Abstract 444: Flotillin-1 Facilitates Toll-like Receptor 3 Signaling In Human Endothelial Cells

Abstract

Objective: Endothelial cells are important elements in the vascular response to danger associated molecules signaling through Toll-like receptors (TLRs). Flotillin-1 and -2 are markers of membrane rafts but their true endothelial function is unknown. We hypothesized that flotillins are required for TLR signaling in human umbilical vein endothelial cells (HUVECs). Approach and Results: Knockdown of flotillin-1 by shRNA decreased the TLR3 mediated poly-I:C-induced but not the TLR4-mediated LPS-induced inflammatory activation of HUVEC. As TLR3 but not TLR4 signal through the endosomal compartment, flotillin-1 might be involved in the transport of poly-I:C to its receptor. Consistently, uptake of poly-I:C was attenuated by flotillin-1 knockdown and probably involved the scavenger receptor SCARA4 as revealed by knockdown of this receptor. To determine the underlying mechanism, SILAC proteomics was performed. Down-regulation of flotillin-1 lead to a reduction of the structural caveolae proteins caveolin-1, cavin-1 and -2, suggesting a role of flotillin-1 in caveolae formation. Flotillin-1 and caveolin-1 colocalized within the cell, and knockdown of flotillin-1 decreased caveolin-1 expression. Importantly, downregulation of caveolin-1 also attenuated TLR3-induced signaling. To demonstrate the importance of this finding, cell adhesion was studied. Flotillin-1 shRNA attenuated the poly-I:C-mediated induction of the adhesion molecules VCAM-1 and ICAM-1. As a consequence, the oly-I:C-induced adhesion of peripheral blood mononuclear cells onto HUVECs was significantly attenuated by flotillin-1 shRNA. Conclusion: Interaction between flotillin-1 and caveolin-1 may facilitate the transport of TLR3-ligands to its intracellular receptor and enables inflammatory TLR3 signaling.