顔写真

オオネダ キヌコ
大根田 絹子
Kinuko Ohneda
所属
東北メディカル・メガバンク機構 バイオバンク部門
職名
教授
学位
  • 博士(医学)(東北大学)

研究分野 1

  • ライフサイエンス / 分子生物学 /

論文 70

  1. Effect of Nicotinamide Mononucleotide Concentration in Human Milk on Neurodevelopmental Outcome: The Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study. 国際誌

    Yoshie Saito, Keigo Sato, Shinji Jinno, Yoshitaka Nakamura, Takahiro Nobukuni, Soichi Ogishima, Satoshi Mizuno, Seizo Koshiba, Shinichi Kuriyama, Kinuko Ohneda, Masashi Morifuji

    Nutrients 16 (1) 2023年12月31日

    DOI: 10.3390/nu16010145  

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    (1) Background: Breast milk is the only source of nutrition for breastfed infants, but few studies have examined the relationship between breast milk micronutrients and infant neurodevelopmental outcome in exclusively breastfed infants. The aim of this study was to characterize the association between nicotinamide adenine dinucleotide (NAD)-related compounds in the breast milk of Japanese subjects and infant neurodevelopmental outcome. (2) Methods: A total of 150 mother-child pairs were randomly selected from the three-generation cohort of the Tohoku Medical Megabank in Japan. Infants were exclusively breastfed for up to 6 months. Breast milk was collected at 1 month postpartum, and the quantity of NAD-related substances in the breast milk was quantified. The mothers also completed developmental questionnaires at 6, 12, and 24 months. The relationship between the concentration of NAD-related substances in breast milk and developmental indicators was evaluated via ordinal logistic regression analysis. (3) Results: Nicotinamide mononucleotide (NMN) was quantified as the major NAD precursor in breast milk. The median amount of NMN in the breast milk was 9.2 μM. The NMN concentration in breast milk was the only NAD-related substance in breast milk that showed a significant positive correlation with neurodevelopmental outcome in infants at 24 months. (4) Conclusions: The results suggest that NMN in human milk may be an important nutrient for early childhood development.

  2. ゲノムコホート研究参加者5万人を対象としたBRCA1/2遺伝情報の回付と医療への連携

    濱中 洋平, 大根田 絹子, 川目 裕, 布施 昇男, 長神 風二, 鈴木 洋一, 山口 由美, 多田 寛, 原田 成美, 宮下 穣, 江幡 明子, 佐藤 未来, 柳垣 美歌, 山本 雅之, 石田 孝宣

    日本乳癌学会総会プログラム抄録集 31回 89-89 2023年6月

    出版者・発行元:(一社)日本乳癌学会

  3. Returning individual genomic results to population-based cohort study participants with BRCA1/2 pathogenic variants.

    Kinuko Ohneda, Yohei Hamanaka, Hiroshi Kawame, Nobuo Fuse, Fuji Nagami, Yoichi Suzuki, Yumi Yamaguchi-Kabata, Muneaki Shimada, Atsushi Masamune, Yoko Aoki, Takanori Ishida, Masayuki Yamamoto

    Breast cancer (Tokyo, Japan) 30 (1) 110-120 2023年1月

    DOI: 10.1007/s12282-022-01404-7  

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    BACKGROUND: Recent advances in human genome research have provided evidence for genotype-phenotype associations, pathogenicity, and clinical actionability of variants and genomic risk prediction of disease. However, the return of individual genomic results to healthy individuals is fraught with ethical and practical complexity. METHODS: Individual genomic results were returned to BRCA1/2 pathogenic variant (PV) carriers of the Tohoku Medical Megabank cohort study participants with an information on hereditary breast and ovarian cancer syndrome (HBOC). One hundred and eighty participants, including 9 BRCA1/2 PV carriers, were asked about their willingness to receive individual genomic results, without revealing the gene name and related disorders, prior to the study. Of the 142 participants who responded, 103 showed willingness to know their genomic information. Each of the six BRCA1/2 PV carriers who consented to participate in the study received information about HBOC in person and underwent validation testing with blood resampling. RESULTS: All participants were in their 60s or 70s; of the four females and two males, two had a history of breast cancer and five had a family history of HBOC-related cancers. All participants appreciated the information, without remarkable negative psychological impact of the return, and intended to undergo clinical risk surveillance. Five participants were accompanied by family members while receiving the results, and three first-degree female relatives wished to undergo genomic testing at the hospital. CONCLUSIONS: Our results suggest that returning actionable genomic information to participants in a population-based genome cohort study is beneficial for preventing or providing early-stage intervention for associated diseases.

  4. マスト細胞における転写因子GATA2の高度転写活性化機序の解析

    大森 慎也, 高井 淳, 森口 尚, 森 哲哉, 大根田 絹子

    日本生化学会大会プログラム・講演要旨集 95回 1P-248 2022年11月

    出版者・発行元:(公社)日本生化学会

  5. The Il6 -39 kb enhancer containing clustered GATA2- and PU.1-binding sites is essential for Il6 expression in murine mast cells. 国際誌

    Shin'ya Ohmori, Jun Takai, Satoshi Uemura, Akihito Otsuki, Tetsuya Mori, Kinuko Ohneda, Takashi Moriguchi

    iScience 25 (9) 104942-104942 2022年9月16日

    DOI: 10.1016/j.isci.2022.104942  

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    Mast cells serve as a first-line defense of innate immunity. Interleukin-6 (IL-6) induced by bacterial lipopolysaccharide (LPS) in mast cells plays a crucial role in antibacterial protection. The zinc finger transcription factor GATA2 cooperatively functions with the ETS family transcription factor PU.1 in multiple mast cell activities. However, the regulatory landscape directed by GATA2 and PU.1 under inflammation remains elusive. We herein showed that a large proportion of GATA2-binding peaks were closely located with PU.1-binding peaks in distal cis-regulatory regions of inflammatory cytokine genes in mast cells. Notably, GATA2 and PU.1 played crucial roles in promoting LPS-mediated inflammatory cytokine production. Genetic ablation of GATA2-PU.1-clustered binding sites at the Il6 -39 kb region revealed its central role in LPS-induced Il6 expression in mast cells. We demonstrate a novel collaborative activity of GATA2 and PU.1 in cytokine induction upon inflammatory stimuli via the GATA2-PU.1 overlapping sites in the distal cis-regulatory regions.

  6. 全ゲノム/全エキソーム解析による生殖細胞系列多型の探索 一般住民コホートにおけるBRCA遺伝子バリアントの探索及び結果の回付事業について(Exploration of BRCA1/2 gene variants in a general population cohort and return of genomic results to the participants)

    徳永 英樹, 安田 純, 島田 宗昭, 濱中 洋平, 重田 昌吾, 布施 昇男, 勝岡 史城, 荻島 創一, 山口 由美, 寳澤 篤, 川目 裕, 大根田 絹子, 青木 洋子, 山本 雅之, 八重樫 伸生

    日本癌学会総会記事 81回 S8-1 2022年9月

    出版者・発行元:(一社)日本癌学会

    ISSN:0546-0476

  7. A Pilot Study for Return of Individual Pharmacogenomic Results to Population-Based Cohort Study Participants.

    Kinuko Ohneda, Masahiro Hiratsuka, Hiroshi Kawame, Fuji Nagami, Yoichi Suzuki, Kichiya Suzuki, Akira Uruno, Mika Sakurai-Yageta, Yohei Hamanaka, Makiko Taira, Soichi Ogishima, Shinichi Kuriyama, Atsushi Hozawa, Hiroaki Tomita, Naoko Minegishi, Junichi Sugawara, Inaho Danjoh, Tomohiro Nakamura, Tomoko Kobayashi, Yumi Yamaguchi-Kabata, Shu Tadaka, Taku Obara, Eiji Hishimuma, Nariyasu Mano, Masaki Matsuura, Yuji Sato, Masateru Nakasone, Yohei Honkura, Jun Suzuki, Yukio Katori, Yoichi Kakuta, Atsushi Masamune, Yoko Aoki, Masaharu Nakayama, Shigeo Kure, Kengo Kinoshita, Nobuo Fuse, Masayuki Yamamoto

    JMA journal 5 (2) 177-189 2022年4月15日

    DOI: 10.31662/jmaj.2021-0156  

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    Introduction: Pharmacogenomic (PGx) testing results provide valuable information on drug selection and appropriate dosing, maximization of efficacy, and minimization of adverse effects. Although the number of large-scale, next-generation-sequencing-based PGx studies has recently increased, little is known about the risks and benefits of returning PGx results to ostensibly healthy individuals in research settings. Methods: Single-nucleotide variants of three actionable PGx genes, namely, MT-RNR1, CYP2C19, and NUDT15, were returned to 161 participants in a population-based Tohoku Medical Megabank project. Informed consent was obtained from the participants after a seminar on the outline of this study. The results were sent by mail alongside sealed information letter intended for clinicians. As an exception, genetic counseling was performed for the MT-RNR1 m.1555A > G variant carriers by a medical geneticist, and consultation with an otolaryngologist was encouraged. Questionnaire surveys (QSs) were conducted five times to evaluate the participants' understanding of the topic, psychological impact, and attitude toward the study. Results: Whereas the majority of participants were unfamiliar with the term PGx, and none had undergone PGx testing before the study, more than 80% of the participants felt that they could acquire basic PGx knowledge sufficient to understand their genomic results and were satisfied with their potential benefit and use in future prescriptions. On the other hand, some felt that the PGx concepts or terminology was difficult to fully understand and suggested that in-person return of the results was desirable. Conclusions: These results collectively suggest possible benefits of returning preemptive PGx information to ostensibly healthy cohort participants in a research setting.

  8. The return of individual genomic results to research participants: design and pilot study of Tohoku Medical Megabank Project. 国際誌

    Hiroshi Kawame, Akimune Fukushima, Nobuo Fuse, Fuji Nagami, Yoichi Suzuki, Mika Sakurai-Yageta, Jun Yasuda, Yumi Yamaguchi-Kabata, Kengo Kinoshita, Soichi Ogishima, Takako Takai, Shinichi Kuriyama, Atsushi Hozawa, Naoki Nakaya, Tomohiro Nakamura, Naoko Minegishi, Junichi Sugawara, Kichiya Suzuki, Hiroaki Tomita, Akira Uruno, Tomoko Kobayashi, Yayoi Aizawa, Tomoharu Tokutomi, Kayono Yamamoto, Kinuko Ohneda, Shigeo Kure, Yoko Aoki, Hideki Katagiri, Yasushi Ishigaki, Shojiro Sawada, Makoto Sasaki, Masayuki Yamamoto

    Journal of human genetics 67 (1) 9-17 2022年1月

    DOI: 10.1038/s10038-021-00952-8  

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    Certain large genome cohort studies attempt to return the individual genomic results to the participants; however, the implementation process and psychosocial impacts remain largely unknown. The Tohoku Medical Megabank Project has conducted large genome cohort studies of general residents. To implement the disclosure of individual genomic results, we extracted the potential challenges and obstacles. Major challenges include the determination of genes/disorders based on the current medical system in Japan, the storage of results, prevention of misunderstanding, and collaboration of medical professionals. To overcome these challenges, we plan to conduct multilayer pilot studies, which deal with different disorders/genes. We finally chose familial hypercholesterolemia (FH) as a target disease for the first pilot study. Of the 665 eligible candidates, 33.5% were interested in the pilot study and provided consent after an educational "genetics workshop" on the basic genetics and medical facts of FH. The genetics professionals disclosed the results to the participants. All positive participants were referred to medical care, and a serial questionnaire revealed no significant psychosocial distress after the disclosure. Return of genomic results to research participants was implemented using a well-prepared protocol. To further elucidate the impact of different disorders, we will perform multilayer pilot studies with different disorders, including actionable pharmacogenomics and hereditary tumor syndromes.

  9. 温故知新;古くて新しいGATA転写因子研究の最前線 炎症制御因子としてのGATA2

    高井 淳, 大森 慎也, 大根田 絹子, 上村 聡志, 山本 雅之, 森口 尚

    日本生化学会大会プログラム・講演要旨集 94回 [2S12m-02] 2021年11月

    出版者・発行元:(公社)日本生化学会

  10. マウスマスト細胞における新規Il6遺伝子制御領域(Il6-DRE)の同定と解析

    大森 慎也, 森口 尚, 高井 淳, 森 哲哉, 大根田 絹子

    日本生化学会大会プログラム・講演要旨集 94回 [P-633] 2021年11月

    出版者・発行元:(公社)日本生化学会

  11. マウスマスト細胞における新規Il6遺伝子制御領域(Il6-DRE)の同定と解析

    大森 慎也, 森口 尚, 高井 淳, 森 哲哉, 大根田 絹子

    日本生化学会大会プログラム・講演要旨集 94回 [P-633] 2021年11月

    出版者・発行元:(公社)日本生化学会

  12. 温故知新;古くて新しいGATA転写因子研究の最前線 炎症制御因子としてのGATA2

    高井 淳, 大森 慎也, 大根田 絹子, 上村 聡志, 山本 雅之, 森口 尚

    日本生化学会大会プログラム・講演要旨集 94回 [2S12m-02] 2021年11月

    出版者・発行元:(公社)日本生化学会

  13. ゲノムコホート研究におけるBRCA1/2遺伝情報返却とその後の医療機関との連携の取組み

    濱中 洋平, 多田 寛, 宮下 穣, 原田 成美, 佐藤 章子, 江幡 明子, 大根田 絹子, 布施 昇男, 川目 裕, 鈴木 洋一, 長神 風二, 鈴木 吉也, 佐藤 政文, 平塚 真弘, 櫻井 美佳, 宇留野 晃, 山口 由美, 平良 摩紀子, 山本 雅之, 石田 孝宣

