Faculty Profiles - TAKARADA Takeshi

Papers - TAKARADA Takeshi

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[Role of glutamine transporter expressed in astroglia cells in glutamic acid-induced neuronal death]. Reviewed

Safuru, A., Kokura, M., Takarada, T., Yoneta, Y.

Nihon shinkei seishin yakurigaku zasshi = Japanese journal of psychopharmacology 31 ( 2 ) 91 - 93 2011.4

Glutamate Preferentially Suppresses Osteoblastogenesis Than Adipogenesis Through the Cystine/Glutamate Antiporter in Mesenchymal Stem Cells Reviewed

Mika Takarada-Iemata, Takeshi Takarada, Yukari Nakamura, Eri Nakatani, Osamu Hori, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 226 ( 3 ) 652 - 665 2011.3

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

We have shown that glutamate (Glu) signaling machineries, such as receptors (GluR) and transporters, are functionally expressed by mesenchymal stem cells, in addition to by their progeny cells such as osteoblasts and chondrocytes. Sustained exposure to Glu induced significant decreases in alkaline phosphatase (ALP) staining and osteoblastic marker gene expression in the mesenchymal C3H10T1/2 stem cells infected with runt-related transcription factor-2 (Runx2) adenovirus, without markedly affecting Oil Red O staining for adipocytes in cells cultured with adipogenic inducers. In cells with Runx2 adenovirus, the cystine/Glu antiporter substrate cystine significantly prevented the decreases by Glu in both ALP staining and osteoblastic marker gene expression, with GluR agonists being ineffective. In cells with Runx2 adenovirus, Glu significantly decreased [C-14] cystine uptake, intracellular glutathione (GSH) level, Runx2 recruitment to osteocalcin promoter and nuclear Runx2 protein level, respectively. Cystine again significantly prevented the decreases by Glu in both GSH levels and Runx2 recruitment. In mouse bone marrow stromal cells, Glu and a GSH depleter significantly decreased ALP staining without affecting Oil Red O staining. Knockdown of the cystine/Glu antiporter led to markedly decreased ALP staining and GSH levels, with concomitant prevention of the decrease by Glu, in cells with Runx2 adenovirus. These results suggest that Glu may play a role as a negative regulator at an early differentiation stage into osteoblasts than adipocytes through a mechanism relevant to nuclear translocation of Runx2 after regulation of intracellular GSH levels by the cystine/Glu antiporter expressed in mesenchymal stem cells. J. Cell. Physiol. 226: 652-665, 2011. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jcp.22390

Exacerbated vulnerability to oxidative stress in astrocytic C6 glioma cells with stable overexpression of the glutamine transporter slc38a1 Reviewed

Masato Ogura, Takeshi Takarada, Noritaka Nakamichi, Hirofumi Kawagoe, Aya Sako, Ryota Nakazato, Yukio Yoneda

NEUROCHEMISTRY INTERNATIONAL 58 ( 4 ) 504 - 511 2011.3

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

We have previously demonstrated the functional expression of glutamine (Gln) transporter (GlnT) believed to predominate in neurons for the neurotransmitter glutamate pool by rat neocortical astrocytes devoid of neuronal marker expression, with exacerbated vulnerability to oxidative stress after transient overexpression. To evaluate molecular mechanisms underlying the exacerbation, we established stable GlnT transfectants in rat astrocytic C6 glioma cells. In two different clones of stable transfectants with increased intracellular Gln levels, exposure to hydrogen peroxide (H(2)O(2)) and A23187, but not to tunicamycin or 2,4-dinitrophenol, led to significant exacerbation of the cytotoxicity compared to cells with empty vector (EV). Stable GlnT overexpression led to a significant increase in heme oxygenase-1 protein levels in a manner sensitive to H(2)O(2), whereas H(2)O(2) was significantly more effective in increasing NO(2) accumulation and reactive oxygen species (ROS) generation in stable GlnT transfectants than in EV cells. Moreover, exposure to A23187 led to a more effective increase in the generation of ROS in stable GlnT transfectants than in stable EV transfectants. These results suggest that GlnT may play a role in the mechanisms underlying the determination of cellular viability in astrocytes through modulation of intracellular ROS generation. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2011.01.007

