FUKUSHIMA Nobuyuki

Researcher Information

J-Global ID

• 200901063822948189

Research Areas

• Life sciences / Pharmacology
• Life sciences / Pharmaceuticals - health and biochemistry
• Life sciences / Developmental biology
• Life sciences / Cell biology
• Life sciences / Neuroscience - general

Published Papers

• Oleic acid stimulates cell proliferation and BRD4-L-MYC-dependent glucose transporter transcription through PPARα activation in ovarian cancer cells

  Kado T; Kusakari N; Tamaki T; Murota K; Tsujiuchi T; Fukushima N

  Biochem Biophys Res Commun 657 (21) 24 - 34 2023 [Refereed]
  
• The Japanese lamprey (Lethenteron camtschaticum) expresses functional lysophosphatidic acid receptors.

  Takeru Tamaki; Nao Kagawa; Nobuyuki Fukushima

  Biochemical and biophysical research communications 568 1 - 7 2021/06 [Refereed]
   

  Lysophosphatidic acid (LPA) signaling plays diverse roles in the development of various vertebrates such as mammals and fish. The lamprey is a fish that retains ancestral features of vertebrates, but information regarding lamprey LPA receptor genes is limited. Here, using information from the lamprey genome database, we cloned two LPA receptor genes, Lpar1 and Lpar5, from the Japanese lamprey (Lethenteron camtschaticum). Lamprey Lpar1 had a high amino acid identity to mouse and medaka fish Lpar1, whereas Lpar5 amino acid sequences were more diverse between species. Our functional analyses using a heterologous expression system demonstrated that Lpar1 and Lpar5 responded to LPA treatment with G12/13-associated cellular responses, which are indicative of cytoskeletal actions. The existence of functional LPA receptors in the Japanese lamprey suggests that LPA receptor-dependent signals contribute to lamprey growth and development.
• Expression of lysophosphatidic acid receptor 1 in the adult female mouse pituitary gland.

  Ryutaro Moriyama; Nobuyuki Fukushima

  Neuroscience letters 741 135506 - 135506 2021/01 [Refereed]
   

  Lysophosphatidic acid receptor 1 (LPA1) is a receptor of lysophosphatidic acid (LPA). The present study investigated Lpar1 mRNA expression in the mouse pituitary gland by RT-PCR, in situ hybridization, and immunohistochemistry. Lpar1 mRNA was abundantly expressed in the pituitary gland. In situ hybridization and immunohistochemistry revealed over 90 % of a common glycoprotein α-subunit, luteinizing hormone β-subunit, and thyroid-stimulating hormone β-subunit immunoreactive cells co-expressed Lpar1 mRNA in the anterior pituitary gland, but few growth hormone, adrenocorticotropic hormone, and prolactin cells co-expressed Lpar1. Furthermore, Lpar1 mRNA levels in the pituitary gland were increased after ovariectomy and decreased after E2 administration. These results demonstrate that LPA1-mediated signaling may play physiological roles in gonadotropes and thyrotropes in the mouse pituitary gland.
• Cytosine arabinoside induces phosphorylation of histone H2AX in hippocampal neurons via a noncanonical pathway.

  Saki Nakayama; Miyu Adachi; Misaki Hatano; Noriyuki Inahata; Tetsuji Nagao; Nobuyuki Fukushima

  Neurochemistry international 142 104933 - 104933 2021/01 [Refereed]
   

  Cytosine arabinoside (Ara-C), an anticancer drug, is known to inhibit DNA replication in mitotic cells. Ara-C is also considered to induce DNA damage, leading to neuronal cell death. To identify the mechanism by which Ara-C kills neurons, we assessed the levels of phosphorylated histone H2AX (γ-H2AX), a marker for DNA double-strand breaks (DSBs), in hippocampal neurons cultured for 48 h with Ara-C. There was a time-dependent increase in the percentage of cells accumulating γ-H2AX, but TUNEL staining did not indicate the formation of DSBs. The nuclear spread of γ-H2AX remained after Ara-C was withdrawn. These features of Ara-C-induced γ-H2AX formation were quite distinct from those observed in proliferating pheochromocytoma cells. Furthermore, Ara-C-induced γ-H2AX formation appeared to utilize cyclin-dependent kinase 7, but not ataxia telangiectasia mutated (ATM) or ATM and Rad3 related, which are well-known kinases in γ-H2AX formation. Taken together, our findings indicated that Ara-C stimulated γ-H2AX formation in neurons without DSB formation and utilization of canonical kinases, leading to neuronal cell death.
• Involvement of FFA1 and FFA4 in the regulation of cellular functions during tumor progression in colon cancer cells

  Kaede Takahashi; Kaori Fukushima; Yuka Onishi; Kanako Minami; Shiho Otagaki; Kaichi Ishimoto; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  Experimental Cell Research Elsevier Inc. 369 (1) 54 - 60 1090-2422 2018/08 [Refereed]
   

  Free fatty acid receptor 1 (FFA1) and FFA4 mediate a variety of biological responses through binding of medium- and long-chain free fatty acids. The aim of this study was to investigate an involvement of FFA1 and FFA4 in the regulation of cellular functions during tumor progression in colon cancer cells. The long-term fluorouracil (5-FU) and cisplatin (CDDP) treated cells were generated from DLD1 cells (DLD-5FU and DLD-CDDP cells, respectively). FFAR1 expressions were lower in DLD-5FU and DLD-CDDP cells than in DLD1 cells. In contrast, DLD-5FU and DLD-CDDP cells showed the high FFAR4 expressions, compared with DLD1 cells. The cell motile activities of DLD-5FU and DLD-CDDP cells were reduced by GW9508 which is an agonist of FFA1 and FFA4. Moreover, GW1100, an antagonist of FFA1, inhibited the cell motile activities of DLD-5FU and DLD-CDDP cells. To evaluate whether FFA1 and FFA4 regulate the enhancement of cell motility, invasion and colony formation, highly migratory (hmDLD1) cells were established from DLD1 cells. FFAR1 expression was significantly higher in hmDLD1 cells than in DLD1 cells, but no change of FFAR4 expression was observed. The elevated cell motile and invasive activities and colony formation of hmDLD1 cells were suppressed by FFA1 inhibition. These results suggest that FFA1 and FFA4 are involved in the regulation of cellular functions during tumor progression in colon cancer DLD1 cells.
• Lysophosphatidic acid receptor-2 (LPA₂) and LPA₅ regulate cellular functions during tumor progression in fibrosarcoma HT1080 cells.

  Takahashi K; Minami K; Otagaki S; Ishimoto K; Fukushima K; Fukushima N; Honoki K; Tsujiuchi T

  Biochemical and biophysical research communications 0006-291X 2018/08 [Refereed]
  
• Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells.

  Fukushima K; Takahashi K; Kusaka M; Ishimoto K; Minami K; Otagaki S; Fukushima N; Honoki K; Tsujiuchi T

  Journal of receptor and signal transduction research 1 - 5 1079-9893 2018/08 [Refereed]
  
• Promotion of cell-invasive activity through the induction of LPA receptor-1 in pancreatic cancer cells.

  Fukushima K; Otagaki S; Takahashi K; Minami K; Ishimoto K; Fukushima N; Honoki K; Tsujiuchi T

  Journal of receptor and signal transduction research 38 (4) 367 - 371 1079-9893 2018/08 [Refereed]
  
• Involvement of LPA receptor-5 in the enhancement of cell motile activity by phorbol ester and anticancer drug treatments in melanoma A375 cells

  Kaori Fukushima; Kaede Takahashi; Aya Kurokawa; Kaichi Ishimoto; Shiho Otagaki; Kanako Minami; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  Biochemical and Biophysical Research Communications Elsevier B.V. 496 (1) 225 - 230 1090-2104 2018/01 [Refereed]
   

  Lysophosphatidic acid (LPA) signaling through six subtypes of LPA receptors (LPA1 to LPA6) regulates a variety of biological responses in cancer cells. The aim of our study was to evaluate an involvement of LPA receptors in the activation of cell motility by phorbol ester and anticancer drug treatments in melanoma A375 cells. Cells were treated with 12-O-tetradecanoylphorbol- 13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu) for 3 days. The cell motile activity of TPA treated cells was significantly higher than that of PDBu treated cells, correlating with LPAR5 expression levels. LPA5 knockdown suppressed the high cell motile activity induced by TPA. To assess whether the cell motile activity of A375 cells is stimulated through LPA5 induced by anticancer drugs, the long-term cisplatin (CDDP) and dacarbazine (DTIC) treated cells were generated from A375 cells (A375-CDDP and A375-DTIC cells, respectively). The expression levels of LPA receptor genes were changed in A375-CDDP and A375-DTIC cells. In particular, CDDP and DTIC treatment markedly elevated LPAR5 expressions. The cell motile activities of A375-CDDP and A375-DTIC cells were significantly higher than that of untreated cells. These results suggest that the cell motile activity is regulated through the induction of LPA5 by phorbol ester and anticancer drug treatments in A375 cells.
• Effects of LPA1 and LPA6 on the regulation of colony formation activity in colon cancer cells treated with anticancer drugs

  Kaede Takahashi; Kaori Fukushima; Shiho Otagaki; Kaichi Ishimoto; Kanako Minami; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  Journal of Receptors and Signal Transduction Taylor and Francis Ltd 38 (1) 71 - 75 1532-4281 2018/01 [Refereed]
   

  Lysophosphatidic acid (LPA) is a simple physiological lipid and exhibits a variety of cellular responses via the activation of G protein-coupled transmembrane LPA receptors (LPA receptor-1 (LPA1) to LPA6). The aim of our study was to investigate effects of LPA receptors on soft agar colony formation in colon cancer cells treated with anticancer drugs. DLD1 cells were treated with fluorouracil (5-FU) or cisplatin (CDDP) for at least six months (DLD-5FU and DLD-CDDP cells, respectively). LPAR1 gene expression was markedly elevated in DLD-5FU cells. In contrast, DLD-CDDP cells showed the high expression of LPAR6 gene. In colony formation assay, DLD-5FU cells formed markedly large-sized colonies, while no colony formation was observed in DLD1 and DLD-CDDP cells. The large-sized colonies formed in DLD-5FU cells were suppressed by LPA1 knockdown. In contrast, LPA6 knockdown increased the size of colonies. In addition, DLD-5FU cells were further treated with CDDP for three months (DLD-C-F cells). DLD-CDDP cells were also treated with 5-FU (DLD-F-C cells). DLD-C-F cells formed large-sized colonies, but not DLD-F-C cells, correlating with LPAR1 and LPAR6 gene expression levels. These results suggest that LPA1 and LPA6 may regulate the colony formation activity in DLD1 cells treated with anticancer drugs.
• Involvement of LPA signaling via LPA receptor-2 in the promotion of malignant properties in osteosarcoma cells

  Kaede Takahashi; Kaori Fukushima; Kosuke Tanaka; Kanako Minami; Kaichi Ishimoto; Shiho Otagaki; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  Experimental Cell Research Elsevier Inc. 369 (2) 316 - 324 1090-2422 2018 [Refereed]
   

  Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors mediates various biological effects in cancer cells. This study aimed to investigate the roles of LPA receptors in the regulation of cellular functions during tumor progression in osteosarcoma cells. Long-term cisplatin (CDDP)-treated MG63-C and MG63-R7-C cells were generated from osteosarcoma MG-63 and highly-migratory MG63-R7 cells, respectively. LPAR2 and LPAR3 expression levels were significantly higher in MG63-C cells than in MG-63 cells, while LPAR1 expression was reduced. MG63-C cells were highly motile, compared with MG-63 cells. MG63-C cell motility was suppressed by LPA2 knockdown and enhanced by the LPA1/LPA3 antagonist, dioctanoylglycerol pyrophosphate. LPAR2 and LPAR3 expression levels were significantly elevated in MG63-R7-C cells in comparison with MG63-R7 cells. MG63-R7-C cells were found to be highly invasive, correlating with metalloproteinase-2 activation. MG63-R7-C cells formed large colonies, whereas colony formation was absent from MG63-R7 cells. Notably, MG63-R7-C cell activities were inhibited by LPA2 knockdown. These results suggest that LPA signaling via LPA2 plays an important role in the acquisition of malignant properties during tumor progression in MG-63 cells.
• Exposure to bisphenol A affects GABAergic neuron differentiation in neurosphere cultures

  Nobuyuki Fukushima; Tetsuji Nagao

  NeuroReport Lippincott Williams and Wilkins 29 (9) 712 - 717 1473-558X 2018 [Refereed]
   

  Endocrine-disrupting chemicals (EDCs) influence not only endocrine functions but also neuronal development and functions. In-vivo studies have suggested the relationship of EDC-induced neurobehavioral disorders with dysfunctions of neurotransmitter mechanisms including γ-Aminobutyric acid (GABA)ergic mechanisms. However, whether EDCs affect GABAergic neuron differentiation remains unclear. In the present study, we show that a representative EDC, bisphenol A (BPA), affects GABAergic neuron differentiation. Cortical neurospheres prepared from embryonic mice were exposed to BPA for 7 days, and then neuronal differentiation was induced. We found that BPA exposure resulted in a decrease in the ratio of GABAergic neurons to total neurons. However, the same exposure stimulated the differentiation of neurons expressing calbindin, a calcium-binding protein observed in a subpopulation of GABAergic neurons. These findings suggested that BPA might influence the formation of an inhibitory neuronal network in developing cerebral cortex involved in the occurrence of neurobehavioral disorders.
• Polyunsaturated fatty acids induce ovarian cancer cell death through ROS-dependent MAP kinase activation

  Aiko Tanaka; Akane Yamamoto; Kaeko Murota; Toshifumi Tsujiuchi; Masao Iwamori; Nobuyuki Fukushima

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 493 (1) 468 - 473 0006-291X 2017/11 [Refereed]
   

  Free fatty acids not only play a role in cell membrane construction and energy production but also exert diverse cellular effects through receptor and non-receptor mechanisms. Moreover, epidemiological and clinical studies have so far suggested that polyunsaturated fatty acids (PUFAs) could have health benefits and the advantage as therapeutic use in cancer treatment. However, the underlying mechanisms of PUFA-induced cellular effects remained to be cleared. Here, we examined the effects of omega-3 and to omega-6 PUFAs on cell death in ovarian cancer cell lines. omega-3 PUFA, docosahexaenoic acid (DHA) and omega-6 PUFA, gamma-linolenic acid (gamma-LNA) induced cell death in KF28 cells at the levels of physiological concentrations, but not HAC2 cells. Pharmacological and biochemical analyses demonstrated that cell death induced by DHA and gamma-LNA was correlated with activation of JNK and p38 MAP kinases, and further an upstream MAP kinase kinase, apoptosis signal -regulating kinase 1, which is stimulated by reactive oxygen species (ROS). Furthermore, an antioxidant vitamin E attenuated PUFA-induced cell death and MAP kinase activation. These findings indicate that PUFA-induced cell death involves ROS-dependent MAP kinase activation and is a cell type -specific action. A further study of the underlying mechanisms for ROS-dependent cell death induced by PUFAs will lead to the discovery of a new target for cancer therapy or diagnosis. (C) 2017 Elsevier Inc. All rights reserved.
• Enhanced cellular functions through induction of LPA(2) by cisplatin in fibrosarcoma HT1080 cells

  Kaede Takahashi; Kaori Fukushima; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  MOLECULAR AND CELLULAR BIOCHEMISTRY SPRINGER 431 (1-2) 29 - 35 0300-8177 2017/07 [Refereed]
   

  Lysophosphatidic acid (LPA) is a simple biophysical lipid which interacts with at least six subtypes of G protein-coupled LPA receptors (LPA(1)-LPA(6)). In cancer cells, LPA signaling via LPA receptors is involved in the regulation of malignant properties, such as cell growth, motility, and invasion. The aim of this study was to assess whether LPA receptors regulate cellular functions of fibrosarcoma cells treated with anticancer drug. HT1080 cells were maintained by the stepwise treatment of cisplatin (CDDP) at a range of 0.01 to 1.0 A mu M for approximately 6 months. The cell motile and invasive activities of long-term CDDP-treated (HT-CDDP) cells were significantly stimulated by LPA treatment, while HT-CDDP cells in the static state showed the low cell motile and invasive activities in comparison with HT1080 cells. Since the expression level of LPAR2 gene was markedly elevated in HT-CDDP cells, LPA(2) knockdown cells were generated from HT-CDDP cells. The cell motile and invasive activities of HT-CDDP cells were reduced by LPA(2) knockdown. In colony assay, large-sized colonies formed by long-term CDDP treatment were suppressed by LPA(2) knockdown. In addition, LPA(2) knockdown cells reduced LPA production by autotaxin (ATX), correlating with ATX expression level. These results suggest that LPA signaling via LPA(2) may play an important role in the regulation of cellular functions in HT1080 cells treated with CDDP.
• Functional characterization of lysophosphatidic acid receptor 1 mutants identified in rat cancer tissues

  Shoichi Ishii; Toshifumi Tsujiuchi; Nobuyuki Fukushima

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 486 (3) 767 - 773 0006-291X 2017/05 [Refereed]
   

  Lysophosphatidic acid (LPA), an extracellular lipid mediator, exerts various cellular effects through activation of LPA receptors, LPA(1)-LPA(6), in many types of cells including cancer cells. We recently found several missense mutations of Lpar1 in rat cancer tissues. One of these mutations is located at the extracellular tip of the seventh transmembrane domain of LPA(1), and another three mutations are found within the NPXXY motif in the seventh transmembrane domain. These mutants are designated F295S LPA(1) and P308S, 1310T, and Y311H LPA(1), respectively. Here, we examined the functions of these LPA(1) mutants. Compared with wild-type (WT) LPA(1), F295S, P308S, and 1310T LPA(1) showed decreased maximal responses in inhibition of cAMP formation, Ca2+ mobilization, and cytoskeletal changes. Y311H LPA(1) failed to show LPA-induced cellular responses. However, these LPA(1) mutants were internalized in response to LPA exposure. Finally, while WT and F295S LPA(1) showed a similar, broad distribution throughout the cell, P308S, 1310T, and Y311H LPA(1) displayed a restricted cellular distribution and co-localized with the endoplasmic reticulum. These data suggest that the LPA(1) mutants perturb LPA signaling in cancer tissues. (C) 2017 Elsevier Inc. All rights reserved.
• Different effects of G -protein -coupled receptor 120 (GPR120) and GPR40 on cell motile activity of highly migratory osteosarcoma cells

  Kaede Takahashi; Kaori Fukushima; Yuka Onishi; Yusuke Node; Karin Inui; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 484 (3) 675 - 680 0006-291X 2017/03 [Refereed]
   

  G-protein-coupled receptor 120 (GPR120) and GPR40 are members of free fatty acid (FFA) receptors and mediate a variety of biological responses through binding of medium- and long -chain FFAs. Recently, it has been reported that GPR120 and GPR40 regulated cellular functions of cancer cells. In the present study, to assess whether GPR120 and GPR40 are involved in the enhancement of cell motile activity of osteosarcoma cells, we established highly migratory (MG63-R7) cells from osteosarcoma MG -63 cells. The expression level of GPR120 gene was significantly higher in MG63-R7 cells than in MG -63 cells, while no change of GPR40 expression was observed. In cell motility assay, the cell motile activity of MG63-R7 cells was approximately 200 times higher than that of MG-63 cells. The cell motile activity of MG63-R7 cells was stimulated by GW9508, which is an agonist of GPR120 and GPR40. Moreover, a GPR40 antagonist GW1100 elevated the cell motile activity of MG63-R7 cells in the presence of GW9508. To confirm the effects of GPR120 and GPR40 on the cell motile activity of MG63-R7 cells, GPR120 knockdown cells were generated from MG63-R7 cells. The cell motile activity of MG63-R7 cells was markedly suppressed by GPR120 knockdown. These results indicated that GPR120 enhanced and GPR40 inhibited the cell motile activity of highly migratory osteosarcoma cells. (C) 2017 Elsevier Inc. All rights reserved.
• Lysophosphatidic acid signaling via LPA(1) and LPA(3) regulates cellular functions during tumor progression in pancreatic cancer cells

  Kaori Fukushima; Kaede Takahashi; Eri Yamasaki; Yuka Onishi; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  EXPERIMENTAL CELL RESEARCH ELSEVIER INC 352 (1) 139 - 145 0014-4827 2017/03 [Refereed]
   

  Lysophosphatidic acid (LPA) signaling via G protein -coupled LPA receptors exhibits a variety of biological effects, such as cell proliferation, motility and differentiation. The aim of this study was to evaluate the roles of LPA(1) and LPA(3) in cellular functions during tumor progression in pancreatic cancer cells. LPA(1) and LPA(3) knockdown cells were generated from PANC-1 cells. The cell motile and invasive activities of PANC-1 cells were inhibited by LPA(1) and LPA(3) knockdown. In gelatin zymography, LPA(1) and LPA(3) knockdown cells indicated the low activation of matrix metalloproteinase-2 (MMP-2) in the presence of LPA. Next, to assess whether LPA1 and LPA3 regulate cellular functions induced by anticancer drug, PANC-1 cells were treated with cisplatin (CDDP) for approximately 6 months. The cell motile and invasive activities of long-term CDDP treated cells were markedly higher than those of PANC-1 cells, correlating with the expression levels of LPARI and LPAR3 genes. In soft agar assay, the long-term CDDP treated cells formed markedly large sized colonies. In addition, the cell motile and invasive activities enhanced by CDDP were significantly suppressed by LPA(1) and LPA(3) knockdown as well as colony formation. These results suggest that LPA signaling via LPA(1) and LPA(3) play an important role in the regulation of cellular functions during tumor progression in PANC-1 cells.
• Lysophosphatidic acid (LPA) signaling via LPA4 and LPA6 negatively regulates cell motile activities of colon cancer cells.

