Faculty Profiles - TAKARADA Takeshi

Papers - TAKARADA Takeshi

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SGCRNA: Spectral Clustering-Guided Co-Expression Network Analysis Without Scale-Free Constraints for Multi-Omic Data

Tatsunori Osone, Tomoka Takao, Shigeo Otake, Takeshi Takarada

bioRxiv 2025.4

Modeling human limb skeletal development using human pluripotent stem cell-derived skeletal assembloids

Tomoka Takao, Tatsunori Osone, Kohei Sato, Daisuke Yamada, Yuki Fujisawa, Masaya Hagiwara, Eiji Nakata, Toshifumi Ozaki, Junya Toguchida, Takeshi Takarada

bioRxiv 2025.1

PRRX1 upregulates PD-L1 in human mesenchymal stem cells

Taro Osawa, Daisuke Yamada, Tomoko Takao, Lu Ming, Takeshi Takarada

In Vitro Cellular & Developmental Biology - Animal 2024.12

Efficient Production of Chondrocyte Particles from Human iPSC-Derived Chondroprogenitors Using a Plate-Based Cell Self-Aggregation Technique. International journal

Shojiro Hanaki, Daisuke Yamada, Tomoka Takao, Ryosuke Iwai, Takeshi Takarada

International journal of molecular sciences 25 ( 22 ) 2024.11

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

The limited capacity of articular cartilage for self-repair is a critical challenge in orthopedic medicine. Here, we aimed to develop a simplified method of generating chondrocyte particles from human-induced pluripotent stem cell-derived expandable limb-bud mesenchymal cells (ExpLBM) using a cell self-aggregation technique (CAT). ExpLBM cells were induced to form chondrocyte particles through a stepwise differentiation protocol performed on a CAT plate (prevelex-CAT®), which enables efficient and consistent production of an arbitrary number of uniformly sized particles. Histological and immunohistochemical analyses confirmed that the generated chondrocyte particles expressed key cartilage markers, such as type II collagen and aggrecan, but not hypertrophic markers, such as type X collagen. Additionally, when these particles were transplanted into osteochondral defects in rats with X-linked severe combined immunodeficiency, they demonstrated successful engraftment and extracellular matrix production, as evidenced by Safranin O and Toluidine Blue staining. These data suggest that the plate-based CAT system offers a robust and scalable approach to produce a large number of chondrocyte particles in a simplified and efficient manner, with potential application to cartilage regeneration. Future studies will focus on refining the system and exploring its clinical applications to the treatment of cartilage defects.

DOI： 10.3390/ijms252212063

悪性末梢神経鞘腫瘍における悪性化を促進する新規メカニズム転写因子PRRX1とTOP2Aのタンパク質間相互作用の発見

たき平将太, 山田大祐, 大曽根達則, 高尾知佳, 板野拓人, 藤原智洋, 中田英二, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 98 ( 8 ) S1957 - S1957 2024.9

悪性末梢神経鞘腫瘍におけるPRRX1とTOP2Aの相互作用による悪性化メカニズムの新規解明

たき平将太, 中田英二, 板野拓人, 藤原智洋, 国定俊之, 大曽根達則, 山田大祐, 高尾知佳, 宝田剛志, 尾崎敏文

日本整形外科学会雑誌 98 ( 6 ) S1529 - S1529 2024.6

ヒトiPS細胞由来軟骨組織を利用した小児気管疾患に対する再生治療法の開発

花木祥二朗, 高尾知佳, 藤澤佑樹, 山田大祐, 岩井良輔, 宝田剛志

日本小児外科学会雑誌 60 ( 3 ) 509 - 509 2024.4

ヒトiPS細胞由来軟骨組織を利用した小児気管疾患に対する再生治療法の開発

花木祥二朗, 高尾知佳, 藤澤佑樹, 山田大祐, 岩井良輔, 宝田剛志

日本小児外科学会雑誌 60 ( 3 ) 509 - 509 2024.4

PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumours. International journal

Shota Takihira, Daisuke Yamada, Tatsunori Osone, Tomoka Takao, Masakiyo Sakaguchi, Michiyuki Hakozaki, Takuto Itano, Eiji Nakata, Tomohiro Fujiwara, Toshiyuki Kunisada, Toshifumi Ozaki, Takeshi Takarada

British journal of cancer 2024.3

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

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

悪性末梢神経鞘腫瘍において転写因子PRRX1はTOP2Aと相互作用し悪性化を促進させる

たき平将太, 中田英二, 板野拓人, 藤原智洋, 国定俊之, 大曽根達則, 山田大祐, 高尾知佳, 宝田剛志, 尾崎敏文

日本整形外科学会雑誌 98 ( 2 ) S76 - S76 2024.3

悪性末梢神経鞘腫瘍において転写因子PRRX1はTOP2Aと相互作用し悪性化を促進させる

たき平将太, 中田英二, 板野拓人, 藤原智洋, 国定俊之, 大曽根達則, 山田大祐, 高尾知佳, 宝田剛志, 尾崎敏文

日本整形外科学会雑誌 98 ( 2 ) S76 - S76 2024.3

神経線維腫症1型患者由来ヒトiPS細胞株の樹立

大澤太郎, 中田英二, 岡本真幸, 山田大祐, 二川摩周, 高尾知佳, 平沢晃, 尾崎敏文, 宝田剛志

日本レックリングハウゼン病学会学術大会プログラム・抄録集 15回 24 - 24 2024.2

Antitumor activity of α-pinene in T-cell tumors. International journal

Masaya Abe, Noboru Asada, Maiko Kimura, Chie Fukui, Daisuke Yamada, Ziyi Wang, Masayuki Miyake, Takeshi Takarada, Mitsuaki Ono, Michinori Aoe, Wataru Kitamura, Masayuki Matsuda, Takashi Moriyama, Akifumi Matsumura, Yoshinobu Maeda

Cancer science 2024.1

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

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

ヒトiPS細胞由来肢芽間葉系細胞から作製した軟骨組織体を用いた骨再生

中田英二, 佐藤浩平, 高尾知佳, 藤澤祐樹, 山田大祐, 上原健敬, 藤原智洋, 尾崎敏文, 宝田剛志

移植 58 ( 3 ) 293 - 293 2023.12

Alpha-Pinene As a Novel Therapeutic Agent for T-Cell Tumors

Masaya Abe, Noboru Asada, Maiko Kimura, Chie Fukui, Daisuke Yamada, Ziyi Wang, Masayuki Miyake, Takeshi Takarada, Mitsuaki Ono, Michinori Aoe, Wataru Kitamura, Masayuki Matsuda, Takashi Moriyama, Akifumi Matsumura, Yoshinobu Maeda

Blood 142 ( Supplement 1 ) 1441 - 1441 2023.11

Publishing type：Research paper (scientific journal) Publisher：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 &lt; 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 &lt; 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 &lt; 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

Development of cartilage tissue using a stirred bioreactor and human iPSC-derived limb bud mesenchymal cells. International journal

Yuki Fujisawa, Tomoka Takao, Daisuke Yamada, Takeshi Takarada

Biochemical and biophysical research communications 687 149146 - 149146 2023.10

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

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

Fabrication of shape-designable cartilage from human induced pluripotent stem cell-derived chondroprogenitors using a cell self-aggregation technique. International journal

Tomoyuki Ota, Tomoka Takao, Ryosuke Iwai, Takeshi Moriwaki, Yohei Kitaguchi, Yuki Fujisawa, Daisuke Yamada, Yoshihiro Kimata, Takeshi Takarada

Biomedical materials (Bristol, England) 2023.10

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

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

ヒトiPS細胞由来肢芽間葉系細胞の発生機序の検討

高尾知佳, 大曽根達則, 山田大祐, 中田英二, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 8 ) S1599 - S1599 2023.8

ヒトiPS細胞由来肢芽間葉系細胞を用いた骨再建法の開発

佐藤浩平, 高尾知佳, 中田英二, 藤澤佑樹, 山田大祐, 上原健敬, 藤原智洋, 依光正則, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 8 ) S1868 - S1868 2023.8

悪性末梢神経鞘腫瘍におけるPRRX1の悪性化因子としての役割

たき平将太, 中田英二, 板野拓人, 片山晴喜, 藤原智洋, 国定俊之, 山田大祐, 高尾知佳, 宝田剛志, 尾崎敏文

日本整形外科学会雑誌 97 ( 8 ) S1890 - S1890 2023.8

ヒトiPS細胞由来肢芽間葉系細胞の発生機序の検討

高尾知佳, 大曽根達則, 山田大祐, 中田英二, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 8 ) S1599 - S1599 2023.8

ヒトiPS細胞由来肢芽間葉系細胞を用いた骨再建法の開発

佐藤浩平, 高尾知佳, 中田英二, 藤澤佑樹, 山田大祐, 上原健敬, 藤原智洋, 依光正則, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 8 ) S1868 - S1868 2023.8

悪性末梢神経鞘腫瘍における治療標的PRRX1の同定と新規創薬開発の可能性

たき平将太, 山田大祐, 岡本真幸, 高尾知佳, 中田英二, 板野拓人, 藤原智洋, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 6 ) S1428 - S1428 2023.6

ヒト多能性幹細胞から誘導した肢芽間葉系細胞と、その拡大培養法の開発

中田英二, 山田大祐, 高尾知佳, たき平将太, 藤原智洋, 尾崎敏文, 宝田剛志

移植 57 ( 4 ) 363 - 363 2023.4

ヒトiPS細胞から誘導した肢芽間葉系細胞による軟骨シートの作製

藤澤佑樹, 高尾知佳, 佐藤正人, 豊田恵利子, 山田大祐, 中田英二, 尾崎敏文, 宝田剛志

移植 57 ( 4 ) 362 - 362 2023.4

悪性末梢神経鞘腫瘍におけるPRRX1の治療標的分子としての可能性

たき平将太, 中田英二, 大曽根達則, 山田大祐, 高尾知佳, 佐藤浩平, 畑利彰, 藤原智洋, 国定俊之, 宝田剛志, 尾崎敏文

日本整形外科学会雑誌 97 ( 3 ) S1040 - S1040 2023.3

A novel chondrocyte sheet fabrication using human-induced pluripotent stem cell-derived expandable limb-bud mesenchymal cells. International journal

