GSK-3β在人類腦神經膠質瘤細胞株誘導細胞自噬作用所扮演的角色

Roles of GSK-3β in the induction of autophagy in U87-MG human glioma cells

曹玫芬(Mei-Fen Tsao)

指導教授 ： 李宏謨

臺北醫學大學/醫學院/醫學科學研究所/碩士(2008年)

摘要

GSK-3β（Glycogen Synthase Kinase 3β） 對於細胞週期的進行、細胞凋亡、細胞自噬作用和侵襲性有著深遠的影響。然而，GSK-3β在其中所扮演的角色卻未完全了解。本研究利用各種激酶活性的GSK3β突變種（mock、 wild type、 S9A 和 K85R）轉染U87-MG人類腦神經膠質瘤細胞株，藉此分子探子來研究GSK-3β對於細胞週期的進行、細胞凋亡、細胞自噬作用和侵襲性的影響。本實驗發現wild type (GSK-3β) 或 kinase dead (GSK-3β K85R)增加了 GSK-3β本身在 serine9 的磷酸化。GSK-3β K85R持久表達的U87-MG細胞株細胞有促細胞增生的情形，然而constitutive active (GSK-3β S9A)卻沒有影響。細胞週期進行需cyclins, Cdks and Cdk inhibitors的調控，由實驗得知GSK-3β S9A持久表達的U87-MG細胞株p27蛋白表現增加。本研究並探討GSK-3β活性對於U87-MG細胞株是否會產生抗凋亡的特性，研究結果顯示Bcl-xL, Bcl and Mcl-1等抗凋亡蛋白及Bax and Bad 等促凋亡蛋白的表現，並沒有顯著差異，推測GSK-3β活化可能不會導致或抑制引發細胞凋亡的產生。此外，近來的研究證實了在MEF 和 COS-7 兩種細胞株中，當活化GSK-3β時，會誘導細胞自噬作用(macroautophagy)。本研究中，接著探討當活化GSK-3β時，在U87-MG人類腦神經膠質瘤細胞株中是否會刺激細胞自體吞噬作用的產生。利用持續保持激酶活性的GSK3β?n?n-S9A轉染U87-MG細胞株時，LC3-II的蛋白表現量增加；若利用失去激酶活性的GSK3β-K85R，轉染U87-MG細胞株時，LC3-II的蛋白表現量減少。所以，我們推測GSK3β?n?n的活化和自體吞噬小體（autophagosome）的形成有相關。進一步利用帶有綠色螢光蛋白的LC3的質體（GFP-LC3），再次轉染這些持續表現不同GSK3β突變株的U87-MG人類腦神經膠質瘤細胞株，觀察到GSK3β被抑制時，自噬小體形成也跟著減少。由這些研究結果，我們可以得知GSK3β在誘導細胞自體吞噬作用的過程，扮演著很重要的角色，並藉此探討GSK3β下游的基因是否有受到影響。而這樣的結果也可能是引起Doxorubicin這個化療用藥，常在乳癌的病人身上產生抗藥性的原因。先前研究已知GSK3β在Wnt訊息傳遞路徑扮演重要的角色，而本研究由轉染GSK3β?n?n-S9A U87-MG細胞株的侵襲性比wild type降低，推測GSK3β的活性亦直接影響了癌細胞的侵襲力。綜合以上數據本研究證實GSK3β的活性可調控U87-MG人類腦神經膠質瘤細胞株細胞週期的進行、細胞自噬作用和侵襲性，但對細胞凋亡沒有影響。

關鍵字

GSK-3β ； 細胞自噬作用 ； 細胞週期細胞增生

並列摘要

GSK-3β exerts profound effects on cell cycle progression, apoptosis, autophagy and invasion. However, the mechanism by which GSK-3β exerts these functions are not completely understood. In the present study, we constructs GSK-3β mutants and transfected into U87-MG human glioma cells. U87-MG cells transfected with mock, wild type, S9A and K85R express various kinase activities were used to examine the effects of GSK-3β on cell cycle progression, apoptosis, autophagy and invasion. Ectopic expressing wild type (GSK-3β) or kinase dead (GSK-3β K85R) increased GSK-3β serine 9 phosphorylation. Expression of GSK-3β K85R increased cell proliferation, whereas constitutive active (GSK-3β S9A) has not effect on cell proliferation. Cell cycle progression is regulated by cyclins, Cdks and Cdk inhibitors. In U87-MG cells, expression of GSK-3β S9A increased the protein level of p27Kip-1. We next examined whether GSK-3β affect cell apoptosis in U87-MG cells. Permanent expression of various GSK-3β mutants did not alter apoptotic cell death. In agreement, transfection of GSK-3β did not alter the expression of pro-apoptotic proteins including Bax and Bad or anti-apoptotic proteins such as Bcl-xL, Bcl and Mcl-1. We next investigated whether activation of GSK-3β stimulates autophagy while permanent expression of GSK-3β S9A increased LC3-II level, GSK-3β K85R decreased LC3-II. Suggesting that GSK-3β activation is linked to autophagosome formation. Taken together, these data suggest that GSK-3β may play a role in the induction of autophagy, which is important in Doxorubicin drug resistance. In conclusion, these results suggest that GSK-3β may regulate cell cycle progression, autophagy and invasion but not apoptosis.

並列關鍵字

GSK-3β ； autophagy ； cell cycle ； cell proliferation

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