  Takahashi K; Fukushima K; Onishi Y; Fukushima N; Honoki K; Tsujiuchi T

  Biochem Biophys Res Commun. 483 652 - 657 2017 [Refereed]
  
• Different Induction of LPA Receptors by Chemical Liver Carcinogens Regulates Cellular Functions of Liver Epithelial WB-F344 Cells

  Miku Hirane; Shuhei Ishii; Ayaka Tomimatsu; Kaori Fukushima; Kaede Takahashi; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  MOLECULAR CARCINOGENESIS WILEY-BLACKWELL 55 (11) 1573 - 1583 0899-1987 2016/11 [Refereed]
   

  Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) mediates a variety of cellular functions, including cell motility. In the present study, we investigated the effects of LPA receptors on cell motile activity during multi-stage hepatocarcinogenesis in rat liver epithelial WB-F344 cells treated with chemical liver carcinogens. Cells were treated with a initiator (N-nitrosodiethylamine (DEN)) and three promoters (phenobarbital (PB), okadaic acid (OA) and clofibrate) every 24 h for 2 days. Cell motile activity was elevated by DEN, correlating with Lpar3 expression. PB, OA, and clofibrate elevated Lpar1 expression and inhibited cell motile activity. To evaluate the effects of long-term treatment on cell motility, cells were treated with DEN and/or PB for at least 6 months. Lpar3 expression and cell motile activity were significantly elevated by the long-term DEN treatment with or without further PB treatment. In contrast, long-term PB treatment with or without further DEN elevated Lpar1 expression and inhibited cell motility. When the synthesis of extracellular LPA was blocked by a potent ATX inhibitor S32826 before cell motility assay, the cell motility induced by DEN and PB was markedly suppressed. These results suggest that activation of the different LPA receptors may regulate the biological functions of cells treated with chemical carcinogens. (C) 2015 Wiley Periodicals, Inc.
• Negative Effects of G-Protein-Coupled Free Fatty Acid Receptor GPR40 on Cell Migration and Invasion in Fibrosarcoma HT1080 Cells

  Shuhei Ishii; Yuka Kitamura; Miku Hirane; Ayaka Tomimatsu; Kaori Fukushima; Kaede Takahashi; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  MOLECULAR CARCINOGENESIS WILEY-BLACKWELL 55 (11) 1553 - 1559 0899-1987 2016/11 [Refereed]
   

  G-protein-coupled receptor 40 (GPR40) and GPR120 mediate a variety of biological functions by the binding of long and medium chain free fatty acids. In the present study, we investigated a role of GPR40 in the pathogenesis of fibrosarcoma HT1080 cells. The GPR40 gene expression was detected in HT1080 cells, but not the GPR120 gene. The cell motile and invasive activities were markedly enhanced by GPR40 knockdown, compared with control cells. To evaluate whether GPR40 is involved in the cellular functions of HT1080 cells during anticancer drug treatment, HT1080 cells were maintained in condition medium containing cisplatin (CDDP) (0.01-1.0 mu M) for 6 mo. The expression levels of the GPR40 gene was elevated by the long-term CDDP treatment in HT1080 cells, while the GPR120 gene expression remained unchanged. The cell motile and invasive activities of HT1080 cells treated with CDDP were significantly lower than those of untreated cells. In gelatin zymography, the activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 of HT1080 cells were enhanced by the long-term CDDP treatment. In addition, GW9508 which is an agonist of GPR40 and GPR120 suppressed the cell motile and invasive activities of HT1080 cells treated with CDDP as well as the MMP activation. These results suggest that GPR40 negatively regulates the tumor progression of fibrosarcoma cells. (C) 2015 Wiley Periodicals, Inc.
• Different effects of GPR120 and GPR40 on cellular functions stimulated by 12-O-tetradecanoylphorbol-13- acetate in melanoma cells.

  FUKUSHIMA Nobuyuki

  Biochem. Biophys. Res. Commun. 475 25 - 30 2016/07 [Refereed]
  
• Diverse effects of G-protein-coupled free fatty acid receptors on the regulation of cellular functions in lung cancer cells

  Tsubasa Kita; Yui Kadochi; Kaede Takahashi; Kaori Fukushima; Eri Yamasaki; Taiki Uemoto; Miku Hirane; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  EXPERIMENTAL CELL RESEARCH ELSEVIER INC 342 (2) 193 - 199 0014-4827 2016/03 [Refereed]
   

  Free fatty acids (FFAs) are dietary nutrients which mediate a variety of biological effects through binding to G-protein-coupled FFA receptors (FFARs). G-protein-coupled receptor 120 (GPR120) and GPR40 are identified as FFARs for long- and medium-chain fatty acids. Here we investigated whether GPR120 and GPR40 are involved in the acquisition of malignant properties in lung cancer cells. Three lung cancer RLCNR, LL/2 and A549 cells used in this study expressed GPR120 and GPR40 genes. The cell motile activities of all cells were significantly suppressed by a GPR40 antagonist GW1100. In addition, GPR40 knockdown inhibited the cell motile activity of A549 cells. In gelatin zymography, matrix metalloproteinase-2 (MMP-2) activity in GPR40 knockdown was significantly lower than that in control cells. Next, to evaluate effects of GPR120 and GPR40 on cellular functions induced by anti-cancer drug, the long-term cisplatin (CDDP) treated (A549-CDDP) cells were generated. The expression levels of GPR120 and GPR40 were significantly decreased in A549-CDDP cells. While A549-CDDP cells showed the high cell motile activity, GW1100 suppressed the cell motile activity of A549-CDDP cells. These results demonstrate that GPR120 negatively and GPR40 positively regulate cellular functions during tumor progression in lung cancer cells. (C) 2016 Elsevier Inc. All rights reserved.
• LPA signaling through LPA receptors regulates cellular functions of endothelial cells treated with anticancer drugs

  Shiori Mori; Mutsumi Araki; Shuhei Ishii; Miku Hirane; Kaori Fukushima; Ayaka Tomimatsu; Kaede Takahashi; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  MOLECULAR AND CELLULAR BIOCHEMISTRY SPRINGER 408 (1-2) 147 - 154 0300-8177 2015/10 [Refereed]
   

  Lysophosphatidic acid (LPA) signaling via LPA receptors provides a variety of cellular functions, including angiogenesis. In this study, to assess an involvement of LPA receptors in cell motile activities of endothelial cells during chemotherapy, F-2 cells were treated with cisplatin (CDDP) and doxorubicin (DOX) at a concentration of 0.01 mu M every 24 h for at least 1 month. The treatment of CDDP and DOX inhibited the expression levels of the LPA receptor-1 (Lpar1), Lpar2, and Lpar3 genes in F-2 cells. The cell motile activities of CDDP and DOX treated cells were relatively lower than those of untreated cells. Next, we investigated whether cancer cells could stimulate the cell motile activities of F-2 cells treated with CDDP and DOX. For cell motility assay, CDDP- and DOX-treated cells were co-cultured with pancreatic cancer PANC-1 cells. The cell motile activities of CDDP- and DOX-treated cells were significantly enhanced by the existence of PANC-1 cells, correlating with the LPA receptor expressions. In addition, the elevated cell motile activities were suppressed by the pretreatment of an autotaxin inhibitor S32826. These results suggest that LPA signaling via LPA receptors may regulate the cell motile activities of F-2 cells treated with anticancer drugs.
• Different roles of GPR120 and GPR40 in the acquisition of malignant properties in pancreatic cancer cells

  Kaori Fukushima; Eri Yamasaki; Shuhei Ishii; Ayaka Tomimatsu; Kaede Takahashi; Miku Hirane; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 465 (3) 512 - 515 0006-291X 2015/09 [Refereed]
   

  Free fatty acids (FFAs) act as extracellular signaling molecules through binding to G-protein-coupled FFA receptors (FFARs). GPR120 and GPR40 are identified as FFARs for medium- and long-chain fatty acids. In the present study, we investigated roles of GPR120 and GPR40 in cellular functions of pancreatic cancer PANC-1 cells, using GPR120 and GPR40 knockdown cells (PANC-sh120 and PANC-sh40 cells respectively). In cell motility assay, PANC-sh120 cells showed the low cell motility, compared with control cells. In contrast, the cell motility of PANC-sh40 cells was significantly higher than that of control cells. Activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. While PANC-sh120 cells indicated the reduced MMP-2 activity, MMP-2 activity in PANC-sh40 cells was significantly higher than that in control cells. On the other hand, no activation of MMP-9 was detected in all cells. In colony assay, the large sized colonies were markedly formed in PANC-sh40 cells. No colony formation was observed in PANC-sh120 cells as well as control cells. These results suggest that distinct effects of GPR120 and GPR40 are involved in the acquisition of malignant property in pancreatic cancer cells. (C) 2015 Elsevier Inc. All rights reserved.
• Comparative analyses of lysophosphatidic acid receptor-mediated signaling

  Nobuyuki Fukushima; Shoichi Ishii; Toshifumi Tsujiuchi; Nao Kagawa; Kazutaka Katoh

  CELLULAR AND MOLECULAR LIFE SCIENCES SPRINGER BASEL AG 72 (12) 2377 - 2394 1420-682X 2015/06 [Refereed]
   

  Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish.
• Diverse effects of LPA(4), LPA(5) and LPA(6) on the activation of tumor progression in pancreatic cancer cells

  Shuhei Ishii; Miku Hirane; Kaori Fukushima; Ayaka Tomimatsu; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 461 (1) 59 - 64 0006-291X 2015/05 [Refereed]
   

  Lysophosphatidic acid (LPA) is an extracellular biological lipid which interacts with G protein-coupled LPA receptors (LPA(1) to LPA(6)). LPA signaling via LPA receptors mediates several cellular responses. In the present study, to assess the roles of LPA(4), LPA(6) and LPA(6) in cellular functions of pancreatic cancer cells, we generated LEA receptor knockdown cells from PANC-1 cells (PANC-sh4, PANC-sh5 and PANC-sh6 cells, respectively). In cell motility assay, PANC-sh4 and PANC-sh5 cells enhanced the cell motile activities, compared with control cells. In contrast, the cell motile activity of PANC-sh6 cells was suppressed. The invasive activities of PANC-sh4 and PANC-sh5 cells were markedly stimulated, while PANC-sh6 cells showed the low invasive activity. In colony assay, PANC-sh4 and PANC-sh5 cells formed the large sized colonies, but not PANC-sh6 cells. When endothelial cells were incubated with supernatants from PANC-sh4 and PANC-sh5 cells, the cell motility and tube formation of endothelial cells were significantly induced, but not PANC-sh6 cells. These results suggest that the diverse roles of LPA(4), LPA(6) and LPA(6) are involved in the activation of tumor progression in pancreatic cancer cells. (C) 2015 Elsevier Inc. All rights reserved.
• Role of GPR120 in cell motile activity induced by 12-O-tetradecanoylphorbol-13-acetate in liver epithelial WB-F344 cells

  Shuhei Ishii; Miku Hirane; Yuka Kitamura; Shiori Mori; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  MOLECULAR AND CELLULAR BIOCHEMISTRY SPRINGER 400 (1-2) 145 - 151 0300-8177 2015/02 [Refereed]
   

  G-protein-coupled receptor 120 (GPR120) is identified as a G-protein-coupled receptor for unsaturated long-chain free fatty acids that mediates insulin signaling and anti-inflammatory effects. Recently, it has been reported that GPR120 promotes the cell motile activity and angiogenesis in cancer cells. In this study, we assessed the role of GPR120 in the cell motile activity induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in rat liver epithelial WB-F344 cells. Cells were treated with TPA at a concentration of 5 nM for 72 h. The expression level of the Gpr120 gene was measured by quantitative real-time RT-PCR analysis. Cells treated with TPA showed the elevated Gpr120 expression, in comparison with untreated cells. In cell motility assays, the cell motile activity of cells treated with TPA was significantly higher than that of untreated cells. To confirm whether GPR120 is involved in the cell motile activity mediated by TPA, we generated GPR120 knockdown cells from WB-F344 cells. The cell motile activity induced by TPA was significantly suppressed by GPR120 knockdown. These results suggest that GPR120 plays an important role in the cell motile activity induced by TPA in WB-F344 cells.
• Expression of the long-chain fatty acid receptor GPR120 in the gonadotropes of the mouse anterior pituitary gland

  Ryutaro Moriyama; Chikaya Deura; Shingo Imoto; Kazuhiro Nose; Nobuyuki Fukushima

  HISTOCHEMISTRY AND CELL BIOLOGY SPRINGER 143 (1) 21 - 27 0948-6143 2015/01 [Refereed]
   

  G-protein-coupled receptor 120 (GPR120) has been known to be a receptor of long-chain fatty acids. Here, we investigated GPR120 expression in the mouse pituitary gland via real-time PCR, in situ hybridization, and immunohistochemistry. GPR120 mRNA was abundantly expressed in the pituitary gland of ad-lib fed animals. In situ hybridization and immunohistochemistry revealed GPR120 expression in the gonadotropes of the anterior pituitary gland, but not in thyrotropes, somatotropes, lactotropes, corticotropes, melanotropes, and the posterior pituitary gland. Furthermore, 24 h of fasting induced an increase in GPR120 mRNA expression in the pituitary gland. These results demonstrate that GPR120 in mouse pituitary gonadotropes is upregulated by fasting and that it may play a role in controlling gonadotropin secretion.
• Opposite effects of GPR120 and GPR40 on cell motile activity induced by ethionine in liver epithelial cells

  Shuhei Ishii; Miku Hirane; Sayumi Kato; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 456 (1) 135 - 138 0006-291X 2015/01 [Refereed]
   

  Free fatty acids (FFAs) are dietary nutrients which act as ligands for FFAs receptors. G-protein-coupled receptor 120 (GPR120) and GPR40 are activated by long and medium chain FFAs. In the present study, we investigated the role of the GPR120 and GPR40 in cell motile activity stimulated by ethionine in rat liver epithelial WB-F344 cells. Cells were treated with ethionine at a concentration of 10 mu M every 24 h for 2 days. The expression levels of the Gpr120 and Gpr40 genes in WB-F344 cells treated ethionine were significantly higher than those in untreated cells. In cell motility assay, the cell motile activity of WB-F344 cells was markedly elevated by ethionine, compared with untreated cells. To evaluate the effects of GPR120 on the cell motile activity by ethionine, we established GPR120 knockdown cells from WB-F344 cells. The cell motile activity stimulated by ethionine was significantly suppressed by GPR120 knockdown. In addition, a potent GPR40 antagonist GW1100 enhanced the cell motile activity by ethionine. These results suggest that opposite effects of GPR120 and GPR40 may be involved in the cell motile activity stimulated by ethionine in WB-F344 cells. (C) 2014 Elsevier Inc. All rights reserved.
• Functional lysophosphatidic acid receptors expressed in Oryzias latipes

  Yuji Morimoto; Shoichi Ishii; Jun-ichi Ishibashi; Kazutaka Katoh; Toshifumi Tsujiuchi; Nao Kagawa; Nobuyuki Fukushima

  GENE ELSEVIER SCIENCE BV 551 (2) 189 - 200 0378-1119 2014/11 [Refereed]
   

  Lysophosphatidic acid (LPA) signaling is known to play biological and pathophysiological roles in many types of animals. Medaka (Oryzias latipes) is an experimental fish that can be easily maintained, propagated, and analyzed, and whose genome has been completely sequenced. However, there is limited information available regarding medaka LPA receptors. Here, using information from the medaka genome database, we examine the genomic structures, expression, and functions of six LPA receptor genes, Lpar1-Lpar6. Our analyses reveal that the genomic structures of Lpar1 and Lpar4 are different from those deduced from the database. Functional analyses using a heterologous expression system demonstrate that all medaka LPA receptors except for LPA(5b) respond to LPA treatment with cytoskeletal changes. These findings provide useful information on the structure and function of medaka LPA receptor genes, and identify medaka as a useful experimental model for exploration of the biological significance of LPA signaling. (C) 2014 Elsevier B.V. All rights reserved.
• Lysophosphatidic acid receptor-5 negatively regulates cell motile and invasive activities of human sarcoma cell lines

  Yan Dong; Miku Hirane; Mutsumi Araki; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  MOLECULAR AND CELLULAR BIOCHEMISTRY SPRINGER 393 (1-2) 17 - 22 0300-8177 2014/08 [Refereed]
   

  LPA signaling via LPA receptors [LPA receptor-1 (LPA(1))-LPA(6)] mediates the several cellular responses in cancer cells, including cell motility and invasion. In the present study, to investigate a role of LPA(5) in the cell motile and invasive activities of sarcoma cells, LPAR5 knockdown (HOSL5 and HT1080L5) cells were generated from human osteosarcoma HOS and fibrosarcoma HT1080 cells, respectively. In cell motility assays with cell culture inserts, HOSL5 and HT1080L5 cells indicated the high cell motile activities, compared with control cells. The cell invasive activities of HOSL5 and HT1080L5 cells were significantly higher than those of control cells. Moreover, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by gelatin zymography. MMP-2 was significantly activated in HOSL5 cells, but not MMP-9. The elevated activities of MMP-2 and MMP-9 were found in HT1080L5 cells, in comparison with control cells. These results suggest that LPA signaling via LPA(5) negatively regulates the cell motile and invasive activities of human sarcoma cells.
• Long-term exposure of 3T3 fibroblast cells to endocrine disruptors alters sensitivity to oxidative injury

  Yuka Nishimura; Yasuyoshi Nakai; Aiko Tanaka; Tetsuji Nagao; Nobuyuki Fukushima

  CELL BIOLOGY INTERNATIONAL WILEY-BLACKWELL 38 (7) 868 - 874 1065-6995 2014/07 [Refereed]
   

  When Swiss 3T3 fibroblasts were exposed to bisphenol A (BPA) or nonylphenol (NP) within a range of 0.1-100 nM for 30-45 days, increased resistance to oxidative injury was found. Western blot analysis indicated concomitant increased expression of bcl-2 protein and reduced histone methylation levels in cells after BPA or NP exposure. Using a heterologous expression system, both chemicals could stimulate G protein-coupled receptor 30 (GPR30), a transmembrane estrogen receptor predominantly expressed in 3T3 cells, at lower concentrations, which gave increased survival. Taken together, these results suggest that BPA or NP exposure might cause alterations in cellular activity against oxidative stress, possibly through GPR30.
• Diverse effects of LPA receptors on cell motile activities of cancer cells

  Toshifumi Tsujiuchi; Miku Hirane; Yan Dong; Nobuyuki Fukushima

  JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION INFORMA HEALTHCARE 34 (3) 149 - 153 1079-9893 2014/06 [Refereed]
   

  Lysophosphatidic acid (LPA) is a bioactive lipid that interacts with G protein-coupled transmembrane LPA receptors (LPA(1) to LPA(6)). LPA mediates a variety of cellular responses in normal cells, including cell growth, motility, differentiation, morphogenesis, and prevention from apoptosis. Furthermore, LPA signaling via LPA receptors is involved in the pathogenesis of several diseases, including cancer. Cell motility is one of the important properties during the progression of cancer cells. In recent studies, it has been demonstrated that LPA receptors have the diverse effects in the cell motile activities of cancer cells, depending on the cell types involved. In this review, we provide the current knowledge for the biological roles of LPA receptors in the cell motile activities of cancer cells.
• Effects of bisphenol A and 4-nonylphenol on cellular responses through the different induction of LPA receptors in liver epithelial WB-F344 cells

  Yan Dong; Mutsumi Araki; Miku Hirane; Eriko Tanabe; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION INFORMA HEALTHCARE 34 (3) 201 - 204 1079-9893 2014/06 [Refereed]
   

  Lysophosphatidic acid (LPA) signaling via G protein-coupled transmembrane LPA receptors (LPA(1) to LPA(6)) mediates a variety of cellular functions, including cell proliferation, migration, morphogenesis, and differentiation. Recently, we demonstrated that the different induction of LPA receptors by estrogens regulates cell motile activity of rat liver epithelial WB-F344 cells. In the present study, to assess whether endocrine disruptors (EDs) are involved in cellular functions through LPA signaling, we measured cell motile activity and LPA receptor expressions in WB-F344 cells treated with bisphenol A (BPA) and 4-nonylphenol (4-NP). Using quantitative real time RT-PCR analysis, the Lpar1 expression was elevated in BPA-treated cells, whereas the Lpar3 expression was decreased. In contrast, 4-NP increased the Lpar3 expression, but not the Lpar1 and Lpar2. For cell motility assay with a Cell Culture Insert, cell motile activity of BPA-treated cells was significantly lower than that of untreated cells. In contrast, 4-NP markedly enhanced cell motile activity. The effects of BPA and 4-NP on cell motility were inhibited by the Lpar1 or Lpar3 knockdown. These results suggest that BPA and 4-NP may regulate cell motile activity through the different induction of LPA receptors in WB-F344 cells.
• Inhibitory effects of lysophosphatidic acid receptor-5 on cellular functions of sarcoma cells

  Mutsumi Araki; Misaho Kitayoshi; Yan Dong; Miku Hirane; Shuhei Ozaki; Shiori Mori; Nobuyuki Fukushima; Kanya Honoki; Toshifumi Tsujiuchi

  GROWTH FACTORS INFORMA HEALTHCARE 32 (3-4) 117 - 122 0897-7194 2014/06 [Refereed]
   

  Lysophosphatidic acid (LPA) is a bioactive lipid that interacts with G protein-coupled LPA receptors (LPA receptor-1 (LPA(1)) to LPA(6)). Here, we investigated the effects of LPA signaling via LPA(5) on cellular functions of sarcoma cells by generating Lpar5 overexpressing and Lpar5 knockdown cells from rat osteosarcoma and malignant fibrous histiocytoma cells, respectively. The cell motility activity of Lpar5 overexpressing cells was significantly lower, while Lpar5 knockdown cells showed high cell motility, compared with respective controls. Gelatin zymography showed that LPA(5) suppressed the activation of matrix metalloproteinase-2. LPA(5) also inhibited the cell motility activity of endothelial cells, correlating with the expression levels of vascular endothelial growth factor genes. These results suggest that LPA signaling via LPA(5) negatively regulates the cellular functions of rat sarcoma cells.
• Long-term pre-exposure of pheochromocytoma PC12 cells to endocrine-disrupting chemicals influences neuronal differentiation

  Yuka Nishimura; Tetsuji Nagao; Nobuyuki Fukushima

  NEUROSCIENCE LETTERS ELSEVIER IRELAND LTD 570 1 - 4 0304-3940 2014/06 [Refereed]
   

  This study examined the effects of exposure to low concentrations (0.1-100 nM) of bisphenol A (BPA) or nonylphenol (NP) on neuronal differentiation in pheochromocytoma PC12 cells. Pre-exposure to BPA for a week or a month, but not for a day, decreased neuronal differentiation in PC12 cells. Likewise, one week's pre-exposure to NP also inhibited neuronal differentiation in these cells. The inhibitory effects were still observed when PC12 cells were treated with BPA or NP for a week, followed by a week's withdrawal. These findings suggest that long-term exposure of PC12 cells to low concentrations of BPA or NP leads to changes in the cellular machinery responsible for neuronal differentiation, and these changes might be retained within PC12 cells. (C) 2014 Elsevier Ireland Ltd. All rights reserved.
• Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

  Yan Dong; Miku Hirane; Mutsumi Araki; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 446 (2) 585 - 589 0006-291X 2014/04 [Refereed]
   

  Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA(1)-LPA(6)) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA(1) inhibited the cell motile activities of mouse fibroblast 313 cells. In the present study, to evaluate a role of LPA(5) in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA(1) and LPA(5) on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA(5) may act as a negative regulator of cellular responses in mouse fibroblast 313 cells, similar to the case for LPA(1). (C) 2014 Elsevier Inc. All rights reserved.
• Lysophosphatidic acid receptors in cancer pathobiology

  Toshifumi Tsujiuchi; Mutsumi Araki; Miku Hirane; Yan Dong; Nobuyuki Fukushima

  HISTOLOGY AND HISTOPATHOLOGY F HERNANDEZ 29 (3) 313 - 321 0213-3911 2014/03 [Refereed]
   

  Lysophosphatidic acid (LPA) receptors (LPA(1) to LPA(6)) are G protein-coupled transmembrane and mediate a variety of biological responses through the binding of LPA, such as cell proliferation, migration, morphogenesis and differentiation. Previously, high secretion levels of LPA were found in blood and ascites from patients with aggressive ovarian cancer. So far, numerical studies have demonstrated that LPA signaling via LPA receptors contributes to the acquisition of malignant potency by several cancer cells. Moreover, genetic and epigenetic alterations of LPA receptor genes have been detected in cancer cells. Therefore, it is suggested that LPA signaling may be a target molecule for the establishment of chemoprevention agents in clinical cancer approaches. Here, we review the current knowledge for the biological roles of LPA signaling via LPA receptors in the pathogenesis of cancer cells.
• Ethionine regulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

  Serina Inoue; Eriko Tanabe; Ayano Shibata; Miku Hirane; Mutsumi Araki; Yan Dong; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  MOLECULAR AND CELLULAR BIOCHEMISTRY SPRINGER 383 (1-2) 173 - 177 0300-8177 2013/11 [Refereed]
   

