研究者詳細 - 寳田　剛志

Inhibition of the glutamine transporter SNAT1 confers neuroprotection in mice by modulating the mTOR-autophagy system. 査読国際誌

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

Communications biology 2 346 - 346 2019年

記述言語：英語掲載種別：研究論文（学術雑誌）

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

DOI： 10.1038/s42003-019-0582-4

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

Nakamichi, N., Takarada, T.

Yakugaku Zasshi 139 ( 6 ) 845 - 846 2019年

RUNX2 Promotes Malignant Progression in Glioma. 査読国際誌

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

Neurochemical research 43 ( 11 ) 2047 - 2054 2018年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

DOI： 10.1007/s11064-018-2626-4

The MAPK Erk5 is necessary for proper skeletogenesis involving a Smurf-Smad-Sox9 molecular axis. 査読国際誌

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

Development (Cambridge, England) 145 ( 14 ) 2018年7月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

DOI： 10.1242/dev.164004

Radiobrominated benzimidazole-quinoline derivatives as Platelet-derived growth factor receptor beta (PDGFRβ) imaging probes. 査読国際誌

Effendi N, Mishiro K, Takarada T, Makino A, Yamada D, Kitamura Y, Shiba K, Kiyono Y, Odani A, Ogawa K

Scientific reports 8 ( 1 ) 10369 - 10369 2018年7月

Physiological role of urothelial cancer-associated one long noncoding RNA in human skeletogenic cell differentiation. 査読国際誌

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

Journal of cellular physiology 233 ( 6 ) 4825 - 4840 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

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

DOI： 10.1002/jcp.26285

生後の骨・軟骨におけるアグリカンの役割の解明

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

日本結合組織学会学術大会プログラム・抄録集 50回 155 - 155 2018年6月

Core Binding Factor β Expression in Ovarian Granulosa Cells Is Essential for Female Fertility. 査読国際誌

Lee-Thacker S, Choi Y, Taniuchi I, Takarada T, Yoneda Y, Ko C, Jo

Endocrinology 159 ( 5 ) 2094 - 2109 2018年5月

Type IV collagen α6 chain is a regulator of keratin 10 in keratinization of oral mucosal epithelium. 査読国際誌

Komori T, Ono M, Hara ES, Ueda J, Nguyen HTT, Nguyen HT, Yonezawa T, Maeba T, Kimura-Ono A, Takarada T, Momota R, Maekawa K, Kuboki T, Oohashi T

Scientific reports 8 ( 1 ) 2612 - 2612 2018年2月

Disruption of Bmal1 Impairs Blood-Brain Barrier Integrity via Pericyte Dysfunction. 査読国際誌

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

The Journal of neuroscience : the official journal of the Society for Neuroscience 37 ( 42 ) 10052 - 10062 2017年10月

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

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

DOI： 10.1523/JNEUROSCI.3639-16.2017

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

Effendi N, Ogawa K, Mishiro K, Takarada T, Yamada D, Kitamura Y, Shiba K, Maeda T, Odani A

Bioorganic & medicinal chemistry 25 ( 20 ) 5576 - 5585 2017年10月

Deletion of Runx2 in Articular Chondrocytes Decelerates the Progression of DMM-Induced Osteoarthritis in Adult Mice. 査読国際誌

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

Scientific reports 7 ( 1 ) 2371 - 2371 2017年5月

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

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

DOI： 10.1038/s41598-017-02490-w

Scopus

Bone Resorption Is Regulated by Circadian Clock in Osteoblasts 査読

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

Journal of Bone and Mineral Research 32 ( 4 ) 872 - 881 2017年4月

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

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

DOI： 10.1002/jbmr.3053

Scopus

Bone Resorption Is Regulated by Circadian Clock in Osteoblasts. 査読国際誌

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 32 ( 4 ) 872 - 881 2017年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY

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

DOI： 10.1002/jbmr.3053

The transcriptional modulator Ifrd1 controls PGC-1α expression under short-term adrenergic stimulation in brown adipocytes. 査読

Park G, Horie T, Kanayama T, Fukasawa K, Iezaki T, Onishi Y, Ozaki K, Nakamura Y, Yoneda Y, Takarada T, Hinoi E

The FEBS journal 284 ( 5 ) 784 - 795 2017年3月

The intrinsic microglial clock system regulates interleukin-6 expression. 査読国際誌

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

Glia 65 ( 1 ) 198 - 208 2017年1月

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

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

DOI： 10.1002/glia.23087

Transcriptional Modulator Ifrd1 Regulates Osteoclast Differentiation through Enhancing the NF-κB/NFATc1 Pathway. 査読

Iezaki T, Fukasawa K, Park G, Horie T, Kanayama T, Ozaki K, Onishi Y, Takahata Y, Nakamura Y, Takarada T, Yoneda Y, Nakamura T, Vacher J, Hinoi E

Molecular and cellular biology 36 ( 19 ) 2451 - 2463 2016年10月

ATF3 deficiency in chondrocytes alleviates osteoarthritis development 査読

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

JOURNAL OF PATHOLOGY 239 ( 4 ) 426 - 437 2016年8月

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

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

DOI： 10.1002/path.4739