PowerPoint Slideshow about ' 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' - yardley
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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.