KINDAI UNIVERSITY


*A space between the first name and last name, please enter

FUKUSHIMA Nobuyuki

Profile

FacultyDepartment of Life Science / Graduate School of Science and Engineering Research
PositionProfessor
Degree
Commentator Guidehttps://www.kindai.ac.jp/meikan/656-fukushima-nobuyuki.html
URL
Mail
Last Updated :2020/09/30

Research Activities

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

  • 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, Biochemical and biophysical research communications, Aug. 2018 , 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, Journal of receptor and signal transduction research, 1 - 5, Aug. 2018 , 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, Journal of receptor and signal transduction research, 38(4), 367 - 371, Aug. 2018 , 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, Biochemical and Biophysical Research Communications, 496(1), 225 - 230, Jan. 29 2018 , Refereed
    Summary: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.
  • 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., Biochem Biophys Res Commun., 483, 652 - 657, 2017 , Refereed
  • Different effects of GPR120 and GPR40 on cellular functions stimulated by 12-O-tetradecanoylphorbol-13- acetate in melanoma cells., FUKUSHIMA Nobuyuki, 475, 25 - 30, Jul. 2016 , Refereed
  • 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, JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION, 32(4), 209 - 213, Aug. 2012 , Refereed
    Summary: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.
  • 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., Biochem Biophys Res Commun., 349(3), 1151 - 1155, Oct. 2006 , Refereed
  • 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, J Toxicol Pathol, 19, 137 - 142, 2006 , Refereed
  • Lysophospholipid receptors: signaling and biology., Ishii I, Fukushima N, Ye X, Chun J, Annu Rev Biochem, Annu Rev Biochem, 73(1), 321-354 - 354, 2004 , Refereed
    Summary: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
  • Lysophospholipid receptors., Fukushima N, Ishii I, Contos JJ, Weiner JA, Chun J, Annu Rev Pharmacol Toxicol, Annu Rev Pharmacol Toxicol, 41(1), 507-534 - 534, 2001 , Refereed
    Summary: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
  • G(i1) and G(oA) differentially determine kinetic efficacies of agonists for ?-opioid receptor, Kohno, M., Fukushima, N., Yoshida, A., Ueda, H., FEBS Letters, FEBS Letters, 473(1), 101 - 105, 2000 , Refereed
    Summary: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, 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, Mol Pharmacol, 58(5), 895-902, 2000 , Refereed
  • 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, Journal of Neurochemistry, 66(2), 845 - 851, 1996 , Refereed
    Summary: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.
  • 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, Neuroscience Letters, 176, 55 - 58, Jul. 18 1994
    Summary:[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, NeuroReport, 5, 1985 - 1988, Jan. 01 1994
    Summary: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, FEBS LETTERS, 333(3), 311 - 314, Nov. 1993
    Summary: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).
  • 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, Neuroscience Letters, 81, 309 - 313, Oct. 1987 , Refereed
    Summary: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.
  • 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, Experimental Cell Research, 369(1), 54 - 60, Aug. 01 2018 , Refereed
    Summary: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.
  • 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, Journal of Receptors and Signal Transduction, 38(1), 71 - 75, Jan. 02 2018 , Refereed
    Summary: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, Experimental Cell Research, 369(2), 316 - 324, 2018 , Refereed
    Summary: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, NeuroReport, 29(9), 712 - 717, 2018 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 493(1), 468 - 473, Nov. 2017 , Refereed
    Summary: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, MOLECULAR AND CELLULAR BIOCHEMISTRY, 431(1-2), 29 - 35, Jul. 2017 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 486(3), 767 - 773, May 2017 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 484(3), 675 - 680, Mar. 2017 , Refereed
    Summary: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, EXPERIMENTAL CELL RESEARCH, 352(1), 139 - 145, Mar. 2017 , Refereed
    Summary: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.
  • 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, MOLECULAR CARCINOGENESIS, 55(11), 1573 - 1583, Nov. 2016 , Refereed
    Summary: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, MOLECULAR CARCINOGENESIS, 55(11), 1553 - 1559, Nov. 2016 , Refereed
    Summary: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.
