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研究生(外文):Shi-Jeih Tsai
論文名稱(外文):Signal Mechanisms of GDNF/RET-Induced Migration in MDCK Cells
指導教授(外文):Ming-Jer Tang
外文關鍵詞:RETGDNFGDNFR-alphaCell MigrationPhosphatidylinositol 3-kinase
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RET(Rearrangement-induced Transformation)是一種受體酪氨酸激化脢(Receptor tyrosine kinase),目前已知與腎臟及周邊神經系統之發育有關。活化RET需要其他兩種分子同時存在:膠質細胞衍生性神經營養因子(Glial cell line-Derived Neurotrophic Factor,GDNF)及GDNFR-alpha,一種醣磷脂醯肌醇連結之細胞表面受體(Glycosyl phosphatidylinositol-linked cell surface receptor)。為了研究RET在細胞內之訊息傳遞機制,我們建立了一個將RET大量表現在MDCK細胞株的系統;在此系統中已經證實了RET之活化與細胞移行(Migration)、散生(Scattering)、以及細胞伸出偽足(Filopodia and lamellipodia)等現象有關。在此,我們又建立了一些表現突變RET之MDCK細胞株,如正常之RET(Ret9,Ret2),激化脢失去功能之RET(Kinase mutant, km),及一些將不同位置之酪氨酸(Tyrosine,Y)置換為苯丙氨酸(Phenylalanine,F)之RET如M1062(Y1062F)、M1090(Y1090F)、M2(Y1090F and Y1096F)、M708(Y660F, Y687F, Y826F, Y1015F, Y1029F, Y1062F, and Y1096F)及M816(Y660F, Y687F, Y826F, Y1015F, Y1029F, Y1062F, Y1090F, and Y1096F)。在這些表現不同突變RET之細胞中,Ret9細胞經GDNF/GDNFR-處理可促進其細胞增生、移行能力及散生增加。而m和M816在GDNF/GDNFR-alpha的刺激下並沒有任何型態、移行及增生方面的改變。有趣的是,GDNF/GDNFR-alpha會使M708細胞散生及移行增加,但不會促進生長。另外,M1062及M2兩株細胞的細胞移行能力較正常RET之細胞如Ret9,Ret2等之移行來的低,但M1090細胞之移行能力則非常顯著,因此我推測Tyr1096可分別正向調控Tyr1062及負向調控Tyr1090這兩個酪氨酸送出影響細胞移行的訊息。我們也發現了RET的活化可以引起磷脂醯肌醇-3激化脢(Phosphatidylinositol 3-Kinase ,PI3-kinase)的活性。而用磷脂醯肌醇-3激化脢之抑制劑LY294002來處理Ret9、M708及M1090三種細胞則可抑制GDNF/RET所引發之細胞移行。綜合上述之結果,我們認為由RET之Tyr1062、Tyr1090及Tyr1096所下傳的訊息與細胞移行有關,且這些引起細胞移行的訊息可能是透過磷脂醯肌醇-3激化脢所調控的。

RET (Rearrangement-induced Transformation) is a receptor tyrosine kinase involved in the development of kidney and certain parts of the peripheral nervous system. The activation of RET requires GDNF (Glial cell line-Derived Neurotrophic Factor) and GDNFR-, a glycosyl phosphatidylinositol-linked cell surface receptor. It has been demonstrated that activation of RET induces cell migration, scattering and filopodia and lamellipodia formation in RET overexpressing MDCK cells. To investigate the signal pathway mechanisms of RET activation, we employed MDCK cells stably transfected with wild type RET (Ret9, Ret2), kinase dead mutant (Km), and various mutants in which different tyrosine residues (Y) were substituted with phenylalanine (F) including M1090 (Y1090F), M2 (Y1090F and Y1096F), M1062 (Y1062F), M708 (Y660F, Y687F, Y826F, Y1015F, Y1029F, Y1062F, and Y1096F) and M816 (Y660F, Y687F, Y826F, Y1015F, Y1029F, Y1062F, Y1090F, and Y1096F). Among these transfectants, neither Km nor M816 cells showed any morphological or growth effects upon RET activation by addition of GDNF/GDNFR-alpha. Interestingly, M708 exhibited marked increase in migration and scattering, but no change in proliferation in response to GDNF/GDNFR-alpha indicating the role of Tyr1090 in cell migration. In addition, M2 and M1062 exhibited decreased migration capacity whereas M1090 migrated significantly, suggesting that Tyr1062 plays roles in cell migration and Tyr1096 may serve as a positive regulator for Tyr1062 and inhibitor for Tyr1090 in GDNF/RET-induced cell motility. We also found that GDNF/RET interactions resulted in enhanced phosphatidylinositol 3-kinase (PI3-kinase) activity. LY294002, a PI3-kinase inhibitor, blocked GDNF- induced migration effects in Ret9 and M708 cells. Taken together, Tyr1062, Tyr1090 and Tyr1096 of RET may mediate GDNF/RET-induced cell migration through activation of PI3-kinase.


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