Abstract 912: Spleen Tyrosine Kinase Regulates TLR4 dependent Macropinocytosis And Lipid Accumulation In Macrophages

Abstract

Toll-like receptors play a key role in the signaling pathways of innate immune response and have been recently shown to contribute to vascular inflammation and atherosclerosis. Oxidation of LDL is considered a leading mechanism of atherogenesis, and in our previous studies, we demonstrated that minimally modified LDL (mmLDL) induced TLR4-dependent chemokine secretion as well as robust actin polymerization and spreading of macrophages. We noticed that these mmLDL-induced cytoskeletal rearrangements led to extensive vacuolization, characteristic of macropinocytosis. In this study, we examined the mechanisms of TLR4-induced actin polymerization and macropinocytosis. It has been reported that spleen tyrosine kinase (Syk) regulates actin cytoskeleton and that it may interact with TLR4. We found in the immunoprecipitation experiments that mmLDL induced Syk association with TLR4 as well as Syk phosphorylation. Next we developed a J774 macrophage cell line, which stably expressed Syk shRNA and had the Syk expression reduced by 70% or more. Using this Syk-deficient macrophage cell line and specific Syk inhibitors, we demonstrated that Syk is necessary for mmLDL-induced activation of Ras and phosphorylation of Raf-MEK1-ERK1/2. The ERK1/2 activation resulted in phosphorylation of paxillin and an increase in the formation of N-WASP/Arp2 complex, which initiates actin polymerization. Taken together, these results suggest that Syk is a critical kinase in the mmLDL macrophage activation and that the mmLDL-induced cytoskeleton signaling. In addition, we demonstrated that mmLDL induced macrophage uptake of native LDL and of fluorescent 10 KDa dextran, and that this effect was Syk-dependent. These data suggest a novel pathway for the mmLDL-induced macropinocytosis in macrophages, which may constitute a mechanism for macrophage lipid accumulation and foam cell formation, a characteristic step in the development of atherosclerosis.