E-Selectin-Dependent Signaling Via the Mitogen-Activated Protein Kinase Pathway in Vascular Endothelial Cells

Abstract

E-selectin, a cytokine-inducible adhesion molecule, supports rolling and stable arrest of leukocytes on activated vascular endothelium. Previous studies have suggested that this transmembrane protein can also transduce signals into the endothelial cell. We now demonstrate activation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured HUVEC in response to E-selectin-dependent leukocyte adhesion and Ab-mediated cross-linking of cell surface E-selectin. Adhesion of increasing numbers of HL60 cells to IL-1β-activated HUVEC stimulated robust increases in MAPK activity that were abrogated by an E-selectin blocking Ab. Cross-linking of cell surface E-selectin with Abs, as a mimic of multivalent ligand engagement, strongly stimulated MAPK/extracellular signal-related kinase (ERK) kinase (MEK)-dependent MAPK activation and concomitant up-regulation of mRNA for c-fos, an immediate early response gene, whereas Ab cross-linking of HLA class I molecules (present at comparable density) failed to do so. Coimmunoprecipitation documented Ras, Raf-1 and, phospho-MEK complex formation. Unactivated HUVEC transduced with a full-length adenoviral E-selectin construct also exhibited cross-link-induced MAPK activation, macromolecular complex formation, and c-fos up-regulation, whereas HUVEC transduced with a cytoplasmic domain deletion mutant failed to respond. These observations indicate that E-selectin can transduce an activating stimulus via the MAPK cascade into the endothelial cell during leukocyte adhesion.

Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases

“…The notion that E-selectin induces a forward signalling in endothelial cells has previously been supported by studies showing that the adhesion of LS174T colon adenocarcinoma to E-selectin-expressing HUVEC initiate the endocytosis of E-selectin (Burdick et al, 2003). It is also supported by studies reporting that activation of E-selectin by crosslinking activating antibodies or adhesion of HL-60 leukaemia cell induces the clustering of E-selectin and triggers the activation of ERK and p38 in endothelial cells (Yoshida et al, 1996;Hu et al, 2000Hu et al, , 2001Yoshida et al, 2003). Activation of ERK derives from the phosphorylation of Tyr603 within E-selectin by an unknown kinase (Hu et al, 2001).…”

“…We next verified whether the adhesion of colon cancer cells HT-29 to HUVEC also initiated the activation of E-selectin and then of ERK and p38 in the endothelial cells. In this set of experiments, HT-29 cells were previously fixed with 2% paraformaldehyde to highlight the activation of the MAP kinases of solely endothelial cells (Hu et al, 2000;Laferrie`re et al, 2004). Then, fixed HT-29 cells were added to E-selectin-expressing HUVEC for increasing intervals of time and the activation of p38 and ERK was determined.…”

“…It requires specific interactions between adhesion receptors present on vascular endothelial cells and their counter-receptors on cancer cells (Haier and Nicolson, 2001). These adhesive interactions induce a reverse signalling in the cancer cells that increases their motile potential and a forward signalling in endothelial cells that elicits breaches in the endothelium layer (Hu et al, 2000;Laferrie`re et al, 2001;Weis et al, 2004). However, in both cases the signalling mechanisms involved are still ill defined.…”

“…More recently, the p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases have been identified as important molecules that regulate cellular tension and integrity of endothelial junctions (Kevil et al, 1998(Kevil et al, , 2001Verin et al, 2000;Wang and Doerschuk, 2001;Yao et al, 2001;Borbiev et al, 2004). Moreover, several studies have shown that the activation of E-selectin induces signalling in the endothelial cells that leads to activation of ERK and p38 (Hu et al, 2000;Hu et al, 2001;Yoshida et al, 2003). However, nothing is known as to whether or not E-selectin-mediated signalling in endothelial cells controls transendothelial permeability and migration of cancer cells.…”

Clustering endothelial E-selectin in clathrin-coated pits and lipid rafts enhances leukocyte adhesion under flow

“…In flow-free conditions, adhesion of leukocytes to cytokine-activated endothelial cells induces association of E-selectin with lipid raft domains, cytoskeletal components, and signaling molecules. [23][24][25][26][27] Indeed, it has been suggested that E-selectin is primarily associated with lipid rafts even before it engages leukocytes. 27 If this were true, E-selectin would probably not cluster in clathrin-coated pits, as lipid rafts are not known to mix with clathrin-enriched structures.…”