Diacylglycerol kinase-ε is required for the formation of GPI-anchored CD14 and modulates the LPS-induced proinflammatory responses of macrophages

Abstract

Diacylglycerol kinase-{varepsilon} (DGK{varepsilon}) is a unique member of the DGK family with strict specificity toward DAG containing stearic and arachidonic fatty acid residues, called SAG, and producing phosphatidic acid used for the synthesis of phosphatidylinositol (PI). PI and its derivatives, both phosphorylated and non-phosphorylated ones, regulate a multitude of processes, including the signaling of diverse plasma membrane receptors. To the latter belong Toll-like receptor 4 (TLR4) and its accessory CD14 protein activated in macrophages by bacterial lipopolysaccharide (LPS). To assess the role of DGK{varepsilon} in the LPS-induced pro-inflammatory responses, we obtained Raw264 cells stably depleted of DGK{varepsilon} and subsequently rescued them with DGK{varepsilon}-Myc expressed at a level similar to the native one. As a result, SAG phosphorylation was markedly decreased and then restored in those cells, with the activity of other DGKs unaffected. The depletion of DGK{varepsilon} nullified the LPS-induced pro-inflammatory signaling of TLR4 dependent on CD14-mediated internalization of TLR4 and the TRIF engagement in endosomes. In contrast, the MyD88-dependent signaling pathway, for which CD14 involvement can be dispensible, was inhibited only partially. In accordance, no mature, GPI-anchored form of CD14 was produced in the DGK{varepsilon}-depleted cells and no CD14 was found on the cell surface. The reintroduction of DGK{varepsilon} restored both the abundance of GPI-CD14 and the CD14-dependent signaling of TLR4. These results indicate that the DGK{varepsilon}-dependent phosphorylation of SAG controls the synthesis of the pool of PI that serves for the biosynthesis of the GPI moiety of CD14. We thereby have identified DGK{varepsilon} as a key factor determining the sensitivity of macrophages to LPS.