Faculty Profiles - TAKARADA Takeshi

Papers - TAKARADA Takeshi

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A critical importance of polyamine site in NMDA receptors for neurite outgrowth and fasciculation at early stages of P19 neuronal differentiation Reviewed

Danko Georgiev, Hideo Taniura, Yuki Kambe, Takeshi Takarada, Yukio Yoneda

EXPERIMENTAL CELL RESEARCH 314 ( 14 ) 2603 - 2617 2008.8

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

We have investigated the role of N-methyl-D-aspartate receptors (NMDARs) and gamma-aminobutyric acid receptors type A (CABAARs) at an early stage of P19 neuronal differentiation. The subunit expression was profiled in 24-hour intervals with RT-PCR and functionality of the receptors was verified via fluo-3 imaging of Ca2+ dynamics in the immautre P19 neurons showing that both NMDA and GABA excite neuronal bodies, but only polyamine-site sensitive NMDAR stimulation leads to enhanced Ca2+ signaling in the growth cones. Inhibition of NR1/NR2B NMDARs by 1 mu M ifenprodil severely impaired P19 neurite extension and fasciculation, and this negative effect was fully reversible by polyamine addition. In contrast, GABAAR antagonism by a high dose of 200 mu M bicuculline had no observable effect on P19 neuronal differentiation and fasciculation. Except for the differential NMDAR and GABAAR profiles of Ca2+ signaling Within the immature P19 neurons, we have also shown that inhibition of NR1/NR2B NMDARs strongly decreased mRNA level of NCAM-180, which has been previously implicated as a regulator of neuronal growth cone protrusion and neurite extension. Our data thus suggest a critical role of NR1/NR2B NMDARs during the process of neuritogenesis and fasciculation of P19 neurons via differential control of local growth cone Ca2+ surges and NCAM-180 signaling. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2008.06.009

GABAシグナルによるマウス神経系前駆細胞の増殖と分化の調節(Modulation of proliferation and differentiation by GABAergic signaling in murine neural progenitor cells)

小澤秀介, 福井正樹, 寳田剛志, 谷浦秀夫, 中道範隆, 米田幸雄

神経化学 47 ( 2-3 ) 225 - 225 2008.8

Differential regulation of cellular maturation in chondrocytes and osteoblasts by glycine Reviewed

Yoshifumi Takahata, Takeshi Takarada, Masato Osawa, Eiichi Hinoi, Yukari Nakamura, Yukio Yoneda

CELL AND TISSUE RESEARCH 333 ( 1 ) 91 - 103 2008.7

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

Previous studies have demonstrated the functional expression, by osteoblasts, of N-methyl-D-aspartate (NMDA) receptors responsible for the promotion of cellular differentiation in bone. We have now evaluated the possible role of the endogenous co-agonist of NMDA receptors, glycine (Gly), in chondrogenesis. In ex vivo organotypic cultures of fetal mouse tibias, proximal and distal cartilaginous primordia were significantly increased in the presence of Gly, with the osteogenic center being unchanged. Exposure to Gly drastically increased mRNA expression of the calcified chondrocyte marker osteopontin, without markedly affecting that of a proliferating chondrocyte marker or a hypertrophic chondrocyte marker, as shown in organotypic cultures by in situ hybridization analysis. Gly significantly increased Ca(2+) accumulation, osteopontin mRNA expression, and alkaline phosphatase activity in cultured rat costal chondrocytes, without significantly affecting those in cultured rat calvarial osteoblasts. The increase induced by Gly was significantly prevented by an NMDA receptor channel blocker and an antagonist at the Gly site on NMDA receptors, but not by an inhibitory Gly receptor antagonist or a Gly transporter inhibitor, in cultured chondrocytes. Constitutive mRNA expression was seen for NR1, NR2D, and NR3A subunits of NMDA receptors, but not for Gly receptors and transporters, in cultured chondrocytes. Corresponding immunoreactive proteins were detected for NR1 and NR2D subunits in cartilaginous zones of fetal mouse tibias. Thus, Gly might, at least in part, play a role as a trophic factor in the mechanisms associated with chondral calcification through the Gly site of NMDA receptors functionally expressed by chondrocytes in rodent cartilage.