Tomoka Takao, Masato Sato, Yuki Fujisawa, Eriko Toyoda, Daisuke Yamada, Yukio Hitsumoto, Eiji Nakata, Toshifumi Ozaki, Takeshi Takarada

Stem cell research & therapy 14 ( 1 ) 34 - 34 2023.2

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

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

悪性末梢神経鞘腫瘍におけるPRRX1の悪性化因子としての役割

たき平将太, 中田英二, 山田大祐, 片山晴喜, 畑利彰, 藤原智洋, 高尾知佳, 国定俊之, 宝田剛志, 尾崎敏文

日本レックリングハウゼン病学会学術大会プログラム・抄録集 14回 16 - 16 2023.2

A protocol to induce expandable limb-bud mesenchymal cells from human pluripotent stem cells. International journal

Tomoka Takao, Daisuke Yamada, Takeshi Takarada

STAR protocols 3 ( 4 ) 101786 - 101786 2022.12

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

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

Establishment of a human pluripotent stem cell-derived MKX-td Tomato reporter system. International journal

Yuki Fujisawa, Lu Ming, Daisuke Yamada, Tomoka Takao, Takeshi Takarada

Stem cell research & therapy 13 ( 1 ) 515 - 515 2022.11

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

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

Fabrication of scaffold-free mesenchyme tissue bands by cell self-aggregation technique for potential use in tissue regeneration. International journal

Tomoyuki Ota, Ryosuke Iwai, Yohei Kitaguchi, Takeshi Takarada, Yoshihiro Kimata

Biomedical materials (Bristol, England) 17 ( 6 ) 2022.11

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

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

ヒトiPS細胞来肢芽間葉系細胞を用いた硝子軟骨シートの作製

高尾知佳, 佐藤正人, 豊田恵利子, 山田大祐, 中田英二, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 96 ( 8 ) S1557 - S1557 2022.9

悪性末梢神経鞘腫瘍におけるPRRX1の悪性化因子としての役割

たき平将太, 山田大祐, 高尾知佳, 中田英二, 近藤宏也, 佐藤浩平, 畑利彰, 藤原智洋, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 96 ( 8 ) S1768 - S1768 2022.9

ヒトiPS細胞来肢芽間葉系細胞を用いた硝子軟骨シートの作製

高尾知佳, 佐藤正人, 豊田恵利子, 山田大祐, 中田英二, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 96 ( 8 ) S1557 - S1557 2022.9

悪性末梢神経鞘腫瘍におけるPRRX1の悪性化因子としての役割

たき平将太, 山田大祐, 高尾知佳, 中田英二, 近藤宏也, 佐藤浩平, 畑利彰, 藤原智洋, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 96 ( 8 ) S1768 - S1768 2022.9

Vestigial-Like 3 Plays an Important Role in Osteoblast Differentiation by Regulating the Expression of Osteogenic Transcription Factors and BMP Signaling. International journal

Haoze Yuan, Mika Ikegame, Yoko Fukuhara, Fumiko Takemoto, Yaqiong Yu, Jumpei Teramachi, Yao Weng, Jiajie Guo, Daisuke Yamada, Takeshi Takarada, Ying Li, Hirohiko Okamura, Bin Zhang

Calcified tissue international 111 ( 3 ) 331 - 344 2022.6

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

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

Identification of Surface Antigens That Define Human Pluripotent Stem Cell-Derived PRRX1+Limb-Bud-like Mesenchymal Cells. International journal

Daisuke Yamada, Tomoka Takao, Masahiro Nakamura, Toki Kitano, Eiji Nakata, Takeshi Takarada

International journal of molecular sciences 23 ( 5 ) 2022.2

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

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

Mouse Model for Optogenetic Genome Engineering.

Tomoka Takao, Daisuke Yamada, Takeshi Takarada

Acta medica Okayama 76 ( 1 ) 1 - 5 2022.2

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

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

Establishment of high-throughput drug screening system using human limb bud mesenchymal cells derived from skeletal dysplasia specific iPSC

Yamada Daisuke, Takao Tomoka, Nakamura Masahiro, Ming Lu, Toguchida Junya, Takarada Takeshi

Proceedings for Annual Meeting of The Japanese Pharmacological Society 95 2-O-087 2022

Language：Japanese Publisher：Japanese Pharmacological Society

[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

O‐GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics‐oriented study

Yao Weng, Ziyi Wang, Yoko Fukuhara, Airi Tanai, Mika Ikegame, Daisuke Yamada, Takeshi Takarada, Takashi Izawa, Satoru Hayano, Kaya Yoshida, Hiroshi Kamioka, Hirohiko Okamura

BioFactors 47 ( 6 ) 992 - 1015 2021.8

Induction and expansion of human PRRX1+ limb-bud-like mesenchymal cells from pluripotent stem cells. International journal

Daisuke Yamada, Masahiro Nakamura, Tomoka Takao, Shota Takihira, Aki Yoshida, Shunsuke Kawai, Akihiro Miura, Lu Ming, Hiroyuki Yoshitomi, Mai Gozu, Kumi Okamoto, Hironori Hojo, Naoyuki Kusaka, Ryosuke Iwai, Eiji Nakata, Toshifumi Ozaki, Junya Toguchida, Takeshi Takarada

Nature biomedical engineering 5 ( 8 ) 926 - 940 2021.8

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

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

悪性末梢神経鞘腫瘍におけるPRRX1の悪性化因子としての役割

たき平将太, 山田大祐, 高尾知佳, 中田英二, 近藤宏也, 佐藤浩平, 畑利彰, 藤原智洋, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 95 ( 8 ) S1554 - S1554 2021.8

Oncogenic potential of human pluripotent stem cell-derived lung organoids with HER2 overexpression. International journal

Akihiro Miura, Daisuke Yamada, Masahiro Nakamura, Shuta Tomida, Dai Shimizu, Yan Jiang, Tomoka Takao, Hiromasa Yamamoto, Ken Suzawa, Kazuhiko Shien, Masaomi Yamane, Masakiyo Sakaguchi, Shinichi Toyooka, Takeshi Takarada

International journal of cancer 149 ( 8 ) 1593 - 1604 2021.6

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

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

RUNX2 regulates leukemic cell metabolism and chemotaxis in high-risk T cell acute lymphoblastic leukemia. International journal

Filip Matthijssens, Nitesh D Sharma, Monique Nysus, Christian K Nickl, Huining Kang, Dominique R Perez, Beatrice Lintermans, Wouter Van Loocke, Juliette Roels, Sofie Peirs, Lisa Demoen, Tim Pieters, Lindy Reunes, Tim Lammens, Barbara De Moerloose, Filip Van Nieuwerburgh, Dieter L Deforce, Laurence C Cheung, Rishi S Kotecha, Martijn Dp Risseeuw, Serge Van Calenbergh, Takeshi Takarada, Yukio Yoneda, Frederik W van Delft, Richard B Lock, Seth D Merkley, Alexandre Chigaev, Larry A Sklar, Charles G Mullighan, Mignon L Loh, Stuart S Winter, Stephen P Hunger, Steven Goossens, Eliseo F Castillo, Wojciech Ornatowski, Pieter Van Vlierberghe, Ksenia Matlawska-Wasowska

The Journal of clinical investigation 131 ( 6 ) 2021.3

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

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

PRRX1 promotes malignant properties in human osteosarcoma. International journal

Ryoji Joko, Daisuke Yamada, Masahiro Nakamura, Aki Yoshida, Shota Takihira, Tomoka Takao, Ming Lu, Kohei Sato, Tatsuo Ito, Toshiyuki Kunisada, Eiji Nakata, Toshifumi Ozaki, Takeshi Takarada

Translational oncology 14 ( 1 ) 100960 - 100960 2021.1

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

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

BMP-2/β-TCP Local Delivery for Bone Regeneration in MRONJ-Like Mouse Model. International journal

Akihiro Mikai, Mitsuaki Ono, Ikue Tosa, Ha Thi Thu Nguyen, Emilio Satoshi Hara, Shuji Nosho, Aya Kimura-Ono, Kumiko Nawachi, Takeshi Takarada, Takuo Kuboki, Toshitaka Oohashi

International journal of molecular sciences 21 ( 19 ) 2020.9

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

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

Core Binding Factors are essential for ovulation, luteinization, and female fertility in mice. International journal

Somang Lee-Thacker, Hayce Jeon, Yohan Choi, Ichiro Taniuchi, Takeshi Takarada, Yukio Yoneda, CheMyong Ko, Misung Jo

Scientific reports 10 ( 1 ) 9921 - 9921 2020.6

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

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

Establishment of a tTA-dependent photoactivatable Cre recombinase knock-in mouse model for optogenetic genome engineering. Reviewed International journal

Tomoka Takao, Yuichi Hiraoka, Kenji Kawabe, Daisuke Yamada, Lu Ming, Kohichi Tanaka, Moritoshi Sato, Takeshi Takarada

Biochemical and biophysical research communications 526 ( 1 ) 213 - 217 2020.5

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

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

A RUNX2 stabilization pathway mediates physiologic and pathologic bone formation. International journal

Jung-Min Kim, Yeon-Suk Yang, Kwang Hwan Park, Xianpeng Ge, Ren Xu, Na Li, Minkyung Song, Hyunho Chun, Seoyeon Bok, Julia F Charles, Odile Filhol-Cochet, Brigitte Boldyreff, Teresa Dinter, Paul B Yu, Ning Kon, Wei Gu, Takeshi Takarada, Matthew B Greenblatt, Jae-Hyuck Shim

Nature communications 11 ( 1 ) 2289 - 2289 2020.5

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

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

骨肉腫におけるPRRX1の発現は悪性化に関与する

上甲良二, 山田大祐, 中田英二, たき平将太, 尾崎敏文, 宝田剛志

中部日本整形外科災害外科学会雑誌 63 ( 春季学会 ) 212 - 212 2020.4

Glutamatergic neurons in the medial prefrontal cortex mediate the formation and retrieval of cocaine-associated memories in mice. Reviewed International journal