  Lysophosphatidic acid (LPA) receptors (LPA(1) to LPA(6)) indicate a variety of cellular responses, such as cell proliferation, migration, differentiation, and morphogenesis. However, the role of each LPA receptor is not functionally equivalent. Ethionine, an ethyl analog of methionine, is well known to be one of the potent liver carcinogens in rats. In this study, to assess whether ethionine may regulate cell motile activity through LPA receptors, rat liver epithelial (WB-F344) cells were treated with ethionine for 48 h. In cell motility assay with a cell culture insert, the treatment of ethionine at 1.0 and 10 mu M enhanced significantly high cell motile activity, compared with untreated cells. The expression levels of LPA receptor genes in cells treated with ethionine were measured by quantitative real time RT-PCR analysis. The expression of the Lpar3 gene in ethionine-treated cells was significantly higher than that in untreated cells. Furthermore, to confirm an involvement of LPA(3) on cell motility increased by ethionine, the Lpar3 knockdown cells were also used. The cell motile activity by ethionine was completely suppressed in the Lpar3 knockdown cells. These results suggest that LPA signaling through LPA(3) may be involved in cell motile activity stimulated by ethionine in WB-F344 cells.
• Inhibitory effects of LPA(1) on cell motile activities stimulated by hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone in fibroblast 3T3 cells

  Miku Hirane; Mutsumi Araki; Yan Dong; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 441 (1) 47 - 52 0006-291X 2013/11 [Refereed]
   

  Reactive oxygen species (ROS) are known to mediate a variety of biological responses, including cell motility. Recently, we indicated that lysophosphatidic acid (LPA) receptor-3 (LPA(3)) increased cell motile activity stimulated by hydrogen peroxide. In the present study, we assessed the role of LPA(1) in the cell motile activity mediated by ROS in mouse fibroblast 3T3 cells. 3T3 cells were treated with hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) at concentrations of 0.1 and 1 mu M for 48 h. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3 cells treated with hydrogen peroxide and DMNQ were significantly higher than those of untreated cells. 3T3 cells treated with hydrogen peroxide and DMNQ showed elevated expression levels of the Lpar3 gene, but not the Lpar1 and Lpar2 genes. To investigate the effects of LPA(1) on the cell motile activity induced by hydrogen peroxide and DMNQ, Lpar1-overexpressing (3T3-a1) cells were generated from 3T3 cells and treated with hydrogen peroxide and DMNQ. The cell motile activities stimulated by hydrogen peroxide and DMNQ were markedly suppressed in 3T3-a1 cells. These results suggest that LPA signaling via LPA(1) inhibits the cell motile activities stimulated by hydrogen peroxide and DMNQ in 3T3 cells. (C) 2013 Elsevier Inc. All rights reserved.
• Downregulation of activation factors of endothelia and fibroblasts via lysophosphatidic acid signaling in a mouse lung cancer LL/2 cell line

  Eriko Tanabe; Misaho Kitayoshi; Miku Hirane; Mutsumi Araki; Yan Dong; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  Journal of Receptors and Signal Transduction 33 (5) 286 - 290 1079-9893 2013/10 [Refereed]
   

  Angiogenesis stimulates the invasive and metastatic process of cancer cells. It is also known that activated fibroblasts promote cancer cell growth and enhance invasive and metastatic potential. Lysophosphatidic acid (LPA) is a biological mediator and interacts with G protein-coupled transmembrane LPA receptors (LPA1 to LPA6). In this study, to assess an involvement of LPA3 on angiogenesis and fibroblast activation, the Lpar3-expressing cells were generated from mouse lung cancer LL/2 cells, which unexpressed LPA3. The Lpar3-expressing cells were maintained in serum-free Dulbecco's modified Eagle's medium for 48h, and cell motility assay was performed with a cell culture Insert. When endothelial F-2 cells and 3T3 fibroblasts were cultured with conditioned medium from the Lpar3-expressing cells, their cell motile activities were significantly lower than the Lpar3-unexpressing (control) cells. Expression levels of vascular endothelial growth factor (Vegf) and fibroblast growth factor (Fgf) genes in the Lpar3-expressing cells were measured by quantitative real time reverse transcription polymerase chain reaction analysis. The expressions of Vegf-A. Fgfa and Fgfb genes in the Lpar3-expressing cells were significantly lower than those in control cells, correlating with the effects on cell motile activities of F-2 and 3T3 cells. These results suggest that LPA signaling through LPA3 may inhibit angiogenesis and fibroblast activation in mouse lung cancer cells. © 2013 Informa Healthcare USA, Inc.
• Extracellular lipid metabolism influences the survival of ovarian cancer cells

  Shohei Kuwata; Kei Ohkubo; Shun Kumamoto; Naoto Yamaguchi; Naoki Izuka; Kaeko Murota; Toshifumi Tsujiuchi; Masao Iwamori; Nobuyuki Fukushima

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 439 (2) 280 - 284 0006-291X 2013/09 

  Lysophosphatidic acid (LPA) is an extracellular lipid mediator consisting of a fatty acid and a phosphate group linked to the glycerol backbone. Here, we show that 1-oleoyl- and 1-palmitoyl-LPA, but not 1-stearoyl- or alkyl-LPA, enhance HNOA ovarian cancer cell survival. Other lysophospholipids with oleic or lauric acid, but not stearic acid, also induce the survival effects. HNOA cells have the lipase activities that cleave LPA to generate fatty acid. Oleic acid stimulates HNOA cell survival via increased glucose utilization. Our findings suggest that extracellular lysolipid metabolism might play an important role in HNOA cell growth. (C) 2013 Elsevier Inc. All rights reserved.
• Extracellular lipid metabolism influences the survival of ovarian cancer cells

  Shohei Kuwata; Kei Ohkubo; Shun Kumamoto; Naoto Yamaguchi; Naoki Izuka; Kaeko Murota; Toshifumi Tsujiuchi; Masao Iwamori; Nobuyuki Fukushima

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 439 (2) 280 - 284 0006-291X 2013/09 [Refereed]
   

  Lysophosphatidic acid (LPA) is an extracellular lipid mediator consisting of a fatty acid and a phosphate group linked to the glycerol backbone. Here, we show that 1-oleoyl- and 1-palmitoyl-LPA, but not 1-stearoyl- or alkyl-LPA, enhance HNOA ovarian cancer cell survival. Other lysophospholipids with oleic or lauric acid, but not stearic acid, also induce the survival effects. HNOA cells have the lipase activities that cleave LPA to generate fatty acid. Oleic acid stimulates HNOA cell survival via increased glucose utilization. Our findings suggest that extracellular lysolipid metabolism might play an important role in HNOA cell growth. (C) 2013 Elsevier Inc. All rights reserved.
• Hydrogen peroxide stimulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

  Ayano Shibata; Eriko Tanabe; Serina Inoue; Misaho Kitayoshi; Souta Okimoto; Miku Hirane; Mutsumi Araki; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  Biochemical and Biophysical Research Communications 433 (3) 317 - 321 0006-291X 2013/04 [Refereed]
   

  Hydrogen peroxide which is one of reactive oxygen species (ROS) mediates a variety of biological responses, including cell proliferation and migration. In the present study, we investigated whether lysophosphatidic acid (LPA) signaling is involved in cell motile activity stimulated by hydrogen peroxide. The rat liver epithelial WB-F344 cells were treated with hydrogen peroxide at 0.1 or 1μM for 48h. In cell motility assays, hydrogen peroxide treated cells showed significantly high cell motile activity, compared with untreated cells. To measure the expression levels of LPA receptor genes, quantitative real time RT-PCR analysis was performed. The expressions of LPA receptor-3 (Lpar3) in hydrogen peroxide treated cells were significantly higher than those in control cells, but not Lpar1 and Lpar2 genes. Next, to assess the effect of LPA3 on cell motile activity, the Lpar3 knockdown cells from WB-F344 cells were also treated with hydrogen peroxide. The cell motile activity of the knockdown cells was not stimulated by hydrogen peroxide. Moreover, in liver cancer cells, hydrogen peroxide significantly activated cell motility of Lpar3-expressing cells, but not Lpar3-unexpressing cells. These results suggest that LPA signaling via LPA3 may be mainly involved in cell motile activity of WB-F344 cells stimulated by hydrogen peroxide. © 2013 Elsevier Inc.
• Lysophosphatidic acid receptor-3 increases tumorigenicity and aggressiveness of rat hepatoma RH7777 cells

  Kyoko Okabe; Mai Hayashi; Kohei Kato; Mai Okumura; Rie Fukui; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  MOLECULAR CARCINOGENESIS WILEY-BLACKWELL 52 (4) 247 - 254 0899-1987 2013/04 [Refereed]
   

  Lysophosphatidic acid (LPA), which interacts with G protein-coupled transmembrane LPA receptors exhibits several biological effects, such as cell proliferation, migration, and differentiation. Recently, it has been reported that alteration of LPA receptor genes occurs in several cancer cells. In this study, to assess the biological role of LPA receptor-3 (LPA3) in the pathogenesis of tumor cells, we generated the Lpar3-expressing cells (RHa3B12 and RHa3G8) from rat hepatoma RH7777 cells, and examined their abilities of cell migration and tumorigenicity, compared with the Lpar3-unexpressing cells. In cell motility and invasion assays, RHa3B12 and RHa3G8 cells showed significantly higher intrinsic activity without LPA treatment than control RH7777AB cells. LPA treatment further increased cell motility and invasion of these cells. The cell motility of RHa3B12 and RHa3G8 cells stimulated by LPA treatment was significantly suppressed by pretreatment with inhibitors of Gi or Gq proteins. In a soft agar assay, the large sized colonies were formed in RHa3B12 and RHa3G8 cells, but not in RH7777AB cells. The cell survival of RHa3G8 cells treated with cisplatin (CDDP) or doxorubicin (DOX) was higher than that of RH7777AB cells, correlating with the elevated expression levels of multidrug-resistance related genes, Mdr1a, Mdr1b, and Gstp1. These results suggest that LPA3 may be involved in progression and aggressiveness of rat hepatoma RH7777 cells. (c) 2011 Wiley Periodicals, Inc.
• Neural Effects of Lysophosphatidic Acid (LPA) Signaling

  Nobuyuki Fukushima

  Lysophospholipid Receptors: Signaling and Biochemistry John Wiley and Sons 399 - 418 2013/02 [Refereed]
  
• Involvement of oncogenic K-ras on cell migration stimulated by lysophosphatidic acid receptor-2 in pancreatic cancer cells

  Kyohei Yoshikawa; Eriko Tanabe; Ayano Shibata; Serina Inoue; Misaho Kitayoshi; Souta Okimoto; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  EXPERIMENTAL CELL RESEARCH ELSEVIER INC 319 (3) 105 - 112 0014-4827 2013/02 [Refereed]
   

  Lysophosphatidic acid (LPA) mediates a variety of cellular responses with atleast six G protein-coupled transmembrane receptors (LPA receptor-1 (LPA(1)-LPA(6))). The interaction between LPA receptors and other cellular molecules on the biological function is not fully understood. Recently, we have reported that LPA(1) suppressed and LPA(3) stimulated cell migration of pancreatic cancer cells. In the present study, to evaluate the function of LPA(2) on motile and invasive activities of pancreatic cancer cells, we generated Lpar2 knockdown (HPD-sh2) cells from hamster pancreatic cancer cells and measured their cell migration ability. In cell motility and invasive assays with an uncoated Cell Culture Insert, HPD-sh2 cells showed significantly lower intrinsic activity than control (HPD-GFP) cells. Since K-ras mutations were frequently detected in pancreatic cancer, we next investigated whether oncogenic K-ras is involved in cell migration induced by LPA2 using K-ras knockdown (HPD-K2) cells. The cell motile ability of HPD-K2 cells was significantly lower than that of control cells. To confirm LPA2 increases cell migration activity, cells were pretreated with dioctylglycerol pyrophosphate (DGPP) which is the antagonist of LPA(1)/LPA(3). The cell motile and invasive abilities of DGPP -treated HPD-GFP cells were markedly higher than those of untreated cells, but DGPP did not stimulate cell migration of HPD-K2 cells. These results suggest that cell migration activity of pancreatic cancer cells stimulated by LPA2 may be enhanced by oncogenic K-ras. (c) 2012 Elsevier Inc. All rights reserved.
• Negative regulation of cell motile and invasive activities by lysophosphatidic acid receptor-3 in colon cancer HCT116 cells

  Rie Fukui; Eriko Tanabe; Misaho Kitayoshi; Kyohei Yoshikawa; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  TUMOR BIOLOGY SPRINGER 33 (6) 1899 - 1905 1010-4283 2012/12 [Refereed]
   

  Lysophosphatidic acid (LPA) mediates a wide range of biological responses with G protein-coupled transmembrane receptors (LPA receptors). So far, at least six types of LPA receptors (LPA receptor-1 (LPA(1)) to LPA(6)) have been identified. Recently, it has been reported that LPA(3) indicates opposite effects on cellular functions of cancer cells. In the present study, to assess a biological role of LPA(3) on cell migration ability of colon cancer cells, we generated LPA receptor-3 (LPAR3) knockdown (HCT-sh3-3) cells from HCT116 and measured cell motile and invasion activities. In motility assay with a cell culture insert, HCT-sh3-3 cells showed significantly high cell motile activity, compared with control cells. For invasion assay, the filter was coated with Matrigel. The invasive activity of HCT-sh3-3 cells was significantly higher than that of control cells. Furthermore, we also examined the effects of LPAR3 knockdown on the interaction between colon cancer cells and endothelial F-2 cells. When F-2 cells were cultured with serum-free DMEM containing a supernatant from HCT-sh3-3 cells, the cell growth rate and migration activity of F-2 cells were significantly stimulated, associating with the elevated expressions of vascular endothelial growth factor (VEGF)-A and VEGF-C genes in HCT-sh3-3 cells. These results suggest that LPA(3) may act as a negative regulator on cell motile and invasive abilities of colon cancer HCT116 cells.
• Loss of lysophosphatidic acid receptor-3 suppresses cell migration activity of human sarcoma cells

  Eriko Tanabe; Misaho Kitayoshi; Kyohei Yoshikawa; Ayano Shibata; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION INFORMA HEALTHCARE 32 (6) 328 - 334 1079-9893 2012/12 [Refereed]
   

  Lysophosphatidic acid (LPA) interacts with at least six G protein-coupled transmembrane LPA receptors (LPA(1)-LPA(6)). Recently, we have reported that LPA(3) indicated opposite effects on cell migration, depending on the cell types. In the present study, to assess an involvement of LPA(3) on cell migration of sarcoma cells, we generated LPA receptor-3 (LPAR3)-knockdown (HT1080-sh3 and HOS-sh3, respectively) cells from fibrosarcoma HT1080 and osteosarcoma HOS cells, and measured their cell migration abilities. In cell motility assay with a Cell Culture Insert, both LPAR3-knockdown cells showed significantly lower cell motile activities than control cells. Next, to investigate the effect of LPAR3-knockdown on invasion activity, which degraded the extracellular matrices, the Matrigel-coated filter was used. HT1080-sh3 cells showed significantly low invasive activity compared with control cells, while no invasive activity was found in HOS-sh3 cells. In gelatin zymography, no significant difference of matrix metalloproteinase (MMP)-2 and MMP-9 activities were detected in all cells. The results indicated that LPA(3) acts as a positive regulator of cell motility and invasion in sarcoma cells, suggesting that LPA signaling pathway via LPA(3) may be involved in the progression of sarcoma cells.
• Regulation of cell motile activity through the different induction of LPA receptors by estrogens in liver epithelial WB-F344 cells

  Eriko Tanabe; Ayano Shibata; Serina Inoue; Misaho Kitayoshi; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 428 (1) 105 - 109 0006-291X 2012/11 [Refereed]
   

  Lysophosphatidic acid (LPA) interacts with G protein-coupled transmembrane LPA receptors (LPA receptors; LPA(1)-LPA(6)). Recently, we demonstrated that each LPA receptor acts as a positive or negative regulator of cell migration ability. It is known that estrogens indicate a variety of biological functions, including cell motility. In the present study, to assess whether LPA signaling is involved in cell motile activity stimulated by estrogens, we measured cell motile activity and LPA receptor expressions of rat liver epithelial WB-F344 cells treated with 17 beta-estradiol (E-2), ethinyl estradiol (EE) and diethylstilbestrol (DES) at concentrations of 0.1 and 1.0 mu M for 48 h. The cell motility of E-2 and EE treated cells was significantly higher than that of untreated cells. By contrast, DES markedly inhibited cell motile activity. Using quantitative real time RT-PCR analysis, Lpar1 and Lpar3 expressions in E-2 treated cells were significantly higher than those in untreated cells. In EE treated cells, Lpar3 expression was markedly elevated, whereas Lpar1 expression was decreased. On the other hand, Lpar1 expression was significantly increased in DES treated cells. Interestingly, the effects of E-2, EE and DES on cell motility were suppressed by Lpar1 or Lpar3 knockdown. These results suggest that the different induction of LPA receptors by estrogens may regulate cell motile activity of WB-F344 cells. (C) 2012 Elsevier Inc. All rights reserved.
• Opposite roles of LPA(1) and LPA(3) on cell motile and invasive activities of pancreatic cancer cells

  Kohei Kato; Kyohei Yoshikawa; Eriko Tanabe; Misaho Kitayoshi; Rie Fukui; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  TUMOR BIOLOGY SPRINGER 33 (5) 1739 - 1744 1010-4283 2012/10 [Refereed]
   

  Lysophosphatidic acid (LPA) interacts with at least six G protein-coupled transmembrane LPA receptors. Recently, it has been demonstrated that each LPA receptor acts as a positive or negative regulator of cellular function. In the present study, to assess a biological role of LPA receptors on cell migration of pancreatic cancer cells, we generated LPA receptor-1 (LPA(1)) and LPA(3) knockdown cells from hamster pancreatic cancer cells by transfection with short hairpin RNA plasmids and measured their cell motile and invasive abilities. In cell motility and invasion assay, a Cell Culture Insert, coated with or without a Matrigel, was used. While the cell motility and invasion of Lpar1 knockdown cells were markedly enhanced than those of control cells, Lpar3 knockdown cells showed significantly lower cell motility and invasion. Moreover, to investigate an involvement of LPA(1) and LPA(3) in the development of pancreatic cancers, we also measured the expression levels of Lpar1 and Lpar3 genes in hamster pancreatic duct adenocarcinomas (PDAs) induced by a nitroso compound. The expressions of Lpar1 gene in PDAs were significantly lower than those in normal pancreatic tissues. By contrast, the elevated expressions of Lpar3 gene were detected in PDAs. We thus demonstrate that LPA(1) and LPA(3) play the different roles on cell migration ability of pancreatic cancer cells, suggesting the opposite effects via LPA(1) and LPA(3) may contribute to the pathogenesis of pancreatic cancers in hamsters.
• Different effects on cell proliferation and migration abilities of endothelial cells by LPA(1) and LPA(3) in mammary tumor FM3A cells

  Misaho Kitayoshi; Rie Fukui; Eriko Tanabe; Kohei Kato; Kyohei Yoshikawa; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION INFORMA HEALTHCARE 32 (4) 209 - 213 1079-9893 2012/08 [Refereed]
   

  Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors and mediate a variety of cellular responses through the binding of LPA. So far, six types of LPA receptors (LPA receptor-1 (LPA(1)) to LPA(6)) have been identified. Recently, it has been demonstrated that each LPA receptor has opposite effects on malignant property of cancer cells. In this study, to evaluate an involvement of LPA receptors on angiogenic process in mammary tumor cells, we generated Lpar1- and Lpar3-expressing (FM3A-a1 and FM3A-a3A9, respectively) cells from FM3A cells, and investigated the effects on cell proliferation and migration abilities of endothelial F-2 cells by those cells. In Vegf-A and Vegf-C genes, FM3A-a1 cells indicated high expression and FM3A-a3A9 cells showed low expression, compared with control cells. When F-2 cells were cultured with a supernatant from FM3A-a1 cells, the cell growth rate and migration ability of F-2 cells was significantly higher than control cells. By contrast, a supernatant from FM3A-a3A9 cells significantly inhibited those abilities of F-2 cells. These results suggest that LPA(1) and LPA(3) may play opposite roles on the regulation of endothelial cells in mouse mammary tumor FM3A cells.
• Enhancement of endothelial cell migration by constitutively active LPA(1)-expressing tumor cells

  Misaho Kitayoshi; Kohei Kato; Eriko Tanabe; Kyohei Yoshikawa; Rie Fukui; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 422 (2) 339 - 343 0006-291X 2012/06 [Refereed]
   

  Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA(1) to LPA(6)). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA(1) induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA(1) on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA(1) than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA(1). These results suggest that activation of LPA signaling via mutated LPA(1) may play an important role in the promotion of angiogenesis in rat neuroblastoma cells. (C) 2012 Elsevier Inc. All rights reserved.
• Lysophosphatidic acid induces neurite branch formation through LPA(3)

  Daisuke Furuta; Masayuki Yamane; Toshifumi Tsujiuchi; Ryutaro Moriyama; Nobuyuki Fukushima

  MOLECULAR AND CELLULAR NEUROSCIENCE ACADEMIC PRESS INC ELSEVIER SCIENCE 50 (1) 21 - 34 1044-7431 2012/05 [Refereed]
   

  Although neurite branching is crucial for neuronal network formation after birth, its underlying mechanisms remain unclear. Here, we demonstrate that lysophosphatidic acid (LPA) stimulates neurite branching through a novel signaling pathway. Treatment of neuronal cell lines with LPA resulted in neurite branch formation when LPA(3) receptor was introduced. The effects of LPA were blocked by inhibition of G(q) signaling. Furthermore, expression of inhibitory mutants of the small GTPase Rnd2/Rho7 or an Rnd2 effector rapostlin abolished LPA(3)-mediated neurite branching. The LPA(3) agonist 2(S)-OMPT or LPA also induced axonal branch formation in hippocampal neurons, which was blocked by G(q) and Rnd2 pathway inhibition or LPA(3) knockdown. These findings suggest that the novel signaling pathway involving LPA(3), G(q), and Rnd2 may play an important role in neuronal network formation. (c) 2012 Elsevier Inc. All rights reserved.
• Differential function of lysophosphatidic acid receptors in cell proliferation and migration of neuroblastoma cells

  Mai Hayashi; Kyoko Okabe; Kohei Kato; Mai Okumura; Rie Fukui; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  CANCER LETTERS ELSEVIER IRELAND LTD 316 (1) 91 - 96 0304-3835 2012/03 [Refereed]
   

  Lysophosphatidic acid (LPA) is a bioactive lipid mediator that induces diverse cellular biological effects and interacts with G protein-coupled transmembrane LPA receptors. In the present study, to assess biological roles of LPA receptors in the pathogenesis of tumor cells, each LPA receptor (Lpar1, Lpar2 or Lpar3)-expressing rat neuroblastoma B103 cells (Ipa1-1, Ipa2-2 or Ipa3-3-2 cells, respectively) were used. In cell motility and invasion assay, Ipa2-2 and Ipa3-3-2 cells showed significant higher intrinsic activity without LPA treatment than LPA receptor-unexpressing AB2-1 bf cells. LPA treatment further increased cell motility of these cells, which was suppressed by the pretreatment with inhibitors of Gi, Gq protein, or ROCK. By contrast, Ipa1-1 cells markedly decreased intrinsic cell motility and invasion, compared with AB2-1 bf cells. Constitutively active mutant Lpar1-expressing cells (Ipa1 Delta-1) showed significant high motility, comparable with those of Ipa2-2 and Ipa3-3-2. In soft agar assay. Ipa3-3-2 and Ipa1 Delta-1 cells showed colony formation, but other cells failed. These results suggest that LPA receptors may play different roles in cell proliferation and migration of rat neuroblastoma cells. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
• Constitutively active lysophosphatidic acid receptor-1 enhances the induction of matrix metalloproteinase-2

