Researchers Database

kaneda ruri

    Support Center for Clinical Investigation Associate Professor
Last Updated :2021/12/08

Researcher Information

J-Global ID

Research Interests

  • 心不全   ヒストン修飾酵素阻害薬   エピジェネティクス   

Research Areas

  • Life sciences / Cardiology

Published Papers

  • Tomohiro Matsuhashi, Takako Hishiki, Heping Zhou, Tomohiko Ono, Ruri Kaneda, Tatsuya Iso, Aiko Yamaguchi, Jin Endo, Yoshinori Katsumata, Anzai Atsushi, Tsunehisa Yamamoto, Kohsuke Shirakawa, Xiaoxiang Yan, Ken Shinmura, Makoto Suematsu, Keiichi Fukuda, Motoaki Sano
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 82 116 - 124 0022-2828 2015/05 [Refereed][Not invited]
    Dichloroacetate (DCA) promotes pyruvate entry into the Krebs cycle by inhibiting pyruvate dehydrogenase (PDH) kinase and thereby maintaining PDH in the active dephosphorylated state. DCA has recently gained attention as a potential metabolic-targeting therapy for heart failure but the molecular basis of the therapeutic effect of DCA in the heart remains a mystery. Once-daily oral administration of DCA alleviates pressure overload-induced left ventricular remodeling. We examined changes in the metabolic fate of pyruvate carbon (derived from glucose) entering the Krebs cycle by metabolic interventions of DCA. C-13(6)-glucose pathway tracing analysis revealed that instead of being completely oxidized in the mitochondria for ATP production, DCA-mediated PDH dephosphorylation results in an increased acetyl-CoA pool both in control and pressure-overloaded hearts. DCA induces hyperacetylation of histone H3K9 and H4 in a dose-dependent manner in parallel to the dephosphorylation of PDH in cultured cardiomyocytes. DCA administration increases histone H3K9 acetylation in in vivo mouse heart. Interestingly, DCA-dependent histone acetylation was associated with an up-regulation of 23% of genes (545 out of 23,474 examined). Gene ontology analysis revealed that these genes are highly enriched in transcription-related categories. This evidence suggests that sustained activation of PDH by DCA results in an overproduction of acetyl-CoA, which exceeds oxidation in the Krebs cycle and results in histone acetylation. We propose that DCA-mediated PDH activation has the potential to induce epigenetic remodeling in the heart, which, at least in part, forms the molecular basis for the therapeutic effect of DCA in the heart. (C) 2015 Elsevier Ltd. All rights reserved.
  • Naoto Muraoka, Hiroyuki Yamakawa, Kazutaka Miyamoto, Taketaro Sadahiro, Tomohiko Umei, Mari Isomi, Hanae Nakashima, Mizuha Akiyama, Rie Wada, Kohei Inagawa, Takahiko Nishiyama, Ruri Kaneda, Toru Fukuda, Shu Takeda, Shugo Tohyama, Hisayuki Hashimoto, Yoshifumi Kawamura, Naoki Goshima, Ryo Aeba, Hiroyuki Yamagishi, Keiichi Fukuda, Masaki Ieda
    EMBO JOURNAL 33 (14) 1565 - 1581 0261-4189 2014/07 [Refereed][Not invited]
    Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.
  • Yu Liu, Ruri Kaneda, Thomas W. Leja, Tatiana Subkhankulova, Oleg Tolmachov, Gabriella Minchiotti, Robert J. Schwartz, Mauricio Barahona, Michael D. Schneider
    STEM CELLS 32 (6) 1515 - 1526 1066-5099 2014/06 [Refereed][Not invited]
    Cardiac muscle differentiation in vivo is guided by sequential growth factor signals, including endoderm-derived diffusible factors, impinging on cardiogenic genes in the developing mesoderm. Previously, by RNA interference in AB2.2 mouse embryonic stem cells (mESCs), we identified the endodermal transcription factor Sox17 as essential for Mesp1 induction in primitive mesoderm and subsequent cardiac muscle differentiation. However, downstream effectors of Sox17 remained to be proven functionally. In this study, we used genome-wide profiling of Sox17-dependent genes in AB2.2 cells, RNA interference, chromatin immunoprecipitation, and luciferase reporter genes to dissect this pathway. Sox17 was required not only for Hhex (a second endodermal transcription factor) but also for Cer1, a growth factor inhibitor from endoderm that, like Hhex, controls mesoderm patterning in Xenopus toward a cardiac fate. Suppressing Hhex or Cer1 blocked cardiac myogenesis, although at a later stage than induction of Mesp1/2. Hhex was required but not sufficient for Cer1 expression. Over-expression of Sox17 induced endogenous Cer1 and sequence-specific transcription of a Cer1 reporter gene. Forced expression of Cer1 was sufficient to rescue cardiac differentiation in Hhex-deficient cells. Thus, Hhex and Cer1 are indispensable components of the Sox17 pathway for cardiopoiesis in mESCs, acting at a stage downstream from Mesp1/2.