DOI： 10.1007/s00441-008-0607-7

Serine racemase suppresses chondrogenic differentiation in cartilage in a Sox9-dependent manner Reviewed

Takeshi Takarada, Eiichi Hinoi, Yoshifumi Takahata, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 215 ( 2 ) 320 - 328 2008.5

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 N-methyl-D-aspartate (NMDA) receptors, from L-serine (L-Ser) in the central nervous system. In the present study, we investigated the role of SIR in the regulation of chondrogenic differentiation in cartilage. On in situ hybridization analysis of tibia from neonatal rats,SR mRNA was ubiquitously expressed in all cell layers of proliferating to hypertrophic chondrocytes. In the pre-chondrogenic cell line ATDC5 cells, mRNA expression was seen with SR irrespective of the cellular maturity, with no mRNA expression of the NRI subunit essential for the heteromeric assembly of functional NMDA receptor channels. In ATDC5 cells stably overexpressing SR, significant inhibition was found with the maturation-dependent temporal increases in Alcian blue staining, alkaline phosphatase (ALP) activity and mRNA expression of type 11 and type X collagens. Stable overexpression of SR significantly impaired the sry-type HMG box 9 (Sox9) transcriptinal activity in ATDC5 cells, while Sox9 transcriptional activity was significantly inhibited in COS7 cells with co-introduction of SIR and Sox9. However, no significant inhibition was seen with Sox9 transcriptional activity in COS7 cells co-introduced of either SRK56G defective of D-Ser formation ability or 3-phosphoglycerate dehydrogenase essential for D-Ser biosynthesis. The co-introduction of SR with Sox9 significantly decreased the Sox9 protein level with that of Sox9 mRNA being unchanged. These results suggest that SR may negatively regulate cellular differentiation through the inhibition of Sox9 transcriptional activity in chondrocytes.

DOI： 10.1002/icp.21310

Glutamate as a signal mediator in bone Reviewed

Takeshi Takarada, Yukio Yoneda

JOURNAL OF PHARMACOLOGICAL SCIENCES 106 ( 4 ) 536 - 541 2008.4

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

The view that L-glutamate (Glu) is an excitatory amino acid nurotransmitter in the mammalian central nervous system is prevailing on the basis of successful cloning of a number of genes encoding different signaling molecules, such as Glu receptors for the signal input, Glu transporters for the signal termination and vesicular Glu transporters for the signal output through exocytotic release. Little attention has been paid to an extracellular transmitter role of Glu in peripheral neuronal and non-neuronal tissues, by contrast, whereas recent molecular biological and pharmacological analyses including ours give rise to a novel function for Glu as an autocrine and/or paracrine signal mediator in bone comprised of osteoblasts, osteoclasts and osteocytes, in addition to other peripheral tissues including pancreas, adrenal and pituitary glands. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying the maintenance of cellular homeostasis as an excitatory neurotransmitter in the central nervous system and as an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, therefore, we would outline the possible signaling system for Glu to play a role as an extracellular signal mediator in mechanisms underlying maintenance of the cellular homeostasis in bone.

DOI： 10.1254/jphs.FM0070243

A role of the clock gene Period1 in chondrogenic differentiation Reviewed

Ayumi Kodama, Koichi Sahara, Takeshi Takarada, Yukio Yoneda

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 128 142 - 144 2008

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：PHARMACEUTICAL SOC JAPAN

The view that circadian rhythm involves different proteins relevant to clock genes, including Period (Per), Cryptochrome, Clock, Bmal1 and Dec, in the suprachiasmatic nucleus in mammals is prevailing. In line with recent studies on expression of these clock genes in particular peripheral tissues such as heart and liver, we have shown transient promotion by parathyroid hormone (PTH) of Per1 mRNA expression in cultured chondrocytes. In order to elucidate a possible functional role of Per1 in chondrocytes, in the present study, we have attempted to establish the mouse chondrogenic cell line ATDC5 cells stably transfected with Per1 expression vector. For this purpose, ATDC5 cells were transfected with pcDNA3.1 containing the full-length coding region of Per1 (ATDC5-Per1) or empty vector (ATDC5-EV), followed by selection of particular clones with G418. Pools of clones of ATDC5-Per1 cells were isolated for determination of expression levels of Per1 by real-time-PCR. In ATDC5 cells stably overexpressing Per1, significant inhibition was found with the maturation-dependent temporal increases in Alcian blue staining and mRNA expression of type II and type X collagens. An attempt was next made to elucidate underlying mechanisms for the suppression by Per1 overexpression of chondrogenic differentiation with the reporter assay using a 4x48-p89-Luc, 4 tandem copies of Sox9 allies (Sox5, Sox6 and Sox9) binding site linked to the minimal Col II gene promoter in the luciferase reporter plasmid. The 4x48-p89-Luc plasmid was transiently transfected into ATDC5-Per1 and ATDC5-EV cells, followed by determination of luciferase activity 48 h after transfection. Luciferase activity was significantly inhibited in ATDC5-Per1 cells compared with that in ATDC5-EV cells. Moreover, overexpression of Per1 decreased the Sox6 mRNA level along with a decreased Sox6 promoter activities required for Sox6 mRNA expression in ATDC5 cells. These results suggest that PTH may suppress chondrogenesis through the promotion of Per1 expression toward repression of Sox6 mRNA expression.

