Journal of leukocyte biology

Contribution of CD14 and TLR4 to changes of the PI(4,5)P2 level in LPS-stimulated cells.

PMID 27418354


LPS binds sequentially to CD14 and TLR4/MD2 receptor triggering production of proinflammatory mediators. The LPS-induced signaling is controlled by a plasma membrane lipid PI(4,5)P2 and its derivatives. Here, we show that stimulation of murine peritoneal macrophages with LPS induces biphasic accumulation of PI(4,5)P2 with peaks at 10 and 60-90 min that were still seen after silencing of TLR4 expression. In contrast, the PI(4,5)P2 elevation was abrogated when CD14 was removed from the cell surface. To assess the contribution of CD14 and TLR4 to the LPS-induced PI(4,5)P2 changes, we used HEK293 transfectants expressing various amounts of CD14 and TLR4. In cells with a low content of CD14 and high of TLR4, no accumulation of PI(4,5)P2 occurred. With an increasing amount of CD14 and concomitant decrease of TLR4, 2 peaks of PI(4,5)P2 accumulation appeared, eventually approaching those found in LPS-stimulated cells expressing CD14 alone. Mutation of the signaling domain of TLR4 let us conclude that the receptor activity can modulate PI(4,5)P2 accumulation in cells when expressed in high amounts compared with CD14. Among the factors limiting PI(4,5)P2 accumulation are its hydrolysis, phosphorylation, and availability of its precursor, PI(4)P. Inhibition of PLC and PI3K or overexpression of PI4K IIα that produces PI(4)P promoted PI(4,5)P2 elevation in LPS-stimulated cells. The elevation of PI(4,5)P2 was dispensable for TLR4 signaling yet enhanced its magnitude. Taken together, these data suggest that LPS-induced accumulation of PI(4,5)P2 that maximizes TLR4 signaling is controlled by CD14, whereas TLR4 can fine tune the process by affecting the PI(4,5)P2 turnover.