Tong Zhang, Junko Yanagida, Hironori Kamii, Shintaro Wada, Masaki Domoto, Hitoki Sasase, Satoshi Deyama, Takeshi Takarada, Eiichi Hinoi, Kenji Sakimura, Akihiro Yamanaka, Takashi Maejima, Michihiro Mieda, Takeshi Sakurai, Naoya Nishitani, Kazuki Nagayasu, Shuji Kaneko, Masabumi Minami, Katsuyuki Kaneda

Addiction biology 25 ( 1 ) e12723 2020.1

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

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

【脳の半分を占めるグリア細胞脳と心と体をつなぐ"膠"】(第2章)グリア細胞と神経免疫・臓器連関ペリサイト機能欠損による血液脳関門の破綻

中里亮太, 山田大祐, 宝田剛志

実験医学 37 ( 17 ) 2854 - 2860 2019.11

Language：Japanese Publisher：(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)

Postnatal Runx2 deletion leads to low bone mass and adipocyte accumulation in mice bone tissues. Reviewed International journal

Ikue Tosa, Daisuke Yamada, Misa Yasumatsu, Eiichi Hinoi, Mitsuaki Ono, Toshitaka Oohashi, Takuo Kuboki, Takeshi Takarada

Biochemical and biophysical research communications 516 ( 4 ) 1229 - 1233 2019.9

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

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

時期特異的Runx2欠損マウスを用いた成体におけるRunx2の機能解析

土佐郁恵, 河邊憲司, 大野充昭, 窪木拓男, 宝田剛志

日本骨代謝学会学術集会プログラム抄録集 37回 216 - 216 2019.9

Runx2 is required for postnatal intervertebral disc tissue growth and development. Reviewed International journal

Lifan Liao, Hua Jiang, Yunshan Fan, Ronald S Lu, Changli Wei, Takeshi Takarada, Shisheng He, Di Chen

Journal of cellular physiology 234 ( 5 ) 6679 - 6687 2019.5

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

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

骨髄間葉系幹細胞におけるRunx2の発現低下は骨および骨髄の加齢様変化をもたらす

土佐郁恵, 山田大祐, 大野充昭, 大橋俊孝, 窪木拓男, 宝田剛志

日本補綴歯科学会誌 11 ( 特別号 ) 129 - 129 2019.5

Glutamatergic neurons in the medial prefrontal cortex mediate the formation and retrieval of cocaine-associated memories in mice

Zhang, Tong, Yanagida, Junko, Kamii, Hironori, Wada, Shintaro, Domoto, Masaki, Sasase, Hitoki, Deyama, Satoshi, Takarada, Takeshi, Hinoi, Eiichi, Sakimura, Kenji, Yamanaka, Akihiro, Maejima, Takashi, Mieda, Michihiro, Sakurai, Takeshi, Nishitani, Naoya, Nagayasu, Kazuki, Kaneko, Shuji, Minami, Masabumi, Kaneda, Katsuyuki

Addiction biology Epub 2019.2

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

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

Bone Marrow Cells Inhibit BMP-2-Induced Osteoblast Activity in the Marrow Environment. Reviewed International journal

Ha Thi Nguyen, Mitsuaki Ono, Yasutaka Oida, Emilio Satoshi Hara, Taishi Komori, Kentaro Akiyama, Ha Thi Thu Nguyen, Kyaw Thu Aung, Hai Thanh Pham, Ikue Tosa, Takeshi Takarada, Koichi Matsuo, Toshihide Mizoguchi, Toshitaka Oohashi, Takuo Kuboki

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 34 ( 2 ) 327 - 332 2019.2

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

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

Design, synthesis, and biological evaluation of radioiodinated benzo[d]imidazole-quinoline derivatives for platelet-derived growth factor receptor β (PDGFRβ) imaging. Reviewed International journal

Nurmaya Effendi, Kenji Mishiro, Takeshi Takarada, Daisuke Yamada, Ryuichi Nishii, Kazuhiro Shiba, Seigo Kinuya, Akira Odani, Kazuma Ogawa

Bioorganic & medicinal chemistry 27 ( 2 ) 383 - 393 2019.1

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

Several malignant tumors and fibrotic diseases are associated with PDGFRβ overexpression and excessive signaling, making this receptor attractive for molecular targeting and imaging approaches. A series of benzo[d]imidazole-quinoline derivatives were designed and synthesized to develop radioiodinated compounds as PDGFRβ-specific imaging probes. The structure activity relationship (SAR) evaluation of the designed compounds was performed. Among them, 2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (5a) and 4-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (5d) exhibited a relatively high PDGFRβ-TK inhibitory potency, whereas iodinated 5a derivative 5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (8) exhibited a superior inhibitory potency as PDGFRβ inhibitor than iodinated 5d derivative 4-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (11). Furthermore, [125I]8 and [125I]11 were synthesized and evaluated for PDGFRβ radioligand ability, both in vitro and in vivo. Cellular uptake experiments showed that [125I]8 had a higher uptake in BxPC3-luc cells as PDGFRβ-positive cells than [125I]11. Incubation of [125I]8 after pretreatment of PDGFRβ ligands significantly reduced the uptake of [125I]8. In biodistribution experiments using tumor-bearing mice, [125I]8 accumulation in the tumor 1 h postinjection was higher than that of the benzo[d]imidazol-quinoline derivative [125I]IIQP, used in our previous research. These results indicate that [125I]8 could be a promising PDGFRβ imaging agent. Although its clinical application requires further structural modifications, the results obtained in this research may be useful for the development of PDGFRβ-specific radioligands.

DOI： 10.1016/j.bmc.2018.12.016

Molecular understanding of hierarchy and lineage of mesenchymal stem cells in vivo Reviewed

Kawabe, K., Takarada, T.

Folia Pharmacologica Japonica 153 ( 2 ) 67 - 72 2019

[Dissecting the Hierarchy and Lineage of Mesenchymal Stem Cells Using Mouse Genetics as a Step toward Drug Discovery and Regenerative Medicine]. Reviewed

Takeshi Takarada

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan 139 ( 6 ) 867 - 871 2019

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

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

Inhibition of the glutamine transporter SNAT1 confers neuroprotection in mice by modulating the mTOR-autophagy system. Reviewed International journal

Daisuke Yamada, Kenji Kawabe, Ikue Tosa, Shunpei Tsukamoto, Ryota Nakazato, Miki Kou, Koichi Fujikawa, Saki Nakamura, Mitsuaki Ono, Toshitaka Oohashi, Mari Kaneko, Shioi Go, Eiichi Hinoi, Yukio Yoneda, Takeshi Takarada

Communications biology 2 346 - 346 2019

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

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

Elucidation of the mechanisms underlying regulation of somatic stem cell function: Possible application to the treatment of neuropsychiatric, metabolic bone, and lifestyle diseases Reviewed

Nakamichi, N., Takarada, T.

Yakugaku Zasshi 139 ( 6 ) 845 - 846 2019

RUNX2 Promotes Malignant Progression in Glioma. Reviewed International journal

Daisuke Yamada, Koichi Fujikawa, Kenji Kawabe, Takuya Furuta, Mitsutoshi Nakada, Takeshi Takarada

Neurochemical research 43 ( 11 ) 2047 - 2054 2018.11

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

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

The MAPK Erk5 is necessary for proper skeletogenesis involving a Smurf-Smad-Sox9 molecular axis. Reviewed International journal

Takashi Iezaki, Kazuya Fukasawa, Tetsuhiro Horie, Gyujin Park, Samuel Robinson, Michio Nakaya, Hiroyuki Fujita, Yuki Onishi, Kakeru Ozaki, Takashi Kanayama, Manami Hiraiwa, Yuka Kitaguchi, Katsuyuki Kaneda, Yukio Yoneda, Takeshi Takarada, X Edward Guo, Hitoshi Kurose, Eiichi Hinoi

Development (Cambridge, England) 145 ( 14 ) 2018.7

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

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

Radiobrominated benzimidazole-quinoline derivatives as Platelet-derived growth factor receptor beta (PDGFRβ) imaging probes. Reviewed International journal

Nurmaya Effendi, Kenji Mishiro, Takeshi Takarada, Akira Makino, Daisuke Yamada, Yoji Kitamura, Kazuhiro Shiba, Yasushi Kiyono, Akira Odani, Kazuma Ogawa

Scientific reports 8 ( 1 ) 10369 - 10369 2018.7

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

Platelet-derived growth factor receptor beta (PDGFRβ) affects in numerous human cancers and has been recognized as a promising molecular target for cancer therapies. The overexpression of PDGFRβ could be a biomarker for cancer diagnosis. Radiolabeled ligands having high affinity for the molecular target could be useful tools for the imaging of overexpressed receptors in tumors. In this study, we aimed to develop radiobrominated PDGFRβ ligands and evaluate their effectiveness as PDGFRβ imaging probes. The radiolabeled ligands were designed by modification of 1-{2-[5-(2-methoxyethoxy)-1H- benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-amine (1), which shows selective inhibition profile toward PDGFRβ. The bromine atom was introduced directly into C-5 of the quinoline group of 1, or indirectly by the conjugation of 1 with the 3-bromo benzoyl group. [77Br]1-{5-Bromo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinoline-8-yl}piperidin-4-amine ([77Br]2) and [77Br]-N-3-bromobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}-piperidin-4-amine ([77Br]3) were prepared using a bromodestannylation reaction. In a cellular uptake study, [77Br]2 and [77Br]3 more highly accumulatd in BxPC3-luc cells (PDGFRβ-positive) than in MCF7 cells (PDGFRβ-negative), and their accumulation was significantly reduced by pretreatment with inhibitors. In biodistribution experiments, [77Br]2 accumulation was higher than [77Br]3 accumulation at 1 h postinjection. These findings suggest that [76Br]2 is more promising for positron emission tomography (PET) imaging of PDGFRβ than [76Br]3.

