance: Our results indicate that GABA(B)R1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner of heterodimeric assembly to the functional GABA(B)R.

DOI： 10.1371/journal.pone.0020167

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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-BLACKWELL  

We have shown that glutamate (Glu) signaling machineries, such as receptors (GluR) and transporters, are functionally expressed by mesenchymal stem cells, in addition to by their progeny cells such as osteoblasts and chondrocytes. Sustained exposure to Glu induced significant decreases in alkaline phosphatase (ALP) staining and osteoblastic marker gene expression in the mesenchymal C3H10T1/2 stem cells infected with runt-related transcription factor-2 (Runx2) adenovirus, without markedly affecting Oil Red O staining for adipocytes in cells cultured with adipogenic inducers. In cells with Runx2 adenovirus, the cystine/Glu antiporter substrate cystine significantly prevented the decreases by Glu in both ALP staining and osteoblastic marker gene expression, with GluR agonists being ineffective. In cells with Runx2 adenovirus, Glu significantly decreased [C-14] cystine uptake, intracellular glutathione (GSH) level, Runx2 recruitment to osteocalcin promoter and nuclear Runx2 protein level, respectively. Cystine again significantly prevented the decreases by Glu in both GSH levels and Runx2 recruitment. In mouse bone marrow stromal cells, Glu and a GSH depleter significantly decreased ALP staining without affecting Oil Red O staining. Knockdown of the cystine/Glu antiporter led to markedly decreased ALP staining and GSH levels, with concomitant prevention of the decrease by Glu, in cells with Runx2 adenovirus. These results suggest that Glu may play a role as a negative regulator at an early differentiation stage into osteoblasts than adipocytes through a mechanism relevant to nuclear translocation of Runx2 after regulation of intracellular GSH levels by the cystine/Glu antiporter expressed in mesenchymal stem cells. J. Cell. Physiol. 226: 652-665, 2011. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jcp.22390

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

We have previously demonstrated the functional expression of glutamine (Gln) transporter (GlnT) believed to predominate in neurons for the neurotransmitter glutamate pool by rat neocortical astrocytes devoid of neuronal marker expression, with exacerbated vulnerability to oxidative stress after transient overexpression. To evaluate molecular mechanisms underlying the exacerbation, we established stable GlnT transfectants in rat astrocytic C6 glioma cells. In two different clones of stable transfectants with increased intracellular Gln levels, exposure to hydrogen peroxide (H(2)O(2)) and A23187, but not to tunicamycin or 2,4-dinitrophenol, led to significant exacerbation of the cytotoxicity compared to cells with empty vector (EV). Stable GlnT overexpression led to a significant increase in heme oxygenase-1 protein levels in a manner sensitive to H(2)O(2), whereas H(2)O(2) was significantly more effective in increasing NO(2) accumulation and reactive oxygen species (ROS) generation in stable GlnT transfectants than in EV cells. Moreover, exposure to A23187 led to a more effective increase in the generation of ROS in stable GlnT transfectants than in stable EV transfectants. These results suggest that GlnT may play a role in the mechanisms underlying the determination of cellular viability in astrocytes through modulation of intracellular ROS generation. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2011.01.007

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

We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the bidirectional cystine/Glu antiporter in undifferentiated osteoblastic MC3T3-E1 cells. In this study, we investigated the expression profile of the xCT subunit of the antiporter as well as the master regulator of osteoblastogenesis runt-related transcription factor-2 (Runx2) in ovariectomized mouse bone. In spinal columns isolated 28 days after ovariectomy, a marked reduction was seen with the intensity of Von Kossa staining used as an index of ossification. In femurs of these ovariectomized mice, a significant decrease was seen in mRNA and protein levels of Runx2 along with increased expression of both mRNA and the corresponding protein for the xCT subunit. To evaluate the possible role of the antiporter in osteoblastogenesis, stable transfectants were established with the xCT subunit toward the culture with osteoblastic differentiation inducers in MC3T3-E1 cells. In stable xCT transfectants cultured under differentiation conditions, marked decreases were seen in nodule formation, Ca2+ accumulation, and osteoblastic marker gene expression, in addition to downregulation of both mRNA and the corresponding protein for Runx2. Runx2 promoter activity was markedly stimulated in MC3T3-E1 cells transfected with a responsive promoter plasmid after the culture under differentiation conditions, while transient and stable transfection with xCT expression vector invariably prevented the stimulation through an activator protein-1 site. These results suggest that Runx2 expression would be negatively regulated by the cystine/glutamate antiporter expressed by osteoblastic cells at the level of gene transactivation.

DOI： 10.1254/jphs.10310FP

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記述言語：日本語   出版者・発行元：(公社)日本薬理学会  

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

We have previously shown the functional expression of GABA(B) receptors (GABA(B)R) composed of GABA(B)R1 and GABA(B)R2 subunits with ability to promote proliferation and neuronal differentiation in cultured neural progenitor cells (NPC) isolated from embryonic mouse brains. In this study, we evaluated postnatal changes in the expression profiles of different markers for progenitor, neuronal, astroglial, and microglial cells in brains of GABA(B)R1-null mice. Consistent with undifferentiated murine NPC cultured with epidermal growth factor, a significant and selective decrease was seen in mRNA expression of the proneural gene Mash1 in brains of GABA(B)R1-null mice at I day after birth. The expression of several NPC marker proteins was similarly decreased in brains of both wild-type and GABA(B)R1-null mice from 1 to 7 days after birth, while slight changes were induced in both mRNA and proteins for neuronal, astroglial, and microglial markers between wild-type and GABA(B)R1-null mouse brains within this developmental stage. In particular discrete brain structures of adult GABA(B)R1-null mice at 56 days after birth, a significant decrease was seen in neuronal marker protein levels along with a significant increase in both astroglial and microglial marker protein expression. Although no significant difference was found in mRNA expression of the partner GABA(B)R2 subunit between wild-type and GABA(B)R1-null mouse brains, GABA(B)R2 subunit protein levels were gradually declined during postnatal development within 56 days after birth in GABA(B)R1-null mouse brains. These results suggest that GABA(B)R2 protein levels are closely correlated with the partner subunit GABA(B)R1 protein levels in mouse brains during postnatal development in vivo.

DOI： 10.1254/jphs.10254FP

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

Conventional N-methyl-D-aspartate (NMDA) receptor (NMDAR) is a heteromeric complex between the essential NR1 subunit and one of NR2A-D subunits toward functional channels permeable to Ca2+ rather than Na+ ions. Although recent studies identified dominant negative NR3A and NR3B subunits, whether these subunits inhibit Ca2+ mobilization through NMDAR channels into mitochondria is not clarified so far. In this study, we investigated Ca2+ influx across acquired NMDAR channels composed of different NR subunits artificially expressed in HEK293 cells. The addition of NMDA markedly increased intracellular free Ca2+ levels determined by Fluo-3 in cells transfected with either NR2A or NR2B subunit together with NR1 subunit. Further addition of dizocilpine completely inhibited the increase by NMDA in both types of acquired channels, while the NR2B subunit selective antagonist ifenprodil drastically inhibited the increase by NMDA in cells expressing NR1/NR2B, but not NR1/NR2A, subunits. Similar pharmacological profiles were invariably seen with cell death by NMDA. Introduction of both NR3A and NR3B subunits significantly inhibited the increase by NMDA in intracellular free Ca2+ levels in both acquired channels, while introduction of either NR3A or NR3B alone was ineffective. Co-expression of both NR3A and NR3B subunits was also required for the prevention of increased mitochondrial free Ca2+ levels determined by Rhod-2, as well as decreased cellular viability, in cells expressing NR1/NR2A or NR1/NR2B subunits upon exposure to NMDA. These results suggest that co-expression of both NR3A and NR3B subunits is essential for the dominant negative properties on Ca2+ mobilization through acquired functional NMDAR channels into mitochondria. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2010.08.009

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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-BLACKWELL  