    日本乳癌学会総会プログラム抄録集 29回 21-21 2021年7月

    出版者・発行元:(一社)日本乳癌学会

  14. ゲノムコホート研究におけるBRCA1/2遺伝情報返却とその後の医療機関との連携の取組み

    濱中 洋平, 多田 寛, 宮下 穣, 原田 成美, 佐藤 章子, 江幡 明子, 大根田 絹子, 布施 昇男, 川目 裕, 鈴木 洋一, 長神 風二, 鈴木 吉也, 佐藤 政文, 平塚 真弘, 櫻井 美佳, 宇留野 晃, 山口 由美, 平良 摩紀子, 山本 雅之, 石田 孝宣

    日本乳癌学会総会プログラム抄録集 29回 21-21 2021年7月

    出版者・発行元:(一社)日本乳癌学会

  15. Rejuvenation of mesenchymal stem cells by extracellular vesicles inhibits the elevation of reactive oxygen species. 国際誌

    Vuong Cat Khanh, Toshiharu Yamashita, Kinuko Ohneda, Chiho Tokunaga, Hideyuki Kato, Motoo Osaka, Yuji Hiramatsu, Osamu Ohneda

    Scientific reports 10 (1) 17315-17315 2020年10月14日

    DOI: 10.1038/s41598-020-74444-8  

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    Aging induces numerous cellular disorders, such as the elevation of reactive oxygen species (ROS), in a number type of cells, including mesenchymal stem cells (MSCs). However, the correlation of ROS and impaired healing abilities as well as whether or not the inhibition of elevating ROS results in the rejuvenation of elderly MSCs is unclear. The rejuvenation of aged MSCs has thus recently received attention in the field of regenerative medicine. Specifically, extracellular vesicles (EVs) act as a novel tool for stem cell rejuvenation due to their gene transfer ability with systemic effects and safety. In the present study, we examined the roles of aging-associated ROS in the function and rejuvenation of elderly MSCs by infant EVs. The data clearly showed that elderly MSCs exhibited the downregulation of superoxide dismutase (SOD)1 and SOD3, which resulted in the elevation of ROS and downregulation of the MEK/ERK pathways, which are involved in the impairment of the MSCs' ability to decrease necrotic area in the skin flap model. Furthermore, treatment with the antioxidant Edaravone or co-overexpression of SOD1 and SOD3 rescued elderly MSCs from the elevation of ROS and cellular senescence, thereby improving their functions. Of note, infant MSC-derived EVs rejuvenated elderly MSCs by inhibiting ROS production and the acceleration of cellular senescence and promoting the proliferation and in vivo functions in both type 1 and type 2 diabetic mice.

  16. コホート調査参加者に対するゲノム薬理学(PGx)遺伝情報の返却(回付) 個別化予防・医療の確立を目指して

    大根田 絹子, 布施 昇男, 川目 裕, 長神 風二, 平塚 真弘, 櫻井 美佳, 濱中 洋平, 鈴木 吉也, 鈴木 洋一, 山本 雅之

    日本薬学会年会要旨集 140年会 27K-pm08 2020年3月

    出版者・発行元:(公社)日本薬学会

    ISSN:0918-9823

  17. GATA2 and PU.1 Collaborate To Activate the Expression of the Mouse <i>Ms4a2</i> Gene, Encoding FcεRIβ, through Distinct Mechanisms. 国際誌 査読有り

    Ohmori S, Ishijima Y, Numata S, Takahashi M, Sekita M, Sato T, Chugun K, Yamamoto M, Ohneda K

    Molecular and cellular biology 39 (22) 2019年11月15日

    DOI: 10.1128/MCB.00314-19  

    ISSN:0270-7306

  18. Mouse Tryptase Gene Expression is Coordinately Regulated by GATA1 and GATA2 in Bone Marrow-Derived Mast Cells. 国際誌 査読有り

    Kinuko Ohneda, Shin'ya Ohmori, Masayuki Yamamoto

    International journal of molecular sciences 20 (18) 2019年9月17日

    DOI: 10.3390/ijms20184603  

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    Mast cell tryptases have crucial roles in allergic and inflammatory diseases. The mouse tryptase genes represent a cluster of loci on chromosome 16p3.3. While their functional studies have been extensively performed, transcriptional regulation of tryptase genes is poorly understood. In this study, we examined the molecular basis of the tryptase gene expression in bone marrow-derived mast cells (BMMCs) of C57BL/6 mice and in MEDMC-BRC6 mast cells. The expression of the Tpsb2 and Tpsg1 genes, which reside at the 3'-end of the tryptase locus, is significantly decreased by the reduction of the GATA transcription factors GATA1 or GATA2. Chromatin immunoprecipitation assays have shown that the GATA factors bind at multiple regions within the locus, including 1.0 and 72.8 kb upstream of the Tpsb2 gene, and that GATA1 and GATA2 facilitate each other's DNA binding activity to these regions. Deletion of the -72.8 kb region by genome editing significantly reduced the Tpsb2 and Tpsg1 mRNA levels in MEDMC-BRC6 cells. Furthermore, binding of CTCF and the cohesin subunit Rad21 was found upstream of the -72.8 kb region and was significantly reduced in the absence of GATA1. These results suggest that mouse tryptase gene expression is coordinately regulated by GATA1 and GATA2 in BMMCs.

  19. The Gata2 repression during 3T3-L1 preadipocyte differentiation is dependent on a rapid decrease in histone acetylation in response to glucocorticoid receptor activation. 国際誌 査読有り

    Yasushi Ishijima, Shin'ya Ohmori, Ai Uneme, Yusuke Aoki, Miki Kobori, Terutoshi Ohida, Momoko Arai, Misa Hosaka, Kinuko Ohneda

    Molecular and cellular endocrinology 483 39-49 2019年3月1日

    DOI: 10.1016/j.mce.2019.01.002  

    ISSN:0303-7207

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    The transcription factor GATA2 is an anti-adipogenic factor whose expression is downregulated during adipocyte differentiation. The present study attempted to clarify the molecular mechanism underlying the GATA2 repression and found that the repression is dependent on the activation of the glucocorticoid receptor (GR) during 3T3-L1 preadipocyte differentiation. Although several recognition sequences for GR were found in both the proximal and distal regions of the Gata2 locus, the promoter activity was not affected by the GR activation in the reporter assays, and the CRISPR-Cas9-mediated deletion of the two distal regions of the Gata2 locus was not involved in the GR-mediated Gata2 repression. Notably, the level of histone acetylation was markedly reduced at the Gata2 locus during 3T3-L1 differentiation, and the GR-mediated Gata2 repression was significantly relieved by histone deacetylase inhibition. These results suggest that GR regulates the Gata2 gene by reducing histone acetylation in the early phase of adipogenesis.

  20. Uremic Toxins Affect the Imbalance of Redox State and Overexpression of Prolyl Hydroxylase 2 in Human Adipose Tissue-Derived Mesenchymal Stem Cells Involved in Wound Healing. 国際誌 査読有り

    Vuong Cat Khanh, Kinuko Ohneda, Toshiki Kato, Toshiharu Yamashita, Fujio Sato, Kana Tachi, Osamu Ohneda

    Stem cells and development 26 (13) 948-963 2017年7月1日

    出版者・発行元:MARY ANN LIEBERT, INC

    DOI: 10.1089/scd.2016.0326  

    ISSN:1557-8534

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    Chronic kidney disease (CKD) results in a delay in wound healing because of its complications such as uremia, anemia, and fluid overload. Mesenchymal stem cells (MSCs) are considered to be a candidate for wound healing because of the ability to recruit many types of cells. However, it is still unclear whether the CKD-adipose tissue-derived MSCs (CKD-AT-MSCs) have the same function in wound healing as healthy donor-derived normal AT-MSCs (nAT-MSCs). In this study, we found that uremic toxins induced elevated reactive oxygen species (ROS) expression in nAT-MSCs, resulting in the reduced expression of hypoxia-inducible factor-1α (HIF-1α) under hypoxic conditions. Consistent with the uremic-treated AT-MSCs, there was a definite imbalance of redox state and high expression of ROS in CKD-AT-MSCs isolated from early-stage CKD patients. In addition, a transplantation study clearly revealed that nAT-MSCs promoted the recruitment of inflammatory cells and recovery from ischemia in the mouse flap model, whereas CKD-AT-MSCs had defective functions and the wound healing process was delayed. Of note, the expression of prolyl hydroxylase domain 2 (PHD2) is selectively increased in CKD-AT-MSCs and its inhibition can restore the expression of HIF-1α and the wound healing function of CKD-AT-MSCs. These results indicate that more studies about the functions of MSCs from CKD patients are required before they can be applied in the clinical setting.

  21. Uremic Toxins Affect the Imbalance of Redox State and Overexpression of Prolyl Hydroxylase 2 in Human Adipose Tissue-Derived Mesenchymal Stem Cells Involved in Wound Healing 査読有り

    Vuong Cat Khanh, Kinuko Ohneda, Toshiki Kato, Toshiharu Yamashita, Fujio Sato, Kana Tachi, Osamu Ohneda

    STEM CELLS AND DEVELOPMENT 26 (13) 948-963 2017年7月

    出版者・発行元:MARY ANN LIEBERT, INC

    DOI: 10.1089/scd.2016.0326  

    ISSN:1547-3287

    eISSN:1557-8534

    詳細を見る 詳細を閉じる

    Chronic kidney disease (CKD) results in a delay in wound healing because of its complications such as uremia, anemia, and fluid overload. Mesenchymal stem cells (MSCs) are considered to be a candidate for wound healing because of the ability to recruit many types of cells. However, it is still unclear whether the CKD-adipose tissue-derived MSCs (CKD-AT-MSCs) have the same function in wound healing as healthy donor-derived normal AT-MSCs (nAT-MSCs). In this study, we found that uremic toxins induced elevated reactive oxygen species (ROS) expression in nAT-MSCs, resulting in the reduced expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) under hypoxic conditions. Consistent with the uremic-treated AT-MSCs, there was a definite imbalance of redox state and high expression of ROS in CKD-AT-MSCs isolated from early-stage CKD patients. In addition, a transplantation study clearly revealed that nAT-MSCs promoted the recruitment of inflammatory cells and recovery from ischemia in the mouse flap model, whereas CKD-AT-MSCs had defective functions and the wound healing process was delayed. Of note, the expression of prolyl hydroxylase domain 2 (PHD2) is selectively increased in CKD-AT-MSCs and its inhibition can restore the expression of HIF-1 alpha and the wound healing function of CKD-AT-MSCs. These results indicate that more studies about the functions of MSCs from CKD patients are required before they can be applied in the clinical setting.

  22. Microvesicles derived from Alde-Low EPCs support the wound healing capacity of AT-MSCs. 国際誌 査読有り

    Tu TC, Yamashita T, Kato T, Nagano M, Trinh NT, Hamada H, Sato F, Ohneda K, Matsuo-Takasaki M, Ohneda O

    Biochemical and biophysical research communications 477 (1) 68-75 2016年8月12日

    出版者・発行元:None

    DOI: 10.1016/j.bbrc.2016.06.022  

    ISSN:0006-291X

    eISSN:1090-2104

  23. Increased Expression of EGR-1 in Diabetic Human Adipose Tissue-Derived Mesenchymal Stem Cells Reduces Their Wound Healing Capacity. 国際誌 査読有り

    Nhu-Thuy Trinh, Toshiharu Yamashita, Kinuko Ohneda, Kenichi Kimura, Georgina To'a Salazar, Fujio Sato, Osamu Ohneda

    Stem cells and development 25 (10) 760-73 2016年5月15日

    出版者・発行元:MARY ANN LIEBERT, INC

    DOI: 10.1089/scd.2015.0335  

    ISSN:1547-3287

    eISSN:1557-8534

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    The prevalence of type 2 diabetes mellitus (T2DM), which leads to diabetic complications, has been increasing worldwide. The possible applications of T2DM-derived stem cells in cell therapy are limited because their characteristics are still not fully understood. In this study, we characterized adipose tissue-derived mesenchymal stem cells (AT-MSCs) from diabetic patients (dAT-MSCs) and found that insulin receptor substrate-1 (IRS-1) was highly phosphorylated at serine 636/639 in dAT-MSCs. Moreover, we found that early growth response factor-1 (EGR-1) and its target genes of PTEN and GGPS1 were highly expressed in dAT-MSCs in comparison to healthy donor-derived AT-MSCs (nAT-MSCs). We observed impaired wound healing after the injection of dAT-MSCs in the ischemic flap mouse model. The expressions of EGR-1 and its target genes were diminished by small hairpin RNA-targeted EGR-1 (shEGR-1) and treatment with a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) inhibitor (PD98059). Importantly, dAT-MSCs with shEGR-1 were able to restore the wound healing ability in the mouse model. Interestingly, under hypoxic conditions, hypoxia-inducible factor-1α (HIF-1α) can bind to the EGR-1 promoter in dAT-MSCs, but not in nAT-MSCs. Together, these results demonstrate that the expression of EGR-1 was upregulated in dAT-MSCs through two pathways: the main regulatory pathway is the MAPK/ERK pathway, the other is mediated by HIF-1α through direct transcriptional activation at the promoter region of the EGR1 gene. Our study suggests that dAT-MSCs may contribute to microvascular damage and delay wound healing through the overexpression of EGR-1. Interrupting the expression of EGR-1 in dAT-MSCs may be a useful treatment for chronic wounds in diabetic patients.