  • Rie Wada, Naoto Muraoka, Kohei Inagawa, Hiroyuki Yamakawa, Kazutaka Miyamoto, Taketaro Sadahiro, Tomohiko Umei, Ruri Kaneda, Tomoyuki Suzuki, Kaichiro Kamiya, Shugo Tohyama, Shinsuke Yuasa, Kiyokazu Kokaji, Ryo Aeba, Ryohei Yozu, Hiroyuki Yamagishi, Toshio Kitamura, Keiichi Fukuda, Masaki Ieda
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 (31) 12667 - 12672 0027-8424 2013/07 [Refereed][Not invited]
    Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca2+ oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.
  • Yohei Ohno, Shinsuke Yuasa, Toru Egashira, Tomohisa Seki, Hisayuki Hashimoto, Shugo Tohyama, Yuki Saito, Akira Kunitomi, Kenichiro Shimoji, Takeshi Onizuka, Toshimi Kageyama, Kojiro Yae, Tomofumi Tanaka, Ruri Kaneda, Fumiyuki Hattori, Mitsushige Murata, Kensuke Kimura, Keiichi Fukuda
    STEM CELLS INTERNATIONAL 2013 659739  1687-966X 2013 [Refereed][Not invited]
    Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.
  • Kohei Inagawa, Kazutaka Miyamoto, Hiroyuki Yamakawa, Naoto Muraoka, Taketaro Sadahiro, Tomohiko Umei, Rie Wada, Yoshinori Katsumata, Ruri Kaneda, Koji Nakade, Chitose Kurihara, Yuichi Obata, Koichi Miyake, Keiichi Fukuda, Masaki Ieda
    CIRCULATION RESEARCH 111 (9) 1147 - 1156 0009-7330 2012/10 [Refereed][Not invited]
    Rationale: After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. Objective: To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. Methods and Results: Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with-cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into alpha-myosin heavy chain (alpha MHC)-GFP transgenic mouse hearts induced the expression of alpha-MHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2-A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric alpha-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2-A-transduced cells expressed cardiac-specific genes. Conclusions: GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts. (Circ Res. 2012; 111: 1147-1156.)
  • Takahiko Nishiyama, Ruri Kaneda, Tomohiko Ono, Shugo Tohyama, Hisayuki Hashimoto, Jin Endo, Hikaru Tsuruta, Shinsuke Yuasa, Masaki Ieda, Shinji Makino, Keiichi Fukuda
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 425 (4) 755 - 761 0006-291X 2012/09 [Refereed][Not invited]
    MicroRNAs (miRNAs) play a pivotal role during embryonic development and are required for proper organogenesis, including hematopoiesis. Recent studies suggest that, in the early mesoderm, there is an interaction between the hematopoietic and cardiac lineages. However, whether miRNAs can affect other lineages remains unknown. Therefore, we investigated whether hematopoietic miR-142-3p modulated the mesoderm formation. We report that knockdown (KD) of miR-142-3p, a hematopoietic-specific miRNA, in zebrafish resulted in loss of hematopoiesis during embryonic development. Intriguingly, we observed abnormal cardiac phenotypes and insufficiency of somitegenesis in KD-morphants. In the early developmental stage, a tiny heart, contractile dysfunction in the ventricle, cardiac arrhythmia (e.g. a 2:1 ratio of atrial:ventricular beating), and bradycardia were consistently observed. Histological examination revealed severe hypoplasia of the ventricle and disrupted muscle alignment. To determine the mechanism, we performed DNA microarray analysis. The results revealed that the expression of several mesodermal genes essential for the formation of cardiac and somatic mesoderm, such as no tail, T-box gene 16, mesoderm posterior a, one eye pinhead, and rho-associated, coiled-coil containing protein kinase (Rock2a), were increased in miR-142-3p KD-morphants. The luciferase reporter assay revealed that miR-142-3p repressed luciferase activity on the Rock2a 3'-UTR. The findings of the present study indicate that miR-142-3p plays a critical role in hematopoiesis, cardiogenesis, and somitegenesis in the early stage of mesoderm formation via regulation of Rock2a. (C) 2012 Elsevier Inc. All rights reserved.
  • Mie Hara, Shinsuke Yuasa, Kenichiro Shimoji, Takeshi Onizuka, Nozomi Hayashiji, Yohei Ohno, Takahide Arai, Fumiyuki Hattori, Ruri Kaneda, Kensuke Kimura, Shinji Makino, Motoaki Sano, Keiichi Fukuda
    JOURNAL OF EXPERIMENTAL MEDICINE 208 (4) 715 - 727 0022-1007 2011/04 [Refereed][Not invited]
    After skeletal muscle injury, neutrophils, monocytes, and macrophages infiltrate the damaged area; this is followed by rapid proliferation of myoblasts derived from muscle stem cells (also called satellite cells). Although it is known that inflammation triggers skeletal muscle regeneration, the underlying molecular mechanisms remain incompletely understood. In this study, we show that granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is expressed in developing somites. G-CSFR and G-CSF were expressed in myoblasts of mouse embryos during the midgestational stage but not in mature myocytes. Furthermore, G-CSFR was specifically but transiently expressed in regenerating myocytes present in injured adult mouse skeletal muscle. Neutralization of endogenous G-CSF with a blocking antibody impaired the regeneration process, whereas exogenous G-CSF supported muscle regeneration by promoting the proliferation of regenerating myoblasts. Furthermore, muscle regeneration was markedly impaired in G-CSFR-knockout mice. These findings indicate that G-CSF is crucial for skeletal myocyte development and regeneration and demonstrate the importance of inflammation-mediated induction of muscle regeneration.