In our previous studies, particular phenolic ingredients, such as 2-methoxy-4-methylphenol (2M4MP), of the antidiarrheic drug wood creosote significantly prevented cell death by both hydrogen peroxide and glutamate in cultured rat hippocampal neurons. In this study, we further evaluated the pharmacological properties of 2M4MP on Ca2+ influx across native and acquired N-methyl-D-aspartate (NMDA) receptor (NMDAR) channels. The addition of 2M4MP significantly prevented the loss of cellular viability and the increase in intracellular free Ca2+ levels as determined by Fluo-3 in cultured rat hippocampal neurons briefly exposed to NMDA. Brief exposure to NMDA also led to a marked increase in mitochondrial free Ca2+ levels determined by Rhod-2, in addition to intracellular free Ca2+ levels, in HEK293 cells expressing either NR1/NR2A or NR1/NR2B subunit channels. The further addition of the general NMDAR channel blocker dizocilpine similarly inhibited the increase of intracellular Ca2+ levels by NMDA in both types of acquired NMDAR channels, whereas the NR2B subunit selective antagonist ifenprodil drastically inhibited the increase by NMDA in HEK293 cells expressing NR1/NR2B, but not NR1/NR2A, subunits. Similarly, 2M4MP significantly and selectively inhibited the NMDA-induced influx of Ca2+ across acquired NR1/NR2B channels in a concentration-dependent manner. Moreover, prior daily oral administration of 2M4MP significantly reduced the infarct volume in the ipsilateral cerebral hemisphere in rats with middle cerebral artery occlusion 1 day after reperfusion. These results suggest that 2M4MP may protect neurons from excitotoxicity through preferential inhibition of Ca2+ influx across NMDAR channels composed of NR1/NR2B subunits. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jnr.22399

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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-BLACKWELL  

We have previously shown differential vulnerabilities to glutamate (Glu) excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptor (NMDAR) between rat cortical and rat hippocampal neurons in culture. In this study, we evaluated the possible induced tolerance to NMDA neurotoxicity in cultured rat striatal neurons with prior sustained activation of NMDAR. Brief exposure to Glu or NMDA for 1 hr led to a significant decrease in cellular vitality determined 24 hr later in cultured rat striatal neurons, whereas no marked loss was seen in cellular survival after exposure to Glu or NMDA in striatal neurons previously cultured with Glu or NMDA. Sustained culture with Glu or NMDA invariably led to a significant decrease in protein levels of NR2, but not NR1, subunits without affecting their mRNA levels. Similar induced tolerance was seen to the excitotoxicity of NMDA in hippocampal neurons in a manner sensitive to an NMDAR antagonist. Prior culture with NMDA induced less effective alterations in both intracellular free Ca2+ levels and mitochondrial membrane potentials after the addition of NMDA in striatal neurons. However, calpain inhibitor-I significantly prevented the decreased NR2B and NR2C protein levels in striatal neurons cultured with NMDA. These results suggest that prior tonic activation of NMDAR would induce tolerance to the excitotoxicity mediated by NMDAR through a mechanism related to calpain-induced down-regulation of particular NR2 subunits in rat striatal neurons. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jnr.22388

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

Pharmacological properties were evaluated for the antidiarrheic wood creosote ingredient 2-methoxy-4-ethylphenol (2M4EP), which was shown to be protective against neurotoxicity of N-methyl-D-aspartate (NMDA), to modulate Ca2+ influx across acquired and native NMDA receptor (NMDAR) channels. NMDA markedly increased intracellular free Ca2+ levels in HEK293 cells transfected with the expression vector of either NR2A or NR2B subunit together with the essential NR1 subunit vector. Further addition of dizocilpine inhibited the increase by NMDA in intracellular Ca2+ levels in both types of acquired NMDAR channels, while 2M4EP and the NR2B-subunit selective antagonist ifenprodil were more effective in inhibiting the increase by NMDA in HEK293 cells expressing NR1/NR2B subunits than in those with NR1/NR2A subunits. 2M4EP significantly prevented the increased intracellular Ca2+ levels by NMDA in cultured rat hippocampal neurons. Brief exposure to NMDA led to a drastic decrease in cellular viability 24 h later in cultured hippocampal neurons, while 2M4EP significantly prevented the loss of cellular vitality by NMDA. Similarly, 2M4EP more efficiently protected HEK293 cells with NR1/NR2B subunits than those with NR1/NR2A subunits. These results suggest that 2M4EP may protect neurons from excitotoxicity through inhibition of Ca2+ influx across NMDAR channels composed of NR1/NR2B, rather than NR1/NR2A, subunits.

DOI： 10.1254/jphs.09294FP

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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  

In contrast to osteoblasts, little attention has been paid to the functional expression of adrenergic signaling machineries in chondrocytes. Expression of mRNA was for the first time demonstrated for different adrenergic receptor (AdR) subtypes in chondrogenic ATDC5 cells and mouse metatarsals isolated before vascularization in culture, but not for other molecules related to adrenergic signaling. In neonatal mouse tibial sections, beta(2)AdR and alpha(2a)AdR mRNA expression was found in chondrocytes at different developmental stages by in situ hybridization. Exposure to adrenaline significantly suppressed expression of several maturation markers through the cAMP/protein kinase A pathway activated by beta(2)AdR without affecting cellular proliferation in both Cultured ATDC5 cells and metatarsals. Adrenaline also significantly inhibited gene transactivation by sry-type HMG box 9 (Sox9) family members essential for chondrogenic differentiation in a manner prevented by the general beta AdR antagonist propranolol, with a concomitant significant decrease in the levels of Sox6 mRNA and corresponding protein, in ATDC5 cells and primary cultured Mouse costal chondrocytes. Systemic administration of propranolol significantly promoted the increased expression of mRNA for collagen I and collagen X, but not for collagen 11, in callus of fractured femur in mice. These results suggest that adrenaline may interfere with chondrogenic differentiation through downregulation of Sox6 expression for subsequent suppression of gene transactivation mediated by Sox9 family members after activation Of beta(2)AdR expressed by chondrocytes. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2009.05.004

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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  

Serine racemase (SR) is responsible for the biosynthesis of D-serine (D-Ser), an endogenous co-agonist for the glycine (Gly)-binding site on N-methyl-D-aspartate (NMDA) receptors, from L-Ser in the brain. We have previously demonstrated high expression of SR by chondrocytes in cartilage. In this study, we attempted to elucidate the possible functional role of D-Ser in chondrogenesis. Expression of mRNA and corresponding protein was seen for SR in cultured rat costal chondrocytes, while the addition of L-Ser significantly increased intracellular and extracellular levels of D-Ser. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, SIR mRNA was highly localized in hypertrophic and calcified chondrocytes. Exposure to D-Ser not only suppressed several chondrocytic maturation markers, including alkaline phosphatase (ALP) activity, Ca(2+) accumulation, nodule formation, and osteopontin expression, in rat chondrocytes, but also delayed chondral mineralization in mouse metatarsals. Either NMDA or Gly alone significantly increased Ca(2+) accumulation in cultured chondrocytes, whereas D-Ser significantly prevented Ca(2+) accumulation by Gly, but not by NMDA. Gly alone also significantly increased gene transactivation by the introduction of runt-related transcription factor-2 (Runx2) in COS7 cells transfected with NR1 and NR3A subunits,while D-Ser significantly prevented the increase by Gly without affecting the promoter activity of Runx2. In both cultured chondrocytes and metatarsals from NR1-null mice, significant decreases were seen in ALP activity and chondral mineralization, respectively. These results suggest that D-Ser may negatively regulate cellular differentiation through inhibiting NMDA receptors composed of NR1 and NR3A subunits in a manner related to Runx2 transcriptional activity in chondrocytes. J. Cell. Physiol. 220: 756-764, 2009. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jcp.21821

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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-BLACKWELL PUBLISHING, INC  

We have shown preferential expression of both mRNA and corresponding protein for myosin VI (Myo6) in the murine hippocampus within 24 h after the extreme traumatic experience, water-immersion restraint stress (WIRS), prior to a drastic decrease in neural progenitor proliferation in the dentate gyrus. Myosin (Myo6) protein levels were significantly increased in hippocampus within 24 h after flashback experience in mice previously exposed to WIRS. Myo6 protein was ubiquitously distributed in discrete mouse brain regions with exceptionally high expression in olfactory bulb, whereas Myo6 protein was expressed in cultured rat astroglia and neurons, in addition to Myo6 mRNA expression by cultured neural progenitors. In mouse embryonal carcinoma P19 cells endowed to proliferate and differentiate, Myo6 protein was expressed in line with astroglial marker protein expression. Transient over-expression of Myo6 induced a significant decrease in the size of clustered aggregates as an index of self-replication in P19 cells. Immunoprecipitation analysis revealed the interaction between Myo6 and the RNA-binding protein, translocated in liposarcoma (TLS), while TLS was predominantly expressed by neurons in the cortex, striatum, cerebellum, and hippocampus. These results suggest that Myo6 may play a pivotal role in the mechanism underlying the suppressed adult neurogenesis after traumatic stress in association with TLS.

