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系統識別號 U0026-1108201900231500
論文名稱(中文) 探討MDCK細胞中Ha-RasV12及Caveolin-1如何影響循環拉伸介導的細胞垂直排列、高度及存活率改變
論文名稱(英文) Effects of Ha-RasV12 and Caveolin-1 in cyclic stretch-induced alterations of cell perpendicular alignment, cell height and cell survival in MDCK cells
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 107
學期 2
出版年 108
研究生(中文) 李倫維
研究生(英文) Lun-Wei Lee
學號 S36064101
學位類別 碩士
語文別 英文
論文頁數 45頁
口試委員 指導教授-湯銘哲
口試委員-林錫慧
口試委員-邱文泰
口試委員-涂庭源
中文關鍵字 Ha-RasV12  微囊蛋白1  循環性拉伸  肌凝蛋白II  焦點黏著蛋白  c-Jun氨基末端激酶 
英文關鍵字 Ha-RasV12  Cav1  cyclic stretch  myosin II  focal adhesion  JNK 
學科別分類
中文摘要 正常上皮細胞和癌細胞對於機械力的反應截然不同。我們過去發現癌細胞不只比正常細胞來得軟,同時也對軟性基質介導的細胞死亡具有抗性,此現象我們稱作失去感知機械力。細胞內的收縮單元包含焦點黏著蛋白及肌動球蛋白複合物,對於細胞面對機械力會產生相對應的局部收縮是很重要的,過去的研究顯示癌細胞失去感知機械力可能是因為收縮單元的功能缺失。為了測試癌細胞對機械力反應是否會與正常細胞不同,我們在具有過度表現Ha-RasV12系統的狗腎臟上皮細胞施予動態的循環拉伸,發現過度表現Ha-RasV12的細胞會失去拉伸力介導的細胞垂直排列。為了探討收縮單元在拉伸介導的垂直排列扮演甚麼角色,我們測試了肌凝蛋白II抑制劑、敲除微囊蛋白1以及焦點黏著蛋白激酶抑制劑。結果顯示抑制肌凝蛋白II的活性以及抑制焦點黏著蛋白激酶磷酸化可以降低拉伸之後的垂直排列。敲除微囊蛋白1無法減緩拉伸介導的垂直排列,反而促進垂直排列。相反的,過度表現微囊蛋白1增強了帶狀肌動蛋白的收縮力,細胞高度也增加,顯示在拉伸過程中微囊蛋白1主要作用在黏著型連結,而焦點黏著蛋白可能才是改變細胞形狀的機械力感受器。為了探討循環拉伸力是否會對細胞生長產生影響,我們接著測試了細胞增殖及凋亡,實驗結果顯示過度表現Ha-RasV12的細胞會不易適應動態機械力進而造成凋亡細胞的數量增加。更進一步的是,循環拉伸介導c-Jun氨基末端激酶磷酸化只會在細胞全滿的情況下觀察到,指出連接點上的收縮單元及焦點黏著蛋白主導的收縮單元可能會個別影響細胞的存活率。
綜合以上所述,細胞的收縮力對於在動態拉伸的情況下維持細胞是很重要的,進而導致細胞方向改變。循環拉伸可以促進正常細胞增生,卻會讓癌細胞凋亡。
英文摘要 Mechanoresponse is different between normal cells and cancer cells. We previously reported that cancer cells were not only softer than their normal cell type but also resistant to soft matrix-induced cell death, so called loss of mechanosensing. Contractile units (CUs) composed of focal adhesion complex and actomyosin subunits are crucial for providing local contraction in response to mechanical forces. It has been demonstrated that loss of mechanosensing in cancer cells may be due to disfunction of CUs. In order to examine whether cancer cells may respond to mechanical stretch differently from normal cells, we applied a dynamic cyclic stretch force on MK4 cells harboring inducible Ha-RasV12 overexpression system. The results showed that Ha-RasV12 overexpressing cells lost stretch-induced perpendicular alignment. To investigate the role of CUs in stretch induced perpendicular alignment, we tested the effects of myosin II inhibitor, caveolin-1 (Cav-1) knockdown and FAK inhibitor. The results showed that inhibition of myosin II activity and FAK phosphorylation suppressed perpendicular alignment after stretch. Knockdown of Cav1 did not ameliorate stretch-induced perpendicular alignment, but instead promoted perpendicular alignment. In contrast, overexpression of Cav1 enhanced actin belt constriction and increased the cell height, suggesting that Cav1 functioned predominantly in adherens junction during stretch and FA might be the mechanosensor to change the cell orientation. To investigate whether cyclic stretch exerted effects on cell growth, we checked cell proliferation and apoptosis. The results showed that cyclic stretch triggered cell apoptosis in Ha-RasV12 overexpressing cells, but not normal cells. Moreover, cyclic stretch induced phosphorylation of JNK was observed only at the cell confluency, indicating junctional CUs and FA-dominated CUs might influenced the cell survival differently.
