Acid Sphingomyelinase is Required for Lipid Raft TLR4 Complex Formation

摘要

Background: Lipid rafts, composed of sphingolipids, are critical to Toll-like receptor 4 (TLR4) assembly during lipopolysaccharide (LPS) exposure as a result of phosphokinase C (PKC)-ζ activation. However, the mechanism responsible for these events remains unknown. Purpose: We determined whether LPS-induced TLR4 assembly and activation are dependent on the sphingolipid metabolite ceramide, produced by acid sphingomyelinase following the initial binding of LPS to CD14. Methods: Cultured THP-1 cells were stimulated with LPS, exogenous C_2 ceramide, or both. Selected cells were pretreated with the acid sphingomyelinase inhibitor imipramine or CD14 neutralizing antibody. Results: Exposure to LPS led to activation of acid sphingomyelinase, production of ceramide, phosphorylation of PKC-ζ, and assembly of the TLR4 receptor within lipid rafts. This was followed by activation of the MAPK family of products and the liberation of tumor necrosis factor-α. Pretreatment with imipramine or CD14 blockade was associated with attenuation of all of these LPS-induced events. Simultaneous treatment with C_2 ceramide and LPS reversed all the inhibitory effects induced by imipramine, but not those associated with CD14 blockade. Conclusion: Assembly and activation of the TLR4 receptor following LPS binding to CD14 requires the production of ceramide by acid sphingomyelinase.