Phosphatidylcholine (PC)-specific Phospho-Lipase C (PC-PLC) is required for LPS-mediated macrophage activation

Abstract

Lipid rafts are sphingolipid-rich membrane microdomains critical to the assembly of the TLR4 receptor complex during LPS exposure within the macrophage. Previously we have demonstrated that activation of the atypical PKC, PKC-ζ, is required for LPS-mediated TLR4 receptor assembly and activation. However, the mechanism in which this kinase is activated following LPS exposure leading to TLR4 receptor assembly and activation remains unknown. The purpose of this study is to determine if the sphingolipid metabolite ceramide produced by PC-PLC is required for activation of this process by LPS. Methods. Differentiated THP-1 cells were stimulated with 100 ng/ml of LPS. Selected cells were pretreated with 100 μM of the PC-PLC inhibitor tricylodecan-9-yl xanthogentate (D609). Lipid raft and cellular protein were extracted and analyzed for TLR4, and PKC-ζ and MAPK activation. TLR4 mobilization to lipid rafts was verified by immunohistochemistry. Harvested supernatants were analyzed by ELISA for TNF-μ production. Results. LPS led to the phosphorylation of PKC-ζ and the mobilization of TLR4 to lipid rafts as demonstrated by immunoblotting and immunohistochemistry. This receptor complex assembly was followed by the activation of the MAPK family, consisting of ERK 1/2, p38, and JNK, and the production of TNF-μ. Pretreatment with D609 resulted in abolished LPS-mediated PKC-ζ phosphorylation, and attenuated LPS-mediated TLR4 lipid raft mobilization (figure), MAPK activation, and TNF-μ production ( P > 0.05). Conclusion. TLR4 receptor assembly occurs following LPS exposure. Assembly of the TLR4 receptor complex requires the generation of ceramide by PC-PLC. Thus, modulation of sphingholipid conversion to ceramide by PC-PLC may serve as a therapeutic target for modulation of macrophage activation during sepsis.