Protective effect of notoginsenoside R1 in Panax notoginseng on glutamate-induced neurotoxicity Reviewed

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

YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 128 184 - 187 2008

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：PHARMACEUTICAL SOC JAPAN

This study was aimed to investigate pharmacological properties of notoginsenoside R1 (NG-R1), an ingredient in Panax notoginseng, against the neurotoxicity of glutamate (Glu) in primary cultured mouse neocortical neurons. Cerebral cortex was isolated from fetal mice at 15 days after gestation, followed by culture in DMEM in the presence of fetal calf serum (FCS) for the initial 3 days and subsequent culture in DMEM in the absence of FCS with medium change every 3 days. Brief exposure to Glu drastically reduced cell viability 24 h later in cortical neurons cultured for 7 days, while simultaneous addition of NG-R1 led to significant protection against the loss of cellular viability by Glu. NG-R1 inhibited the elevation of intracellular Ca2+ ions, the generation of intracellular reactive oxygen species (ROS) and the depolarization of mitochondrial membrane potential in cultured cortical neurons exposed to Glu. Glu decreased the expression of Bcl-2 and increased that of Bax in cultured cortical neurons, whereas further addition of NG-R1 resulted in significant protection against the Glu-induced changes in Bcl-2 and Bax expressions. These results suggest that particular notoginsenosides may protect neurons from the neurotoxicity of Glu through a mechanism related to the suppression of the elevation of intracellular Ca2+ ions, generation of intracellular ROS, depolarization of mitochondrial membrane potential and induction of apoptosis.

Osteoblast protects osteoclast devoid of sodium-dependent vitamin C transporters from oxidative cytotoxicity of ascorbic acid Reviewed

Takeshi Takarada, Eiichi Hinoi, Yuki Kambe, Koichi Sahara, Shintaro Kurokawa, Yoshifumi Takahata, Yukio Yoneda

EUROPEAN JOURNAL OF PHARMACOLOGY 575 ( 1-3 ) 1 - 11 2007.12

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

The view that ascorbic acid indirectly benefits osteoclastogenesis through expression of receptor activator of nuclear factor-kappa B (NF-kappa B) ligand (RANKL) by osteoblasts is prevailing. In this study, we have examined the direct effect of ascorbic acid on osteoclastogenesis in cultured mouse osteoclasts differentiated from bone marrow precursors. The absence of alkaline phosphatase and ostcoblastic marker genes validated the usefulness of isolation procedures. Sustained exposure to ascorbic acid, but not to dehydroascorbic acid, significantly reduced the number of multinucleated cells positive to tartrate-resistant acid pbosphatase (TRAP) staining. In cultured osteoclasts, mRNA expression was seen for glucose transporter-1 involved in membrane transport of dehydroascorbic acid, but not for sodium-dependent vitamin C transporters-1 and -2 that are both responsible for the transport of ascorbic acid. The inhibition by ascorbic acid was completely prevented by catalase, while ascorbic acid or hydrogen peroxide drastically increased the number of cells stained with propidium iodide and the generation of reactive oxygen species, in addition to inducing mitochondrial membrane depolarization in cultured osteoclasts. In pre-osteoclastic cell line RAW264.7 cells, ascorbic acid similarly inhibited the formation of TRAP-positive multinucleated cells, with a significant decrease in RANKL-induced NF-kappa B transactivation. Moreover, co-culture with osteoblastic MC3T3-E1 cells significantly prevented the ascorbic acid-induced decrease in the number of TRAP-positive multinucleated cells in RAW264.7 cells. These results suggest that ascorbic acid may play a dual repulsive role in osteoclastogenesis toward bone remodeling through the direct cytotoxicity mediated by oxidative stress to osteoclasts, in addition to the indirect trophism mediated by RANKL from osteoblasts. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2007.07.041

Suppression by glutamate of proliferative activity through glutathione depletion mediated by the Cystine/Glutamate antiporter in mesenchymal C3H10T1/2 stem cells Reviewed

Mika Iemata, Takeshi Takarada, Eiichi Hinoi, Hideo Taniura, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 213 ( 3 ) 721 - 729 2007.12