  Kohei Kato; Rie Fukui; Kyoko Okabe; Eriko Tanabe; Misaho Kitayoshi; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 417 (2) 790 - 793 0006-291X 2012/01 [Refereed]
   

  Lysophosphatidic acid (LPA) is a simple phospholipid which interacts with at least six G protein-coupled transmembrane LPA receptors (LPA(1)-LPA(6)). In rat neuroblastoma B103 cells, we have recently reported that each LPA receptor indicates the different cellular functions, including cell motility, invasion and tumorigenicity. Especially, mutated and constitutively active LPA(1) enhanced these cellular effects in B103 cells. In the present study, to better understand a role of mutated LPA(1) underlying progression of cancer cells, we measured the expression and activity levels of matrix metalloproteinases (MMPs) in constitutively active mutant Lpar1-expressing B103 cells (lpa1 Delta-1), compared with each wild-type LPA receptor-expressing cells. LPA receptor-unexpressing cells were also used as control. In quantitative real time RT-PCR analysis, the expressions of Mmp-9 were detected at the same levels in all cells. By contrast, Mmp-2 expressions of lpa1 Delta-1 were significantly higher than those of other cells. In gelatin zymography, proMmp-9 was observed at the same levels in all cells. Interestingly, markedly high levels of proMmp-2 and Mmp-2 were detected in lpa1 Delta-1 cells, whereas no activation was in other cells. The increased expression and activity of Mmp-2 in lpa1 Delta-1 cells were suppressed by the pretreatment with a Gq protein inhibitor. These results suggest that mutated LPA(1) may involve in the enhancement of Mmp-2 expression and activation in rat neuroblastoma cells. (C) 2011 Elsevier Inc. All rights reserved.
• Enhancement of drug resistance by lysophosphatidic acid receptor-3 in mouse mammary tumor fm3a cells

  Rie Fukui; Kohei Kato; Kyoko Okabe; Misaho Kitayoshi; Eriko Tanabe; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  Journal of Toxicologic Pathology 25 (3) 225 - 228 0914-9198 2012 [Refereed]
   

  Lysophosphatidic acid (LPA) acts as a simple phospholipid that interacts with G protein-coupled transmembrane LPA recep- tors. Recently, it has been reported that each LPA receptor plays different biological roles in acquisition of the malignant property of tumor cells. In this study, to assess the involvement of LPA receptor-3 (LPA 3) in cell survival after treatment with anticancer drugs, we generated Lpar3-expressing FM3A-a3A9 cells from mouse mammary tumor FM3A cells and examined the cell survival rate after treatment with anticancer drugs compared with Lpar3-unexpressing cells. Cells were treated with 0.005 to 10 μM of cisplatin (CDDP) or doxorubicin (DOX) for 3 days. For the CDDP and DOX treatments, the cell survival rate of FM3A-a3A9 cells was significantly higher than that of Lpar3-unexpressing cells. The expression level of the Mdrla gene in FM3A-a3A9 cells was higher than that of Lpar3-unexpressing cells, whereas no significant difference in multidrug resistance 1b (Mdr1b) and glutathione S-transferase mu1 (Gstm1) expressions was found. These results suggest that LPA 3 may enhance the cell survival rate after treatment with anticancer drugs in mouse mammary tumor cells, correlating with increased expression of the Mdr1 gene. © 2012 The Japanese Society of Toxicologic Pathology.
• Involvement of ERK in NMDA receptor-independent cortical neurotoxicity of hydrogen sulfide

  Yuko Kurokawa; Fumiko Sekiguchi; Satoko Kubo; Yoshiko Yamasaki; Sachi Matsuda; Yukari Okamoto; Teruki Sekimoto; Anna Fukatsu; Hiroyuki Nishikawa; Toshiaki Kume; Nobuyuki Fukushima; Akinori Akaike; Atsufumi Kawabata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 414 (4) 727 - 732 0006-291X 2011/11 [Refereed]
   

  Hydrogen sulfide (H(2)S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H(2)S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypan blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c(1) leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H(2)S involves activation of the MEK/ERK pathway and some apoptotic mechanisms. (C) 2011 Elsevier Inc. All rights reserved.
• Genetic and epigenetic alterations of lysophosphatidic acid receptor genes in rodent tumors by experimental models

  Toshifumi Tsujiuchi; Kyoko Okabe; Nobuyuki Fukushima

  Journal of Toxicologic Pathology 24 (3) 143 - 148 0914-9198 2011/10 [Refereed]
   

  Lysophosphatidic acid (LPA) is a bioactive mediator and induces several biological effects, including cell proliferation, migration, morphogenesis and differentiation. LPA interacts with at least six G protein-coupled receptors (GPCRs), including LPA receptor-1 (LPA 1), LPA 2, LPA 3, LPA 4, LPA 5 and LPA 6. These receptors show different biological functions through the binding of LPA, depending on the type of cells. In human malignancies, a high level of LPA production was found in plasma and ascites in ovarian cancer cases. Moreover, aberrant expression levels of LPA receptor genes were detected in some cancer cells. Therefore, it is suggested that LPA receptors may be involved in the pathogenesis of tumor cells as well as LPA per se. Recently, we have reported that alterations of LPA receptor genes also occur in rodent tumors. In this review, we summarize the recent evidence in the investigations of LPA receptor alterations in rodent tumors by experimental models. © 2011 The Japanese Society of Toxicologic Pathology.
• No involvement of lysophosphatidic acid receptor-3 in cell migration of mouse lung tumor cells stimulatedby 12-O-tetradecanoylphorbol-13-acetate

  Mai Okumura; Kohei Kato; Rie Fukui; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  Journal of Toxicologic Pathology 24 (3) 183 - 186 0914-9198 2011/10 [Refereed]
   

  The tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates cell migration of several tumor cells. Recently, we reported that loss of lysophosphatidic acid (LPA) receptor-3 (LPA3) enhanced cell migration of murine lung tumor LL/2 cells. In the present study, we investigated whether LPA3 is involved in cell migration of mouse lung tumor cells stimulated by TPA. Exogenous LPA3 gene (Lpar3)-expressing (LL/2-a3) cells and LL/2-AB cells as a vector control generated from LL/2 cells were used. In a cell migration assay, TPA treatment significantly stimulated cell migration of LL/2-AB and LL/2-a3 cells, while the cell migration abilities of LL/2-a3 were markedly lower than those of LL/2-AB cells. Using quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis, no effect of TPA treatment on the expression levels of LPA1, LPA2 and LPA3 genes was detected in either type of cells. These results suggest that the LPA3 may not be involved in the enhanced migration ability by TPA in mouse lung tumor cells. ©2011 The Japanese Society of Toxicologic Pathology. © 2011 The Japanese Society of Toxicologic Pathology.
• Distinct DNA methylation patterns of lysophosphatidic acid receptor genes during rat hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined diet

  Kyoko Okabe; Mai Hayashi; Ikuma Yoshida; Kazuki Nishimura; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  ARCHIVES OF TOXICOLOGY SPRINGER HEIDELBERG 85 (10) 1303 - 1310 0340-5761 2011/10 [Refereed]
   

  Altered expressions of lysophosphatidic acid (LPA) receptor genes have been reported in tumor cells of human and rats. Recently, we detected the frequent mutations of LPA receptor-1 (LPA1) gene in rat hepatocellular carcinomas (HCCs) induced by a choline-deficient L-amino acid-defined (CDAA) diet. In this study, the DNA methylation patterns of LPA receptor genes and their expression levels during rat hepatocarcinogenesis induced by the CDAA diet were investigated. Six-week-old F344 male rats were continuously fed with the CDAA diet, and animals were then killed at 7 days and 2, 12, 20, and 75 weeks, respectively. Genomic DNAs were extracted from livers and HCCs for the assessment of methylation status by bisulfite sequencing, comparing to normal livers. The livers of rats fed the CDAA diet were unmethylated in LPA1 and LPA2 genes as well as normal livers. In LPA3 gene, although normal livers were unmethylated, the livers at 7 days and 2 and 12 weeks weakly or moderately methylated and those at 20 weeks markedly methylated. Moreover, 4 HCCs were completely methylated in LPA3 gene. Expression levels of LPA receptor genes in the livers of rats fed the CDAA diet and HCCs were correlating with DNA methylation status. These results indicate that DNA methylation status of the LPA3 gene was disturbed in the livers of rats fed the CDAA diet and established HCCs, suggesting that alterations of the LPA receptor genes might be involved during rat hepatocarcinogenesis induced by the CDAA diet.
• Induction of lysophosphatidic acid receptor-3 by 12-O-tetradecanoylphorbol-13-acetate stimulates cell migration of rat liver cells

  Kyoko Okabe; Kohei Kato; Miki Teranishi; Mai Okumura; Rie Fukui; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  CANCER LETTERS ELSEVIER IRELAND LTD 309 (2) 236 - 242 0304-3835 2011/10 [Refereed]
   

  12-O-tetradecanoylphorbol-13-acetate (TPA) which is one of tumor promoting agents stimulates cell migration ability of several tumor cells. In the present study, we investigated whether lysophosphatidic acid (LPA) receptors are involved in cell migration of rat liver cells stimulated by TPA. The rat liver epithelial WB-F344 and hepatoma RH7777 cells were treated by TPA for 48 h. The expression levels of LPA receptor genes in those cells were measured by real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis. The expressions of the LPA receptor-3 (Lpar3) gene were significantly elevated in WB-F344 and RH7777 cells treated by TPA, but not Lpar1 and Lpar2 genes. In cell migration assay, TPA treatment showed markedly high cell migration in both cells. The pretreatment with inhibitors of Gi protein suppressed those migration abilities. We next generated the Lpar3 knockdown cells from WB-F344 cells and investigated the effect on cell migration. Interestingly, the cell migration of the knockdown cells was not stimulated by TPA. These results suggest that TPA-stimulated cell migration of rat liver cells may be mainly dependent on the LPA(3)-mediated effect. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
• INHIBITION OF T-TYPE CALCIUM CHANNELS AND HYDROGEN SULFIDE-FORMING ENZYME REVERSES PACLITAXEL-EVOKED NEUROPATHIC HYPERALGESIA IN RATS

  K. Okubo; T. Takahashi; F. Sekiguchi; D. Kanaoka; M. Matsunami; T. Ohkubo; J. Yamazaki; N. Fukushima; S. Yoshida; A. Kawabata

  NEUROSCIENCE PERGAMON-ELSEVIER SCIENCE LTD 188 148 - 156 0306-4522 2011/08 [Refereed]
   

  Hydrogen sulfide (H,S), a gasotransmitter, facilitates pain sensation by targeting Ca(v)3.2 T-type calcium channels. The H(2)S/Ca(v)3.2 pathway appears to play a role in the maintenance of surgically evoked neuropathic pain. Given evidence that chemotherapy-induced neuropathic pain is blocked by ethosuximide, known to block T-type calcium channels, we examined if more selective T-type calcium channel blockers and also inhibitors of cystathionine-gamma-lyase (CSE), a major H(2)S-forming enzyme in the peripheral tissue, are capable of reversing the neuropathic pain evoked by paclitaxel, an anti-cancer drug. It was first demonstrated that T-type calcium channel blockers, NNC 55-0396, known to inhibit Ca(v)3.1, and mibefradil inhibited T-type currents in Ca(v)3.2-transfected HEK293 cells. Repeated systemic administration of paclitaxel caused delayed development of mechanical hyperalgesia, which was reversed by single intraplantar administration of NNC 550396 or mibefradil, and by silencing of Ca(v)3.2 by antisense oligodeoxynucleotides. Systemic administration of DL-propargylglycine and p-cyanoalanine, irreversible and reversible inhibitors of CSE, respectively, also abolished the established neuropathic hyperalgesia. In the paclitaxel-treated rats, upregulation of Ca(v)3.2 and CSE at protein levels was not detected in the dorsal root ganglia (DRG), spinal cord or peripheral tissues including the hindpaws, whereas H(2)S content in hindpaw tissues was significantly elevated. Together, our study demonstrates the effectiveness of NNC 55-0396 in inhibiting Ca(v)3.2, and then suggests that paclitaxel-evoked neuropathic pain might involve the enhanced activity of T-type calcium channels and/or CSE in rats, but not upregulation of Ca(v)3.2 and CSE at protein levels, differing from the previous evidence for the neuropathic pain model induced by spinal nerve cutting in which Ca(v)3.2 was dramatically upregulated in DRG. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
• Possible Involvement of Lysophosphatidic Acid Receptor-5 Gene in the Acquisition of Growth Advantage of Rat Tumor Cells

  Kyoko Okabe; Mai Hayashi; Yasuna Yamawaki; Miki Teranishi; Kanya Honoki; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  MOLECULAR CARCINOGENESIS WILEY-BLACKWELL 50 (8) 635 - 642 0899-1987 2011/08 [Refereed]
   

  Aberrant expressions of lysophosphatidic acid (LPA) receptor genes have been reported in tumor cells. Here, we measured the expression levels of the Lpa5 gene and its DNA methylation status in rat tumor cells, and investigated cell growth effects of LPA in Lpa5 expressed cells. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis revealed that increased expressions of the Lpa5 gene were detected in rat liver-derived hepatoma RH7777 and lung-derived adenocarcinoma RLCNR cells. For the analysis of methylation status, bisulfite sequencing was performed with RH7777 and RLCNR cells and compared with other tumor cells and liver epithelial cells. The Lpa5 gene in Lpa5 unexpressed cells and liver epithelial cells were highly methylated in the 50 upstream region. In contrast, the Lpa5 gene in RH7777 and RLCNR cells was unmethylated, correlating with increased expressions of Lpa5. In the assays for cell growth effects of LPA, LPA enhanced cell proliferation and motility in RH7777 and RLCNR cells. LPA also stimulated cell invasion in RLCNR, but not in RH7777 cells. In rat liver and lung tumors induced by nitroso-compounds, 4 out of 6 hepatocellular carcinomas and 5 out of 6 lung adenocarcinomas indicated increased expressions of Lpa5 with unmethylated status. These results suggest that increased Lpa5 expressions due to aberrant DNA methylation may be involved in the acquisition of growth advantage of rat tumor cells. (C) 2011 Wiley-Liss, Inc.
• Coordinated interactions between actin and microtubules through crosslinkers in neurite retraction induced by lysophosphatidic acid

  Nobuyuki Fukushima; Daisuke Furuta; Toshifumi Tsujiuchi

  NEUROCHEMISTRY INTERNATIONAL PERGAMON-ELSEVIER SCIENCE LTD 59 (2) 109 - 113 0197-0186 2011/08 [Refereed]
   

  Neurite development requires rearrangement of cytoskeletal elements, which are mechanically and functionally integrated with each other. Although the process of how an extracellular signal induces rearrangement of a single element has been closely examined, the mechanisms by which the signal regulates cytoskeletal integration during cell shape changes are poorly understood. We previously reported that lysophosphatidic acid (LPA) induces actin polymerization-dependent microtubule (MT) rearrangement, leading to neurite retraction in cultured neurons. Here we examined whether the crosslinker proteins were involved in LPA-induced neurite retraction using immortalized mouse neuroblast TR cells. When the MT-binding domains of MACF (MT actin-crosslinking factor) were exogenously expressed in TR cells, MTs were found to be stabilized and become resistant to exposure to LPA. On the other hand, expression of MT-associated protein 2c showed no effect on LPA-induced neurite retraction. These findings suggest that MACF is involved in actin-dependent MT rearrangement during LPA-induced neurite retraction. (C) 2011 Elsevier BM. All rights reserved.
• Loss of lysophosphatidic acid receptor-3 enhances cell migration in rat lung tumor cells

  Mai Hayashi; Kyoko Okabe; Yasuna Yamawaki; Miki Teranishi; Kanya Honoki; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 405 (3) 450 - 454 0006-291X 2011/02 [Refereed]
   

  Lysophosphatidic acid (LPA) indicates several biological effects, such as cell proliferation, differentiation and migration. LPA interacts with G protein-coupled transmembrane LPA receptors. In our previous report, we detected that loss of the LPA receptor-1 (Lpar1) expression is due to its aberrant DNA methylation in rat tumor cell lines. In this study, to assess an involvement of the other LPA receptor, Lpar3, in the pathogenesis of rat lung tumor cells, we measured the expression levels of the Lpar3 gene and its DNA methylation status by reverse transcription (RT)-polymerase chain reaction (PCR) and bisulfite sequencing analyses, respectively. RLCNR lung adenocarcinoma cells showed reduced expression of the Lpar3, compared with normal lung tissues. In the 5' upstream region of the Lpar3, normal lung tissues were unmethylated. By contrast, RLCNR cells were highly methylated, correlating with reduced expressions of the Lpar3. Based on these results, we generated the Lpar3-expressing RLCNR-a3 cells and measured the cell migration ability. Interestingly, the cell migration of RLCNR-a3 cells was significantly lower than that of RLCNR cells. This study suggests that loss of the Lpar3 due to aberrant DNA methylation may be involved in the progression of rat lung tumor cells. (C) 2011 Elsevier Inc. All rights reserved.
• Differential expressions and DNA methylation patterns of lysophosphatidic acid receptor genes in human colon cancer cells

  Megumu Tsujino; Minako Fujii; Kyoko Okabe; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  VIRCHOWS ARCHIV SPRINGER 457 (6) 669 - 676 0945-6317 2010/12 [Refereed]
   

  Lysophosphatidic acid (LPA), which is a bioactive phospholipid, interacts with specific G protein-coupled transmembrane receptors. Recently, alterations of LPA receptor genes have been reported in some tumor cells. In this study, we examined the expression profiles and DNA methylation status of LPA receptor 1-5 (LPA1-5) genes in human colon cancer cells and also looked for the mutations. Reverse transcription-polymerase chain reaction (PCR) and bisulfite sequencing analyses were carried out. While LPA1, LPA2, and LPA4 genes were expressed in DLD1, SW480, HCT116, CaCo-2, SW48, and LoVo cells, the expressions of LPA3 and LPA5 genes were various. These expression levels were correlated with DNA methylation status in the 5' upstream regions of the LPA receptor genes. Mutation analysis was also performed using a PCR-single-strand conformation polymorphism method. Although no mutations in LPA1, LPA3 and LPA5 genes were found in all types of cells, LPA2 mutations in DLD1 and SW48 cells, and LPA4 mutation were found in DLD1 cells. On the basis of the present results, we demonstrate that these colon cancer cells will be available to understanding the molecular pathway through LPA receptors in the development of tumor cells, and that LPA receptors may be new molecular targets for therapeutic approaches and chemoprevention.
• Lysophosphatidic acid influences initial neuronal polarity establishment

  Masayuki Yamane; Daisuke Furuta; Nobuyuki Fukushima

  NEUROSCIENCE LETTERS ELSEVIER IRELAND LTD 480 (2) 154 - 157 0304-3940 2010/08 [Refereed]
   

  Neuronal polarity is specified by neurite determination into axons and dendrites. Its establishment requires both extrinsic signals, which regulate axon and dendrite development through repulsive or attractive actions, and intrinsic cellular mechanisms, which include rearrangement and selective transport of the cytoskeleton and localization of intracellular organelles. However, it remains unclear how extrinsic signals activate intrinsic cellular mechanisms to establish neuronal polarity. Here, we examine the effects of lysophosphatidic acid (LPA), a signaling lipid that induces cytoskeletal rearrangement in neuronal cells, on neuronal polarity establishment. In hippocampal neuronal cultures where a concentration gradient of LPA was formed, the bases of axons were located predominantly at the side distal to the LPA source. Furthermore, Golgi apparatus were also positioned distally as early as 1 h after exposure to the LPA source, suggesting that LPA signaling is involved in the initial determination of the area where an axon sprouts, and thereby the establishment of neuronal polarity. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
• No mutations of lysophosphatidic acid receptor genes in lung adenocarcinomas induced by N-Nitrosobis(2-hydroxypropyl)amine in rats

  Naoko Wakabayashi; Megumu Tsujino; Masaki Tajiri; Midori Taki; Ayumi Koshino; Hiroko Ikeda; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  Journal of Toxicologic Pathology 23 (1) 63 - 66 0914-9198 2010/04 [Refereed]
   

  Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation and migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, frequent mutations of the LPA receptor-1 (LPA1) gene were detected in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). In this study, to evaluate the involvement of other LPA receptor gene alterations during lung carcinogenesis, we investigated mutations of the LPA2, LPA3, LPA4 and LPA5 genes in lung adenocarcinomas induced by BHP in rats. Fifteen male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks, and 15 adenocarcinomas were obtained. Genomic DNAs were extracted from frozen tissues, and the LPA2, LPA3, LPA4 and LPA5 genes were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No mutations of LPA2, LPA3, LPA4 and LPA5 were detected in the 15 adenocarcinomas. These results suggest that alterations due to LPA2, LPA3, LPA4 and LPA5 gene mutations might not be involved in the development of lung adenocarcinomas induced by BHP in rats.
• Mutations of Lysophosphatidic Acid Receptor Genes in Human Osteosarcoma Cells

  Kyoko Okabe; Mai Hayashi; Minako Fujii; Kanya Honoki; Toshio Mori; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  PATHOBIOLOGY KARGER 77 (5) 278 - 282 1015-2008 2010 [Refereed]
   

  Objective: Lysophosphatidic acid (LPA), which is a bioactive phospholipid, interacts with specific G protein-coupled transmembrane receptors. Recently, alterations in LPA receptor genes have been reported in some tumor cells. In this study, to assess an involvement of LPA receptor genes in the development of human cancer cells, we looked for the presence of mutations in LPA receptor 1-6 (LPA1-6) genes in MG63 osteosarcoma, HT1080 fibrosarcoma, A549 lung adenocarcinoma, MCF-7 breast carcinoma, and G-361 melanoma cells. Methods: Genomic DNAs were extracted from each cell and polymerase chain reaction-single-strand conformation polymorphism analysis was performed to identify the mutations. Results: MG63 showed mutations in LPA1 and LPA3 genes, while no mutations in the LPA receptor genes were found in HT1080, A549, MCF-7 and G-361 cells. Sequence analysis revealed a CGC to CGT (Arg to Arg) transition at codon 314 in LPA1, and a GCG to GTG (Ala to Val) transition at codon 247 in LPA3. Conclusion: These results indicated that the mutations in LPA1 and LPA3 genes occur in MG63 cells, suggesting that the alterations in LPA receptor genes may play some role in the pathogenesis in human osteosarcoma cells. Copyright (C) 2010 S. Karger AG, Basel
• Different Expressions and DNA Methylation Patterns of Lysophosphatidic Acid Receptor Genes in Mouse Tumor Cells

  Kyoko Okabe; Mai Hayashi; Naoko Wakabayashi; Yasuna Yamawaki; Miki Teranishi; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  PATHOBIOLOGY KARGER 77 (6) 309 - 314 1015-2008 2010 [Refereed]
   

