Faculty Profiles - TAKARADA Takeshi

Papers - TAKARADA Takeshi

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Circadian Clock Regulates Bone Resorption in Mice Reviewed

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

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

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

The circadian clock controls many behavioral and physiological processes beyond daily rhythms. Circadian dysfunction increases the risk of cancer, obesity, and cardiovascular and metabolic diseases. Although clinical studies have shown that bone resorption is controlled by circadian rhythm, as indicated by diurnal variations in bone resorption, the molecular mechanism of circadian clock-dependent bone resorption remains unknown. To clarify the role of circadian rhythm in bone resorption, aryl hydrocarbon receptor nuclear translocator-like (Bmal1), a prototype circadian gene, was knocked out specifically in osteoclasts. Osteoclast-specific Bmal1-knockout mice showed a high bone mass phenotype due to reduced osteoclast differentiation. A cell-based assay revealed that BMAL1 upregulated nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (Nfatc1) transcription through its binding to an E-box element located on the Nfatc1 promoter in cooperation with circadian locomotor output cycles kaput (CLOCK), a heterodimer partner of BMAL1. Moreover, steroid receptor coactivator (SRC) family members were shown to interact with and upregulate BMAL1: CLOCK transcriptional activity. Collectively, these data suggest that bone resorption is controlled by osteoclastic BMAL1 through interactions with the SRC family and binding to the Nfatc1 promoter. (C) 2016 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.2803

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

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

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

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

This study evaluates the pathological role of the stress sensor activating transcription factor-3 (ATF3) in ischemic neurotoxicity. Upregulation of the transcript and protein for ATF3 was seen 2-10 hr after reperfusion in the ipsilateral cerebral hemisphere of mice with transient middle cerebral artery occlusion for 2 hr. Immunohistochemical analysis confirmed the expression of ATF3 by cells immunoreactive for a neuronal marker in neocortex, hippocampus, and striatum within 2 hr after reperfusion. In murine neocortical neurons previously cultured under ischemic conditions for 2 hr, transient upregulation of both Atf3 and ATF3 expression was similarly found during subsequent culture for 2-24 hr under normoxia. Lentiviral overexpression of ATF3 ameliorated the neurotoxicity of glutamate (Glu) in cultured murine neurons along with a slight but statistically significant inhibition of both Fluo-3 and rhodamine-2 fluorescence increases by N-methyl-D-aspartate. Similarly, transient upregulation was seen in Atf3 and ATF3 expression during the culture for 48 hr in neuronal Neuro2A cells previously cultured under ischemic conditions for 2 hr. Luciferase reporter analysis with ATF3 promoter together with immunoblotting revealed the possible involvement of several transcription factors responsive to extracellular and intracellular stressors in the transactivation of the Atf3 gene in Neuro2A cells. ATF3 could be upregulated to play a role in mechanisms underlying mitigation of the neurotoxicity mediated by the endogenous neurotoxin Glu at an early stage after ischemic signal inputs. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/jnr.23723

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

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

Biochemistry and Biophysics Reports 5 89 - 95 2016.3

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

We have shown marked promotion of both proliferation and neuronal differentiation in pluripotent P19 cells exposed to the green tea amino acid theanine, which is a good substrate for SLC38A1 responsible for glutamine transport. In this study, we evaluated the activity of the mammalian target of rapamycin (mTOR) kinase pathway, which participates in protein translation, cell growth and autophagy in a manner relevant to intracellular glutamine levels, in murine neural progenitor cells exposed to theanine. Exposure to theanine promoted the phosphorylation of mTOR and downstream proteins in neurospheres from embryonic mouse neocortex. Although stable overexpression of SLC38A1 similarly facilitated phosphorylation of mTOR-relevant proteins in undifferentiated P19 cells, theanine failed to additionally accelerate the increased phosphorylation in these stable transfectants. Theanine accelerated the formation of neurospheres from murine embryonic neocortex and adult hippocampus, along with facilitation of both 5-bromo-2'-deoxyuridine incorporation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction in embryonic neurospheres. In embryonic neurospheres previously exposed to theanine, a significant increase was seen in the number of cells immunoreactive for a neuronal marker protein after spontaneous differentiation. These results suggest that theanine activates the mTOR signaling pathway for proliferation together with accelerated neurogenesis in murine undifferentiated neural progenitor cells.

