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(Circulation Research. 2006;98:394.)
© 2006 American Heart Association, Inc.

Cellular Biology

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

Lin Yang, Jennifer R. Kowalski, Xi Zhan, Sheila M. Thomas, Francis W. Luscinskas

From the Center for Excellence in Vascular Biology (L.Y., F.W.L.), Departments of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass; Departments of Medicine (J.R.K., S.M.T.), Beth Israel-Deaconess Medical Center and Harvard Medical School, Boston, Mass; Biological and Biomedical Sciences Program (J.R.K.), Harvard Medical School, Boston, Mass; Department of Pathology (X.Z.), Marlene and Stewart Greenbaum Cancer Center and Center of Vascular and Inflammatory Diseases, University of Maryland, Baltimore.

Correspondence to F.W. Luscinskas, Brigham and Women’s Hospital, 77 Ave Louis Pasteur, NRB752, Boston, MA 02115. E-mail fluscinskas{at}rics.bwh.harvard.edu

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 45±8% and 36±6%, 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

Key Words: leukocytes • endothelium • transendothelial migration

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