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

Although previous studies including ours have demonstrated the functional expression of different glutamate (Glu) signaling machineries such as Glu receptors (GluRs) and transporters in osteoblasts and chondrocytes, little attention has been paid to the role of Glu in their ancestral mesenchymal stem cells to date. In the present study, we have evaluated the possible functionality of Glu in cultured mouse mesenchymal stem cell line C3H10T1/2 cells endowed to proliferate for the self-renewal and to differentiate toward osteoblast, chondrocyte, adipocyte, and myocyte lineages. Expression of mRNA was for the first time shown with the cystine/Glu antiporter composed of xCT and 4F2hc subunits, in addition to particular excitatory amino acid transporter (EAAT) isoforms and ionotropic GluRs, in undifferentiated C3H10T1/2 cells. Glu significantly suppressed the proliferation activity at a concentration over 500 mu M without inducing cell death or differentiation, while the suppression occurred in a manner sensitive to the prevention by cystine and reduced glutathione (GSH), but not by EAAT inhibitors. A significant decrease was seen in intracellular GSH levels in C3H10T1/2 cells cultured with Glu, whereas the cellular proliferation activity was drastically decreased by the addition of the GSH depleter cyclohexene-l-one and the GSH biosynthesis inhibitor L-buthionine-[S,R]-sulfoximine, respectively. Transient overexpression of both xCT and 4F2hc subunits led to an increased basal proliferative activity in C3H10T1/2 cells. These results suggest that Glu could suppress the cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the cystine/Glu antiporter in C3H10T1/2 cells.

DOI： 10.1002/jcp.21145

Glutamate is a determinant of cellular proliferation through modulation of nuclear factor E2 p45-related factor-2 expression in osteoblastic MC3T3-E1 cells Reviewed

Kyosuke Uno, Takeshi Takarada, Eiichi Hinoi, Yukio Yoneda

JOURNAL OF CELLULAR PHYSIOLOGY 213 ( 1 ) 105 - 114 2007.10

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

Activation of particular glutamate (Glu) receptors is shown to promote cellular differentiation toward maturation during osteoblastogenesis. In the present study, we have evaluated the possible modulation by Glu of cellular proliferation in osteoblastic cells endowed to proliferate for self-renewal and to differentiate toward matured osteoblasts. Exposure to Glu significantly suppressed the proliferation activity at a concentration over 500 mu M without inducing cell death in osteoblastic MC3T3-E1 cells before differentiation. The suppression by Glu occurred in a manner sensitive to the prevention by either cystine or reduced glutathione. Expression of mRNA was for the first time shown with the cystine/Glu antiporter composed of xCT and 4F2hc subunits in these undifferentiated osteoblastic cells. A significant decrease was seen in intracellular total glutathione levels in undifferentiated MC3T3-E1 cells cultured with Glu, indeed, whereas the cellular proliferation activity was drastically decreased by the addition of the glutathione depleter cyclohexene-1-one and the glutathione biosynthesis inhibitor L-buthionine-[S,R]-sulfoximine, respectively. Exposure to Glu led to a significant increase in mRNA expression of nuclear factor E2 p45-related factor 2 (Nrf2) together with the generation of reactive oxygen species, while a significant decrease was seen in the proliferation activity in MC3T3-E1 cells with stable overexpression of Nrf2. These results suggest that Glu could suppress the cellular proliferation toward self-renewal through a mechanism associated with the upregulation of Nrf2 expression in association with the depletion of intracellular glutathione after promoting the retrograde operation of the cystine/Glu antiporter in undifferentiated MC3T3-E1 cells.

DOI： 10.1002/jcp.21095

Glutamate suppresses osteoclastogenesis through the cystine/glutamate antiporter Reviewed

Eiichi Hinoi, Takeshi Takarada, Kyosuke Uno, Maki Inoue, Yasuhiro Murafuji, Yukio Yoneda

