体内時計の光位相同調とメラトニン出力系の調節メカニズム

• Project/Area Number: 11233202
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: The University of Tokyo
• Principal Investigator: 深田 吉孝 東京大学, 大学院・理学系研究科, 教授 (80165258)
• Co-Investigator(Kenkyū-buntansha): 岡野 俊行 東京大学, 大学院・理学系研究科, 講師 (40272471) ; 森山 芳則 岡山大学, 薬学部, 教授 (10150658)
• Project Period (FY): 1999 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥62,100,000 (Direct Cost: ¥62,100,000); Fiscal Year 2002: ¥15,300,000 (Direct Cost: ¥15,300,000); Fiscal Year 2001: ¥15,300,000 (Direct Cost: ¥15,300,000); Fiscal Year 2000: ¥15,300,000 (Direct Cost: ¥15,300,000); Fiscal Year 1999: ¥16,200,000 (Direct Cost: ¥16,200,000)
• Keywords: 松果体 / 概日リズム / 光応答配列 / サーカディアンリズム / MAPキナーゼ / クリプトクローム / ゼブラフィッシュ / 転写因子 / E4BP4 / 時計遺伝子 / 転写調節 / BMAL / リン酸化 / 概日時計 / メラトニン / シナプス小胞様オルガネラ / グルタミン酸作動系 / 小胞型グルタミン酸トランスポーター / 光受容 / G蛋白質 / 光誘導 / グルタミン酸受容体 / セロトニン

Research Abstract

交付申請書に記載の研究実施計画に沿って研究を遂行し、下記の成果を得た。
1.光依存的転写調節に関わる遺伝子配列(光応答エレメント)を同定するため、光に応答するピノプシン遺伝子のプロモータの解析を行った。その結果、18塩基からなる光応答エレメントを同定することに成功した。また、ゲルシフト解析の結果、この光エレメントに作用する因子は、肝臓等の種々の臓器に発現する転写抑制因子であることがわかった。
2.概日時計発振系におけるMAPキナーゼの生理的役割の解明を目指して、咋年度に続きMAPキナーゼの標的分子を探索した。In vitroでのキナーゼアッセイの結果、BMAL1に加え、クリプトクローム(CRY1およびCRY2)がMAPキナーゼによってリン酸化されることが判明した。転写アッセイに基づく機能解析の結果、MAPキナーゼによるリン酸化に伴ってクリプトクロームの転写抑制能が修飾されることが分かった。そこで、リン酸化部位を特異的に認識する抗体を作製し、このようなリン酸化が生体内でおこっているかどうかを調べた。その結果、培養細胞において強制発現させたCRY1およびCRY2の特定のアミノ酸残基がMAPキナーゼによってリン酸化されることが明らかとなった。
3.エクソロドプシンプロモータは、ゼブラフィッシュにおいて松果体特異的な遺伝子発現を誘導する。このプロモータを利用し、概日時計の発振における時計遺伝子産物(BMAL1およびBMAL2)の機能を明らかにすべく、これらBMALのドミナントネガティブ体を松果体特異的に発現するトランスジェニックゼブラフィッシュを複数系統樹立した。現在、これら個体のメラトニンリズムの解析を進めている。これと並行して、エクソロドプシンプロモータにおいて、松果体特異的な遺伝子発現を誘導するエレメントを決定しPIPE(Pineal Expression Promoting Element)と命名した。

Research Products

• [Publications] Sanada, K: "Mitogen-activated protein kinase phosphorylates and negatively regulates basic helix-loop-helix-PAS transcription factor BMAL1"J.Biol.Chem.. 277. 267-271 (2002)
• [Publications] Takanaka, Y: "A negative regulatory element required for light-dependent pinopsin gene expression"J.Neurosci.. 22. 4357-4363 (2002)
• [Publications] Doi, M: "Light-dependent changes in the chick pineal temperature and the expression of cHsp90α gene : A potential contribution of in vivo temperature change to the photic-entrainment of the chick pineal circadian clock"Zool.Sci.. 19. 633-641 (2002)
• [Publications] Kasahara, T: "Opsin-G11-mediated signaling pathway for photic entrainment of the chicken pineal circadian clock"J.Neurosci.. 22. 7321-7325 (2002)
• [Publications] Hirota, T: "Glucose down-regulates Per1 and Per2 mRNA levels and induces circadian gene expression in cultured rat-1 fibroblasts"J.Biol.Chem.. 277. 44244-44251 (2002)
• [Publications] Asaoka, Y: "Pineal expression-promoting element (PIPE), a cis-acting element, direcs pineal-specific gene expression in zebrafish"Proc.Natl.Acad.Sci.USA.. 99. 15456-15461 (2002)
• [Publications] Shimizu, F: "Purification and immunohistochemical analysis of calcium-binding proteins expressed in the chick pineal gland"J.Pineal Res.. (in press). (2003)
• [Publications] Shimizu, K: "SCOP, a Novel Binding Partner of K-Ras in the Membrane Rafts, Negatively Regulates MAPK Pathway"J.Biol.Chem.. (in press). (2003)
• [Publications] Fukada, Y: "Circadian clock system in the pineal gland"Mol.Neurobiol.. 25. 19-30 (2002)
• [Publications] 深田吉孝: "脳内光受容と体内時計の時刻調節"現代化学. 370. 24-29 (2002)