DOI： 10.1016/j.bbrep.2015.09.021

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

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

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

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

Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Although interferon-related developmental regulator 1 (Ifrd1) has been identified as a transcriptional coactivator/repressor in various cells, little attention has been paid to its role in osteoblastogenesis and bone homeostasis thus far. Here, we show that Ifrd1 is a critical mediator of both the cell-autonomous regulation of osteoblastogenesis and osteoblast-dependent regulation of osteoclastogenesis. Osteoblast-specific deletion of murine Ifrd1 increased bone formation and decreased bone resorption, causing high bone mass. Ifrd1 deficiency enhanced osteoblast differentiation and maturation along with increased expression of Runx2 and osterix (Osx). Mechanistically, Ifrd1 deficiency increased the acetylation status of p65, a component of NF-B, at residues K122 and K123 via the attenuation of the interaction between p65 and histone deacetylase (HDAC). This led to the nuclear export of p65 and a decrease in NF-B-dependent Smad7 expression and the subsequent enhancement of Smad1/Smad5/Smad8-dependent transcription. Moreover, a high bone mass phenotype in the osteoblast-specific deletion of Ifrd1 was markedly rescued by the introduction of one Osx-floxed allele but not of Runx2-floxed allele. Coculture experiments revealed that Ifrd1-deficient osteoblasts have a higher osteoprotegerin (OPG) expression and a lower ability to support osteoclastogenesis. Ifrd1 deficiency attenuated the interaction between -catenin and HDAC, subsequently increasing the acetylation of -catenin at K49, leading to its nuclear accumulation and the activation of the -catenin-dependent transcription of OPG. Collectively, the expression of Ifrd1 in osteoblasts repressed osteoblastogenesis and activated osteoclastogenesis through modulating the NF-B/Smad/Osx and -catenin/OPG pathways, respectively. These findings suggest that Ifrd1 has a pivotal role in bone homeostasis through its expression in osteoblasts in vivo and represents a therapeutic target for bone diseases. (c) 2015 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.2720

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

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

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

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER/PLENUM PUBLISHERS

We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100 mu M in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.

DOI： 10.1007/s11064-015-1591-4

Genetic analysis of Runx2 function during intramembranous ossification Reviewed

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

DEVELOPMENT 143 ( 2 ) 211 - 218 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：COMPANY OF BIOLOGISTS LTD

Runt-related transcription factor 2 (Runx2) is an essential transcriptional regulator of osteoblast differentiation and its haploinsufficiency leads to cleidocranial dysplasia because of a defect in osteoblast differentiation during bone formation through intramembranous ossification. The cellular origin and essential period for Runx2 function during osteoblast differentiation in intramembranous ossification remain poorly understood. Paired related homeobox 1 (Prx1) is expressed in craniofacial mesenchyme, and Runx2 deficiency in cells of the Prx1 lineage (in mice referred to here as Runx2(prx1)(-/-)) resulted in defective intramembranous ossification. Runx2 was heterogeneously expressed in Prx1-GFP(+) cells located at the intrasutural mesenchyme in the calvaria of transgenic mice expressing GFP under the control of the Prx1 promoter. Double-positive cells for Prx1-GFP and stem cell antigen-1 (Sca1) (Prx1(+)Sca1(+) cells) in the calvaria expressed Runx2 at lower levels and were more homogeneous and primitive than Prx1(+)Sca1(-) cells. Osterix (Osx) is another transcriptional determinant of osteoblast lineages expressed by osteoblast precursors; Osx is highly expressed by Prx1(-)Runx2(+) cells at the osteogenic front and on the surface of mineralized bone in the calvaria. Runx2 deficiency in cells of the Osx lineage (in mice referred to here as Runx2(osx)(-/-)) resulted in severe defects in intramembranous ossification. These findings indicate that the essential period of Runx2 function in intramembranous ossification begins at the Prx1(+)Sca1(+) mesenchymal stem cell stage and ends at the Osx(+)Prx1(-)Sca1(-) osteoblast precursor stage.