AMERICAN JOURNAL OF PATHOLOGY 170 ( 4 ) 1277 - 1290 2007.4

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC

Previous studies have demonstrated functional expression of different glutamate receptor subtypes (GluRs) in both osteoblasts and osteoclasts. in the present study, we investigated the possible functional expression by osteoclasts of different glutamatergic signaling machineries including GluRs. In disagreement with the aforementioned prevailing view, no mRNA expression was found for all GluRs examined in primary cultured mouse osteoclasts differentiated from bone marrow precursors. Constitutive expression of mRNA was seen with glutamate transporters, such as excitatory amino acid transporters and cystine/glutamate antiporter, in primary osteoclasts. Glutamate significantly inhibited osteoclastogenesis at a concentration over 500 mu mol/L in both primary osteoclasts and preosteoclastic RAW264.7 cells without affecting the cell viability in a manner sensitive to the antiporter inhibitor. in RAW264.7 cells stably overexpressing the cystine/glutamate antiporter, the inhibition by glutamate was more conspicuous than in cells transfected with empty vector alone. The systemic administration of glutamate significantly prevented the decreased bone mineral density in both femur and tibia in addition to increased osteoclastic indices in ovariectomized mice in vivo. These results suggest that glutamate may play a pivotal role in mechanisms associated with osteoclastogenesis through the cystine/glutamate antiporter functionally expressed by osteoclasts devoid of any GluRs cloned to date.

DOI： 10.2353/ajpath.2007.061039

Nuclear factor E2 p45-related factor 2 negatively regulates chondrogenesis Reviewed

Eiichi Hinoi, Takeshi Takarada, Sayumi Fujimori, Liyang Wang, Mika Iemata, Kyosuke Uno, Yukio Yoneda

BONE 40 ( 2 ) 337 - 344 2007.2

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

The transcription factor nuclear factor E2 p45-related factor 2 (Nrf2) forms heterodimers with small musculoaponeurotic fibrosarcoma (Maf) proteins for the selective recognition of the antioxidant responsive element on target genes, followed by the regulation of gene expression of phase II detoxifying enzymes as well as oxidative-stress-inducible proteins in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of chondrocyte differentiation as well as the expression pattern of Nrf2 in cartilage. In tibia from embryonic mice at E15.5, Nrf2 mRNA expression was restricted to both proliferating and pre-hypertrophic chondrocytes, with few signals in early and late hypertrophic chondrocytes expressing both type X collagen and osteopontin. On in situ hybridization analysis of tibia from neonatal mice at 1 day after birth, by contrast, Nrf2 was expressed in all chondrocytic layers in addition to osteoblasts attached to cancellous bone. In pre-chondrogenic cell line ATDC5 cells, furthermore, expression of Nrf2 mRNA was also confirmed together with mRNA expression of the Kelch-like ECH associating protein 1 and small Maf proteins. In ATDC5 cells stably transfected with Nrf2, significant inhibition was seen in the differentiation-dependent induction of alkaline phosphatase and increase in the Alcian blue staining intensity. Furthermore, stable overexpression of Nrf2 significantly decreased mRNA expression of several chondrocyte differentiation markers such as type II collagen, type X collagen and osteopontin. These data suggest that Nrf2 may be a negative regulator of the cellular differentiation toward maturation in chondrocytes. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2006.08.016

Oral administration of phenolic antidiarrheic ingredients prevents ovariectomy-induced bone loss Reviewed

Nobuaki Moriguchi, Eiichi Hinoi, Takeshi Takarada, Nobuyuki Matsushima, Kyosuke Uno, Yukio Yoneda

BIOCHEMICAL PHARMACOLOGY 73 ( 3 ) 385 - 393 2007.2

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

In the present study, we have attempted to evaluate the pharmacological actions of three major phenolic antidiarrheic ingredients, including 2-methoxyphenol (2MP), 2-methoxy-4-methylphenol (2M4MP) and 2-methoxy-4-ethyphenol (2M4EP), on the functionality and integrity of bone by in vitro and in vivo experimental techniques. Intermittent oral administration of 2M4MP and 2M4EP, but not 2MP, significantly prevented reductions of bone mineral density in total femur, distal femur and tibia, in addition to alterations of several osteoclastic parameters on histomorphometric analysis, when determined 28 days after ovariectomy in mice. All three phenolic ingredients examined significantly inhibited the developmental increase in the number of multinucleated cells positive to tartrate-resistant acid phosphatase staining in cultured mouse osteoclasts differentiated from bone marrow precursors in the presence of both macrophage-colony stimulating factor and receptor activator of nuclear factor-kappa B ligand, which occurred in a concentration-dependent manner at a concentration range of 1 mu M-1 mM without inducing cell death. Moreover, both 2M4MP and 2M4EP at 1 mM not only prevented the cell death induced by 0.5 mM H2O2 in cultured rat calvarial osteoblasts, but also suppressed the generation of intracellular reactive oxygen species in osteoblasts exposed to H2O2, with a radical scavenging action as revealed by electron spin resonance analysis. These results suggest that particular phenolic antidiarrheic ingredients may prevent ovariectomy-induced bone loss through a mechanism related to the inhibition of osteoclastogenesis in association with an anti-oxidative property in osteoblasts. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bcp.2006.09.025

