1994 Fiscal Year Annual Research Report

平滑筋細胞分化過程における分子生物学的解析

Research Project

Project/Area Number	05454157
Research Institution	Osaka University
Principal Investigator	祖父江憲治大阪大学, 医学部, 教授 (20112047)
Co-Investigator(Kenkyū-buntansha)	林謙一郎大阪大学, 医学部, 助手 (90238105) 乾誠大阪大学, 医学部, 助教授 (70223237)
Keywords	平滑筋細胞 / 平滑筋細胞形質転換 / アクチン-ミオシン相互作用 / カルデスモン / 接着因子 / プロモーター / 転写制御
Research Abstract	本研究は平滑筋及び非筋細胞に広範に分布するアクトミオシン系調節蛋白質であるカルデスモン(CaD)の平滑筋細胞の形質転換(分化・脱分化)に伴う高分子型(h‐CaD)と低分子型(l‐CaD)のアイソフォーム間での発現変換と発現量の変化を指標として、初代平滑筋細胞を用いた分化型形質維持システムを確立し、平滑筋細胞分化誘導のメカニズムを解析することを目的として行った。初代平滑筋細胞の培養条件を種々の細胞接着因子を用いて検討を加えた結果、α1β1インテグリンを介した細胞内情報伝達系が平滑筋細胞の脱分化を抑制し、分化型形質を維持させることを見い出した。この培養条件下ではh‐CaDのみならず高分子型トロポミオシン、及びメタビンキュリン等の分化型平滑筋細胞の分子指標となる蛋白質の発現も認められた。また、分化した平滑筋細胞の接着構造(デンスメンブレン)に、α1β1インテグリンが局在することを見い出し、これらの遺伝子群がα1β1インテグリンを介した細胞内情報伝達系の制御下で発現調節されている可能性が示唆された。さらに、この培養システムを用いて、平滑筋細胞におけるCaD遺伝子の転写調節機構の解析を行った。CaDプロモーターの転写活性は分化型平滑筋細胞において極めて高く、培養を重ねた脱分化型平滑筋細胞では前者の70〜40%程度に低下した。また、骨格筋細胞由来のC2C12細胞及びHeLa細胞での転写活性は極低いものであった。これらの結果はそれぞれの細胞でのCaD蛋白質の発現量と一致しており、CaDの組織特異的な発現は転写レベルで制御されていることが明らかになった。(CCAAAAAAGG)に依存しており、この配列と前後6bpを含む配列、CArG1と特異的に結合する核蛋白質が平滑筋細胞核抽出液中に存在することを見出した。以上の結果、このCArG box様配列はCaDの平滑筋特異的な発現を制御している唯一のシス因子であることが明かになった。

Research Products
(21 results)

All Other

All Publications (21 results)

[Publications] Tnaka,J.: "Morphological and biochemical analyses of contractile proteins(actin,myosin,caldesmon and tropomyosin)in normal and transformed cells." J.Cell Sci.104. 595-606 (1993)
[Publications] Tanaka,J.: "Gelsolin is localized in neuronal growth cones." Dev.Brain Res.76. 268-271 (1993)
[Publications] Sobue,K.: "Molecular approach for smooth muscle differentiation:association of caldesmon isoformal interconversion with phenotypic modulation of smooth muscle cells." Biomedical Res.suppl.14. 147-153 (1993)
[Publications] Sobue,K.: "Actin‐based cytoskeleton in growth cone activity" Neurosci.Res.18. 91-102 (1993)
[Publications] Haruna,M.: "Common structural and expressional properties of vertebrate caldesmon genes." Biochemi.Biophys.Res.Commun,. 145-153 (1993)
[Publications] Nakamura,N.: "Rous sercoma virus‐transformed cells develop peculiar adhesive structures along the cell periphery." J.Cell Sci.106. 1057-1069 (1993)
[Publications] Tanaka,J.: "Localization and characterization of gelsolin in nervous tissues‐‐gelsolin is specifically enriched in myelin‐forming cells‐‐" J.Neurosci.14. 1038-1052 (1994)
[Publications] Yano,H.: "Identification of two distinct promoters in the chicken caldesmon gene." Biochemi,Biophys.Res.Commun.201.618-626 (1994)
[Publications] Inui,M.: "Annexin VI binds to a synaptic vesicle protein,synapsin I." J.Neurochem.63. 1917-1923 (1994)
[Publications] Watanabe,T.: "Annexin VI‐binding proteins in brain^*l interaction of annexin VI with a membrane skeletal proteins,calspectin(brain spectrin or fordrin)." J.Biol.Chem.269. 17656-17662 (1994)
[Publications] Kira,M.: "Caldesmon and low Mr isoform of tropomyosin are localized in neuronal growth cones." J.Neurosci.Res.(in press). (1995)
[Publications] Yano,H.: "Transcriptional regulation of the chicken caldesmon gene:activation of gizzard‐type caldesmon promoter a CArG box‐like motif." J.Biol.Chem.(in press). (1995)
[Publications] Yoshida,K.: "Reperfusion of rat heart after brief ischemia induces proteolysis of calspectin(Non‐erythroid spectrin or fodrin)by calpain." Circulation Res.(in press). (1995)
[Publications] Ozawa,K.: "Translocation of cortactin(p80/85)to the actin‐based cytoskeleton during thrombin receptor‐mediated platelet activation." J.Cell Sci.(in press). (1995)
[Publications] Nakamura,N.: "Differential localization of a2,av,b2 and b5 integrins in human osteoclasts:their involvement in cell adhesion and bone resorption." J.Cell Sci.(in press). (1995)
[Publications] Tanaka,J.: "Cell/substratum adhesion and movement of cultured rat astrocytes." Dev.Brain Res.(in press). (1995)
[Publications] Sobue,K.: "Ca2+‐dependent regulation of the neuronal cytoskeleton.in Neuronal Cytoskeleton." CRC Press, 335 (1993)
[Publications] Inui,M.: "Annexin VI‐binding proteins in brain.in Neuronal Cytoskeleton." CRC Press, 335 (1993)
[Publications] Tanaka,J.: "Gelsolin is involved in myelin formation.in Neuronal Cytoskeleton." CRC Press, 335 (1993)
[Publications] Ozawa,K.: "Compartmentation of p60c‐src complex in developing rat forebrain membrane.in Neuronal Cytoskeleton." CRC Press, 335 (1993)
[Publications] Hayashi,K.: "Expressional regulation of caldesmon isoforms in assoceation with phenotypic modulation of smooth muscle cells.in Smooth Muscle Contraction." Japan Sci.Soc.Press, 159 (1995)