DOI： 10.1111/j.1471-4159.2009.06225.x

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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  

We have previously shown significant potentiation of Ca(2+) influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of static magnetism on the functionality of neural progenitor cells endowed to proliferate for self-replication and differentiate into neuronal, astroglial, and oligodendroglial lineages. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Static magnetism not only significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for an astroglial marker, irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism, a significant increase was seen in mRNA expression of several activator-type proneural genes, such as Mash 1, Math 1, and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of activator-type proneural genes by progenitor cells in fetal rat brain. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jnr.22087

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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  

Notoginsenoside R1 (NTR1) is the main active ingredient in Panax notoginseng, a herbal medicine widely used in Asia for years. The purpose of this study was to investigate pharmacological properties of NTR1 on neurotoxicity of glutamate (Glu) in primary cultured mouse cortical neurons along with its possible mechanism of action. We found that NTR1 significantly protected neurons from the loss of cellular viability caused by brief exposure to 10 mu M Glu for 1 hr in a dose-dependent manner at concentrations from 0.1 to 10 mu M, without affecting the viability alone. NTR1 significantly inhibited the increased number of cells positive to propidium iodide (PI) staining, increase of intracellular free Ca(2+) ions, overproduction of intracellular reactive oxygen species, and depolarization of mitochondrial membrane potential in cultured neurons exposed to Glu, in addition to blocking decreased Bcl-2 and increased Bax expression levels. We further evaluated the target site at which NTR1 protects neurons from Glu toxicity by using the acquired expression strategy of N-methyl-D-aspartate (NMDA) receptor subunits in human embryonic kidney 293 cells. We found that 10 mu M NTR1 protected NR1/NR2B subunit expressing cells from cell death by 100 mu M NMDA, but not cells expressing NR1/NR2A subunits, when determined by PI staining. These results suggest that NTR1 may preferentially protect neurons from Glu excitotoxicity mediated by NMDA receptor composed of an NR1/NR2B subunit assembly in the brain. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jnr.22021

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記述言語：英語   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

In this review, we will summarize our ongoing Studies on the functionality of both gamma-aminobutyric acid (GABA) and glutamate receptors expressed by undifferentiated neural progenitor cells isolated from embryonic rodent brains. Cells were cultured with growth factors for the formation of round spheres by clustered cells under floating conditions, whereas a reverse transcription polymerase chain reaction analysis revealed expression of mRNA for particular subtypes of different ionotropic and metabotropic GABA and glutamate receptors in undifferentiated progenitors and neurospheres. Moreover, sustained exposure to either GABAergic or glutamatergic agonists not only modulated the size of neurospheres formed, but also affected spontaneous and induced differentiation of neural progenitor cells into particular progeny cell lineages Such as neurons and astroglia. Both GABA and glutamate could play a pivotal role in the mechanisms underlying proliferation for self-replication along with the determination of Subsequent differentiation fate toward particular progeny lineages through activation of their receptor subtypes functionally expressed by undifferentiated neural progenitor cells. Accordingly, neurogenesis seems to be also under control by GABAergic and glutamatergic signaling in developing brains as seen with neurotransmission in adult brains.

DOI： 10.1254/jphs.08R03CR

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記述言語：英語   出版者・発行元：日本神経化学会  

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

Tacrolimus (FK506) has been used as a therapeutic drug beneficial for the treatment of rheumatoid arthritis in humans. In this study, we investigated the effects of FK506 on cellular differentiation in cultured chondrogenic cells. Culture with FK506 led to a significant and concentration-dependent increase in Alcian blue staining for matrix proteoglycan at 0.1 to 1,000 ng/ml, but not in alkaline phosphatase (ALP) activity, in ATDC5 cells, a mouse prechondrogenic cell line, cultured for 7 to 28 days, while the non-steroidal anti-inflammatory drug indomethacin significantly decreased Alcian blue staining in a concentration-dependent manner, without altering ALP activity. FK506 significantly increased the expression of mRNA for both type 11 and type X collagen, but not for osteopontin, in ATDC5 cells. Similar promotion was seen in chondrogenic differentiation in both mouse metatarsals and chondrocytes cultured with FK506. However, FK506 failed to significantly affect transcriptional activity of the reporter construct for either sry-type HMG box 9 (Sox9) or runt-related transcription factor-2 (Runx2), which are both transcription factors responsible for chondrocytic maturation as a master regulator. These results suggest that FK506 may predominantly promote cellular differentiation into proliferating chondrocytes through a mechanism not relevant to the transactivation by either Sox9 or Runx2 in chondrogenic cells.

DOI： 10.1254/jphs.08315FP

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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 IRELAND LTD  

We have previously shown functional expression by osteoblasts of signaling machineries required for neurotransmission in the brain. In this study, we have evaluated possible functional expression of different osseous genes in the brain. In embryonic and adult mouse brains, mRNA expression was invariably seen for the master regulator of osteoblastic differentiation Runt related factor-2 (Runx2), in addition to the partner protein core binding factor-beta and their targets such as osteopontin (OPN) and matrix metalloproteinase-13 (MMP13), but not for collagen-I or osteocalcin. In pluripotent P19 progenitor cells, Runx2 mRNA expression was drastically increased along with mRNA expression of an astrocytic marker. but not with neuronal marker mRNA expression. Both mRNA and corresponding protein were detected for Runx2 in cultured rat neocortical astrocytes and astrocytic C6 glioma cells. In C6 glioma cells, transient overexpression of Runx2 significantly increased mRNA expression of MMP13, but not of OPN. Moreover, transient overexpression of Runx2 significantly increased luciferase activity in C6 glioma cells transfected with the reporter plasmid linked to a wild-type Runx2 binding element in the MMP13 promoter, but not in cells with a mutated element. These results suggest that Runx2 signal input may lead to transactivation of MMP13 gene without affecting OPN expression in astrocytes. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2008.12.037

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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-BLACKWELL  

Adrenaline is believed to play a dual role as a neurotransmitter in the central nervous system and an adrenomedullary hormone in the peripheral tissues. In contrast to accumulating evidence for the involvement in endochondral ossification, osteolblastogenesis, and osteoclastogenesis, little attention has been paid to the role of adrenergic signals in the mechanisms underlying proliferation and differentiation of mesenchymal stem cells with self-renewal capacity and multi-potentiality to differentiate into osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was seen for different adrenergic receptor (AdR) subtypes, including beta(2)AdR, in the mesenchymal stem cell line C3H10T1/2 cells and mouse bone marrow mesenchymal stem cells before differentiation. Exposure to adrenaline not only increased cAMP formation, phosphorylation of cAMP responsive element (CRE) binding protein (CREB) on serine 133 and CRE reporter activity in a manner sensitive to propranolol, but also rendered C3H10T1/2 cells resistant to the cytotoxicity of hydrogen peroxide, but not of either 2,4-dinitirophenol or tunicamycin. Adrenaline induced a rapid but transient increase in mRNA expression of the antioxidative gene nuclear factor E2 p45-related factor-2 (Nrf2) along with an increase in the cystine/glutamate antiporter subunit xCT mRNA expression. Hydrogen peroxide was less cytotoxic in cells overexpressing Nrf2, moreover, while adrenaline significantly increased xCT promoter activity with an increase in endogenous glutathione levels. These results suggest that adrenaline may selectively protect mesenchymal C3H10T1/2 cells from oxidative stress through a mechanism related to the promoted biosynthesis of glutathione in association with transient Nrf2 expression after activation of beta(2)AdR.