  24. Microvesicles enhance the mobility of human diabetic adipose tissue-derived mesenchymal stem cells in vitro and improve wound healing in vivo. 国際誌 査読有り

    Trinh NT, Yamashita T, Tu TC, Kato T, Ohneda K, Sato F, Ohneda O

    Biochemical and biophysical research communications 473 (4) 1111-1118 2016年5月13日

    出版者・発行元:None

    DOI: 10.1016/j.bbrc.2016.04.025  

    ISSN:0006-291X

    eISSN:1090-2104

  25. A Chemokine Receptor, CXCR4, Which Is Regulated by Hypoxia-Inducible Factor 2α, Is Crucial for Functional Endothelial Progenitor Cells Migration to Ischemic Tissue and Wound Repair. 国際誌 査読有り

    Tu TC, Nagano M, Yamashita T, Hamada H, Ohneda K, Kimura K, Ohneda O

    Stem cells and development 25 (3) 266-76 2016年2月1日

    出版者・発行元:None

    DOI: 10.1089/scd.2015.0290  

    ISSN:1547-3287

    eISSN:1557-8534

  26. GATA2 is critical for the maintenance of cellular identity in differentiated mast cells derived from mouse bone marrow. 国際誌 査読有り

    Shin'ya Ohmori, Takashi Moriguchi, Yuki Noguchi, Muneharu Ikeda, Kota Kobayashi, Nazuki Tomaru, Yasushi Ishijima, Osamu Ohneda, Masayuki Yamamoto, Kinuko Ohneda

    Blood 125 (21) 3306-15 2015年5月21日

    出版者・発行元:AMER SOC HEMATOLOGY

    DOI: 10.1182/blood-2014-11-612465  

    ISSN:0006-4971

    eISSN:1528-0020

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    GATA2 plays a crucial role for the mast cell fate decision. We herein demonstrate that GATA2 is also required for the maintenance of the cellular identity in committed mast cells derived from mouse bone marrow (BMMCs). The deletion of the GATA2 DNA binding domain (GATA2ΔCF) in BMMCs resulted in a loss of the mast cell phenotype and an increase in the number of CD11b- and/or Ly6G/C-positive cells. These cells showed the ability to differentiate into macrophage- and neutrophil-like cells but not into eosinophils. Although the mRNA levels of basophil-specific genes were elevated, CD49b, a representative basophil marker, never appeared on these cells. GATA2 ablation led to a significant upregulation of C/EBPα, and forced expression of C/EBPα in wild-type BMMCs phenocopied the GATA2ΔCF cells. Interestingly, simultaneous deletion of the Gata2 and Cebpa genes in BMMCs restored the aberrant increases of CD11b and Ly6G/C while retaining the reduced c-Kit expression. Chromatin immunoprecipitation assays indicated that GATA2 directly binds to the +37-kb region of the Cebpa gene and thereby inhibits the RUNX1 and PU.1 binding to the neighboring region. Upregulation of C/EBPα following the loss of GATA2 was not observed in cultured mast cells derived from peritoneal fluid, whereas the repression of c-Kit and other mast cell-specific genes were observed in these cells. Collectively, these results indicate that GATA2 maintains cellular identity by preventing Cebpa gene activation in a subpopulation of mast cells, whereas it plays a fundamental role as a positive regulator of mast cell-specific genes throughout development of this cell lineage.

  27. The Human GATA1 Gene Retains a 5' Insulator That Maintains Chromosomal Architecture and GATA1 Expression Levels in Splenic Erythroblasts. 国際誌 査読有り

    Takashi Moriguchi, Lei Yu, Jun Takai, Makiko Hayashi, Hironori Satoh, Mikiko Suzuki, Kinuko Ohneda, Masayuki Yamamoto

    Molecular and cellular biology 35 (10) 1825-37 2015年5月

    出版者・発行元:AMER SOC MICROBIOLOGY

    DOI: 10.1128/MCB.00011-15  

    ISSN:0270-7306

    eISSN:1098-5549

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    GATA1 is a key transcription factor for erythropoiesis. GATA1 gene expression is strictly regulated at the transcriptional level. While the regulatory mechanisms governing mouse Gata1 (mGata1) gene expression have been studied extensively, how expression of the human GATA1 (hGATA1) gene is regulated remains to be elucidated. To address this issue, we generated hGATA1 bacterial artificial chromosome (BAC) transgenic mouse lines harboring a 183-kb hGATA1 locus covering the hGATA1 exons and distal flanking sequences. Transgenic hGATA1 expression coincides with endogenous mGata1 expression and fully rescues hematopoietic deficiency in mGata1 knockdown mice. The transgene exhibited copy number-dependent and integration position-independent expression of hGATA1, indicating the presence of chromatin insulator activity within the transgene. We found a novel insulator element at 29 kb 5' to the hGATA1 gene and refer to this element as the 5' CCCTC-binding factor (CTCF) site. Substitution mutation of the 5' CTCF site in the hGATA1 BAC disrupted the chromatin architecture and led to a reduction of hGATA1 expression in splenic erythroblasts under conditions of stress erythropoiesis. Our results demonstrate that expression of the hGATA1 gene is regulated through the chromatin architecture organized by 5' CTCF site-mediated intrachromosomal interactions in the hGATA1 locus.

  28. Progenitor stage-specific activity of a cis-acting double GATA motif for Gata1 gene expression. 国際誌 査読有り

    Takashi Moriguchi, Mikiko Suzuki, Lei Yu, Jun Takai, Kinuko Ohneda, Masayuki Yamamoto

    Molecular and cellular biology 35 (5) 805-15 2015年3月

    出版者・発行元:AMER SOC MICROBIOLOGY

    DOI: 10.1128/MCB.01011-14  

    ISSN:0270-7306

    eISSN:1098-5549

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    GATA1 is a master regulator of erythropoiesis, expression of which is regulated by multiple discrete cis-acting elements. In this study, we examine the activity of a promoter-proximal double GATA (dbGATA) motif, using a Gata1 bacterial artificial chromosome (BAC)-transgenic green fluorescent protein (GFP) reporter (G1BAC-GFP) mouse system. Deletion of the dbGATA motif led to significant reductions in GFP expression in hematopoietic progenitors, while GFP expression was maintained in erythroblasts. Consistently, in mice with a germ line deletion of the dbGATA motif (Gata1(ΔdbGATA) mice), GATA1 expression in progenitors was significantly decreased. The suppressed GATA1 expression was associated with a compensatory increase in GATA2 levels in progenitors. When we crossed Gata1(ΔdbGATA) mice with Gata2 hypomorphic mutant mice (Gata2(fGN/fGN) mice), the Gata1(ΔdbGATA)::Gata2(fGN/fGN) compound mutant mice succumbed to a significant decrease in the progenitor population, whereas both groups of single mutant mice maintained progenitors and survived to adulthood, indicating the functional redundancy between GATA1 and GATA2 in progenitors. Meanwhile, the effects of the dbGATA site deletion on Gata1 expression were subtle in erythroblasts, which showed increased GATA1 binding and enhanced accumulation of active histone marks around the 1st-intron GATA motif of the ΔdbGATA locus. These results thus reveal a novel role of the dbGATA motif in the maintenance of Gata1 expression in hematopoietic progenitors and a functional compensation between the dbGATA site and the 1st-intron GATA motif in erythroblasts.

  29. Hypoxia-inducible factor-3α promotes angiogenic activity of pulmonary endothelial cells by repressing the expression of the VE-cadherin gene. 国際誌 査読有り

    Kobayashi S, Yamashita T, Ohneda K, Nagano M, Kimura K, Nakai H, Poellinger L, Ohneda O

    Genes to cells : devoted to molecular & cellular mechanisms 20 (3) 224-41 2015年3月

    出版者・発行元:None

    DOI: 10.1111/gtc.12215  

    ISSN:1356-9597

    eISSN:1365-2443

  30. Transcription factor GATA1 is dispensable for mast cell differentiation in adult mice. 国際誌 査読有り

    Kinuko Ohneda, Takashi Moriguchi, Shin'ya Ohmori, Yasushi Ishijima, Hironori Satoh, Sjaak Philipsen, Masayuki Yamamoto

    Molecular and cellular biology 34 (10) 1812-26 2014年5月

    出版者・発行元:AMER SOC MICROBIOLOGY

    DOI: 10.1128/MCB.01524-13  

    ISSN:0270-7306

    eISSN:1098-5549

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    Although previous studies have shown that GATA1 is required for mast cell differentiation, the effects of the complete ablation of GATA1 in mast cells have not been examined. Using conditional Gata1 knockout mice (Gata1(-/y)), we demonstrate here that the complete ablation of GATA1 has a minimal effect on the number and distribution of peripheral tissue mast cells in adult mice. The Gata1(-/y) bone marrow cells were capable of differentiating into mast cells ex vivo. Microarray analyses showed that the repression of GATA1 in bone marrow mast cells (BMMCs) has a small impact on the mast cell-specific gene expression in most cases. Interestingly, however, the expression levels of mast cell tryptases in the mouse chromosome 17A3.3 were uniformly reduced in the GATA1 knockdown cells, and GATA1 was found to bind to a 500-bp region at the 5' end of this locus. Revealing a sharp contrast to that observed in the Gata1-null BMMCs, GATA2 deficiency resulted in a significant loss of the c-Kit(+) FcεRIα(+) mast cell fraction and a reduced expression of several mast cell-specific genes. Collectively, GATA2 plays a more important role than GATA1 in the regulation of most mast cell-specific genes, while GATA1 might play specific roles in mast cell functions.

  31. The Gata1 5' region harbors distinct cis-regulatory modules that direct gene activation in erythroid cells and gene inactivation in HSCs. 国際誌 査読有り

    Jun Takai, Takashi Moriguchi, Mikiko Suzuki, Lei Yu, Kinuko Ohneda, Masayuki Yamamoto

    Blood 122 (20) 3450-60 2013年11月14日

    DOI: 10.1182/blood-2013-01-476911  

    ISSN:0006-4971

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    GATA1 is a master regulator of hematopoietic differentiation, but Gata1 expression is inactivated in hematopoietic stem cells (HSCs). Using a bacterial artificial chromosome containing the Gata1 gene modified with green fluorescent protein (GFP) reporter, we explored the function of the 3.7-kb Gata1 upstream region (GdC region) that harbors 3 core cis-elements: Gata1 hematopoietic enhancer, double GATA-motif, and CACCC-motif. Transgenic GFP expression directed by the Gata1-BAC faithfully recapitulated the endogenous Gata1 expression pattern. However, deletion of the GdC-region eliminated reporter expression in all hematopoietic cells. To test whether the combination of the core cis-elements represents the regulatory function of the GdC-region, we replaced the region with a 659-bp minigene that linked the three cis-elements (MG-GFP). The GFP reporter expression directed by the MG-GFP BAC fully recapitulated the erythroid-megakaryocytic Gata1 expression. However, the GFP expression was aberrantly increased in the HSCs and was associated with decreases in DNA methylation and abundant GATA2 binding to the transgenic MG-GFP allele. The 3.2-kb sequences interspaced between the Gata1 hematopoietic enhancer and the double GATA-motif were able to recruit DNA methyltransferase 1, thereby exerting a cis-repressive function in the HSC-like cell line. These results indicate that the 3.2-kb interspacing sequences inactivate Gata1 by maintaining DNA-methylation in the HSCs.

  32. GATA factor switching from GATA2 to GATA1 contributes to erythroid differentiation. 国際誌 査読有り

    Mikiko Suzuki, Maki Kobayashi-Osaki, Shuichi Tsutsumi, Xiaoqing Pan, Shin'ya Ohmori, Jun Takai, Takashi Moriguchi, Osamu Ohneda, Kinuko Ohneda, Ritsuko Shimizu, Yasuharu Kanki, Tatsuhiko Kodama, Hiroyuki Aburatani, Masayuki Yamamoto

    Genes to cells : devoted to molecular & cellular mechanisms 18 (11) 921-33 2013年11月

    DOI: 10.1111/gtc.12086  

    ISSN:1356-9597

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    Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the 'GATA factor switching'. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1-knockdown embryos that concomitantly express Gata2-GFP reporter, high-level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1-binding sites widely overlap with the sites pre-occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient-phase genes.

  33. Establishment of erythroleukemic GAK14 cells and characterization of GATA1 N-terminal domain. 国際誌 査読有り

    Harumi Y Mukai, Mikiko Suzuki, Masumi Nagano, Shin'ya Ohmori, Akihito Otsuki, Kouhei Tsuchida, Takashi Moriguchi, Kinuko Ohneda, Ritsuko Shimizu, Osamu Ohneda, Masayuki Yamamoto

    Genes to cells : devoted to molecular & cellular mechanisms 18 (10) 886-98 2013年10月

    出版者・発行元:WILEY-BLACKWELL

    DOI: 10.1111/gtc.12084  

    ISSN:1356-9597

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    GATA1 is a transcription factor essential for erythropoiesis and megakaryopoiesis. It has been found that Gata1 gene knockdown heterozygous female (Gata1(G1.05/+)) mice spontaneously develop erythroblastic leukemias. In this study, we have generated a novel Gata1 knockdown erythroblastic cell line, designated GAK14, from the leukemia cells in the Gata1(G1.05/+) mice. Although GAK14 cells maintain immature phenotype on OP9 stromal cells in the presence of erythropoietin and stem cell factor, the cells produce Gr-1-, Mac1-, B220-, CD3e- or CD49b-positive hematopoietic cells when co-cultured with DAS104-8 feeder cells. However, GAK14 cells did not produce erythroid and megakaryocytic lineages, perhaps due to the absence of GATA1. Indeed, GAK14 cells became capable of differentiating into mature erythroid cells when complemented with full-length GATA1 and co-cultured with fetal liver-derived FLS5 stromal cells. This differentiation potential was impaired when GATA1 lacking the N-terminal domain was complemented. The N-terminal domain is known to contribute to the pathogenesis of transient abnormal myelopoiesis and acute megakaryoblastic leukemia related to Down syndrome. These results thus showed that GAK14 cells will serve as a powerful tool for dissecting domain function of GATA1 and that the GATA1 N-terminal domain is essential for the erythroid differentiation of GAK14 cells.

