1995 Fiscal Year Annual Research Report

アポトーシス誘導の膜脂質情報変換とその分子機構

Research Project

Project/Area Number	07457036
Research Category	Grant-in-Aid for General Scientific Research (B)
Research Institution	Gifu University
Principal Investigator	野澤義則岐阜大学, 医学部, 教授 (10021362)
Co-Investigator(Kenkyū-buntansha)	坂野喜子岐阜大学, 医学部, 助手 (50116852) 中島茂岐阜大学, 医学部, 講師 (60188935)
Keywords	アポトーシス / C2セラミド / ホスホリパーゼ / 情報変換系
Research Abstract	TNF(腫瘍壊死因子)によるアポトーシス誘発に各種膜脂質情報変換酵素(PLA2,PI-PLC,PC-PLC,PLD,SMアーゼ)が関与しているとされているが、PLA_2,PI-PLC(イノシトールリン脂質特異性PLC)を除いて、他の酵素の活性調節機構はほとんど明らかにされていない。なかでも、アポトーシスで重要視されているSMアーゼに関してはごく限られた知見しかない。そこで、本年度はアポトーシスを引き起こすことが知られているセラミド(新規セカンドメッセンジャー)の下流シグナルについてラット好塩基球性白血病細胞(RBL)を用いて詳しく検討した。膜透過性C2セラミド(合成品)を用い、SMaseで生じたセラミドの下流シグナルを明らかにするために、RBL細胞に抗原刺激を行いホスホリパーゼD(PLD)の活性化におけるセラミドの影響について検討した。パルミチン酸標識したRBL細胞をC2セラミドで前処理し、抗原刺激を行うと対照に比してPLD活性化により産生されるホスファチジルブタノール(PBut)の量が著しく減少したこの原因を明らかにするために、抗原刺激によるPLDの活性化に関与する因子について、特にCa^<2+>の上昇、プロテインキナーゼC(PKC)の活性化について調べたところ、細胞内カルシウムの上昇が阻止されていることが細胞内カルシウム濃度の測定により分かった。細胞内カルシウムの上昇はイノシトール代謝によるIP3によることが明らかにされているが、C2セラミドによりIP3産生は影響なく、細胞外からの流入の阻害によるものであることが判明した。またPLDの活性化因子としてPKCの活性化(細胞膜へのトランスロケーション)が明らかにされており、C2セラミドによる各種PKCアイソザイムの膜へのトランスロケーションの影響を調べたところ、PKCαのトランスロケーションが特異的に阻害されることがわかり、これがPLD活性化の阻害の原因であることが判明した。以上の結果は、C2セラミドの下流シグナルにPLD活性化によるシグナル連携が関与していることを示唆するものであり、今後アポトーシスとPLD活性化についてさらに検討する。

Research Products

(16 results)

All Other

All Publications (16 results)

[Publications] Alam S.: "Defective phospholipase Dactivation in Ki-ras-tansformed NIH3T3 cells: Evidence for downstream effect of PLC-γ1" Biochem. Biophys. Res. Commun.207. 460-466 (1995)
[Publications] Banno Y.: "Endogenous cleavage of phospholipase Cβ3 by agonist-induced activation of calpain in human platelets" J. Biol. Chem.270. 4318-4323 (1995)
[Publications] Nakamura Y.: "Effects of an anti allergic drug, permirolast potassium on tyrosine phosphorylation and MAP kinase activation in antigen-stimulated rat basophilic leukemia(RBL-2H3) cells" Jpn. J. Allergol.44. 34-44 (1995)
[Publications] Nagata K.: "Possible interaction of hemoglobin with a low Mr GTP-binding protein ram p25" Biochem. Mol. Biol. Int.35. 507-515 (1995)
[Publications] Fujita H.: "Functions of mutant gelsolin, His 321 isolated from a flat revertant of Ras-transformed cells" Eur. J. Biochem.229. 615-620 (1995)
[Publications] Nagata K.: "A low molecular GTP-binding protein, Rapl, in human platelets: Localization, translocation and phosphorylation by cyclic AMP-dependent protein kinase" Brit. J. Haematol.90. 180-186 (1995)
[Publications] Ohguchi K.: "Activation of membrane-bound phospholipase D by protein kinase C in HL-60 cells: Synergistic action of a small GTP-binding protein RhoA" Biochem. Biophys. Res. Commun.211. 306-311 (1995)
[Publications] Nakashima S.: "Delection and site-directed mutagenesis of EF-hand domain of phospholipase C-delta 1: Effects on its activity" Biochem. Biophys. Res. Commun.211. 364-369 (1995)
[Publications] Adachi T.: "Roles of prostaglandin production and MAP kinase activation in hepatocyte growth factor-meiated rat hepatocyte proliferation" Hepatology. 21. 1668-1674 (1995)
[Publications] Kumada T.: "Antigen-mediated phospholipase D activation in rat basophilic leukemia (RBL-2H3) cells: Involvement of calcium/calmodulin" Biochem. Biophys. Acta. 1258. 107-114 (1995)
[Publications] Liu B.: "Implication of protein kinase C α in PAF-stimulated phospholipase D (PLD) activation in Chinese hamster ovary (CHO) cells expressing PAF receptor" Biochem. Biophys. Res. Commun.214. 418-423 (1995)
[Publications] Nagata K.: "Identification of heterotrimeric GTP-binding Proteins in human megakaryoblastic leukemia cell, MEG-O1, and their alteration during cellular differentiation" Life Sci.57. 1675-1681 (1995)
[Publications] Nakamura Y.: "C2-ceramide inhibits IgG-mediated phospholipase D but not C activation in rat basophilic leukemia(RBL-2H3) cells" J.Immunol.156. 256-262 (1996)
[Publications] Ohguchi K.: "Regulation of membrane-bound phospholipase D by Protein kinase C in HL-60 cells: Synergistic action of small GTP-binding protein RhoA" J. Biol. Chem.271. 4366-4373 (1996)
[Publications] Nagata K.: "Identification and localization of gane expression of a low Mr GTP-binding protein, Ram p25 in pituitary gland" Biochem. Biophys. Res. Commun.217. 1223-1230 (1996)
[Publications] Nakashima S.: "Platelet phospholipase C, D. Handbook of Experimental Pharmacology,“Platelets and their factors"(Bruchhausen, V. and Walter, U., eds.)" Springer-Verlag, Berlin, Heiderberg(in press), (1996)

