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)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 Iα and Iγ (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 Iα and PIP5K Iγ. Silencing of PIP5K Iα and PIP5K Iγ, 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.

Association of Lyn kinase with membrane rafts determines its negative influence on LPS-induced signaling

“…Real-time quantitative PCR analysis was performed essentially as described by Plociennikowska et al (2015b).…”

Early innate immune responses to bacterial LPS

“…In macrophages, LPS induces aggregation of CD14 in plasma membrane lipid rafts (Fig. 1B) [33]. This CD14 aggregation promotes increased PI(4,5)P2 synthesis and drives increased inflammatory signaling.…”

“…As with microglial cells, PI(4,5)P2 enrichment after LPS stimulation was dependent on PIP5K isoforms, however both PIP5Kα and γ were shown to contribute to this process (Fig. 1B) [33]. Since CD14 does not have a cytosolic tail for direct signaling to intracellular regulators, the mechanism by which CD14 signals to promote PIP5K α and γ activation is not immediately clear.…”

Lipopolysaccharide Detection across the Kingdoms of Life

“…Several regulators of the unique signaling pathway activated by CD14 have been defined [26, 32, 34–37], but the molecular details of this new pathway are unclear. The third consequence of LPS-CD14 interactions is the rapid modification of membrane phosphoinositides, in particular leading to the generation of PI(4,5)P2 [38, 39]. LPS detection by CD14 leads to the activation of the cytosolic phosphoinositide kinases PIP5K Iα and PIP5K Iγ, which phosphorylate the 5′ phosphate present in plasma membrane-localized PI(4)P to generate PI(4,5)P2 [38, 39].…”

“…The third consequence of LPS-CD14 interactions is the rapid modification of membrane phosphoinositides, in particular leading to the generation of PI(4,5)P2 [38, 39]. LPS detection by CD14 leads to the activation of the cytosolic phosphoinositide kinases PIP5K Iα and PIP5K Iγ, which phosphorylate the 5′ phosphate present in plasma membrane-localized PI(4)P to generate PI(4,5)P2 [38, 39]. This process (which is independent of TLR4 signaling) is perhaps most notable, as PI(4,5)P2 is a central regulator of TIRAP localization and the process of endocytosis (and phagocytosis) [19, 20, 40].…”