Neutral Sphingomyelinase和Ceramide在Peptidoglycan刺激RAW264.7巨噬細胞引發環氧酵素-2表現之角色探討

1. Neutral Sphingomyelinase和Ceramide在Peptidoglycan刺激RAW264.7巨噬細胞引發環氧酵素-2表現之角色探討 • Peptidoglycan (PGN) 為革蘭氏陽性菌細胞壁上的主要成分， 會活化宿主的免疫系統並引發發炎物質的釋放。有關於PGN引發cyclooxygenase-2 (COX-2) 表現的訊息傳遞路徑目前瞭解的並不多，於是在本篇論文中，我們將探討PGN誘導neutral sphingomyelinase和ceramide活化的訊息傳遞路徑及它們在PGN誘導RAW 264.7巨噬細胞NF-kB活化和COX-2表現的訊息傳遞路徑中所扮演的角色。PGN誘導COX-2的表現會被phosphatidylcholine-phospholipase C (PC-PLC) 抑制劑 (D609), protein kinase C (PKC) 抑制劑 (Go 6976, Ro 31-8220)和neutral sphingomyelinase (nSMase)抑制劑3-O-methyl-sphingomyeline (3-OMe-SM)所抑制，但此反應不被phosphatidylinositol-phospholipase C (PI-PLC) 抑制劑 (U-73122) 和acid sphingomyelinase (aSMase) 抑制劑 (imipramine) 所抑制。以PGN刺激細胞會造成PKC活性增加，此作用會被D609, Go 6976和Ro 31-8220所抑制，但卻不被U-73122所影響。另外PGN會有時間相關性的增加nSMase活性及造成ceramide的產生，但卻不影響aSMase的活性；PGN所誘導的nSMase活性增加及ceramide的產生會被D609、Go 6976、Ro 31-8220和3-OMe-SM所抑制。以C2-ceramide, bacterial SMase和ceramidase抑制劑 (N-oleylethanolamine) 皆會使p38 MAPK活化且會增加PGN所誘導的COX-2表現。3-OMe-SM會抑制PGN引發的p38 MAPK活化，但不會影響JNK和ERK的活化。由PGN引發之p38 MAPK活化和kB-luciferase的活性皆可被D609, Go 6976和3-OMe-SM所抑制。接著我們更進一步證實PGN誘導COX-2的表現會被MKK3 dominant negative mutant (MKK3 DN), MKK6 DN和p38 MAPKa DN所抑制。由我們以上的結果證實，在RAW 264.7巨噬細胞中，PGN會經由活化PC-PLC/PKC路徑而導致neutral sphingomyelinase活化和ceramide的產生，進一步經由MKK3/6和p38 MAPK的作用促使NF-kB活化，最後引發COX-2的表現。

2. Role of Neutral Sphingomyelinase and Ceramide in Peptidoglycan Induced Cyclooxygenase-2 Expression in RAW 264.7 Macrophages • Peptidoglycan (PGN), a main cell wall component of Gram positive bacteria, activates the immune system of host and induces release of inflammatory mediators. However, the PGN-induced COX-2 expression is still unclear. This study investigated the signaling pathway involved in PGN-induced activation of neutral sphingomyelinase (nSMase)/ceramide and their role in PGN-induced nuclear factor-kB (NF-kB) activation and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. The PGN-induced COX-2 expression was attenuated by the phosphatidylcholine-phospholipase C (PC-PLC) inhibitor (D609), the PKC inhibitors (Go 6976 and Ro 31-8220), the neutral sphingomyelinase (nSMase) inhibitor (3-OMe-SM), but not by the phosphatidylinositol-phospholipase C (PI-PLC) inhibitor (U-73122) and the acidic sphingomyelinase (aSMase) inhibitor (imipramine). Treatment of cells with PGN caused an increase in PKC activity; this effect was inhibited by D609, Go 6976, and Ro 31-8220, but not by U-73122. PGN caused time-dependent increases in nSMase activity and ceramide formation, but not aSMase activity. The PGN-induced nSMase activation and ceramide formation were inhibited by D-609, Go 6976, Ro 31-8220, and 3-OMe-SM. C2 ceramide, bacterial SMase, and N-oleylethanolamine (a ceramidase inhibitor) all induced p38 MAPK activation, and enhanced the PGN-induced COX-2 expression. 3-OMe-SM inhibited the PGN-induced p38 MAPK activation, while it had no effect on PGN-induced activations of extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK). The PGN-mediated increases in p38 MAPK activation and kB-luciferase activity were also inhibited by D609, Go 6976 and 3-OMe-SM. Furthermore, the PGN-induced increase in COX-2 expression were inhibited by MKK3 dominant negative mutant (MKK3 DN), MKK6 DN and p38 MAPKa DN. Our results demonstrated that PGN activates the PC-PLC/PKC pathway to induce nSMase activation and ceramide formation, which in turn initiates the activations of MKK3/6, p38 MAPK and NF-kB, and ultimately induces COX-2 expression in RAW 264.7 macrophages.