  34. GATA2はマスト細胞の分化形質の維持に必須である

    大森 慎也, 石嶋 康史, 森口 尚, 山本 雅之, 大根田 絹子

    日本生化学会大会プログラム・講演要旨集 86回 1T14a-08 2013年9月

    出版者・発行元:(公社)日本生化学会

  35. Ablation of Mina53 in mice reduces allergic response in the airways. 査読有り

    Tetsuya Mori, Kengo Okamoto, Yuji Tanaka, Kwesi Teye, Toshiyuki Umata, Kinuko Ohneda, Kenichi Tokuyama, Masaru Okabe, Makoto Tsuneoka

    Cell structure and function 38 (2) 155-67 2013年

    eISSN:1347-3700

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    The mina53 (myc-induced nuclear antigen with a 53 kDa molecular mass; also known as mina) was identified as a direct transcriptional target of the oncoprotein Myc and encodes a conserved protein in vertebrates. While Mina53 is known to be associated with tumorigenesis, it is not clear what role Mina53 plays in non-neoplastic tissues. To directly address the roles of Mina53 in non-neoplastic tissues, we created mina53-deficient mice. Both male and female mina53-deficient mice reached adulthood and were fertile, suggesting that Mina53 is dispensable for the basic developmental processes. Since we found that Mina53 was expressed in cells responsible for immune responses, we investigated whether Mina53 was involved in immune responses. When mice were exposed intranasally to house dust mites as an allergen, the airway tract showed hyperresponsiveness to methacholine in wild-type mice but not in mina53-deficient mice. The mina53-deficient mice also showed a significantly reduced migration of immune cells, including eosinophils, into bronchoalveolar lavage fluid compared with wild-type mice. The levels of Th2 cytokines, IL-4 and IL-5, produced in response to house dust mites were lower in the mina53-deficient mice than in wild-type mice. The level of IFN-γ in bronchoalveolar lavage fluid was significantly decreased by exposure to house dust mites in wild-type mice but not in the mina53-deficient mice. These results suggest that Mina53 plays a role in the allergic response to inhaled allergens, possibly through controlling IL-4 production.

  36. Mast cell deficiency results in the accumulation of preadipocytes in adipose tissue in both obese and non-obese mice. 国際誌 査読有り

    Yasushi Ishijima, Shin'ya Ohmori, Kinuko Ohneda

    FEBS open bio 4 18-24 2013年

    出版者・発行元:ELSEVIER SCIENCE LONDON

    DOI: 10.1016/j.fob.2013.11.004  

    ISSN:2211-5463

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    Mast cells have been suggested to play key roles in adipogenesis. We herein show that the expression of preadipocyte, but not adipocyte, marker genes increases in the white adipose tissue of mast cell-deficient (Kit(W-sh/W-sh) ) mice under both obese and non-obese conditions. In vitro culturing with adipogenic factors revealed increased adipocytes differentiated from the Kit(W-sh/W-sh) stromal vascular fraction, suggesting the accumulation of preadipocytes. Moreover, the increased expression of preadipocyte genes was restored by mast cell reconstitution in the Kit(W-sh/W-sh) mice. These results suggest positive effects of mast cells on the preadipocyte to adipocyte transition under both physiological and pathological conditions.

  37. Regulation of GATA factor expression is distinct between erythroid and mast cell lineages. 国際誌 査読有り

    Shin'ya Ohmori, Jun Takai, Yasushi Ishijima, Mikiko Suzuki, Takashi Moriguchi, Sjaak Philipsen, Masayuki Yamamoto, Kinuko Ohneda

    Molecular and cellular biology 32 (23) 4742-55 2012年12月

    DOI: 10.1128/MCB.00718-12  

    ISSN:0270-7306

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    The zinc finger transcription factors GATA1 and GATA2 participate in mast cell development. Although the expression of these factors is regulated in a cell lineage-specific and differentiation stage-specific manner, their regulation during mast cell development has not been clarified. Here, we show that the GATA2 mRNA level was significantly increased while GATA1 was maintained at low levels during the differentiation of mast cells derived from mouse bone marrow (BMMCs). Unlike in erythroid cells, forced expression or small interfering RNA (siRNA)-mediated knockdown of GATA1 rarely affected GATA2 expression, and vice versa, in mast cells, indicating the absence of cross-regulation between Gata1 and Gata2 genes. Chromatin immunoprecipitation assays revealed that both GATA factors bound to most of the conserved GATA sites of Gata1 and Gata2 loci in BMMCs. However, the GATA1 hematopoietic enhancer (G1HE) of the Gata1 gene, which is essential for GATA1 expression in erythroid and megakaryocytic lineages, was bound only weakly by both GATA factors in BMMCs. Furthermore, transgenic-mouse reporter assays revealed that the G1HE is not essential for reporter expression in BMMCs and peritoneal mast cells. Collectively, these results demonstrate that the expression of GATA factors in mast cells is regulated in a manner quite distinct from that in erythroid cells.

  38. GATA transcription factors are involved in IgE-dependent mast cell degranulation by enhancing the expression of phospholipase C-γ1. 国際誌 査読有り

    Ishijima Y, Ohmori S, Uenishi A, Ohneda K

    Genes to cells : devoted to molecular & cellular mechanisms 17 (4) 285-301 2012年4月

    DOI: 10.1111/j.1365-2443.2012.01588.x  

    ISSN:1356-9597

  39. Identification of human placenta-derived mesenchymal stem cells involved in re-endothelialization. 国際誌 査読有り

    Tu Cam Tran, Kenichi Kimura, Masumi Nagano, Toshiharu Yamashita, Kinuko Ohneda, Haruhiko Sugimori, Fujio Sato, Yuzuru Sakakibara, Hiromi Hamada, Hiroyuki Yoshikawa, Son Nghia Hoang, Osamu Ohneda

    Journal of cellular physiology 226 (1) 224-35 2011年1月

    出版者・発行元:WILEY-BLACKWELL

    DOI: 10.1002/jcp.22329  

    ISSN:0021-9541

    eISSN:1097-4652

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    Human placenta is an attractive source of mesenchymal stem cells (MSC) for regenerative medicine. The cell surface markers expressed on MSC have been proposed as useful tools for the isolation of MSC from other cell populations. However, the correlation between the expression of MSC markers and the ability to support tissue regeneration in vivo has not been well examined. Here, we established several MSC lines from human placenta and examined the expression of their cell surface markers and their ability to differentiate toward mesenchymal cell lineages. We found that the expression of CD349/frizzled-9, a receptor for Wnt ligands, was positive in placenta-derived MSC. So, we isolated CD349-negative and -positive fractions from an MSC line and examined how successfully cell engraftment repaired fractured bone and recovered blood flow in ischemic regions using mouse models. CD349-negative and -positive cells displayed a similar expression pattern of cell surface markers and facilitated the repair of fractured bone in transplantation experiments in mice. Interestingly, CD349-negative, but not CD349-positive cells, showed significant effects on recovering blood flow following vascular occlusion. We found that induction of PDGFβ and bFGF mRNAs by hypoxia was greater in CD349-negative cells than in CD349-positive cells while the expression of VEGF was not significantly different in CD349-negative and CD349-positive cells. These findings suggest the possibility that CD349 could be utilized as a specialized marker for MSC isolation for re-endothelialization.

  40. Hypoxia responsive mesenchymal stem cells derived from human umbilical cord blood are effective for bone repair. 国際誌 査読有り

    Masumi Nagano, Kenichi Kimura, Toshiharu Yamashita, Kinuko Ohneda, Daisuke Nozawa, Hiromi Hamada, Hiroyuki Yoshikawa, Naoyuki Ochiai, Osamu Ohneda

    Stem cells and development 19 (8) 1195-210 2010年8月

    出版者・発行元:MARY ANN LIEBERT INC

    DOI: 10.1089/scd.2009.0447  

    ISSN:1547-3287

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    Mesenchymal stem cells (MSCs) are highly useful in a variety of cell therapies owing to their multipotential differentiation capability. MSCs derived from umbilical cord blood are generally isolated by their plastic adherence without using specific cell surface markers and examined for their osteogenic, adipogenic, and chondrogenic differentiation properties retrospectively. Here, we report 2 subpopulations of MSCs, separated based on aldehyde dehydrogenase (ALDH) activity. MSCs with a high ALDH activity (Alde-High) proliferated more than those with a low ALDH activity (Alde-Low). Alde-High MSCs had a greater ability to differentiate than Alde-Low MSCs in in vitro culture. Transplantation of Alde-High MSCs into fractured mouse femurs enabled early repair of tissues and rapid bone substitution. Alde-High MSCs were also more responsive to hypoxia than Alde-Low MSCs, with the upregulation of Flt-1, CXCR4, and Angiopoietin-2. Thus, MSCs with a high ALDH activity might serve as an effective therapeutic tool for healing fractures within a short period of time.

  41. Fractionation of mature eosinophils in GATA-reporter transgenic mice. 査読有り

    Kibom Kim, Norio Suzuki, Kinuko Ohneda, Naoko Minegishi, Masayuki Yamamoto

    The Tohoku journal of experimental medicine 220 (2) 127-38 2010年2月

    DOI: 10.1620/tjem.220.127  

    eISSN:1349-3329

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    Eosinophils contribute to the pathophysiology of allergic and infectious diseases, albeit their molecular functions remain unknown. Mature eosinophils are identified by their unique morphology and staining characteristics. However, it is difficult to fractionate living eosinophils by flow cytometry because these granulocytes express multiple cell surface markers that are shared by other cells of hematopoietic or non-hematopoietic origin. In this study, we describe a flow cytometry-based method to enumerate and fractionate eosinophils by developmental stages. To fractionate these cell types, we used transgenic mouse lines that express fluorescent proteins under control of the Gata1 gene hematopoietic regulatory region (Gata1-HRD), which is exclusively active in Gata1-expressing hematopoietic cells, including eosinophils. As expected, mature eosinophils were highly enriched in the fluorescent reporter-expressing subfraction of bone marrow myeloid cells that were negatively selected by using multiple antibodies against B220, CD4, CD8, Ter119, c-Kit and CD71. Cytochemical analyses of flow-sorted cells identified the cells in this fraction as eosinophils harboring eosinophilic granules. Additionally, expression of eosinophil-specific genes, for instance eosinophil enzymes and the IL-5 receptor alpha gene, were specifically detected in this fraction. The expression of these eosinophil-specific genes increased as the cells differentiated. This method for enrichment of bone marrow eosinophils is applicable to fractionation of eosinophils and bronchoalveolar lavage fluid from transgenic mice with atopic asthma. Thus, both pathological and developmental stages of eosinophils are efficiently fractionated by this flow cytometry-based method using Gata1-HRD transgenic reporter mice. This study, therefore, proposes a useful means to study the experimental allergic and inflammatory systems.

  42. Characterization of a functional ZBP-89 binding site that mediates Gata1 gene expression during hematopoietic development. 国際誌 査読有り

    Kinuko Ohneda, Shin'ya Ohmori, Yasushi Ishijima, Mayu Nakano, Masayuki Yamamoto

    The Journal of biological chemistry 284 (44) 30187-99 2009年10月30日

    DOI: 10.1074/jbc.M109.026948  

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    GATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5' region of the enhancer. However, the importance of the GC-rich region adjacent to the 3'-end of this GATA site has been also suggested. In this study, we show that this GC-rich region contains five contiguous deoxyguanosine residues (G(5) string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G(5) string (G(4) mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89 to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89 activates the Gata1 enhancer depending on the G(5) string sequence. Finally, transgenic mouse studies reveal that the G(4) mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G(5) string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region.

  43. Differential contribution of the Gata1 gene hematopoietic enhancer to erythroid differentiation. 国際誌 査読有り

    Mikiko Suzuki, Takashi Moriguchi, Kinuko Ohneda, Masayuki Yamamoto

    Molecular and cellular biology 29 (5) 1163-75 2009年3月

    DOI: 10.1128/MCB.01572-08  

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    GATA1 is a key regulator of erythroid cell differentiation. To examine how Gata1 gene expression is regulated in a stage-specific manner, transgenic mouse lines expressing green fluorescent protein (GFP) reporter from the Gata1 locus in a bacterial artificial chromosome (G1BAC-GFP) were prepared. We found that the GFP reporter expression faithfully recapitulated Gata1 gene expression. Using GFP fluorescence in combination with hematopoietic surface markers, we established a purification protocol for two erythroid progenitor fractions, referred to as burst-forming units-erythroid cell-related erythroid progenitor (BREP) and CFU-erythroid cell-related erythroid progenitor (CREP) fractions. We examined the functions of the Gata1 gene hematopoietic enhancer (G1HE) and the highly conserved GATA box in the enhancer core. Both deletion of the G1HE and substitution mutation of the GATA box caused almost complete loss of GFP expression in the BREP fraction, but the CREP stage expression was suppressed only partially, indicating the critical contribution of the GATA box to the BREP stage expression of Gata1. Consistently, targeted deletion of G1HE from the chromosomal Gata1 locus provoked suppressed expression of the Gata1 gene in the BREP fraction, which led to aberrant accumulation of BREP stage hematopoietic progenitor cells. These results demonstrate the physiological significance of the dynamic regulation of Gata1 gene expression in a differentiation stage-specific manner.

  44. A Chemokine Receptor CXCR4 Plays a Crucial Role for Repairing Ischemic Tissue through Regulation of HIF-2a 査読有り

    Nagano, Masumi, Kimura, Ken-ichi, Yamashita, Toshiharu, Hamada, Hiromi, Ohneda, Kinuko, Yoshikawa, Hiroyuki, Ohneda, Osamu

    BLOOD 112 (11) 1336-1337 2008年11月

    出版者・発行元:AMER SOC HEMATOLOGY

    ISSN:0006-4971

  45. The microenvironment for erythropoiesis is regulated by HIF-2alpha through VCAM-1 in endothelial cells. 国際誌 査読有り

    Toshiharu Yamashita, Osamu Ohneda, Ai Sakiyama, Fumiko Iwata, Kinuko Ohneda, Yoshiaki Fujii-Kuriyama

    Blood 112 (4) 1482-92 2008年8月15日

    出版者・発行元:AMER SOC HEMATOLOGY

    DOI: 10.1182/blood-2007-11-122648  

    ISSN:0006-4971

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    Erythropoiesis is a dynamic process regulated by oxygen in vertebrates. Recent evidence has indicated that erythropoietin (Epo) expression is regulated by hypoxia-inducible transcription factors (HIFs), HIF-2alpha in particular. In this study, we report that knockdown mutation of HIF-2alpha in mice (kd/kd) results in normocytic anemia, despite Epo induction in response to hypoxia not being severely affected. Transplantation analyses clearly demonstrated that the hematopoietic microenvironment, but not the hematopoietic cells, was altered in kd/kd. Furthermore, cell-type specific recovery of HIF-2alpha expression in endothelial cells (ECs) abrogated the anemic condition of the kd/kd mice, indicating that HIF-2alpha in EC plays an essential role in supporting erythropoiesis. In the absence of HIF-2alpha, the expression of vascular adhesion molecule-1 (VCAM-1) was reduced significantly and restoration of VCAM-1 expression in kd/kd ECs enhanced the development of erythroid progenitors. Finally, a chromatin immunoprecipitation assay and a reporter assay indicated that VCAM-1 gene transcription is directly regulated by HIF-2alpha. These data suggest that the hematopoietic microenvironment required for erythropoiesis is dynamically regulated by oxygen through the functions of HIF-2alpha in ECs.