DOI： 10.1002/jcp.21594

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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 INC  

We have investigated the role of N-methyl-D-aspartate receptors (NMDARs) and gamma-aminobutyric acid receptors type A (CABAARs) at an early stage of P19 neuronal differentiation. The subunit expression was profiled in 24-hour intervals with RT-PCR and functionality of the receptors was verified via fluo-3 imaging of Ca2+ dynamics in the immautre P19 neurons showing that both NMDA and GABA excite neuronal bodies, but only polyamine-site sensitive NMDAR stimulation leads to enhanced Ca2+ signaling in the growth cones. Inhibition of NR1/NR2B NMDARs by 1 mu M ifenprodil severely impaired P19 neurite extension and fasciculation, and this negative effect was fully reversible by polyamine addition. In contrast, GABAAR antagonism by a high dose of 200 mu M bicuculline had no observable effect on P19 neuronal differentiation and fasciculation. Except for the differential NMDAR and GABAAR profiles of Ca2+ signaling Within the immature P19 neurons, we have also shown that inhibition of NR1/NR2B NMDARs strongly decreased mRNA level of NCAM-180, which has been previously implicated as a regulator of neuronal growth cone protrusion and neurite extension. Our data thus suggest a critical role of NR1/NR2B NMDARs during the process of neuritogenesis and fasciculation of P19 neurons via differential control of local growth cone Ca2+ surges and NCAM-180 signaling. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2008.06.009

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記述言語：英語   出版者・発行元：日本神経化学会  

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

Previous studies have demonstrated the functional expression, by osteoblasts, of N-methyl-D-aspartate (NMDA) receptors responsible for the promotion of cellular differentiation in bone. We have now evaluated the possible role of the endogenous co-agonist of NMDA receptors, glycine (Gly), in chondrogenesis. In ex vivo organotypic cultures of fetal mouse tibias, proximal and distal cartilaginous primordia were significantly increased in the presence of Gly, with the osteogenic center being unchanged. Exposure to Gly drastically increased mRNA expression of the calcified chondrocyte marker osteopontin, without markedly affecting that of a proliferating chondrocyte marker or a hypertrophic chondrocyte marker, as shown in organotypic cultures by in situ hybridization analysis. Gly significantly increased Ca(2+) accumulation, osteopontin mRNA expression, and alkaline phosphatase activity in cultured rat costal chondrocytes, without significantly affecting those in cultured rat calvarial osteoblasts. The increase induced by Gly was significantly prevented by an NMDA receptor channel blocker and an antagonist at the Gly site on NMDA receptors, but not by an inhibitory Gly receptor antagonist or a Gly transporter inhibitor, in cultured chondrocytes. Constitutive mRNA expression was seen for NR1, NR2D, and NR3A subunits of NMDA receptors, but not for Gly receptors and transporters, in cultured chondrocytes. Corresponding immunoreactive proteins were detected for NR1 and NR2D subunits in cartilaginous zones of fetal mouse tibias. Thus, Gly might, at least in part, play a role as a trophic factor in the mechanisms associated with chondral calcification through the Gly site of NMDA receptors functionally expressed by chondrocytes in rodent cartilage.

DOI： 10.1007/s00441-008-0607-7

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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  

Serine racemase (SR) is responsible for the biosynthesis of D-serine (D-Ser), an endogenous co-agonist for N-methyl-D-aspartate (NMDA) receptors, from L-serine (L-Ser) in the central nervous system. In the present study, we investigated the role of SIR in the regulation of chondrogenic differentiation in cartilage. On in situ hybridization analysis of tibia from neonatal rats,SR mRNA was ubiquitously expressed in all cell layers of proliferating to hypertrophic chondrocytes. In the pre-chondrogenic cell line ATDC5 cells, mRNA expression was seen with SR irrespective of the cellular maturity, with no mRNA expression of the NRI subunit essential for the heteromeric assembly of functional NMDA receptor channels. In ATDC5 cells stably overexpressing SR, significant inhibition was found with the maturation-dependent temporal increases in Alcian blue staining, alkaline phosphatase (ALP) activity and mRNA expression of type 11 and type X collagens. Stable overexpression of SR significantly impaired the sry-type HMG box 9 (Sox9) transcriptinal activity in ATDC5 cells, while Sox9 transcriptional activity was significantly inhibited in COS7 cells with co-introduction of SIR and Sox9. However, no significant inhibition was seen with Sox9 transcriptional activity in COS7 cells co-introduced of either SRK56G defective of D-Ser formation ability or 3-phosphoglycerate dehydrogenase essential for D-Ser biosynthesis. The co-introduction of SR with Sox9 significantly decreased the Sox9 protein level with that of Sox9 mRNA being unchanged. These results suggest that SR may negatively regulate cellular differentiation through the inhibition of Sox9 transcriptional activity in chondrocytes.

DOI： 10.1002/icp.21310

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

The view that L-glutamate (Glu) is an excitatory amino acid nurotransmitter in the mammalian central nervous system is prevailing on the basis of successful cloning of a number of genes encoding different signaling molecules, such as Glu receptors for the signal input, Glu transporters for the signal termination and vesicular Glu transporters for the signal output through exocytotic release. Little attention has been paid to an extracellular transmitter role of Glu in peripheral neuronal and non-neuronal tissues, by contrast, whereas recent molecular biological and pharmacological analyses including ours give rise to a novel function for Glu as an autocrine and/or paracrine signal mediator in bone comprised of osteoblasts, osteoclasts and osteocytes, in addition to other peripheral tissues including pancreas, adrenal and pituitary glands. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying the maintenance of cellular homeostasis as an excitatory neurotransmitter in the central nervous system and as an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, therefore, we would outline the possible signaling system for Glu to play a role as an extracellular signal mediator in mechanisms underlying maintenance of the cellular homeostasis in bone.

DOI： 10.1254/jphs.FM0070243

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PHARMACEUTICAL SOC JAPAN  

This study was aimed to investigate pharmacological properties of notoginsenoside R1 (NG-R1), an ingredient in Panax notoginseng, against the neurotoxicity of glutamate (Glu) in primary cultured mouse neocortical neurons. Cerebral cortex was isolated from fetal mice at 15 days after gestation, followed by culture in DMEM in the presence of fetal calf serum (FCS) for the initial 3 days and subsequent culture in DMEM in the absence of FCS with medium change every 3 days. Brief exposure to Glu drastically reduced cell viability 24 h later in cortical neurons cultured for 7 days, while simultaneous addition of NG-R1 led to significant protection against the loss of cellular viability by Glu. NG-R1 inhibited the elevation of intracellular Ca2+ ions, the generation of intracellular reactive oxygen species (ROS) and the depolarization of mitochondrial membrane potential in cultured cortical neurons exposed to Glu. Glu decreased the expression of Bcl-2 and increased that of Bax in cultured cortical neurons, whereas further addition of NG-R1 resulted in significant protection against the Glu-induced changes in Bcl-2 and Bax expressions. These results suggest that particular notoginsenosides may protect neurons from the neurotoxicity of Glu through a mechanism related to the suppression of the elevation of intracellular Ca2+ ions, generation of intracellular ROS, depolarization of mitochondrial membrane potential and induction of apoptosis.

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PHARMACEUTICAL SOC JAPAN  

The view that circadian rhythm involves different proteins relevant to clock genes, including Period (Per), Cryptochrome, Clock, Bmal1 and Dec, in the suprachiasmatic nucleus in mammals is prevailing. In line with recent studies on expression of these clock genes in particular peripheral tissues such as heart and liver, we have shown transient promotion by parathyroid hormone (PTH) of Per1 mRNA expression in cultured chondrocytes. In order to elucidate a possible functional role of Per1 in chondrocytes, in the present study, we have attempted to establish the mouse chondrogenic cell line ATDC5 cells stably transfected with Per1 expression vector. For this purpose, ATDC5 cells were transfected with pcDNA3.1 containing the full-length coding region of Per1 (ATDC5-Per1) or empty vector (ATDC5-EV), followed by selection of particular clones with G418. Pools of clones of ATDC5-Per1 cells were isolated for determination of expression levels of Per1 by real-time-PCR. In ATDC5 cells stably overexpressing Per1, significant inhibition was found with the maturation-dependent temporal increases in Alcian blue staining and mRNA expression of type II and type X collagens. An attempt was next made to elucidate underlying mechanisms for the suppression by Per1 overexpression of chondrogenic differentiation with the reporter assay using a 4x48-p89-Luc, 4 tandem copies of Sox9 allies (Sox5, Sox6 and Sox9) binding site linked to the minimal Col II gene promoter in the luciferase reporter plasmid. The 4x48-p89-Luc plasmid was transiently transfected into ATDC5-Per1 and ATDC5-EV cells, followed by determination of luciferase activity 48 h after transfection. Luciferase activity was significantly inhibited in ATDC5-Per1 cells compared with that in ATDC5-EV cells. Moreover, overexpression of Per1 decreased the Sox6 mRNA level along with a decreased Sox6 promoter activities required for Sox6 mRNA expression in ATDC5 cells. These results suggest that PTH may suppress chondrogenesis through the promotion of Per1 expression toward repression of Sox6 mRNA expression.

