Peptidoglycan刺激小鼠巨噬細胞環氧酵素-2表現的訊號傳遞路徑探討

1. Peptidoglycan刺激小鼠巨噬細胞環氧酵素-2表現的訊號傳遞路徑探討Peptidoglycan刺激小鼠巨噬細胞環氧酵素-2表現的訊號傳遞路徑探討 • Peptidoglycan (PGN)為革蘭氏陽性菌細胞壁主要的一種成分。PGN會活化宿主的天然免疫系統和誘發發炎物質的釋放。而有關於PGN引發cyclooxgenase-2 (COX-2)表現的路徑目前所知不多。本篇論文，我們將探討在RAW 264.7巨噬細胞中，PGN引發COX-2表現的訊號傳遞路徑。PGN以劑量-相關及時間-相關的方式誘發COX-2的表現。PGN引發COX-2的表現會明顯的被轉錄抑制劑actinomycin D及轉譯抑制劑cyclohexamide 所抑制。而polymyxin B (會與內毒素結合並使內毒素失去活性)也會降低LPS引發COX-2的表現量，而不影響PGN所引發的反應。 Ras抑制劑(manumycin A)、Raf抑制劑(GW 5074)或MEK抑制劑(PD 98059)抑制了PGN引發COX-2的表現。而PGN也會活化p44/42 mitogen-activated-protein-kinase (p44/42 MAPK)，且manumycin A、GW 5074或PD 98059會抑制p44/42 MAPK的活化，但此反應不被p38 mitogen-activated-protein-kinase (p38 MAPK)抑制劑(SB 203580)抑制。Tyrosine kinase抑制劑(genistein)或p38 MAPK抑制劑(SB 203580)也會抑制 PGN引發COX-2的表現。而PGN也會活化p38 MAPK，且genistein、manumycin A或SB 203580會抑制p38 MAPK的活化。此外， protein kinase C (PKC)抑制劑(Go 6976)也減少PGN引發COX-2表現。另一方面，轉錄因子NF-kB的抑制劑pyrrolidine dithiocarbamate (PDTC)或IkB protease 的抑制劑L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), calpain inhibitor也同樣地抑制了PGN所引發的 COX-2表現。PGN也會使細胞質中的IkBa磷酸化、IkBa的降解和p65 NF-kB從細胞質轉位到細胞核。由electrophoretic mobility shift assay (EMSA)結果發現， PGN刺激活化NF-kB。而PGN所活化的NF-kB會被Go 6976抑制，但不被manumycin A、GW 5074、PD 98059、genistein或SB 203580抑制。綜合上述實驗結果，PGN經由p44/42 MAPK、p38 MAPK及PKC 的活化皆引發COX-2表現，但PGN活化NF-kB的路徑可經由PKC，而非經由p44/42 MAPK及p38 MAPK。

2. Studies on the Signaling Pathway of Peptidoglycan-Mediated Cyclooxygenase-2 Expression in RAW 264.7 Macrophages • Peptidoglycan (PGN), a main cell wall component of Gram-positive bacteria, activates the innate immune system of host and induces release of inflammatory molecules. The signaling pathway in PGN-mediated cyclooxgenase-2 (COX-2) expression is poorly understood. In this study, we investigated the signaling pathway of PGN-induced COX-2 expression in murine RAW 264.7 macrophages. PGN caused a concentration- and time-dependent increase in COX-2 expression. The PGN-induced increase in COX-2 expression was significantly inhibited by transcription inhibitor (actinomycin D) or translation inhibitor (cyclohexamide). Polymyxin B, which binds and inactivates endotoxin, attenuated lipopolysaccharie (LPS)-induced COX-2 expression, while it has no effect on PGN-induced effect. The Ras inhibitor (manumycin A), Raf inhibitor (GW 5074) or MEK inhibitor (PD 98059) reduced the PGN-induced increases in COX-2 expression. Treatment of RAW 264.7 macrophages with PGN caused an activation of p44/42 mitogen-activated protein kinase (p44/42 MAPK); this effect was inhibited by manumycin A, GW 5074 or PD 98059, but not by the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, SB 203580. The tyrosine kinase inhibitor (genistein) and p38 MAPK inhibitor (SB 203580) decreased the PGN-induced increase in COX-2 expression. Treatment of RAW 264.7 macrophages with PGN caused an activation of p38 MAPK; this effect was inhibited by genistein, manumycin A or p38 MAPK inhibitor. The protein kinase C (PKC) inhibitor (Go 6976) also attenuated PGN-induced COX-2 expression. On the other hand, the NF-kB inhibitor (pyrrolidine dithiocarbamate, PDTC) or IkB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone, TPCK and Calpain inhibitor Ι) also decreaseed PGN-induced increases in COX-2 expression. Exposure of RAW 264.7 macrophages to PGN caused a translocation of p65 NF-kB from the cytosol to the nucleus and a degradation of IkBa in the cytosol. Treatment of RAW 264.7 macrophages with PGN caused NF-kB activation by detecting the formation of NF-kB-specific DNA-protein complex in the nucleus; this effect was inhibited by Go 6976, but not by manumycin A, GW 5074, PD 98059, genistein, or SB 203580. These results indicated that the activation of p44/42 MAPK, p38 MAPK, and PKC involved in the PGN-mediated COX-2 expression. The PGN-mediated PKC involves NF-kB activation, rather than by p44/42 MAPK or p38.