  46. Hypoxia-inducible transcription factor-2alpha in endothelial cells regulates tumor neovascularization through activation of ephrin A1. 国際誌 査読有り

    Toshiharu Yamashita, Kinuko Ohneda, Masumi Nagano, Chika Miyoshi, Naomi Kaneko, Yoshihiro Miwa, Masayuki Yamamoto, Osamu Ohneda, Yoshiaki Fujii-Kuriyama

    The Journal of biological chemistry 283 (27) 18926-36 2008年7月4日

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.M709133200  

    ISSN:0021-9258

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    The hypoxia-inducible transcription factors (HIF)-1alpha and -2alpha mediate responses to hypoxia, such as tumor neovascularization. To determine the function of HIF-2alpha in vascular endothelial cells (ECs), we examined vascular formation in HIF-2alpha knockdown (kd/kd) mice transplanted with tumors. We observed that both the tumor size and the number of large vessels growing within transplanted melanomas were significantly reduced in kd/kd recipients compared with wild-type (WT) mice. In contrast, we observed a similar extent of vascular formation within fibrosarcomas transplanted from either kd/kd or WT mice into WT recipients. Thus, HIF-2alpha expression in host animal ECs, but not in the tumor cells, is crucial for tumor neovascularization. HIF-2alpha may function through ephrin A1 as the expression of ephrin A1 and related genes was markedly reduced in kd/kd ECs, and HIF-2alpha specifically bound a hypoxia-response element sequence in the ephrin A1 promoter. Treatment of WT ECs with an ephrin A1 inhibitor (ephrin A1-Fc) also impaired neovascularization. We conclude that in ECs, HIF-2alpha plays an essential role in vascular remodeling during tumor vascularization through activation of at least ephrin A1.

  47. Ablation of Gata1 in adult mice results in aplastic crisis, revealing its essential role in steady-state and stress erythropoiesis. 国際誌 査読有り

    Laura Gutiérrez, Saho Tsukamoto, Mikiko Suzuki, Harumi Yamamoto-Mukai, Masayuki Yamamoto, Sjaak Philipsen, Kinuko Ohneda

    Blood 111 (8) 4375-85 2008年4月15日

    DOI: 10.1182/blood-2007-09-115121  

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    The transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon- or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre-mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cre-mediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human.

  48. Abnormal heart development and lung remodeling in mice lacking the hypoxia-inducible factor-related basic helix-loop-helix PAS protein NEPAS. 国際誌 査読有り

    Toshiharu Yamashita, Osamu Ohneda, Masumi Nagano, Motoyuki Iemitsu, Yuichi Makino, Hirotoshi Tanaka, Takashi Miyauchi, Katsutoshi Goto, Kinuko Ohneda, Yoshiaki Fujii-Kuriyama, Lorenz Poellinger, Masayuki Yamamoto

    Molecular and cellular biology 28 (4) 1285-97 2008年2月

    DOI: 10.1128/MCB.01332-07  

    eISSN:1098-5549

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    Hypoxia-inducible factors (HIFs) are crucial for oxygen homeostasis during both embryonic development and postnatal life. Here we show that a novel HIF family basic helix-loop-helix (bHLH) PAS (Per-Arnt-Sim) protein, which is expressed predominantly during embryonic and neonatal stages and thereby designated NEPAS (neonatal and embryonic PAS), acts as a negative regulator of HIF-mediated gene expression. NEPAS mRNA is derived from the HIF-3alpha gene by alternative splicing, replacing the first exon of HIF-3alpha with that of inhibitory PAS. NEPAS can dimerize with Arnt and exhibits only low levels of transcriptional activity, similar to that of HIF-3alpha. NEPAS suppressed reporter gene expression driven by HIF-1alpha and HIF-2alpha. By generating mice with a targeted disruption of the NEPAS/HIF-3alpha locus, we found that homozygous mutant mice (NEPAS/HIF-3alpha(-)(/)(-)) were viable but displayed enlargement of the right ventricle and impaired lung remodeling. The expression of endothelin 1 and platelet-derived growth factor beta was increased in the lung endothelial cells of NEPAS/HIF-3alpha-null mice. These results demonstrate a novel regulatory mechanism in which the activities of HIF-1alpha and HIF-2alpha are negatively regulated by NEPAS in endothelial cells, which is pertinent to lung and heart development during the embryonic and neonatal stages.

  49. Identification of functional endothelial progenitor cells suitable for the treatment of ischemic tissue using human umbilical cord blood. 国際誌 査読有り

    Masumi Nagano, Toshiharu Yamashita, Hiromi Hamada, Kinuko Ohneda, Ken-ichi Kimura, Tomoki Nakagawa, Masabumi Shibuya, Hiroyuki Yoshikawa, Osamu Ohneda

    Blood 110 (1) 151-60 2007年7月1日

    出版者・発行元:AMER SOC HEMATOLOGY

    DOI: 10.1182/blood-2006-10-047092  

    ISSN:0006-4971

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    Umbilical cord blood (UCB) has been used as a potential source of various kinds of stem cells, including hematopoietic stem cells, mesenchymal stem cells, and endothelial progenitor cells (EPCs), for a variety of cell therapies. Recently, EPCs were introduced for restoring vascularization in ischemic tissues. An appropriate procedure for isolating EPCs from UCB is a key issue for improving therapeutic efficacy and eliminating the unexpected expansion of nonessential cells. Here we report a novel method for isolating EPCs from UCB by a combination of negative immunoselection and cell culture techniques. In addition, we divided EPCs into 2 subpopulations according to the aldehyde dehydrogenase (ALDH) activity. We found that EPCs with low ALDH activity (Alde-Low) possess a greater ability to proliferate and migrate compared to those with high ALDH activity (Alde-High). Moreover, hypoxia-inducible factor proteins are up-regulated and VEGF, CXCR4, and GLUT-1 mRNAs are increased in Alde-Low EPCs under hypoxic conditions, while the response was not significant in Alde-High EPCs. In fact, the introduction of Alde-Low EPCs significantly reduced tissue damage in ischemia in a mouse flap model. Thus, the introduction of Alde-Low EPCs may be a potential strategy for inducing rapid neovascularization and subsequent regeneration of ischemic tissues.

  50. Dynamic regulation of Gata factor levels is more important than their identity. 国際誌 査読有り

    Rita Ferreira, Albert Wai, Ritsuko Shimizu, Nynke Gillemans, Robbert Rottier, Marieke von Lindern, Kinuko Ohneda, Frank Grosveld, Masayuki Yamamoto, Sjaak Philipsen

    Blood 109 (12) 5481-90 2007年6月15日

    DOI: 10.1182/blood-2006-11-060491  

    ISSN:0006-4971

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    Three Gata transcription factors (Gata1, -2, and -3) are essential for hematopoiesis. These factors are thought to play distinct roles because they do not functionally replace each other. For instance, Gata2 messenger RNA (mRNA) expression is highly elevated in Gata1-null erythroid cells, yet this does not rescue the defect. Here, we test whether Gata2 and -3 transgenes rescue the erythroid defect of Gata1-null mice, if expressed in the appropriate spatiotemporal pattern. Gata1, -2, and -3 transgenes driven by beta-globin regulatory elements, directing expression to late stages of differentiation, fail to rescue erythropoiesis in Gata1-null mutants. In contrast, when controlled by Gata1 regulatory elements, directing expression to the early stages of differentiation, Gata1, -2, and -3 do rescue the Gata1-null phenotype. The dramatic increase of endogenous Gata2 mRNA in Gata1-null progenitors is not reflected in Gata2 protein levels, invoking translational regulation. Our data show that the dynamic spatiotemporal regulation of Gata factor levels is more important than their identity and provide a paradigm for developmental control mechanisms that are hard-wired in cis-regulatory elements.

  51. GATA-1 self-association controls erythroid development in vivo. 国際誌 査読有り

    Ritsuko Shimizu, Cecelia D Trainor, Keizo Nishikawa, Makoto Kobayashi, Kinuko Ohneda, Masayuki Yamamoto

    The Journal of biological chemistry 282 (21) 15862-71 2007年5月25日

    DOI: 10.1074/jbc.M701936200  

    ISSN:0021-9258

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    GATA-1 is the key transcription factor for the development of the erythroid, megakaryocytic, eosinophilic, and mast cell lineages. GATA-1 possesses the ability to self-associate, and this characteristic has been suggested to be important for GATA-1 function. To elucidate the roles self-associated GATA-1 plays during hematopoietic cell development in vivo, in this study we prepared GATA-1 mutants in which three lysine residues potentially contributing to the self-association (Lys-245, Lys-246, and Lys-312) are substituted in combination with alanines. Of the mutants, 3KA harboring alanine substitutions in all three lysines showed reduced self-association activity without considerable interference in the modification of GATA-1 by acetylation. We generated transgenic mouse lines that express these GATA-1 mutants utilizing the Gata1 hematopoietic regulatory domain, and crossed the mice to Gata1 knockdown (GATA-1.05) mutant mice. Although NKA (K245A and K246A) and CKA (K312A) mutants almost fully rescued the GATA-1.05 mice from anemia and embryonic lethality, the 3KA mutant only partially rescued the GATA-1.05 mutant mice. Even with the higher than endogenous level expression, GATA-1.05/Y::3KA embryos were prone to die at various stages in mid-to-late gestation. Live birth and an anemic phenotype were restored in some embryos depending on the expression level of the 3KA transgene. The expression of the transferrin receptor and heme biosynthesis enzymes was impaired in the yolk sac and liver of the 3KA-rescued embryos. Immature erythroid cells with insufficient expression of the transferrin receptor accumulated in the livers of 3KA-rescued embryos. These results provide the first convincing line of evidence that the self-association of GATA-1 is important for proper mammalian erythroid development in vivo.

  52. GATA-4 incompletely substitutes for GATA-1 in promoting both primitive and definitive erythropoiesis in vivo. 国際誌 査読有り

    Sakie Hosoya-Ohmura, Naomi Mochizuki, Mikiko Suzuki, Osamu Ohneda, Kinuko Ohneda, Masayuki Yamamoto

    The Journal of biological chemistry 281 (43) 32820-30 2006年10月27日

    DOI: 10.1074/jbc.M605735200  

    ISSN:0021-9258

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    Vertebrate GATA transcription factors have been classified into two subgroups; GATA-1, GATA-2, and GATA-3 are expressed in hematopoietic cells, whereas GATA-4, GATA-5, and GATA-6 are expressed in mesoendoderm-derived tissues. We previously discovered that expression of GATA-2 or GATA-3 under the transcriptional control for the Gata1 gene eliminates lethal anemia in Gata1 germ line mutant mice (Gata1.05/Y). Here, we show that the GATA-4 expression by the same regulatory cassette prolongs the life span of Gata1.05/Y embryos from embryonic day 12.5 to 15.5 but fails to abrogate its embryonic lethality. Gata1.05/Y mice bearing the GATA-4 transgene showed impaired maturation of both primitive and definitive erythroid cells and defective erythroid cell expansion in fetal liver. Moreover, the incidence of apoptosis was observed prominently in primitive erythroid cells. In contrast, a GATA-4-GATA-1 chimeric protein prepared by linking the N-terminal region of GATA-4 to the C-terminal region of GATA-1 significantly promoted the differentiation and survival of primitive erythroid cells, although this protein is still insufficient for rescuing Gata1.05/Y embryos from lethal anemia. These data thus show a functional incompatibility between hematopoietic and endodermal GATA factors in vivo and provide evidence indicating specific roles of the C-terminal region of GATA-1 in primitive erythropoiesis.

  53. Real-time monitoring of stress erythropoiesis in vivo using Gata1 and beta-globin LCR luciferase transgenic mice. 国際誌 査読有り

    Mikiko Suzuki, Kinuko Ohneda, Sakie Hosoya-Ohmura, Saho Tsukamoto, Osamu Ohneda, Sjaak Philipsen, Masayuki Yamamoto

    Blood 108 (2) 726-33 2006年7月15日

    DOI: 10.1182/blood-2005-10-4064  

    ISSN:0006-4971

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    Erythroid progenitors have the potential to proliferate rapidly in response to environmental stimuli. This process is referred to as stress erythropoiesis, with erythropoietin (EPO) playing central roles in its promotion. In this study, we wanted to elucidate the molecular mechanisms governing the regulation of stress erythropoiesis and the maintenance of red-cell homeostasis. This was achieved by our development of a noninvasive real-time monitoring system for erythropoiesis using transgenic mouse lines expressing luciferase under the control of the mouse Gata1 hematopoietic regulatory domain (G1-HRD-luc) or human beta-globin locus control region (Hbb-LCR-luc). Optical bioluminescence images revealed that the luciferase was specifically expressed in spleen and bone marrow and was induced rapidly in response to anemia and hypoxia stimuli. The G1-HRD-luc activity tracked the emergence and disappearance of proerythroblast-stage progenitors, whereas the Hbb-LCR-luc activity tracked erythroblasts and later stage erythroid cells. Increased plasma EPO concentration preceded an increase in G1-HRD-luc, supporting our contention that EPO acts as the key upstream signal in stress erythropoiesis. Hence, we conclude that G1-HRD-luc and Hbb-LCR-luc reporters are differentially activated during stress erythropoiesis and that the transgenic mouse lines used serve as an important means for understanding the homeostatic regulation of erythropoiesis.