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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 BV  

The view that ascorbic acid indirectly benefits osteoclastogenesis through expression of receptor activator of nuclear factor-kappa B (NF-kappa B) ligand (RANKL) by osteoblasts is prevailing. In this study, we have examined the direct effect of ascorbic acid on osteoclastogenesis in cultured mouse osteoclasts differentiated from bone marrow precursors. The absence of alkaline phosphatase and ostcoblastic marker genes validated the usefulness of isolation procedures. Sustained exposure to ascorbic acid, but not to dehydroascorbic acid, significantly reduced the number of multinucleated cells positive to tartrate-resistant acid pbosphatase (TRAP) staining. In cultured osteoclasts, mRNA expression was seen for glucose transporter-1 involved in membrane transport of dehydroascorbic acid, but not for sodium-dependent vitamin C transporters-1 and -2 that are both responsible for the transport of ascorbic acid. The inhibition by ascorbic acid was completely prevented by catalase, while ascorbic acid or hydrogen peroxide drastically increased the number of cells stained with propidium iodide and the generation of reactive oxygen species, in addition to inducing mitochondrial membrane depolarization in cultured osteoclasts. In pre-osteoclastic cell line RAW264.7 cells, ascorbic acid similarly inhibited the formation of TRAP-positive multinucleated cells, with a significant decrease in RANKL-induced NF-kappa B transactivation. Moreover, co-culture with osteoblastic MC3T3-E1 cells significantly prevented the ascorbic acid-induced decrease in the number of TRAP-positive multinucleated cells in RAW264.7 cells. These results suggest that ascorbic acid may play a dual repulsive role in osteoclastogenesis toward bone remodeling through the direct cytotoxicity mediated by oxidative stress to osteoclasts, in addition to the indirect trophism mediated by RANKL from osteoblasts. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2007.07.041

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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-BLACKWELL  

Although previous studies including ours have demonstrated the functional expression of different glutamate (Glu) signaling machineries such as Glu receptors (GluRs) and transporters in osteoblasts and chondrocytes, little attention has been paid to the role of Glu in their ancestral mesenchymal stem cells to date. In the present study, we have evaluated the possible functionality of Glu in cultured mouse mesenchymal stem cell line C3H10T1/2 cells endowed to proliferate for the self-renewal and to differentiate toward osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was for the first time shown with the cystine/Glu antiporter composed of xCT and 4F2hc subunits, in addition to particular excitatory amino acid transporter (EAAT) isoforms and ionotropic GluRs, in undifferentiated C3H10T1/2 cells. Glu significantly suppressed the proliferation activity at a concentration over 500 mu M without inducing cell death or differentiation, while the suppression occurred in a manner sensitive to the prevention by cystine and reduced glutathione (GSH), but not by EAAT inhibitors. A significant decrease was seen in intracellular GSH levels in C3H10T1/2 cells cultured with Glu, whereas the cellular proliferation activity was drastically decreased by the addition of the GSH depleter cyclohexene-l-one and the GSH biosynthesis inhibitor L-buthionine-[S,R]-sulfoximine, respectively. Transient overexpression of both xCT and 4F2hc subunits led to an increased basal proliferative activity in C3H10T1/2 cells. These results suggest that Glu could suppress the cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the cystine/Glu antiporter in C3H10T1/2 cells.

DOI： 10.1002/jcp.21145

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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  

Activation of particular glutamate (Glu) receptors is shown to promote cellular differentiation toward maturation during osteoblastogenesis. In the present study, we have evaluated the possible modulation by Glu of cellular proliferation in osteoblastic cells endowed to proliferate for self-renewal and to differentiate toward matured osteoblasts. Exposure to Glu significantly suppressed the proliferation activity at a concentration over 500 mu M without inducing cell death in osteoblastic MC3T3-E1 cells before differentiation. The suppression by Glu occurred in a manner sensitive to the prevention by either cystine or reduced glutathione. Expression of mRNA was for the first time shown with the cystine/Glu antiporter composed of xCT and 4F2hc subunits in these undifferentiated osteoblastic cells. A significant decrease was seen in intracellular total glutathione levels in undifferentiated MC3T3-E1 cells cultured with Glu, indeed, whereas the cellular proliferation activity was drastically decreased by the addition of the glutathione depleter cyclohexene-1-one and the glutathione biosynthesis inhibitor L-buthionine-[S,R]-sulfoximine, respectively. Exposure to Glu led to a significant increase in mRNA expression of nuclear factor E2 p45-related factor 2 (Nrf2) together with the generation of reactive oxygen species, while a significant decrease was seen in the proliferation activity in MC3T3-E1 cells with stable overexpression of Nrf2. These results suggest that Glu could suppress the cellular proliferation toward self-renewal through a mechanism associated with the upregulation of Nrf2 expression in association with the depletion of intracellular glutathione after promoting the retrograde operation of the cystine/Glu antiporter in undifferentiated MC3T3-E1 cells.

DOI： 10.1002/jcp.21095

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC INVESTIGATIVE PATHOLOGY, INC  

Previous studies have demonstrated functional expression of different glutamate receptor subtypes (GluRs) in both osteoblasts and osteoclasts. in the present study, we investigated the possible functional expression by osteoclasts of different glutamatergic signaling machineries including GluRs. In disagreement with the aforementioned prevailing view, no mRNA expression was found for all GluRs examined in primary cultured mouse osteoclasts differentiated from bone marrow precursors. Constitutive expression of mRNA was seen with glutamate transporters, such as excitatory amino acid transporters and cystine/glutamate antiporter, in primary osteoclasts. Glutamate significantly inhibited osteoclastogenesis at a concentration over 500 mu mol/L in both primary osteoclasts and preosteoclastic RAW264.7 cells without affecting the cell viability in a manner sensitive to the antiporter inhibitor. in RAW264.7 cells stably overexpressing the cystine/glutamate antiporter, the inhibition by glutamate was more conspicuous than in cells transfected with empty vector alone. The systemic administration of glutamate significantly prevented the decreased bone mineral density in both femur and tibia in addition to increased osteoclastic indices in ovariectomized mice in vivo. These results suggest that glutamate may play a pivotal role in mechanisms associated with osteoclastogenesis through the cystine/glutamate antiporter functionally expressed by osteoclasts devoid of any GluRs cloned to date.

DOI： 10.2353/ajpath.2007.061039

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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  

The transcription factor nuclear factor E2 p45-related factor 2 (Nrf2) forms heterodimers with small musculoaponeurotic fibrosarcoma (Maf) proteins for the selective recognition of the antioxidant responsive element on target genes, followed by the regulation of gene expression of phase II detoxifying enzymes as well as oxidative-stress-inducible proteins in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of chondrocyte differentiation as well as the expression pattern of Nrf2 in cartilage. In tibia from embryonic mice at E15.5, Nrf2 mRNA expression was restricted to both proliferating and pre-hypertrophic chondrocytes, with few signals in early and late hypertrophic chondrocytes expressing both type X collagen and osteopontin. On in situ hybridization analysis of tibia from neonatal mice at 1 day after birth, by contrast, Nrf2 was expressed in all chondrocytic layers in addition to osteoblasts attached to cancellous bone. In pre-chondrogenic cell line ATDC5 cells, furthermore, expression of Nrf2 mRNA was also confirmed together with mRNA expression of the Kelch-like ECH associating protein 1 and small Maf proteins. In ATDC5 cells stably transfected with Nrf2, significant inhibition was seen in the differentiation-dependent induction of alkaline phosphatase and increase in the Alcian blue staining intensity. Furthermore, stable overexpression of Nrf2 significantly decreased mRNA expression of several chondrocyte differentiation markers such as type II collagen, type X collagen and osteopontin. These data suggest that Nrf2 may be a negative regulator of the cellular differentiation toward maturation in chondrocytes. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2006.08.016

