Phosphatidylcholine-specific phospholipase C (PC-PLC) is required for LPS-mediated macrophage activation through CD14

Abstract

Lipid rafts, composed of sphingolipids, are critical to Toll-like receptor 4 (TLR4) assembly during lipopolysaccharide (LPS) exposure, as a result of protein kinase C (PKC)-zeta activation. However, the mechanism responsible for this remains unknown. The purpose of this study is to determine if LPS-induced TLR4 assembly and activation are dependent on the sphingolipid metabolite ceramide produced by phosphatidylcholine-specific phospholipase C (PC-PLC) or CD14. To study this, THP-1 cells were stimulated with LPS. Selected cells were pretreated with the PC-PLC inhibitor D609, exogenous C2 ceramide, CD14 neutralizing antibody, or TLR4 neutralizing antibody. LPS led to production of ceramide, phosphorylation of PKC-zeta, and assembly of the TLR4 within lipid rafts. This was followed by activation of the mitogen-activated protein kinase family and the liberation of cytokines. Pretreatment with D609 or CD14 blockade was associated with attenuated LPS-induced ceramide production, TLR4 assembly on lipid rafts, and cytokine production. Pretreatment with TLR4 blockade did not affect LPS-induced ceramide production but was associated with significant attenuation in cytokine production. Treatment with C2 ceramide prior to LPS reversed the inhibitory effects induced by D609 but not of CD14 or TLR4 blockade. C2 ceramide alone induced the activation of PKC-zeta and the assembly of TLR4 but was not associated with cytokine liberation. This study demonstrates that TLR4 assembly and activation following LPS exposure require the production of ceramide by PC-PLC, which appears to be CD14-dependent.

Mechanism of Glycosaminoglycan-Mediated Bone and Joint Disease

“…In addition, MPS synovial cells have elevated levels of the prosurvival lipid, S1P, contributing to the enhanced proliferation rate. Changes in the production of the signaling lipids, ceramide and S1P, are known consequences of LPS signaling, 26,27,37 supporting our central hypothesis. Osteopenia, in addition to other bone lesions, has been observed in some of the MPS disorders, including MPS VI.…”

Eritoran Suppresses Colon Cancer by Altering a Functional Balance in Toll-like Receptors That Bind Lipopolysaccharide

“…Previous studies have indicated that the binding of LPS to CD14 activates a cascade of lipid messengers and protein kinase Cz (PKCz) prior to the recruitment of TLR4 into lipid rafts (9,10). Single and double gene silencing of TLR4, CD14, and/or PKCz in spheroids was specific and did not alter any proteins other than the target molecules ( Supplementary Fig.…”

“…The binding of LPS to CD14 on the lipid raft domain activates a cascade of lipid messengers and protein kinase Cz (PKCz) to recruit TLR4 to form a complex with CD14 (9,10). The transfer of LPS to TLR4/MD2 subsequently initiates cytoplasmic signaling, resulting in the production of proinflammatory cytokines, such as myeloid differentiation factor (MyD88), MAPK, and inhibitor of kB (IkB)/nuclear factor-kB (NFkB; refs.…”

Functional role of phosphatidylcholine‐specific phospholipase C in regulating CD16 membrane expression in natural killer cells

“…Furthermore, inhibition of the enzyme by D609 leads to the disappearance of both the CD16 receptor and PC-PLC from these cholesterol-rich structures. D609 is a wellknown inhibitor of PC-PLC and is widely used to investigate the role of this enzyme in several cellular processes, such as LPS-mediated macrophage activation and B lymphocyte activation through CD38 signaling [24,25].…”