A Negative Correlation Between Expression Profiles of Runt-Related Transcription Factor-2 and Cystine/Glutamate Antiporter xCT Subunit in Ovariectomized Mouse Bone Reviewed

Kyosuke Uno, Takeshi Takarada, Yukari Nakamura, Hiroyuki Fujita, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 115 ( 3 ) 309 - 319 2011.3

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

We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the bidirectional cystine/Glu antiporter in undifferentiated osteoblastic MC3T3-E1 cells. In this study, we investigated the expression profile of the xCT subunit of the antiporter as well as the master regulator of osteoblastogenesis runt-related transcription factor-2 (Runx2) in ovariectomized mouse bone. In spinal columns isolated 28 days after ovariectomy, a marked reduction was seen with the intensity of Von Kossa staining used as an index of ossification. In femurs of these ovariectomized mice, a significant decrease was seen in mRNA and protein levels of Runx2 along with increased expression of both mRNA and the corresponding protein for the xCT subunit. To evaluate the possible role of the antiporter in osteoblastogenesis, stable transfectants were established with the xCT subunit toward the culture with osteoblastic differentiation inducers in MC3T3-E1 cells. In stable xCT transfectants cultured under differentiation conditions, marked decreases were seen in nodule formation, Ca2+ accumulation, and osteoblastic marker gene expression, in addition to downregulation of both mRNA and the corresponding protein for Runx2. Runx2 promoter activity was markedly stimulated in MC3T3-E1 cells transfected with a responsive promoter plasmid after the culture under differentiation conditions, while transient and stable transfection with xCT expression vector invariably prevented the stimulation through an activator protein-1 site. These results suggest that Runx2 expression would be negatively regulated by the cystine/glutamate antiporter expressed by osteoblastic cells at the level of gene transactivation.

DOI： 10.1254/jphs.10310FP

GABAB R1サブユニット欠損マウスにおける神経系前駆細胞の機能変化

福井正樹, 小澤秀介, 宝田剛志, 米田幸雄

日本薬理学雑誌 137 ( 3 ) 29P - 29P 2011.3

Gradual Downregulation of Protein Expression of the Partner GABA(B)R2 Subunit During Postnatal Brain Development in Mice Defective of GABA(B)R1 Subunit Reviewed

Masaki Fukui, Shusuke Ozawa, Noritaka Nakamichi, Ryota Nakazato, Takeshi Takarada, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 115 ( 1 ) 45 - 55 2011.1

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

We have previously shown the functional expression of GABA(B) receptors (GABA(B)R) composed of GABA(B)R1 and GABA(B)R2 subunits with ability to promote proliferation and neuronal differentiation in cultured neural progenitor cells (NPC) isolated from embryonic mouse brains. In this study, we evaluated postnatal changes in the expression profiles of different markers for progenitor, neuronal, astroglial, and microglial cells in brains of GABA(B)R1-null mice. Consistent with undifferentiated murine NPC cultured with epidermal growth factor, a significant and selective decrease was seen in mRNA expression of the proneural gene Mash1 in brains of GABA(B)R1-null mice at I day after birth. The expression of several NPC marker proteins was similarly decreased in brains of both wild-type and GABA(B)R1-null mice from 1 to 7 days after birth, while slight changes were induced in both mRNA and proteins for neuronal, astroglial, and microglial markers between wild-type and GABA(B)R1-null mouse brains within this developmental stage. In particular discrete brain structures of adult GABA(B)R1-null mice at 56 days after birth, a significant decrease was seen in neuronal marker protein levels along with a significant increase in both astroglial and microglial marker protein expression. Although no significant difference was found in mRNA expression of the partner GABA(B)R2 subunit between wild-type and GABA(B)R1-null mouse brains, GABA(B)R2 subunit protein levels were gradually declined during postnatal development within 56 days after birth in GABA(B)R1-null mouse brains. These results suggest that GABA(B)R2 protein levels are closely correlated with the partner subunit GABA(B)R1 protein levels in mouse brains during postnatal development in vivo.

