Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is necessary for Toll-like receptor 4 (TLR4) signalling during a lipopolysaccharide(LPS)-induced pro-inflammatory response. At the same time, the raft protein CD14 facilitates activation of cascades downstream of TLR4; however, it is unclear whether functions of CD14 and PI(4,5)P2 are connected. On page , Katarzyna Kwiatkowska and colleagues address the effect treatment of J774 macrophage-like cells with LPS has on the generation of CD14 and PI(4,5)P2. The authors find that stimulation with LPS causes CD14 to form clusters in the plasma membrane and observe an increase of PI(4,5)P2 levels and PI(4,5)P2 accumulation in the proximity of CD14 clusters. Importantly, crosslinking of CD14 at the cell surface is sufficient for PI(4,5)P2 levels to rise, thus indicating that PI(4,5)P2 is generated in response to CD14 clustering. Moreover, in HEK293 cells that do not have endogenous TLR4 or CD14, PI(4,5)P2 is also generated in response to clustering of exogenous CD14. The authors then identify that type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) isoforms Iα and Iγ are responsible for the generation of PI(4,5)P2 because both colocalise with CD14 upon stimulation with LPS, and associate with the raft fraction containing CD14 and newly generated PI(4,5)P2, whereas their knockdown inhibits activation of NF-κB, a downstream target of TLR4, and decreases subsequent cytokine production. Taken together, these results reveal that CD14 clustering and concomitant PI(4,5)P2 generation in rafts play a pivotal role during the pro-inflammatory response.