DOI： 10.1038/s41598-018-28529-0

Physiological role of urothelial cancer-associated one long noncoding RNA in human skeletogenic cell differentiation. Reviewed International journal

Takanori Ishikawa, Takashi Nishida, Mitsuaki Ono, Takeshi Takarada, Ha Thi Nguyen, Shinnosuke Kurihara, Takayuki Furumatsu, Yurika Murase, Masaharu Takigawa, Toshitaka Oohashi, Hiroshi Kamioka, Satoshi Kubota

Journal of cellular physiology 233 ( 6 ) 4825 - 4840 2018.6

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

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

生後の骨・軟骨におけるアグリカンの役割の解明

鳥原秀美, 大野充昭, 栗原伸之介, 枝松緑, 宝田剛志, 上岡寛, 大橋俊孝

日本結合組織学会学術大会プログラム・抄録集 50回 155 - 155 2018.6

Core Binding Factor β Expression in Ovarian Granulosa Cells Is Essential for Female Fertility. Reviewed International journal

Somang Lee-Thacker, Yohan Choi, Ichiro Taniuchi, Takeshi Takarada, Yukio Yoneda, CheMyong Ko, Misung Jo

Endocrinology 159 ( 5 ) 2094 - 2109 2018.5

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

Core binding factor β (CBFβ) is a non-DNA-binding partner of all RUNX proteins and critical for transcription activity of CBF transcription factors (RUNXs/CBFβ). In the ovary, the expression of Runx1 and Runx2 is highly induced by the luteinizing hormone (LH) surge in ovulatory follicles, whereas Cbfb is constitutively expressed. To investigate the physiological significance of CBFs in the ovary, the current study generated two different conditional mutant mouse models in which granulosa cell expression of Cbfb and Runx2 was reduced by Cre recombinase driven by an Esr2 promoter. Cbfbgc-/- and Cbfbgc-/- × Runx2gc+/- mice exhibited severe subfertility and infertility, respectively. In the ovaries of both mutant mice, follicles develop normally, but the majority of preovulatory follicles failed to ovulate either in response to human chorionic gonadotropin administration in pregnant mare serum gonadotropin-primed immature animals or after the LH surge at 5 months of age. Morphological and physiological changes in the corpus luteum of these mutant mice revealed the reduced size, progesterone production, and vascularization, as well as excessive lipid accumulation. In granulosa cells of periovulatory follicles and corpora lutea of these mice, the expression of Edn2, Ptgs1, Lhcgr, Sfrp4, Wnt4, Ccrl2, Lipg, Saa3, and Ptgfr was also drastically reduced. In conclusion, the current study provided in vivo evidence that CBFβ plays an essential role in female fertility by acting as a critical cofactor of CBF transcription factor complexes, which regulate the expression of specific key ovulatory and luteal genes, thus coordinating the ovulatory process and luteal development/function in mice.

DOI： 10.1210/en.2018-00011

Type IV collagen α6 chain is a regulator of keratin 10 in keratinization of oral mucosal epithelium. Reviewed International journal

Taishi Komori, Mitsuaki Ono, Emilio Satoshi Hara, Junji Ueda, Ha Thi Thu Nguyen, Ha Thi Nguyen, Tomoko Yonezawa, Takahiro Maeba, Aya Kimura-Ono, Takeshi Takarada, Ryusuke Momota, Kenji Maekawa, Takuo Kuboki, Toshitaka Oohashi

Scientific reports 8 ( 1 ) 2612 - 2612 2018.2

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

Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue, and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva. Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells. Therefore, first, to identify the keratinized mucosa-specific basement membrane components, immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice. No difference in the expression pattern of type IV collagen α1(IV) and α2(IV) chains was observed in the keratinized and non-keratinized mucosa. Interestingly, however, type IV collagen α5(IV) and α6(IV) chains specifically were strongly detected in the keratinized mucosa. To analyze the functional roles of the type IV collagen isoform α6(IV) in oral mucosa keratinization, we analyzed Col4a6-knockout mice. Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice. Additionally, in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of α6(IV) chain in epithelial keratinization. These findings indicate that α112:α556 (IV) network, which is the only network that includes the α6(IV) chain, is one regulator of KRT10 expression in keratinization of oral mucosal epithelium.

DOI： 10.1038/s41598-018-21000-0

Disruption of Bmal1 Impairs Blood-Brain Barrier Integrity via Pericyte Dysfunction. Reviewed International journal

Ryota Nakazato, Kenji Kawabe, Daisuke Yamada, Shinsuke Ikeno, Michihiro Mieda, Shigeki Shimba, Eiichi Hinoi, Yukio Yoneda, Takeshi Takarada

The Journal of neuroscience : the official journal of the Society for Neuroscience 37 ( 42 ) 10052 - 10062 2017.10

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

Synthesis and evaluation of radioiodinated 1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-amine derivatives for platelet-derived growth factor receptor β (PDGFRβ) imaging. Reviewed International journal

Nurmaya Effendi, Kazuma Ogawa, Kenji Mishiro, Takeshi Takarada, Daisuke Yamada, Yoji Kitamura, Kazuhiro Shiba, Takehiko Maeda, Akira Odani

Bioorganic & medicinal chemistry 25 ( 20 ) 5576 - 5585 2017.10

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

Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane tyrosine kinase receptor and it is upregulated in various malignant tumors. Radiolabeled PDGFRβ inhibitors can be a convenient tool for the imaging of tumors overexpressing PDGFRβ. In this study, [125I]-1-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinoline-8-yl}piperidin-4-amine ([125I]IIQP) and [125I]-N-3-iodobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}-piperidin-4-amine ([125I]IB-IQP) were designed and synthesized, and their potential as PDGFRβ imaging agents was evaluated. In cellular uptake experiments, [125I]IIQP and [125I]IB-IQP showed higher uptake by PDGFRβ-positive cells than by PDGFRβ-negative cells, and the uptake in PDGFRβ-positive cells was inhibited by co-culture with PDGFRβ ligands. The biodistribution of both radiotracers in normal mice exhibited hepatobiliary excretion as the main route. In mice inoculated with BxPC3-luc (PDGFRβ-positive), the tumor uptake of radioactivity at 1h after the injection of [125I]IIQP was significantly higher than that after the injection of [125I]IB-IQP. These results indicated that [125I]IIQP can be a suitable PDGFRβ imaging agent. However, further modification of its structure will be required to obtain a more appropriate PDGFRβ-targeted imaging agent with a higher signal/noise ratio.

DOI： 10.1016/j.bmc.2017.08.025

Deletion of Runx2 in Articular Chondrocytes Decelerates the Progression of DMM-Induced Osteoarthritis in Adult Mice. Reviewed International journal

Lifan Liao, Shanxing Zhang, Jianhong Gu, Takeshi Takarada, Yukio Yoneda, Jian Huang, Lan Zhao, Chun-do Oh, Jun Li, Baoli Wang, Meiqing Wang, Di Chen

Scientific reports 7 ( 1 ) 2371 - 2371 2017.5

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

Bone Resorption Is Regulated by Circadian Clock in Osteoblasts Reviewed

Takeshi Takarada, Cheng Xu, Hiroki Ochi, Ryota Nakazato, Daisuke Yamada, Saki Nakamura, Ayumi Kodama, Shigeki Shimba, Michihiro Mieda, Kazuya Fukasawa, Kakeru Ozaki, Takashi Iezaki, Koichi Fujikawa, Yukio Yoneda, Rika Numano, Akiko Hida, Hajime Tei, Shu Takeda, Eiichi Hinoi

Journal of Bone and Mineral Research 32 ( 4 ) 872 - 881 2017.4

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

Bone Resorption Is Regulated by Circadian Clock in Osteoblasts. Reviewed International journal

Takeshi Takarada, Cheng Xu, Hiroki Ochi, Ryota Nakazato, Daisuke Yamada, Saki Nakamura, Ayumi Kodama, Shigeki Shimba, Michihiro Mieda, Kazuya Fukasawa, Kakeru Ozaki, Takashi Iezaki, Koichi Fujikawa, Yukio Yoneda, Rika Numano, Akiko Hida, Hajime Tei, Shu Takeda, Eiichi Hinoi

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 32 ( 4 ) 872 - 881 2017.4

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

The transcriptional modulator Ifrd1 controls PGC-1 alpha expression under short-term adrenergic stimulation in brown adipocytes Reviewed

Gyujin Park, Tetsuhiro Horie, Takashi Kanayama, Kazuya Fukasawa, Takashi Iezaki, Yuki Onishi, Kakeru Ozaki, Yukari Nakamura, Yukio Yoneda, Takeshi Takarada, Eiichi Hinoi

FEBS JOURNAL 284 ( 5 ) 784 - 795 2017.3

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

Sympathetic tone activates the function of classical brown adipocytes, which constitutively exist in the brown adipose tissue (BAT), and inducible brown adipocytes (so-called beige adipocytes), which sporadically reside within the white adipose tissue (WAT). Here we identified the transcriptional modulator interferon-related developmental regulator 1 (Ifrd1) as a negative regulator of thermogenic and mitochondrial gene expression in brown adipocytes. Ifrd1 expression was markedly induced by cold exposure and administration of CL-316243 (a beta 3 adrenergic agonist) in interscapular brown adipose and inguinal subcutaneous WATs, but not in epididymal visceral WAT, in vivo. Adrenergic stimulation also induced Ifrd1 expression in brown adipocytes in a cAMP responsive element binding protein-dependent manner in vitro. CL-316243 injection markedly elevated thermogenic and mitochondrial gene expression, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc1 alpha) in the subcutaneous WAT of Ifrd1 knockout mice compared with gene expression in wild-type mice. Pgc1a promoter activity enhanced by the transcription factor specificity protein 1 (Sp1) was markedly repressed by co-introduction of Ifrd1 in brown adipocytes, whereas the repression was markedly prevented by the addition of trichostatin A, a histone deacetylase inhibitor. Moreover, adrenergic stimulation induced complex formation between Ifrd1, Sp1 and mSIN3B, which is a component of the SIN complex containing histone deacetylase, in brown adipocytes. These findings, therefore, suggest that Ifrd1 could be a pivotal negative regulator of sympathetic regulation of thermogenic and mitochondrial gene expression in brown adipocytes by interacting with Sp1 and the mSIN3 complex.