DOI： 10.1254/jphs.10254FP

Requirement of both NR3A and NR3B subunits for dominant negative properties on Ca2+ mobilization mediated by acquired N-methyl-D-aspartate receptor channels into mitochondria Reviewed

Ryo Fukumori, Takeshi Takarada, Noritaka Nakamichi, Yuki Kambe, Hirofumi Kawagoe, Ryota Nakazato, Yukio Yoneda

NEUROCHEMISTRY INTERNATIONAL 57 ( 7 ) 730 - 737 2010.12

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

Conventional N-methyl-D-aspartate (NMDA) receptor (NMDAR) is a heteromeric complex between the essential NR1 subunit and one of NR2A-D subunits toward functional channels permeable to Ca2+ rather than Na+ ions. Although recent studies identified dominant negative NR3A and NR3B subunits, whether these subunits inhibit Ca2+ mobilization through NMDAR channels into mitochondria is not clarified so far. In this study, we investigated Ca2+ influx across acquired NMDAR channels composed of different NR subunits artificially expressed in HEK293 cells. The addition of NMDA markedly increased intracellular free Ca2+ levels determined by Fluo-3 in cells transfected with either NR2A or NR2B subunit together with NR1 subunit. Further addition of dizocilpine completely inhibited the increase by NMDA in both types of acquired channels, while the NR2B subunit selective antagonist ifenprodil drastically inhibited the increase by NMDA in cells expressing NR1/NR2B, but not NR1/NR2A, subunits. Similar pharmacological profiles were invariably seen with cell death by NMDA. Introduction of both NR3A and NR3B subunits significantly inhibited the increase by NMDA in intracellular free Ca2+ levels in both acquired channels, while introduction of either NR3A or NR3B alone was ineffective. Co-expression of both NR3A and NR3B subunits was also required for the prevention of increased mitochondrial free Ca2+ levels determined by Rhod-2, as well as decreased cellular viability, in cells expressing NR1/NR2A or NR1/NR2B subunits upon exposure to NMDA. These results suggest that co-expression of both NR3A and NR3B subunits is essential for the dominant negative properties on Ca2+ mobilization through acquired functional NMDAR channels into mitochondria. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuint.2010.08.009

Preferential Inhibition by Antidiarrheic 2-Methoxy-4-Methylphenol of Ca2+ Influx Across Acquired N-Methyl-D-Aspartate Receptor Channels Composed of NR1/NR2B Subunit Assembly Reviewed

Noritaka Nakamichi, Ryo Fukumori, Takeshi Takarada, Yuki Kambe, Tomomi Yamamoto, Nobuyuki Matsushima, Nobuaki Moriguchi, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 88 ( 11 ) 2483 - 2493 2010.8

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

In our previous studies, particular phenolic ingredients, such as 2-methoxy-4-methylphenol (2M4MP), of the antidiarrheic drug wood creosote significantly prevented cell death by both hydrogen peroxide and glutamate in cultured rat hippocampal neurons. In this study, we further evaluated the pharmacological properties of 2M4MP on Ca2+ influx across native and acquired N-methyl-D-aspartate (NMDA) receptor (NMDAR) channels. The addition of 2M4MP significantly prevented the loss of cellular viability and the increase in intracellular free Ca2+ levels as determined by Fluo-3 in cultured rat hippocampal neurons briefly exposed to NMDA. Brief exposure to NMDA also led to a marked increase in mitochondrial free Ca2+ levels determined by Rhod-2, in addition to intracellular free Ca2+ levels, in HEK293 cells expressing either NR1/NR2A or NR1/NR2B subunit channels. The further addition of the general NMDAR channel blocker dizocilpine similarly inhibited the increase of intracellular Ca2+ levels by NMDA in both types of acquired NMDAR channels, whereas the NR2B subunit selective antagonist ifenprodil drastically inhibited the increase by NMDA in HEK293 cells expressing NR1/NR2B, but not NR1/NR2A, subunits. Similarly, 2M4MP significantly and selectively inhibited the NMDA-induced influx of Ca2+ across acquired NR1/NR2B channels in a concentration-dependent manner. Moreover, prior daily oral administration of 2M4MP significantly reduced the infarct volume in the ipsilateral cerebral hemisphere in rats with middle cerebral artery occlusion 1 day after reperfusion. These results suggest that 2M4MP may protect neurons from excitotoxicity through preferential inhibition of Ca2+ influx across NMDAR channels composed of NR1/NR2B subunits. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jnr.22399