DOI： 10.1242/dev.128793

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

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

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

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

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

DOI： 10.1002/jcp.24988

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

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

CELL 161 ( 7 ) 1576 - 1591 2015.6

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

The synthesis of type I collagen, the main component of bone matrix, precedes the expression of Runx2, the earliest determinant of osteoblast differentiation. We hypothesized that the energetic needs of osteoblasts might explain this apparent paradox. We show here that glucose, the main nutrient of osteoblasts, is transported in these cells through Glut1, whose expression precedes that of Runx2. Glucose uptake favors osteoblast differentiation by suppressing the AMPK-dependent proteasomal degradation of Runx2 and promotes bone formation by inhibiting another function of AMPK. While RUNX2 cannot induce osteoblast differentiation when glucose uptake is compromised, raising blood glucose levels restores collagen synthesis in Runx2-null osteoblasts and initiates bone formation in Runx2-deficient embryos. Moreover, RUNX2 favors Glut1 expression, and this feedforward regulation between RUNX2 and Glut1 determines the onset of osteoblast differentiation during development and the extent of bone formation throughout life. These results reveal an unexpected intricacy between bone and glucose metabolism.

DOI： 10.1016/j.cell.2015.05.029

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

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

PLOS ONE 10 ( 5 ) e0127421 2015.5

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

Background
Several chemicals have been widely used to evaluate the involvement of free Ca2+ in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca2+-sensitive reagents in diverse cultured cells.
Methodology/Principal Findings
In rat astrocytic C6 glioma cells loaded with the fluorescent Ca2+ dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca2+ chelator EGTA irrespective of added CaCl2. The addition of the Ca2+ ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn2+ ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn2+ chelator N, N, N', N'-tetrakis(2-pyridylmethyl) ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters.
Conclusions/Significance
Taken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses mediated by Ca2+ in diverse cells enriched of Zn2+.

DOI： 10.1371/journal.pone.0127421

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

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

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

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

Posttraumatic stress disorder is a long-lasting psychiatric disease with the consequence of hippocampal atrophy in humans exposed to severe fatal stress. We demonstrated a positive correlation between the transient decline of 5-bromo-2 '-deoxyuridine (BrdU) incorporation in the hippocampal dentate gyrus (DG) and long-lasting behavioral abnormalities in mice with traumatic stress. Here, we investigated pharmacological properties of theanine on the declined BrdU incorporation and abnormal behaviors in mice with traumatic stress. Prior daily oral administration of theanine at 50-500 mg/kg for 5 days significantly prevented the decline of BrdU incorporation, while theanine significantly prevented the decline in the DG even when administered for 5 days after stress. Consecutive daily administration of theanine significantly inhibited the prolonged immobility in mice with stress in forced swimming test seen 14 days later. Although traumatic stress significantly increased spontaneous locomotor activity over 30 min even when determined 14 days later, the increased total locomotion was significantly ameliorated following the administration of theanine at 50 mg/kg for 14 days after stress. These results suggest that theanine alleviates behavioral abnormalities together with prevention of the transient decline of BrdU incorporation in the hippocampal DG in adult mice with severe traumatic stress. (C) 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

DOI： 10.1016/j.jphs.2014.12.018

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

Takarada, T.

Folia Pharmacologica Japonica 144 ( 4 ) 178 - 184 2014.10

Recent advances in Runx2 research Reviewed

Takarada, T.

Folia Pharmacologica Japonica 144 ( 2 ) 98 - 98 2014.8

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

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

NEUROCHEMISTRY INTERNATIONAL 74 24 - 35 2014.7

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

Runt-related transcription factor-2 (Runx2) is the master regulator of osteoblastogenesis with an ability to promote differentiation of mesenchymal stem cells into the osteoblastic lineage. We have previously shown constitutive and functional expression of Runx2 by astroglial cells. In this study, we investigated the possible expression of Runx2 by both murine microglia and microglial cell line BV-2 cells. Runx2 expression was seen in cultured microglia and BV-2 cells, while sustained exposure to 1 mM ATP led to a significant but transient increase in mRNA and corresponding protein expression of Runx2 within 24 h. The increase in Runx2 expression was invariably prevented by several chemicals with antagonistic properties for P2X7 purinergic receptor, calmodulin and calcineurin in BV-2 cells, with a P2X7 receptor agonist more than quadrupling Runx2 expression. A significant increase in Runx2 expression was seen in osteoclastic cells, but not in osteoblastic or chondrocytic cells, when exposed to a high concentration of ATP. In BV2-cells with control siRNA, a significant decrease was found in the number of cells with at least one process within 3 h after the exposure to 1 mM ATP, followed by an increase up to 24 h. However, Runx2 siRNA significantly deteriorated the property to induce delayed process extension during 6-24 h after exposure to ATP along with drastically decreased Runx2 protein levels. These results suggest that Runx2 is constitutively and functionally expressed by microglial cells with responsiveness to ATP for upregulation in the murine brain. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2014.04.010