Possible expression of a particular gamma-aminobutyric acid transporter isoform responsive to upregulation by hyperosmolarity in rat calvarial osteoblasts Reviewed

Sayumi Fujimori, Eiichi Hinoi, Takeshi Takarada, Mika Iemata, Yoshifumi Takahata, Yukio Yoneda

EUROPEAN JOURNAL OF PHARMACOLOGY 550 ( 1-3 ) 24 - 32 2006.11

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

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the brain, but widely distributed in different peripheral organs. We have previously shown the functional expression of GABAB receptors required for GABAergic signal input by cultured rat calvarial osteoblasts. This study focused on the possible functional expression of the machinery required for GABAergic signal termination such as GABA transporters. In rat calvarial osteoblasts cultured for 7 days, [H-3]GABA accumulation was observed in a temperature-, sodium- and chloride-dependent manner, consisting of a single component with a K-m value of 789.6 +/- 9.0 mu M and a V-max value of 4.4 +/- 0.1 nmol/min/mg protein, respectively. Both nipecotic and L-2,4-diaminobutyric acids significantly inhibited [H-3]GABA accumulation in a concentration-dependent manner. Constitutive expression was seen with mRNA for the betaine/GABA transporter-1 (BGT-1) and taurine transporter (TauT), while hyperosmotic cultivation led to significant increases in both [3H]GABA accumulation and BGT-1 mRNA expression without affecting TauT mRNA expression. Highly immunoreactive cells were detected for the BGT-1 isoforin at the surface of trabecular bone of neonatal rat tibias. Sustained exposure to GABA significantly inhibited alkaline phosphatase (ALP) activity, but not cellular viability, at concentrations above 0.1 mM in osteoblasts cultured for 3 to 28 days. Nipecotic acid not only decreased ALP activity alone, but also further decreased ALP activity in osteoblasts cultured in the presence of GABA. These results suggest that the BGT-1 isoforin may be functionally expressed by rat calvarial osteoblasts to play a hitherto unidentified role in mechanisms underlying hyperosmotic regulation of osteoblastogenesis. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ejphar.2006.08.088

A molecular mechanism of pyruvate protection against cytotoxicity of reactive oxygen species in osteoblasts Reviewed

Eiichi Hinoi, Takeshi Takarada, Yuriko Tsuchihashi, Sayumi Fujimori, Nobuaki Moriguchi, Liyang Wang, Kyosuke Uno, Yukio Yoneda

MOLECULAR PHARMACOLOGY 70 ( 3 ) 925 - 935 2006.9

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS

We demonstrated previously that exogenous pyruvate has a protective action against cell death by hydrogen peroxide in cultured osteoblasts through a mechanism associated with its antioxidative property. In the present study, we have evaluated possible participation of monocarboxylate transporters (MCTs) responsible for the bidirectional membrane transport of pyruvate in the cytoprotective property in osteoblasts. Expression of the MCT2 isoform was found in cultured rat calvarial osteoblasts and in osteoblasts located on mouse tibia at both mRNA and protein levels. The accumulation of [C-14] pyruvate occurred in a temperature- and pH-dependent manner in osteoblasts cultured for 7 days with high sensitivity to a specific MCT inhibitor, whereas pyruvate was released into extracellular spaces from cultured osteoblasts in a fashion sensitive to the MCT inhibitor. Transient overexpression of the MCT2 isoform led to reduced vulnerability to the cytotoxicity of hydrogen peroxide with an increased activity of [C-14] pyruvate accumulation in murine osteoblastic MC3T3-E1 cells. Ovariectomy significantly decreased the content of pyruvate in femoral bone marrows in mice in vivo, whereas daily i.p. administration of pyruvate at 0.25 g/kg significantly prevented alterations of several histomorphometric parameters as well as cancellous bone loss in femurs by ovariectomy on 28 days after the operation. These results suggest that MCTs may be functionally expressed by osteoblasts to play a pivotal role in mechanisms related to the cytoprotective property of pyruvate.

