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系統識別號 U0026-0812200911165364
論文名稱(中文) 膠原蛋白的物理特性透過lipid raft 誘發細胞伸展、爬行以及ERK1/2活化之探討
論文名稱(英文) Mechanical property of collagen fiber-induced cell spreading and migration are mediated by phosphorylation of ERK 1/2 via lipid raft
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 92
學期 2
出版年 93
研究生(中文) 徐裕智
研究生(英文) Yu-Chih Hsu
學號 s3691406
學位類別 碩士
語文別 英文
論文頁數 39頁
口試委員 指導教授-湯銘哲
召集委員-蔡少正
口試委員-陳鴻震
中文關鍵字 細胞爬行  膠原蛋白凝交的軟硬度  膠原蛋白凝膠 
英文關鍵字 collagen gel  rigidity  ERK  lipid raft 
學科別分類
中文摘要 中文摘要

  對於生物物理訊息如何調控一個細胞的行為仍是很少人瞭解,但是一個有趣的問題。我們將狗腎臟上皮細胞株-Madin-Darby canine kidney (MDCK) 培養在膠原蛋白凝膠、塗有膠原蛋白的培養皿以及一般的培養皿上來研究膠原蛋白纖維的軟硬如何影響細胞生理機能。我們發現膠原蛋白凝膠可以刺激細胞爬行並且有大量的ERK1/2磷酸化的現象產生,而且這樣磷酸化的情況可以維持到八個小時之久,此現象並未在於培養在塗有膠原蛋白的培養皿和培養皿的細胞上出現,由此可知,這種ERK1/2活化和細胞爬行的現象是由基質的軟硬不同所造成的。利用PD98059 和U0126 (MEK1 inhibitors)去抑制軟性基質所引發的ERK1/2磷酸化會造成細胞的伸展能力下降並且抑制細胞的爬行,但不影響細胞在塗有膠原蛋白的培養皿和培養皿的伸展。有趣的是,我們同時發現被磷酸化的ERK1/2可以表現在細胞核以及focal adhesion的位置。表示細胞可以藉由磷酸化的ERK出現在focal adhesion進而造成細胞伸展在軟性基質上。以lipid raft的抑制劑或是caveolin-1siRNA的實驗證明lipid raft是調控ERK1/2磷酸化的主要來源。總而言之,軟性基質可以誘發細胞中的ERK1/2磷酸化藉此促使細胞伸展和爬行於軟性質上,而這種ERK1/2磷酸化主要是從lipid raft來,而不是caveolae。

英文摘要 Abstract
  How mechanical signals affect cell behaviors is a less studied issue. In this study, we cultured Madin-Darby canine kidney (MDCK) cells under different conditions, collagen gel, collagen gel-coated dish and dish, to investigate how rigidity of collagen fiber affected the cellular behaviors. I found that collagen gel induced cell migration and phosphorylation of ERK1/2 within 1 h and the induction could last for 8 h. Because collagen gel coating did not alter ERK 1/2 activity, collagen gel-induced cell migration and activation of ERK1/2 are mediated by physical property, i.e. rigidity. Inhibition of collagen gel-induced ERK1/2 phosphorylation by MEK inhibitors, PD98059 and U0126, resulted in cell round up and reduction of cell migration. Interestingly, the collagen gel-induced ERK1/2 phosphorylation was present in nuclei and focal adhesion site, indicating that ERK1/2 activation leads to cell spreading and migration. Moreover, filipin III, a lipid raft inhibitor, prevented low rigidity-induced ERK1/2 phosphorylation. On the other hand, caveolin-1 siRNA did not block low rigidity-induced cell round up. Taken together, low rigidity of collagen fiber induces activation of ERK1/2 which facilitates cell spreading and migration on collagen gel, which may be mediated by lipid raft, but not caveolae.

論文目次 Contents
誌謝……………………………………………………………3
List of Figures……………………………………………5
Abstract………………………………………………………6
中文摘要………………………………………………………8
Introduction…………………………………………………10
Materials and Methods……………………………………14
Results………………………………………………………20
Discussion……………………………………………………29
References……………………………………………………35
Figures
作者簡歷………………………………………………………52