Taken together, cellular contractility is important for the maintenance of cells during dynamic stretching, leading to cell orientation change. Cyclic stretch promotes normal cell proliferation, but triggers transformed cell apoptosis.
論文目次 中文摘要 I
Abstract III
誌謝 V
Content VI
List of figures and legends VII
Introduction 1
Materials and methods 7
Results 11
Discussion 18
Conclusion 22
References 23
Figures 26
參考文獻 1. Lin HH, Lin HK, Lin IH, Chiou YW, Chen HW, Liu CY, et al. Mechanical phenotype of cancer cells: cell softening and loss of stiffness sensing. Oncotarget. 2015;6(25):20946-58.
2. Ringer P, Colo G, Fassler R, Grashoff C. Sensing the mechano-chemical properties of the extracellular matrix. Matrix Biol. 2017;64:6-16.
3. Tee SY, Fu J, Chen CS, Janmey PA. Cell shape and substrate rigidity both regulate cell stiffness. Biophys J. 2011;100(5):L25-7.
4. Yeh YC, Ling JY, Chen WC, Lin HH, Tang MJ. Mechanotransduction of matrix stiffness in regulation of focal adhesion size and number: reciprocal regulation of caveolin-1 and beta1 integrin. Sci Rep. 2017;7(1):15008.
5. Sethi K, Cram EJ, Zaidel-Bar R. Stretch-induced actomyosin contraction in epithelial tubes: Mechanotransduction pathways for tubular homeostasis. Semin Cell Dev Biol. 2017;71:146-52.
6. Jufri NF, Mohamedali A, Avolio A, Baker MS. Mechanical stretch: physiological and pathological implications for human vascular endothelial cells. Vasc Cell. 2015;7:8.
7. Ives CL, Eskin SG, McIntire LV. Mechanical effects on endothelial cell morphology: in vitro assessment. In Vitro Cell Dev Biol. 1986;22(9):500-7.
8. Huang C, Miyazaki K, Akaishi S, Watanabe A, Hyakusoku H, Ogawa R. Biological effects of cellular stretch on human dermal fibroblasts. J Plast Reconstr Aesthet Surg. 2013;66(12):e351-61.
9. Hsu HJ, Lee CF, Kaunas R. A dynamic stochastic model of frequency-dependent stress fiber alignment induced by cyclic stretch. PLoS One. 2009;4(3):e4853.
10. Hamzeh MT, Sridhara R, Alexander LD. Cyclic stretch-induced TGF-beta1 and fibronectin expression is mediated by beta1-integrin through c-Src- and STAT3-dependent pathways in renal epithelial cells. Am J Physiol Renal Physiol. 2015;308(5):F425-36.
11. Duda M, Kirkland NJ, Khalilgharibi N, Tozluoglu M, Yuen AC, Carpi N, et al. Polarization of Myosin II Refines Tissue Material Properties to Buffer Mechanical Stress. Dev Cell. 2019;48(2):245-60.
12. Peverali FA, Basdra EK, Papavassiliou AG. Stretch-mediated activation of selective MAPK subtypes and potentiation of AP-1 binding in human osteoblastic cells. Mol Med. 2001;7(1):68-78.
13. Orr AW, Helmke BP, Blackman BR, Schwartz MA. Mechanisms of mechanotransduction. Dev Cell. 2006;10(1):11-20.
14. Martinac B. Mechanosensitive ion channels: molecules of mechanotransduction. J Cell Sci. 2004;117(Pt 12):2449-60.
15. Tijore A, Yao M, Wang YH, Nematbakhsh Y, Hariharan A, Lim CT, et al. Mechanical stretch kills transformed cancer cells. bioRxiv. 2018;1-38.
16. Yadav S, Vadivelu R, Ahmed M, Barton M, Nguyen NT. Stretching cells - An approach for early cancer diagnosis. Exp Cell Res. 2019;378(2):191-7.
17. Iskratsch T, Wolfenson H, Sheetz MP. Appreciating force and shape-the rise of mechanotransduction in cell biology. Nat Rev Mol Cell Biol. 2014;15(12):825-33.