  Objective: Lysophosphatidic acid (LPA) receptors act as several biological effectors through LPA, which is a bioactive phospholipid. Recently, aberrant expressions of LPA receptor genes due to DNA methylation have been detected in several tumor cells. In this study, we measured expression levels and DNA methylation status of LPA receptor genes in mouse tumor cells, LL/2 lung carcinoma, B16F0 melanoma, FM3A mammary carcinoma and L1210 leukemia cells, compared with normal tissues. Methods: Total RNAs were extracted and RT-PCR analysis was performed. For DNA methylation status, bisulfite sequencing analysis was carried out, comparing outcomes with other tumor cells and normal tissues. Results: The expressions of LPA1 gene were shown in LL/2, but not in B16F0, FM3A and L1210 cells. While the LPA2 gene was expressed in all 4 tumor cells, the LPA3 gene was unexpressed in them. The LPA1 and LPA3 unexpressed cells were highly methylated, although normal tissues were all unmethylated. The DNA methylation status was correlated with gene expression levels in cancer cells. Conclusion: The present results demonstrate that DNA methylation patterns of LPA receptor genes are dependent on cancer cell types, suggesting that LPA receptors may be new molecular targets for therapeutic approaches and chemoprevention. Copyright (C) 2011 S. Karger AG, Basel
• Post-translational modifications of tubulin in the nervous system

  Nobuyuki Fukushima; Daisuke Furuta; Yuji Hidaka; Ryutaro Moriyama; Toshifumi Tsujiuchi

  JOURNAL OF NEUROCHEMISTRY WILEY 109 (3) 683 - 693 0022-3042 2009/05 [Refereed]
   

  Many studies have shown that microtubules (MTs) interact with MT-associated proteins and motor proteins. These interactions are essential for the formation and maintenance of the polarized morphology of neurons and have been proposed to be regulated in part by highly diverse, unusual post-translational modifications (PTMs) of tubulin, including acetylation, tyrosination, detyrosination, Delta 2 modification, polyglutamylation, polyglycylation, palmitoylation, and phosphorylation. However, the precise mechanisms of PTM generation and the properties of modified MTs have been poorly understood until recently. Recent PTM research has uncovered the enzymes mediating tubulin PTMs and provided new insights into the regulation of MT-based functions. The identification of tubulin deacetylase and discovery of its specific inhibitors have paved the way to understand the roles of acetylated MTs in kinesin-mediated axonal transport and neurodegenerative diseases such as Huntington's disease. Studies with tubulin tyrosine ligase (TTL)-null mice have shown that tyrosinated MTs are essential in normal brain development. The discovery of TTL-like genes encoding polyglutamylase has led to the finding that polyglutamylated MTs which accumulate during brain development are involved in synapse vesicle transport or neurite outgrowth through interactions with motor proteins or MT-associated proteins, respectively. Here we review current exciting topics that are expected to advance MT research in the nervous system.
• Biochemical re-evaluation of GFP-tubulin expressed in mammalian cells

  Furuta Daisuke; Moriyama Ryutaro; Fukushima Nobuyuki

  Science and technology 近畿大学理工学総合研究所 21 (21) 19 - 23 0916-2054 2009 

  Green fluorescent protein (GFP)-tagged tubulin has been widely employed to examine tubulin dynamics in living cells. In the present study, we biochemically re-evaluate the property of GFP-tubulin expressed in cells. When expressed in mammalian cells, antibodies against GFP or the amino-terminal region of tubulin detected GFP-tubulin in western blot analyses. By contrast, antibodies against the carboxyl terminal region of tubulin failed to recognize exogenous GFP-tubulin, but not endogenous tubulin. These results indicate that GFP-tubulin is partially truncated at the carboxyl terminal region and suggest that data of experiments using GFP-tubulin should be carefully analyzed.
• Frequent mutations of lysophosphatidic acid receptor-1 gene in rat liver tumors

  Yumi Obo; Takanori Yamada; Mami Furukawa; Mayuko Hotta; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS ELSEVIER SCIENCE BV 660 (1-2) 47 - 50 0027-5107 2009/01 [Refereed]
   

  Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with Specific G protein-coupled transmembrane receptors, including LPA1 to LPA5. In the present study, to clarify an involvement of LPA I gene alterations in the development of hepatocellular carcinomas (HCCs) we investigated the LPA1 mutations in rat HCCs induced by exogenous and endogenous liver carcinogenesis models. We induced HCCs in rats with N-nitrosodiethylamine (DEN) and a choline-deficient L-amino acid-defined (CDAA) diet. RNAs were extracted from 15 HCCS induced by DEN and 12 HCCs induced by the CDAA diet. To identify LPA1 mutations, reverse transcription (RT) - polymerase chain reaction (PCR) - single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Missense mutations were detected in 7 out of 15 HCCs (46.7%,) induced by DEN. Five Out of 12 HCCs (41.7%,) induced by the CDAA diet also showed missense mutations. These results demonstrated that mutations in LPA1 gene occur in rat HCCs induced by DEN and the CDAA diet, suggesting that LPA1 mutations may be essentially involved ill rat liver carcinogenesis. (c) 2008 Elsevier B.V. All rights reserved.
• Mutations of lysophosphatidic acid receptor-1 gene during progression of lung tumors in rats

  Takanori Yamada; Yumi Obo; Mami Furukawa; Mayuko Hotta; Ayako Yamasaki; Kanya Honoki; Nobuyuki Fukushima; Toshifumi Tsujiuchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ACADEMIC PRESS INC ELSEVIER SCIENCE 378 (3) 424 - 427 0006-291X 2009/01 [Refereed]
   

  Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. In this study, mutations of lysophosphatidic acid receptor-1 (LPA1) gene were investigated to clarify the possible molecular mechanisms underlying the development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age. were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks. Genomic DNAs were extracted from paraffin-embedded tissues and exons 2-4 were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No LPA1 mutations were detected in 15 hyperplasias, but 2 out of 12 adenomas (16.7%) and 7 Out Of 17 adenocarcinomas (41.2%). These results suggest that mutations of LPA1 gene may be involved in the acquisition of growth advantage from adenomas to adenocarcinomas in lung carcinogenesis induced in rats by BHP. (C) 2008 Elsevier Inc. All rights reserved.
• Infrequent Mutation of Lysophosphatidic Acid Receptor-1 Gene in Hamster Pancreatic Duct Adenocarcinomas and Established Cell Lines

  Toshifumi Tsujiuchi; Mami Furukawa; Yumi Obo; Ayako Yamasaki; Mayuko Hotta; Chie Kusunoki; Naoko Suyama; Toshio Mori; Kanya Honoki; Nobuyuki Fukushima

  JOURNAL OF TOXICOLOGIC PATHOLOGY JAPANESE SOC TOXICOLOGIC PATHOLOGY 22 (1) 89 - 92 0914-9198 2009 [Refereed]
   

  To evaluate the involvement of lysophosphatidic acid receptor-1 (LPA1) gene alteration in pancreatic carcinogenesis, we investigated mutations in the LPA1 gene in hamster pancreatic duct adenocarcinomas (PDAs) and established cell lines. Female Syrian golden hamsters received 30 mg/kg of N-nitrosobis(2-oxopropyl) amine (BOP) followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine and then L-methionine and a further administration of 20 mg/kg BOP. A total of 10 PDAs obtained 10 weeks after beginning the experiment and three cell lines established from subcutaneously transplantable PDAs in syngeneic hamsters were examined for mutations using reverse transcription-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) analysis. A mutation was detected in only one PDA (1/10, 10%) in the form of a GGA to GTA (Gly to Val) transversion at codon 355, and no mutations were detected in the three cell lines. These results suggest that the LPA1 gene mutation may play roles in a limited fraction of BOP-induced pancreatic duct carcinogenesis in hamsters. (J Toxicol Pathol 2009; 22: 89-92)
• 長鎖脂肪酸はゴナドトロフのGnRHレセプターmRNA発現量を制御する

  出浦 慎哉; 福嶋 伸之; 森山 隆太郎

  日本内分泌学会雑誌 (一社)日本内分泌学会 84 (2) 526 - 526 0029-0661 2008/09
• マウス下垂体のリゾホスファチジン酸受容体1はエストロジェン依存的に発現が抑制される

  十河 由紀; 宮里 公子; 福嶋 伸之; 森山 隆太郎

  The Journal of Reproduction and Development (公社)日本繁殖生物学会 54 (Suppl.) j106 - j106 0916-8818 2008/08
• A lysophosphatidic acid receptor lacking the PDZ-binding domain is constitutively active and stimulates cell proliferation

  Shinya Shano; Kazuki Hatanaka; Shinsuke Ninose; Ryutaro Moriyama; Toshifumi Tsujiuchi; Nobuyuki Fukushima

  BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH ELSEVIER SCIENCE BV 1783 (5) 748 - 759 0167-4889 2008/05 

  Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates cell proliferation, survival, and motility of normal and cancer cells. These effects are produced through G protein-coupled LPA receptors, LPA(1) to LPA(5). We generated an LPA(1) mutant lacking the SerValVal sequence of the C-terminal PDZ-binding domain to examine the role of this domain in intracellular signaling and other cellular functions. B103 neuroblastoma cells expressing the mutant LPA(1) showed rapid cell proliferation and tended to form colonies under serum-free conditions. The enhanced cell proliferation of the mutant cells was inhibited by exogenous expression of the plasmids inhibiting G proteins including G(beta gamma), G(alpha i) and G(alpha q) or G(alpha 12/13), or treatment with pertussis toxin, phosphoinositide 3-kinase (PI3K) inhibitors or a Rho inhibitor. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting increases in phosphorylated Akt in western blot analyses or by directly measuring Rho activity. Interestingly, expression of the mutant LPA(1) in non-tumor mouse fibroblasts induced colony formation in a clonogenic soft agar assay, indicating that oncogenic pathways were activated. Taken together, these observations suggest that the mutant LPA(1) constitutively activates the G protein signaling leading to PI3K-Akt and Rho pathways, resulting in enhanced cell proliferation. (C) 2007 Elsevier B.V. All rights reserved.
• A lysophosphatidic acid receptor lacking the PDZ-binding domain is constitutively active and stimulates cell proliferation.

  Shinya Shano; Kazuki Hatanaka; Shinsuke Ninose; Ryutaro Moriyama; Toshifumi Tsujiuchi; Nobuyuki Fukushima

  Biochimica et biophysica acta 1783 (5) 748 - 59 0006-3002 2008/05 

  Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates cell proliferation, survival, and motility of normal and cancer cells. These effects are produced through G protein-coupled LPA receptors, LPA(1) to LPA(5). We generated an LPA(1) mutant lacking the SerValVal sequence of the C-terminal PDZ-binding domain to examine the role of this domain in intracellular signaling and other cellular functions. B103 neuroblastoma cells expressing the mutant LPA(1) showed rapid cell proliferation and tended to form colonies under serum-free conditions. The enhanced cell proliferation of the mutant cells was inhibited by exogenous expression of the plasmids inhibiting G proteins including G(betagamma), G(alphai) and G(alphaq) or G(alpha12/13), or treatment with pertussis toxin, phosphoinositide 3-kinase (PI3K) inhibitors or a Rho inhibitor. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting increases in phosphorylated Akt in western blot analyses or by directly measuring Rho activity. Interestingly, expression of the mutant LPA(1) in non-tumor mouse fibroblasts induced colony formation in a clonogenic soft agar assay, indicating that oncogenic pathways were activated. Taken together, these observations suggest that the mutant LPA(1) constitutively activates the G protein signaling leading to PI3K-Akt and Rho pathways, resulting in enhanced cell proliferation.
• Lysophosphatidic acid stimulates astrocyte proliferation through LPA(1)

  Shinya Shano; Ryutaro Moriyama; Jerold Chun; Nobuyuki Fukushima

  NEUROCHEMISTRY INTERNATIONAL PERGAMON-ELSEVIER SCIENCE LTD 52 (1-2) 216 - 220 0197-0186 2008/01 

  Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions through multiple types of LPA receptors. Here we explore the role of LPA receptor subtypes in cortical astrocyte functions. Astrocytes cultured under serum-free conditions were found to express the genes of five LPA receptor subtypes, lpa(1) to lpa(5). When astrocytes were treated with dibutyryl cyclic adenosine monophosphate, a reagent inducing astrocyte differentiation or activation, lpa(1) expression levels remained unchanged, but those of other LPA receptor subtypes were relatively reduced. LPA stimulated DNA synthesis in both undifferentiated and differentiated astrocytes, but failed to do so in astrocytes prepared from mice lacking lpa(1) gene. LPA also inhibited [H-3]-glutamate uptake in both undifferentiated and differentiated astrocytes; and LPA-induced inhibition of glutamate uptake was still observed in lpa(1)-deficient astrocytes. Taken together, these observations demonstrate that LPA(1) mediates LPA-induced stimulation of cell proliferation but not inhibition of glutamate uptake in astrocytes. (C) 2007 Elsevier Ltd. All rights reserved.
• Estrogen induces suppression of lysophosphatidic acid receptor 1 mRNA expression level in the mouse anterior pituitary gland

  Sogo Yuki; Miyazato Takako; Fukushima Nobuyuki; Moriyama Ryutaro

  The Journal of Reproduction and Development Supplement THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 101 (0) 528 - 528 2008 

  【目的】リゾホスファチジン酸 (LPA) は，細胞増殖促進や細胞形態変化などの生理活性を有するリゾリン脂質メディエーターである。LPA受容体はGタンパク質共役型で，5種類のサブタイプ (LPA1～LPA5) が知られている。また，LPAは成熟したほ乳類の脳内および血漿中にも存在するが，その生理的役割はあまり解明されていない。これまでの研究から，48時間の絶食によりマウス下垂体のLpa1 mRNA発現量が増加することを明らかにしている。本研究では，マウス下垂体のLpa1 mRNA発現細胞を同定し，さらにその発現制御因子について検討した。【方法】実験には8週齢の雌雄マウスを用いた。下垂体におけるLPA1発現細胞を同定するためin situ hybridization法と免疫組織化学的手法を用いた。また，エストロジェンがLpa1 mRNA発現量を制御する因子であるか検討するため卵巣除去 (OVX) およびOVX後，エストラジオール17β (OVX+E₂) を代償投与したマウスを作成し，Lpa1 mRNA発現量を定量した。【結果】In situ hybridizationを行った結果，下垂体前葉細胞の細胞質特異的にLpa1 mRNA発現が観察された。Lpa1 mRNA発現細胞の一部はα-subunitとLHβ免疫陽性であった。また，OVXにより発情終止期のマウスに比べLpa1 mRNA発現量は有意に増加し，OVX+E₂群ではE₂濃度依存的にLpa1 mRNA発現量が減少した。以上より，マウス下垂体のLpa1 mRNA発現量はエストロジェン依存的に抑制され，少なくとも一部のLpa1 mRNAは性腺刺激ホルモン産生細胞に局在していることが明らかとなった。
• グルコースと長鎖脂肪酸の濃度変化はマウス下垂体前葉のゴナドトロフで長鎖脂肪酸受容体GPR120 mRNA発現量を制御する

  森山 隆太郎; 出浦 慎哉; 小浜 梓; 能勢 和浩; 福島 伸之

  The Journal of Reproduction and Development (公社)日本繁殖生物学会 53 (Suppl.) j157 - j157 0916-8818 2007/09
• Lysophosphatidic acid stimulates neuronal differentiation of cortical neuroblasts through the LPA(1)-G(i/o) pathway

  Nobuyuki Fukushima; Shinya Shano; Ryutaro Moriyama; Jerold Chun

  NEUROCHEMISTRY INTERNATIONAL PERGAMON-ELSEVIER SCIENCE LTD 50 (2) 302 - 307 0197-0186 2007/01 

  Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates cortical development. Here we examined how LPA influences the cell fate of cortical neuroblasts using a neurosphere culture system. We generated neurospheres in the presence of basic fibroblast growth factor (bFGF). Treatment with LPA throughout the culture period significantly reduced the number of cells in the neurospheres. When dissociated single cells derived from neurospheres were induced to differentiate by adherence on coverslips, the proportion of MAP2-positive neurons was higher in LPA-treated neurospheres than in those treated with bFGF alone, and the proportion of myelin basic protein-positive oligodendrocytes was lower. Consistent with this finding, LPA raised the ratio of beta-tubulin type III-positive young neurons and reduced the ratio of CD140a-positive oligodendrocyte precursors in neurospheres. These effects of LPA were inhibited by pretreatment of neurospheres with pertussis toxin or an LPA(1)-preferring antagonist, Ki16425. Moreover, LPA-induced enhancement of neuronal differentiation was not observed in neurospheres derived from lpa(1)-null mice. These results suggest that LPA promotes the commitment of neuroblasts to the neural lineage through the LPA(1)-G(i/o) pathway. (c) 2006 Elsevier Ltd. All rights reserved.
• The change in concentration of glucose and long-chain fatty acid regulates GPR120 mRNA expression in mouse anterial pituitary gonadotrophs

  Moriyama Ryutaro; Deura Chikaya; Kohama Azusa; Nose Kazuhiro; Fukushima Nobuyuki

  The Journal of Reproduction and Development Supplement THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 100 (0) 20032 - 20032 2007 

  【目的】G protein-coupled receptor 120 (GPR120) は長鎖脂肪酸をリガンドとするGタンパク質共役型受容体である。これまでに、マウス下垂体前葉の性腺刺激ホルモン産生細胞 (ゴナドトロフ) で24時間の絶食によりGPR120 mRNA発現量が統計的優位に増加することを報告している。本研究の目的は、絶食時のゴナドトロフでGPR120 mRNA発現量を増加させる因子を明らかとすることにある。【方法】実験には8週齢の成熟雄マウス (照明点灯6時30分;消灯18時30分) とゴナドトロフ株化細胞LβT2を用いた。下垂体前葉よりRNAを抽出し、GPR120 mRNA発現量の日内変動をリアルタイムPCR法により観察した。さらに、in vitroの実験として、培養液中のグルコース濃度を5mMから2mMに低下した群と、培養液中に0.1mMパルミチン酸を溶解した群を作成し、24時間培養後、GPR120 mRNA発現量を観察した。【結果】下垂体前葉のGPR120 mRNA発現量は明期後半の15時から増加し始めて17時にピークへ達し、暗期には一定のレベルを保った。また、LβT2細胞を用いたin vitroの実験において、グルコース濃度を2mMに低下した群と0.1mMパルミチン酸に暴露した群で無血清コントロール群に比べてGPR120 mRNA発現量が増加した。以上の結果より、グルコースや長鎖脂肪酸などエネルギー基質の濃度変化が、ゴナドトロフのGPR120 mRNA発現量を制御する因子の一つであることが示唆された。
• Promoting effects of bioactive contaminants in a plasma-derived thrombin reagent on NGF-induced neurite outgrowth of a rat pheochromocytoma cell line, PC12.

  Takeshi Tarui; Keita Nagasawa; Nobuyuki Fukushima; Hiroyuki Nishikawa; Atsufumi Kawabata

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN PHARMACEUTICAL SOC JAPAN 127 163 - 165 0031-6903 2007 [Refereed]
   

  Thrombin modulates neuronal functions via proteinase-activated receptors (PARs) in the CNS. In the present study, we examined effect of a human plasma-derived thrombin reagent (hp-thrombin) on neurite outgrowth in a rat pheochromocytoma cell line, PC12. Although it alone had no effect, hp-thrombin promoted NGF-induced neurite outgrowth in PC12 cells. This effect was neither inhibited by hirudin, a specific inhibitor of thrombin, and nor mimicked by receptor-activating peptides for PARI or PAR4. In addition, human recombinant thrombin did not cause facilitation of neurite outgrowth. Finally, the effect of hp-thrombin on NGF-evoked neurite outgrowth was specifically blocked by a Src-selective tyrosine kinase inhibitor. Collectively, unknown bioactive contaminants in hp-thrombin appear to facilitate neurite outgrowth, an indicator for neural differentiation, and the underlying mechanisms would involve activation of Src kinase.
• Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines.

  Tsujiuchi T; Shimizu K; Onishi M; Sugata E; Fujii H; Mori T; Honoki K; Fukushima N

  Biochem Biophys Res Commun. 349 (3) 1151 - 1155 2006/10 [Refereed]
  
• マウス下垂体のゴナドトロフに発現する長鎖脂肪酸受容体GPR120

  森山 隆太郎; 井本 慎吾; 車野 慎哉; 福嶋 伸之

  The Journal of Reproduction and Development (公社)日本繁殖生物学会 52 (Suppl.) j54 - j54 0916-8818 2006/08
• Actomyosin-dependent microtubule rearrangement in lysophosphatidic acid-induced neurite remodeling of young cortical neurons

  Nobuyuki Fukushima; Yuka Morita

  BRAIN RESEARCH ELSEVIER SCIENCE BV 1094 65 - 75 0006-8993 2006/06 

  It has been shown that lysophosphatidic acid (LPA), a signaling phospholipid, induces neurite retraction and the formation of retraction fibers in young cortical neurons by actin rearrangement. This study examined the rearrangement of microtubules (MTs) during LPA-induced neurite remodeling by immunostaining with antibodies against several types of tubulin. The results showed that alpha-tubulin was present in growing neurites as well as in cell bodies with various localization profiles. Exposure of neurons to LPA resulted in neurite retraction, accompanied by the rearrangement of MTs in neurites and the accumulation of MTs in cell bodies, without significant changes in the total amount of MTs in the cytoskeletal fraction of cultured neurons. Similar findings were obtained when young neurons were stained for other types of tubulin, including beta-tubulin type III and posttranslationally acetylated and tyrosinated tubulin. LPA-induced MT rearrangement was accompanied by accumulation of myosin IIB and polymerized actin at the base of retraction fibers. These effects of LPA on MTs and myosin 1113 were blocked by pretreatment with inhibitors of the actomyosin and Rho pathways (cytochalasin D, blebbistatin, and Y27632), but not by an MT stabilizer (taxol), whereas taxol inhibited neurite retraction and MT depolymerization induced by nocodazole. Furthermore, neurofilaments also showed rearrangement in response to LPA, which was blocked by cytochalasin D and Y27632, but not taxol. Taken together, these results suggested that LPA did not induce MT depolymerization and that LPA-induced actomyosin activation produced MT and neurofilament rearrangement, leading to neurite remodeling. (c) 2006 Elsevier B.V. All rights reserved.
• Long-chain fatty acid receptor GPR120 expression in gonadotropes of the mouse pituitary gland.

  Moriyama Ryutaro; Imoto Shingo; Shano Shinya; Fukushima Nobuyuki

  The Journal of Reproduction and Development Supplement THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT 99 (0) 19 - 19 2006 

  【目的】G protein-coupled receptor 120 (GPR120) は長鎖脂肪酸をリガンドとするGタンパク質共役型受容体である。これまでに回腸や直腸にGPR120 mRNAが発現すること、腸内分泌細胞でglucagon-like peptide-1分泌に関与することが報告されている。しかし、腸以外での生理的役割は解明されていない。絶食時には血中遊離脂肪酸濃度が上昇することから、我々は絶食時にはGPR120を介した摂食や生殖の制御メカニズムが存在すると仮説提唱した。本研究の目的は、絶食時の性腺機能抑制に関与するGPR120の発現部位を解明することにある。【方法】実験には 8週齢のICR系雄マウスを用いた。正常給餌、24および 48 時間絶食した動物の視床下部、延髄、下垂体、精巣、回腸、直腸より抽出したRNA用いて、リアルタイム PCR法によりGPR120 mRNA発現量の変化を調べた。次に、凍結切片を用いた in situ hybridization法および免疫組織化学法により、GPR120 mRNAとタンパク質の発現細胞を観察した。【結果】GPR120 mRNAは下垂体、精巣、回腸、直腸で高い発現が観察された。24および48時間の絶食により、下垂体と直腸のGPR120 mRNA発現量が、正常給餌群に比べて統計的有意 (p<0.05) に増加した。下垂体組織を用いた in situ hybridization法の結果、下垂体前葉の細胞でGPR120 mRNA発現が観察された。蛍光2重染色の結果、GPR120様免疫陽性細胞は下垂体前葉で観察され、LHβ、FSHβおよびαサブユニットと共存していた。しかし、TSHβとの共存は観察されなかった。以上より、GPR120は下垂体のゴナドトロフと精巣に存在することが示された。また、マウス下垂体のゴナドトロフにおいてGPR120は絶食時のLH合成または分泌抑制に関与することが示唆された。
• Aberrant expressions of lysophosphatidic acid receptor genes in lung and liver tumors of rats.