DOI： 10.1111/febs.14019

The intrinsic microglial clock system regulates interleukin-6 expression. Reviewed International journal

Ryota Nakazato, Shogo Hotta, Daisuke Yamada, Miki Kou, Saki Nakamura, Yoshifumi Takahata, Hajime Tei, Rika Numano, Akiko Hida, Shigeki Shimba, Michihiro Mieda, Eiichi Hinoi, Yukio Yoneda, Takeshi Takarada

Glia 65 ( 1 ) 198 - 208 2017.1

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

Transcriptional Modulator Ifrd1 Regulates Osteoclast Differentiation through Enhancing the NF-kappa B/NFATc1 Pathway Reviewed

Takashi Iezaki, Kazuya Fukasawa, Gyujin Park, Tetsuhiro Horie, Takashi Kanayama, Kakeru Ozaki, Yuki Onishi, Yoshifumi Takahata, Yukari Nakamura, Takeshi Takarada, Yukio Yoneda, Takashi Nakamura, Jean Vacher, Eiichi Hinoi

MOLECULAR AND CELLULAR BIOLOGY 36 ( 19 ) 2451 - 2463 2016.10

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

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Here, we show that the transcriptional coactivator/repressor interferon-related developmental regulator 1 (Ifrd1) is expressed in osteoclast lineages and represents a component of the machinery that regulates bone homeostasis. Ifrd1 expression was transcriptionally regulated in preosteoclasts by receptor activator of nuclear factor kappa B (NF-kappa B) ligand (RANKL) through activator protein 1. Global deletion of murine Ifrd1 increased bone formation and decreased bone resorption, leading to a higher bone mass. Deletion of Ifrd1 in osteoclast precursors prevented RANKL-induced bone loss, although no bone loss was observed under normal physiological conditions. RANKL-dependent osteoclastogenesis was impaired in vitro in Ifrd1-deleted bone marrow macrophages (BMMs). Ifrd1 deficiency increased the acetylation of p65 at residues K122 and K123 via the inhibition of histone deacetylase-dependent deacetylation in BMMs. This repressed the NF-kappa B-dependent transcription of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), an essential regulator of osteoclastogenesis. These findings suggest that an Ifrd1/NF-kappa B/NFATc1 axis plays a pivotal role in bone remodeling in vivo and represents a therapeutic target for bone diseases.

DOI： 10.1128/MCB.01075-15

ATF3 deficiency in chondrocytes alleviates osteoarthritis development Reviewed

Takashi Iezaki, Kakeru Ozaki, Kazuya Fukasawa, Makoto Inoue, Shigetaka Kitajima, Takeshi Muneta, Shu Takeda, Hiroyuki Fujita, Yuki Onishi, Tetsuhiro Horie, Yukio Yoneda, Takeshi Takarada, Eiichi Hinoi

JOURNAL OF PATHOLOGY 239 ( 4 ) 426 - 437 2016.8

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

ATF3 controls proliferation of osteoclast precursor and bone remodeling Reviewed

Kazuya Fukasawa, Gyujin Park, Takashi Iezaki, Tetsuhiro Horie, Takashi Kanayama, Kakeru Ozaki, Yuki Onishi, Yoshifumi Takahata, Yukio Yoneda, Takeshi Takarada, Shigetaka Kitajima, Jean Vacher, Eiichi Hinoi

SCIENTIFIC REPORTS 6 30918 2016.8

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

Circadian Clock Regulates Bone Resorption in Mice Reviewed

Cheng Xu, Hiroki Ochi, Toru Fukuda, Shingo Sato, Satoko Sunamura, Takeshi Takarada, Eiichi Hinoi, Atsushi Okawa, Shu Takeda

JOURNAL OF BONE AND MINERAL RESEARCH 31 ( 7 ) 1344 - 1355 2016.7

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

Protective upregulation of activating transcription factor-3 against glutamate neurotoxicity in neuronal cells under ischemia Reviewed

Takeshi Takarada, Miki Kou, Miho Hida, Ryo Fukumori, Saki Nakamura, Takaya Kutsukake, Nobuyuki Kuramoto, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 94 ( 5 ) 378 - 388 2016.5

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

Possible activation by the green tea amino acid theanine of mammalian target of rapamycin signaling in undifferentiated neural progenitor cells in vitro Reviewed

Takeshi Takarada, Noritaka Nakamichi, Ryota Nakazato, Takami Kakuda, Hiroshi Kokubo, Shinsuke Ikeno, Saki Nakamura, Nobuyuki Kuramoto, Eiichi Hinoi, Yukio Yoneda

Biochemistry and Biophysics Reports 5 89 - 95 2016.3

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

The Transcriptional Modulator Interferon-Related Developmental Regulator 1 in Osteoblasts Suppresses Bone Formation and Promotes Bone Resorption Reviewed

Takashi Iezaki, Yuki Onishi, Kakeru Ozaki, Kazuya Fukasawa, Yoshifumi Takahata, Yukari Nakamura, Koichi Fujikawa, Takeshi Takarada, Yukio Yoneda, Yui Yamashita, Go Shioi, Eiichi Hinoi

JOURNAL OF BONE AND MINERAL RESEARCH 31 ( 3 ) 573 - 584 2016.3

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

Upregulation of Slc38a1 Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine Reviewed

Takeshi Takarada, Masato Ogura, Noritaka Nakamichi, Takami Kakuda, Ryota Nakazato, Hiroshi Kokubo, Shinsuke Ikeno, Saki Nakamura, Takaya Kutsukake, Eiichi Hinoi, Yukio Yoneda

NEUROCHEMICAL RESEARCH 41 ( 1-2 ) 5 - 15 2016.2

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

Genetic analysis of Runx2 function during intramembranous ossification Reviewed

Takeshi Takarada, Ryota Nakazato, Azusa Tsuchikane, Koichi Fujikawa, Takashi Iezaki, Yukio Yoneda, Eiichi Hinoi

DEVELOPMENT 143 ( 2 ) 211 - 218 2016.1

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

Upregulation of Runt-Related Transcription Factor-2 Through CCAAT Enhancer Binding Protein-beta Signaling Pathway in Microglial BV-2 Cells Exposed to ATP Reviewed

Ryota Nakazato, Takeshi Takarada, Shinsuke Ikeno, Saki Nakamura, Takaya Kutsukake, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 230 ( 10 ) 2510 - 2521 2015.10

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

We have shown constitutive expression of the master regulator of osteoblastogenesis, runt-related transcription factor-2 (Runx2), by microglia cells outside bone. Here, we attempted to evaluate the pathological significance of Runx2 in microglial BV-2 cells exposed to ATP at a high concentration. Marked upregulation of Runx2 transcript and protein expression was seen in cells exposed to 1 mM ATP for a period longer than 30 min without inducing cytotoxicity. The Runx2 upregulation by ATP was prevented by extracellular and intracellular Ca2+ chelators, while thapsigargin upregulated Runx2 expression alone without affecting the upregulation by ATP. A calmodulin antagonist prevented the upregulation by ATP, with calcineurin inhibitors being ineffective. Although ATP markedly increased nuclear levels of nuclear factor of activated T cell-2 (NFAT2), Runx2 promoter activity was not simulated by the introduction of either NFAT1 or NFAT2, but facilitated by that of CCAAT enhancer binding protein-alpha (C/EBP alpha), C/EBP beta and nuclear factor (erythroid-derived 2)-like-2 (Nrf2). Exposure to ATP up-regulated C/EBP beta and Nrf2, but not C/EBP alpha, expression, in addition to increasing nuclear levels of respective corresponding proteins. Runx2 upregulation by ATP was deteriorated by knockdown of C/EBP beta but not by that of Nrf2, however, while exposure to ATP up-regulated matrix metalloproteinase-13 (Mmp13) expression in a Runx2-dependent manner. Overexpression of Runx2 up-regulated Mmp13 expression with promoted incorporation of fluorescent beads into BV-2 cells without ATP. These results suggest that extracellular ATP up-regulates Runx2 expression through activation of the C/EBP beta signaling in a calmodulin-dependent manner to play a pivotal role in phagocytosis in microglial BV-2 cells. (C) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/jcp.24988

Glucose Uptake and Runx2 Synergize to Orchestrate Osteoblast Differentiation and Bone Formation Reviewed

Jianwen Wei, Junko Shimazu, Munevver P. Makinistoglu, Antonio Maurizi, Daisuke Kajimura, Haihong Zong, Takeshi Takarada, Takashi Lezaki, Jeffrey E. Pessin, Eiichi Hinoi, Gerard Karsenty

CELL 161 ( 7 ) 1576 - 1591 2015.6

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

Potential Interactions of Calcium-Sensitive Reagents with Zinc Ion in Different Cultured Cells Reviewed

Koichi Fujikawa, Ryo Fukumori, Saki Nakamura, Takaya Kutsukake, Takeshi Takarada, Yukio Yoneda

PLOS ONE 10 ( 5 ) e0127421 2015.5

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

Daily oral intake of theanine prevents the decline of 5-bromo-2 '-deoxyuridine incorporation in hippocampal dentate gyrus with concomitant alleviation of behavioral abnormalities in adult mice with severe traumatic stress Reviewed

Takeshi Takarada, Noritaka Nakamichi, Takami Kakuda, Ryota Nakazato, Hiroshi Kokubo, Shinsuke Ikeno, Saki Nakamura, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 127 ( 3 ) 292 - 297 2015.3

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

Analysis of the signaling cascade of transcription factors in joint tissue with the aim of drug discovery Reviewed

Takarada, T.

Folia Pharmacologica Japonica 144 ( 4 ) 178 - 184 2014.10

Recent advances in Runx2 research Reviewed

Takarada, T.

