骨芽細胞のwound healingにおける非受容体型チロシンキナーゼの関与
Src family kinase（SFK）は，細胞の分化，形態形成，運動に関与するなど，多彩な機能をもつ非受容体型チロシンキナーゼである．ノックアウトマウスを用いた実験からSrcは破骨細胞の機能発現に不可欠であることは知られているが，SFKの骨芽細胞における機能はほとんど知られていない．今回，我々は，骨芽細胞の増殖，遊走に対するSFKの関与を明らかにするために，骨芽細胞株MC3T3-E1のwound healingに，SFK阻害剤であるPP2添加が及ぼす影響を検討した．Wound healing assay では，PP2添加によりwound healing は抑制されたが，細胞増殖活性には影...
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| Published in | 日本再生歯科医学会誌 Vol. 4; no. 1; pp. 3 - 14 |
|---|---|
| Main Authors | , , |
| Format | Journal Article |
| Language | Japanese |
| Published |
日本再生歯科医学会
2006
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1348-9615 1880-0815 |
| DOI | 10.11223/jard.4.3 |
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| Abstract | Src family kinase（SFK）は，細胞の分化，形態形成，運動に関与するなど，多彩な機能をもつ非受容体型チロシンキナーゼである．ノックアウトマウスを用いた実験からSrcは破骨細胞の機能発現に不可欠であることは知られているが，SFKの骨芽細胞における機能はほとんど知られていない．今回，我々は，骨芽細胞の増殖，遊走に対するSFKの関与を明らかにするために，骨芽細胞株MC3T3-E1のwound healingに，SFK阻害剤であるPP2添加が及ぼす影響を検討した．Wound healing assay では，PP2添加によりwound healing は抑制されたが，細胞増殖活性には影響しなかった．ウエスタンブロッティング法による細胞内シグナル伝達分子の検討では，PP2添加によりAKT，ERK1/2のリン酸化が阻害された．また，PI3-kinase（PI3K）の阻害剤であるLY294002添加でwound healing は抑制された． 以上のことから，MC3T3-E1の遊走にはSFKとPI3Kが関与することが示唆された． |
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| AbstractList | Src family kinase（SFK）は，細胞の分化，形態形成，運動に関与するなど，多彩な機能をもつ非受容体型チロシンキナーゼである．ノックアウトマウスを用いた実験からSrcは破骨細胞の機能発現に不可欠であることは知られているが，SFKの骨芽細胞における機能はほとんど知られていない．今回，我々は，骨芽細胞の増殖，遊走に対するSFKの関与を明らかにするために，骨芽細胞株MC3T3-E1のwound healingに，SFK阻害剤であるPP2添加が及ぼす影響を検討した．Wound healing assay では，PP2添加によりwound healing は抑制されたが，細胞増殖活性には影響しなかった．ウエスタンブロッティング法による細胞内シグナル伝達分子の検討では，PP2添加によりAKT，ERK1/2のリン酸化が阻害された．また，PI3-kinase（PI3K）の阻害剤であるLY294002添加でwound healing は抑制された． 以上のことから，MC3T3-E1の遊走にはSFKとPI3Kが関与することが示唆された． Src family kinase(SFK)は, 細胞の分化, 形態形成, 運動に関与するなど, 多彩な機能をもつ非受容体型チロシンキナーゼである. ノックアウトマウスを用いた実験からSrcは破骨細胞の機能発現に不可欠であることは知られているが, SFKの骨芽細胞における機能はほとんど知られていない. 今回, 我々は, 骨芽細胞の増殖, 遊走に対するSFKの関与を明らかにするために, 骨芽細胞株MC3T3-E1のwound healingに, SFK阻害剤であるPP2添加が及ぽす影響を検討した. Wound healing assayでは, PP2添加によりwound healingは抑制されたが, 細胞増殖活性には影響しなかった. ウエスタンブロッティング法による細胞内シグナル伝達分子の検討では, PP2添加によりAKT, ERK1/2のリン酸化が阻害された. また, PI3-kkmase(PI3K)の阻害剤であるLY294002添加でwound healingは抑制された. 以上のことから, MC3T3-E1の遊走にはSFKとPI3Kが関与することが示唆された. |
| Author | 堂前, 英資 池尾, 隆 合田, 征司 |
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| References | 13)Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA. Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol 1997; 137: 481-492. 7)Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 2005; 6: 56-68. 4)Ishizawar R, Parsons SJ. c-Src and cooperating partners in human cancer. Cancer Cell 2004; 6: 209-214. 26)Laprise P, Langlois MJ, Boucher MJ, Jobin C, Rivard N. Down-regulation of MEK/ERK signaling by E-cadherin- dependent PI3K/Akt pathway in differentiating intestinal epithelial cells. J Cell Physiol 2004; 199: 32-39. 15)Pfeilschifter J, Wolf O, Naumann A, Minne HW, Mundy GR, Ziegler R. Chemotactic response of osteoblastlike cells to transforming growth factor beta. J Bone Miner Res 1990; 5: 825-830. 24)Imai S, Kaksonen M, Raulo E, Kinnunen T, Fages C, Meng X, Lakso M, Rauvala H. Osteoblast recruitment and bone formation enhanced by cell matrix-associated heparin-binding growth-associated molecule (HB-GAM). J Cell Biol 1998; 143: 1113-1128. 11)Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M, Yamamoto T. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 1995; 377: 539-544. 25)Manara MC, Bernard G, Lollini PL, Nanni P, Zuntini M, Landuzzi L, Benini S, Lattanzi G, Sciandra M, Serra M, Colombo MP, Bernard A, Picci P, Scotlandi K. CD99 acts as an oncosuppressor in osteosarcoma. Mol Biol Cell 2006; 17: 1910-1921. 1)Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L, Tassinari MS, Kennedy MB, Pockwinse S, Lian JB, Stein GS. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1990; 143: 420-430. 28)Salazar EP, Rozengurt E. Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397. J Biol Chem 2001; 276: 17788-17795. 