Activation of SRC Tyrosine Kinases in Response to ICAM-1 Ligation in Pulmonary Microvascular Endothelial Cells

Qin Wang,Gordon R Pfeiffer,William A Gaarde

doi:10.1074/jbc.m308466200

Abstract

Previous studies demonstrated that ICAM-1 ligation on human pulmonary microvascular endothelial cells (ECs) sequentially induces activation of xanthine oxidase and p38 MAPK. Inhibition of these signaling events reduces neutrophil migration to the EC borders. This study examined the role of SRC tyrosine kinases in ICAM-1-initiated signaling within these ECs. Cross-linking ICAM-1 on tumor necrosis factor-alpha-pretreated ECs induced an increase in the activity of SRC tyrosine kinases. This increase was inhibited by allopurinol (a xanthine oxidase inhibitor), Me2SO (a hydroxyl radical scavenger), or deferoxamine (an iron chelator). Phenylarsine oxide, a tyrosine phosphatase inhibitor, reduced the base-line activity of SRC as well as the increase in SRC activity induced by ICAM-1 cross-linking. Specific inhibition of the protein expression of the SRC homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) by an antisense oligonucleotide prevented the induced SRC activation but had no effect on the basal SRC activity. Activation of SRC tyrosine kinases was accompanied by tyrosine phosphorylation of ezrin at Tyr-146, which was inhibited by PP2, an SRC tyrosine kinase inhibitor. Moreover, PP2 completely inhibited p38 activation, suggesting a role for SRC tyrosine kinases in p38 activation. These data demonstrate that ICAM-1 ligation activates SRC tyrosine kinases and that this activation requires SHP-2 as well as production of reactive oxygen species generated from xanthine oxidase. Activation of SRC tyrosine kinases in turn leads to tyrosine phosphorylation of ezrin, as well as activation of p38, a kinase previously identified to be required for cytoskeletal changes induced by ICAM-1 ligation and for neutrophil migration along the EC surface.

Highlights

Recent studies have provided evidence that ligation of endothelial cell (EC)1 adhesion molecules including ICAM-1 is capable of initiating outside-in signaling events into endothelial cells (ECs) upon leukocyte adhesion
Our studies demonstrate the following: 1) cross-linking ICAM-1 induces activation of SRC tyrosine kinases; 2) activation of SRC tyrosine kinases requires production of reactive oxygen species generated from xanthine oxidase and the SRC homology 2-containing protein-tyrosine phosphatase-2 (SHP-2); 3) activation of SRC tyrosine kinases is required for tyrosine phosphorylation of ezrin as well as activation of p38 MAPK
This study evaluated the role of SRC tyrosine kinases in ICAM-1-induced signaling events in human pulmonary microvascular ECs

Summary

Introduction

Recent studies have provided evidence that ligation of endothelial cell (EC) adhesion molecules including ICAM-1 is capable of initiating outside-in signaling events into ECs upon leukocyte adhesion (reviewed in Refs. 1 and 2). The mechanisms underlying SRC activation in response to ICAM-1 cross-linking have been examined in rat brain microvascular ECs, where activation of SRC requires activation of phospholipase C [6] In these ECs and human umbilical vein ECs, activation of SRC in turn results in tyrosine phosphorylation of cortactin [5,6,7], an actin-binding protein that modulates the F-actin cytoskeleton as well as EC locomotion (14 – 16). We have demonstrated recently [4, 17] that neutrophil adhesion to TNF-␣-pretreated human pulmonary microvascular ECs induces cytoskeletal changes that require ICAM-1-initiated signaling pathways and can be mimicked by cross-linking ICAM-1 with antibodies.

Methods

Results

Conclusion