  54. Graded levels of GATA-1 expression modulate survival, proliferation, and differentiation of erythroid progenitors. 国際誌 査読有り

    Xiaoqing Pan, Osamu Ohneda, Kinuko Ohneda, Fokke Lindeboom, Fumiko Iwata, Ritsuko Shimizu, Masumi Nagano, Naruyoshi Suwabe, Sjaak Philipsen, Kim-Chew Lim, James D Engel, Masayuki Yamamoto

    The Journal of biological chemistry 280 (23) 22385-94 2005年6月10日

    DOI: 10.1074/jbc.M500081200  

    ISSN:0021-9258

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    Transcription factor GATA-1 plays an important role in gene regulation during the development of erythroid cells. Several reports suggest that GATA-1 plays multiple roles in survival, proliferation, and differentiation of erythroid cells. However, little is known about the relationship between the level of GATA-1 expression and its nature of multifunction to affect erythroid cell fate. To address this issue, we developed in vitro embryonic stem (ES) culture system by using OP9 stromal cells (OP9/ES cell co-culture system), and cultured the mutant (GATA-1.05 and GATA-1-null) and wild type (WT)ES cells, respectively. By using this OP9/ES cell co-culture system, primitive and definitive erythroid cells were developed individually, and we examined how expression level of GATA-1 affects the development of erythroid cells. GATA-1.05 ES-derived definitive erythroid cells were immature with the appearance of proerythroblasts, and highly proliferated, compared with WT and GATA-1-null ES-derived erythroid cells. Extensive studies of cell cycle kinetics revealed that the GATA-1.05 proerythroblasts accumulated in S phase and expressed lower levels of p16(INK4A) than WT ES cell-derived proerythroblasts. We concluded that GATA-1 must achieve a critical threshold activity to achieve selective activation of specific target genes, thereby influencing the developmental decision of an erythroid progenitor cell to undergo apoptosis, proliferation, or terminal differentiation.

  55. [Flies, fishes and frogs turn the tide of hematopoietic research]. 査読有り

    Kinuko Ohneda, Masayuki Yamamoto

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 50 (6 Suppl) 633-7 2005年5月

    ISSN:0039-9450

  56. Essential role of synoviolin in embryogenesis. 国際誌 査読有り

    Naoko Yagishita, Kinuko Ohneda, Tetsuya Amano, Satoshi Yamasaki, Akiko Sugiura, Kaneyuki Tsuchimochi, Hiroshi Shin, Ko-Ichi Kawahara, Osamu Ohneda, Tomohiko Ohta, Sakae Tanaka, Masayuki Yamamoto, Ikuro Maruyama, Kusuki Nishioka, Akiyoshi Fukamizu, Toshihiro Nakajima

    The Journal of biological chemistry 280 (9) 7909-16 2005年3月4日

    DOI: 10.1074/jbc.M410863200  

    ISSN:0021-9258

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    We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno(-/-)) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno(-/-) fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno(-/-) embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno(-/-) mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno(+/+) mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.

  57. Essential role of synoviolin in embryonic hematopoiesis. 査読有り

    Yagishita, N, Amano, T, Yamasaki, S, Tsuchimochi, K, Shin, H, Kawahara, K, Ohneda, K, Ohneda, O, Yamamoto, M, Nishioka, K, Maruyama, I, Fukamizu, A.Nakajima T

    Journal of Biological Chemistry (280) 7909 7916 2005年1月

  58. Transgenic over-expression of GATA-1 mutant lacking N-finger domain causes hemolytic syndrome in mouse erythroid cells. 国際誌 査読有り

    Mayu Nakano, Kinuko Ohneda, Harumi Yamamoto-Mukai, Ritsuko Shimizu, Osamu Ohneda, Sakie Ohmura, Mikiko Suzuki, Saho Tsukamoto, Toru Yanagawa, Hiroshi Yoshida, Yuichi Takakuwa, Masayuki Yamamoto

    Genes to cells : devoted to molecular & cellular mechanisms 10 (1) 47-62 2005年1月

    DOI: 10.1111/j.1365-2443.2005.00814.x  

    ISSN:1356-9597

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    Transcription factor GATA-1 is essential for erythroid cell differentiation. GATA-binding motifs have been found in the regulatory regions of various erythroid-specific genes, suggesting that GATA-1 contributes to gene regulation during the entire process of erythropoiesis. A GATA-1 germ-line mutation results in embryonic lethality due to defective primitive erythropoiesis and GATA-1-null embryonic stem cells fails to differentiate beyond the proerythroblast stage. Therefore, the precise roles of GATA-1 in the later stages of erythropoiesis could not be clarified. Under the control of a GATA-1 gene hematopoietic regulatory domain, a GATA-1 mutant lacking the N-finger domain (DeltaNF mutant) was over-expressed in mice. These mice exhibited abnormal morphology in peripheral red blood cells (RBCs), reticulocytosis, splenomegaly, and erythroid hyperplasia, indicating compensated hemolysis. These mice were extremely sensitive to phenylhydrazine (PHZ), an agent that induces hemolysis, and their RBCs were osmotically fragile. Importantly, the hemolytic response to PHZ was partially restored by the simultaneous expression of wild-type GATA-1 with the DeltaNF mutant, supporting our contention that DeltaNF protein competitively inhibits the function of endogenous GATA-1. These data provide the first in vivo evidence that the NF domain contributes to the gene regulation that is critical for differentiation and survival of mature RBCs in postnatal erythropoiesis.

  59. Leukemogenesis caused by incapacitated GATA-1 function. 国際誌 査読有り

    Ritsuko Shimizu, Takashi Kuroha, Osamu Ohneda, Xiaoqing Pan, Kinuko Ohneda, Satoru Takahashi, Sjaak Philipsen, Masayuki Yamamoto

    Molecular and cellular biology 24 (24) 10814-25 2004年12月

    DOI: 10.1128/MCB.24.24.10814-10825.2004  

    ISSN:0270-7306

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    GATA-1 is essential for the development of erythroid and megakaryocytic lineages. We found that GATA-1 gene knockdown female (GATA-1.05/X) mice frequently develop a hematopoietic disorder resembling myelodysplastic syndrome that is characterized by the accumulation of progenitors expressing low levels of GATA-1. In this study, we demonstrate that GATA-1.05/X mice suffer from two distinct types of acute leukemia, an early-onset c-Kit-positive nonlymphoid leukemia and a late-onset B-lymphocytic leukemia. Since GATA-1 is an X chromosome gene, two types of hematopoietic cells reside within heterozygous GATA-1 knockdown mice, bearing either an active wild-type GATA-1 allele or an active mutant GATA-1.05 allele. In the hematopoietic progenitors with the latter allele, low-level GATA-1 expression is sufficient to support survival and proliferation but not differentiation, leading to the accumulation of progenitors that are easily targeted by oncogenic stimuli. Since such leukemia has not been observed in GATA-1-null/X mutant mice, we conclude that the residual GATA-1 activity in the knockdown mice contributes to the development of the malignancy. This de novo model recapitulates the acute crisis found in preleukemic conditions in humans.

  60. Transgenic rescue of GATA-1 deficient mice with GATA-1 lacking a FOG-1 association site phenocopies patients with X-linked thrombocytopenia 招待有り 査読有り

    SHIMIZU R

    Blood 103 (7) 2560-2567 2004年4月1日

    DOI: 10.1182/blood-2003-07-2514  

  61. GATA-1 testis activation region is essential for Sertoli cell-specific expression of GATA-1 gene in transgenic mouse 招待有り 査読有り

    WAKABAYASHI J.

    Genes Cells 8 (7) 619-630 2003年7月

    DOI: 10.1046/j.1365-2443.2003.00658.x  

  62. A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo 査読有り

    OHNEDA K.

    Genes Cells 7 (12) 1243-1254 2002年12月

    DOI: 10.1046/j.1365-2443.2002.00595.x  

  63. Reduction of spontaneous and irradiation-induced apoptosis in small intestine of IGF-I transgenic mice. 国際誌 査読有り

    Heather R Wilkins, Kinuko Ohneda, Temitope O Keku, A Joseph D'Ercole, C Randall Fuller, Kristen L Williams, P Kay Lund

    American journal of physiology. Gastrointestinal and liver physiology 283 (2) G457-64-G464 2002年8月

    出版者・発行元:AMER PHYSIOLOGICAL SOC

    DOI: 10.1152/ajpgi.00019.2002  

    ISSN:0193-1857

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    Insulin-like growth factor I (IGF-I) may promote survival of putative stem cells in the small intestinal epithelium. Mitosis and apoptosis were quantified in crypts of nonirradiated and irradiated IGF-I transgenic (TG) and wild-type (WT) littermates. The mean apoptotic index was significantly greater in WT vs. TG littermates. After irradiation, apoptotic indexes increased, and WT mice showed a more dramatic increase in apoptosis than TG mice at the location of putative stem cells. After irradiation, no mitotic figures were observed in WT crypts, whereas mitosis was maintained within the jejunal epithelium of TG mice. The abundance and localization of Bax mRNA did not differ between nonirradiated littermates. However, there was more Bax mRNA in TG vs. WT mice after irradiation. Bax mRNA was located along the entire length of the irradiated crypt epithelium, but there was less Bax protein observed in the bottom third of TG mouse crypts compared with WT littermates. IGF-I regulates cell number by stimulating crypt cell proliferation and decreasing apoptosis preferentially within the stem cell compartment.

  64. Roles of hematopoietic transcription factors GATA-1 and GATA-2 in the development of red blood cell lineage 査読有り

    K Ohneda, M Yamamoto

    ACTA HAEMATOLOGICA 108 (4) 237-245 2002年

    出版者・発行元:KARGER

    DOI: 10.1159/000065660  

    ISSN:0001-5792

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    The transcription factors GATA-1 and GATA-2 play key roles in gene regulation during erythropoiesis. Gene ablation studies in mouse revealed that GATA-2 is crucial for the maintenance and proliferation of immature hematopoietic progenitors, whereas GATA-1 is essential for the survival of erythroid progenitors as well as the terminal differentiation of erythroid cells. Both GATA-1 and GATA-2 are regulated in a cell-type-specific manner, their expression being strictly controlled during the development and differentiation of erythroid cells. Closer examination revealed a cross-regulatory mechanism by which GATA-1 can control the expression of GATA-2 and vice versa, possibly via essential GATA binding sites in their cis-acting elements. In addition, recent studies identified several human inherited hematopoietic disorders that are caused by mutations in cis-acting GATA binding motifs or mutations in GATA-1 itself. Copyright (C) 2002 S. Karger AG, Basel.

  65. ALCAM (CD166): its role in hematopoietic and endothelial development 査読有り

    O Ohneda, K Ohneda, F Arai, J Lee, T Miyamoto, Y Fukushima, D Dowbenko, LA Lasky, T Suda

    BLOOD 98 (7) 2134-2142 2001年10月

    出版者・発行元:AMER SOC HEMATOLOGY

    ISSN:0006-4971

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    A critical role for the endothelium of yolk sac and dorsal aorta has been shown in embryonic hematopoiesis. A stromal cell line derived from yolk sac, YSCL-72, has been chosen to search for a novel molecule associated with embryonic hematopoiesis. Analysis between YSCL-72 and an adult aorta-derived endothelial cell line, EOMA, demonstrated that activated leukocyte cell adhesion molecule (ALCAM, or CD166) was specifically expressed in YSCL-72 but not in EOMA. Immunohistochemical study showed that ALCAM was expressed In the endothelium of yolk sac and dorsal aorta but not In adult aorta. ALCAM-transfected EOMA cells supported development of hematopoietic progenitor cells compared with vector-transfected EOMA cells, suggesting that ALCAM appeared to be crucial for hematopoiesis. In addition, ALCAM was found to be involved in capillary tube formation and hemangioblast differentiation. Taken together with these findings, ALCAM is highly associated not only with embryonic hematopoiesis but also vasculoangiogenesis. (C) 2001 by The American Society of Hematology.

  66. In vivo requirements for GATA-1 functional domains during primitive and definitive erythropoiesis 査読有り

    SHIMIZU R.

    EMBO J. 20 (18) 5250-5260 2001年9月17日

    DOI: 10.1093/emboj/20.18.5250  

  67. 成体での血管新生におけるHLFの役割

    守田 匡伸, 高橋 智, 中島 修, 大根田 絹子, 山下 年晴, 依馬 正次, 柴原 茂樹, 鵜殿 徹男, 富田 浩史, 玉井 信

    生化学 73 (8) 918-918 2001年8月

    出版者・発行元:(公社)日本生化学会

    ISSN:0037-1017

  68. The homeodomain of PDX-1 mediates multiple protein-protein interactions in the formation of a transcriptional activation complex on the insulin promoter 査読有り

    K Ohneda, RG Mirmira, JH Wang, JD Johnson, MS German

    MOLECULAR AND CELLULAR BIOLOGY 20 (3) 900-911 2000年2月

    出版者・発行元:AMER SOC MICROBIOLOGY

    ISSN:0270-7306

    詳細を見る 詳細を閉じる

    Activation of insulin gene transcription specifically in the pancreatic beta cells depends on multiple nuclear proteins that interact with each other and with sequences on the insulin gene promoter to build a transcriptional activation complex. The homeodomain protein PDX-1 exemplifies such interactions by binding to the A3/4 region of the rat insulin I promoter and activating insulin gene transcription by cooperating with the basic-helix-loop-helix (bHLH) protein E47/Pan1, which binds to the adjacent E2 site. The present study provides evidence that the homeodomain of PDX-1 acts as a protein-protein interaction domain to recruit multiple proteins, including E47/Pan1, BETA2/NeuroD1, and high-mobility group protein I(Y), to an activation complex on the E2A3/4 minienhancer, The transcriptional activity of this complex results from the clustering of multiple activation domains capable of interacting with coactivators and the basal transcriptional machinery. These interactions are not common to all homeodomain proteins: the LIM homeodomain protein Lmx1.1 can also activate the E2A3/4 minienhancer in cooperation with E47/Pan1 but does so through different interactions. Cooperation between Lmx1.1 and E47/Pan1 results not only in the aggregation of multiple activation domains but also in the unmasking of a potent activation domain on E47/Pan1 that is normally silent in non-beta cells. While more than one activation complex may be capable of activating insulin gene transcription through the E2A3/4 minienhancer, each is dependent on multiple specific interactions among a unique set of nuclear proteins.

