Endothelial Cell Cortactin Phosphorylation by Src Contributes to Polymorphonuclear Leukocyte Transmigration In Vitro

Abstract

Abstract-The underlying mechanisms that regulate leukocyte transendothelial migration through the vascular endothelium remain unclear. Cortactin is a substrate of Src tyrosine kinases and a regulator of cytoskeletal dynamics. Previous studies demonstrated a role for Src phosphorylation of cortactin in clustering of E-selectin and intercellular cell adhesion molecule-1 around adherent leukocytes. In the current study, we used an in vitro flow model to investigate the role of Src-induced cortactin phosphorylation in endothelium during polymorphonuclear leukocyte (PMN) transmigration through human umbilical vein endothelium (HUVEC) monolayers preactivated with tumor necrosis factor-␣. Inhibition of Src in HUVEC using Src kinase inhibitors PP2 and SU6656 reduced PMN transmigration by 45Ϯ8% and 36Ϯ6%, respectively. Live cell imaging of green fluorescent protein-tagged cortactin in HUVEC revealed redistribution of cortactin in the region surrounding transmigrating PMN. Knockdown of cortactin in HUVEC by small interfering RNA also impaired transmigration to a similar degree, and this phenotype was rescued by reexpression of wild-type cortactin. Analysis of the location of initial arrest and locomotion of PMN adherent to HUVEC demonstrated that inhibition of Src tyrosine kinases or pretreatment with cortactin small interfering RNA reduced PMN transmigration at endothelial cell-to-cell junctions and not adhesion. Tyrosine phosphorylation of cortactin was important for transmigration, because expression of a mutant, in which the tyrosine phosphorylation sites were mutated to phenylalanine (cortactin3F), failed to rescue PMN transmigration. Moreover, expression of cortactin3F alone partially blocked PMN transmigration. These data suggest a model whereby tyrosine phosphorylation of cortactin by Src family kinases regulates PMN transmigration (Circ Res. 2006;98:394-402.)Key Words: leukocytes Ⅲ endothelium Ⅲ transendothelial migration A key component of the inflammatory response is leukocyte recruitment to sites of injury, immune response, or infection. Leukocytes interact with the vascular endothelium through sequential, overlapping steps including leukocyte arrest, rolling, firm adhesion, and ultimately transendothelial migration (TEM) through the vascular endothelium to underlying tissues. 1 Leukocyte adhesion or crosslinking of the endothelial adhesion molecules E-selectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, or P-selectin triggers association of these molecules with the endothelial cytoskeleton and transmits outside-in signals that include increases in both intracellular Ca 2ϩ concentration and myosin contractility and activation of p38 kinase, pp 60src (Src), and Rho GTPase family. 2,3 These signals lead to rapid cytoskeletal reorganization. [1][2][3] Previous studies have shown that treatment of endothelium with cytoskeletal disrupting agents or inhibitors of myosin light-chain kinase (MLCK) blocks leukocyte transmigration, 4 suggesting that cytoskeletal reorganization is...

Cortactin Has an Essential and Specific Role in Osteoclast Actin Assembly

“…When expressed in cells with endogenous cortactin, this mutant had only a small dominant negative effect, and the mutant protein localized to podosomes, suggesting that the tyrosine residues have a primary or direct role in cortactin function. Consistent with our results, the 3YF cortactin mutant failed to rescue a cortactin knockdown defect in endothelial cells assayed for the ability to support leukocyte transmigration (Yang et al, 2006).…”

Nifedipine Interferes with Migration of Vascular Smooth Muscle Cells via Inhibition of Pyk2-Src Axis

“…In the present study, PP2 and KI-Src inhibited VSMC migration, indicating that Src kinase activity mediates VSMC migration. Nifedipine potently inhibited PDGF-induced Src activation and tyrosine phosphorylation of Cas, paxillin, and cortactin, all of which have been implicated in cell migration [16][17][18] . Indeed, Kyaw and colleagues reported that Cas is involved in Anginduced VSMC migration 23) , and we previously used RNA interference techniques to demonstrate that Cas, cortactin, and paxillin are required for VSMC migration (unpublished data).…”

“…Src phosphorylates several actin-associated proteins, such as Cas, paxillin, and cortactin 14,15) . Tyrosine phosphorylation of these proteins has been reported to be involved in cell migration [16][17][18] . PDGF increased tyrosine phosphorylation of Cas, paxillin, and cortactin, and nifedipine significantly inhibited PDGF-induced phosphorylation of these proteins (Fig.…”

ICAM-1-Mediated, Src- and Pyk2-Dependent Vascular Endothelial Cadherin Tyrosine Phosphorylation Is Required for Leukocyte Transendothelial Migration

“…Yang et al (22,43) have demonstrated a requirement for Src-mediated phosphorylation of cortactin specifically in paracellular leukocyte TEM. It was also shown that cortactin phosphorylation is required for leukocyte-mediated cytoskeletal remodeling as well as clustering of ICAM-1 (43).…”