DOI： 10.1124/mol.106.024398

Cytoprotection by pyruvate through an anti-oxidative mechanism in cultured rat calvarial osteoblasts Reviewed

N. Moriguchi, E. Hinoi, Y. Tsuchihashi, S. Fujimori, M. Iemata, T. Takarada, Y. Yoneda

HISTOLOGY AND HISTOPATHOLOGY 21 ( 9 ) 969 - 977 2006.9

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

Although we have previously shown drastic cell death by pyruvate deficiency in osteoblasts at the proliferative stage, the exact mechanism remains unclear so far. Cell survivability was significantly decreased in rat calvarial osteoblasts cultured for 0 to 3 days in vitro (DIV) following replacement of the eutrophic alpha-modified minimum essential medium (alpha-MEM) with Dulbecco's modified eagle medium (DMEM) for cultivation. The addition of pyruvate enriched in alpha-MEM, but not in MEM, entirely prevented cell death induced by the medium replacement throughout a culture period from 0 to 3 DIV. Both cysteine and reduced glutathione protected cell death in cells cultured for 3 DIV without significantly affecting that in cells cultured for 1 DIV, however, while none of lactate, acetate and insulin significantly prevented the cell death irrespective of the culture period up to 3 DIV. A marked increase was detected in intracellular reactive oxygen species (ROS) levels 4 h after the medium replacement. In osteoblasts cultured in alpha-MEM for 3 DIV, but not in those for 7 DIV, hydrogen peroxide (H2O2) markedly decreased cell survivability when exposed for 2 to 24 h. Furthermore, H2O2 was effective in significantly decreasing cell survivability in osteoblasts cultured in DMEM for 7 DIV. Pyruvate at 1 mM not only prevented cell death by H2O2, but also suppressed the generation of intracellular ROS in osteoblasts exposed to H2O2. These results suggest that pyruvate could be cytoprotective through a mechanism associated with the anti-oxidative property rather than an energy fuel in cultured rat calvarial osteoblasts.

Up-regulation of per mRNA expression by parathyroid hormone through a protein kinase A-CREB-dependent mechanism in chondrocytes Reviewed

Eiichi Hinoi, Taichi Ueshima, Hironori Hojo, Mika Iemata, Takeshi Takarada, Yukio Yoneda

JOURNAL OF BIOLOGICAL CHEMISTRY 281 ( 33 ) 23632 - 23642 2006.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

In bone, clock genes are involved in the circadian oscillation of bone formation and extracellular matrix expression. However, to date little attention has been paid to circadian rhythm in association with expression of clock genes during chondrogenesis in cartilage. In this study, we investigated the functional expression of different clock genes by chondrocytes in the course of cartilage development. The mRNA expression of types I, II, and X collagens exhibited a 24-h rhythm with a peak at zeitgeber time 6, in addition to a 24-h rhythmicity of all the clock genes examined in mouse femurs in vivo. Marked expression of different clock genes was seen in both osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells in vitro, whereas parathyroid hormone (PTH) transiently increased period 1 (per1) mRNA expression at 1 h inboth cell lines. Similar increases were seen in the mRNA levels for both per1 and per2 in prehypertrophic chondrocytes in metatarsal organotypic cultures within 2 h of exposure to PTH. PTH significantly activated the mouse per1 (mper1) and mper2 promoters but not the mper3 promoter in a manner sensitive to both a protein kinase A inhibitor and deletion of the cAMP-responsive element sequence (CRE) in ATDC5cells. In HEK293 cells, introduction of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (bmal1)/clock enhanced mouse type II collagen first intron reporter activity without affecting promoter activity, with reduction effected by either per1 or per2. These results suggest that PTH directly stimulates mper expression through a protein kinase A-CRE-binding protein signaling pathway for subsequent regulation of bmal1/clock-dependent extracellular matrix expression in cartilage.

DOI： 10.1074/jbc.M512362200

Excitatory amino acid transporters expressed by synovial fibroblasts in rats with collagen-induced arthritis Reviewed

