| Summary: | 碩士 === 臺灣大學 === 生理學研究所 === 98 === Background: Lipopolysaccharide (LPS) is a component of the outer membrane of Gram (-) bacteria. Proinflammatory responses are mounted by immune cells (e.g. monocyte/macrophage) following LPS recognition by its receptor complex (CD14/TLR4/MD2) on cell surface. However, distinct expression patterns of CD14 and TLR4 were characterized on human intestinal epithelial cells, which were suggested as a mechanism to tolerate against commensal bacteria in the gut lumen. Previous studies from our laboratory have demonstrated that luminal LPS challenge induced epithelial cell apoptosis and tight junctional breakage in human intestinal Caco-2 cells1-2, of which the mechanism was independent of TLR4 signaling (unpublished data). Recent evidence on monocytic cell lineages showed that after LPS binding to membranous CD14, a cascade of lipid secondary messenger pathway occurred. Activation of phosphatidylcholine-specific phospholipase C (PC-PLC) caused the generation of diacylglycerol (DAG) which then catalyzed sphingomyelinase to convert sphingolipid to ceramide. Formation of ceramide activated PKCζ leading to recruitment of TLR4 to lipid rafts to form a complex with CD14. Both ceramide and PKCζ were implicated in pro-apoptotic pathways in various epithelial cell types. It remains unknown whether CD14-mediated signals are involved in the mechanism of LPS-induced apoptosis in intestinal epithelial cells. Materials and methods: Caco-2 cells (C2BBe clone) were grown to confluency and apically challenged with LPS at various concentration and time points. In some groups, cells were pretreated with pharmacological inhibitors, such as D609 (a PC-PLC inhibitor), imipramine (a sphingomyelinase inhibitor), Gö6983 (a broad spectrum PKC inhibitor), or inhibitory PKCζ pseudosubstrate prior to LPS challenge. Cell apoptosis was assessed by TUNEL staining and quantified using a Cell Death ELISA kit. The activation status of PKCζ was examined by western blotting and immunofluorescent confocal microscopy. Production of ceramide was detected by Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC—MS/MS). The expression levels of CD14 and TLR4 proteins and mRNA were examined by immunofluorescent staining and RT-PCR. Results: Apical expression of CD14 and absence of TLR4 staining was confirmed on Caco-2 cells before and after LPS challenge. Exposure to luminal LPS increased the level of apoptosis in Caco-2 cells in a time- and dose-dependent manner. Pretreatment with D609, imipramine, Gö6983, and inhibitory PKCζ pseudosubstrate decreased the cell apoptotic levels induced by LPS. The level of ceramide (C16) was significantly elevated after LPS challenge. Moreover, LPS triggered increased phosphorylation and membrane translocation of cytosolic PKCζ to colocalize with tight junctions. The activation of PKCζ was blocked by pretreatment with D609 and imipramine. Conclusion: LPS-induced intestinal epithelial apoptosis is dependent on CD14-mediated signaling pathways such as ceramide and PKCζ.
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