  Tsujiuchi T; Shimizu K; Onishi M; shigemura M; Shano S; Honoki K; Fukushima N

  J Toxicol Pathol 19 137 - 142 2006 [Refereed]
  
• neurosphere培養系におけるリゾホスファチジン酸による神経発生の増強(Increased neurogenesis by lysophosphatidic acid in neurosphere culture system)

  福嶋 伸之; 森山 隆太郎

  神経化学 日本神経化学会 44 (2-3) 217 - 217 0037-3796 2005/08
• Lysophospholipid receptors: signaling and biology.

  Ishii I; Fukushima N; Ye X; Chun J

  Annu Rev Biochem 73 (1) 321-354 - 354 2004 [Refereed]
   

  Isao Ishii, Nobuyuki Fukushima, Xiaoqin Ye, Jerold Chun, 2004, 'LYSOPHOSPHOLIPIDRECEPTORS: Signaling and Biology', <i>Annual Review of Biochemistry</i>, vol. 73, no. 1, pp. 321-354
• Characterization of lpa(2) (Edg4) and lpa(1)/lpa(2) (Edg2/Edg4) lysophosphatidic acid receptor knockout mice: Signaling deficits without obvious phenotypic abnormality attributable to lpa(2)

  JJA Contos; Ishii, I; N Fukushima; MA Kingsbury; XQ Ye; S Kawamura; JH Brown; J Chun

  MOLECULAR AND CELLULAR BIOLOGY AMER SOC MICROBIOLOGY 22 (19) 6921 - 6929 0270-7306 2002/10 [Refereed]
   

  Lysophosphatidic acid (LPA), a bioactive lipid produced by several cell types including postmitotic neurons and activated platelets, is thought to be involved in various biological processes, including brain development. Three cognate G protein-coupled receptors encoded by lpa(1)/lp(A1)/Edg-2/Gpcr26, lpa(2)/lp(A2)/Edg-4, and lpa(3)/lp(A3)/Edg-7 mediate the cellular effects of LPA. We have previously shown that deletion of lpa(1) in mice results in craniofacial dysmorphism, semilethality due to defective suckling behavior, and generation of a small fraction of pups with frontal hematoma. To further investigate the role of these receptors and LPA signaling in the organism, we deleted lpa(2) in mice. Homozygous knockout (lpa(2)((-/-))) mice were born at the expected frequency and displayed no obvious phenotypic abnormalities. Intercrosses allowed generation of lpa(1)((-/-)) lpa(2)((-/-)) double knockout mice, which displayed no additional phenotypic abnormalities relative to lpa(1)((-/-)) mice except for an increased incidence of perinatal frontal hematoma. Histological analyses of lpa(1)((-/-)) lpa(2)((-/-)) embryonic cerebral cortices did not reveal obvious differences in the proliferating cell population. However, many LPA-induced responses, including phospholipase C activation, Ca2+ mobilization, adenylyl cyclase activation, proliferation, JNK activation, Akt activation, and stress fiber formation, were absent or severely reduced in embryonic fibroblasts derived from lpa(1)((-/-)) lpa(2)((-/-)) mice. Except for adenylyl cyclase activation [which was nearly abolished in lpa(1)((-/-)) fibroblasts], these responses were only partially affected in lpa(1)((-/-)) and lpa(2)((-/-)) fibroblasts. Thus, although LPA(2) is not essential for normal mouse development, it does act redundantly with LPA(1) to mediate most LPA responses in fibroblasts.
• Dual regulation of actin rearrangement through lysophosphatidic acid receptor in neuroblast cell lines: Actin depolymerization by Ca2+-alpha-actinin and polymerization by Rho

  N Fukushima; Ishii, I; Y Habara; CB Allen; J Chun

  MOLECULAR BIOLOGY OF THE CELL AMER SOC CELL BIOLOGY 13 (8) 2692 - 2705 1059-1524 2002/08 [Refereed]
   

  Lysophosphatidic acid (LPA) is a potent lipid mediator with actions on many cell types. Morphological changes involving actin polymerization are mediated by at least two cognate G protein-coupled receptors, LPA(1)/EDG-2 or LPA(2)/EDG-4. Herein, we show that LPA can also induce actin depolymerization preceding actin polymerization within single TR mouse immortalized neuroblasts. Actin depolymerization resulted in immediate loss of membrane ruffling, whereas actin polymerization resulted in process retraction. Each pathway was found to be independent: depolymerization mediated by intracellular calcium mobilization, and a-actinin activity and polymerization mediated by the activation of the small Rho GTPase. alpha-Actinin-mediated depolymerization seems to be involved in growth cone collapse of primary neurons, indicating a physiological significance of LPA-induced actin depolymerization. Further evidence for dual regulation of actin rearrangement was found by heterologous retroviral transduction of either lpa(1) or lpa(2) in B103 cells that neither express LPA receptors nor respond to LPA, to confer both forms of LPA-induced actin rearrangements. These results suggest that diverging intracellular signals from a single type of LPA receptor could regulate actin depolymerization, as well as polymerization, within a single cell. This dual actin rearrangement may play a novel, important role in regulation of the neuronal morphology and motility during brain development.
• Lysophosphatidic acid influences the morphology and motility of young, postmitotic cortical neurons

  N Fukushima; JA Weiner; D Kaushal; JJA Contos; SK Rehen; MA Kingsbury; KY Kim; J Chun

  MOLECULAR AND CELLULAR NEUROSCIENCE ACADEMIC PRESS INC ELSEVIER SCIENCE 20 (2) 271 - 282 1044-7431 2002/06 [Refereed]
   

  Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that produces process retraction and cell rounding through its cognate receptors in neuroblastoma cell lines. Although the expression profile of LPA receptors in developing brains suggests a role for LPA in central nervous system (CNS) development, how LPA influences the morphology of postmitotic CNS neurons remains to be determined. Here we have investigated the effects of exogenous LPA on the morphology of young, postmitotic neurons in primary culture. When treated with LPA, these neurons responded by not only retracting processes but also producing retraction fiber "caps" characterized by fine actin filaments emanating from a dense core. Retraction fiber caps gradually vanished due to the outward spread of regrowing membranes along the fibers, suggesting a role for caps as scaffolds for regrowth of retracted processes. Furthermore, LPA also affects neuronal migration in vitro and in vivo. Taken together, these results implicate LPA as an extracellular lipid signal affecting process outgrowth and migration of early postmitotic neurons during development.
• Regulation of Schwann cell morphology and adhesion by receptor-mediated lysophosphatidic acid signaling

  JA Weiner; N Fukushima; JJA Contos; SS Scherer; J Chun

  JOURNAL OF NEUROSCIENCE SOC NEUROSCIENCE 21 (18) 7069 - 7078 0270-6474 2001/09 [Refereed]
   

  In peripheral nerves, Schwann cells (SCs) form contacts with axons, other SCs, and extracellular matrix components that are critical for their migration, differentiation, and response to injury. Here, we report that lysophosphatidic acid (LPA), an extracellular signaling phospholipid, regulates the morphology and adhesion of cultured SCs. Treatment with LPA induces f-actin rearrangements resulting in a "wreath"-like structure, with actin loops bundled peripherally by short orthogonal filaments. The latter appear to anchor the SC to a laminin substrate, because they colocalize with the focal adhesion proteins, paxillin and vinculin. SCs also respond to LPA treatment by forming extensive cell-cell junctions containing N-cadherin, resulting in cell clustering. Pharmacological blocking experiments indicate that LPA-induced actin rearrangements and focal adhesion assembly involve Rho pathway activation via a pertussis toxin-insensitive G-protein. The transcript encoding LPA1, the canonical G-protein-coupled receptor for LPA, is upregulated after sciatic nerve transection, and SCs cultured from Ip(A7)-null mice exhibit greatly diminished morphological responses to LPA. Cultured SCs can release an LPA-like factor implicating SCs as a potential source of endogenous, signaling LPA. These data, together with the previous demonstration of LPA-mediated SC survival, implicate endogenous receptor-mediated LPA signaling in the control of SC development and function.
• Two Novel Xenopus Homologs of Mammalian LPA1/EDG-2 Function as Lysophosphatidic Acid Receptors in Xenopus Oocytes and Mammalian Cells

  Yuka Kimura; Anja Schmitt; Nobuyuki Fukushima; Isao Ishii; Hideo Kimura; Angel R. Nebreda; Jerold Chun

  Journal of Biological Chemistry 276 (18) 15208 - 15215 0021-9258 2001/05 [Refereed]
   

  Lysophosphatidic acid (LPA) induces diverse biological responses in many types of cells and tissues by activating its specific G protein-coupled receptors (GPCRs). Previously, three cognate LPA GPCRs (LP A1/VZG-1/EDG-2, LPA2/EDG-4, and LPA3/EDG-7) were identified in mammals. By contrast, an unrelated GPCR, PSP24, was reported to be a high affinity LPA receptor in Xenopus laevis oocytes, raising the possibility that Xenopus uses a very different form of LPA signaling. Toward addressing this issue, we report two novel Xenopus genes, xlpA1-1 and xlpA1-2, encoding LPA1 homologs (∼90% amino acid sequence identity with mammalian LPA1). Both xlpA1-1 and xlpA1-2 are expressed in oocytes and the nervous system. Overexpression of either gene in oocytes potentiated LPA-induced oscillatory chloride ion currents through a pertussis toxin-insensitive pathway. Injection of antisense oligonucleotides designed to inhibit xlpA1-1 and xlpA1-2 expression in oocytes eliminated their endogenous response to LPA. Furthermore, retrovirus-mediated heterologous expression of xlp A1-1 or xlpA1-2 in B103 rat neuroblastoma cells that are unresponsive to LPA conferred LPA-induced cell rounding and adenylyl cyclase inhibition. These results indicate that XLPA1-1 and XLP A1-2 are functional Xenopus LPA receptors and demonstrate the evolutionary conservation of LPA signaling over a range of vertebrate phylogeny.
• The LPA receptors

  N Fukushima; J Chun

  PROSTAGLANDINS & OTHER LIPID MEDIATORS ELSEVIER SCIENCE INC 64 (1-4) 21 - 32 0090-6980 2001/04 [Refereed]
   

  Lysophosphatidic acid (LPA) is a growth factor-like lipid that produces many cellular responses. These responses, including actin cytoskeletal rearrangements, cell proliferation and inhibition of gap junction communication, have been documented in many cell types over the last 2 decades. Both non-receptor and receptor-mediated mechanisms had been implicated to explain these responses. 4 clear advance in this field was the cloning and functional identification of LPA receptors, and there are currently three high-affinity members, LPA1, LPA2 and LPA3 (synonymous with orphan receptor names edg-2, edg-4 and edg-7, respectively). Here we review the gene structure, expression and functions of LPA receptors. We also discuss the in vivo roles mediated by a single LPA receptor type, based on studies of the nervous system, a major locus of LPA receptor expression. (C) 2001 Elsevier Science Inc. All rights reserved.
• Lysophospholipid receptors.

  Fukushima N; Ishii I; Contos JJ; Weiner JA; Chun J

  Annu Rev Pharmacol Toxicol 41 (1) 507-534 - 534 2001 [Refereed]
   

  Nobuyuki Fukushima, Isao Ishii, James JA Contos, Joshua A Weiner, Jerold Chun, 2001, 'LYSOPHOSPHOLIPIDRECEPTORS', <i>Annual Review of Pharmacology and Toxicology</i>, vol. 41, no. 1, pp. 507-534
• Lysophosphatidic acid (LPA) is a novel extracellular regulator of cortical neuroblast morphology

  N Fukushima; JA Weiner; J Chun

  DEVELOPMENTAL BIOLOGY ACADEMIC PRESS INC 228 (1) 6 - 18 0012-1606 2000/12 [Refereed]
   

  During cerebral cortical neurogenesis, neuroblasts in the ventricular zone (VZ) undergo a shape change termed "interkinetic nuclear migration" whereby cells alternate between fusiform and rounded morphologies. We previously identified Ip(Ai) the first receptor gene for a signaling phospholipid called lysophosphatidic acid (LPA) and showed its enriched expression in the VZ. Here we report that LPA induces changes in neuroblast morphology from fusiform to round in primary culture, accompanied by nuclear movements, and formation of f-actin retraction fibers. These changes are mediated by the activation of the small GTPase, Rho. In explant cultures, where the cerebral cortical architecture remains intact, LPA not only induces cellular and nuclear rounding in the VZ, but also produces an accumulation of rounded nuclei at the ventricular surface. Consistent with a biological role for these responses, utilization of a sensitive and specific bioassay indicates that postmitotic neurons can produce extracellular LPA. These results implicate LPA as a novel factor in cortical neurogenesis and further implicate LPA as an extracellular signal from postmitotic neurons to proliferating neuroblasts. (C) 2000 Academic Press.
• Requirement for the Ip(A1) lysophosphatidic acid receptor gene in normal suckling behavior

  JJA Contos; N Fukushima; JA Weiner; D Kaushal; J Chun

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 97 (24) 13384 - 13389 0027-8424 2000/11 [Refereed]
   

  Although extracellular application of lysophosphatidic acid (LPA) has been extensively documented to produce a variety of cellular responses through a family of specific G protein-coupled receptors, the in vivo organismal role of LPA signaling remains largely unknown. The first identified LPA receptor gene, I-pA1/vzg-1/ edg-2, was previously shown to have remarkably enriched embryonic expression in the cerebral cortex and dorsal olfactory bulb and postnatal expression in myelinating glia including Schwann cells. Here, we show that targeted deletion of I-pA1 results in approximately 50% neonatal lethality, impaired suckling in neonatal pups, and loss of LPA responsivity in embryonic cerebral cortical neuroblasts with survivors showing reduced size, craniofacial dysmorphism, and increased apoptosis in sciatic nerve Schwann cells. The suckling defect was responsible for the death among I-pA1(-/-) neonates and the stunted growth of survivors. Impaired suckling behavior was attributable to defective olfaction, which is likely related to developmental abnormalities in olfactory bulb and/or cerebral cortex. Our results provide evidence that endogenous lysophospholipid signaling requires an Ip receptor gene and indicate that LPA signaling through the LPA1 receptor is required for normal development of an inborn, neonatal behavior.
• Neuronal death inhibitor and its signal transduction

  H Ueda; N Fukushima; A Yoshida; K Mizuno; W Hamabe

  FRONTIERS OF THE MECHANISMS OF MEMORY AND DEMENTIA ELSEVIER SCIENCE BV (1200) 83 - 86 0531-5131 2000 [Refereed]
   

  Cultured cortical neurons cell density-dependently survive under serum-free condition. Cell death in low-density culture was suppressed by the conditioned medium (CM) from high-density culture. Two neuronal death inhibitors (NDIs) were purified from the CM through various chromatographies. The active NDIs finally purified from SDS-PAGE were found at the size of 20 and 22 kDa protein. Pharmacological studies revealed that both NDI20 and NDI22 protect cortical neurons from the cell death through activation of phospholipase C (PLC) and protein kinase C (PKC).
• Serum-free induced neuronal apoptosis-like cell death is independent of caspase activity

  Hamabe, W.; Fukushima, N.; Yoshida, A.; Ueda, H.

  Molecular Brain Research 78 (1-2) 186 - 191 0169-328X 2000 [Refereed]
   

  Cultured cortical neurons survived in a density-dependent manner under serum-free conditions. Low-density cultured cells died in an aurintricarboxylic acid (ATA)-sensitive manner, which was accompanied with marked chromatin condensation and nuclear fragmentation. These features, characteristic for apoptosis, were not attenuated by DEVD-CHO, a caspase-3-specific inhibitor, or zVAD-FMK, a broad range caspase inhibitor, while zVAD-FMK showed a marked inhibition of camptothecin-induced cell death. Therefore, cortical neurons died in an apoptosis-like and a caspase-independent manner under serum-free conditions. Theme: Development and regeneration. Topic: Neuronal death. Copyright (C) 2000 Elsevier Science B.V.
• G(i1) and G(oA) differentially determine kinetic efficacies of agonists for ?-opioid receptor

  Kohno, M.; Fukushima, N.; Yoshida, A.; Ueda, H.

  FEBS Letters 473 (1) 101 - 105 0014-5793 2000 [Refereed]
   

  We examined the diversity of single receptor function by measuring receptor-G protein coupling in the baculovirus-Sf21 expression system. In comparative studies using Sf21 cell membranes expressing κ-opioid receptor (KOR) plus Gα(i1)β1γ2or KOR plus Gα(oA)β1γ2, there was no significant difference between both preparations in the K(i) values of various κ-opioid ligands for the displacement of [3H]U69593 binding. However, a marked difference in the rank order of agonists to stimulate [35S]GTPγS binding was observed between both preparations. These findings suggest that agonist efficacy is dependent on the population of different G proteins expressed in various tissues. Copyright (C) 2000 Federation of European Biochemical Societies.
• Neurobiology of receptor-mediated lysophospholipid signaling. From the first lysophospholipid receptor to roles in nervous system function and development.

  Chun J; Weiner JA; Fukushima N; Contos JJ; Zhang G; Kimura Y; Dubin A; Ishii I; Hecht JH; Akita C; Kaushal D

  Ann N Y Acad Sci 905 110-117 - 117 2000 [Refereed]
  
• Functional comparisons of the lysophosphatidic acid receptors, LP(A1)/VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7 in neuronal cell lines using a retrovirus expression system.

  Ishii I; Contos JJ; Fukushima N; Chun J

  Mol Pharmacol 58 (5) 895-902 - 902 2000 [Refereed]
  
• Comparative analysis of three murine G-protein coupled receptors activated by sphingosine-1-phosphate

  GF Zhang; JJA Contos; JA Weiner; N Fukushima; J Chun

  GENE ELSEVIER SCIENCE BV 227 (1) 89 - 99 0378-1119 1999/02 [Refereed]
   

  The cloning and analysis of the first identified lysophosphatidic acid (LPA) receptor gene, lp(A1) (also referred to as vzg-1 or edg-2), led us to identify homologous murine genes that might also encode receptors for related lysophospholipid ligands. Three murine genomic clones (designated lp(B1), lp(B2), and lp(B3)) were isolated, corresponding to human/rat Edg-1, rat H218/AGR16, and human edg-3, respectively. Based on the amino acid similarities of their predicted proteins (44-52% identical), the three lp(B) genes could be grouped into a separate G-protein coupled receptor subfamily, distinct from that containing the LPA receptor genes lp(A1) and lp(A2). Unlike lp(A1) and lp(A2), which contain multiple coding exons, all Ip, members contained a single coding exon. Heterologous expression of individual Ip, members in a hepatoma cell line (RH7777), followed by S-35-GTP gamma S incorporation assays demonstrated that each of the three LPB receptors conferred sphingosine-1-phosphate-dependent, but not lysophosphatidic acid-dependent, G-protein activation. Northern blot and in situ hybridization analyses revealed overlapping as well as distinct expression patterns in both embryonic and adult tissues. This comparative characterization of multiple sphingosine-l-phosphate receptor genes and their spatiotemporal expression patterns will aid in understanding the biological roles of this enlarging lysophospholipid receptor family. (C) 1999 Elsevier Science B.V. All rights reserved.
• A single receptor encoded by vzg-1/lp(A1)/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid

  N Fukushima; Y Kimura; J Chun

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA NATL ACAD SCIENCES 95 (11) 6151 - 6156 0027-8424 1998/05 [Refereed]
   

  Extracellular lysophosphatidic acid (LPA) produces diverse cellular responses in many cell types. Recent reports of several molecularly distinct G protein coupled receptors have raised the possibility that the responses to LPA stimulation could be mediated by the combination of several uni-functional receptors. To address this issue, we analyzed one receptor encoded by ventricular zone gene-1 (vzg-1) (also referred to as lp(A1)/edg-2) by using heterologous expression in a neuronal and nonneuronal cell line. VZG-1 expression was necessary and sufficient in mediating multiple effects of LPA: [H-3]-LPA binding, G protein activation, stress fiber formation, neurite retraction, serum response element activation, and increased DNA synthesis. These results demonstrate that a single receptor, encoded by vzg-1, can activate multiple LPA-dependent responses in cells from distinct tissue lineages.
• Melittin, a metabostatic peptide inhibiting G(s) activity

  Fukushima, N.; Kohno, M.; Kato, T.; Kawamoto, S.; Okuda, K.; Misu, Y.; Ueda, H.

  Peptides 19 (5) 811 - 819 0196-9781 1998 [Refereed]
   

  Some basic amphiphilic peptides are known to directly stimulate heterotrimeric GTP-binding proteins (G proteins). Mastoparan and melittin are known to stimulate G(i) activities. Here, we found melittin inhibited guanine nucleotide-dependent adenylyl cyclase activity in synaptic membranes of the rat cerebral cortex. However, in insect cell membranes overexpressing specific heterotrimeric G proteins using baculovirus expression system, melittin showed unique effects different from those by mastoparan on G protein activities. This peptide markedly stimulated G(il) and G(il) activities, whereas it did inhibit G(s) activities. Kinetic studies revealed that the inhibition of G(s) activity by melittin is attributed to the inhibition of GDP release in exchange for added guanine nucleotides (or the association of guanine nucleotides). Thus, melittin may be the first metabostatic peptide inhibiting G protein (G(s)) activity, and both mechanisms through the stimulation of G(i) and inhibition of G(s) might be involved in the melittin-induced inhibition of adenylyl cyclase.
• Low-density induced apoptosis of cortical neurons is inhibited by serum factors

  Sasaki, Y.; Fukushima, N.; Yoshida, A.; Ueda, H.

  Cellular and Molecular Neurobiology 18 (5) 487 - 496 0272-4340 1998 [Refereed]
   

  1. We investigated the survival of neurons under serum-free conditions without any exogenous signal molecules, using primary cultures of rat cerebral cortex. 2. Survival activity, measured with Alamar Blue, showed a cell density dependency under serum-free conditions. 3. The addition of fetal bovine serum suppressed the apoptotic cell death accompanied by DNA-laddering and fragmentation specific in low-density cultures, resulting in the disappearance of the cell density dependency of survival. 4. These findings suggest that serum factors may substitute for endogenous survival factors from cortical neurons in high-density cultures.
• A subtype of ?-opioid receptor mediates inhibition of high-affinity GTPase inherent in Gi1in Guinea pig cerebellar membranes

  Ueda, H.; Misawa, H.; Fukushima, N.; Katada, T.; Ui, M.; Satoh, M.

