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Takahito Tamai,
Osamu Yamaguchi,
Shungo Hikoso,
Toshihiro Takeda,
Manabu Taneike,
Takafumi Oka,
Jota Oyabu,
Tomokazu Murakawa,
Hiroyuki Nakayama, Yoshihiro Uno,
Kyoji Horie,
Kazuhiko Nishida,
Nahum Sonenberg,
Ajay M Shah,
Junji Takeda,
Issei Komuro,
Kinya Otsu
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ABSTRACT: Cardiomyocytes proliferate during fetal life, but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb(-/-)). Rheb(-/-) mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb(-/-) was lower than that in the control (Rheb(+/+)) at postnatal day 8. The cell surface area of cardiomycytes isolated from the mouse hearts increased from postnatal day 5 to 8 in Rheb(+/+) mice, but not in Rheb(-/-) mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb(-/-) hearts during the neonatal period. Rheb(-/-) hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1, at postnatal day 3, but showed attenuation at postnatal day 5 or 8 compared to the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb(-/-) hearts at postnatal day 8. Futhermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb(-/-) mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.
Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor
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Chikara Kokubu,
Kyoji Horie,
Koichiro Abe,
Ryuji Ikeda,
Sumi Mizuno, Yoshihiro Uno,
Sanae Ogiwara,
Masato Ohtsuka,
Ayako Isotani,
Masaru Okabe,
Kenji Imai,
Junji Takeda
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ABSTRACT: A large cis-regulatory landscape is a common feature of vertebrate genomes, particularly at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying large genomic regions of interest spanning over hundreds of kilobases are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping trait of the Sleeping Beauty transposon in the mouse genome. We generated embryonic stem cells with a targeted integration of the transposon vector, carrying an enhancer-detecting lacZ reporter and loxP cassette, into the developmentally critical Pax1 gene locus, followed by efficient local transpositions, nested deletion formation and derivation of embryos by tetraploid complementation. Comparative reporter expression analysis among different insertion/deletion embryos substantially facilitated long-range cis-regulatory element mapping in the genomic neighborhood and demonstrated the potential of the transposon-based approach as a versatile tool for exploration of defined genomic intervals of functional or clinical relevance, such as disease-associated microdeletions.
Nature Genetics 09/2009; 41(8):946-52. · 35.53 Impact Factor
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Shungo Hikoso,
Osamu Yamaguchi,
Yuko Nakano,
Toshihiro Takeda,
Shigemiki Omiya,
Isamu Mizote,
Manabu Taneike,
Takafumi Oka,
Takahito Tamai,
Jota Oyabu, Yoshihiro Uno,
Yasushi Matsumura,
Kazuhiko Nishida,
Keiichiro Suzuki,
Mikihiko Kogo,
Masatsugu Hori,
Kinya Otsu
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ABSTRACT: Cardiomyocyte death plays an important role in the pathogenesis of heart failure. The nuclear factor (NF)-kappaB signaling pathway regulates cell death, however, the effect of NF-kappaB pathway on cell death can vary in different cells or stimuli. The purpose of the present study was to clarify the in vivo role of the NF-kappaB pathway in response to pressure overload. First, we subjected C57Bl6/J mice to pressure overload by means of transverse aortic constriction (TAC) and examined the activity of the NF-kappaB pathway in response to pressure overload. IkappaB kinase (IKK) and NF-kappaB were activated after TAC. Then, we investigated the role of the activation using cardiac-specific IKKbeta-deficient mice (CKO). CKO displayed normal global cardiac structure and function compared with control littermates. We subjected CKO and control mice to pressure overload. One week after TAC, CKO showed cardiac dilation, dysfunction, and lung congestion, which are characteristics of heart failure. The number of apoptotic cells in the hearts of CKO mice increased significantly after TAC. The levels of manganese superoxide dismutase mRNA and protein expression in CKO after TAC were significantly attenuated compared with control mice. The levels of oxidative stress and c-Jun N-terminal kinase (JNK) activation in CKO after TAC were significantly greater than those in control mice. Isoproterenol-induced cell death of isolated adult CKO cardiomyocytes was inhibited by treatment with either a manganese superoxide dismutase mimetic or a JNK inhibitor. Thus, the IKKbeta/NF-kappaB signaling pathway plays a protective role in cardiomyocytes because of the attenuation of oxidative stress and JNK activation in a setting of acute pressure overload.
