乙型樣澱粉蛋白經由IKK/FKHR/Bim?徑誘導C6星??經膠質瘤細胞凋亡之探討

β-Amyloid Induced C6 Glioma Cell Apoptosis via IKK/FKHR/Bim Pathway

周恩如(En-Ju Chou)

指導教授 ： 林建煌 ； 陳炳常

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2006年)

摘要

乙型樣澱粉蛋白 (β-Amyloid, Aβ) 被認為是引起許多?經退化性疾病的主要原因，而Aβ和星?細胞的交互作用對?經細胞產生的傷害會?進ㄧ步促進?經的退化。此外，星?細胞又是構成血腦障壁的主要成分之ㄧ，因此星?細胞凋亡對於中樞?經系統以及腦血管退化之病?發展過程是非常重要的。在本?文中，我們將探討Aβ調控星??經膠質瘤細胞凋亡的詳細分子機轉。在C6星??經膠質瘤細胞中，Aβ誘導增加BimEL的表現而?是BimL和BimS。Aβ也可誘導增加Bim報告基因的活性。?用轉染FKHR結合序?突變型Bim報告基因質體可減少Aβ誘導之Bim報告基因的活性。轉染野生型FKHR oligodeoxynucleotides 能抑制Aβ所誘導之Bim的表現和C6星??經膠質瘤細胞凋亡。Aβ誘導FKHR之Ser256的去磷酸化呈現時間相關性，並且藉由DNA-binding affinity pull down assay證實Aβ可以誘導FKHR結合至bim基因起始區上。轉染野生型及持續活化型IKKβ質體可抑制Aβ誘導FKHR去磷酸化、Bim的表現以及C6星??經膠質瘤細胞凋亡。 Aβ也會時間相關性地誘導IKKα/β Ser180/Ser181的去磷酸化，並且藉由蛋白磷酸激?活性的測試顯示Aβ可?低IKKα/β的活性。此外，在C6星??經膠質瘤細胞中，Aβ可誘導IKKα/β、FKHR以及14-3-3的分?。PP2A的抑制劑okadaic acid可以阻斷Aβ誘導的IKK去磷酸化、FKHR去磷酸化、Bim的表現以及C6細胞的死亡。再者，我們發現Aβ可誘導PP2A的活性增加。綜合以上實驗結果推測Aβ可經由PP2A/IKK/FKHR/Bim訊息?徑誘導C6星??經膠質瘤細胞死亡。

關鍵字

乙型樣澱粉蛋白 ； C6星??經膠質瘤細胞 ； 凋亡

並列摘要

β-Amyloid peptide (Aβ) has been implicated as a key molecule in the neurodegenerative diseases. The Aβ-astrocyte interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration. Astrocyte is a cellular component of blood-brain barrier, thus the regulation of astrocyte apoptosis plays a causal role in pathological processes in the CNS and cerebrovascular degeneration. This study was designed to investigate the mechanism of Aβ-induced C6 glioma cell apoptosis. Aβ induced an increase in BimEL, but not BimL and BimS, expression in C6 glioma cells. Aβ also caused an increase in Bim-luciferase activity, which was reduced by transfection with the mutation of forkhead transcription factor (FKHR) site in Bim-luciferase reporter construct. Transfection with the wild type FKHR oligodeoxynucleotides inhibited Aβ-induced BimEL expression and C6 glioma cell apoptosis. Treatment of C6 glioma cells with Aβ induced FKHR dephosphorylation at Ser256 in a time-dependent manner. Aβ induced an increase in FKHR binding to the bim promoter by DNA-binding affinity pull down assay. Furthermore, transfection with the plasmids of wild type IKKβ and constitutively active IKKβ reversed Aβ-induced FKHR dephosphorylation, Bim expression, and C6 glioma cell apoptosis. Aβ also induced IKKα/β dephosphorylation at Ser180/Ser181 and reduced IKKα/β activity in a time-dependent manner. In addition, Aβ induced the dissociation among IKKα/β, FKHR, and 14-3-3 in C6 glioma cells. Okadaic acid, a potent PP2A inhibitor, inhibited Aβ-induced IKKα/β dephosphorylation, FKHR dephosphorylation, Bim expression, and C6 cell apoptosis. Furthermore, Aβ induced an increase in protein phosphatase 2A activity. Taken together, these results suggest that the mechanism of Aβ-induced C6 cell apoptosis involves PP2A activation, IKKα/β dephosphorylation, FKHR activation, and Bim expression.

並列關鍵字

β-Amyloid ； C6 Glioma Cell ； Apoptosis

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