  • 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, EXPERIMENTAL CELL RESEARCH, 342(2), 193 - 199, Mar. 2016 , Refereed
    Summary: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, MOLECULAR AND CELLULAR BIOCHEMISTRY, 408(1-2), 147 - 154, Oct. 2015 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 465(3), 512 - 515, Sep. 2015 , Refereed
    Summary: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, CELLULAR AND MOLECULAR LIFE SCIENCES, 72(12), 2377 - 2394, Jun. 2015 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 461(1), 59 - 64, May 2015 , Refereed
    Summary: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, MOLECULAR AND CELLULAR BIOCHEMISTRY, 400(1-2), 145 - 151, Feb. 2015 , Refereed
    Summary: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, HISTOCHEMISTRY AND CELL BIOLOGY, 143(1), 21 - 27, Jan. 2015 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 456(1), 135 - 138, Jan. 2015 , Refereed
    Summary: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, GENE, 551(2), 189 - 200, Nov. 2014 , Refereed
    Summary: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, MOLECULAR AND CELLULAR BIOCHEMISTRY, 393(1-2), 17 - 22, Aug. 2014 , Refereed
    Summary: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, CELL BIOLOGY INTERNATIONAL, 38(7), 868 - 874, Jul. 2014 , Refereed
    Summary: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, JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION, 34(3), 149 - 153, Jun. 2014 , Refereed
    Summary: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, JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION, 34(3), 201 - 204, Jun. 2014 , Refereed
    Summary: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, GROWTH FACTORS, 32(3-4), 117 - 122, Jun. 2014 , Refereed
    Summary: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, NEUROSCIENCE LETTERS, 570, 1 - 4, Jun. 2014 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 446(2), 585 - 589, Apr. 2014 , Refereed
    Summary: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, HISTOLOGY AND HISTOPATHOLOGY, 29(3), 313 - 321, Mar. 2014 , Refereed
    Summary: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, MOLECULAR AND CELLULAR BIOCHEMISTRY, 383(1-2), 173 - 177, Nov. 2013 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 441(1), 47 - 52, Nov. 2013 , Refereed
    Summary: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, Journal of Receptors and Signal Transduction, 33(5), 286 - 290, Oct. 2013 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 439(2), 280 - 284, Sep. 2013
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 439(2), 280 - 284, Sep. 2013 , Refereed
    Summary: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, Biochemical and Biophysical Research Communications, 433(3), 317 - 321, Apr. 12 2013 , Refereed
    Summary: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, MOLECULAR CARCINOGENESIS, 52(4), 247 - 254, Apr. 2013 , Refereed
    Summary: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, Lysophospholipid Receptors: Signaling and Biochemistry, 399 - 418, Feb. 19 2013 , 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, EXPERIMENTAL CELL RESEARCH, 319(3), 105 - 112, Feb. 2013 , Refereed
    Summary: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, TUMOR BIOLOGY, 33(6), 1899 - 1905, Dec. 2012 , Refereed
    Summary: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, JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION, 32(6), 328 - 334, Dec. 2012 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 428(1), 105 - 109, Nov. 2012 , Refereed
    Summary: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, TUMOR BIOLOGY, 33(5), 1739 - 1744, Oct. 2012 , Refereed
    Summary: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.
  • 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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 422(2), 339 - 343, Jun. 2012 , Refereed
    Summary: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, MOLECULAR AND CELLULAR NEUROSCIENCE, 50(1), 21 - 34, May 2012 , Refereed
    Summary: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, CANCER LETTERS, 316(1), 91 - 96, Mar. 2012 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 417(2), 790 - 793, Jan. 2012 , Refereed
    Summary: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, Journal of Toxicologic Pathology, 25(3), 225 - 228, 2012 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 414(4), 727 - 732, Nov. 2011 , Refereed
    Summary: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, Journal of Toxicologic Pathology, 24(3), 143 - 148, Oct. 13 2011 , Refereed
    Summary: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, Journal of Toxicologic Pathology, 24(3), 183 - 186, Oct. 13 2011 , Refereed
    Summary: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, ARCHIVES OF TOXICOLOGY, 85(10), 1303 - 1310, Oct. 2011 , Refereed
    Summary: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, CANCER LETTERS, 309(2), 236 - 242, Oct. 2011 , Refereed
    Summary: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.
