Flotillin-1 facilitates toll-like receptor 3 signaling in human endothelial cells.

Christian Fork,Ritva Tikkanen,Benjamin J Nichols,Juliane Hitzel,Ralf P Brandes

doi:10.1007/s00395-014-0439-4

Abstract

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). 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 signals 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 led 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 in an endoplasmic reticulum stress-dependent manner. 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 poly-I:C-induced adhesion of peripheral blood mononuclear cells onto HUVECs was significantly attenuated by flotillin-1 shRNA. Collectively, these data suggest that interaction between flotillin-1 and caveolin-1 may facilitate the transport of TLR3-ligands to its intracellular receptor and enables inflammatory TLR3 signaling.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-014-0439-4) contains supplementary material, which is available to authorized users.

Highlights

Endothelial cells are the gate keepers of vascular inflammatory signaling
The role of flotillin-1 in toll-like receptors (TLRs)-induced signaling in human umbilical vein endothelial cells (HUVEC) was studied by shRNA and TLR signaling was initiated by selective agonists
N = 3; *p \ 0.05 with vs. without polyI.C, #p \ 0.05 shRNA against green fluorescent protein (shGFP) vs. shFlot expected, TLR4 activation, which predominantly signals through MyD88 only induced a weak activation of the IRF pathway and importantly, this response was not affected by down-regulation of flotillin-1

Summary

Introduction

Endothelial cells are the gate keepers of vascular inflammatory signaling. In acute inflammation, the endothelium attracts and guides leukocytes to the site of affliction. The concept of membrane rafts is still vigorously debated, there is consensus that proteins of the flotillin family are enriched in this fraction and are markers of rafts [32] Despite their ubiquitous expression and high conservation, the physiological function of flotillins is far from clear and their role in the endothelium has, to our knowledge, not yet been determined. Flotillins modulate epidermal growth factor receptor auto-phosphorylation and activation of downstream target molecules [1]. Considering their prominent positioning on lipid rafts, we here hypothesize that flotillins contribute to vascular inflammatory signaling in response to the endosomal TLR3 and studied this in human umbilical vein endothelial cells (HUVECs)

Materials and methods

Results

Discussion

Conclusion