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

In the present study, we have attempted to evaluate the pharmacological actions of three major phenolic antidiarrheic ingredients, including 2-methoxyphenol (2MP), 2-methoxy-4-methylphenol (2M4MP) and 2-methoxy-4-ethyphenol (2M4EP), on the functionality and integrity of bone by in vitro and in vivo experimental techniques. Intermittent oral administration of 2M4MP and 2M4EP, but not 2MP, significantly prevented reductions of bone mineral density in total femur, distal femur and tibia, in addition to alterations of several osteoclastic parameters on histomorphometric analysis, when determined 28 days after ovariectomy in mice. All three phenolic ingredients examined significantly inhibited the developmental increase in the number of multinucleated cells positive to tartrate-resistant acid phosphatase staining in cultured mouse osteoclasts differentiated from bone marrow precursors in the presence of both macrophage-colony stimulating factor and receptor activator of nuclear factor-kappa B ligand, which occurred in a concentration-dependent manner at a concentration range of 1 mu M-1 mM without inducing cell death. Moreover, both 2M4MP and 2M4EP at 1 mM not only prevented the cell death induced by 0.5 mM H2O2 in cultured rat calvarial osteoblasts, but also suppressed the generation of intracellular reactive oxygen species in osteoblasts exposed to H2O2, with a radical scavenging action as revealed by electron spin resonance analysis. These results suggest that particular phenolic antidiarrheic ingredients may prevent ovariectomy-induced bone loss through a mechanism related to the inhibition of osteoclastogenesis in association with an anti-oxidative property in osteoblasts. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bcp.2006.09.025

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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 BV  

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the brain, but widely distributed in different peripheral organs. We have previously shown the functional expression of GABAB receptors required for GABAergic signal input by cultured rat calvarial osteoblasts. This study focused on the possible functional expression of the machinery required for GABAergic signal termination such as GABA transporters. In rat calvarial osteoblasts cultured for 7 days, [H-3]GABA accumulation was observed in a temperature-, sodium- and chloride-dependent manner, consisting of a single component with a K-m value of 789.6 +/- 9.0 mu M and a V-max value of 4.4 +/- 0.1 nmol/min/mg protein, respectively. Both nipecotic and L-2,4-diaminobutyric acids significantly inhibited [H-3]GABA accumulation in a concentration-dependent manner. Constitutive expression was seen with mRNA for the betaine/GABA transporter-1 (BGT-1) and taurine transporter (TauT), while hyperosmotic cultivation led to significant increases in both [3H]GABA accumulation and BGT-1 mRNA expression without affecting TauT mRNA expression. Highly immunoreactive cells were detected for the BGT-1 isoforin at the surface of trabecular bone of neonatal rat tibias. Sustained exposure to GABA significantly inhibited alkaline phosphatase (ALP) activity, but not cellular viability, at concentrations above 0.1 mM in osteoblasts cultured for 3 to 28 days. Nipecotic acid not only decreased ALP activity alone, but also further decreased ALP activity in osteoblasts cultured in the presence of GABA. These results suggest that the BGT-1 isoforin may be functionally expressed by rat calvarial osteoblasts to play a hitherto unidentified role in mechanisms underlying hyperosmotic regulation of osteoblastogenesis. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2006.08.088

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

We demonstrated previously that exogenous pyruvate has a protective action against cell death by hydrogen peroxide in cultured osteoblasts through a mechanism associated with its antioxidative property. In the present study, we have evaluated possible participation of monocarboxylate transporters (MCTs) responsible for the bidirectional membrane transport of pyruvate in the cytoprotective property in osteoblasts. Expression of the MCT2 isoform was found in cultured rat calvarial osteoblasts and in osteoblasts located on mouse tibia at both mRNA and protein levels. The accumulation of [C-14] pyruvate occurred in a temperature- and pH-dependent manner in osteoblasts cultured for 7 days with high sensitivity to a specific MCT inhibitor, whereas pyruvate was released into extracellular spaces from cultured osteoblasts in a fashion sensitive to the MCT inhibitor. Transient overexpression of the MCT2 isoform led to reduced vulnerability to the cytotoxicity of hydrogen peroxide with an increased activity of [C-14] pyruvate accumulation in murine osteoblastic MC3T3-E1 cells. Ovariectomy significantly decreased the content of pyruvate in femoral bone marrows in mice in vivo, whereas daily i.p. administration of pyruvate at 0.25 g/kg significantly prevented alterations of several histomorphometric parameters as well as cancellous bone loss in femurs by ovariectomy on 28 days after the operation. These results suggest that MCTs may be functionally expressed by osteoblasts to play a pivotal role in mechanisms related to the cytoprotective property of pyruvate.

DOI： 10.1124/mol.106.024398

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：F HERNANDEZ  

Although we have previously shown drastic cell death by pyruvate deficiency in osteoblasts at the proliferative stage, the exact mechanism remains unclear so far. Cell survivability was significantly decreased in rat calvarial osteoblasts cultured for 0 to 3 days in vitro (DIV) following replacement of the eutrophic alpha-modified minimum essential medium (alpha-MEM) with Dulbecco's modified eagle medium (DMEM) for cultivation. The addition of pyruvate enriched in alpha-MEM, but not in MEM, entirely prevented cell death induced by the medium replacement throughout a culture period from 0 to 3 DIV. Both cysteine and reduced glutathione protected cell death in cells cultured for 3 DIV without significantly affecting that in cells cultured for 1 DIV, however, while none of lactate, acetate and insulin significantly prevented the cell death irrespective of the culture period up to 3 DIV. A marked increase was detected in intracellular reactive oxygen species (ROS) levels 4 h after the medium replacement. In osteoblasts cultured in alpha-MEM for 3 DIV, but not in those for 7 DIV, hydrogen peroxide (H2O2) markedly decreased cell survivability when exposed for 2 to 24 h. Furthermore, H2O2 was effective in significantly decreasing cell survivability in osteoblasts cultured in DMEM for 7 DIV. Pyruvate at 1 mM not only prevented cell death by H2O2, but also suppressed the generation of intracellular ROS in osteoblasts exposed to H2O2. These results suggest that pyruvate could be cytoprotective through a mechanism associated with the anti-oxidative property rather than an energy fuel in cultured rat calvarial osteoblasts.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

In bone, clock genes are involved in the circadian oscillation of bone formation and extracellular matrix expression. However, to date little attention has been paid to circadian rhythm in association with expression of clock genes during chondrogenesis in cartilage. In this study, we investigated the functional expression of different clock genes by chondrocytes in the course of cartilage development. The mRNA expression of types I, II, and X collagens exhibited a 24-h rhythm with a peak at zeitgeber time 6, in addition to a 24-h rhythmicity of all the clock genes examined in mouse femurs in vivo. Marked expression of different clock genes was seen in both osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells in vitro, whereas parathyroid hormone (PTH) transiently increased period 1 (per1) mRNA expression at 1 h inboth cell lines. Similar increases were seen in the mRNA levels for both per1 and per2 in prehypertrophic chondrocytes in metatarsal organotypic cultures within 2 h of exposure to PTH. PTH significantly activated the mouse per1 (mper1) and mper2 promoters but not the mper3 promoter in a manner sensitive to both a protein kinase A inhibitor and deletion of the cAMP-responsive element sequence (CRE) in ATDC5cells. In HEK293 cells, introduction of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (bmal1)/clock enhanced mouse type II collagen first intron reporter activity without affecting promoter activity, with reduction effected by either per1 or per2. These results suggest that PTH directly stimulates mper expression through a protein kinase A-CRE-binding protein signaling pathway for subsequent regulation of bmal1/clock-dependent extracellular matrix expression in cartilage.