Circulation Research 06/2009; 105(1):70-9. · 9.49 Impact Factor
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Kojiro Yae,
Vincent W Keng,
Masato Koike,
Kosuke Yusa,
Michiyoshi Kouno, Yoshihiro Uno,
Gen Kondoh,
Takahiro Gotow,
Yasuo Uchiyama,
Kyoji Horie,
Junji Takeda
[show abstract]
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ABSTRACT: The Sleeping Beauty (SB) transposon system has generated many transposon-insertional mutant mouse lines, some of which have resulted in embryonic lethality when bred to homozygosity. Here we report one such insertion mapped to the mouse actin-related protein complex subunit 3 gene (Arpc3). Arpc3 is a component of the Arp2/3 complex, which plays a major role in actin nucleation with Y-shaped branching from the mother actin filament in response to migration signaling. Arpc3 transposon-inserted mutants developed only to the blastocyst stage. In vitro blastocyst culture of Arpc3 mutants exhibited severe spreading impairment of trophoblasts. This phenotype was also observed in compound heterozygotes generated using conventional gene-targeted and transposon-inserted alleles. Arpc3-deficient mutants were shown to lack actin-rich structures in the spreading trophoblast. Electron microscopic analysis demonstrated the lack of mesh-like structures at the cell periphery, suggesting a role of Arpc3 in Y-shaped branching formation. These data indicate the importance of Arpc3 in the Arp2/3 complex for trophoblast outgrowth and suggest that Arpc3 may be indispensable for implantation.
Molecular and Cellular Biology 09/2006; 26(16):6185-96. · 5.53 Impact Factor
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Vincent W Keng,
Kojiro Yae,
Tomoko Hayakawa,
Sumi Mizuno, Yoshihiro Uno,
Kosuke Yusa,
Chikara Kokubu,
Taroh Kinoshita,
Keiko Akagi,
Nancy A Jenkins,
Neal G Copeland,
Kyoji Horie,
Junji Takeda
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ABSTRACT: Recent consolidation of the whole-genome sequence with genome-wide transcriptome profiling revealed the existence of functional units within the genome in specific chromosomal regions, as seen in the coordinated expression of gene clusters and colocalization of functionally related genes. An efficient region-specific mutagenesis screen would greatly facilitate research in addressing the importance of these clusters. Here we use the 'local hopping' phenomenon of a DNA-type transposon, Sleeping Beauty (SB), for region-specific saturation mutagenesis. A transgenic mouse containing both transposon (acts as a mutagen) and transposase (recognizes and mobilizes the transposon) was bred for germ-cell transposition events, allowing us to generate many mutant mice. All genes within a 4-Mb region of the original donor site were mutated by SB, indicating the potential of this system for functional genomic studies within a specific chromosomal region.
Nature Methods 11/2005; 2(10):763-9. · 19.28 Impact Factor
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Kazuhiko Nishida,
Osamu Yamaguchi,
Shinichi Hirotani,
Shungo Hikoso,
Yoshiharu Higuchi,
Tetsuya Watanabe,
Toshihiro Takeda,
Soh Osuka,
Takashi Morita,
Gen Kondoh, [......],
Yasushi Matsumura,
Jun-ichi Miyazaki,
Tatsuhiko Sudo,
Kenichi Hongo,
Yoichiro Kusakari,
Satoshi Kurihara,
Kenneth R Chien,
Junji Takeda,
Masatsugu Hori,
Kinya Otsu
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ABSTRACT: The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38alpha is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38alpha in hearts. First, we generated mice with floxed p38alpha alleles and crossbred them with mice expressing the Cre recombinase under the control of the alpha-myosin heavy-chain promoter to obtain cardiac-specific p38alpha knockout mice. These cardiac-specific p38alpha knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38alpha plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.
Molecular and Cellular Biology 01/2005; 24(24):10611-20. · 5.53 Impact Factor