  • Yuichi Tamura, Keisuke Matsumura, Motoaki Sano, Hidenori Tabata, Kensuke Kimura, Masaki Ieda, Takahide Arai, Yohei Ohno, Hideaki Kanazawa, Shinsuke Yuasa, Ruri Kaneda, Shinji Makino, Kazunori Nakajima, Hideyuki Okano, Keiichi Fukuda
    ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY 31 (3) 582 - 589 1079-5642 2011/03 [Refereed][Not invited]
    Objective-We recently demonstrated that primitive neural crest-derived (NC) cells migrate from the cardiac neural crest during embryonic development and remain in the heart as dormant stem cells, with the capacity to differentiate into various cell types, including cardiomyocytes. Here, we examined the migration and differentiation potential of these cells on myocardial infarction (MI). Methods and Results-We obtained double-transgenic mice by crossing protein-0 promoter-Cre mice with Floxed-enhanced green fluorescent protein mice, in which the NC cells express enhanced green fluorescent protein. In the neonatal heart, NC stem cells (NCSCs) were localized predominantly in the outflow tract, but they were also distributed in a gradient from base to apex throughout the ventricular myocardium. Time-lapse video analysis revealed that the NCSCs were migratory. Some NCSCs persisted in the adult heart. On MI, NCSCs accumulated at the ischemic border zone area (BZA), which expresses monocyte chemoattractant protein-1 (MCP-1). Ex vivo cell migration assays demonstrated that MCP-1 induced NCSC migration and that this chemotactic effect was significantly depressed by an anti-MCP-1 antibody. Small NC cardiomyocytes first appeared in the BZA 2 weeks post-MI and gradually increased in number thereafter. Conclusion-These results suggested that NCSCs migrate into the BZA via MCP-1/CCR2 signaling and contribute to the provision of cardiomyocytes for cardiac regeneration after MI. (Arterioscler Thromb Vasc Biol. 2011;31:582-589.)
  • Yan Zhang, Motoaki Sano, Ken Shinmura, Kayoko Tamaki, Yoshinori Katsumata, Tomohiro Matsuhashi, Shintaro Morizane, Hideyuki Ito, Takako Hishiki, Jin Endo, Heping Zhou, Shinsuke Yuasa, Ruri Kaneda, Makoto Suematsu, Keiichi Fukuda
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 49 (4) 576 - 586 0022-2828 2010/10 [Refereed][Not invited]
    Reactive oxygen species (ROS) attack polyunsaturated fatty acids of the membrane and trigger lipid peroxidation, which results in the generation of alpha,beta-unsaturated aldehydes, such as 4-hydroxy-2-nonenal (4-HNE). There is compelling evidence that high concentrations of aldehydes are responsible for much of the damage elicited by cardiac ischemia-reperfusion injury, while sublethal concentrations of aldehydes stimulate stress resistance pathways, to achieve cardioprotection. We investigated the mechanism of cardioprotection mediated by 4-HNE. For cultured cardiomyocytes, 4-HNE was cytotoxic at higher concentrations (>= 20 mu M) but had no appreciable cytotoxicity at lower concentrations. Notably, a sublethal concentration (5 mu M) of 4-HNE primed cardiomyocytes to become resistant to cytotoxic concentrations of 4-HNE. 4-HNE induced nuclear translocation of transcription factor NF-E2-related factor 2 (Nrf2), and enhanced the expression of gamma-glutamylcysteine ligase (GCL) and the core subunit of the Xc(-) high-affinity cystine transporter (xCT), thereby increasing 1.45-fold the intracellular GSH levels. Cardiomyocytes treated with either Nrf2-specific siRNA or the GCL inhibitor L-buthionine sulfoximine (BSO) were less tolerant to 4-HNE. Moreover, the cardioprotective effect of 4-HNE pretreatment against subsequent glucose-free anoxia followed by reoxygenation was completely abolished in these cells. Intravenous administration of 4-HNE (4 mg/kg) activated Nrf2 in the heart and increased the intramyocardial GSH content, and consequently improved the functional recovery of the left ventricle following ischemia-reperfusion in Langendorff-perfused hearts. This cardioprotective effect of 4-HNE was not observed for Nrf2-knockout mice. In summary, 4-HNE activates Nrf2-mediated gene expression and stimulates GSH biosynthesis, thereby conferring on cardiomyocytes protection against ischemia-reperfusion injury. (c) 2010 Elsevier Ltd. All rights reserved.