DOI： 10.1074/jbc.M512362200

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

Although previous studies have demonstrated increased levels of the brain neurotransmitter glutamate (Glu) in the synovial fluid from patients with arthritis, not much attention has been paid to the possible role of Glu in joint synovial tissues to date. Constitutive expression of mRNA was for the first time shown with glutamate aspartate transporter, glutamate transporter-1 and excitatory amino acid carrier-1 (EAAC1), in addition to with particular ionotropic and metabotropic Gin receptors, in cultured synovial fibroblasts prepared from knee joints of male Lewis rats. Immunohistochemical analysis revealed high localization of immunoreactive EAAC1 at synovial tissues. The accumulation of [H-3]Glu occurred in a temperature- and sodium-dependent manner in cultured synovial fibroblasts, with a Km of 23.1 +/- 1.1 mu M and a V-max of 237.1 +/- 31.1 pmol(mg protein min), respectively. In rats with arthritis induced by immunization to type-II collagen, marked increases were seen in hind paw volume, cytokine mRNA expression and Glu levels in synovial tissues, in addition to histological erosion. In cultured synovial fibroblasts prepared from these arthritic rats, [H-3]Glu accumulation was drastically increased with biochemical and pharmacological profiles similar to those seen in normal synovial fibroblasts. The exposure to Glu at 500 mu M doubled the incorporation of 5-bromo-2'-deoxyuridine in cultured synovial fibroblasts of arthritic but not normal rats, without significantly affecting mRNA expression of different cytokines in both synovial fibroblasts. These results suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation through particular transporters functionally expressed by synovium in rheumatoid arthritis. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bcp.2005.09.010

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

1 Previous studies have demonstrated the functional expression by osteoblasts of glutamate (Glu) signaling machineries responsible for the stimulation of cell proliferation and differentiation in bone, while there is no information available on the expression of the Glu signaling system by cartilage to date.
2 In cultured mouse embryonic metatarsals isolated before vascularization, chondral mineralization was almost completely inhibited in the presence of the group III metabotropic Glu receptor (mGluR) agonist L-(1)-2-amino-4-phosphonobutyrate (L-AP4) in a manner sensitive to an antagonist, with the total length being unchanged.
3 A group II mGluR agonist was similarly more effective in inhibiting the mineralization than a group I mGluR agonist, while none of ionotropic GluR agonists drastically affected the mineralization.
4 Both histological and in situ hybridization analyses revealed that L-AP4 specifically inhibited chondral mineralization, without apoptotic cell death, in cultured metatarsals.
5 In addition to the constitutive expression of mRNA for particular mGluRs in both cultured mouse metatarsals and rat costal chondrocytes, L-AP4 significantly inhibited the accumulation of cyclic AMP by forskolin and parathyroid hormone in a manner sensitive to a group III mGluR antagonist in cultured chondrocytes.
6 Moreover, L-AP4 drastically inhibited the expression of osteopontin mRNA in both cultured metatarsals and chondrocytes.
7 These results suggest that Glu may at least in part play a role as a signal mediator in mechanisms associated with chondral mineralization through the group III mGluR subtype functionally expressed by chondrocytes in rodent cartilage.

DOI： 10.1038/sj.bjp.0706358

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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  

We have directed a polyclonal antibody against an oligopeptide (123-136) of the transcription factor cyclic AMP responsive element-binding protein (CREB) including the serine residue at 133. Rabbit sera were purified by ammonium sulfate precipitation, followed by affinity chromatography to homogeneity on one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified antibody not only induced marked supershift of CREB binding, without affecting binding of activator protein-1 on gel retardation electrophoresis, but also differentiated between CREB and CREB phosphorylated at serine133 in brain nuclear fractions on Western blotting. Immunoreactive CREB was detected in both cytosolic and nuclear fractions of discrete murine brain structures but was more highly condensed in cerebellum than in neocortex and hippocampus. Incubation of brain nuclear fractions led to a marked export of immunoreactive CREB in a temperature-dependent manner, whereas the temperature-dependent export activity was significantly lower in cerebellum than in other brain structures. Suppression of general new protein synthesis by cycloheximide (500 mg/kg, i.p.) in vivo resulted in a significant decrease in the nuclear CREB level, with a concomitant increase in the cytosolic level in hippocampus, but not in cerebellum. These results suggest that the nuclear export activity might vary from region to region in murine brains through a hitherto unidentified mechanism other than the nuclear localization signal, to result in different nuclear condensation ratios for subsequent elicitation of differential transcriptional activities by the constitutive transcription factor CREB in the nucleus. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/jnr.20504

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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-BLACKWELL  

In rat hippocampal neurons cultured with the antagonist for N-methyl-D-aspartate (NMDA) receptors dizocilpine (MK-801) for 8 days in vitro (DIV), a significant decrease was seen in the expression of microtubule-associated protein-2 (MAP-2) as well as mRNA for both brain-derived neurotrophic factor (BDNF) and growth-associated protein-43 (GAP-43), in addition to decreased viability. MK-801 not only decreased the expression of the NRII subunit of NMDA receptors but also increased NR2A expression, without affecting NR2B expression. Repetitive daily exposure to static magnetic fields at 100 mT for 15 min led to a decrease in the expression of MAP-2, without significantly affecting cell viability or the expression of neuronal nuclei (NeuN) and GAP-43. However, the repetitive magnetism prevented decreases in both BDNF mRNA and MAP-2 and additionally increased the expression of NR2A subunit, without altering NR1 expression in neurons cultured in the presence of MK-801. Repetitive magnetism was also effective in preventing the decrease by MK-801 in the ability of NMDA to increase intracellular free Ca2+ ions, without affecting the decrease in the maximal response. These results suggest that repetitive magnetism may at least in part counteract the neurotoxicity of MK-801 through modulation of the expression of particular NMDA receptor subunits in cultured rat hippocampal neurons. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/jnr.20497

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記述言語：英語   出版者・発行元：2  

The L-glutamate (Glu) has been hypothesized as an excitatory amino acid neurotransmitter in the mammalian central nervous system after successful cloning and identification of a number of genes encoding signaling machineries required for the neurocrine at synapses in the brain. These include excitatory amino acid transporters (EAATs) for signal termination and vesicular Glu transporters (VGLUTs) for signal output through exocytotic release, in addition to Glu receptors (GluRs) for signal input. These Glu signaling molecules not only play key roles in mechanisms associated with synaptic plasticity such as learning and memory, but also participate in the etiology and pathology of different neuropsychiatric disorders and neuronal cell death seen in various neurodegenerative diseases. Of the aforementioned Glu signaling molecules, EAATs are essential for the termination of signal transmission mediated by Glu as well as for the prevention of neurotoxicity mediated by this endogenous excitotoxin, while VGLUTs are crucial for the storage of Glu in synaptic vesicles to suffice for the definition of a glutamatergic phenotype. Many early desperate efforts were devoted to the search and development of novel compounds with a therapeutic window toward GluRs, while relatively little attention was paid to either EAATs or VGLUTs in this aspect. In this review, therefore, we will summarize the classification and functionality of EAATs and VGLUTs with a focus on their possibilities as potential therapeutic targets for different neurodegenerative and neuropsychiatric disorders related to malfunction of Glu signaling in human beings. © 2005 Bentham Science Publishers Ltd.

DOI： 10.2174/1568007053544093

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

In the present study, we have attempted to demonstrate constitutive and functional expression in bone of particular glutamate transporters (GluTs) required for signal termination in glutamatergic signaling process. Reverse transcription polymerase chain reaction revealed constitutive expression of mRNA for the neuronal GluT subtype excitatory amino acid carrier-1, in addition to glial subtypes such as glutamate aspartate transporter and glutamate transporter-1, in rat calvarial osteoblasts cultured for 7-21 days in vitro (DIV). The accumulation of [H-3]glutamate (Glu) occurred in a temperature- and sodium-dependent manner with pharmacological profiles similar to those for brain GluTs in osteoblasts cultured for 7 DIV, while three different agonists at ionotropic Glu receptors significantly inhibited the accumulation of [H-3]Glu in osteoblasts. Although [H-3]Glu accumulation consisted of a single component with a K-m value of 26.0 +/- 15.8 muM and a V-max value of 960 +/- 122 nmol/(min mg protein), respectively, in osteoblasts cultured for 7 DIV, in vitro maturation led to a significant decrease in V-max value to 290 +/- 33 nmol/(min mg protein) without significantly affecting K-m values on 21 DIV. These results suggest that Glu could be incorporated into intracellular locations through glial and/or neuronal GluT subtypes expressed in cultured rat calvarial osteoblasts. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bcp.2004.03.020

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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 ENDOCRINOLOGY  

Neither expression nor functionality is clear in peripheral tissues with the molecular machineries required for excitatory neurotransmitter signaling by L-glutamate (Glu) in the central nervous system, while a recent study has shown that several Glu receptors are functionally expressed in the rat testis. This fact prompted us to explore the possible functional expression in the rat testis of the Glu transporters usually responsible for the regulation of extracellular Gin concentrations in the brain. RT-PCR revealed the expression, in the rat testis, of mRNA for five different subtypes of Glu transporters, in addition to that for particular subtypes of ionotropic and metabotropic Glu receptors. Glutamate transporter-1 (GLT-1) was different in the brain from that in the testis in terms of molecular sizes on Northern and Western blot analyses. Iu situ hybridization aswell as immunohistochemical analysis showed localized expression of glutamate aspartate transporter at interstitial spaces and GLT-1 at elongated spermatids in the rat testis respectively. The expression of mRNA was localized for excitatory amino acid transporter-5 at the basal compartment of the seminiferous tubule in the rat testis. [H-3]Glu was accumulated in testicular crude mitochondrial fractions in a temperature- and sodium-dependent saturable manner with pharmacological profiles similar to those shown in brain crude mitochondrial fractions. These results suggested that particular subtypes of central Glu transporters for the regulation of extracellular Glu concentrations in the rat testis could be constitutively and functionally expressed.