  Journal of Neurochemistry 66 (2) 845 - 851 0022-3042 1996 [Refereed]
   

  The κ-opioid receptor agonists including U-50,488H and dynorphin A (1-17) in ranges of 0.1-100 nM inhibited the hydrolysis of GTP to GDP (Pirelease) inherent in GTP-binding proteins (G proteins) in guinea pig cerebellar membranes. U-50,488H inhibited only high-affinity GTPase activity, not low-affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations >1 μM. The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations >1 μg/ml but not with preactivated cholera toxin (30 μg/ml). Similar blockade of κ-receptor-mediated inhibition was also observed when membranes were pretreated with a low concentration (8 μM) of N-ethylmaleimide (NEM) at low temperature (4°C), which alkylates the cysteine residue to be ADP-ribosylated by PTX; but this treatment caused no significant change in κ-agonist binding. When purified Gi1, but not Go, was reconstituted into membranes pretreated with NEM, the κ-receptor-mediated inhibition was recovered. These findings suggest that a subtype of κ-opioid receptor is coupled to inhibition of intrinsic activity of Gi1.
• PROTEIN-KINASE-C INVOLVEMENT IN HOMOLOGOUS DESENSITIZATION OF DELTA-OPIOID RECEPTOR-COUPLED TO G(I1)-PHOSPHOLIPASE C ACTIVATION IN XENOPUS OOCYTES

  H UEDA; T MIYAMAE; C HAYASHI; S WATANABE; N FUKUSHIMA; Y SASAKI; T IWAMURA; Y MISU

  JOURNAL OF NEUROSCIENCE OXFORD UNIV PRESS INC 15 (11) 7485 - 7499 0270-6474 1995/11 [Refereed]
   

  We have developed the coexpression system of both delta-opiold receptor (DOR1) and M(2)-muscarinic receptor (M(2)) which mediate agonist-evoked currents due to common post-receptor mechanisms including G(i1) and phospholipase C (PLC) activation in Xenopus oocytes reconstituted with G(i1)alpha. The DOR1-currents by 100 nM D-Ser(2)-leu-enkephalin-Thr(6) (DSLET) were selectively desensitized by 10 nM phorbol 12-myristate 13-acetate (PMA). The PMA-desensitization of DSLET-currents was abolished in the presence of calphostin C, a protein kinase C inhibitor, or reversed by an intracellular injection of calcineurin, a protein phosphatase 2B. When a higher concentration (3 mu M) of DSLET was used, DSLET-currents were rapidly desensitized by repeated challenges of DSLET itself. However, repeated challenges of 10 mu M ACh caused no influence on such DSLET- or M(2)-currents. The desensitization of DSLET-currents was selectively reversed by protein kinase C inhibitors. Similar results were also obtained with various delta-opioid agonists. These resutis suggest that protein kinase C is involved in the homologous desensitization of delta-opioid receptors.
• Protein kinase C inhibitor potentiates the agonist-induced GTPase activity in COS cell membranes expressing δ-opioid receptor

  Chifumi Hayashi; Hiroshi Ueda; Nobuyuki Fukushima; Takashi Katayama; Yoshimi Misu

  Molecular Brain Research 33 (2) 347 - 350 0169-328X 1995 

  In COS-7 cell membranes expressing cloned δ-opioid receptor, [d-Ser2, Leu5]enkephalin-Thr6, an opioid δ-agonist, showed no significant stimulation of high-affinity GTPase, while this agonist binding showed a guanine nucleotide sensitivity. Significant stimulation of GTPase activity by this agonist was observed only when the cells were pretreated with 0.1 μM calphostin C, a protein kinase C inhibitor, and when this inhibitor was further added to the reaction mixture at 1 μM. These findings suggest that protein kinase C is involved in the heterologous desensitization of δ-opioid receptor in the cells. © 1995.
• Evidence for a metabostatic opioid κ-receptor inhibiting pertussis toxin-sensitive metabotropic glutamate receptor-currents in Xenopus oocytes

  Ueda, H.; Miyamae, T.; Nobuyuki, F.; Watanabe, S.; Misu, Y.

  FEBS Letters 375 (3) 201 - 205 0014-5793 1995 [Refereed]
   

  Glutamate evoked pertussis toxin-sensitive currents in Xenopus oocytes expressing metabotropic glutamate receptor subtype 1 (mGluR1) and exogenous Gilα. The mGluR1-currents were completely blocked by U-73122, a phospholipase C (PLC) inhibitor and by niflumic acid, a chloride channel blocker. In the oocyte further coinjected with poly(A)+RNA from the guinea pig cerebellum, the mGluR1-currents were inhibited by U-50488H, an opioid κ-agonist, and this inhibition was blocked by norbinaltorphimine, an opioid κ-antagonist. These findings suggest that the mRNA encoding a novel subtype of opioid κ-receptor which inhibits Gi1-PLC-mediated currents is present in guinea pig cerebellar poly(A)+fractions. © 1995.
• Opioid μ- and κ-receptor mediate phospholipase C activation through Gi1 in Xenopus oocytes

  Hiroshi Ueda; Takeaki Miyamae; Nobuyuki Fukushima; Hiroshi Takeshima; Kazuhiko Fukuda; Yukio Sasaki; Yoshimi Misu

  Molecular Brain Research 32 166 - 170 0169-328X 1995/01 

  In the Xenopus oocytes expressing μ or κ-opioid receptors, agonist-induced currents were observed only when the oocyte was coinjected with Gi1 α RNA and pretreated with K-252a, a potent inhibitor of protein kinases. The evoked currents were abolished by i intracellular injection of EGTA or inositol 1,4,5-trisphosphate and the current-voltage relationship revealed that they are mediated through typical calcium-dependent chloride channels. These findings suggest that the μ- and κ-receptors mediate phospholipase C activation through Gi1 α, and that these receptor mechanisms including downstream signalings might be inhibited by phosphorylation in vivo in the Xenopus oocyte. © 1995.
• Species and age-dependent differences of functional coupling between opioid δ-receptor and G-proteins and possible involvement of protein kinase C in striatal membranes

  Nobuyuki Fukushima; Hiroshi Ueda; Chifumi Hayashi; Takashi Katayama; Takeaki Miyamae; Yoshimi Misu

  Neuroscience Letters 176 55 - 58 0304-3940 1994/07 

  [d-Ser2,Leu5]-enkephalin-Thr6(DSLET) and [d-Pen2,5]-enkephalin (DPDPE), both δ-agonists, stimulated the high affinity GTPase in the rat striatal membranes in a naltrindole-reversible manner. Similar stimulation was also observed in the striatal membrane preparations of 16-week-old guinea pigs, while not in those preparations of 4-week-old ones. When calphostin C, a protein kinase C inhibitor, was added to the reaction mixture, DSLET showed a marked stimulation in this activity in 4-week guinea pig striatal membranes. There was no effect of KT5720, a cyclic AMP-dependent protein kinase inhibitor, on such δ-opioid-mediated responses. These findings suggest that protein kinase C is locally involved in the functional uncoupling of δ-receptors to G-proteins in the striatum of young guinea pigs. © 1994.
• Protein kinase inhibitor potentiates opioid δ–receptor currents in xenopus oocytes

  Hiroshi Ueda; Takeaki Miyamae; Nobuyuki Fukushima; Yoshimi Misu

  NeuroReport 5 1985 - 1988 0959-4965 1994/01 

  In Xenopus oocytes injected with RNA coding for the 5–opioid receptor a small chloride current was evoked by [D–Ala2, Ser5]leucine–enkephaline–Thr6(DSLET), a 5,–2opioid agonist. The evoked currents were rapidly reduced upon repeated challenges of DSLET. When Gila RNA was co–injected into the oocyte, the evoked currents were increased 3.8–fold and became constant after at least three repeated challenges. In oocytes injected with RNAs coding for 5–receptor and Gila, pretreatment with K–252a, a potent inhibitor of protein kinases, further potentiated the 5–receptor–mediated current responses, compared with those without the inhibitor. These results suggest that signalling involving the 5–opioid receptor is inactivated through in vivo phosphorylation in the Xeno– pus oocyte. © Rapid Communications of Oxford Ltd.
• DELTA-OPIOID RECEPTOR MEDIATES PHOSPHOLIPASE-C ACTIVATION VIA G(I) IN XENOPUS-OOCYTES

  T MIYAMAE; N FUKUSHIMA; Y MISU; H UEDA

  FEBS LETTERS ELSEVIER SCIENCE BV 333 (3) 311 - 314 0014-5793 1993/11 

  Cloned mouse delta-subtype opioid receptor (DOR 1) was expressed in Xenopus oocytes to study the signal transduction. Opioid delta-agonists evoked a calcium-dependent chloride current in oocytes injected with mRNA derived from DOR1, together with that from the alpha subunit of G(i)1. The delta-agonist-induced current was blocked by naltrindol, a delta-specific antagonist. The delta-agonist evoked no or very weak currents in oocytes with the alpha subunit of G(q) or G(o). These findings indicate the functional coupling between the opioid delta-receptor and phospholipase C through an activation of G(i).
• The kyotorphin (tyrosine-arginine) receptor and a selective reconstitution with purified Gi, measured with GTPase and phospholipase C assays

  H. Ueda; Y. Yoshihara; H. Misawa; N. Fukushima; T. Katada; M. Ui; H. Takagi; M. Satoh

  Journal of Biological Chemistry 264 3732 - 3741 0021-9258 1989/02 [Refereed]
   

  We attempted to identify the kyotorphin receptor and the post receptor mechanisms mediated by GTP-binding proteins (G-proteins), using reconstitution techniques. The specific binding of [3H]kyotorphin in rat brain membranes was composed of high affinity (K(d) = 0.34 nM) and low affinity (K(d) = 9.07 nM) binding. As the high affinity binding disappeared in the presence of guanosine 5'-O-(3-thiotriphosphate) and MgCl2, we investigated the kyotorphin receptor-mediated changes in membrane G-protein activity by measuring low K(m) GTPase activity. Kyotorphin produced a stimulation of low K(m) GTPase, and this stimulation was antagonized by Leu-Arg, a synthetic dipeptide which showed a potent displacement of [3H]kyotorphin binding, yet in itself had no effect on the low K(m) GTPase. The kyotorphin stimulation of low K(m) GTPase was abolished by pretreating membranes with islet-activating protein, pertussis toxin, and was recovered by reconstitution with purified G-protein, G(i), but not with G(o). Similar evidence of selective coupling of kyotorphin receptor to G(i) was obtained with the phospholipase C assay. Kyotorphin-induced stimulation of phospholipase C was also abolished by islet-activating protein-treatment and recovered by reconstitution with G(i) but not with G(o). These findings indicate that specific high and low affinity kyotorphin receptors exist in the rat brain and that the kyotorphin receptor is functionally coupled to stimulation of phospholipase C, through G(i). This study provides the first evidence of a selective involvement of G(i) in the receptor-mediated activation of phospholipase C.
• Presynaptic opioid kappa-receptor and regulation of the release of Met-enkephalin in the rat brainstem

  Hiroshi Ueda; Nobuyuki Fukushima; Ming Ge; Hiroshi Takagi; Masamichi Satoh

  Neuroscience Letters 81 309 - 313 0304-3940 1987/10 [Refereed]
   

  Opioid κ-agonists had much more potent inhibitory effects on the high K+-evoked Met-enkephalin release from rat brain slices than did the μ- or δ-agonists. The opioid κ-antagonist, MR2266 enhanced the evoked release of Met-enkephalin to a greater extent than did μ- or δ-antagonists in vitro and had a potent analgesia in mice in vivo. These findings suggest that the release of Met-enkephalin may be regulated in vitro and in vivo, mainly by presynaptic κ-receptor-mediated mechanisms. © 1987.
• A Met-enkephalin releaser (kyotorphin)-induced release of plasma membrane-bound Ca2+ from rat brain synaptosomes

  Hiroshi Ueda; Nobuyuki Fukushima; Yoshihiro Yoshihara; Hiroshi Takagi

  Brain Research 419 197 - 200 0006-8993 1987/09 [Refereed]
   

  The neuropeptide kyotorphin (Tyr-Arg) released45Ca2+from synaptosomal membrane preparations of the rat lower brainstem. This dipeptide also decreased plasma membrane-bound Ca2+([Ca2+]m), determined using chlorotetracycline, in the slice and synaptosome of the rat lower brainstem. Taken together with other data indicating that kyotorphin induces an increase in synaptosomal intracellular Ca2+([Ca2+]i) in a Ca2+channel-independent manner, it is suggested that kyotorphin may mobilize the [Ca2+]mand thus increase the [Ca2+]iin the nerve terminals of the rat lower brainstem. © 1987.
• Kyotorphin (tyrosine-arginine) synthetase in rat brain synaptosomes.

  H. Ueda; Y. Yoshihara; N. Fukushima; H. Shiomi; A. Nakamura; H. Takagi

  Journal of Biological Chemistry 262 8165 - 8173 0021-9258 1987/06 [Refereed]
   

  Kyotorphin (Tyr-Arg) is a unique neuropeptide which produces analgesia by releasing Met-enkephalin from slices of the brain and spinal cord. Recent studies revealed that kyotorphin possesses the properties of neurotransmitter/neuroregulator. In the present study, we identified a kyotorphin synthetase in the soluble fraction of rat brain synaptosomes (synaptosol) and characterized it. The enzyme partially purified with Sephacryl S-300 showed an absolute requirement for ATP, MgCl2, tyrosine, and arginine. The optimal pH was 7.5-9.0 and the pI was determined to be 6.1-6.2 by isoelectric focusing. The Km was 25.6 microM for tyrosine, 926 microM for arginine, 294 microM for ATP, and 442 microM for MgCl2. The Vmax was 34.0 pmol/mg of protein/h. The apparent molecular size of this "kyotorphin synthetase" further purified by the DE52 column was 240,000-245,000 daltons, estimated using TSKgel G4000SW column chromatography. The enzyme reaction is represented by the following equation: Tyr + Arg + ATP + MgCl2 + kyotorphin synthetase----Tyr-Arg (kyotorphin) + AMP + PPi + MgCl2 + kyotorphin synthetase. The regional distribution and subcellular localization of the synthetase showed a close correlation to that of kyotorphin levels in the rat brain. The amounts of kyotorphin formed from amino acids by the synthetase in the dialyzed synaptosol was 3.0-4.0 times higher than that from precursor proteins by processing enzymes within the 30 min incubation.
• THE SPECIFIC OPIOID KAPPA-AGONIST U-50,488H INHIBITS LOW KM GTPASE

  H UEDA; H MISAWA; N FUKUSHIMA; H TAKAGI

  EUROPEAN JOURNAL OF PHARMACOLOGY ELSEVIER SCIENCE BV 138 (1) 129 - 132 0014-2999 1987/06 

  DAGO, a specific opioid μ-agonists stimulated the low Km GTPase activity of membranes of the guinea-pig striatum at 1-1000 nM and this effect was antagonized by naloxone 10-100 nM. On the other hand, U-50,488H, a specific opioid κ-agonist inhibited the low Km GTPase activity of membranes of the guinea-pig cerebellum at 1-100 nM. As this effect was antagonized by MR2266 but not by naloxone, the findings suggest that opioid μ- and κ-receptor stimulation is probably linked to the stimulation and inhibition of low Km GTPase, respectively. © 1987.
• A novel proenkephalin processing carboxypeptidase and its activation by cyclic AMP dependent protein kinase

  Hiroshi Ueda; Nobuyuki Fukushima; Hiroshi Takagi

  Biochemical and Biophysical Research Communications 142 595 - 602 0006-291X 1987/01 [Refereed]
   

  Carboxypeptidase B-like enzymes cleaving Met-enkephalin-Arg from synaptosomes of the rat striatum purified using a DEAE-cellulose column and Met-Arg-CH-Sepharose 4B affinity column proved to be different from enkephalin-convertase, lysosomal carboxypeptidase B-like enzyme, pancreas carboxy-peptidase B and carboxypeptidase Y, in effects of inhibitors and activators, pH optimum (7.5 - 8.5) and molecular size (50,000). This enzyme, named "Processin CP-E" was activated by cAMP dependent protein kinase, and the Vmax was increased from 4.3 to 13.3 μM/min/mg protein, while the Km (28.2 μM) was unchanged. © 1987.
• Uptake and release of kyotorphin in rat brain synaptosomes

  Hiroshi Ueda; Sumie Matsumoto; Yoshihiro Yoshihara; Nobuyuki Fukushima; Hiroshi Takagi

  Life Sciences 38 2405 - 2411 0024-3205 1986/06 [Refereed]
   

  Uptake and release of kyotorphin (TyrArg) in rat brain synaptosomes were studied. Synthetic kyotorphin was taken up into crude synaptosomes (P2), in a temperature-dependent manner. The Km and Vmax of the uptake were 1.31 ± 0.12 × 10-4M and 5.9 ± 0.5 pmol/mg protein/min, respectively. Metabolic inhibitors such as dinitrophenol and iodoacetamide and ouabain which is known as an inhibitor of Na+dependent uptake mechanism significantly inhibited the uptake. When the synaptosomes previously preloaded with synthetic kyotorphin at 10-4M were exposed to high K+medium, kyotorphin was released in a Ca2+-dependent manner. These findings support the view that kyotorphin plays a role as neurotransmitter/neuroregulator. © 1986.
• Pertussis toxin (IAP) enhances maitotoxin (a putative Ca2+ channel agonist)-induced Ca2+ entry into synaptosomes

  Hiroshi Ueda; Shigeki Tamura; Nobuyuki Fukushima; Hirodhi Takagi

  European journal of pharmacology 122 379 - 380 0014-2999 1986/04 [Refereed]
  
• Low doses of naloxone produce analgesia in the mouse brain by blocking presynaptic autoinhibition of enkephalin release

  Hiroshi Ueda; Nobuyuki Fukushima; Tadaaki Kitao; Ming Ge; Hiroshi Takagi

  Neuroscience Letters 65 247 - 252 0304-3940 1986/04 [Refereed]
   

  The involvement of presynaptic autoinhibition of Met-enkephalin release in naloxone-induced analgesia was studied. In both acetic acid writhing and tail-flick tests in mice, naloxone produced biphasic effects, analgesia at very low doses (1 μg/kg s.c. or 1 ng intracisternal) and hyperalgesia at higher doses (100 μg/kg s.c. or 100 ng intracisternal). Morphine at 10-6to 10-5M depressed the high K+-evoked release of Met-enkephalin from slices of the rat brainstem by 12.5-55.9% of control, while naloxone at 10-6M significantly enhanced the release by 80.6%. These findings strongly suggest that in the mouse brain a very low dose of naloxone produces analgesia by blocking autoinhibition of enkephalin release. © 1986.

Books etc

• Lysophosphatidic acid receptor. Encyclopedia of SIgnaling Molecules,

  Fukushima, N; Kado T; Tsujiuchi, T Springer 2017
• Neural effects of LPA signaling. In Lysophospholipid Receptors: Signaling and Biochemistry.

  FUKUSHIMA Nobuyuki (Single work)Wiley 2013
• Cytoskeleton of the Nervous System, Advances in Neurobiology, Microtubules in the Nervous System.

  Fukushima, N (Single work)Springer 2011 

  神経系における微小管についてまとめた。

Conference Activities & Talks

• オレイン酸はRMG-1卵巣がん細胞のグルタミン代謝を調節し細胞増殖を促進させる  [Not invited]

  玉置 彪流; 福嶋伸之

  第94回日本生化学会大会  2021/11
• CD36 mediates oleic acid-induced CDK activation to stimulate cell cycle progression and glucose transporter transcription in ovarian cancer cells  [Not invited]

  門剛史; 岩森正男; 福嶋伸之

  第92回生化学会大会  2019/09
• シトシンアラビノシドを暴露したマウス海馬神経細胞におけるヒストンH2AXリン酸化の新規メカニズム

  中山 紗希; 福嶋 伸之

  Neuro2019  2019/07
• GENOTOXIC CHEMICAL-INDUCED NEURONAL DEGENERATION IS ACCOMPANIED BY HISTONE H2A.X PHOSPHORYLATION  [Not invited]

  Fukushima, N; Nakayama, S

  2019AD/PD  2019/03
• Genotoxic chemical-induced histone H2AX phosphorylation and its restoration in postmitotic mouse hippocampal neurons  [Not invited]

  中山紗希; 福嶋伸之

  第41回日本神経科学大会  2018/07
• 海馬神経細胞におけるシトシンアラビノシド誘発性DNA損傷はデオキシシチジンにより阻害されるのみならず回復される  [Not invited]

  稲畑則幸; 足立 実優; 福嶋伸之

  第40回日本神経科学大会  2017/07
• 神経系細胞におけるシトシンアラビノシドのDNA損傷作用

  足立 実優; 畑野 弥咲; 福嶋伸之

  第59回日本神経化学会大会  2016/09
• Cytosine arabinoside induces DNA double strand breaks in hippocampal neurons in culture

  Adachi, M; Hatano, M; Fukushima, N

  Neuro2016  2016/07
• Molecular mechanism of LPA3-mediated axonal branching formation in cultured hippocampal neurons  [Not invited]

  古田大祐; 山根昌之; 森山 隆太郎; 辻内俊文; 福嶋 伸之; 立命館大学薬学部

  第83回生化学会大会  2010/12  神戸  第83回生化学会大会
  
• Lysophosphatidic acid receptor 3 activation induces axonal branch formation in cultured hippocampal neurons  [Not invited]

  福嶋 伸之; 古田大祐; 山根昌之; 辻内俊文; 森山 隆太郎

  Neuro2010 （第33回神経科学会、第53回神経化学会）  2010/09  神戸  Neuro2010 （第33回神経科学会、第53回神経化学会）
  
• 長鎖脂肪酸はマウス下垂体のGpr120 mRNA発現量を増加させる.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第25回下垂体研究会学術集会  2010/08  伊良湖、伊良湖ガーデンホテル  第25回下垂体研究会学術集会
  
• Palmitate regulates GnRH receptor mRNA expression level in the gonadotrope of the mouse anterior pituitary gland.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  14th International Congress of Endocrinology  2010/03  kyoto  14th International Congress of Endocrinology
  
• グルコースの利用阻害は下垂体の性腺刺激ホルモン産生細胞でGnRHレセプターおよびFSHβ mRNA発現量を抑制する.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第102回日本繁殖生物学会大会  2010  奈良、近畿大学  第102回日本繁殖生物学会大会
  
• 神経発達とリゾホスファチジン酸シグナル  [Not invited]

  福嶋 伸之

  脂質メディエーターワークショップ  2009/12  草津  脂質メディエーターワークショップ
  
• Lysophosphatidic acid receptor 3 is involved in neurite branching formation in hippocampal neurons  [Not invited]

  古田大祐; 山根昌之; 森山 隆太郎; 辻内俊文; 福嶋 伸之

  第82回日本生化学会大会  2009/10  神戸  第82回日本生化学会大会
  
• 高脂肪食負荷がゴナドトロフの長鎖脂肪酸受容体Gpr120と性腺刺激ホルモンmRNA発現に与える影響.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第102回日本繁殖生物学会大会  2009/09  奈良、近畿大学  第102回日本繁殖生物学会大会
  
• 高脂肪食給餌が成熟雄マウス下垂体のゴナドトロフに局在する長鎖脂肪酸受容体GPR120と性腺刺激ホルモンmRNA発現に与える影響.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第24回下垂体研究会学術集会  2009/08  三沢、小牧温泉 青森屋  第24回下垂体研究会学術集会
  
• グルコースの利用阻害はゴナドトロフ株化細胞LβT2で性腺刺激ホルモン放出ホルモン受容体 (GnRH-R) と卵胞刺激ホルモンβ鎖 (FSHβ) mRNA発現量を抑制する.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第24回下垂体研究会学術集会  2009/08  三沢、小牧温泉 青森屋  第24回下垂体研究会学術集会
  
• LPA induces neurite shape changes through LPA3  [Not invited]