  69. WECHE: A novel hematopoietic regulatory factor

    O Ohneda, K Ohneda, H Nomiyama, Z Zheng, SA Gold, F Arai, T Miyamoto, BE Taillon, RA McIndoe, RA Shimkets, DA Lewin, T Suda, LA Lasky

    IMMUNITY 12 (2) 141-150 2000年2月

    出版者・発行元:CELL PRESS

    ISSN:1074-7613

    詳細を見る 詳細を閉じる

    Previously, we described AGM-derived endothelial cell lines that either inhibited or permitted the development of erythroid or B cells. We utilized a differential gene expression method to isolate a chemokine, termed WECHE, from one of these cell lines. WECHE inhibited the formation of erythroid cells but had no effect on either myeloid or B cell formation. WECHE repressed BFU-E development from either mouse fetal liver or bone marrow progenitor cells but had no effect on colony formation induced by IL-3 or IL-7. WECHE reduced HPP-CFC production from fetal liver-derived stem cells. WECHE hindered the growth of yolk sac-derived endothelial cells. WECHE was also chemotactic for bone marrow cells. Thus, WECHE is a novel chemokine that regulates hematopoietic differentiation.

  70. WECHE: A novel hematopoietic regulatory factor

    Osamu Ohneda, Kinuko Ohneda, Hisayuki Nomiyama, Zhong Zheng, Steven A. Gold, Fumio Arai, Takeshi Miyamoto, Bruce E. Taillon, Richard A. McIndoe, Richard A. Shimkets, David A. Lewin, Toshio Suda, Laurence A. Lasky

    Journal of Cultural Heritage 1 141-150 2000年1月1日

    ISSN:1296-2074

    詳細を見る 詳細を閉じる

    Previously, we described AGM-derived endothelial cell lines that either inhibited or permitted the development of erythroid or B cells. We utilized a differential gene expression method to isolate a chemokine, termed WECHE, from one of these cell lines. WECHE inhibited the formation of erythroid cells but had no effect on either myeloid or B cell formation. WECHE repressed BFU-E development from either mouse fetal liver or bone marrow progenitor cells but had no effect on colony formation induced by IL-3 or IL-7. WECHE reduced HPP-CFC production from fetal liver-derived stem cells. WECHE hindered the growth of yolk sac-derived endothelial cells. WECHE was also chemotactic for bone marrow cells. Thus, WECHE is a novel chemokine that regulates hematopoietic differentiation.

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MISC 19

  1. ゲノムコホート調査におけるゲノム薬理学(PGx)遺伝情報返却(回付)のパイロット研究

    濱中 洋平, 大根田 絹子, 布施 昇男, 川目 裕, 長神 風二, 平塚 真弘, 宇留野 晃, 櫻井 美佳, 平良 摩紀子, 鈴木 吉也, 鈴木 洋一, 山本 雅之

    日本遺伝カウンセリング学会誌 41 (2) 122-122 2020年6月

    出版者・発行元:(一社)日本遺伝カウンセリング学会

    ISSN:1347-9628

  2. GATA2によるマスト細胞の分化と維持 (特集 マスト細胞の分化と機能の制御)

    大森 慎也, 大根田 絹子

    臨床免疫・アレルギー科 = Clinical immunology & allergology 64 (4) 352-359 2015年10月

    出版者・発行元:科学評論社

    ISSN:1881-1930

  3. GATA2がCebpaの転写を抑制することが骨髄マスト細胞の分化形質維持に必要である

    大森慎也, 登丸菜月, 石嶋康史, 森口尚, 山本雅之, 大根田絹子

    日本分子生物学会年会プログラム・要旨集(Web) 37th 3P-0588 (WEB ONLY) 2014年

  4. BACトランスジェニックマウスを用いた血球分化マスター転写因子GATA1の遺伝子発現制御機構の解析

    高井 淳, 森口 尚, 鈴木 未来子, 大根田 絹子, 山本 雅之

    生化学 85 (8) 720-720 2013年8月

    出版者・発行元:(公社)日本生化学会

    ISSN:0037-1017

    eISSN:2189-0544

  5. GATA1は骨髄マスト細胞(BMMCs)の増殖と生存に必要である

    植栗 幸洋, 大森 慎也, 石嶋 康史, 平林 優奈, 森口 尚, フィリップセン・シャック, 山本 雅之, 大根田 絹子

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 2P-0545 2010年12月

    出版者・発行元:(公社)日本生化学会

  6. Gata1遺伝子発現制御機構のBACトランスジェニックマウスを用いた解析

    高井淳, 森口尚, 鈴木未来子, 大根田絹子, 山本雅之

    生化学 83回・33回 2P-0548 2010年12月

    出版者・発行元:(公社)日本生化学会

    ISSN:0037-1017

  7. 成体Gata1欠損マウスにおける赤血球分化異常

    大森 慎也, 大根田 絹子

    血液・腫瘍科 57 (6) 658-663 2008年12月

    出版者・発行元:科学評論社

    ISSN:0915-8529

  8. BACトランスジェニックマウスを用いたGata1遺伝子発現制御機構の解析

    高井淳, 森口尚, 鈴木美来子, 大根田絹子, 山本雅之

    生化学 81回・31回 2T3-11 2008年11月

    出版者・発行元:(公社)日本生化学会

    ISSN:0037-1017

  9. Gata1遺伝子発現制御領域の血球分化段階特異的な機能的貢献

    森口尚, 鈴木未来子, 高井淳, 大根田絹子, 山本雅之

    生化学 2008年

    ISSN:0037-1017

  10. G1HE-core contributes to the stage-specific expression of the Gata1 gene during erythroid development

    Mikiko Suzuki, Kinuko Ohneda, Masayuki Yamamoto

    BLOOD CELLS MOLECULES AND DISEASES 38 (2) 180-180 2007年3月

    出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE

    DOI: 10.1016/j.bcmd.2006.10.135  

    ISSN:1079-9796

  11. Dynamic regulation of gata factor levels is more important than their identity

    Ferreira Rita, Wai Albert, Shimizu Ritsuko, Gillemans Nynke, Rottier Robbert, von Lindern Marieke, Ohneda Kinuko, Grosveld Frank, Yamamoto Masayuki, Philipsen Sjaak

    BLOOD CELLS MOLECULES AND DISEASES 38 (2) 171-172 2007年3月

    DOI: 10.1016/j.bcmd.2006.10.117  

    ISSN:1079-9796

  12. モデル生物が切り開く造血発生研究の新展開 (発生システムのダイナミクス) -- (組織・器官形成)

    大根田 絹子, 山本 雅之

    蛋白質核酸酵素 50 (6) 633-637,495 2005年5月

    出版者・発行元:共立出版

    ISSN:0039-9450

  13. Self-association of GATA-1 is required for erythropoiesis in vivo.

    R Shimizu, K Ohneda, K Nishikawa, CD Trainor, M Yamamoto

    BLOOD CELLS MOLECULES AND DISEASES 34 (2) 122-122 2005年3月

    出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE

    ISSN:1079-9796

  14. GATA1 function, a paradigm for transcription factors in hematopoiesis. 国際誌 査読有り

    Rita Ferreira, Kinuko Ohneda, Masayuki Yamamoto, Sjaak Philipsen

    Molecular and cellular biology 25 (4) 1215-27 2005年2月

    出版者・発行元:AMER SOC MICROBIOLOGY

    DOI: 10.1128/MCB.25.4.1215-1227.2005  

    ISSN:0270-7306

  15. A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo.

    K Ohneda, R Shimizu, S Takahashi, JD Enge, M Yamamoto

    BLOOD CELLS MOLECULES AND DISEASES 31 (1) 157-157 2003年7月

    出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE

    ISSN:1079-9796

  16. mafAノックアートマウスの解析

    ZHANG C, 森口尚, 梶原美和子, 大石久史, 浜田理人, 森戸直記, 大根田絹子, DOUGLAS ENGEL J, 山本雅之

    日本分子生物学会年会プログラム・講演要旨集 26th 2003年

  17. Chronic overexpression of IGF-I and inhibition of spontaneous and irradiation-induced apoptosis in the mouse jejunum

    HR Wilkins, K Ohneda, KL Williams, CR Fuller, AJ D'Ercole, PK Lund

    GASTROENTEROLOGY 122 (4) A371-A371 2002年4月

    出版者・発行元:W B SAUNDERS CO

    ISSN:0016-5085

  18. Regulation of insulin gene transcription

    OHNEDA K.

    Sem Cell Dev Biol 11 227-233 2000年

  19. Increased expression of nucleoside diphosphate kinases/nm23 in human diploid fibroblasts transformed by SV40 large T antigen or 60Co irradiation

    Kinuko Ohneda, Mitsugu Fukuda, Nobuko Shimada, Naoshi Ishikawa, Tateo Ichou, Kazuhiko Kaji, Takayoshi Toyota, Narimichi Kimura

    FEBS Letters 348 (3) 273-277 1994年7月18日

    DOI: 10.1016/0014-5793(94)00623-7  

    ISSN:0014-5793

    詳細を見る 詳細を閉じる

    When the expression levels of nucleoside diphosphate (NDP) kinase/nm23 were examined in four human normal diploid fibroblast cell lines in comparison with their corresponding immortalized cells transformed by SV40 large T antigen or 60Co irradiation, mRNA levels of the two isoforms (NDP kinase A/nm23-H1, NDP kinase B/nm23-H2) were increased in the immortalized cell lines. The increase was found to be associated with increased translation products. Furthermore, the cell extracts prepared from these immortalized cell lines demonstrated slightly higher enzyme activity than those from their normal counterparts. Neither the growth state nor the in vitro aging largely affected their expression in a normal cell line (TIG-3) examined. The results suggest possible involvement of NDP kinases/nm23 in acquiring an infinite growth property of these cells. © 1994.

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共同研究・競争的資金等の研究課題 12

  1. マスト細胞におけるGata2遺伝子高度活性化の分子機序の解明

    大根田 絹子, 大森 慎也

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (C)

    研究機関:Tohoku University

    2021年4月1日 ~ 2024年3月31日

    詳細を見る 詳細を閉じる

    GATA2は、多くのマスト細胞特異的な遺伝子の発現を制御し、マスト細胞の分化形質維持に必須の転写因子である。ヒトとマウスにおいてGATA2遺伝子は高度に活性化しており、その標的遺伝子であるKit, Tpsb2, Cpa3などの発現量は高く、これらの遺伝子産物はマスト細胞の機能発現に重要である。このことから、Gata2遺伝子の高度活性化は、標的遺伝子の高い発現量を維持し、マスト細胞の機能発現に重要なのではないかと考えた。本研究は、GATA2遺伝子高度活性化の分子機序を明らかにすることを目的としている。 令和3年度は、マウスGata2遺伝子の転写開始点から+53-57kbpに広範囲にH3K27ac修飾がみられる領域(Gata2 downstream regulatory element:Gata2 DRE)に着目し、その機能を解析した。この領域はヒトK562細胞を用いたCRISPRiスクリーニング法で報告されたGATA2エンハンサー(Gasperini, 2019)と相同的な位置に存在するが、その機能は解析されていない。公開ChIP-SEQデータベースで、活性化クロマチン指標(H3K27ac, H3K4me1, ATACなど)を調べたところ、マスト細胞でGata2 DREが強く活性化されていることが確認された。MEDMC-BRC6マスト細胞株においても、Gata2 DREは既知のGata2+9.5kbpエンハンサーと同程度に強く活性化していることが確認された。また同領域にはGATA2とGATA1の結合ピークが認められた。つぎに、ゲノム編集法でGata2 DREを欠失する細胞を作製した。その結果、Gata2の発現量が約50%に低下した。

  2. マスト細胞遺伝子発現制御におけるGata1/Gata2とPU.1の相互作用の解析

    大根田 絹子, 大森 慎也

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (C)

    2018年4月1日 ~ 2021年3月31日

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    本研究の目的は、マスト細胞特異的遺伝子の発現制御にGATA因子(GATA1とGATA2)とPU.1との相互抑制作用がどのように機能しているのかを明らかにすることである。初年度はまず、マスト細胞における PU.1完全欠失の表現型が報告されていなかったため、エストロゲン誘導性にCre-loxPシステムが作動する「条件付きPU.1ノックアウトマウス」から作製したPU.1欠失骨髄由来マスト細胞(BMMCs)を用いて主な表現型を解析した。その際、既に報告されているsiRNAによるPU.1ノックダウン(KD)マスト細胞との比較と、PU.1欠失時のGATA因子の発現変動に注目した。その結果、PU.1を完全に欠失させたBMMCsは、細胞の生存率、細胞の形態、顆粒の染色性、抗原非依存性の脱顆粒能は、野生型BMMCsと同様であったが、高親和性IgE受容体(FceRI)の発現強度の減少、FceRIb mRNAの発現量低下、シグナル分子Sykの発現低下が認められ、抗原刺激によるマスト細胞の脱顆粒反応が低下していた。これらの表現型は、PU.1KDBMMCsの解析で報告されている表現型と一致していた。また、PU.1欠失時には、GATA1とGATA2のmRNA量が中等度に増加していた。一方、GATA因子をKDしたBMMCsやMEDMC-BRC6マスト細胞株(BRC6細胞)でのPU.1mRNA量は、単一のGATA因子をKDしても変化はなく、GATA1とGATA2の双方をKDすると顕著に増加した。この細胞では、PU.1が過剰に発現しているにも関わらず、FceRIb mRNA量は顕著に低下していた。また、FACS解析でのFceRIの発現は細胞表面のFceRIアルファ鎖に対する抗体で検出しているが、PU.1欠失BMMCsでは、FceRIaのmRNA量は逆に増加しており、FceRIbのmRNA量と相関していた。

