Rosiglitazone在大鼠神經膠質瘤細胞中誘導 Mitogen-Activated Protein Kinase Phosphatase-1 表現的作用機制探討

The mechanism of rosiglitazone-induced mitogen-activated protein kinase phosphatase-1 expression in rat C6 glioma cells

廖婉茹(Wan-Ju Liao)

指導教授 ： 李宏謨

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2005年)

摘要

Rosiglitazone（RSG）是一種化學合成的PPAR-γ (peroxisome proliferators-activated receptor-??agonist，可用於第二型糖尿病的治療， 它與PPAR-γ具有很強的親和力，為TZDs (thiazolidinediones)類的藥物。RSG還有許多其他的作用，例如: 抗發炎，但其調控的機制目前還不是很清楚。在過去的研究中指出，MKP-1(Mitogen- Activated Protein Kinase Phosphatase-1)能夠使MAPK(Mitogen- Activated Protein Kinase)去活化。為了探討MKP-1在RSG抗發炎反應中扮演的角色，利用RSG處理大鼠神經膠質瘤細胞(C6 glioma)，發現RSG會誘導MKP-1表現，在兩小時達到最大量，八小時就回復到原背景值。而以MG-132(蛋白分解?抑制劑)處理，能夠使MKP-1蛋白表現延長至八小時。為了探討RSG調控MKP-1是經由轉錄或轉譯作用，以actinomycin D及cyclohexamide處理皆能抑制由RSG引起的MKP-1表現，表示MKP-1是經由de novo合成而來。為了探討其中的訊息傳遞路徑，利用各種藥理性的抑制劑去抑制ROS、PI3-K、ERK 、NF?B,、p38 MAPK、JNK 路徑，發現ERK 1/2抑制劑 (PD098059)，NF-?B 抑制劑(PDTC)、 p38 MAPK 抑制劑(SB203580) 和JNK抑制劑(SP600125)能夠抑制MKP-1表現，但PI3-K 抑制劑(LY294002)和ROS 抑制劑(l-NAC)不能抑制由 RSG誘導的MKP-1表現，而RSG作用後，磷酸化ERK也明顯上升，表示RSG經由ERK的訊息傳遞路徑而活化MKP-1。在其他研究報告發現，LPS(Lipopolysacchride)可以引起發炎反應，而iNOS可以作為其指標，本篇研究發現RSG可以抑制由LPS所誘導的iNOS表現，而以triptolide (MKP-1 inhibitor) 前處理之後，原先抑制的情況即回復，推斷MKP-1在抗發炎的反應中扮演一個很重要的角色。此外，基質金屬蛋白?(Metalloproteinase ,MMPs)為惡性腫瘤細胞的轉移的一個要素，本研究發現RSG能夠抑制C6 glioma cell 所產生的MMP-2。綜觀以上結果可知MKP-1為細胞內訊息傳遞的調控樞紐，而RSG能夠活化MKP-1而抑制iNOS及MMP-2的表現。

關鍵字

大鼠神經膠質瘤細胞

並列摘要

Rosiglitazone(RSG) or thiazolidinediones is a ligand of the peroxisome proliferators-activated receptor-??PPAR-????which has been used in the treatment of type II diabetes. Additionally, RSG has been shown to exert a variety of beneficial effects, such as anti-inflammatory. However, the underneath mechanisms are not clear. MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate and inactivate MAP kinase. To investigate whether MKP-1 plays a role in mediating RSG-induced anti-inflammatory effects. Treatment of C6 glioma cells with RSG, induced a dose-dependent induction of MKP-1, the maximum effect was seen at 2 hours and decreased at 8 hours. Pretreatment with MG-132 prolonged the increasing of MKP-1 protein levels to 8 hours, suggesting RSG may increase MKP-1 stability. To investigate whether RSG regulate the transcription and translation levels of MKP-1, we find that Act. D (actinomycin D) and CHX (cyclohexamide) can inhibit RSG-stimulated MKP-1 expression demonstrated that MKP-1 goes through de novo synthesis. In order to investigate the signaling mechanism, several pharmacological inhibitors were used to block ROS, PI3-K, ERK1/2, NF-?B, p38 MAPK and JNK pathways. Pretreatment cells with ERK inhibitor (PD098059), NF-?B inhibitor (PDTC), p38 MAPK inhibitor (SB203580) and JNK inhibitor (SP600125) but not PI3-K inhibitor (LY294002) nor ROS blocker(l-NAC) inhibit RSG-stimulated MKP-1 expression. After RSG treatment, phosphorylated- ERK rise was detected. To examine the anti- inflammation role of MKP-1, we demonstrated that RSG can inhibit LPS-induced iNOS expression. Blocking MKP-1 with triptolide reversed iNOS expression. In addition, tumor invasion is regulated by metalloproteinase (MMPs), in our experiments, RSG inhibits MMP-2 mRNA expression. Blocking MKP-1 with triptolide, MMP-2 mRNA expression was reversed. We conclude that RSG exerts anti-inflammatory and MMP-2 inhibition effects through MKP-1 modulation.

並列關鍵字

Rosiglitazone ； Mitogen-Activated Protein Kinase Phosphatase-1

參考文獻

(1998). Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012-2018.(2005).

Matrix metalloproteinase activity and osteoclasts in experimental prostate cancer bone metastasis tissue. Am J Pathol 166, 1173-1186.

Asada, K., Sasaki, S., Suda, T., Chida, K., and Nakamura, H. (2004). Antiinflammatory roles of peroxisome proliferator-activated receptor gamma in human alveolar macrophages. Am J Respir Crit Care Med 169, 195-200.

Bhagavathula, N., Nerusu, K. C., Lal, A., Ellis, C. N., Chittiboyina, A., Avery, M. A., Ho, C. I., Benson, S. C., Pershadsingh, H. A., Kurtz, T. W., and Varani, J. (2004). Rosiglitazone inhibits proliferation, motility, and matrix metalloproteinase production in keratinocytes. J Invest Dermatol 122, 130-139.

Butler, G. S., Butler, M. J., Atkinson, S. J., Will, H., Tamura, T., van Westrum, S. S., Crabbe, T., Clements, J., d'Ortho, M. P., and Murphy, G. (1998). The TIMP2 membrane type 1 metalloproteinase "receptor" regulates the concentration and efficient activation of progelatinase A. A kinetic study. J Biol Chem 273, 871-880.