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

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)P-2] 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)P-2 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)P-2 and type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) isoforms I alpha and I. (encoded by Pip5k1a and Pip5k1c, respectively) in the CD14 region. Clustering of CD14 with antibodies, without LPS and TLR4 participation, was sufficient to trigger PI(4,5)P-2 elevation. The newly generated PI(4,5)P-2 accumulated in rafts, which also accommodated CD14 and a large portion of PIP5K Ia and PIP5K I gamma. Silencing of PIP5K Ia and PIP5K I., or application of drugs interfering with PI(4,5)P-2 synthesis and availability, abolished the LPS-induced PI(4,5)P-2 elevation and inhibited downstream pro-inflammatory reactions. Taken together, these data indicate that LPS induces clustering of CD14, which triggers PI(4,5)P-2 generation in rafts that is required for maximal pro-inflammatory signaling of TLR4.