  • 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, MOLECULAR CARCINOGENESIS, 50(8), 635 - 642, Aug. 2011 , Refereed
    Summary: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, NEUROCHEMISTRY INTERNATIONAL, 59(2), 109 - 113, Aug. 2011 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 405(3), 450 - 454, Feb. 2011 , Refereed
    Summary: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, VIRCHOWS ARCHIV, 457(6), 669 - 676, Dec. 2010 , Refereed
    Summary: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, NEUROSCIENCE LETTERS, 480(2), 154 - 157, Aug. 2010 , Refereed
    Summary: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, Journal of Toxicologic Pathology, 23(1), 63 - 66, Apr. 2010 , Refereed
    Summary: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, PATHOBIOLOGY, 77(5), 278 - 282, 2010 , Refereed
    Summary: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, PATHOBIOLOGY, 77(6), 309 - 314, 2010 , Refereed
    Summary: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, JOURNAL OF NEUROCHEMISTRY, 109(3), 683 - 693, May 2009 , Refereed
    Summary: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.
  • 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, MUTATION RESEARCH-FUNDAMENTAL AND MOLECULAR MECHANISMS OF MUTAGENESIS, 660(1-2), 47 - 50, Jan. 2009 , Refereed
    Summary: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, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 378(3), 424 - 427, Jan. 2009 , Refereed
    Summary: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, JOURNAL OF TOXICOLOGIC PATHOLOGY, 22(1), 89 - 92, 2009 , Refereed
    Summary: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)
  • 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, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 1783(5), 748 - 759, May 2008
    Summary: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.
  • Lysophosphatidic acid stimulates astrocyte proliferation through LPA(1), Shinya Shano, Ryutaro Moriyama, Jerold Chun, Nobuyuki Fukushima, NEUROCHEMISTRY INTERNATIONAL, NEUROCHEMISTRY INTERNATIONAL, 52(1-2), 216 - 220, Jan. 2008
    Summary: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.
  • 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, NEUROCHEMISTRY INTERNATIONAL, 50(2), 302 - 307, Jan. 2007
    Summary: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.
  • 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, YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN, 127, 163 - 165, 2007 , Refereed
    Summary: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.
  • Actomyosin-dependent microtubule rearrangement in lysophosphatidic acid-induced neurite remodeling of young cortical neurons, Nobuyuki Fukushima, Yuka Morita, BRAIN RESEARCH, BRAIN RESEARCH, 1094, 65 - 75, Jun. 2006
    Summary: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.
  • 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, MOLECULAR AND CELLULAR BIOLOGY, 22(19), 6921 - 6929, Oct. 2002 , Refereed
    Summary: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, MOLECULAR BIOLOGY OF THE CELL, 13(8), 2692 - 2705, Aug. 2002 , Refereed
    Summary: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, MOLECULAR AND CELLULAR NEUROSCIENCE, 20(2), 271 - 282, Jun. 2002 , Refereed
    Summary: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, JOURNAL OF NEUROSCIENCE, 21(18), 7069 - 7078, Sep. 2001 , Refereed
    Summary: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, Journal of Biological Chemistry, 276(18), 15208 - 15215, May 04 2001 , Refereed
    Summary: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, PROSTAGLANDINS & OTHER LIPID MEDIATORS, 64(1-4), 21 - 32, Apr. 2001 , Refereed
    Summary: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.
  • Lysophosphatidic acid (LPA) is a novel extracellular regulator of cortical neuroblast morphology, N Fukushima, JA Weiner, J Chun, DEVELOPMENTAL BIOLOGY, DEVELOPMENTAL BIOLOGY, 228(1), 6 - 18, Dec. 2000 , Refereed
    Summary: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, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 97(24), 13384 - 13389, Nov. 2000 , Refereed
    Summary: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.