Folia Pharmacologica Japonica 144 ( 2 ) 98 - 98 2014.8

Constitutive and functional expression of runt-related transcription factor-2 by microglial cells Reviewed

Ryota Nakazato, Takeshi Takarada, Takumi Watanabe, Binh Thanh Nguyen, Shinsuke Ikeno, Eiichi Hinoi, Yukio Yoneda

NEUROCHEMISTRY INTERNATIONAL 74 24 - 35 2014.7

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

Recent advances in research on mesenchymal stem cells

Takarada Takeshi

Folia Pharmacologica Japonica 144 ( 6 ) 305 - 305 2014

An Analysis of Skeletal Development in Osteoblast-Specific and Chondrocyte-Specific Runt- Related Transcription Factor-2 ( Runx2) Knockout Mice Reviewed

Takeshi Takarada, Eiichi Hinoi, Ryota Nakazato, Hiroki Ochi, Cheng Xu, Azusa Tsuchikane, Shu Takeda, Gerard Karsenty, Takaya Abe, Hiroshi Kiyonari, Yukio Yoneda

JOURNAL OF BONE AND MINERAL RESEARCH 28 ( 10 ) 2064 - 2069 2013.10

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

Regulatory Mechanisms of Skeletal Tissues by Amino Acid Signaling Reviewed

Takeshi Takarada

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 133 ( 7 ) 799 - 802 2013.7

Language：Japanese Publisher：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

Mitochondrial uncoupling protein-2 in glutamate neurotoxicity. Reviewed

Takarada T, Fukumori R, Yoneda Y

Nihon yakurigaku zasshi. Folia pharmacologica Japonica 142 ( 1 ) 13 - 16 2013.7

Selective Inhibition by Ethanol of Mitochondrial Calcium Influx Mediated by Uncoupling Protein-2 in Relation to N-Methyl-D-Aspartate Cytotoxicity in Cultured Neurons Reviewed

Ryo Fukumori, Takeshi Takarada, Ryota Nakazato, Koichi Fujikawa, Miki Kou, Eiichi Hinoi, Yukio Yoneda

PLOS ONE 8 ( 7 ) e69718 2013.7

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

Myosin VI Reduces Proliferation, but Not Differentiation, in Pluripotent P19 Cells Reviewed

Takeshi Takarada, Miki Kou, Noritaka Nakamichi, Masato Ogura, Yuma Ito, Ryo Fukumori, Hiroshi Kokubo, Gabriela B. Acosta, Eiichi Hinoi, Yukio Yoneda

PLoS ONE 8 ( 5 ) e63947 2013.5

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

A negative correlation between Per1 and Sox6 expression during chondrogenic differentiation in pre-chondrocytic ATDC5 cells Reviewed

Nguyen Quynh Le, Nguyen Thanh Binh, Takeshi Takarada, Mika Takarada-Iemata, Eiichi Hinoi, Yukio Yoneda

Journal of Pharmacological Sciences 122 ( 4 ) 318 - 325 2013

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

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

Possible neuroprotective property of nicotinic acetylcholine receptors in association with predominant upregulation of glial cell line-derived neurotrophic factor in astrocytes Reviewed

Takeshi Takarada, Noritaka Nakamichi, Hirofumi Kawagoe, Masato Ogura, Ryo Fukumori, Ryota Nakazato, Koichi Fujikawa, Miki Kou, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 90 ( 11 ) 2074 - 2085 2012.11

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

Promoted Neuronal Differentiation after Activation of Alpha4/Beta2 Nicotinic Acetylcholine Receptors in Undifferentiated Neural Progenitors Reviewed

Takeshi Takarada, Noritaka Nakamichi, Seiya Kitajima, Ryo Fukumori, Ryota Nakazato, Nguyen Quynh Le, Yeong-Hun Kim, Koichi Fujikawa, Miki Kou, Yukio Yoneda

PLOS ONE 7 ( 10 ) e46177 2012.10

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

Clock Genes Influence Gene Expression in Growth Plate and Endochondral Ossification in Mice Reviewed

Takeshi Takarada, Ayumi Kodama, Shogo Hotta, Michihiro Mieda, Shigeki Shimba, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF BIOLOGICAL CHEMISTRY 287 ( 43 ) 36081 - 36095 2012.10

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

Promotion of Both Proliferation and Neuronal Differentiation in Pluripotent P19 Cells with Stable Overexpression of the Glutamine Transporter slc38a1 Reviewed

Masato Ogura, Takami Kakuda, Takeshi Takarada, Noritaka Nakamichi, Ryo Fukumori, Yeong-Hun Kim, Eiichi Hinoi, Yukio Yoneda

PLOS ONE 7 ( 10 ) e48270 2012.10

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

Osteoclastogenesis is negatively regulated by D-serine produced by osteoblasts Reviewed

Takeshi Takarada, Mika Takarada-Iemata, Yoshifumi Takahata, Daisuke Yamada, Tomomi Yamamoto, Yukari Nakamura, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 227 ( 10 ) 3477 - 3487 2012.10

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

Signaling Factors in a Variety of Cells Derived from Mesenchymal Stem Cells Reviewed

Takeshi Takarada

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 132 ( 10 ) 1145 - 1149 2012.10

Language：Japanese Publisher：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

Possible involvement of mitochondrial uncoupling protein-2 in cytotoxicity mediated by acquired N-methyl-D-aspartate receptor channels Reviewed

Ryo Fukumori, Takeshi Takarada, Yuki Kambe, Ryota Nakazato, Koichi Fujikawa, Yukio Yoneda

NEUROCHEMISTRY INTERNATIONAL 61 ( 4 ) 498 - 505 2012.9

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

Positive Regulation by gamma-Aminobutyric Acid B Receptor Subunit-1 of Chondrogenesis through Acceleration of Nuclear Translocation of Activating Transcription Factor-4 Reviewed

Yoshifumi Takahata, Eiichi Hinoi, Takeshi Takarada, Yukari Nakamura, Shinya Ogawa, Yukio Yoneda

JOURNAL OF BIOLOGICAL CHEMISTRY 287 ( 40 ) 33293 - 33303 2012.9

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

A view that signaling machineries for the neurotransmitter gamma-aminobutyric acid (GABA) are functionally expressed by cells outside the central nervous system is now prevailing. In this study, we attempted to demonstrate functional expression of GABAergic signaling molecules by chondrocytes. In cultured murine costal chondrocytes, mRNA was constitutively expressed for metabotropic GABA(B) receptor subunit-1 (GABA(B)R1), but not for GABA(B)R2. Immunohistochemical analysis revealed the predominant expression of GABABR1 by prehypertrophic to hypertrophic chondrocytes in tibial sections of newborn mice. The GABA(B)R agonist baclofen failed to significantly affect chondrocytic differentiation determined by Alcian blue staining and alkaline phosphatase activity in cultured chondrocytes, whereas newborn mice knocked out of GABA(B)R1 (KO) showed a decreased body size and delayed calcification in hyoid bone and forelimb and hindlimb digits. Delayed calcification was also seen in cultured metatarsals from KO mice with a marked reduction of Indian hedgehog gene (Ihh) expression. Introduction of GABABR1 led to synergistic promotion of the transcriptional activity of activating transcription factor-4 (ATF4) essential for normal chondrogenesis, in addition to facilitating ATF4-dependent Ihh promoter activation. Although immunoreactive ATF4 was negligibly detected in the nucleus of chondrocytes from KO mice, ATF4 expression was again seen in the nucleus and cytoplasm after the retroviral introduction of GABABR1 into cultured chondrocytes from KO mice. In nuclear extracts of KO chondrocytes, a marked decrease was seen in ATF4 DNA binding. These results suggest that GABABR1 positively regulates chondrogenesis through a mechanism relevant to the acceleration of nuclear translocation of ATF4 for Ihh expression in chondrocytes.

DOI： 10.1074/jbc.M112.344051

Delayed Mitochondrial Membrane Potential Disruption by ATP in Cultured Rat Hippocampal Neurons Exposed to N-Methyl-D-Aspartate Reviewed

Koichi Fujikawa, Noritaka Nakamichi, Shunsuke Kato, Ryo Fukumori, Miho Hida, Takeshi Takarada, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 119 ( 1 ) 20 - 29 2012.5

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

Possible Modulation of Process Extension by N-Methyl-D-aspartate Receptor Expressed in Osteocytic MLO-Y4 Cells Reviewed

Hiroyuki Fujita, Eiichi Hinoi, Eri Nakatani, Tomomi Yamamoto, Takeshi Takarada, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 119 ( 1 ) 112 - 116 2012.5

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

Transferrin receptor-1 suppresses neurite outgrowth in neuroblastoma Neuro2A cells Reviewed

Yukary Nakamura, Noritaka Nakamichi, Takeshi Takarada, Kiyokazu Ogita, Yukio Yoneda

NEUROCHEMISTRY INTERNATIONAL 60 ( 5 ) 448 - 457 2012.4

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

Positive Regulation of Osteoclastic Differentiation by Growth Differentiation Factor 15 Upregulated in Osteocytic Cells Under Hypoxia Reviewed

Eiichi Hinoi, Hiroki Ochi, Takeshi Takarada, Eri Nakatani, Takashi Iezaki, Hiroko Nakajima, Hiroyuki Fujita, Yoshifumi Takahata, Shinya Hidano, Takashi Kobayashi, Shu Takeda, Yukio Yoneda

JOURNAL OF BONE AND MINERAL RESEARCH 27 ( 4 ) 938 - 949 2012.4

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

Artificial orchestration of functional NMDAR channels in HEK293 cells Reviewed

Takaya Katsurayama, Ryo Fukumori, Takeshi Takarada, Yukio Yoneda

Japanese Journal of Neuropsychopharmacology 32 ( 2 ) 113 - 114 2012.4

NR2-reactive antibody decreases cell viability through augmentation of Ca2+ influx in systemic lupus erythematosus Reviewed

Takahisa Gono, Takeshi Takarada, Ryo Fukumori, Yasushi Kawaguchi, Hirotaka Kaneko, Masanori Hanaoka, Yasuhiro Katsumata, Yukio Yoneda, Hisashi Yamanaka

ARTHRITIS AND RHEUMATISM 63 ( 12 ) 3952 - 3959 2011.12

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

Negative Regulation of Osteoblastogenesis Through Downregulation of Runt-Related Transcription Factor-2 in Osteoblastic MC3T3-E1 Cells with Stable Overexpression of the Cystine/Glutamate Antiporter xCT Subunit Reviewed

Kyosuke Uno, Takeshi Takarada, Mika Takarada-Iemata, Yukari Nakamura, Hiroyuki Fujita, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 226 ( 11 ) 2953 - 2964 2011.11

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

Osteoblastic gamma-Aminobutyric Acid, Type B Receptors Negatively Regulate Osteoblastogenesis toward Disturbance of Osteoclastogenesis Mediated by Receptor Activator of Nuclear Factor kappa B Ligand in Mouse Bone Reviewed

