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
中文摘要
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。
英文摘要
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.