Induced Tolerance to Glutamate Neurotoxicity Through Down-Regulation of NR2 Subunits of N-Methyl-D-Aspartate Receptors in Cultured Rat Striatal Neurons Reviewed

Yuki Kambe, Noritaka Nakamichi, Takeshi Takarada, Ryo Fukumori, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 88 ( 10 ) 2177 - 2187 2010.8

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

We have previously shown differential vulnerabilities to glutamate (Glu) excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptor (NMDAR) between rat cortical and rat hippocampal neurons in culture. In this study, we evaluated the possible induced tolerance to NMDA neurotoxicity in cultured rat striatal neurons with prior sustained activation of NMDAR. Brief exposure to Glu or NMDA for 1 hr led to a significant decrease in cellular vitality determined 24 hr later in cultured rat striatal neurons, whereas no marked loss was seen in cellular survival after exposure to Glu or NMDA in striatal neurons previously cultured with Glu or NMDA. Sustained culture with Glu or NMDA invariably led to a significant decrease in protein levels of NR2, but not NR1, subunits without affecting their mRNA levels. Similar induced tolerance was seen to the excitotoxicity of NMDA in hippocampal neurons in a manner sensitive to an NMDAR antagonist. Prior culture with NMDA induced less effective alterations in both intracellular free Ca2+ levels and mitochondrial membrane potentials after the addition of NMDA in striatal neurons. However, calpain inhibitor-I significantly prevented the decreased NR2B and NR2C protein levels in striatal neurons cultured with NMDA. These results suggest that prior tonic activation of NMDAR would induce tolerance to the excitotoxicity mediated by NMDAR through a mechanism related to calpain-induced down-regulation of particular NR2 subunits in rat striatal neurons. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/jnr.22388

Inhibition by 2-Methoxy-4-ethylphenol of Ca2+ Influx Through Acquired and Native N-Methyl-D-aspartate-Receptor Channels Reviewed

Ryo Fukumori, Noritaka Nakamichi, Takeshi Takarada, Yuki Kambe, Nobuyuki Matsushima, Nobuaki Moriguchi, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 112 ( 3 ) 273 - 281 2010.3

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

Pharmacological properties were evaluated for the antidiarrheic wood creosote ingredient 2-methoxy-4-ethylphenol (2M4EP), which was shown to be protective against neurotoxicity of N-methyl-D-aspartate (NMDA), to modulate Ca2+ influx across acquired and native NMDA receptor (NMDAR) channels. NMDA markedly increased intracellular free Ca2+ levels in HEK293 cells transfected with the expression vector of either NR2A or NR2B subunit together with the essential NR1 subunit vector. Further addition of dizocilpine inhibited the increase by NMDA in intracellular Ca2+ levels in both types of acquired NMDAR channels, while 2M4EP and the NR2B-subunit selective antagonist ifenprodil were more effective in inhibiting the increase by NMDA in HEK293 cells expressing NR1/NR2B subunits than in those with NR1/NR2A subunits. 2M4EP significantly prevented the increased intracellular Ca2+ levels by NMDA in cultured rat hippocampal neurons. Brief exposure to NMDA led to a drastic decrease in cellular viability 24 h later in cultured hippocampal neurons, while 2M4EP significantly prevented the loss of cellular vitality by NMDA. Similarly, 2M4EP more efficiently protected HEK293 cells with NR1/NR2B subunits than those with NR1/NR2A subunits. These results suggest that 2M4EP may protect neurons from excitotoxicity through inhibition of Ca2+ influx across NMDAR channels composed of NR1/NR2B, rather than NR1/NR2A, subunits.