18. Meacci G, Wolfenson H, Liu S, Stachowiak MR, Iskratsch T, Mathur A, et al. alpha-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions. Mol Biol Cell. 2016;27(22):3471-9.
19. Wolfenson H, Meacci G, Liu S, Stachowiak MR, Iskratsch T, Ghassemi S, et al. Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices. Nat Cell Biol. 2016;18(1):33-42.
20. Chan CE, Odde DJ. Traction dynamics of filopodia on compliant substrates. Science. 2008;322(5908):1687-91.
21. Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR. Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat Rev Mol Cell Biol. 2009;10(11):778-90.
22. Smutny M, Cox HL, Leerberg JM, Kovacs EM, Conti MA, Ferguson C, et al. Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens. Nat Cell Biol. 2010;12(7):696-702.
23. Shutova MS, Asokan SB, Talwar S, Assoian RK, Bear JE, Svitkina TM. Self-sorting of nonmuscle myosins IIA and IIB polarizes the cytoskeleton and modulates cell motility. J Cell Biol. 2017;216(9):2877-89.
24. Wang HB, Dembo M, Wang YL. Substrate flexibility regulates growth and apoptosis of normal but not transformed cells. Am J Physiol Cell Physiol. 2000;279(5):C1345-50.
25. Yang B, Wolfenson H, Nakazawa N, Liu S, Hu J, Sheetz MP. Stopping transformed frowth with cytoskeletal proteins: turning a devil into an angel. bioRxiv. 2017;1-37.
26. Wozniak MA, Modzelewska K, Kwong L, Keely PJ. Focal adhesion regulation of cell behavior. Biochim Biophys Acta. 2004;1692(2-3):103-19.
27. Chen Y, Pasapera AM, Koretsky AP, Waterman CM. Orientation-specific responses to sustained uniaxial stretching in focal adhesion growth and turnover. PNAS. 2013;110(26):E2352.
28. Andersen JI, Pennisi CP, Fink T, Zachar V. Focal adhesion kinase activation is necessary for stretch-induced alignment and enhanced differentiation of myogenic precursor cells. Tissue Engineering Part A. 2017;24(7-8):631-40.
29. Lin HK, Lin HH, Chiou YW, Wu CL, Chiu WT, Tang MJ. Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-Ras(V12) -induced cell transformation. J Cell Mol Med. 2018;22(5):2631-43.
30. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell. 2000;103(2):239-52.
31. Lin A, Dibling B. The true face of JNK activation in apoptosis. Aging Cell. 2002;1(2):112-6.
32. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35(4):495-516.
33. Yang DD, Conze D, Whitmarsh AJ, Barrett T, Davis RJ, Rincon M, et al. Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2. Immunity. 1998;9(4):575-85.
34. Kaunas R, Usami S, Chien S. Regulation of stretch-induced JNK activation by stress fiber orientation. Cell Signal. 2006;18(11):1924-31.
35. Kushida N, Kabuyama Y, Yamaguchi O, Homma Y. Essential role for extracellular Ca2+ in JNK activation by mechanical stretch in bladder smooth muscle cells. Am J Physiol Cell Physiol. 2001;281(4):C1165-C72.
36. Pereira AM, Tudor C, Kanger JS, Subramaniam V, Martin-Blanco E. Integrin-Dependent activation of the JNK signaling pathway by mechanical stress. PLoS One. 2011;6(12):e26182.
37. Codelia VA, Sun G, Irvine KD. Regulation of YAP by mechanical strain through Jnk and Hippo signaling. Current Biology. 2014;24(17):2012-7.
38. Lee CF, Haase C, Deguchi S, Kaunas R. Cyclic stretch-induced stress fiber dynamics - dependence on strain rate, Rho-kinase and MLCK. Biochem Biophys Res Commun. 2010;401(3):344-9.
39.Totsukawa G, Wu Y, Sasaki Y, Hartshorne DJ, Yamakita Y, Yamashiro S, et al. Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts. J Cell Biol. 2004;164(3):427-39.
40. Wang YH, Chiu WT, Wang YK, Wu CC, Chen TL, Teng CF, et al. Deregulation of AP-1 proteins in collagen gel-induced epithelial cell apoptosis mediated by low substratum rigidity. J Biol Chem. 2007;282(1):752-63.
41. Chen B, Kemkemer R, Deibler M, Spatz J, Gao H. Cyclic Stretch Induces Cell Reorientation on Substrates by Destabilizing Catch Bonds in Focal Adhesions. PLoS One. 2012;7(11):e48346.
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