Recent advances in research on mesenchymal stem cells

Takarada Takeshi

Folia Pharmacologica Japonica 144 ( 6 ) 305 - 305 2014

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

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

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

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

Global gene deletion studies in mice and humans have established the pivotal role of runt related transcription factor-2 (Runx2) in both intramembranous and endochondral ossification processes during skeletogenesis. In this study, we for the first time generated mice carrying a conditional Runx2 allele with exon 4, which encodes the Runt domain, flanked by loxP sites. These mice were crossed with 1(I)-collagen-Cre or 1(II)-collagen-Cre transgenic mice to obtain osteoblast-specific or chondrocyte-specific Runx2 deficient mice, respectively. As seen in Runx2(-/-) mice, perinatal lethality was observed in 1(II)-Cre;Runx2(flox/flox) mice, but this was not the case in animals in which 1(I)-collagen-Cre was used to delete Runx2. When using double-staining with Alizarin red for mineralized matrix and Alcian blue for cartilaginous matrix, we observed previously that mineralization was totally absent at embryonic day 15.5 (E15.5) throughout the body in Runx2(-/-) mice, but was found in areas undergoing intramembranous ossification such as skull and clavicles in 1(II)-Cre;Runx2(flox/flox) mice. In newborn 1(II)-Cre;Runx2(flox/flox) mice, mineralization impairment was restricted to skeletal areas undergoing endochondral ossification including long bones and vertebrae. In contrast, no apparent skeletal abnormalities were seen in mutant embryo, newborn, and 3-week-old to 6-week old-mice in which Runx2 had been deleted with the 1(I)-collagen-Cre driver. These results suggest that Runx2 is absolutely required for endochondral ossification during embryonic and postnatal skeletogenesis, but that disrupting its expression in already committed osteoblasts as achieved here with the 1(I)-collagen-Cre driver does not affect overtly intramembranous and endochondral ossification. The Runx2 floxed allele established here is undoubtedly useful for investigating the role of Runx2 in particular cells. (c) 2013 American Society for Bone and Mineral Research.

DOI： 10.1002/jbmr.1945

Regulatory Mechanisms of Skeletal Tissues by Amino Acid Signaling Reviewed

Takeshi Takarada

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

Language：Japanese Publisher：PHARMACEUTICAL SOC JAPAN

In this review, we would outline the possible signaling system for three types of amino acids including glutamate (Glu), gamma-aminobutyric acid (GABA) and D-serine (D-Ser) to play a role as an extracellular signal mediator in mechanisms underlying maintenance of the cellular homeostasis in skeletal tissues. Although Glu and GABA has been thought to be an excitatory/inhibitory amino acid neurotransmitter in the mammalian central nervous system, our molecular biological analyses give rise to a novel function for Glu and GABA as an autocrine and/or paracrine factor in three types of distinct cell types including osteoblasts, osteoclasts and chondrocytes in bone tissues. Moreover, D-Ser plays a pivotal role in osteoclastogenesis through a mechanism related to the incorporation of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts. Accordingly, bone formation and maintenance seems to be under control by amino acid signaling in skeletal tissues as seen with neurotransmission in the brain.