DOI： 10.1254/jphs.09294FP

Interference by adrenaline with chondrogenic differentiation through suppression of gene transactivation mediated by Sox9 family members Reviewed

Takeshi Takarada, Hironori Hojo, Mika Iemata, Koichi Sahara, Ayumi Kodama, Nobuhiro Nakamura, Eiichi Hinoi, Yukio Yoneda

BONE 45 ( 3 ) 568 - 578 2009.9

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

In contrast to osteoblasts, little attention has been paid to the functional expression of adrenergic signaling machineries in chondrocytes. Expression of mRNA was for the first time demonstrated for different adrenergic receptor (AdR) subtypes in chondrogenic ATDC5 cells and mouse metatarsals isolated before vascularization in culture, but not for other molecules related to adrenergic signaling. In neonatal mouse tibial sections, beta(2)AdR and alpha(2a)AdR mRNA expression was found in chondrocytes at different developmental stages by in situ hybridization. Exposure to adrenaline significantly suppressed expression of several maturation markers through the cAMP/protein kinase A pathway activated by beta(2)AdR without affecting cellular proliferation in both Cultured ATDC5 cells and metatarsals. Adrenaline also significantly inhibited gene transactivation by sry-type HMG box 9 (Sox9) family members essential for chondrogenic differentiation in a manner prevented by the general beta AdR antagonist propranolol, with a concomitant significant decrease in the levels of Sox6 mRNA and corresponding protein, in ATDC5 cells and primary cultured Mouse costal chondrocytes. Systemic administration of propranolol significantly promoted the increased expression of mRNA for collagen I and collagen X, but not for collagen 11, in callus of fractured femur in mice. These results suggest that adrenaline may interfere with chondrogenic differentiation through downregulation of Sox6 expression for subsequent suppression of gene transactivation mediated by Sox9 family members after activation Of beta(2)AdR expressed by chondrocytes. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2009.05.004

Interference With Cellular Differentiation by D-Serine Through Antagonism at N-Methyl-D-Aspartate Receptors Composed of NR1 and NR3A Subunits in Chondrocytes Reviewed

Takeshi Takarada, Yoshifumi Takahata, Mika Iemata, Eiichi Hinoi, Kyosuke Uno, Takao Hirai, Tomomi Yamamoto, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 220 ( 3 ) 756 - 764 2009.9

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

Serine racemase (SR) is responsible for the biosynthesis of D-serine (D-Ser), an endogenous co-agonist for the glycine (Gly)-binding site on N-methyl-D-aspartate (NMDA) receptors, from L-Ser in the brain. We have previously demonstrated high expression of SR by chondrocytes in cartilage. In this study, we attempted to elucidate the possible functional role of D-Ser in chondrogenesis. Expression of mRNA and corresponding protein was seen for SR in cultured rat costal chondrocytes, while the addition of L-Ser significantly increased intracellular and extracellular levels of D-Ser. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, SIR mRNA was highly localized in hypertrophic and calcified chondrocytes. Exposure to D-Ser not only suppressed several chondrocytic maturation markers, including alkaline phosphatase (ALP) activity, Ca(2+) accumulation, nodule formation, and osteopontin expression, in rat chondrocytes, but also delayed chondral mineralization in mouse metatarsals. Either NMDA or Gly alone significantly increased Ca(2+) accumulation in cultured chondrocytes, whereas D-Ser significantly prevented Ca(2+) accumulation by Gly, but not by NMDA. Gly alone also significantly increased gene transactivation by the introduction of runt-related transcription factor-2 (Runx2) in COS7 cells transfected with NR1 and NR3A subunits,while D-Ser significantly prevented the increase by Gly without affecting the promoter activity of Runx2. In both cultured chondrocytes and metatarsals from NR1-null mice, significant decreases were seen in ALP activity and chondral mineralization, respectively. These results suggest that D-Ser may negatively regulate cellular differentiation through inhibiting NMDA receptors composed of NR1 and NR3A subunits in a manner related to Runx2 transcriptional activity in chondrocytes. J. Cell. Physiol. 220: 756-764, 2009. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jcp.21821