  • 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, GENE, 227(1), 89 - 99, Feb. 1999 , Refereed
    Summary: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, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 95(11), 6151 - 6156, May 1998 , Refereed
    Summary: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.
  • 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, Biochemical and Biophysical Research Communications, 142, 595 - 602, Jan. 1987 , Refereed
    Summary: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.
  • 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, Neuroscience Letters, 65, 247 - 252, Apr. 1986 , Refereed
    Summary: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, Sole author, Wiley,   2013
  • Cytoskeleton of the Nervous System, Advances in Neurobiology, Microtubules in the Nervous System., Fukushima, N, Sole author, Springer,   2011

Conference Activities & Talks

  • Molecular mechanism of LPA3-mediated axonal branching formation in cultured hippocampal neurons,   2010 12
  • Lysophosphatidic acid receptor 3 activation induces axonal branch formation in cultured hippocampal neurons,   2010 09
  • Palmitate regulates GnRH receptor mRNA expression level in the gonadotrope of the mouse anterior pituitary gland., 14th International Congress of Endocrinology,   2010 03 , 14th International Congress of Endocrinology
  • Lysophosphatidic acid receptor 3 is involved in neurite branching formation in hippocampal neurons,   2009 10
  • LPA induces neurite shape changes through LPA3, PLM2009,   2009 05 , PLM2009
  • Regulation of neurite morphology by LPA, PLM2009,   2009 05 , PLM2009
  • Palmitate regulates GnRH receptor mRNA expression level in the gonadotrope of the mouse pituitary gland.,   2009 05
  • Palmitate regulates GnRH receptor mRNA expression in the gonadotrope of the mouse anterior pituitary,   2008 07
  • Fasting induced increase in long-chain fatty acid receptor GPR120 expression in the gonadotrope of the mouse anterior pituitary., 37th Annual Meeting of Society for Neuroscience,   2007 11 , 37th Annual Meeting of Society for Neuroscience
  • Palmitate-induced increase in long-chain fatty acid receptor GPR120 mRNA level in LβT2 pituitary gonadotrope cells.,   2007 09
  • Fasting induced Long-chain fatty acid receptor GPR120 expression increase in the anterior pituitary of mouse.,   2006 07

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 , https://jglobal.jst.go.jp/detail?JGLOBAL_ID=201802230476896788
  • 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 , http://jglobal.jst.go.jp/public/201302244884897670
  • 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
  • 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 25 , 10.14882/jrds.102.0.1050.0, http://jglobal.jst.go.jp/public/200902234809881965