• [Publications] 岡野俊行: "体内時計の時刻を調節する遺伝子の発見"生物の科学 遺伝. 56. 26-28 (2002)
• [Publications] 深田吉孝: "体内時計とメラトニン合成"現代医療. 34(6). 35-40 (2002)
• [Publications] Hirota, T: "Effect of brefeldin A on melatonin secretion of chick pineal cells"J. Biochem.. 129. 51-59 (2001)
• [Publications] Nakamura, A: "Regulatory mechanism for the stability of the meta II intermediate of pinopsin"J. Biochem.. 129. 329-334 (2001)
• [Publications] Okano, T: "Cloning of mouse BMAL2 and its daily expression profile in the suprachiasmatic nucleus : A remarkable acceleration of Bmal2 sequence divergence after Bmal gene duplication"Neurosci. Lett.. 300. 111-114 (2001)
• [Publications] Hayashi, Y: "Circadian and photic regulation of MAP kinase by Ras-and protein phosphatase-dependent pathways in the chick pineal gland"FEBS Lett.. 491. 71-75 (2001)
• [Publications] Okano, T: "Photoreception and circadian clock system of the chicken pineal gland"Microscopy Reseach and Technique. 53. 72-80 (2001)
• [Publications] Yoshikawa, T: "Immunohistochemical localization of opsins and alpha-subunit of transducin in the pineal complex and deep brain of the Japanese grass lizard, Takydromus tachycdromoides"Zool. Sci.. 18. 325-330 (2001)
• [Publications] Doi, M: "Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene"Proc. Nati. Acad. Sci. USA. 98. 8089-8094 (2001)
• [Publications] Okano, T: "Chicken pineal clock genes : Implication of BMAL2 as a bidirectional regulator in circadian clock oscillation"Genes Cells. 6. 825-836 (2001)
• [Publications] Yamamoto, K: "Chicken pineal Cry genes : Light-dependent up-regulation of eCry1 and cCry2 transcripts"Neurosci. Lett.. 313. 13-16 (2001)
• [Publications] Fukada, Y: "Circadian clock system in the pineal gland"Mol. Neurobiol.. 25. 1-12 (2002)
• [Publications] Sanada, K: "Mitogen-activated protein kinase phosphorylates and negatively regulates basic helix-loop-helix-PAS transcription factor BMAL1"J. Biol. Chem.. 277. 267-271 (2002)
• [Publications] 岡野俊行: "鳥類松果体細胞の概日時計システム"細胞工学. 20(6). 837-842 (2001)
• [Publications] 深田吉孝: "脳内光受容と体内時計の時刻調節"現代化学. 370. 24-29 (2002)
• [Publications] 松田孝彦: "タンパク質の脂質修飾.(分担執筆)シリーズ・バイオサイエンスの新世紀』第4巻「糖と脂質の生物学」(川嵜敏祐・井上圭三編)"共立出版. 220(201-213) (2001)
• [Publications] 深田吉孝: "光を感じる脳〜生物時計の目〜.(分担執筆)『生き物はどのように世界を見ているか』(和田勝編)"学会出版センター. 151(101-124) (2001)
• [Publications] Sanada,K.: "Role of circadian activation of mitogen-activated protein kinase in chicken pineal clock oscillation."J.Neurosci.. 20. 986-991 (2000)
• [Publications] Kojima,D.: "Vertebrate ancient-long opsin : A green-sensitive photoreceptive molecule present in zebrafish deep brain and retinal horizontal cells."J.Neurosci.. 20. 2845-2851 (2000)
• [Publications] Okano,K.: "Diversity of opsin immunoreactivities in the extraretinal tissues of four anuran amphibians."J.Exp.Zool.. 286. 136-142 (2000)
• [Publications] Matsushita,A.: "Colocalization of pinopsin with two types of G-protein α-subunits in the chicken pineal gland."Cell Tissue Res.. 299. 245-251 (2000)
• [Publications] Kasahara,T.: "Rod-type transducin α-subunit mediates a phototransduction pathway in the chicken pineal gland."J.Neurochem.. 75. 217-224 (2000)
• [Publications] Harada,Y.: "Circadian activation of bullfrog retinal mitogen-activated protein kinase associates with oscillator function."J.Biol.Chem.. 275. 37078-37085 (2000)
• [Publications] Wada,Y.: "Phototransduction molecules in the pigeon deep brain."J.Comp.Neurol.. 428. 138-144 (2000)