  • Shinsuke Yuasa, Takeshi Onizuka, Kenichiro Shimoji, Yohei Ohno, Toshimi Kageyama, Sung Han Yoon, Toru Egashira, Tomohisa Seki, Hisayuki Hashimoto, Takahiko Nishiyama, Ruri Kaneda, Mitsushige Murata, Fumiyuki Hattori, Shinji Makino, Motoaki Sano, Satoshi Ogawa, Owen W. J. Prall, Richard P. Harvey, Keiichi Fukuda
    CIRCULATION RESEARCH 106 (6) 1083 - 1091 0009-7330 2010/04 [Refereed][Not invited]
    Rationale: The transcriptional networks guiding heart development remain poorly understood, despite the identification of several essential cardiac transcription factors. Objective: To isolate novel cardiac transcription factors, we performed gene chip analysis and found that Zac1, a zinc finger-type transcription factor, was strongly expressed in the developing heart. This study was designed to investigate the molecular and functional role of Zac1 as a cardiac transcription factor. Methods and Results: Zac1 was strongly expressed in the heart from cardiac crescent stages and in the looping heart showed a chamber-restricted pattern. Zac1 stimulated luciferase reporter constructs driven by ANF, BNP, or alpha MHC promoters. Strong functional synergy was seen between Zac1 and Nkx2-5 on the ANF promoter, which carries adjacent Zac1 and Nkx2-5 DNA-binding sites. Zac1 directly associated with the ANF promoter in vitro and in vivo, and Zac1 and Nkx2-5 physically associated through zinc fingers 5 and 6 in Zac1, and the homeodomain in Nkx2-5. Zac1 is a maternally imprinted gene and is the first such gene found to be involved in heart development. Homozygous and paternally derived heterozygous mice carrying an interruption in the Zac1 locus showed decreased levels of chamber and myofilament genes, increased apoptotic cells, partially penetrant lethality and morphological defects including atrial and ventricular septal defects, and thin ventricular walls. Conclusions: Zac1 plays an essential role in the cardiac gene regulatory network. Our data provide a potential mechanistic link between Zac1 in cardiogenesis and congenital heart disease manifestations associated with genetic or epigenetic defects in an imprinted gene network. (Circ Res. 2010; 106: 1083-1091.)
  • Fumiyuki Hattori, Hao Chen, Hiromi Yamashita, Shugo Tohyama, Yu-suke Satoh, Shinsuke Yuasa, Weizhen Li, Hiroyuki Yamakawa, Tomofumi Tanaka, Takeshi Onitsuka, Kenichiro Shimoji, Yohei Ohno, Toru Egashira, Ruri Kaneda, Mitsushige Murata, Kyoko Hidaka, Takayuki Morisaki, Erika Sasaki, Takeshi Suzuki, Motoaki Sano, Shinji Makino, Shinzo Oikawa, Keiichi Fukuda
    NATURE METHODS 7 (1) 61 - U15 1548-7091 2010/01 [Refereed][Not invited]
    Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched(>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.
  • Tomoaki Wada, Yoshihiro Yamashita, Yasushi Saga, Kayoko Takahashi, Koji Koinuma, Young Lim Choi, Ruri Kaneda, Shin-Ichiro Fujiwara, Manabu Soda, Hideki Watanabe, Kentaro Kurashina, Hisashi Hatanaka, Munehiro Enomato, Shuji Takada, Hiroyuki Mano, Mitsuaki Suzuki
    INTERNATIONAL JOURNAL OF ONCOLOGY 35 (5) 973 - 976 1019-6439 2009/11 [Refereed][Not invited]
    The purpose of this study was to screen for genes involved in ovarian carcinogenesis in an attempt to develop an effective molecular-targeted therapy for ovarian cancer. We constructed retroviral expression libraries for the human ovarian cancer cell lines SHIN-3 and TYK-CPr, and performed a focus formation assay with 3T3 cells. As a result, proteasome subunit beta-type 2 (PSMB2), ubiquitin-specific protease 14 (USP14), and keratin 8 (KRT8) were identified from SHIN-3, and polymerase H RNA subunit (POLR2E), chaperonin containing T-complex polypeptide 1 subunit 4 (CCT4), glia maturation factor beta (GMFB), and neuroblastoma ras viral oncogene homolog (NRAS) from TYK-CPr. NRAS gene analysis revealed a CAA -> AAA substitution at codon 61, resulting in a Glu -> Lys change at position 61. When the mutant NRAS was introduced into fibroblasts for its expression, many transformed foci were generated, confirming the transforming ability of the mutant NRAS.