    Summary:【目的】マウス下垂体の性腺刺激ホルモン産生細胞(ゴナドトロフ)には長鎖脂肪酸受容体G protein-coupled receptor 120 (GPR120)が局在している。これまでに我々は、血中遊離脂肪酸が上昇する絶食時にマウス下垂体の<I>Gpr120</I> mRNA発現量が増加すること、パルミチン酸曝露によりゴナドトロフ株化細胞L&beta;T2で<I>Gpr120</I> mRNA発現量が増加し、<I>GnRH receptor</I> (<I>GnRH-R</I>) mRNA発現量が減少することを報告している(第101回 日本繁殖生物学会)。本研究では、高脂肪食給餌が下垂体の<I>Gpr120</I>、<I>GnRH-R</I>、<I>LH&beta;</I>、<I>FSH&beta;</I> mRNA発現量に与える影響を検討した。【方法】実験には12週齢のICR雄マウスを用いた。飼料に含まれる脂質:炭水化物:タンパク質のエネルギー比率は、10:70:20(コントロール食)と60:20: 20(高脂肪食)だった。1ヶ月間、自由摂食下で飼育した後、下垂体の<I>Gpr120</I>、<I>GnRH-R</I>、<I>LH&beta;</I>、<I>FSH&beta;</I> mRNA発現量をReal-time PCR法で測定した。【結果】1ヶ月間の給餌により高脂肪食群とコントロール群の体重はそれぞれ52.2 &plusmn; 3.4 gと42.2 &plusmn; 0.8 gになった。高脂肪食群ではコントロール群に比べて下垂体の<I>Gpr120</I>、<I>GnRH-R</I>、<I>FSH&beta;</I> mRNA発現量が統計的有意に増加した。また、<I>LH&beta;</I> mRNA発現量もコントロール群に比べて増加する傾向があった。以上の結果から、血中遊離脂肪酸が増加する生理的条件下では、ゴナドトロフの<I>Gpr120</I> mRNA発現量が増加すること、高脂肪食給餌が性腺刺激ホルモン、特にFSHの合成を促進することが示唆された。現在、高脂肪食給餌がマウスの血中LHとFSHのパルス状分泌に影響を与えるか検討している。
  • 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 25 , 10.14882/jrds.102.0.1049.0, http://jglobal.jst.go.jp/public/200902253135496470
  • 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 , http://ci.nii.ac.jp/naid/130007022586
    Summary:【目的】栄養は性腺機能を制御する因子の1つである。本研究ではマウス下垂体の性腺刺激ホルモン産生細胞 (ゴナドトロフ) が性腺機能を制御するエネルギーセンサーとして血糖値を感知すると仮説提唱して実験を行った。本研究の目的は、グルコースの拮抗利用阻害剤である2-deoxy-D-glucose (2DG) によりゴナドトロフの性腺刺激ホルモン放出ホルモン受容体 (GnRH-R) や性腺刺激ホルモン mRNA発現量が低下するか<I>in vitro</I>で検討することにある。【方法】 実験には8週齢のICR雄マウスとゴナドトロフ株化細胞L&beta;T2を用いた。下垂体とL&beta;T2でエネルギーセンシングに重要なAMP活性化プロテインキナーゼ (AMPK)、グルコキナーゼ (GK)、グルコース輸送担体2 (GLUT2)、GLUT4等のmRNAが発現しているかRT-PCR法で測定した。また、L&beta;T2を5mMの2DG に24時間暴露した後、GnRH-R、黄体形成ホルモン&beta;鎖 (LH&beta;)、卵胞刺激ホルモン&beta;鎖 (FSH&beta;) mRNA発現量の変化をreal-time PCR法で測定した。さらに、エネルギーセンサーとして知られるAMPKのリン酸化をwestern blot法で観察した。【結果】下垂体とL&beta;T2にはAMPK、GK、GLUT2、GLUT4等のmRNAが発現していた。また、2DG曝露によりL&beta;T2でAMPKのリン酸化が誘起され、GnRH-RとFSH&beta; mRNA発現量が低下した。しかし、AMPK活性剤であるAICARを投与した実験では、GnRH-RとFSH&beta; mRNA発現量の低下は観察できなかった。以上より、マウス下垂体にはグルコースを含めたエネルギー基質の感知メカニズムが存在すること、また、グルコース利用性の低下はゴナドトロフでGnRH-RとFSH&beta; mRNA発現量を低下させるが、そのメカニズムにAMPKのリン酸化は関与しないことが示唆された。
  • Palmitic acid regulates GnRH-receptor mRNA expression level in the mouse gonadotrope, Deura Chikaya, Hukusima Nobuyuki, Moriyama Ryutaro, The Journal of Reproduction and Development Supplement, 101, 0, 501, 501,   2008 , http://ci.nii.ac.jp/naid/130007022735
    Summary:【目的】長鎖脂肪酸はエネルギー基質であると同時に,シグナル分子としても機能する。これまでにマウス下垂体の性腺刺激ホルモン産生細胞 (ゴナドトロフ) に長鎖脂肪酸受容体G protein-coupled receptor 120 (GPR120) が発現することを明らかとしている。本研究の目的はゴナドトロフにおいて長鎖脂肪酸がGPR120を介して性腺機能を制御するか否か検討することにある。【方法】8週齢のICR雄マウス (点灯6時30分;消灯18時30分),マウス下垂体の初代培養細胞, ゴナドトロフ株化細胞L&beta;T2を用いた。明期にのみ餌を与える時間制限給餌を10日間行い,下垂体の<I>GPR120</I>および<I>GnRH-receptor</I> (<I>GnRH-R</I>) mRNA発現量の日内変動を測定した。ゴナドトロフに対する長鎖脂肪酸の作用を調べるため,下垂体の初代培養細胞とL&beta;T2細胞を用いて,パルミチン酸暴露による<I>GPR120</I>および<I>GnRH-R</I> mRNA発現量の変化を測定した。また,L&beta;T2細胞がパルミチン酸暴露によって受容体を介した細胞活性を起こすか,MAPキナーゼのリン酸化を指標として検討した。【結果】<I>GPR120</I>および<I>GnRH-R</I> mRNA発現量の日内変動を観察した結果, 正常給餌群では明期後半に<I>GPR120</I> mRNA 発現量の増加と,<I>GnRH-R</I> mRNA発現量の減少を観察した。一方,時間制限給餌群では正常給餌群で観察した<I>GPR120</I> mRNA 発現量の増加と<I>GnRH-R</I> mRNA発現量の減少は観察できなかった。パルミチン酸に暴露した下垂体の初代培養細胞とL&beta;T2細胞ではコントロール群に比べて<I>GPR120</I> mRNA 発現量が増加し,<I>GnRH-R</I> mRNA 発現量が減少した。さらに,L&beta;T2細胞においてパルミチン酸暴露後10分をピークとしたERKのリン酸化を検出したが,p38とJNKのリン酸化は検出できなかった。以上より,ゴナドトロフにおいて長鎖脂肪酸は何らかの受容体を介して<I>GnRH-R</I> mRNA発現量を制御することが示唆された。
