Endothelial cells (ECs) respond to TNF-α by altering their F-actin cytoskeleton and junctional permeability through mechanisms that include protein kinase C (PKC) and p38 MAPK. Ezrin, radixin, and moesin (ERM) regulate many cell processes that often require a conformational change of these proteins as a result of phosphorylation on a conserved threonine residue near the C terminus. This study tested the hypothesis that ERM proteins are phosphorylated on this critical threonine residue through TNF-α-induced activation of PKC and p38 and modulate permeability increases in pulmonary microvascular ECs. TNF-α induced ERM phosphorylation on the threonine residue that required activation of p38, PKC isoforms, and phosphatidylinositol-4-phosphate 5-kinase Iα, a major enzyme generating phosphatidylinositol 4,5-bisphosphate, and phosphorylated ERM were prominently localized at the EC periphery. TNF-α-induced ERM phosphorylation was accompanied by cytoskeletal changes, paracellular gap formation, and increased permeability to fluxes of dextran and albumin. These changes required activation of p38 and PKC and were completely prevented by inhibition of ERM protein expression using small interfering RNA. Thus, ERM proteins are phosphorylated through p38 and PKC-dependent mechanisms and modulate TNF-α-induced increases in endothelial permeability. Phosphorylation of ERM likely plays important roles in EC responses to TNF-α by modulating the F-actin cytoskeleton, adhesion molecules, and signaling events.

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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-␣-pretreated ECs induced an increase in the activity of SRC tyrosine kinases. This increase was inhibited by allopurinol (a xanthine oxidase inhibitor), Me 2 SO (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.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 ECs upon leukocyte adhesion (reviewed in Refs. 1 and 2). ICAM-1-initiated signaling events into ECs play important roles in modulating neutrophil migration along and/or across endothelium during inflammatory responses. Sans et al. (3) demonstrated that deletion of the ICAM-1 cytoplasmic domain completely inhibits neutrophil transmigration, but not adhesion, in a reconstituted cell line, suggesting a role for ICAM-1-dependent outside-in signaling in mediating neutrophil migration. In addition, inhibition of ICAM-1-inititated signaling events in pulmonary microvascular ECs reduces neutrophil migration along the EC surface to reach EC borders (4).Within the signaling pathways induced through ligation of ICAM-1, activation of SRC tyrosine kinases has been demonstrated in human umbilical vein ECs and rat brain microvascular ECs (5-7). In addition, ligation of ICAM-1 induces activation of LYN, a member of the SRC family tyrosine kinases, in a B cell lymphoma line (8). Each member of the human SRC tyrosine kinase family is composed of a unique region, an SRC homology 2 and 3 domain, a catalytic domain containing r...