DOI： 10.1248/yakushi.13-00062

Mitochondrial uncoupling protein-2 in glutamate neurotoxicity. Reviewed

Takarada T, Fukumori R, Yoneda Y

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

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

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

PLOS ONE 8 ( 7 ) e69718 2013.7

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

Background: We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca2+ levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca2+ levels in cultured murine neocortical neurons.
Methodology/Principal Findings: In neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca2+ levels determined by Rhod-2 at concentrations of 0.1 to 100 mu M. Further addition of 250 mM ethanol significantly inhibited the increase by glutamate and NMDA in Rhod-2 fluorescence, while similarly potent inhibition of the NMDA-induced increase was seen after exposure to ethanol at 50 to 250 mM in cultured neurons. Lentiviral overexpression of UCP2 significantly accelerated the increase by NMDA in Rhod-2 fluorescence in neurons, without affecting Fluo-3 fluorescence for intracellular Ca2+ levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca2+ levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca2+ levels irrespective of UCP2 overexpression. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by ethanol in neurons exposed to glutamate. In HEK293 cells with NMDAR containing GluN2B subunit, more efficient inhibition was similarly induced by ethanol at 50 and 250 mM on the NMDA-induced increase in mitochondrial Ca2+ levels than in those with GluN2A subunit. Decreased protein levels of GluN2B, but not GluN2A, subunit were seen in immunoprecipitates with UCP2 from neurons with brief exposure to ethanol at concentrations over 50 mM.
Conclusions/Significance: Ethanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca2+ incorporation and cell death after NMDAR activation in neurons.

DOI： 10.1371/journal.pone.0069718

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

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

PLoS ONE 8 ( 5 ) e63947 2013.5

Language：English Publishing type：Research paper (scientific journal) Publisher：5

Background:We have previously shown marked upregulation of the mRNA and corresponding protein for the cellular motor molecule myosin VI (Myo6) after an extremely traumatic stress experience, along with a delayed decrease in 5-bromo-2′-deoxyuridine incorporation in the murine hippocampus, a brain structure believed to undergo adult neurogenesis. In this study, we investigated the role of Myo6 in both proliferation and differentiation in pluripotent P19 cells by using stable transfection and RNA interference techniques.Methodology/Principal Findings:Stable overexpression of Myo6 not only led to significant inhibition of the reducing activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and the size of clustered aggregates in P19 cells, but also resulted in selectively decreased mRNA expression of the repressor type proneural gene Hes5 without affecting the expression of neuronal and astroglial marker proteins. In P19 cells transfected with Myo6 siRNA, by contrast, a significant increase was found in the size of aggregate and MTT reduction along with increased Sox2 protein levels, in addition to marked depletion of the endogenous Myo6 protein. In C6 glioma cells, however, introduction of Myo6 siRNA induced a drastic decrease in endogenous Myo6 protein levels without significantly affecting MTT reduction. The Ca2+ ionophore A23187 drastically increased the luciferase activity in P19 cells transfected with a Myo6 promoter reporter plasmid, but not in HEK293, Neuro2A and C6 glioma cells transfected with the same reporter.Conclusions/Significance:These results suggest that Myo6 may play a predominant pivotal role in the mechanism underlying proliferation without affecting differentiation to progeny lineages in pluripotent P19 cells. © 2013 Takarada et al.

DOI： 10.1371/journal.pone.0063947

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

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

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

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

Pre-chondrocytes undergo cellular differentiation stages during chondrogenesis under the influence by different transcription factors such as sry-type high mobility group box-9 (Sox9) and runt-related transcription factor-2 (Runx2). We have shown upregulation by parathyroid hormone (PTH) of the clock gene Period-1 (Per1) through the cAMP/protein kinase A signaling pathway in pre-chondrocytic ATDC5 cells. Here, we investigated the role of Per1 in the suppression of chondrogenic differentiation by PTH. In ATDC5 cells exposed to 10 nM PTH, a drastic but transient increase in Per1 expression was seen only 1 h after addition together with a prolonged decrease in Sox6 levels. However, no significant changes were induced in Sox5 and Runx2 levels in cells exposed to PTH. In stable Per1 transfectants, a significant decrease in Sox6 levels was seen, with no significant changes in Sox5 and Sox9 levels, in addition to the inhibition of gene transactivation by Sox9 allies. Knockdown of Per1 by siRNA significantly increased the Sox6 and type II collagen levels in cells cultured for 24-60 h. These results suggest that Per1 plays a role in the suppressed chondrocytic differentiation by PTH through a mechanism relevant to negative regulation of transactivation of the Sox6 gene during chondrogenesis. © The Japanese Pharmacological Society.

DOI： 10.1254/jphs.13091FP

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