  • Kaneda R, Fukuda K
    Circulation journal : official journal of the Japanese Circulation Society 73 (8) 1397 - 1398 1346-9843 2009/08 [Refereed][Not invited]
  • Satori Tokudome, Motoaki Sano, Ken Shinmura, Tomohiro Matsuhashi, Shintaro Morizane, Hidenori Moriyama, Kayoko Tamaki, Kentaro Hayashida, Hiroki Nakanishi, Noritada Yoshikawa, Noriaki Shimizu, Jin Endo, Takaharu Katayama, Mitsushige Murata, Shinsuke Yuasa, Ruri Kaneda, Kengo Tomita, Naomi Eguchi, Yoshihiro Urade, Koichiro Asano, Yasunori Utsunomiya, Takeshi Suzuki, Ryo Taguchi, Hirotoshi Tanaka, Keiichi Fukuda
    JOURNAL OF CLINICAL INVESTIGATION 119 (6) 1477 - 1488 0021-9738 2009/06 [Refereed][Not invited]
    Lipocalin-type prostaglandin D synthase (L-PGDS), which was originally identified as an enzyme responsible for PGD(2) biosynthesis in the brain, is highly expressed in the myocardium, including in cardiomyocytes. However, the factors that control expression of the gene encoding L-PGDS and the pathophysiologic role of L-PGDS in cardiomyocytes are poorly understood. in the present study, we demonstrate that glucocorticoids, which act as repressors of prostaglandin biosynthesis in most cell types, upregulated the expression of L-PGDS together with cytosolic calcium-dependent phospholipase A2 and COX2 via the glucocorticoid receptor (GR) in rat cardiomyocytes. Accordingly, PGD2 was the most prominently induced prostaglandin in vivo in mouse hearts and in vitro in cultured rat cardiomyocytes after exposure to GR-selective agonists. In isolated Langendorff-perfused mouse hearts, dexamethasone alleviated ischemia/reperfusion injury. This cardioprotective effect was completely abrogated by either pharmacologic inhibition of COX2 or disruption of the gene encoding L-PGDS. In in vivo ischemia/reperfusion experiments, dexamethasone reduced infarct size in wild-type mice. This cardioprotective effect of dexamethasone was markedly reduced in L-PGDS-deficient mice. In cultured rat cardiomyocytes, PGD2 protected against cell death induced by anoxia/reoxygenation via the D-type prostanoid receptor and the ERK1/2-mediated pathway. Taken together, these results suggest what we believe to be a novel interaction between glucocorticoid-GR signaling and the cardiomyocyte survival pathway mediated by the arachidonic acid cascade.
  • Ruri Kaneda, Shuji Takada, Yoshihiro Yamashita, Young Lim Choi, Mutsuko Nonaka-Sarukawa, Manabu Soda, Yoshio Misawa, Tadashi Isomura, Kazuyuki Shimada, Hiroyuki Mano
    GENES TO CELLS 14 (1) 69 - 77 1356-9597 2009/01 [Refereed][Not invited]
    Epigenetic alterations are implicated in the development of cardiac hypertrophy and heart failure, but little is known of which epigenetic changes in which regions of the genome play such a role. We now show that trimethylation of histone H3 on lysine-4 (K4TM) or lysine-9 (K9TM) is markedly affected in cardiomyocytes in association with the development of heart failure in a rat disease model. High-throughput pyrosequencing performed with ChIP products for K4TM or K9TM prepared from human left ventricular tissue with retained or damaged function also revealed that protein-coding genes located in the vicinity of K4TM marks differ between functional and disabled myocytes, yet both sets of genes encode proteins that function in the same signal transduction pathways for cardiac function, indicative of differential K4TM marking during the development of heart failure. However, K9TM mark-profile was less dependent on the disease status compared to that of K4TM. Our data collectively reveal global epigenetic changes in cardiac myocytes associated with heart failure.
  • Young Lim Choi, Ruri Kaneda, Tomoaki Wada, Shin-ichiro Fujiwara, Manabu Soda, Hideki Watanabe, Kentaro Kurashina, Hisashi Hatanaka, Munehiro Enomoto, Shuji Takada, Yoshihiro Yamashita, Hiroyuki Mano
    LEUKEMIA RESEARCH 31 (2) 203 - 209 0145-2126 2007/02 [Refereed][Not invited]
    To identify transforming genes in acute myeloid leukemia (AML) we here constructed a retroviral cDNA expression library from an AML patient, and then used this library to infect a mouse cell line 32Dcl3-mCAT. cDNA inserts of the cell clones which proliferated in the presence of granulocyte colony-stimulating factor were derived from JAK3 encoding a JAK3 mutant with a valine-to-alanine substitution at codon 674 and two additional amino acid substitutions. The transforming activity of JAK3(V674A) was confirmed by its introduction into 32Dcl3-mCAT. Sequencing of the original JAK3 cDNA derived from the patient, however, failed to detect the V674A mutation. (c) 2006 Elsevier Ltd. All rights reserved.
  • Shuji Takada, Tomoaki Wada, Ruri Kaneda, Young Lim Choi, Yoshihiro Yamashita, Hiroyuki Mano
    MECHANISMS OF DEVELOPMENT 123 (6) 472 - 480 0925-4773 2006/06 [Refereed][Not invited]
    The anti-Mullerian hormone gene (Amh) is responsible for regression in males of the Mullerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species. (c) 2006 Elsevier Ireland Ltd. All rights reserved.
  • S Takada, J Ota, N Kansaku, H Yamashita, T Izumi, M Ishikawa, T Wada, R Kaneda, YL Choi, K Koinuma, SI Fujiwara, H Aoki, H Kisanuki, Y Yamashita, H Mano
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 145 (2) 208 - 213 0016-6480 2006/01 [Refereed][Not invited]
    Sox9 is a member of the Sry-type HMG-box (Sox) gene family. It encodes a transcription factor and is thought to be important for sexual differentiation in chicken. In the present study we have isolated Sox9 cDNAs from quail and duck, and examined the expression patterns of the corresponding genes in early embryonic gonads by whole-mount in situ hybridization. We developed a polymerase chain reaction-based protocol to identify the sex of quail and duck embryos before its morphological manifestation. Sox9 expression was first detected on days 5 and 7 in the gonads of male quail and duck embryos, respectively, and was not apparent in female gonads at these stages. These expression patterns are similar to that of chicken Sox9. Our results thus suggest that the expression of quail and duck Sox9 is associated with testis differentiation. (c) 2005 Elsevier Inc. All rights reserved.
