Acid sphingomyelinase is required for lipid Raft TLR4 complex formation.
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)-zeta activation. However, the mechanism responsible for these events remains unknown.


OBJECTIVE
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 PKCzeta, 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-alpha. 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.


CONCLUSIONS
Assembly and activation of the TLR4 receptor following LPS binding to CD14 requires the production of ceramide by acid sphingomyelinase.
Cuschieri
Joseph
J
Department of Surgery, University of Washington, Harborview Medical Center, 325 Ninth Avenue, Seattle, WA 98104, USA. jcuschie@u.washington.edu
Bulger
Eileen
E
Billgrin
Jens
J
Garcia
Iris
I
Maier
Ronald V
RV
D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007099:Imipramine / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D018613:Microscopy, Confocal; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D000099221:Protein Kinase C zeta
D002518:Ceramides; D004791:Enzyme Inhibitors; D007099:Imipramine; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D020929:Mitogen-Activated Protein Kinase Kinases; D011493:Protein Kinase C; D013108:Sphingomyelin Phosphodiesterase; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D000099221:Protein Kinase C zeta; C495345:TLR4 protein, human