結締組織生長因子刺激C6神經膠瘤細胞引發環氧酵素-2表現之機轉探討

中樞神經系統損傷後會伴隨有神經膠變性 (reactive gliosis) 現象，在神經系統內結締組織生長因子 (connective tissue growth factor, CTGF) 被認為組織修復與gliosis有關。而環氧酵素Cyclooxygenase-2，為中樞神經受損時慢性發炎反應之重要指標。然而對於CTGF如何調控中樞系統內慢性發炎反應，目前仍不清楚。在我們的研究結果中顯示，CTGF會以濃度相關性與時間相關性增加COX-2表現及釋放，同時也會依濃度相關性促進COX-2-luciferase活性增加。CTGF可造成C6神經膠瘤細胞中ERK、JNK蛋白產生磷酸化現象；進一步給予PD98059 (MEK抑制劑)及SP600125 (JNK抑制劑)可抑制經CTGF所誘導之COX-2蛋白表現增加及抑制COX-2-luciferase活性上升。而且給予PD98059前處理後，我們可以發現原本CTGF刺激IKKα/β蛋白磷酸化的現象會被抑制。除此之外，再給予PD98059及SP600125前處理，可以發現CTGF誘發κB-luciferase活性上升的現象亦會被抑制。而在使用SB203580 (p38抑制劑)後並沒有觀察到抑制現象外，在給予CTGF對於p38蛋白的磷酸化也無活化作用。另一方面，我們也發現IκB kinase α/β (IKKα/β)、IκBα及NF-κB轉錄因子也都參與在CTGF誘導COX-2表現的訊息傳遞路徑之中。在給予CTGF刺激後，會依時間相關性誘導IKKα/β和IκBα的磷酸化及IκBα蛋白的降解，亦有時間相關性地誘導p65蛋白磷酸化。在前處理PDTC (NF-κB抑制劑)後可抑制CTGF所誘導COX-2蛋白的表現。另外，分別使用κB mutanted-COX-2 promoter及IκBαM轉染進C6神經膠瘤細胞，我們發現CTGF所誘導上升的COX-2-luciferase activity 或COX-2蛋白表現的現象分別會被抑制。最後使用CHIP技術證明CTGF會誘導NF-κB轉錄因子結合至COX-2 promoter region。綜合以上結果發現，在C6神經膠瘤細胞中，CTGF可活化JNK、ERK / IKKα/β/ IκBα訊息傳遞路徑，進而使得NF-κB轉錄因子活化並結合至COX-2 promoter來調控COX-2表現。

關鍵字

結締組織生長因子

並列摘要

In CNS trauma process, Connective tissue growth factor (CTGF) regulates a wide range of cellular process, including angiogenesis and reactive gliosis. There is growing evidence that increased expression of cyclooxygenase-2 (COX-2) in acute CNS injury is key event in the pathogenesis of CNS injury. However, the molecular mechanisms underlying CTGF-induced COX-2 expression are still undefined In this study, we investigated the involvement of the ERK, JNK, IκBkinase α/β (IKKα/β), and NF-κB signaling pathways in CTGF-induced COX-2 expression in rat C6 glioma cells. Treatment of C6 glioma cell with CTGF caused not only increased COX-2 expression in a concentration- and time-dependent manner but also increased COX-2 -luciferase activity. Treatment of C6 glioma cells with PD98059 (a MEK inhibitor) or SP600125 (a JNK inhibitor) inhibited CTGF-induced COX-2 expression, COX-2-luciferase activity and κB-luciferase acivity. The CTGF-mediated increases in IKKα/β phosphorylation and κB-luciferase activity were inhibited by PD98059 and SP600125. Stimulation of cells with CTGF caused an increase in ERK and JNK phosphorylation in a dose-dependent manner. In addition, treatment of C6 glioma cells with pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor), inhibited CTGF-induced COX-2 expression. The CTGF-induced increase in COX-2-expression or COX-2-luciferase activity were also blocked by the dominant negative of IκBαM and κB-mutanted-COX-2 promoter. Treatment of C6 glioma cells with CTGF induced IKKα/β, IκBα phosphorylation, and IκBα?? degradation. Moreover, CTGF increased binding of p65 and p50 to COX-2 promoter region. Taken together, these results suggest that the JNK- and ERK-dependent/ IKKα/β/ IκBα and NF-κB signaling pathway play important role in CTGF-induced COX-2 expression in C6 glioma cells.

並列關鍵字

CTGF ； C6

參考文獻

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結締組織生長因子刺激C6神經膠瘤細胞引發環氧酵素-2表現之機轉探討

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