  • 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
  • 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 , 10.1016/j.neures.2010.07.2177
  • 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
  • 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
  • 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 , 10.1002/jcb.20093
    Summary:Lysophosphatidic acid (LPA) elicits diverse cellular responses through cell surface LPA receptors in nervous system-derived cells and cell lines. The developing nervous system is one of the major loci for LPA receptor expression. Recent studies have also revealed that metabolic pathways of LPA are present in the nervous system. A growing body of literature suggests a crucial role for LPA in neuronal development processes, including neurogenesis, neuronal migration, neuritogenesis, and myelination. © 2004 Wiley-Liss, Inc.
  • Neurobiology of lysophosphatidic acid signaling, N Fukushima, XQ Ye, J Chun, NEUROSCIENTIST, 8, 6, 540, 550,   2002 12 , 10.1177/1073858402238513
    Summary:Lysophosphatidic acid (LPA), a growth factor-like lysophospholipid, induces diverse cellular responses. The identification of the first LPA receptor gene, through studies of neuroproliferative regions within the embryonic cerebral cortex, has led to the classification of a family of at least eight lysophospholipid receptors with diverse roles in organismal development and function. A growing body of literature has identified roles for LPA signaling under physiological and pathological conditions, particularly within the developing nervous system. Here the authors review features of the LPA receptor family and cellular responses of nervous system-derived cells, and discuss developmental and pathological roles for LPA signaling in the nervous system.
  • Lysophosphatidic acid in neural signaling, XQ Ye, N Fukushima, MA Kingsbury, J Chun, NEUROREPORT, 13, 17, 2169, 2175,   2002 12 , 10.1097/01.wnr.0000044217.09266.2d
    Summary:The physiological and pathological importance of lysophosphatidic acid (LPA) in the nervous system is underscored by its presence, as well as the expression of its receptors in neural tissues. In fact, LPA produces responses in a broad range of cell types related to the function of the nervous system. These cell types include neural cell lines, neural progenitors, primary neurons, oligodendrocytes, Schwann cells, astrocytes, microglia, and brain endothelial cells. LPA-induced cell type-specific effects include changes in cell morphology, promotion of cell proliferation and cell survival, induction of cell death, changes in ion conductance and Ca2+ mobilization, induction of pain transmission, and stimulation of vasoconstriction. These effects are mediated through a number of G protein-coupled LPA receptors that activate various downstream signaling cascades. This review provides a current summary of LPA-induced effects in neural cells in vitro or in vivo in combination with our current understanding of the signaling pathways responsible for these effects.
  • 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