A protein-protein interaction of stress-responsive myosin VI endowed to inhibit neural progenitor self-replication with RNA binding protein, TLS, in murine hippocampus Reviewed

Takeshi Takarada, Keisuke Tamaki, Toru Takumi, Masato Ogura, Yuma Ito, Noritaka Nakamichi, Yukio Yoneda

JOURNAL OF NEUROCHEMISTRY 110 ( 5 ) 1457 - 1468 2009.9

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

We have shown preferential expression of both mRNA and corresponding protein for myosin VI (Myo6) in the murine hippocampus within 24 h after the extreme traumatic experience, water-immersion restraint stress (WIRS), prior to a drastic decrease in neural progenitor proliferation in the dentate gyrus. Myosin (Myo6) protein levels were significantly increased in hippocampus within 24 h after flashback experience in mice previously exposed to WIRS. Myo6 protein was ubiquitously distributed in discrete mouse brain regions with exceptionally high expression in olfactory bulb, whereas Myo6 protein was expressed in cultured rat astroglia and neurons, in addition to Myo6 mRNA expression by cultured neural progenitors. In mouse embryonal carcinoma P19 cells endowed to proliferate and differentiate, Myo6 protein was expressed in line with astroglial marker protein expression. Transient over-expression of Myo6 induced a significant decrease in the size of clustered aggregates as an index of self-replication in P19 cells. Immunoprecipitation analysis revealed the interaction between Myo6 and the RNA-binding protein, translocated in liposarcoma (TLS), while TLS was predominantly expressed by neurons in the cortex, striatum, cerebellum, and hippocampus. These results suggest that Myo6 may play a pivotal role in the mechanism underlying the suppressed adult neurogenesis after traumatic stress in association with TLS.

DOI： 10.1111/j.1471-4159.2009.06225.x

Possible Promotion of Neuronal Differentiation in Fetal Rat Brain Neural Progenitor Cells After Sustained Exposure to Static Magnetism Reviewed

Noritaka Nakamichi, Yukichi Ishioka, Takao Hirai, Shusuke Ozawa, Masaki Tachibana, Nobuhiro Nakamura, Takeshi Takarada, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 87 ( 11 ) 2406 - 2417 2009.8

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

We have previously shown significant potentiation of Ca(2+) influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of static magnetism on the functionality of neural progenitor cells endowed to proliferate for self-replication and differentiate into neuronal, astroglial, and oligodendroglial lineages. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Static magnetism not only significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for an astroglial marker, irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism, a significant increase was seen in mRNA expression of several activator-type proneural genes, such as Mash 1, Math 1, and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of activator-type proneural genes by progenitor cells in fetal rat brain. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jnr.22087

Possible Protection by Notoginsenoside R1 against Glutamate Neurotoxicity Mediated by N-methyl-D-aspartate Receptors Composed of an NR1/NR2B Subunit Assembly Reviewed

Bin Gu, Noritaka Nakamichi, Wen-Sheng Zhang, Yukary Nakamura, Yuki Kambe, Ryo Fukumori, Kazuhiro Takuma, Kiyofumi Yamada, Takeshi Takarada, Hideo Taniura, Yukio Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 87 ( 9 ) 2145 - 2156 2009.7