Yoshifumi Takahata, Takeshi Takarada, Eiichi Hinoi, Yukari Nakamura, Hiroyuki Fujita, Yukio Yoneda

JOURNAL OF BIOLOGICAL CHEMISTRY 286 ( 38 ) 32906 - 32917 2011.9

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The prevailing view is that signaling machineries for the neurotransmitter GABA are also expressed by cells outside the CNS. In cultured murine calvarial osteoblasts, mRNA was constitutively expressed for both subunits 1 and 2 of metabotropic GABA(B) receptor (GABA(B)R), along with inhibition by the GABA(B)R agonist baclofen of cAMP formation, alkaline phosphatase (ALP) activity, and Ca(2+) accumulation. Moreover, baclofen significantly inhibited the transactivation of receptor activator of nuclear factor-kappa B ligand (RANKL) gene in a manner sensitive to a GABA(B)R antagonist, in addition to decreasing mRNA expression of bone morphogenetic protein-2 (BMP2), osteocalcin, and osterix. In osteoblastic MC3T3-E1 cells stably transfected with GABA(B)R1 subunit, significant reductions were seen in ALP activity and Ca(2+) accumulation, as well as mRNA expression of osteocalcin, osteopontin, and osterix. In cultured calvarial osteoblasts from GABA(B)R1-null mice exhibiting low bone mineral density in tibia and femur, by contrast, both ALP activity and Ca(2+) accumulation were significantly increased together with promoted expression of both mRNA and proteins for BMP2 and osterix. No significant change was seen in the number of multinucleated cells stained for tartrate-resistant acid phosphatase during the culture of osteoclasts prepared from GABA(B)R1-null mice, whereas a significant increase was seen in the number of tartrate-resistant acid phosphatase-positive multinucleated cells in co-culture of osteoclasts with osteoblasts isolated from GABA(B)R1-null mice. These results suggest that GABA(B)R is predominantly expressed by osteoblasts to negatively regulate osteoblastogenesis through down-regulation of BMP2 expression toward disturbance of osteoclastogenesis after down-regulation of RANKL expression in mouse bone.

DOI： 10.1074/jbc.M111.253526

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl-D-aspartate receptors in cultured rat hippocampal neurons Reviewed

Yuki Kambe, Noritaka Nakamichi, Takeshi Takarada, Ryo Fukumori, Ryota Nakazato, Eiichi Hinoi, Yukio Yoneda

NEUROCHEMISTRY INTERNATIONAL 59 ( 1 ) 10 - 20 2011.8

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

Selective Upregulation of Pen1 mRNA Expression by ATP Through Activation of P2X7 Purinergic Receptors Expressed in Microglial Cells Reviewed

Ryota Nakazato, Takeshi Takarada, Tomomi Yamamoto, Shogo Hotta, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 116 ( 4 ) 350 - 361 2011.8

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

Selective downregulation of N-methyl-D-aspartate receptor (NMDAR) rather than non-NMDAR subunits in ipsilateral cerebral hemispheres in rats with middle cerebral artery occlusion Reviewed

Takeshi Takarada, Tomoya Hara, Shiho Konishi, Ryota Nakazato, Yukio Yoneda

Japanese Journal of Neuropsychopharmacology 31 ( 4 ) 187 - 194 2011.8

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

Positive Regulation by GABA(B)R1 Subunit of Leptin Expression through Gene Transactivation in Adipocytes Reviewed

Yukari Nakamura, Eiichi Hinoi, Takeshi Takarada, Yoshifumi Takahata, Tomomi Yamamoto, Hiroyuki Fujita, Saya Takada, Syota Hashizume, Yukio Yoneda

PLOS ONE 6 ( 5 ) e20167 2011.5

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

[Role of glutamine transporter expressed in astroglia cells in glutamic acid-induced neuronal death]. Reviewed

Safuru, A., Kokura, M., Takarada, T., Yoneta, Y.

Nihon shinkei seishin yakurigaku zasshi = Japanese journal of psychopharmacology 31 ( 2 ) 91 - 93 2011.4

Glutamate Preferentially Suppresses Osteoblastogenesis Than Adipogenesis Through the Cystine/Glutamate Antiporter in Mesenchymal Stem Cells Reviewed

Mika Takarada-Iemata, Takeshi Takarada, Yukari Nakamura, Eri Nakatani, Osamu Hori, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 226 ( 3 ) 652 - 665 2011.3

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

Exacerbated vulnerability to oxidative stress in astrocytic C6 glioma cells with stable overexpression of the glutamine transporter slc38a1 Reviewed

Masato Ogura, Takeshi Takarada, Noritaka Nakamichi, Hirofumi Kawagoe, Aya Sako, Ryota Nakazato, Yukio Yoneda

NEUROCHEMISTRY INTERNATIONAL 58 ( 4 ) 504 - 511 2011.3

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

A Negative Correlation Between Expression Profiles of Runt-Related Transcription Factor-2 and Cystine/Glutamate Antiporter xCT Subunit in Ovariectomized Mouse Bone Reviewed

Kyosuke Uno, Takeshi Takarada, Yukari Nakamura, Hiroyuki Fujita, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 115 ( 3 ) 309 - 319 2011.3

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

GABAB R1サブユニット欠損マウスにおける神経系前駆細胞の機能変化

福井正樹, 小澤秀介, 宝田剛志, 米田幸雄

日本薬理学雑誌 137 ( 3 ) 29P - 29P 2011.3

Gradual Downregulation of Protein Expression of the Partner GABA(B)R2 Subunit During Postnatal Brain Development in Mice Defective of GABA(B)R1 Subunit Reviewed

Masaki Fukui, Shusuke Ozawa, Noritaka Nakamichi, Ryota Nakazato, Takeshi Takarada, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 115 ( 1 ) 45 - 55 2011.1

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

Requirement of both NR3A and NR3B subunits for dominant negative properties on Ca2+ mobilization mediated by acquired N-methyl-D-aspartate receptor channels into mitochondria Reviewed

Ryo Fukumori, Takeshi Takarada, Noritaka Nakamichi, Yuki Kambe, Hirofumi Kawagoe, Ryota Nakazato, Yukio Yoneda

NEUROCHEMISTRY INTERNATIONAL 57 ( 7 ) 730 - 737 2010.12

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

Preferential Inhibition by Antidiarrheic 2-Methoxy-4-Methylphenol of Ca2+ Influx Across Acquired N-Methyl-D-Aspartate Receptor Channels Composed of NR1/NR2B Subunit Assembly Reviewed

Noritaka Nakamichi, Ryo Fukumori, Takeshi Takarada, Yuki Kambe, Tomomi Yamamoto, Nobuyuki Matsushima, Nobuaki Moriguchi, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 88 ( 11 ) 2483 - 2493 2010.8

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

Induced Tolerance to Glutamate Neurotoxicity Through Down-Regulation of NR2 Subunits of N-Methyl-D-Aspartate Receptors in Cultured Rat Striatal Neurons Reviewed

Yuki Kambe, Noritaka Nakamichi, Takeshi Takarada, Ryo Fukumori, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 88 ( 10 ) 2177 - 2187 2010.8

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

Inhibition by 2-Methoxy-4-ethylphenol of Ca2+ Influx Through Acquired and Native N-Methyl-D-aspartate-Receptor Channels Reviewed

Ryo Fukumori, Noritaka Nakamichi, Takeshi Takarada, Yuki Kambe, Nobuyuki Matsushima, Nobuaki Moriguchi, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 112 ( 3 ) 273 - 281 2010.3

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

Interference by adrenaline with chondrogenic differentiation through suppression of gene transactivation mediated by Sox9 family members Reviewed

Takeshi Takarada, Hironori Hojo, Mika Iemata, Koichi Sahara, Ayumi Kodama, Nobuhiro Nakamura, Eiichi Hinoi, Yukio Yoneda

BONE 45 ( 3 ) 568 - 578 2009.9

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

Interference With Cellular Differentiation by D-Serine Through Antagonism at N-Methyl-D-Aspartate Receptors Composed of NR1 and NR3A Subunits in Chondrocytes Reviewed

Takeshi Takarada, Yoshifumi Takahata, Mika Iemata, Eiichi Hinoi, Kyosuke Uno, Takao Hirai, Tomomi Yamamoto, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 220 ( 3 ) 756 - 764 2009.9

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

A protein-protein interaction of stress-responsive myosin VI endowed to inhibit neural progenitor self-replication with RNA binding protein, TLS, in murine hippocampus Reviewed

Takeshi Takarada, Keisuke Tamaki, Toru Takumi, Masato Ogura, Yuma Ito, Noritaka Nakamichi, Yukio Yoneda

JOURNAL OF NEUROCHEMISTRY 110 ( 5 ) 1457 - 1468 2009.9

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

Possible Promotion of Neuronal Differentiation in Fetal Rat Brain Neural Progenitor Cells After Sustained Exposure to Static Magnetism Reviewed

Noritaka Nakamichi, Yukichi Ishioka, Takao Hirai, Shusuke Ozawa, Masaki Tachibana, Nobuhiro Nakamura, Takeshi Takarada, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 87 ( 11 ) 2406 - 2417 2009.8

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

Possible Protection by Notoginsenoside R1 against Glutamate Neurotoxicity Mediated by N-methyl-D-aspartate Receptors Composed of an NR1/NR2B Subunit Assembly Reviewed

Bin Gu, Noritaka Nakamichi, Wen-Sheng Zhang, Yukary Nakamura, Yuki Kambe, Ryo Fukumori, Kazuhiro Takuma, Kiyofumi Yamada, Takeshi Takarada, Hideo Taniura, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 87 ( 9 ) 2145 - 2156 2009.7

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

Neurogenesis Mediated by gamma-Aminobutyric Acid and Glutamate Signaling Reviewed

Noritaka Nakamichi, Takeshi Takarada, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 110 ( 2 ) 133 - 149 2009.6

Language：English Publisher：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

GABA-B受容体を介した神経系細胞系譜への分化調節(Modulation of differentiation into neural lineages through GABAB receptors)