E Hinoi, R Ohashi, S Miyata, Y Kato, M Iemata, H Hojo, T Takarada, Y Yoneda

BIOCHEMICAL PHARMACOLOGY 70 ( 12 ) 1744 - 1755 2005.12

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

Although previous studies have demonstrated increased levels of the brain neurotransmitter glutamate (Glu) in the synovial fluid from patients with arthritis, not much attention has been paid to the possible role of Glu in joint synovial tissues to date. Constitutive expression of mRNA was for the first time shown with glutamate aspartate transporter, glutamate transporter-1 and excitatory amino acid carrier-1 (EAAC1), in addition to with particular ionotropic and metabotropic Gin receptors, in cultured synovial fibroblasts prepared from knee joints of male Lewis rats. Immunohistochemical analysis revealed high localization of immunoreactive EAAC1 at synovial tissues. The accumulation of [H-3]Glu occurred in a temperature- and sodium-dependent manner in cultured synovial fibroblasts, with a Km of 23.1 +/- 1.1 mu M and a V-max of 237.1 +/- 31.1 pmol(mg protein min), respectively. In rats with arthritis induced by immunization to type-II collagen, marked increases were seen in hind paw volume, cytokine mRNA expression and Glu levels in synovial tissues, in addition to histological erosion. In cultured synovial fibroblasts prepared from these arthritic rats, [H-3]Glu accumulation was drastically increased with biochemical and pharmacological profiles similar to those seen in normal synovial fibroblasts. The exposure to Glu at 500 mu M doubled the incorporation of 5-bromo-2'-deoxyuridine in cultured synovial fibroblasts of arthritic but not normal rats, without significantly affecting mRNA expression of different cytokines in both synovial fibroblasts. These results suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation through particular transporters functionally expressed by synovium in rheumatoid arthritis. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bcp.2005.09.010

Abolition of chondral mineralization by group III metabotropic glutamate receptors expressed in rodent cartilage Reviewed

LY Wang, E Hinoi, A Takemori, T Takarada, Y Yoneda

BRITISH JOURNAL OF PHARMACOLOGY 146 ( 5 ) 732 - 743 2005.11

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

1 Previous studies have demonstrated the functional expression by osteoblasts of glutamate (Glu) signaling machineries responsible for the stimulation of cell proliferation and differentiation in bone, while there is no information available on the expression of the Glu signaling system by cartilage to date.
2 In cultured mouse embryonic metatarsals isolated before vascularization, chondral mineralization was almost completely inhibited in the presence of the group III metabotropic Glu receptor (mGluR) agonist L-(1)-2-amino-4-phosphonobutyrate (L-AP4) in a manner sensitive to an antagonist, with the total length being unchanged.
3 A group II mGluR agonist was similarly more effective in inhibiting the mineralization than a group I mGluR agonist, while none of ionotropic GluR agonists drastically affected the mineralization.
4 Both histological and in situ hybridization analyses revealed that L-AP4 specifically inhibited chondral mineralization, without apoptotic cell death, in cultured metatarsals.
5 In addition to the constitutive expression of mRNA for particular mGluRs in both cultured mouse metatarsals and rat costal chondrocytes, L-AP4 significantly inhibited the accumulation of cyclic AMP by forskolin and parathyroid hormone in a manner sensitive to a group III mGluR antagonist in cultured chondrocytes.
6 Moreover, L-AP4 drastically inhibited the expression of osteopontin mRNA in both cultured metatarsals and chondrocytes.
7 These results suggest that Glu may at least in part play a role as a signal mediator in mechanisms associated with chondral mineralization through the group III mGluR subtype functionally expressed by chondrocytes in rodent cartilage.

DOI： 10.1038/sj.bjp.0706358

Nuclear condensation of cyclic adenosine monophosphate responsive element-binding protein in discrete murine brain structures Reviewed

N Kuramoto, K Kubo, K Ogita, J Platenik, VJ Balcar, T Takarada, N Nakamichi, Y Yoneda

JOURNAL OF NEUROSCIENCE RESEARCH 80 ( 5 ) 667 - 676 2005.6

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

We have directed a polyclonal antibody against an oligopeptide (123-136) of the transcription factor cyclic AMP responsive element-binding protein (CREB) including the serine residue at 133. Rabbit sera were purified by ammonium sulfate precipitation, followed by affinity chromatography to homogeneity on one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified antibody not only induced marked supershift of CREB binding, without affecting binding of activator protein-1 on gel retardation electrophoresis, but also differentiated between CREB and CREB phosphorylated at serine133 in brain nuclear fractions on Western blotting. Immunoreactive CREB was detected in both cytosolic and nuclear fractions of discrete murine brain structures but was more highly condensed in cerebellum than in neocortex and hippocampus. Incubation of brain nuclear fractions led to a marked export of immunoreactive CREB in a temperature-dependent manner, whereas the temperature-dependent export activity was significantly lower in cerebellum than in other brain structures. Suppression of general new protein synthesis by cycloheximide (500 mg/kg, i.p.) in vivo resulted in a significant decrease in the nuclear CREB level, with a concomitant increase in the cytosolic level in hippocampus, but not in cerebellum. These results suggest that the nuclear export activity might vary from region to region in murine brains through a hitherto unidentified mechanism other than the nuclear localization signal, to result in different nuclear condensation ratios for subsequent elicitation of differential transcriptional activities by the constitutive transcription factor CREB in the nucleus. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/jnr.20504

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