1995 Fiscal Year Annual Research Report

アポトーシス誘導の膜脂質情報変換とその分子機構

Principal Investigator

野澤 義則 岐阜大学, 医学部, 教授 (10021362)

Research Products

[Publications] Alam S.: "Defective phospholipase Dactivation in Ki-ras-tansformed NIH3T3 cells: Evidence for downstream effect of PLC-γ1" Biochem. Biophys. Res. Commun.207. 460-466 (1995)

[Publications] Banno Y.: "Endogenous cleavage of phospholipase Cβ3 by agonist-induced activation of calpain in human platelets" J. Biol. Chem.270. 4318-4323 (1995)

[Publications] Nakamura Y.: "Effects of an anti allergic drug, permirolast potassium on tyrosine phosphorylation and MAP kinase activation in antigen-stimulated rat basophilic leukemia(RBL-2H3) cells" Jpn. J. Allergol.44. 34-44 (1995)

[Publications] Nagata K.: "Possible interaction of hemoglobin with a low Mr GTP-binding protein ram p25" Biochem. Mol. Biol. Int.35. 507-515 (1995)

[Publications] Fujita H.: "Functions of mutant gelsolin, His 321 isolated from a flat revertant of Ras-transformed cells" Eur. J. Biochem.229. 615-620 (1995)

[Publications] Nagata K.: "A low molecular GTP-binding protein, Rapl, in human platelets: Localization, translocation and phosphorylation by cyclic AMP-dependent protein kinase" Brit. J. Haematol.90. 180-186 (1995)

[Publications] Ohguchi K.: "Activation of membrane-bound phospholipase D by protein kinase C in HL-60 cells: Synergistic action of a small GTP-binding protein RhoA" Biochem. Biophys. Res. Commun.211. 306-311 (1995)

[Publications] Nakashima S.: "Delection and site-directed mutagenesis of EF-hand domain of phospholipase C-delta 1: Effects on its activity" Biochem. Biophys. Res. Commun.211. 364-369 (1995)

[Publications] Adachi T.: "Roles of prostaglandin production and MAP kinase activation in hepatocyte growth factor-meiated rat hepatocyte proliferation" Hepatology. 21. 1668-1674 (1995)

[Publications] Kumada T.: "Antigen-mediated phospholipase D activation in rat basophilic leukemia (RBL-2H3) cells: Involvement of calcium/calmodulin" Biochem. Biophys. Acta. 1258. 107-114 (1995)

[Publications] Liu B.: "Implication of protein kinase C α in PAF-stimulated phospholipase D (PLD) activation in Chinese hamster ovary (CHO) cells expressing PAF receptor" Biochem. Biophys. Res. Commun.214. 418-423 (1995)

[Publications] Nagata K.: "Identification of heterotrimeric GTP-binding Proteins in human megakaryoblastic leukemia cell, MEG-O1, and their alteration during cellular differentiation" Life Sci.57. 1675-1681 (1995)

[Publications] Nakamura Y.: "C2-ceramide inhibits IgG-mediated phospholipase D but not C activation in rat basophilic leukemia(RBL-2H3) cells" J.Immunol.156. 256-262 (1996)

[Publications] Ohguchi K.: "Regulation of membrane-bound phospholipase D by Protein kinase C in HL-60 cells: Synergistic action of small GTP-binding protein RhoA" J. Biol. Chem.271. 4366-4373 (1996)

[Publications] Nagata K.: "Identification and localization of gane expression of a low Mr GTP-binding protein, Ram p25 in pituitary gland" Biochem. Biophys. Res. Commun.217. 1223-1230 (1996)

[Publications] Nakashima S.: "Platelet phospholipase C, D. Handbook of Experimental Pharmacology,“Platelets and their factors"(Bruchhausen, V. and Walter, U., eds.)" Springer-Verlag, Berlin, Heiderberg(in press), (1996)

野澤義則岐阜大学, 医学部, 教授 (10021362)