參考文獻 References
Baron W, Decker L, Colognato H, ffrench-Constant C. 2003. Regulation of integrin growth factor interactions in oligodendrocytes by lipid raft microdomains. Curr Biol 13:151-155.
Brahmbhatt AA, Klemke RL. 2003. ERK and RhoA differentially regulate pseudopodia growth and retraction during chemotaxis. J Biol Chem 278:13016-13025.
Burgos PV, Klattenhoff C, de la Fuente E, Rigotti A, Gonzalez A. 2004. Cholesterol depletion induces PKA-mediated basolateral-to-apical transcytosis of the scavenger receptor class B type I in MDCK cells. Proc Natl Acad Sci U S A 101:3845-3850.
Carragher NO, Westhoff MA, Fincham VJ, Schaller MD, Frame MC. 2003. A novel role for FAK as a protease-targeting adaptor protein: regulation by p42 ERK and Src. Curr Biol 13:1442-1450.
Chiu SJ, Jiang ST, Wang YK, Tang MJ. 2002. Hepatocyte growth factor upregulates alpha2beta1 integrin in Madin-Darby canine kidney cells: implications in tubulogenesis. J Biomed Sci 9:261-272.
Cho KA, Ryu SJ, Park JS, Jang IS, Ahn JS, Kim KT, Park SC. 2003. Senescent phenotype can be reversed by reduction of caveolin status. J Biol Chem 278:27789-27795.
Cukierman E, Pankov R, Stevens DR, Yamada KM. 2001. Taking cell-matrix adhesions to the third dimension. Science 294:1708-1712.
Ferraro JT, Daneshmand M, Bizios R, Rizzo V. 2004. Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures. Am J Physiol Cell Physiol 286:C831-839.
Fincham VJ, James M, Frame MC, Winder SJ. 2000. Active ERK/MAP kinase is targeted to newly forming cell-matrix adhesions by integrin engagement and v-Src. Embo J 19:2911-2923.
Geiger B, Bershadsky A. 2002. Exploring the neighborhood: adhesion-coupled cell mechanosensors. Cell 110:139-142.
Giancotti FG, Ruoslahti E. 1999. Integrin signaling. Science 285:1028-1032.
Howe AK, Aplin AE, Juliano RL. 2002. Anchorage-dependent ERK signaling--mechanisms and consequences. Curr Opin Genet Dev 12:30-35.
Hunger-Glaser I, Salazar EP, Sinnett-Smith J, Rozengurt E. 2003. Bombesin, lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesion kinase phosphorylation at Ser-910: requirement for ERK activation. J Biol Chem 278:22631-22643.
Hynes RO. 2002. Integrins: bidirectional, allosteric signaling machines. Cell 110:673-687.
Ishibe S, Joly D, Zhu X, Cantley LG. 2003. Phosphorylation-dependent paxillin-ERK association mediates hepatocyte growth factor-stimulated epithelial morphogenesis. Mol Cell 12:1275-1285.
Kawamura S, Miyamoto S, Brown JH. 2003. Initiation and transduction of stretch-induced RhoA and Rac1 activation through caveolae: cytoskeletal regulation of ERK translocation. J Biol Chem 278:31111-31117.
Ku H, Meier KE. 2000. Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells. J Biol Chem 275:11333-11340.
Kurzchalia TV, Parton RG. 1999. Membrane microdomains and caveolae. Curr Opin Cell Biol 11:424-431.
Liang CC, Chen HC. 2001. Sustained activation of extracellular signal-regulated kinase stimulated by hepatocyte growth factor leads to integrin alpha 2 expression that is involved in cell scattering. J Biol Chem 276:21146-21152.
Matsubayashi Y, Ebisuya M, Honjoh S, Nishida E. 2004. ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing. Curr Biol 14:731-735.
Nanjundan M, Sun J, Zhao J, Zhou Q, Sims PJ, Wiedmer T. 2003. Plasma membrane phospholipid scramblase 1 promotes EGF-dependent activation of c-Src through the epidermal growth factor receptor. J Biol Chem 278:37413-37418.
Nguyen DH, Catling AD, Webb DJ, Sankovic M, Walker LA, Somlyo AV, Weber MJ, Gonias SL. 1999. Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner. J Cell Biol 146:149-164.
Palazzo AF, Eng CH, Schlaepfer DD, Marcantonio EE, Gundersen GG. 2004. Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling. Science 303:836-839.
Pelham RJ, Jr., Wang Y. 1997. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci U S A 94:13661-13665.
Rizzo V, Sung A, Oh P, Schnitzer JE. 1998. Rapid mechanotransduction in situ at the luminal cell surface of vascular endothelium and its caveolae. J Biol Chem 273:26323-26329.
Roberts MS, Woods AJ, Shaw PE, Norman JC. 2003. ERK1 associates with alpha(v)beta 3 integrin and regulates cell spreading on vitronectin. J Biol Chem 278:1975-1985.
Sato K, Hattori S, Irie S, Kawashima S. 2003. Spike formation by fibroblasts adhering to fibrillar collagen I gel. Cell Struct Funct 28:229-241.
Simons K, Toomre D. 2000. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31-39.
Sowa G, Pypaert M, Sessa WC. 2001. Distinction between signaling mechanisms in lipid rafts vs. caveolae. Proc Natl Acad Sci U S A 98:14072-14077.
Subauste MC, Pertz O, Adamson ED, Turner CE, Junger S, Hahn KM. 2004. Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility. J Cell Biol 165:371-381.
Vial E, Sahai E, Marshall CJ. 2003. ERK-MAPK signaling coordinately regulates activity of Rac1 and RhoA for tumor cell motility. Cancer Cell 4:67-79.
Wang HB, Dembo M, Hanks SK, Wang Y. 2001. Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proc Natl Acad Sci U S A 98:11295-11300.
Wang HB, Dembo M, Wang YL. 2000. Substrate flexibility regulates growth and apoptosis of normal but not transformed cells. Am J Physiol Cell Physiol 279:C1345-1350.
Wang YK, Wang YH, Wang CZ, Sung JM, Chiu WT, Lin SH, Chang YH, Tang MJ. 2003. Rigidity of collagen fibrils controls collagen gel-induced down-regulation of focal adhesion complex proteins mediated by alpha2beta1 integrin. J Biol Chem 278:21886-21892.
Wickstrom SA, Alitalo K, Keski-Oja J. 2003. Endostatin associates with lipid rafts and induces reorganization of the actin cytoskeleton via down-regulation of RhoA activity. J Biol Chem 278:37895-37901.
Wozniak MA, Desai R, Solski PA, Der CJ, Keely PJ. 2003. ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix. J Cell Biol 163:583-595.
Young RM, Holowka D, Baird B. 2003. A lipid raft environment enhances Lyn kinase activity by protecting the active site tyrosine from dephosphorylation. J Biol Chem 278:20746-20752.
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