  • K Koinuma, R Kaneda, M Toyota, Y Yamashita, S Takada, YL Choi, T Wada, M Okada, F Konishi, H Nagai, H Mano
    CARCINOGENESIS 26 (12) 2078 - 2085 0143-3334 2005/12 [Refereed][Not invited]
    A subset of colorectal carcinomas (CRCs) is associated with microsatellite instability (MSI) of the genome. Although extensive methylation of CpG islands within the promoter regions of DNA mismatch repair genes such as MLH1 is thought to play a central role in tumorigenesis for MSI-positive sporadic CRCs, it has been obscure whether such aberrant epigenetic regulation occurs more widely and affects other cancer-related genes in vivo. Here, by using methylated CpG island amplification coupled with representational difference analysis (MCA-RDA), we screened genomic fragments that are selectively methylated in CRCs positive for MLH1 methylation, resulting in the identification of hundreds of CpG islands containing genomic fragments. Methylation status of such CpG islands was verified for 28 genomic clones in 8 CRC specimens positive for MLH1 methylation and the corresponding paired normal colon tissue as well as in 8 CRC specimens negative for methylation. Many of the CpG islands were preferentially methylated in the MLH1 methylation-positive CRC specimens, although methylation of some of them was more widespread. These data provide insights into the complex regulation of the methylation status of CpG islands in CRCs positive for MSI and MLH1 methylation.
  • S Fujiwara, Y Yamashita, YM Choi, T Wada, R Kaneda, S Takada, Y Maruyama, K Ozawa, H Mano
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 338 (2) 1256 - 1262 0006-291X 2005/12 [Refereed][Not invited]
    Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies. To obtain insight into the molecular pathogenesis of PDC we constructed a retroviral cDNA expression library with total RNA isolated from the PDC cell line MiaPaCa-2. Screening of this library with the use of a focus formation assay with NIH 3T3 mouse fibroblasts resulted in the identification of 13 independent genes with transforming activity. One of the cDNAs thus identified encodes an NH2-terminally truncated form of the lymphotoxin-beta receptor (LTBR). The transforming activity of this short-type LTBR in 3T3 cells was confirmed by both an in vitro assay of cell growth in soft agar and an in vivo assay of turnorigenicity in nude mice. The full-length (wild-type) LTBR protein was also found to manifest similar transforming activity. These observations suggest that LTBR, which belongs to the tumor necrosis factor receptor superfamily of proteins, may contribute to human carcinogenesis. (c) 2005 Elsevier Inc. All rights reserved.
  • R Kaneda, K Kario, S Hoshide, Y Umeda, Y Hoshide, K Shimada
    AMERICAN JOURNAL OF HYPERTENSION 18 (12) 1528 - 1533 0895-7061 2005/12 [Refereed][Not invited]
    Background. Morning blood pressure (BP) surge seems to be a risk factor for cardiovascular events. Although physical activity after arising significantly affects morning BP surge, it has remained unclear whether morning BP surge after controlling for physical activity (morning BP reactivity) is associated with target organ damage. Methods: We performed ambulatory BP monitoring with simultaneous actigraphy and echocardiography in 120 community-dwelling Japanese subjects. We determined the waking time by actigraphy, and defined morning BP surge (MBPS) as the average of systolic BP during the 2 h after awakening minus the average of systolic BPs during the 1 h that included the lowest sleep BP. The ratio of MBPS/(sum of the 2-h physical activity after the arising time)(0.5) was calculated as the morning BP reactivity (MBPR). Results: In all the subjects studied (n = 120), MBPR was positively associated with left ventricular (LV) mass index (r = 0.30, P = .001). The MBPR had a positive association with both 24-h BP variability (SD) (r = 0.373, P < .001) and awake BP variability (r = 0.20, P < .05). The MBP hyper-reactive group (the highest quartile [Q4] of MBPR: n = 30) had significantly higher LV mass index than the nonreactive group (the other quartiles [Q1 to 3]: n = 90) (140 v 113 g/m(2), p < .001). Even after controlling for age, body mass index, gender, and 24-h systolic BP, the MBP hyper-reactive status still remained a strong predictor for LV hypertrophy. Conclusions: Exaggerated MBPS, adjusted for physical activity, is associated with cardiac hypertrophy independent of ambulatory BP level in a community-dwelling population.