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

L-Glutamate (Glu) has been thought to be an excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). The hypothesis is supported by successful cloning of a number of genes encoding different signaling molecules, such as Glu receptors for signal input, Glu transporters for signal termination, and vesicular Glu transporters for signal output through exocytotic release. Limited information is available in the literature with regard to an extracellular transmitter role of Glu in peripheral neuronal and non-neuronal tissues, whereas recent molecular biological analyses including ours give rise to a novel function for Glu as an autocrine and/or paracrine factor in bone comprised of osteoblasts, osteoclasts, and osteocytes, in addition to other peripheral tissues including pancreas, adrenal, and pituitary glands. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying maintenance of cellular homeostasis as an excitatory neurotransmitter in the CNS and as an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, therefore, we summarized the possible signaling by Glu as an extracellular signal mediator in mechanisms underlying maintenance of cellular homeostasis with a focus on bone tissues.

DOI： 10.1254/jphs.94.215

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記述言語：英語   出版者・発行元：BLACKWELL PUBLISHING LTD  

The hypothesis that L-glutamate (Glu) is an excitatory amino acid neurotransmitter in the mammalian central nervous system is now gaining more support after the successful cloning of a number of genes coding for the signaling machinery required for this neurocrine at synapses in the brain. These include Glu receptors (signal detection), Glu transporters (signal termination) and vesicular Glu transporters (signal output through exocytotic release). Relatively little attention has been paid to the functional expression of these molecules required for Glu signaling in peripheral neuronal and non-neuronal tissues; however, recent molecular biological analyses show a novel function for Glu as an extracellular signal mediator in the autocrine and/or paracrine system. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying the maintenance of cellular homeostasis - as an excitatory neurotransmitter in the central neurocrine system and an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, the possible Glu signaling methods are outlined in specific peripheral tissues including bone, testis, pancreas, and the adrenal, pituitary and pineal glands.

DOI： 10.1046/j.1432-1033.2003.03907.x

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掲載種別：研究論文（学術雑誌）  

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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 BV  

Accumulation of [H-3]L-serine in crude synaptosomal fractions freshly prepared from rat brain has been found to be temperature-sensitive and to consist of both Na+-dependent and Na+-independent components. The accumulation of [H-3]L-serine measured at submicromolar concentrations had a distinct substrate selectivity, different from the uptake of [H-3]L-proline, [H-3]L-glutamate and [H-3]GABA. It was fully inhibited by L-glutamine, L-asparagine, L-cysteine, L-alanine, L-leucine, L-isoleucine, L-tyrosine, L-phenylalanine, L-threonine and by the synthetic marker for the large neutral amino acid transport systems 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, but not influenced by P-alanine, taurine, glycine nor was it inhibited by the marker for the A system, L-2-methylamino isobutyric acid. D-Serine at 1 mM concentration produced no significant inhibition of the accumulation of 10 nM [H-3]L-serine. We conclude that L-serine uptake observed in the present study is mediated by at least two distinct transport systems: a Na+-dependent one of lower affinity (K-m in mM range) and a Na+-independent system of higher affinity (K(m)approximate to20-100 muM). Characteristics of [(3) H]L-serine accumulation displayed at low substrate concentrations suggest that it was mediated neither by the typical 'A', nor by the 'large neutral', amino acid transport systems but predominantly by transporters belonging to the recently identified LAT (L-amino acid transporter) family. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S0006-8993(03)03024-5

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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  

Constitutive expression of mRNA was seen for the vesicular glutamate transporter brain-specific Na+-dependent inorganic phosphate cotransporter (BNPI), but not differentiation-associated Na+-dependent inorganic phosphate cotransporter, in rat calvarial osteoblasts cultured for 7 and 21 days in vitro (DIV). Three different agonists for ionotropic glutamate receptors (iGluR) at 1 mM, as well as 50 mM KCl, significantly increased the release of endogenous L-glutamate from osteoblasts cultured for 7 DIV when determined 5 min after the addition by using a high performance liquid chromatograph. The inhibitor of desensitization Of DL-alpha-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors cyclothiazide significantly potentiated and prolonged the release of endogenous L-glutamate evoked by AMPA in a dose-dependent manner. The release evoked by AMPA was significantly prevented by the addition of an AMPA receptor antagonist as well as by the removal of Ca2+ ions. These results suggest that endogenous L-glutamate could be released from intracellular vesicular constituents associated with BNPI through activation of particular iGluR subtypes expressed in cultured rat calvarial osteoblasts. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02223-4

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記述言語：日本語   出版者・発行元：(公社)日本整形外科学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY  

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記述言語：日本語   出版者・発行元：(株)羊土社  

ペリサイト(周皮細胞)は、血管を構成する血管内皮細胞の周囲に接着し、血管の安定化や機能維持において重要な役割を担う。特に、中枢神経系においては、血液脳関門の形成・維持においてペリサイトの存在が不可欠であることが知られている。正常な神経機能を維持するうえで、血液脳関門は必須な保護機構であり、加齢などに伴う血液脳関門の機能低下はさまざまな神経変性疾患を引き起こす原因の1つであると考えられている。これらの理由から、近年、ペリサイトによる血液脳関門の機能維持メカニズムは大きな注目を集めている。(著者抄録)

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記述言語：日本語   出版者・発行元：日本臨床精神神経薬理学会・日本神経精神薬理学会  

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記述言語：日本語   出版者・発行元：日本臨床精神神経薬理学会・日本神経精神薬理学会  

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記述言語：日本語   出版者・発行元：(株)ニュー・サイエンス社  

睡眠など、多くの生体機能は生物が持つ「体内時計」と呼ばれるシステムにより、約24時間周期のリズムを刻むことが知られている。この体内時計の部品となるのが「時計遺伝子」と呼ばれる遺伝子群であり、これらの遺伝子活性のリズムが体内時計を生み出すと考えられている。我々はこの時計遺伝子の1つである「Bmal1」の欠失が血液と脳組織の物質の移動を制限するシステムである「血液脳関門(Blood-Brain Barrier、BBB)」の恒常性維持機能を減弱させることを発見し、さらに、これは脳血管周囲に存在する「ペリサイト」の機能破綻によるものであることを見出した。これらの研究成果は体内時計と血液脳関門の機能維持が密接に関連していることを示しており、アルツハイマー病などの神経変性疾患では血液脳関門の破綻が認められることからも、睡眠障害と中枢神経変性疾患の関係性を解き明かす上で重要な知見をもたらすと考えられる。(著者抄録)

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記述言語：英語   出版者・発行元：CELL PRESS  

DOI： 10.1016/j.cell.2015.08.018

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J-GLOBAL

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：OXFORD UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等  

Systemic lupus erythematosus (SLE) is characterized by the presence of several autoantibodies, including anti-dsDNA. Among types of SLE, neuropsychiatric (NP)-SLE accounts for significant morbidity and mortality. Cerebrovascular disease, which could account for most of the serious permanent neurological damage, is a common presentation of NP-SLE. The pathophysiology of NP-SLE involves several factors, including vasculitis, thrombosis, and inflammation and/or apoptosis of neuronal and glial cells. The current treatment strategy is immunosuppressive therapy, which is occasionally insufficient for patients with NP-SLE. Recent studies have revealed that autoantibodies, such as anti-NR2, pass from the peripheral blood to the brain through the blood-brain barrier, cross-react with human brain tissue and cause increased intracellular Ca2+ in SLE. Regulating blood-brain barrier permeability, inhibiting autoantibody deposition in tissues and modulating intracellular Ca2+ may be new concepts for the treatment with NP-SLE. © 2013 Future Medicine Ltd.

DOI： 10.2217/ijr.13.48

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：日本語  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：日本語   出版者・発行元：金原一郎記念医学医療振興財団  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.1584

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.983

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.2147

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.1637

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：CAMBRIDGE UNIV PRESS  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：JAPANESE PHARMACOLOGICAL SOC  

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