• [Publications] Moriyama,Y.: "Synaptic-like microvesicles,synaptic vesicle couterparts in endocrine cells, are involved in a novel regulatory mechanism for hormonal synthesis and secretion."J.Exp.Biol.. 203. 117-125 (2000)
• [Publications] Yatsushiro,S.: "Ionotropic glutamate receptors expressed in rat pinealocytes are involved in exocytosis of L-glutamate."J.Neurochem.. 75. 288-297 (2000)
• [Publications] Yatsushiro,S.: "Glutamate receptor subunit δ2 is highly expressed in a novel population of glial-like cells in rat pineal glands in culture."J.Neurochem.. 75. 1115-1122 (2000)
• [Publications] Takeda,M.: "Ionotropic glutamate receptors expressed in human retinoblastoma Y79 cells."Neurosci.Lett.. 294. 97-100 (2000)
• [Publications] Hayashi,M.: "Vacular H^+-ATPase localizad in plasma membranes of malaria parasite cells,Plasmodium falciparum, is in volved in regional acidification of parasaitized erythrocytes."J.Biol.Chem.. 275. 34353-34358 (2000)
• [Publications] Hirota,T.: "Effect of brefeldin A on malatonin secretion of chick pineal cells."J.Biochem.. 129. 51-59 (2001)
• [Publications] Nakamura,A.: "Regulatory mechanism for stability of meta II intermediate of pinopsin."J.Biochem. 129. 329-334 (2001)
• [Publications] Okano,T.: "Cloning of mouse BMAL2 and its daily expression profile in the suprachiasmatic nucleus : a remarkable acceleration of Bmal2 sequence divergence after Bmal gene duplication."Neurosci.Lett.. 300. 111-114 (2001)
• [Publications] Okano,T.: "Photoreceptor and circadian clock system of the chicken pineal gland."Microscopy Research and Technique. (in press). (2001)
• [Publications] Yoshikawa,T.: "Immunohistochemical localization of opsins and alpha-subunit of transducin in the pineal complex and deep brain of the Japanese grass lizard, Takydromus tachydromoides."Zool.Sci.. (in press). (2001)
• [Publications] Hayashi,Y.: "Circadian and photic regulation of MAP kinase by Ras-and protein phosphatase-dependent pathway in the chick pineal gland."FEBS Lett.. (in press). (2001)
• [Publications] Yamada,H.: "Ca^<2+>-dependent exocytosis of L-glutamate by αTC6, clonal mouse pancreatic αcells."Diabetes. (in press). (2001)
• [Publications] 吉川朋子: "脳内光受容分子."脳の科学特集『生物時計』. 22(5). 527-532 (2000)
• [Publications] 深田吉孝: "松果体とサーカディアンリズム."Clinical Neuroscience特集「サーカディアンリズムのしくみと働き」. 18(10). 1147-1149 (2000)
• [Publications] Okano,T.: "Photoreceptors in pineal gland and brain : Cloning, localization and over-expression.(分担執筆)"Methods in Enzymology"on"Vertebrate Phototransduction and the Visual Cycle"(K.Palczewski,ed.)"Academic Press. 849(278-291) (2000)
• [Publications] Matsuda,T.: "Functional analysis of farnesylation and methylation of transducin."Methods in Enzymology"on"Vertebrate Phototransduction and the Visual Cycle"(K.Palczewski,ed.)"Academic Press. 849(465-481) (2000)
• [Publications] 深田吉孝: "光を感じる脳.(分担執筆)『生き物はどのように世界を見ているか』(和田勝 編)"学会出版センター(in press). (2001)
• [Publications] Yamao, M.: "Differentiation of pinopsin-immunoreactive cells in the developing quail pineal organ : in vivo and in vitro immunohistochemical study"Cell Tissue Res.. 296. 667-671 (1999)
• [Publications] Shouno, O.: "Characterization of N-acylation of Goα purified from bovine retinas"NeuroRepot. 10. 2999-3002 (1999)
• [Publications] Nakamura, A.: "Chimeric nature of pinopsin between rod and cone visual pigments"Biochemistry. 38. 14738-14745 (1999)