小澤秀介, 福井正樹, 宝田剛志, 米田幸雄

神経化学 48 ( 2-3 ) 212 - 212 2009.6

Predominant Promotion by Tacrolimus of Chondrogenic Differentiation to Proliferating Chondrocytes Reviewed

Yukari Nakamura, Takeshi Takarada, Ayumi Kodama, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 109 ( 3 ) 413 - 423 2009.3

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

Transactivation by Runt related factor-2 of matrix metalloproteinase-13 in astrocytes Reviewed

Takeshi Takarada, Yukio Yoneda

NEUROSCIENCE LETTERS 451 ( 2 ) 99 - 104 2009.2

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

Functional Expression of beta(2) Adrenergic Receptors Responsible for Protection Against Oxidative Stress Through Promotion of Glutathione Synthesis After Nrf2 Upregulation in Undifferentiated Mesenchymal C3H10T1/2 Stem Cells Reviewed

Yoshifumi Takahata, Takeshi Takarada, Mika Iemata, Tomomi Yamamoto, Yukary Nakamura, Ayumi Kodama, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 218 ( 2 ) 268 - 275 2009.2

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

A critical importance of polyamine site in NMDA receptors for neurite outgrowth and fasciculation at early stages of P19 neuronal differentiation Reviewed

Danko Georgiev, Hideo Taniura, Yuki Kambe, Takeshi Takarada, Yukio Yoneda

EXPERIMENTAL CELL RESEARCH 314 ( 14 ) 2603 - 2617 2008.8

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

GABAシグナルによるマウス神経系前駆細胞の増殖と分化の調節(Modulation of proliferation and differentiation by GABAergic signaling in murine neural progenitor cells)

小澤秀介, 福井正樹, 寳田剛志, 谷浦秀夫, 中道範隆, 米田幸雄

神経化学 47 ( 2-3 ) 225 - 225 2008.8

Differential regulation of cellular maturation in chondrocytes and osteoblasts by glycine Reviewed

Yoshifumi Takahata, Takeshi Takarada, Masato Osawa, Eiichi Hinoi, Yukari Nakamura, Yukio Yoneda

CELL AND TISSUE RESEARCH 333 ( 1 ) 91 - 103 2008.7

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

Serine racemase suppresses chondrogenic differentiation in cartilage in a Sox9-dependent manner Reviewed

Takeshi Takarada, Eiichi Hinoi, Yoshifumi Takahata, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 215 ( 2 ) 320 - 328 2008.5

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

Glutamate as a signal mediator in bone Reviewed

Takeshi Takarada, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 106 ( 4 ) 536 - 541 2008.4

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

A role of the clock gene Period1 in chondrogenic differentiation Reviewed

Ayumi Kodama, Koichi Sahara, Takeshi Takarada, Yukio Yoneda

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 128 142 - 144 2008

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

Protective effect of notoginsenoside R1 in Panax notoginseng on glutamate-induced neurotoxicity Reviewed

Bin Gu, Yukary Nakamura, Yuki Kambe, Kazuhiro Takuma, Kiyofumi Yamada, Wen-Sheng Zhang, Takeshi Takarada, Noritaka Nakamichi, Yukio Yoneda

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 128 184 - 187 2008

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：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.

Osteoblast protects osteoclast devoid of sodium-dependent vitamin C transporters from oxidative cytotoxicity of ascorbic acid Reviewed

Takeshi Takarada, Eiichi Hinoi, Yuki Kambe, Koichi Sahara, Shintaro Kurokawa, Yoshifumi Takahata, Yukio Yoneda

EUROPEAN JOURNAL OF PHARMACOLOGY 575 ( 1-3 ) 1 - 11 2007.12

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

Suppression by glutamate of proliferative activity through glutathione depletion mediated by the Cystine/Glutamate antiporter in mesenchymal C3H10T1/2 stem cells Reviewed

Mika Iemata, Takeshi Takarada, Eiichi Hinoi, Hideo Taniura, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 213 ( 3 ) 721 - 729 2007.12

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

Glutamate is a determinant of cellular proliferation through modulation of nuclear factor E2 p45-related factor-2 expression in osteoblastic MC3T3-E1 cells Reviewed

Kyosuke Uno, Takeshi Takarada, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 213 ( 1 ) 105 - 114 2007.10

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

Glutamate suppresses osteoclastogenesis through the cystine/glutamate antiporter Reviewed

Eiichi Hinoi, Takeshi Takarada, Kyosuke Uno, Maki Inoue, Yasuhiro Murafuji, Yukio Yoneda

AMERICAN JOURNAL OF PATHOLOGY 170 ( 4 ) 1277 - 1290 2007.4

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

Nuclear factor E2 p45-related factor 2 negatively regulates chondrogenesis Reviewed

Eiichi Hinoi, Takeshi Takarada, Sayumi Fujimori, Liyang Wang, Mika Iemata, Kyosuke Uno, Yukio Yoneda

BONE 40 ( 2 ) 337 - 344 2007.2

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

Oral administration of phenolic antidiarrheic ingredients prevents ovariectomy-induced bone loss Reviewed

Nobuaki Moriguchi, Eiichi Hinoi, Takeshi Takarada, Nobuyuki Matsushima, Kyosuke Uno, Yukio Yoneda

BIOCHEMICAL PHARMACOLOGY 73 ( 3 ) 385 - 393 2007.2

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

Possible expression of a particular gamma-aminobutyric acid transporter isoform responsive to upregulation by hyperosmolarity in rat calvarial osteoblasts Reviewed

Sayumi Fujimori, Eiichi Hinoi, Takeshi Takarada, Mika Iemata, Yoshifumi Takahata, Yukio Yoneda

EUROPEAN JOURNAL OF PHARMACOLOGY 550 ( 1-3 ) 24 - 32 2006.11

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

A molecular mechanism of pyruvate protection against cytotoxicity of reactive oxygen species in osteoblasts Reviewed

Eiichi Hinoi, Takeshi Takarada, Yuriko Tsuchihashi, Sayumi Fujimori, Nobuaki Moriguchi, Liyang Wang, Kyosuke Uno, Yukio Yoneda

MOLECULAR PHARMACOLOGY 70 ( 3 ) 925 - 935 2006.9

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

Cytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts Reviewed

N. Moriguchi, E. Hinoi, Y. Tsuchihashi, S. Fujimori, M. Iemata, T. Takarada, Y. Yoneda

HISTOLOGY AND HISTOPATHOLOGY 21 ( 9 ) 969 - 977 2006.9

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

Up-regulation of per mRNA expression by parathyroid hormone through a protein kinase A-CREB-dependent mechanism in chondrocytes Reviewed

Eiichi Hinoi, Taichi Ueshima, Hironori Hojo, Mika Iemata, Takeshi Takarada, Yukio Yoneda

JOURNAL OF BIOLOGICAL CHEMISTRY 281 ( 33 ) 23632 - 23642 2006.8

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

Excitatory amino acid transporters expressed by synovial fibroblasts in rats with collagen-induced arthritis Reviewed

E Hinoi, R Ohashi, S Miyata, Y Kato, M Iemata, H Hojo, T Takarada, Y Yoneda

BIOCHEMICAL PHARMACOLOGY 70 ( 12 ) 1744 - 1755 2005.12

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

Abolition of chondral mineralization by group III metabotropic glutamate receptors expressed in rodent cartilage Reviewed

LY Wang, E Hinoi, A Takemori, T Takarada, Y Yoneda

BRITISH JOURNAL OF PHARMACOLOGY 146 ( 5 ) 732 - 743 2005.11

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

Nuclear condensation of cyclic adenosine monophosphate responsive element-binding protein in discrete murine brain structures Reviewed

N Kuramoto, K Kubo, K Ogita, J Platenik, VJ Balcar, T Takarada, N Nakamichi, Y Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 80 ( 5 ) 667 - 676 2005.6

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

Counteraction by repetitive daily exposure to static magnetism against sustained blockade of N-methyl-D-aspartate receptor channels in cultured rat hippocampal neurons Reviewed

T Hirai, H Taniura, Y Goto, K Tamaki, H Oikawa, Y Kambe, M Ogura, Y Ohno, T Takarada, Y Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 80 ( 4 ) 491 - 500 2005.5

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

Glutamate transporters as drug targets Reviewed

Eiichi Hinoi, Takeshi Takarada, Yuriko Tsuchihashi, Yukio Yoneda

Current Drug Targets: CNS and Neurological Disorders 4 ( 2 ) 211 - 220 2005.4

Language：English Publisher：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

Accumulation of [H-3]glutamate in cultured rat calvarial osteoblasts Reviewed

T Takarada, E Hinoi, S Fujimori, Y Tsuchihashi, T Ueshima, H Taniura, Y Yoneda

BIOCHEMICAL PHARMACOLOGY 68 ( 1 ) 177 - 184 2004.7

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

Possible expression of functional glutamate transporters in the rat testis Reviewed

T Takarada, E Hinoi, VJ Balcar, H Taniura, Y Yoneda

JOURNAL OF ENDOCRINOLOGY 181 ( 2 ) 233 - 244 2004.5

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

Glutamate signaling system in bone Reviewed

E Hinoi, T Takarada, Y Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 94 ( 3 ) 215 - 220 2004.3

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

Glutamate signaling in peripheral tissues Reviewed

E Hinoi, T Takarada, T Ueshima, Y Tsuchihashi, Y Yoneda

EUROPEAN JOURNAL OF BIOCHEMISTRY 271 ( 1 ) 1 - 13 2004.1

Language：English Publisher：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

Modulation by static magnetism of neuronal activity

Hirai, T., Goto, Y., Takarada, T., Taniura, H., Yoneda, Y.

Biomedical Reviews 15 ( 1 ) 2004

Uptake of [H-3]L-serine in rat brain synaptosomal fractions Reviewed

T Takarada, VJ Balcar, K Baba, A Takamoto, GB Acosta, K Takano, Y Yoneda

BRAIN RESEARCH 983 ( 1-2 ) 36 - 47 2003.9

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

Facilitation of glutamate release by ionotropic glutamate receptors in osteoblasts Reviewed

E Hinoi, S Fujimori, T Takarada, H Taniura, Y Yoneda

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 297 ( 3 ) 452 - 458 2002.9

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