  • Ishikawa J, Kario K, Matsui Y, Shibasaki S, Morinari M, Kaneda R, Hoshide S, Eguchi K, Hojo Y, Shimada K
    Hypertension research : official journal of the Japanese Society of Hypertension 28 (12) 995 - 1001 0916-9636 2005/12 [Refereed][Not invited]
  • R Kaneda, K Kario
    AMERICAN JOURNAL OF HYPERTENSION 18 (10) 1327 - 1328 0895-7061 2005/10 [Refereed][Not invited]
  • H Kisanuki, YL Choi, T Wada, R Moriuchi, S Fujiwara, R Kaneda, K Koinuma, M Ishikawa, S Takada, Y Yamashita, H Mano
    EUROPEAN JOURNAL OF CANCER 41 (14) 2170 - 2175 0959-8049 2005/09 [Refereed][Not invited]
    Pancreatic ductal carcinoma (PDC) remains one of the most intractable malignancies in humans. In order to clarify the molecular events underlying the carcinogenesis in PDC, we constructed a retroviral cDNA expression library from a PDC cell line, and used it to screen transforming genes in PDC by a focus formation assay with mouse 3T3 fibroblasts. We could obtain a total of 30 transformed cell foci in the screening, and one of the cDNA inserts harvested from such cell clones turned out to encode a wild-type human ARAF1. Unexpectedly, a long terminal repeat-driven overexpression of ARAF1 mRNA was confirmed to induce transformed foci in fibroblasts. The oncogenic potential of ARAF1 was examined by injecting the transformed fibroblasts into athymic nude mice. Importantly, ARAF1 mRNA was highly expressed in pancreatic ductal cell specimens purified from patients with PDC. These results have unveiled the transforming potential of ARAF1 protein, and also suggest that quantity of intracellular ARAF1 may be important in carcinogenesis of various human cancers. (c) 2005 Elsevier Ltd. All rights reserved.
  • YL Choi, R Moriuchi, M Osawa, A Iwama, H Makishima, T Wada, H Kisanuki, R Kaneda, J Ota, K Koinuma, M Ishikawa, S Takada, Y Yamashita, K Oshimi, H Mano
    LEUKEMIA RESEARCH 29 (8) 943 - 949 0145-2126 2005/08 [Refereed][Not invited]
    Aggressive natural killer cell leukemia (ANKL) is an intractable malignancy that is characterized by the outgrowth of NK cells. To identify transforming genes in ANKL, we constructed a retroviral cDNA expression library from an ANKL cell line KHYG-1. Infection of 3T3 cells with recombinant retroviruses yielded 33 transformed foci. Nucleotide sequencing of the DNA inserts recovered from these foci revealed that 31 of them encoded KRAS2 with a glycine-to-alanine mutation at codon 12. Mutation-specific PCR analysis indicated that the KRAS mutation was present only in KHYG-1 cells, not in another ANKL cell line or in clinical specimens (n = 8). (c) 2005 Elsevier Ltd. All rights reserved.
  • R Kaneda, S Ueno, Y Yamashita, YL Choi, K Koinuma, S Takada, T Wada, K Shimada, H Mano
    CIRCULATION RESEARCH 97 (3) 210 - 218 0009-7330 2005/08 [Refereed][Not invited]
    The acetylation status of core histones in cardiomyocytes has been linked to the development of cardiac hypertrophy and heart failure. Little is known, however, of the genes affected by abnormal histone acetylation in such pathological conditions. We recently developed a genome-wide screening method, differential chromatin scanning (DCS), to isolate genomic fragments associated with histones subject to differential acetylation. We have now applied DCS to H9C2 rat embryonic cardiomyocytes incubated with or without trichostatin A (TSA), a specific inhibitor of histone deacetylase (HDAC) activity. About 200 genomic fragments were readily isolated by DCS on the basis of the preferential acetylation of associated histones in TSA-treated cells. Quantitation of the amount of DNA in chromatin immunoprecipitates prepared with antibodies to acetylated histone H3 revealed that 37 of 38 randomly chosen DCS clones were preferentially precipitated from the TSA-treated cells, thus verifying the high fidelity of DCS. Epigenetic regulation of DCS clones was further confirmed in cells treated with sodium butyrate, another HDAC inhibitor, as well as in cardiac myocytes isolated from neonatal rats. The mRNA level of 9 (39%) of 23 genes corresponding to DCS clones changed in parallel with the level of histone acetylation in H9C2 cells. Furthermore, a physiological hypertrophic stimulus, cardiotrophin-1, affected the acetylation level of histones associated with genomic regions corresponding to certain DCS clones. Our data thus establish a genome-wide profile of HDAC targets in cardiomyocytes, which should provide a basis for further investigations into the role of epigenetic modification in cardiac disorders.
  • M Ishikawa, K Yoshida, Y Yamashita, J Ota, S Takada, H Kisanuki, K Koinuma, YL Choi, R Kaneda, T Iwao, K Tamada, K Sugano, H Mano
    CANCER SCIENCE 96 (7) 387 - 393 1347-9032 2005/07 [Refereed][Not invited]
    Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies, mainly because of the lack of sensitive detection methods. Although gene expression profiling by DNA microarray analysis is a promising tool for the development of such detection systems, a simple comparison of pancreatic tissues may yield misleading data that reflect only differences in cellular composition. To directly compare PDC cells with normal pancreatic ductal cells, we purified MUC1-positive epithelial cells from the pancreatic juices of 25 individuals with a normal pancreas and 24 patients with PDC. The gene expression profiles of these 49 specimens were determined with DNA microarrays containing > 44000 probe sets. Application of both Welch's analysis of variance and effect size-based selection to the expression data resulted in the identification of 21 probe sets corresponding to 20 genes whose expression was highly associated with clinical diagnosis. Furthermore, correspondence analysis and 3-D projection with these probe sets resulted in separation of the transcriptomes of pancreatic ductal cells into distinct but overlapping spaces corresponding to the two clinical classes. To establish an accurate transcriptome-based diagnosis system for PDC, we applied supervised class prediction algorithms to our large data set. With the expression profiles of only five predictor genes, the weighted vote method diagnosed the class of samples with an accuracy of 81.6%. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC.
