LPS-induced clustering of CD14 triggers generation of PI(4,5)P2

Abstract

Bacterial lipopolysaccharide (LPS) induces strong pro-inflammatory reactions after sequential binding to CD14 protein and TLR4 receptor. Here we show that CD14 controls generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in response to LPS binding. In J774 cells and HEK293 cells expressing CD14 exposed to 10-100 ng/ml LPS, the level of PI(4,5)P2 rose in a biphasic manner with peaks at 5-10 min and 60 min. After 5-10 min of LPS stimulation, CD14 underwent prominent clustering in the plasma membrane accompanied by accumulation of PI(4,5)P2 and type I phosphatidylinositol 4-phosphate 5-kinases (PIP5K) Iα and Iγ in CD14 vicinity. Clustering of CD14 with antibodies, without LPS and TLR4 participation, was sufficient to trigger PI(4,5)P2 elevation. The newly generated PI(4,5)P2 was accumulated in rafts, which also accommodated CD14 and most of PIP5K Iα and Iγ. Silencing of PIP5K Iα and Iγ, or application of drugs interfering with PI(4,5)P2 synthesis and availability, abolished the LPS-induced PI(4,5)P2 elevation and inhibited down-stream pro-inflammatory reactions. Taken together, these data indicate that LPS induces clustering of CD14, which triggers PI(4,5)P2 generation in rafts required for maximal pro-inflammatory signaling of TLR4.