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

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.

Flotillin-dependent lipid-raft microdomains are required for functional phagolysosomes against fungal infections

“…By contrast, wild-type conidia did not show different phagocytosis rates because these conidia via their DHN-melanin layer most likely already disturbed flotillins in the membrane forming the phagocytic cup. In line, recently it was shown that flotillin-1 facilitates inflammatory toll-like receptor 3 signaling in human endothelial cells (Fork, Hitzel et al, 2014).…”

Class-A scavenger receptor function and expression in the rainbow trout (Oncorhynchus mykiss) epithelial cell lines RTgutGC and RTgill-W1

“…SCARA4 has previously been shown to be expressed in mammalian epithelial cells [28]. Although its ability to bind dsRNA is not yet elucidated in rainbow trout, SCARA4 is likely a surface receptor for dsRNA in mammals [10,33]. It is not however a surface receptor for acLDL in mammals [34] and neither could acLDL bind SCARA4 (CL-P1) in zebrafish [11].…”

Flotillins: At the Intersection of Protein S-Palmitoylation and Lipid-Mediated Signaling

“…An involvement of flotillin-1 and -2 in modulating the content of caveolin-1 and annexin-2 in exosomes has also been indicated [55]. Notably, other studies suggest that changes of the exosomal content of caveolin-1 can follow changes of its synthesis at the endoplasmic reticulum that is affected by flotillin-1 [13], as described below.…”

“…A contribution of flotillin-1 to proper functioning of the endoplasmic reticulum has also been indicated by studies of Fork et al [13] yet without addressing the role of its S-palmitoylation. Those authors found that a downregulation of flotillin-1 but not of flotillin-2 with shRNA led in human umbilical vein endothelial cells to a reduction of the level of several other proteins, including caveolin-1.…”

“…EGF receptor signaling leading to cell adhesion [8] EGF receptor clustering and phosphorylation, ERK1/2 and Akt phosphorylation; flotillins as MAP kinase scaffolding proteins [9] EGF receptor expression in breast cancer cells [10] activation of H-Ras in breast cancer cells [11] EGF receptor sorting and lysosomal degradation [12] TLR3 ligand internalization [13] TCR raft association and recycling [14][15][16][17] PrP [Ca +2 ] increase, Fyn and ERK1/2 activation, N-cadherin trafficking [18,19] IgE receptor [Ca +2 ] increase, phosphorylation of IgE receptor γ chain and ERK1/2 [20] insulin receptor insulin-induced glucose uptake via Glut4 transporter [21] Gq protein-coupled receptors p38 phosphorylation [22] not determined signaling leading to axon regeneration: Rho GTPase activation, formation of N-WASP-Arp3/cortactin complexes, p38, ERK1/2 and FAK kinase phosphorylation [23] IGF-1 receptor* IGF-1 receptor transport from endoplasmic reticulum to plasma membrane [24] integrins ERK2 and FAK kinase phosphorylation [25] ACTIN CYTOSKELETON REMODELING Affected processes filopodia formation [26] cell spreading [8] proper localization of Vav during T cell spreading [14] F-actin binding [27] axon regeneration [23] uropod formation in neutrophils [28] uropod formation in T cells, activation of ezrin/radix/moesin, localization of PIP5KIγ to uropod [29][30][31] formation of lamellipodia at the growth cone of neurons [32] cell adhesion and migration via α-actinin binding [25] ENDOCYTOSIS Cargo fluid phase (magnetic dextran, AF488-dextran) [33,34] GPI-anchored proteins [18,33,[35]…”