1994 Fiscal Year Annual Research Report

平滑筋細胞分化過程における分子生物学的解析

Principal Investigator

祖父江 憲治 大阪大学, 医学部, 教授 (20112047)

Research Products

[Publications] Tnaka,J.: "Morphological and biochemical analyses of contractile proteins(actin,myosin,caldesmon and tropomyosin)in normal and transformed cells." J.Cell Sci.104. 595-606 (1993)

[Publications] Tanaka,J.: "Gelsolin is localized in neuronal growth cones." Dev.Brain Res.76. 268-271 (1993)

[Publications] Sobue,K.: "Molecular approach for smooth muscle differentiation:association of caldesmon isoformal interconversion with phenotypic modulation of smooth muscle cells." Biomedical Res.suppl.14. 147-153 (1993)

[Publications] Sobue,K.: "Actin‐based cytoskeleton in growth cone activity" Neurosci.Res.18. 91-102 (1993)

[Publications] Haruna,M.: "Common structural and expressional properties of vertebrate caldesmon genes." Biochemi.Biophys.Res.Commun,. 145-153 (1993)

[Publications] Nakamura,N.: "Rous sercoma virus‐transformed cells develop peculiar adhesive structures along the cell periphery." J.Cell Sci.106. 1057-1069 (1993)

[Publications] Tanaka,J.: "Localization and characterization of gelsolin in nervous tissues‐‐gelsolin is specifically enriched in myelin‐forming cells‐‐" J.Neurosci.14. 1038-1052 (1994)

[Publications] Yano,H.: "Identification of two distinct promoters in the chicken caldesmon gene." Biochemi,Biophys.Res.Commun.201.618-626 (1994)

[Publications] Inui,M.: "Annexin VI binds to a synaptic vesicle protein,synapsin I." J.Neurochem.63. 1917-1923 (1994)

[Publications] Watanabe,T.: "Annexin VI‐binding proteins in brain^*l interaction of annexin VI with a membrane skeletal proteins,calspectin(brain spectrin or fordrin)." J.Biol.Chem.269. 17656-17662 (1994)

[Publications] Kira,M.: "Caldesmon and low Mr isoform of tropomyosin are localized in neuronal growth cones." J.Neurosci.Res.(in press). (1995)

[Publications] Yano,H.: "Transcriptional regulation of the chicken caldesmon gene:activation of gizzard‐type caldesmon promoter a CArG box‐like motif." J.Biol.Chem.(in press). (1995)

[Publications] Yoshida,K.: "Reperfusion of rat heart after brief ischemia induces proteolysis of calspectin(Non‐erythroid spectrin or fodrin)by calpain." Circulation Res.(in press). (1995)

[Publications] Ozawa,K.: "Translocation of cortactin(p80/85)to the actin‐based cytoskeleton during thrombin receptor‐mediated platelet activation." J.Cell Sci.(in press). (1995)

[Publications] Nakamura,N.: "Differential localization of a2,av,b2 and b5 integrins in human osteoclasts:their involvement in cell adhesion and bone resorption." J.Cell Sci.(in press). (1995)

[Publications] Tanaka,J.: "Cell/substratum adhesion and movement of cultured rat astrocytes." Dev.Brain Res.(in press). (1995)

[Publications] Sobue,K.: "Ca2+‐dependent regulation of the neuronal cytoskeleton.in Neuronal Cytoskeleton." CRC Press, 335 (1993)

[Publications] Inui,M.: "Annexin VI‐binding proteins in brain.in Neuronal Cytoskeleton." CRC Press, 335 (1993)

[Publications] Tanaka,J.: "Gelsolin is involved in myelin formation.in Neuronal Cytoskeleton." CRC Press, 335 (1993)

[Publications] Ozawa,K.: "Compartmentation of p60c‐src complex in developing rat forebrain membrane.in Neuronal Cytoskeleton." CRC Press, 335 (1993)

[Publications] Hayashi,K.: "Expressional regulation of caldesmon isoforms in assoceation with phenotypic modulation of smooth muscle cells.in Smooth Muscle Contraction." Japan Sci.Soc.Press, 159 (1995)

祖父江憲治大阪大学, 医学部, 教授 (20112047)