  3. GATA転写因子によるマスト細胞プロテアーゼ遺伝子の統括的な発現制御機構の解明

    大根田 絹子, 石嶋 康史, 大森 慎也

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (C)

    研究機関:Takasaki University of Health and Welfare

    2015年4月1日 ~ 2018年3月31日

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    マスト細胞の機能発現に重要なトリプターゼ遺伝子群(Tpsg1、Tpsb2、Tpsab1)は、マウス17番染色体A3.3に存在している。骨髄由来マスト細胞において、GATA転写因子(GATA1・GATA2)を欠失させると、トリプターゼ遺伝子群の発現が減少した。GATA因子はTpsb2から離れた2つの領域 (region A・B)に結合し、その間に結合するCTCFとコヒーシンのDNA結合を促進していた。また、ゲノム編集によりRegion Aを欠失させるとTpsb2の発現が顕著に減少した。これらの結果から、GATA因子はトリプターゼ遺伝子の活性化型クロマチン高次構造の形成に関与することが示唆された。

  4. 骨髄球系細胞の系列転換における「遷移細胞」の解析

    大根田 絹子

    2013年4月1日 ~ 2015年3月31日

    詳細を見る 詳細を閉じる

    研究代表者らは、以前、マウス骨髄マスト細胞は、GATA2欠失によりマスト細胞の分化形質を失い未分化な形態を示すことを見いだした。このGATA2欠失細胞は、正常な血球分化の過程にはみられない不安定な「遷移状態」にあると考えられた。本研究は、この細胞を解析し、骨髄球系細胞の細胞系列転換機構を明らかにすることを目的として行った。研究結果の概要は、以下のとおりである。 GATA2欠失細胞は、マスト細胞にはほとんど発現していないC/EBPαを高発現しており、培養条件によってマクロファージや好中球様の細胞に系列転換した。一方、好塩基球や好酸球には分化しなかったが、一部の好塩基球特異的な遺伝子群の発現が増加していた。野生型BMMCにC/EBPαを過剰発現させると、C/EBPα の発現量依存的にGATA2mRNAの発現量が低下し、GATA2欠失細胞の表現型が一部再現された。GATA2とC/EBPαを同時に欠失させると、マクロファージや好中球に発現する遺伝子の発現増加は見られなくなったが、マスト細胞特異的な遺伝子の発現低下は観察された。BMMCとは異なり、腹腔細胞由来の培養マスト細胞(PMC)では、GATA2を欠失させてもC/EBPαの増加は見られなかった。これらの結果から、BMMCにおいて、GATA2とC/EBPαはお互いの発現を抑制している可能性が示唆された。GATA2欠失BMMCが正常な好塩基球に系列転換できなかったことから、好塩基球への分化にはGATA2が必要であると考えられた。

  5. GATA因子による成熟マスト細胞の機能制御機構の解明

    大根田 絹子, 石嶋 康史, 大森 慎也

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (C)

    研究機関:Takasaki University of Health and Welfare

    2012年4月1日 ~ 2015年3月31日

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    マスト細胞の分化と機能におけるGATA因子の役割を解明するために、成体マウスでGata1を誘導的に欠失させた。その結果、骨髄由来マスト細胞(BMMC)は正常に分化し、末梢組織マスト細胞もほぼ正常であった。BMMCのマイクロアレイ解析では、トリプターゼ遺伝子群の発現が減少していたが、多くのマスト細胞関連遺伝子の発現はGATA1発現低下の影響を受けなかった。一方、BMMCでGATA2を誘導的に機能欠失すると、フローサイトメトリーでマスト細胞の分画が著明に減少した。これらの結果は、マスト細胞分化形質の維持にはGATA2がより重要であり、GATA1の欠失はGATA2によって代償されたことを示している。

  6. 造血系転写因子によるマスト細胞分化決定機構の解明

    大根田 絹子

    2011年4月1日 ~ 2013年3月31日

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    本研究は、血球系転写因子によるマスト細胞の分化制御機構を解明することを目的としている。前年度は、条件付Gata1ノックアウトマウスを用いた解析により、GATA1は分化したマスト細胞の形質維持に必須ではないことを明らかにした。一方、マウス骨髄マスト細胞(BMMC)では細胞分化とともに GATA2の発現が増加することから、GATA1の機能欠失をGATA2が代償している可能性が考えられた。 今年度は、この可能性を検証するために更に解析を進めた。野生型BMMCを用いたChIP解析では、保存されたGATA配列を有するマスト細胞特異的な遺伝子のプロモーター領域に、GATA1とGATA2の両方が結合していた。また、siRNAでGATA2の発現を抑制すると、GATA2を正常に発現している細胞では見られなかった、GATA1欠失によるマスト細胞特異的な遺伝子発現の低下が観察された。さらに、GATA2のDNA結合ドメインを誘導的に欠失するマウスからBMMCを作成し、GATA2の機能欠失解析を行ったところ、BMMCは、GATA2の機能欠失によりマスト細胞のマーカーであるc-KitとFceRIaの発現を消失し、未熟な骨髄球系細胞に特徴的なGr1やMac1を発現する細胞へと系列転換した。細胞の形態も明らかに変化し、マスト細胞に特徴的な顆粒が失われていた。GATA2機能欠失BMMCでは、GATA1の発現増加はみられず、Scl、 PU.1、 Runx1などの血球系転写因子の発現も変化しなかったが、野生型BMMCには発現していないC/EBPalphaの発現が著しく増加していた。 これらの結果から、GATA2は、GATA1と協調してマスト細胞特異的な遺伝子の発現を制御するのみならず、C/EBPalphaの発現を抑制することにより他の骨髄球系細胞への分化を抑制してマスト細胞の形質を維持していると考えられた。

  7. 成熟マスト細胞におけるGATA-1の機能解析

    大根田 絹子, 石嶋 康史, 大森 慎也

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (C)

    研究機関:Takasaki University of Health and Welfare

    2009年 ~ 2011年

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    本研究では、赤血球と巨核球の分化に必須であることが知られている転写因子GATA1について、その成熟マスト細胞での役割を検証した。成熟マスト細胞のモデルとしてRBL-2H3細胞を用いた実験では、GATA1はGATA2とともにホスホリパーゼC-γ1(PLC-γ1)遺伝子の発現を制御することによって細胞内カルシウム濃度を調整し、抗原刺激による脱顆粒反応を促進していると考えられた。一方マウス骨髄マスト細胞ではGATA1ではなく主にGATA2がPLC-γ1の発現を制御していることが示唆された。

  8. 細胞の癌化とその抑制における転写因子の役割

    山本 雅之, 清水 律子, 大根田 絹子

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research on Priority Areas

    2005年 ~ 2009年

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    本研究では、転写因子の機能と、転写因子自身の制御機構に着目して、転写因子群の発癌抑制における機能貢献を検証した。その結果、異物代謝系および酸化ストレス応答系遺伝子群の発現を統一的に誘導制御するNrf2と、その応答制御を調節するKeap1の機能異常が生体防御系を撹乱し、発癌及び癌悪性化に寄与すること、および、造血系の増殖・分化・細胞死に関わる遺伝子発現を包括的に制御するGATA因子の機能不均衡が、恒常性維持機構を破綻し、白血病発症の誘因となることを見いだした。

  9. 生後の赤血球分化・増殖における転写因子GATA-1の機能解明

    大根田 絹子, 石嶋 康史, 大森 慎也

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (C)

    研究機関:Takasaki University of Health and Welfare

    2007年 ~ 2008年

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    転写因子GATA-1 の生後の造血における役割を検証するため、条件付gata1ノックアウトマウスと誘導的にCre リコンビナーゼを発現するマウスを用いて、成体造血組織においてgata1 を欠失させて解析した。その結果、赤血球分化は赤芽球前駆細胞の段階から障害され、好塩基性赤芽球以降に分化した細胞はほとんどみられなかった。また、これらのマウスに貧血を誘発し赤血球造血を刺激しても代償性造血は観察されなかった。したがって、GATA-1は胎生期のみならず成体においても赤血球分化に必須であることが示唆された。

  10. 細胞分化に伴うマウスGATA-1遺伝子発現の量的制御機構の解明

    大根田 絹子, 大根田 修

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (C)

    2005年 ~ 2006年

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    (1)BACトランスジェニックマウスを用いたin vivoでの定量的なレポーター解析系の確立 Gata1遺伝子の翻訳領域をGFP cDNAに置換したマウス大腸菌人工染色体(BAC)を用いてトランスジェニック(TG)マウス(GATA-1BAC GFP TGマウス)を4ライン樹立した。GFPの発現は内因性Gata1遺伝子の発現様式を忠実に再現しており、GFPmRNA量および蛍光強度はトランスジーンのコピー数に依存していた。この系によって、Gata1遺伝子に存在するシスエレメントがGata1発現量にどの程度貢献しているかについて、マウス個体を用いて解析することが可能となった。 (2)Gata1遺伝子のシスエレメントを欠失させたBAC GFP TGマウスの解析 (1)の系を用いてGata1遺伝子の発現において強力なエンハンサーとして機能するGata1 hematopoietic enhancer (G1HE)領域に欠失や変異を加えたTGマウスを作成した。その結果、骨髄での赤血球系細胞にGFP陽性細胞の減少とGFP発現強度の低下が観察された。赤血球の分化段階を追って解析した結果、Gata1の発現量が最大となる前赤芽球では、GFP強度は1/5程度にまで減少していたもののGFP陽性細胞の数は保たれていた。一方、前赤芽球より未熟な細胞や分化した細胞ではGFP陽性細胞数が著しく減少していた。これらの結果から、前赤芽球ではG1HEに依存しない制御様式が存在しているが、分化の初期および後期ではG1HEはGata1の発現に必須であることが示唆された。さらに、G1HEに含まれるGATA boxに変異を加えただけでほぼ同様の結果が再現されたことから、GATA-1,GATA-2によるGata1遺伝子の自律的な制御がエンハンサー機能の中心的な役割を担っていると考えられた。

  11. 細胞癌化とその抑制における転写因子の役割

    山本 雅之, 大根田 絹子, 清水 律子

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research on Priority Areas

    研究機関:University of Tsukuba

    2003年 ~ 2004年

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    細胞癌化とその抑制における転写因子の役割の解明に,血球系転写因子と白血病をモデル系として選択して取り組んだ.転写因子GATA-1による細胞増殖抑制・分化誘導・細胞死抑制の3つの作用は,赤血球前駆細胞から成熟赤血球への分化に必須の貢献を行っている.私たちは,胚幹細胞の解析から,GATA-1発現が野生型の5%レベルに低下した際には,赤血球系への分化誘導能や細胞増殖抑制能は不十分であったが,細胞死抑制能については,同レベルのGATA-1発現で十分であることを見出した.さらに,GATA-1発現を野生型の5%にまで減弱させたノックダウンヘテロマウスは2種類の白血病を発症すること,しかし,GATA-1完全欠失ヘテロマウスはそのような白血病を発症しないことを見出した.GATA-1はX染色体上に位置するため、X染色体の不活化により,GATA-1ノックダウンヘテロマウスには変異アリルが活性化した赤血球前駆細胞と野生型アリルが活性化した赤血球前駆細胞の2種類が存在するが,同マウスでは,前者が分化も細胞死もできないまま増殖蓄積し,高率に白血病を発症する母地となっている.さらに,ダウン症候群患児に好発する巨核芽球性白血病にはN末端領域を欠失したGATA-1がほぼ全例で発現しているが,同白血病を発症した患児より樹立した細胞株に,野生型GATA-1を遺伝子導入すると赤血球系細胞に分化する.一方,N末端領域欠失変異GATA-1(ΔNT-GATA-1)を導入しても同細胞は分化しなかった.すなわち,変異GATA-1の赤血球形質への分化誘導機能の欠失が白血病発症の病因の一つであることが強く示唆される.

  12. 転座関連遺伝子及び転写因子による細胞分化とがん化の分子機構

    瀬戸 加大, 森下 和広, 山本 雅之, 大根田 絹子, 鈴木 律朗

    2000年 ~ 2004年

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    瀬戸は、粘膜関連リンパ組織(MALT)リンパ腫に特異的に認められる染色体転座、t(11,18)(q21,q21)に関与する遺伝子API2-MALT1について、造血器細胞における機能の解析をするために細胞株や骨髄幹細胞にAPI2-MALT1遺伝子を導入した。Hela細胞株や増殖因子依存性の細胞株ではUV刺激、抗がん剤などによるアポトーシスを抑制する能力があることが明らかとなった。また、API2-MALT1と相互作用する蛋白としてTRAF2、smac、HtrA2などアポトーシスを制御する蛋白を明らかにした。また、悪性リンパ腫のゲノム異常をアレイCGHで検索したところ、病型特異的なゲノム異常が存在することが明らかとなった。 森下は、Evi1転座関連遺伝子の機能を明らかにするためにEvi1遺伝子欠損マウスの造血発生異常を検討し、造血幹細胞の維持・増殖異常及び、血管発生異常を同定した。遺伝子発現解析にてGATA-2遺伝子の転写低下、プロモーター解析によりEvi1はGATA-2の直接の転写調節に係わることがわかった。Evi1は白血病細胞内にてGATA-2転写を亢進させ、白血病幹細胞の増殖維持に関係していることが示唆され白血病発症機構の一端を解明した。t(1,3)転座関連遺伝子としてEVI1遺伝子ファミリーであるMEL1遺伝子を単離し、機能解析によりEVI1と同様の転写因子機能を持っていることがわかり白血病発症機構も類似することが示唆された。

︎全件表示 ︎最初の5件までを表示