Molecular Events in Transmembrane Signaling via E-selectin

Abstract

E-selectin is a cytokine-inducible adhesion molecule that is expressed by activated endothelial cells at sites of inflammation. In addition to supporting rolling and stable arrest of leukocytes, there is increasing evidence that E-selectin functions in transmembrane signaling into endothelial cells during these adhesive interactions. We have previously shown that adhesion of HL-60 cells (which express ligands for E-selectin), or antibodymediated cross-linking of E-selectin, results in formation of a Ras/Raf-1/phospho-MEK macrocomplex, extracellular signal-regulated protein kinase (ERK1/2) activation, and c-fos up-regulation. All of these downstream signaling events appear to require an intact cytoplasmic domain of E-selectin. Here we demonstrate that tyrosine 603 in the cytoplasmic domain of E-selectin is required for the E-selectin-dependent ERK1/2 activation. Tyrosine 603 plays an important role in mediating the association of E-selectin with SHP2, and the catalytic domain of SHP2 is, in turn, critical for E-selectin-dependent ERK1/2 activation. An adapter protein complex consisting of Shc⅐Grb2⅐Sos bridges between SHP2 and the Ras⅐Raf⅐phospho-MEK macrocomplex. These molecular events thus outline a mechanism by which cross-linking of E-selectin by engagement of ligands on adherent leukocytes can initiate a multifunctional signaling pathway in the activated endothelial cell at sites of inflammation. E-selectin is an inducible adhesion molecule that is expressed at relatively high density on the surface of cultured endothelial cells that have been activated by proinflammatory cytokines, such as interleukin-1b (IL-1␤), 1 tumor necrosis factor-␣, or bacterial endotoxin. It is detectable in vivo at the sites of inflammation (1-3). In addition to supporting the rolling and stable arrest of leukocytes on activated endothelium, there is increasing evidence that E-selectin can transduce outside-in signals (4 -6). Recently our laboratory demonstrated that leukocyte adhesion to cell surface E-selectin-activated extracellular signal-regulated protein kinase (ERK1/2), formed a macrocomplex containing Ras/Raf-1/phospho-MEK, and resulted in the up-regulation of c-fos expression (7). In this study, we have investigated the molecular events occurring immediately upstream of Ras/Raf/phospho-MEK and downstream of cell surface E-selectin. The cytoplasmic domain of E-selectin has been implicated in transmembrane signaling (5, 7); however, the molecular mechanisms involved have not been well defined. The cytoplasmic domain of E-selectin consists of 32 amino acids (1), including two tyrosine residues. It has been well documented that phosphorylation of tyrosine residues in the cytoplasmic domains of various types of receptors can play an important role in receptor-mediated transmembrane signal transduction, especially ERK1/2 activation. Therefore, it is reasonable to hypothesize that the two tyrosine residues on the cytoplasmic domain of E-selectin may be involved in the E-selectin-dependent ERK1/2 activation described by our laborat...

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).…”

“…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). Our finding that HT-29 cells also leads to an E-selectin-dependent activation of p38 and ERK in endothelial cells is the first demonstration that E-selectin is activated in response to adhesion of cancer cells from solid tumours.…”

“…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.…”

Hepatitis C Virus and HIV Envelope Proteins Collaboratively Mediate Interleukin-8 Secretion through Activation of p38 MAP Kinase and SHP2 in Hepatocytes

“…We found a similar interaction of Grb2 and Shc with SHP2 during HCV-E2 and HIV-gp120 co-stimulation (data not shown). The association of SHP2, Shc, and Grb2 has also been observed to activate MAP kinase in activated endothelial cells at sites of inflammation (50). To determine the specificity of interaction, immunoprecipitated p38 MAP kinase was probed with various antibodies of signaling molecules such as LDL receptor, phosphatidylinositol 3-kinase, Bad, FasL, c-Jun, and c-Fos.…”

Inhibition of the Tyrosine Phosphatase SHP-2 Suppresses Angiogenesis in vitro and in vivo

“…During the development of hematopoietic cells, it maintains the critical balance between apoptosis, proliferation and differentiation [5,6,7]. In endothelial cells, SHP-2 interacts with adhesion molecules, such as PECAM-1 and VE-cadherin, thereby participating in mechanotransduction and cell motility [8,9,10]. Mobilization of endothelial cells is a prerequisite for angiogenesis, during which the prevention of endothelial apoptosis or anoikis (programmed cell death due to detachment from the extracellular matrix) [11,12,13] is of major importance in order to enable the formation of new vessels from already existing ones.…”