  • J Ishikawa, K Kario, S Hoshide, K Eguchi, M Morinari, R Kaneda, Y Umeda, S Ishikawa, T Kuroda, Y Hojo, K Shimada
    AMERICAN JOURNAL OF HYPERTENSION 18 (7) 958 - 965 0895-7061 2005/07 [Refereed][Not invited]
    Background: Morning blood pressure (BP) surge in ambulatory BP monitoring was a risk factor for stroke in our previous study. We studied the determinants of the morning minus evening systolic BP difference (ME difference) in self-measured BP monitoring, as a possible risk factor for stroke in medicated hypertensive patients. Methods: Nine hundred sixty-nine hypertensive outpatients receiving stable antihypertensive drug treatment were studied using self-measured BP monitoring in the morning and evening. Results: The ME difference ranged from -37.3 to 53.3 mm Hg (mean 7.9 mm Hg). The highest quartile (Q4) of the ME difference group (> 15.0 mm Hg) had older age (68.0 +/- 9.8 years v 66.2 +/- 10.3 years, P =.01) and higher prevalence of men (48.3% v 39.9%, P =.02), regular alcohol drinkers (34.7% v 26.0%, P =.01) and beta-blocker use (26.9% v 19.9%, P =.03) than the other quartile groups (Q1 to Q3), whereas there was no significant difference in the average of morning and evening (ME average) BP. In logistic regression analysis controlling for ME average and other confounding factors, independent risks for Q4 of ME difference were older age (10 years older: odds ratio [OR] 1.21, P =.01, 95% confidence interval (CI) 1.04-1.42), regular alcohol drinker (OR 1.51, P =.04, 95% CI 1.01-2.26), and beta-blocker use (OR 1.50, P =.02, 95% CI 1.06-2.12). Conclusions: Older age, beta-blocker use, and regular alcohol drinking were significant determinants of the exaggerated ME difference in medicated hypertensive patients.
  • R Kaneda, M Toyota, Y Yamashita, K Koinuma, YL Choi, J Ota, H Kisanuki, M Ishikawa, S Takada, K Shimada, H Mano
    GENES TO CELLS 9 (12) 1167 - 1174 1356-9597 2004/12 [Refereed][Not invited]
    Although acetylation-deacetylation of histones contributes to regulation of gene expression, few methods have been available to determine the whole-genome histone acetylation profile in specific cells or tissues. We have now developed a genome-wide screening method, differential chromatin scanning (DCS), to isolate genome fragments embedded in histones subject to differential acetylation. This DCS screening was applied to a human gastric cancer cell line incubated with or without an inhibitor of histone deacetylase (HDAC) activity, resulting in the rapid identification of more than 250 genome fragments. Interestingly, a number of cancer-related genes were revealed to be the targets of HDAC in the cancer cells, including those for tumour protein 73 and cell division cycle 34. Such differential acetylation of histone was also shown to be linked to the regulation of transcriptional activity of the corresponding genes. Among the isolated genome fragments, 94% (32/34) of them were confirmed to be bound to differentially acetylated histones, and the genes corresponding to 78% (7/9) of them exhibited differential transcriptional activity consistent with the level of histone acetylation. With its high fidelity, the DCS method should open a possibility to rapidly compare the genome-wide histone acetylation profiles and to provide novel insights into molecular carcinogenesis.
  • C Tsutsumi, M Ueda, Y Miyazaki, Y Yamashita, YL Choi, J Ota, R Kaneda, K Koinuma, S Fujiwara, H Kisanuki, M Ishikawa, K Ozawa, M Tomonaga, H Mano
    EXPERIMENTAL HEMATOLOGY 32 (9) 828 - 835 0301-472X 2004/09 [Refereed][Not invited]
    Objective. Acute myeloid leukemia (AML) develops de novo or secondarily to either myelodysplastic syndrome (MDS) or anticancer treatment (therapy-related leukemia, TRL). Prominent dysplasia of blood cells is apparent in individuals with MDS-related AML as well as in some patients with TRL or even with de novo AML. The clinical entity of AML with multilineage dysplasia (AML-MLD) is likely to be an amalgamation of MDS-related AML and de novo AML-MLD. The aim of this study was to clarify, by the use of high-density oligonucleotide microarrays, whether these subcategories of AML are intrinsically distinct from each other. Materials and Methods. The AC133(+) hematopoietic stem cell-like fractions were purified from the bone marrow of individuals with de novo AML without dysplasia (n = 15), AML-MLD (n = 11), MDS-related AML (n = 11), or TRL (n = 2), and were subjected to the synthesis of cRNA which was subsequently hybridized to microarray harboring oligonucleotide corresponding to more than 12,000 probe sets. Results. We could identify many genes whose expression was specific to these various subcategories of AML. Furthermore, with the correspondence analysis/three-dimensional projection strategy, we were able to visualize the independent, yet partially overlapping, nature of current AML subcategories on the basis of their transcriptomes. Conclusion. Our data indicate the possibility of subclassification of AML based on gene expression profiles of leukemic blasts. (C) 2004 International Society for Experimental Hematology. Published by Elsevier Inc.

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