Apoptosis Signal Regulated Kinase 1在Peptidoglycan誘導RAW 264.7巨噬細胞Cyclooxygenase-2表現的角色探討

The Role of Apoptosis Signal Regulated Kinase 1 in Peptidoglycan-Induced Cyclooxygenase-2 Expression in RAW 264.7 Macrophages

張家凱(Chia-Kai Chang)

指導教授 ： 陳炳常 ； 林建煌

臺北醫學大學/醫學院/藥理學研究所/碩士(2005年)

摘要

摘要 我們之前的研究證實?聚醣 (peptidoglycan, PGN) 刺激RAW 264.7 巨噬細胞環氧化?-2 (cyclooxygenase-2, COX-2)表現是經由活化Ras/Raf-1/ERK 及 NF-?B 的訊息傳遞路徑而來 (J. Biol. Chem., 279: 20889-20897, 2004)。在本篇論文中，我們進一步探討 apoptosis signal regulated kinase 1 (ASK1)、c-jun N-terminal kinase (JNK)、activator protein-1 (AP-1) 及 CCAAT/enhancer binding protein ? (C/EBP?) 在PGN 刺激 RAW 264.7 巨噬細胞COX-2 蛋白表現中所扮演的角色。PGN誘導 COX-2 的表現會被 ASK1 dominant negative mutant (ASK1DN)、JNK抑制劑 (SP600125)、JNK1 dominant negative mutant (JNK1DN)、JNK2DN 以及 AP-1 抑制劑 (curcumin) 所抑制。 PGN 會時間依賴性地誘導 TRAF6 及 ASK1 聚集至TLR2。RAW 264.7 巨噬細胞受到 PGN 刺激後會時間依賴性地誘導 14-3-3脫離ASK1使得ASK1去磷酸化而進一步導致ASK1的活化及其下游的JNK及 c-jun 的活化。而 PGN 所誘導 JNK 的活化則會被 ASK1DN 及SP600125 所抑制。另一方面，細胞在給予 PGN 刺激後也可見到 AP-1 會和特定的 DNA 序列結合，同時在報告基因 (reporter assay) 的實驗也可見到 AP-1轉錄活性增加。而 PGN 所誘導的 AP-1 報告基因活性的增加會被 SP600125及 curcumin 所抑制。C/EBP??蛋白的表現在 PGN 刺激下也會時間依賴性的增加，同時伴隨著 C/EBP??和特定的 DNA序列結合。而在報告基因實驗更發現PGN所誘導的 COX-2 報告基因活性的增加會因其基因序列上C/EBP 結合位置突變而被抑制。綜合以上實驗結果，我們發現在RAW264.7巨噬細胞中PGN會經由引發TLR2和TRAF6的結合，而導致TRAF6/ASK1/JNK/AP-1路徑的活化，使得 C/EBP表現增加並活化，最終誘導 COX-2 的表現。

並列摘要

Abstract The Ras/Raf-1/extracellular signal-regulated kinase (ERK) pathway is known to be involved in peptidoglycan (PGN)-induced nuclear factor-?B (NF-?B) activation and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages (J. Biol. Chem. 2004, 297: 20889-20897). In this study, we further investigated the roles of apoptosis signal regulated kinase 1 (ASK1), c-jun N-terminal kinase (JNK), activator protein-1 (AP-1), and CCAAT/enhancer binding protein ? (C/EBP?) in PGN-induced COX-2 expression. The PGN-induced COX-2 expression was attenuated by the ASK1 dominant negative mutant (ASK1DN), a JNK inhibitor (SP600125), the JNK1 dominant negative mutant (JNK1DN), the JNK2DN, and the AP-1 inhibitor (curcumin). PGN induced the recruitment of TNFR-associated factor 6 (TRAF6) and ASK1 to toll-like receptor 2 (TLR2) in a time-dependent manner. Treatment of RAW 264.7 macrophages with PGN caused time-dependent dissociation of 14-3-3 with ASK1 and dephosphorylation of ASK1 which resulted in activations of ASK1 and further activates JNK and c-jun. The PGN-induced JNK activation was inhibited by ASK1DN and SP600125. Stimulation of cells with PGN activated the formation of AP-1 specific-DNA protein complex and AP-1-luciferase activity. The PGN-mediated increase in AP-1-luciferase activity was inhibited by SP600125, and curcumin.? Treatment of macrophages with PGN caused time-dependent C/EBP? expression. Stimulation of cells with PGN caused the formation of C/EBP?-specific DNA-protein complex. Furthermore, PGN-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the C/EBP site mutation of COX-2-construct. Our data demonstrated that PGN induced the recruitment of TRAF6 to TLR2 to activate the ASK1/JNK/AP-1 pathway, which in turn initiates CEBP? expression and activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.

並列關鍵字

apoptosis signal-regulating kinase1 ； CCAAT/enhancer binding protein ? ； TNF-? receptor associated factor 2

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