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

Notoginsenoside R1 (NTR1) is the main active ingredient in Panax notoginseng, a herbal medicine widely used in Asia for years. The purpose of this study was to investigate pharmacological properties of NTR1 on neurotoxicity of glutamate (Glu) in primary cultured mouse cortical neurons along with its possible mechanism of action. We found that NTR1 significantly protected neurons from the loss of cellular viability caused by brief exposure to 10 mu M Glu for 1 hr in a dose-dependent manner at concentrations from 0.1 to 10 mu M, without affecting the viability alone. NTR1 significantly inhibited the increased number of cells positive to propidium iodide (PI) staining, increase of intracellular free Ca(2+) ions, overproduction of intracellular reactive oxygen species, and depolarization of mitochondrial membrane potential in cultured neurons exposed to Glu, in addition to blocking decreased Bcl-2 and increased Bax expression levels. We further evaluated the target site at which NTR1 protects neurons from Glu toxicity by using the acquired expression strategy of N-methyl-D-aspartate (NMDA) receptor subunits in human embryonic kidney 293 cells. We found that 10 mu M NTR1 protected NR1/NR2B subunit expressing cells from cell death by 100 mu M NMDA, but not cells expressing NR1/NR2A subunits, when determined by PI staining. These results suggest that NTR1 may preferentially protect neurons from Glu excitotoxicity mediated by NMDA receptor composed of an NR1/NR2B subunit assembly in the brain. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/jnr.22021

Neurogenesis Mediated by gamma-Aminobutyric Acid and Glutamate Signaling Reviewed

Noritaka Nakamichi, Takeshi Takarada, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 110 ( 2 ) 133 - 149 2009.6

Language：English Publisher：JAPANESE PHARMACOLOGICAL SOC

In this review, we will summarize our ongoing Studies on the functionality of both gamma-aminobutyric acid (GABA) and glutamate receptors expressed by undifferentiated neural progenitor cells isolated from embryonic rodent brains. Cells were cultured with growth factors for the formation of round spheres by clustered cells under floating conditions, whereas a reverse transcription polymerase chain reaction analysis revealed expression of mRNA for particular subtypes of different ionotropic and metabotropic GABA and glutamate receptors in undifferentiated progenitors and neurospheres. Moreover, sustained exposure to either GABAergic or glutamatergic agonists not only modulated the size of neurospheres formed, but also affected spontaneous and induced differentiation of neural progenitor cells into particular progeny cell lineages Such as neurons and astroglia. Both GABA and glutamate could play a pivotal role in the mechanisms underlying proliferation for self-replication along with the determination of Subsequent differentiation fate toward particular progeny lineages through activation of their receptor subtypes functionally expressed by undifferentiated neural progenitor cells. Accordingly, neurogenesis seems to be also under control by GABAergic and glutamatergic signaling in developing brains as seen with neurotransmission in adult brains.

DOI： 10.1254/jphs.08R03CR

GABA-B受容体を介した神経系細胞系譜への分化調節(Modulation of differentiation into neural lineages through GABAB receptors)

小澤秀介, 福井正樹, 宝田剛志, 米田幸雄

神経化学 48 ( 2-3 ) 212 - 212 2009.6

Predominant Promotion by Tacrolimus of Chondrogenic Differentiation to Proliferating Chondrocytes Reviewed

Yukari Nakamura, Takeshi Takarada, Ayumi Kodama, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 109 ( 3 ) 413 - 423 2009.3

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

Tacrolimus (FK506) has been used as a therapeutic drug beneficial for the treatment of rheumatoid arthritis in humans. In this study, we investigated the effects of FK506 on cellular differentiation in cultured chondrogenic cells. Culture with FK506 led to a significant and concentration-dependent increase in Alcian blue staining for matrix proteoglycan at 0.1 to 1,000 ng/ml, but not in alkaline phosphatase (ALP) activity, in ATDC5 cells, a mouse prechondrogenic cell line, cultured for 7 to 28 days, while the non-steroidal anti-inflammatory drug indomethacin significantly decreased Alcian blue staining in a concentration-dependent manner, without altering ALP activity. FK506 significantly increased the expression of mRNA for both type 11 and type X collagen, but not for osteopontin, in ATDC5 cells. Similar promotion was seen in chondrogenic differentiation in both mouse metatarsals and chondrocytes cultured with FK506. However, FK506 failed to significantly affect transcriptional activity of the reporter construct for either sry-type HMG box 9 (Sox9) or runt-related transcription factor-2 (Runx2), which are both transcription factors responsible for chondrocytic maturation as a master regulator. These results suggest that FK506 may predominantly promote cellular differentiation into proliferating chondrocytes through a mechanism not relevant to the transactivation by either Sox9 or Runx2 in chondrogenic cells.