3)Tanaka Y. Inflammatory cytokines for osteoclastogenesis. Nippon Rinsho 2005; 63: 1535-1540. 10)Liu Z, Falola J, Zhu X, Gu Y, Kim LT, Sarosi GA, Anthony T, Nwariaku FE. Antiproliferative effects of Src inhibition on medullary thyroid cancer. J Clin Endocrinol Metab 2004; 89: 3503-3509. 17)Kaplan KB, Swedlow JR, Morgan DO, Varmus HE. c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism. Genes Dev 1995; 9: 1505-1517. 9)Lee M, Kim JY, Koh WS. Apoptotic effect of PP2 a Src tyrosine kinase inhibitor, in murine B cell leukemia. J Cell Biochem 2004; 93: 629-638. 5)Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway in human cancer: rationale and promise. Cancer Cell 2003; 4: 257-262. 6)Nishimoto S, Nishida E. MAPK signalling: ERK5 versus ERK1/2. EMBO Rep 2006; 7: 782-786. 14)Tsukamoto T, Matsui T, Fukase M, Fujita T. Platelet-derived growth factor B chain homodimer enhances chemotaxis and DNA synthesis in normal osteoblast-like cells (MC3T3-E1). Biochem Biophys Res Commun 1991; 175: 745-751. 22)Tilghman RW, Slack-Davis JK, Sergina N, Martin KH, Iwanicki M, Hershey ED, Beggs HE, Reichardt LF, Parsons JT. Focal adhesion kinase is required for the spatial organization of the leading edge in migrating cells. J Cell Sci 2005; 118: 2613-2623. 20)Todaro GJ, Lazar GK, Green H. The initiation of cell division in a contact-inhibited mammalian cell line. J Cell Physiol 1965; 66: 325-333. 19)Klinghoffer RA, Sachsenmaier C, Cooper JA, Soriano P. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J 1999; 18: 2459-2471. 23)Katz RW, Teng SY, Thomas S, Landesberg R. Paracrine activation of extracellular signal-regulated kinase in a simple in vitro model of wounded osteoblasts. Bone 2002; 31: 288-295. 18)Talamonti MS, Roh MS, Curley SA, Gallick GE. Increase in activity and level of pp60c-src in progressive stages of human colorectal cancer. J Clin Invest 1993; 91: 53-60. 16)Parsons SJ, Parsons JT. Src family kinases, key regulators of signal transduction. Oncogene 2004; 23: 7906-7909. 12)Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR. Cell migration: integrating signals from front to back. Science 2003; 302: 1704-1709. 21)Zhao M, Song B, Pu J, Wada T, Reid B, Tai G, Wang F, Guo A, Walczysko P, Gu Y, Sasaki T, Suzuki A, Forrester JV, Bourne HR, Devreotes PN, McCaig CD, Penninger JM. Electrical signals control wound healing through phosphatidylinositol- 3-OH kinase-gamma and PTEN. Nature 2006; 442: 457-460. 2)Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature 2003; 423: 349-355. 27)Fujita T, Azuma Y, Fukuyama R, Hattori Y, Yoshida C, Koida M, Ogita K, Komori T. Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. J Cell Biol 2004; 166: 85-95. 8)Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 1983; 96: 191-198. |
| References_xml | – reference: 2)Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature 2003; 423: 349-355. – reference: 15)Pfeilschifter J, Wolf O, Naumann A, Minne HW, Mundy GR, Ziegler R. Chemotactic response of osteoblastlike cells to transforming growth factor beta. J Bone Miner Res 1990; 5: 825-830. – reference: 17)Kaplan KB, Swedlow JR, Morgan DO, Varmus HE. c-Src enhances the spreading of src-/- fibroblasts on fibronectin by a kinase-independent mechanism. Genes Dev 1995; 9: 1505-1517. – reference: 12)Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR. Cell migration: integrating signals from front to back. Science 2003; 302: 1704-1709. – reference: 7)Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 2005; 6: 56-68. – reference: 8)Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 1983; 96: 191-198. – reference: 24)Imai S, Kaksonen M, Raulo E, Kinnunen T, Fages C, Meng X, Lakso M, Rauvala H. Osteoblast recruitment and bone formation enhanced by cell matrix-associated heparin-binding growth-associated molecule (HB-GAM). J Cell Biol 1998; 143: 1113-1128. – reference: 11)Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M, Yamamoto T. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 1995; 377: 539-544. – reference: 18)Talamonti MS, Roh MS, Curley SA, Gallick GE. Increase in activity and level of pp60c-src in progressive stages of human colorectal cancer. J Clin Invest 1993; 91: 53-60. – reference: 21)Zhao M, Song B, Pu J, Wada T, Reid B, Tai G, Wang F, Guo A, Walczysko P, Gu Y, Sasaki T, Suzuki A, Forrester JV, Bourne HR, Devreotes PN, McCaig CD, Penninger JM. Electrical signals control wound healing through phosphatidylinositol- 3-OH kinase-gamma and PTEN. Nature 2006; 442: 457-460. – reference: 5)Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway in human cancer: rationale and promise. Cancer Cell 2003; 4: 257-262. – reference: 6)Nishimoto S, Nishida E. MAPK signalling: ERK5 versus ERK1/2. EMBO Rep 2006; 7: 782-786. – reference: 19)Klinghoffer RA, Sachsenmaier C, Cooper JA, Soriano P. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J 1999; 18: 2459-2471. – reference: 22)Tilghman RW, Slack-Davis JK, Sergina N, Martin KH, Iwanicki M, Hershey ED, Beggs HE, Reichardt LF, Parsons JT. Focal adhesion kinase is required for the spatial organization of the leading edge in migrating cells. J Cell Sci 2005; 118: 2613-2623. – reference: 10)Liu Z, Falola J, Zhu X, Gu Y, Kim LT, Sarosi GA, Anthony T, Nwariaku FE. Antiproliferative effects of Src inhibition on medullary thyroid cancer. J Clin Endocrinol Metab 2004; 89: 3503-3509. – reference: 4)Ishizawar R, Parsons SJ. c-Src and cooperating partners in human cancer. Cancer Cell 2004; 6: 209-214. – reference: 16)Parsons SJ, Parsons JT. Src family kinases, key regulators of signal transduction. Oncogene 2004; 23: 7906-7909. – reference: 9)Lee M, Kim JY, Koh WS. Apoptotic effect of PP2 a Src tyrosine kinase inhibitor, in murine B cell leukemia. J Cell Biochem 2004; 93: 629-638. – reference: 3)Tanaka Y. Inflammatory cytokines for osteoclastogenesis. Nippon Rinsho 2005; 63: 1535-1540. – reference: 23)Katz RW, Teng SY, Thomas S, Landesberg R. Paracrine activation of extracellular signal-regulated kinase in a simple in vitro model of wounded osteoblasts. Bone 2002; 31: 288-295. – reference: 28)Salazar EP, Rozengurt E. Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397. J Biol Chem 2001; 276: 17788-17795. – reference: 27)Fujita T, Azuma Y, Fukuyama R, Hattori Y, Yoshida C, Koida M, Ogita K, Komori T. Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling. J Cell Biol 2004; 166: 85-95. – reference: 26)Laprise P, Langlois MJ, Boucher MJ, Jobin C, Rivard N. Down-regulation of MEK/ERK signaling by E-cadherin- dependent PI3K/Akt pathway in differentiating intestinal epithelial cells. J Cell Physiol 2004; 199: 32-39. – reference: 14)Tsukamoto T, Matsui T, Fukase M, Fujita T. Platelet-derived growth factor B chain homodimer enhances chemotaxis and DNA synthesis in normal osteoblast-like cells (MC3T3-E1). Biochem Biophys Res Commun 1991; 175: 745-751. – reference: 1)Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L, Tassinari MS, Kennedy MB, Pockwinse S, Lian JB, Stein GS. Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 1990; 143: 420-430. – reference: 20)Todaro GJ, Lazar GK, Green H. The initiation of cell division in a contact-inhibited mammalian cell line. J Cell Physiol 1965; 66: 325-333. – reference: 25)Manara MC, Bernard G, Lollini PL, Nanni P, Zuntini M, Landuzzi L, Benini S, Lattanzi G, Sciandra M, Serra M, Colombo MP, Bernard A, Picci P, Scotlandi K. CD99 acts as an oncosuppressor in osteosarcoma. Mol Biol Cell 2006; 17: 1910-1921. – reference: 13)Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA. Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol 1997; 137: 481-492. |
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| Snippet | Src family... Src family kinase(SFK)は, 細胞の分化, 形態形成, 運動に関与するなど, 多彩な機能をもつ非受容体型チロシンキナーゼである. ノックアウトマウスを用いた実験からSrcは破骨細胞の機能発現に不可欠であることは知られているが, SFKの骨芽細胞における機能はほとんど知られていない. 今回,... |
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| SubjectTerms | PI3-kinase Src family kinase wound healing 骨芽細胞 |
| Title | 骨芽細胞のwound healingにおける非受容体型チロシンキナーゼの関与 |
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