  古田大祐; 山根昌之; 森山 隆太郎; 辻内俊文; 福嶋 伸之

  PLM2009  2009/05  Tokyo  PLM2009
  
• Regulation of neurite morphology by LPA  [Not invited]

  福嶋 伸之

  PLM2009  2009/05  Tokyo  PLM2009
  
• Palmitate regulates GnRH receptor mRNA expression level in the gonadotrope of the mouse pituitary gland.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第5回武田科学振興財団薬科学シンポジウム  2009/05  東京、グランドプリンスホテル高輪  第5回武田科学振興財団薬科学シンポジウム
  
• Palmitate regulates GnRH receptor mRNA expression level in the gonadotrope of the mouse pituitary gland.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  4th International Conference on Phospholipase A2 and Lipid Mediators  2009/05  Tokyo  4th International Conference on Phospholipase A2 and Lipid Mediators
  
• 硫化水素により誘起される大脳皮質神経細胞死のメカニズムの解析：特にMEK/ERK系の関与について．  [Not invited]

  川畑 篤史; 関口 富美子; 福嶋 伸之; 赤池昭紀; 久米

  第82回日本薬理学会年会  2009/03  横浜  第82回日本薬理学会年会
  
• Long-chain fatty acids regulate GnRH receptor mRNA expression in the gonadotrope of the mouse anterior pituitary gland.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  38th Annual Meeting of Society for Neuroscience  2008/11  Washington, DC., U. S. A.  38th Annual Meeting of Society for Neuroscience
  
• マウス胎児大脳皮質神経細胞における内因性ガスメッセンジャー硫化水素の神経細胞毒性はMEK-ERK系の活性化に依存する．  [Not invited]

  川畑 篤史; 福嶋 伸之; 関口 富美子; 赤池昭紀; 久米

  第114回日本薬理学会近畿部会  2008/11  神戸  第114回日本薬理学会近畿部会
  
• マウス下垂体のリゾホスファチジン酸受容体1はエストロジェン依存的に発現が抑制される.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第101回日本繁殖生物学会大会  2008/09  福岡、九州大学  第101回日本繁殖生物学会大会
  
• パルミチン酸はゴナドトロフのGnRHレセプターmRNA発現量を制御する.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第101回日本繁殖生物学会大会  2008/09  福岡、九州大学  第101回日本繁殖生物学会大会
  
• 長鎖脂肪酸はゴナドトロフのGnRHレセプター mRNA発現量を制御する.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第35回 日本神経内分泌学会・第23 回日本下垂体研究会合同学術集会  2008/08  東京、政策研究大学院大学  第35回 日本神経内分泌学会・第23 回日本下垂体研究会合同学術集会
  
• Palmitate regulates GnRH receptor mRNA expression in the gonadotrope of the mouse anterior pituitary  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第31回日本神経科学大会  2008/07  東京、東京国際フォーラム  第31回日本神経科学大会
  
• Fasting induced increase in long-chain fatty acid receptor GPR120 expression in the gonadotrope of the mouse anterior pituitary.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  37th Annual Meeting of Society for Neuroscience  2007/11  San Diego, U. S. A.  37th Annual Meeting of Society for Neuroscience
  
• グルコースと長鎖脂肪酸の濃度変化はマウス下垂体前葉のゴナドトロフで長鎖脂肪酸受容体GPR120 mRNA発現量を制御する.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第100回日本繁殖生物学会大会  2007/10  東京、東京大学  第100回日本繁殖生物学会大会
  
• ラット褐色細胞腫由来細胞PC12におけるNGF誘起神経突起伸長に対する血漿由来thrombin試薬中夾雑生理活性物質の促進効果．  [Not invited]

  川畑 篤史; 福嶋 伸之

  生体機能と創薬シンポジウム2007金沢  2007/09  石川  生体機能と創薬シンポジウム2007金沢
  
• Palmitate-induced increase in long-chain fatty acid receptor GPR120 mRNA level in LβT2 pituitary gonadotrope cells.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第30回日本神経科学大会  2007/09  横浜、パシフィコ横浜  第30回日本神経科学大会
  
• マウスのゴナドトロフで長鎖脂肪酸受容体GPR120 mRNA発現量を制御する因子の検討  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第22回下垂体研究会学術集会  2007/08  葉山、湘南国際村センター  第22回下垂体研究会学術集会
  
• マウスのゴナドトロフに発現する長鎖脂肪酸受容体GPR120.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第99回日本繁殖生物学会大会  2006/09  名古屋、名古屋大学  第99回日本繁殖生物学会大会
  
• Fasting induced Long-chain fatty acid receptor GPR120 expression increase in the anterior pituitary of mouse.  [Not invited]

  森山 隆太郎; 福嶋 伸之

  第29回日本神経科学大会  2006/07  京都、国立京都国際会館  第29回日本神経科学大会
  

MISC

• がんゲノム医療時代の遺伝カウンセラーの養成:文部科学省がんプロ養成プランへの参画

  田村和朗; 長尾哲二; 南武志; 日高雄二; 辻内俊文; 巽純子; 福嶋伸之; 加川尚; 西郷和真; 島本茂; 川下理日人; 福岡和也; 中川和彦  日本人類遺伝学会大会プログラム・抄録集  63rd-  380  2018
• マウス下垂体のα‐subunit含有細胞に局在するリゾホスファチジン酸受容体1mRNAの発現量は去勢により増加する

  森山隆太郎; 松本絵美; 原玲奈; 宮里公子; 十河由紀; 福嶋伸之  日本下垂体研究会学術集会プログラム・講演要旨集  28th-  57  2013
• Gonadal Steroids Down-Regulate the Long-Chain Fatty Acid Receptor GPR120 mRNA Expression Levels in Gonadotrophs of the Mouse Anterior Pituitary Gland

  Ryutaro Moriyama; Mari Toyonaga; Kimiko Miyazato; Yuki Sogo; Nobuyuki Fukushima  BIOLOGY OF REPRODUCTION  85-  2011/07
• 培養海馬神経細胞におけるLPA3を介する軸索分岐形成の分子メカニズム(Molecular mechanism of LPA3-Mediated axonal branching formation in cultured hippocampal neurons)

  古田 大祐; 山根 昌之; 森山 隆太郎; 藤田 典久; 辻内 俊文; 福嶋 伸之  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  83回・33回-  2P  -0682  2010/12
• LPA3受容体活性化は海馬神経細胞の軸索分岐形成を引き起こす(Lysophosphatidic acid receptor 3 activation induces axonal branch formation in cultured hippocampal neurons)

  福嶋 伸之; 古田 大祐; 山根 昌之; 辻内 俊文; 森山 隆太郎  神経化学  49-  (2-3)  540  -540  2010/08
• 2-deoxy-D-glucose (2DG) induced glucoprivation suppressed gonadotropin-releasing hormone receptor (GnRH-R) and follicular stimulating hormone beta subunit (FSH beta) mRNA expression levels in the L beta T2 gonadotroph cells

  Ryutaro Moriyama; Ai Nakai; Nobuyuki Fukushima  ENDOCRINE JOURNAL  57-  S605  -S605  2010/03
• Palmitate regulates GnRH receptor mRNA expression levels in the gonadotrope of the mouse anterior-pituitary gland

  Chikaya Deura; Nobuyuki Fukushima; Ryutaro Moriyama  ENDOCRINE JOURNAL  57-  S604  -S605  2010/03
• Long-Chain Fatty Acids Regulate GnRH Receptor mRNA Expression Level in the Gonadotrope of the Mouse Anterior Pituitary Gland.

  Ryutaro Moriyama; Chikaya Deura; Nobuyuki Fukushima  BIOLOGY OF REPRODUCTION  174  -174  2010
• Lysophosphatidic acid receptor 3 activation induces axonal branch formation in cultured hippocampal neurons

  Nobuyuki Fukushima; Daisuke Furuta; Masayuki Yamane; Toshifumi Tsujiuchi; Ryutaro Moriyama  NEUROSCIENCE RESEARCH  68-  E137  -E137  2010
• 培養海馬ニューロンにおいてリゾホスファチジン酸受容体3は神経突起分枝と関係がある(Lysophosphatidic acid receptor 3 is involved in neurite branching formation in cultured hippocampal neurons)

  古田 大祐; 山根 昌之; 森山 隆太郎; 辻内 俊文; 福嶋 伸之  日本生化学会大会プログラム・講演要旨集  82回-  4P  -415  2009/09
• 高脂肪食給餌が性腺刺激ホルモン産生細胞の長鎖脂肪酸受容体Gpr120と性腺刺激ホルモンmRNA発現に与える影響

  出浦慎哉; 福嶋伸之; 森山隆太郎  J Reprod Dev  55-  (Supplement)  J130  -j130  2009/08
• グルコースの利用阻害は下垂体の性腺刺激ホルモン産生細胞でGnRHレセプターおよびFSHβmRNA発現量を抑制する

  森山隆太郎; 中井愛; 福嶋伸之  J Reprod Dev  55-  (Supplement)  J129  -j129  2009/08
• Characterization of H2S-induced neuronal death in cultured cortical neurons: involvement of the MEK/ERK pathway

  Yuko Kurokawa; Satoko Kubo; Yoshiko Yamasaki; Fumiko Sekiguchi; Yukari Okamoto; Yuma Maeda; Toshiaki Kume; Nobuyuki Fukushima; Akinori Akaike; Atsuhumi Kawadata  JOURNAL OF PHARMACOLOGICAL SCIENCES  109-  189P  -189P  2009
• glucose availability suppresses GnRH receptor and FSHbeta mRNA expression in the gonadotrophs of the mouse pituitary gland

  MORIYAMA Ryutaro; NAKAI Ai; FUKUSHIMA Nobuyuki  The Journal of Reproduction and Development Supplement  102-  (0)  1049  -1049  2009
• Palmitate regulates GnRH receptor mRNA expression in the gonadotrope of the mouse anterior pituitary

  Ryutaro Moriyama; Chikaya Oeura; Nobuyuki Fukushima  NEUROSCIENCE RESEARCH  61-  S111  -S111  2008
• Molecular mechanism of LPA-induced neurite branch formation

  Nobuyuki Fukushima; Masayuki Yamane; Daisuke Furuta; Ryutaro Moriyama  NEUROSCIENCE RESEARCH  61-  S91  -S91  2008
• Palmitic acid regulates GnRH-receptor mRNA expression level in the mouse gonadotrope

  Deura Chikaya; Fukusima Nobuyuki; Moriyama Ryutaro  The Journal of Reproduction and Development Supplement  101-  (0)  501  -501  2008
• LPA3を介した神経突起分岐促進作用とその細胞内情報伝達機構

  福嶋伸之; 福嶋伸之; 山根昌之; 古田大助; 徳永亮; 森山隆太郎  生化学  2007
• PDZ結合ドメインを欠損したリゾホスファチジン酸受容体の性質

  車野慎哉; 畑中一貴; 二瀬慎介; 森山隆太郎; 辻内俊文; 辻内俊文; 福嶋伸之; 福嶋伸之; 福嶋伸之  生化学  2007
• Palmitate-induced increase in long-chain fatty acid receptor GPR120 mRNA level in L beta T2 pituitary gonadotrope cells

  Ryutaro Moriyama; Kazuhiro Nose; Azusa Kohama; Nobuyuki Fukushima  NEUROSCIENCE RESEARCH  58-  S223  -S223  2007
• Lysophosphatidic acid stimulates astrocyte proliferation through LPA(1)

  Yuki Sogo; Shano Shinya; Ryutaro Moriyama; Jerold Chun; Nobuyuki Fukushima  NEUROSCIENCE RESEARCH  58-  S197  -S197  2007
• Lysophosphatidic acid stimulates neuronal differentiation through LPA

  Shinsuke Ninose; Shinya Shano; Ryutaro Moriyama; Jerold Chun; Nobuyuki Fukushima  NEUROSCIENCE RESEARCH  58-  S143  -S143  2007
• Lysophosphatidic acid receptor 3 mediates neuritogenesis and branch formation in pheochromocytomal 12 cells

  Nobuyuki Fukushima; Masumi Nakashima; Ryon Tokunaga; Ryutaro Moriyama  NEUROSCIENCE RESEARCH  55-  S183  -S183  2006
• Fasting induced Long-chain fatty acid receptor GPR120 expression in the anterior pituitary of mouse

  Ryutaro Moriyama; Shingo Imoto; Shinya Shano; Nobuyuki Fukushima  NEUROSCIENCE RESEARCH  55-  S226  -S226  2006
• LPA in neural cell development

  Nobuyuki Fukushima  Journal of Cellular Biochemistry  92-  (5)  993  -1003  2004
• リゾフォスファチジン酸と神経系細胞のダイナミクス制御

  福嶋 伸之  神経化学  42-  (1)  61  -73  2003/03
• Neurobiology of lysophosphatidic acid signaling

  N Fukushima; XQ Ye; J Chun  NEUROSCIENTIST  8-  (6)  540  -550  2002/12
• Lysophosphatidic acid in neural signaling

  XQ Ye; N Fukushima; MA Kingsbury; J Chun  NEUROREPORT  13-  (17)  2169  -2175  2002/12
• Lysophospholipid receptors (vol 41, pg 507, 2001)

  N Fukushima; Ishii, I; JJA Contos; JA Weiner; J Chun  ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY  42-  VII  -VII  2002
• A family of lysophospholipid receptors

  FUKUSHIMA Nobuyuki  生化学  72-  (9)  1176  -1179  2000/09
• APOPTOSIS-INHIBITORY FACTORS, AIF-20 AND AIF-22, AND ITS SIGNAL TRANSDUCTION

  FUKUSHIMA Nobuyuki; UEDA Hiroshi  神経化学  35-  (3)  230  -231  1996/09
• 神経系の受容体とリン酸化

  福嶋 伸之; 植田 弘師  最新医学  51-  (7)  1243  -1251  1996/07
• Inositol 1,4,5-trisphosphate-gated calcium transport through plasma membranes in nerve terminals

  H Ueda; S Tamura; N Fukushima; T Katada; M Ui; M Satoh  JOURNAL OF NEUROSCIENCE  16-  (9)  2891  -2900  1996/05
• OPIOID RECEPTOR COUPLING TO GI PHOSPHOLIPASE-C ACTIVATION IN XENOPUS OOCYTES AND HOMOLOGOUS DESENSITIZATION MECHANISMS THROUGH PHOSPHORYLATION

  H UEDA; T MIYAMAE; N FUKUSHIMA; Y MISU  REGULATORY PEPTIDES  54-  (1)  305  -306  1994/11
• INOSITOL 1,4,5-TRISPHOSPHATE ACTIVATES CA2+ CHANNELS IN THE PLASMA-MEMBRANES OF RAT-BRAIN NERVE-TERMINALS

  M SATOH; H UEDA; S TAMURA; Y YOSHIHARA; N FUKUSHIMA  NEURORECEPTOR MECHANISMS IN BRAIN  287-  97  -110  1991
• INOSITOL 1,4,5-TRISPHOSPHATE ACTIVATES CA-2+ CHANNELS IN THE PLASMA-MEMBRANES OF RAT-BRAIN NERVE-TERMINALS

  M SATOH; H UEDA; S TAMURA; N FUKUSHIMA  EUROPEAN JOURNAL OF PHARMACOLOGY  183-  (3)  765  -765  1990/07
• KYOTORPHIN INDUCES MET-ENKEPHALIN FROM SYNAPTOSOMES VIA TRANSLOCALIZING MEMBRANE-BOUND CA-2+ INTO THE CYTOSOLIC SPACE

  H UEDA; Y YOSHIHARA; N FUKUSHIMA; H TAKAGI  FOLIA PHARMACOLOGICA JAPONICA  88-  (1)  P7  -P7  1986/07
• PURIFICATION OF MET-ENKEPHALIN CONVERTASE AND ITS ACTIVATION BY CAMP DEPENDENT KINASE

  N FUKUSHIMA; H UEDA; H TAKAGI  JAPANESE JOURNAL OF PHARMACOLOGY  40-  P79  -P79  1986
• EVIDENCE FOR THE PRESENCE OF PRESYNAPTIC AUTOREGULATION OF MET-ENKEPHALIN IN THE BRAIN AND EFFECTS OF KAPPA-OPIOID ANTAGONISTS THEREON

  H UEDA; N FUKUSHIMA; H TAKAGI  JAPANESE JOURNAL OF PHARMACOLOGY  40-  P80  -P80  1986

Awards & Honors

• 日本神経化学会奨励賞（2002）
  

Research Grants & Projects

• 細胞外脂肪酸によるがん細胞のグルタミン代謝制御機構の解明と健康科学への発展

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  Date (from‐to) : 2022/04 -2025/03 

  Author : 福嶋 伸之
• オレイン酸によるがん細胞増殖機構を解明し健康科学に発展させる基盤研究

  日本学術振興会：科学研究費助成事業 基盤研究

  Date (from‐to) : 2019/04 -2022/03 

  Author : 福嶋 伸之
• Neurodevelopmental toxicities of chemicals are related to an increased microglial activation and neuroinflammation in the neocortex

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2017/04 -2020/03 

  Author : NAGAO Tetsuji

   

  Microglia are the immune cells of the central nervous system (CNS), play a critical role in its development and are involved in neural stem cells proliferation, differentiation, and migration in the developing CNS. To evaluate the microglial morphology and the gene expression of pro-inflammatory cytokines in the neocortex, mice were exposed to BPA or VPA at the critical period. Offspring exposed showed an increased number of amoeboid-type microglia, a microglial differentiation disruption, and an abnormal expression of genes encoding pro-inflammatory factors. The abnormalities induced by VPA exposure were ameliorated by co-treatment with anti-inflammatory drug. The findings suggest that the cytoarchitecture of the neocortex can be restored via treatment with anti-inflammatory agents that target microglial abnormalities and neuroinflammation. Anti-inflammatory therapeutics may provide a novel preventive approach for the abnormalities after the exposure to chemicals.
• Functional analysis of LPA receptors in the acquisition of malignant properties in cancer cells.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2015/03 

  Author : TSUJIUCHI Toshifumi; HONOKI Kanya; FUKUSHIMA Nobuyuki

   

  Lysophosphatidic acid (LPA) is one of extracellular physiological lipids and interacts with G protein-coupled transmembrane LPA receptors (LPA receptor-1 (LPA1) to LPA6). LPA receptors regulate a variety of biological functions through the binding of LPA, such as cell proliferation, migration, morphogenesis and differentiation. In this study, to assess an involvement of LPA receptors in the acquisition of malignant potency, we generated LPA receptor knockdown cells and measured cell motile and invasive activities, tumorigenicity and angiogenic activity of cancer cells. The present results demonstrate that each LPA receptor acts as a positive or negative regulator of tumor progression, dependent on types of cancer cells. Therefore, it is suggested that LPA signaling via LPA receptors may be a target molecule for the establishment of chemoprevention agents in clinical cancer therapy.
• 神経内分泌系におけるLPAシグナルの役割解明に向けた基盤研究

  日本学術振興会：科学研究費補助金

  Date (from‐to) : 2013/04 -2015/03 

  Author : 福嶋 伸之
• リゾホスファチジン酸受容体のシグナル異常とがん

  日本学術振興会：学術研究助成基金助成金

  Date (from‐to) : 2012/04 -2015/03 

  Author : 福嶋 伸之
• リゾホスファチジン酸受容体3を介した神経突起分岐形成の分子機構解明

  日本学術振興会：科学研究費補助金

  Date (from‐to) : 2009/04 -2012/03 

  Author : 福嶋 伸之
• 神経発生期の遺伝子不安定性制御破綻と神経系疾患発症との関連解明に向けた基盤研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2004 -2005 

  Author : 福嶋 伸之

   

  神経発生は分裂能力を有する神経幹細胞が非分裂細胞である神経細胞に分化する基本的生命現象である。最近遺伝子非相同組み換えに関わる分子が神経発生時になんらかの役割を果たしていることが示された。このことは神経発生時においても遺伝子が積極的に不安定(DNA2重鎖の切断[DNA double strand break/DSB]および修復/再会合)となる可能性を示唆している。これらの遺伝子のうちいくつかは神経疾患の原因遺伝子であることから遺伝子不安定性の制御破綻が神経疾患の発症に関連していることをも示している。またある種の内分泌撹乱物質が遺伝子の不安定化に影響する可能性も示唆されている。 昨年度の研究より、マウス胎仔脳におけるDSB修復にはDNAリガーゼ活性だけでなくDNAポリメラーゼ活性も関わっていること、大脳皮質由来の培養神経細胞の生存率はDNA再結合活性と相関していること、が示された。用いたDNA再結合活性の測定法は半定量的であったので、本年度では、定量的かつ高感度な方法の開発を試みた。96ウエルプレートに2本鎖DNAオリゴヌクレオチドを固相化し、続いてビオチン標識2本差DNAおよびT4DNAリガーゼを添加した。16℃で18時間反応させた後、結合したビオチン標識DNA量をアビジン-ビオチン-パーオキシダーゼ複合体(ABC)および発色試薬TMBを用いて測定した。バックグランドのさらなる低減化が課題であるが、6Weiss unitの検出が可能であった。今後、細胞や組織抽出液を用いてDNA再結合活性を比較検討する。 また代表的な内分泌撹乱物質(ビスフェノール、ノニルフェノール)の神経発生に及ぼす効果を調べた。これらの物質をあらかじめPheochromocytoma12細胞に暴露すると、細胞死を誘導することなく神経成長因子による神経分化を阻害することが分かった。今後、これらの物質の暴露によりDSB生成やDSB修復が影響を受けているかどうかについて検討する。
• 神経回路形成時の微小管ダイナミクスに対する脂質メディエーターの効果

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2003 -2004 

  Author : 福嶋 伸之

   

  リゾフォスファチジン酸(LPA)は多様な生理活性を引き起こす細胞外脂質メディエーターである。これまでLPAは幼若神経細胞の神経突起を一時的に退縮させることが示されている。この反応は微小管を安定化させるタクソールでは影響を受けないことがわかった。さらにLPA処理前後において細胞骨格画分におけるチューブリン量に変動がなかったことから、LPAの微小管に対する作用は脱重合の促進ではないと考えられた。むしろLPAは重合微小管の細胞体への輸送を促進していると思われる。さらにLPAによる微小管再編成には重合アクチンが必要であること、少なくとも部分的に微小管アクチン架橋因子(Microtubule actin-crosslinking factor/MACF)が関わっていることが示された。一方、微小管を構成するチューブリンは様々な様式の翻訳後修飾(チロシン化、アセチル化、ポリグルタミン酸化など)を受けることが知られているが、LPA処理によりいずれのチューブリンも再編成されることがわかった。これらのうち、ポリグルタミン酸化チューブリンは他のチューブリンとは異なりアクチンと共存していることが初めて示された。この種のチューブリンは細胞の運動性などに関与していると考えられるため、アクチンとの相互作用を介して細胞形態や運動を制御している可能性が考えられる。実際にポリグルタミン酸化されない変異体チューブリンを神経細胞内に導入すると神経細胞形態が異常になることが示された。今後これらの点を詳細に検討し、細胞外メディエーターによる細胞骨格制御がどのように神経回路形成に影響しているかについて明らかにする。

Others

• 2015/04 -2017/03  ゲノム編集技術を活用した細胞特異的遺伝子欠損メダカの創出とその解析 

  近畿大学学内研究助成金 研究種目；21世紀研究開発奨励金（共同研究助成金） 課題番号；KD02 研究内容；ゲノム編集技術により細胞特異的な脂質受容体欠損メダカを作製する。

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