DOI： 10.1254/jphs.08315FP

Transactivation by Runt related factor-2 of matrix metalloproteinase-13 in astrocytes Reviewed

Takeshi Takarada, Yukio Yoneda

NEUROSCIENCE LETTERS 451 ( 2 ) 99 - 104 2009.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

We have previously shown functional expression by osteoblasts of signaling machineries required for neurotransmission in the brain. In this study, we have evaluated possible functional expression of different osseous genes in the brain. In embryonic and adult mouse brains, mRNA expression was invariably seen for the master regulator of osteoblastic differentiation Runt related factor-2 (Runx2), in addition to the partner protein core binding factor-beta and their targets such as osteopontin (OPN) and matrix metalloproteinase-13 (MMP13), but not for collagen-I or osteocalcin. In pluripotent P19 progenitor cells, Runx2 mRNA expression was drastically increased along with mRNA expression of an astrocytic marker. but not with neuronal marker mRNA expression. Both mRNA and corresponding protein were detected for Runx2 in cultured rat neocortical astrocytes and astrocytic C6 glioma cells. In C6 glioma cells, transient overexpression of Runx2 significantly increased mRNA expression of MMP13, but not of OPN. Moreover, transient overexpression of Runx2 significantly increased luciferase activity in C6 glioma cells transfected with the reporter plasmid linked to a wild-type Runx2 binding element in the MMP13 promoter, but not in cells with a mutated element. These results suggest that Runx2 signal input may lead to transactivation of MMP13 gene without affecting OPN expression in astrocytes. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2008.12.037

Functional Expression of beta(2) Adrenergic Receptors Responsible for Protection Against Oxidative Stress Through Promotion of Glutathione Synthesis After Nrf2 Upregulation in Undifferentiated Mesenchymal C3H10T1/2 Stem Cells Reviewed

Yoshifumi Takahata, Takeshi Takarada, Mika Iemata, Tomomi Yamamoto, Yukary Nakamura, Ayumi Kodama, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 218 ( 2 ) 268 - 275 2009.2

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

Adrenaline is believed to play a dual role as a neurotransmitter in the central nervous system and an adrenomedullary hormone in the peripheral tissues. In contrast to accumulating evidence for the involvement in endochondral ossification, osteolblastogenesis, and osteoclastogenesis, little attention has been paid to the role of adrenergic signals in the mechanisms underlying proliferation and differentiation of mesenchymal stem cells with self-renewal capacity and multi-potentiality to differentiate into osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was seen for different adrenergic receptor (AdR) subtypes, including beta(2)AdR, in the mesenchymal stem cell line C3H10T1/2 cells and mouse bone marrow mesenchymal stem cells before differentiation. Exposure to adrenaline not only increased cAMP formation, phosphorylation of cAMP responsive element (CRE) binding protein (CREB) on serine 133 and CRE reporter activity in a manner sensitive to propranolol, but also rendered C3H10T1/2 cells resistant to the cytotoxicity of hydrogen peroxide, but not of either 2,4-dinitirophenol or tunicamycin. Adrenaline induced a rapid but transient increase in mRNA expression of the antioxidative gene nuclear factor E2 p45-related factor-2 (Nrf2) along with an increase in the cystine/glutamate antiporter subunit xCT mRNA expression. Hydrogen peroxide was less cytotoxic in cells overexpressing Nrf2, moreover, while adrenaline significantly increased xCT promoter activity with an increase in endogenous glutathione levels. These results suggest that adrenaline may selectively protect mesenchymal C3H10T1/2 cells from oxidative stress through a mechanism related to the promoted biosynthesis of glutathione in association with transient Nrf2 expression after activation of beta(2)AdR.

DOI： 10.1002/jcp.21594

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