• [Publications] Mano, H.: "Exo-rhodopsin : A novel rhodopsin expressed in the zebrafish pineal gland"Mol. Brain Res.. 73. 110-118 (1999)
• [Publications] Hayashi, M.: "Vesicular monoamine transporter 1 is responsible for storage of 5-hydroxytryptamine in rat pinealocytes"J. Neurochem.. 73. 2538-2545 (1999)
• [Publications] Ishio, S.: "Hydroxyindole-O-methyltransferase is another target for L-glutamate-evoked inhibition of melatonin synthesis in rat pinealocytes"Brain Res.. 850. 73-78 (1999)
• [Publications] Yatsushiro, S.: "Functional expression of metabotropic glutamate receptor type 5 in rat pinealocytes"Neuroreport. 10. 1599-1603 (1999)
• [Publications] Sanada, K.: "Role of circadian activation of mitogen-activated protein kinase in chicken pineal clock oscillation"J. Neurosci.. 20. 986-991 (2000)
• [Publications] Moriyama, Y.: "Synaptic-like microvesicles, synaptic vesicle counterparts in endocrine cells, are involved in a novel regulatory mechanism for hormonal synthesis and secretion"J. Exp. Biol.. 203. 117-125 (2000)
• [Publications] Okano, K.: "Diversity of opsin immunoreactivities in the extraretianl tissues of four anuran amphibians"J. Exp. Zool.. (in press). (2000)
• [Publications] Matsushita, A.: "Colocalization of pinopsin with two types of G-protein α-subunits in the chicken pineal gland"Cell Tissue Res.. (in press). (2000)
• [Publications] Kojima, D.: "VAL-opsin : A green-sensitive photoreceptive molecule present in zebrafish deep brain and retinal horizontal cells"J. Neurosci.. (in press). (2000)
• [Publications] 深田吉孝: "脂質共有結合による蛋白質機能調節"蛋白質・核酸・酵素増刊号 『脂質の分子生物学と病態生化学』(井上圭三、西島正弘編集). 44(8). 1319-1320 (1999)
• [Publications] 萩原健一: "脂肪酸アシル化よびイソプレニル化による蛋白質機能の調節"蛋白質・核酸・酵素増刊号 『脂質の分子生物学と病態生化学』(井上圭三、西島正弘編集). 44(8). 1337-1345 (1999)
• [Publications] 深田吉孝: "日周リズムと松果体の光受容分子"生体の科学 特集『時間生物学の新たな展開』. 50(3). 221-227 (1999)
• [Publications] 小島大輔: "新規オプシンの同定:ロドプシンファミリーの多様性"細胞工学. 18(8). 1196-1204 (1999)
• [Publications] 岡野俊行: "脳内光レセプター"生物物理. 39(4). 246-249 (1999)
• [Publications] 岡野俊行: "色覚の進化"生物の科学遺伝、特集:色覚のしくみと色覚異常. 53(9). 39-44 (1999)
• [Publications] Kojima, D.: "Non-visual photoreception by a variety of vertebrate opsins (分担執筆) in : "Rhodopsins and Photo-transduction" (J.A.Goode, ed.)"John Wiley & Sons, Chichester, UK (265-279). 306 (1999)
• [Publications] Matsuda, T.: "Functional analysis of farnesylation and methylation of transducin (分担執筆) "Methods in Enzymology" on "Vertebrate Phototransduction and the Visulal Cycle" (K. Palczewski, ed)"Academic Press (in press). (2000)
• [Publications] Okano, T.: "Photoreceptors in pineal glandandbrain : Cloning, localization andover-expression (分担執筆) "Methods in Enzymology" on "Vertebrate Phototransduction and the Visulal Cycle" (K. Palczewski, ed)"Academic Press (in press). (2000)
• [Publications] 深田吉孝: "松果体の光受容(分担執筆) 『生物時計の分子生物学』(海老原史樹文、深田吉孝編)"シュプリンガーフェアラーク東京 (139-150). 201 (1999)
• [Publications] 深田吉孝: "脳内光受容タンパク質ピノプシン(分担執筆) 『光シグナルトランスダクション』(蓮沼仰嗣、木村成道、徳永史生編)"シュプリンガーフェアラーク東京 (146-151). 195 (1999)
• [Publications] 深田吉孝: "視細胞の光信号伝達を担うG蛋白質(分担執筆)『光シグナルトランスダクション』(蓮沼仰嗣、木村成道、徳永史生編)"シュプリンガーフェアラーク東京 (165-171). 195 (1999)
• [Publications] 深田吉孝: "7回膜貫通型/G蛋白質共役レセプター/RGS.『細胞内シグナル伝達』改訂第2版(山本雅偏)"羊土社(62-63). 217 (1999)
• [Publications] 深田吉孝: "脳内光レセプターの多様性(分担執筆)『遺伝子で生物の進化を考える』(第13回「大学と科学」公開シンポジウム組織委員会編)"クバプロ(79-90). 164 (1999)
• [Publications] 深田吉孝: "脳内光受容タンパク質とその役割(分担執筆)、シリーズ・光が拓く生命科学、第1巻:生物の光環境センサー(津田基之編)"共立出版(40-52). 165 (1999)