Endothelial cell cortactin phosphorylation by Src contributes to leukocyte transendothelial migration in vitro

• Published in: The FASEB journal 2006-03, Vol.20 (5), p.A863-A863
• Main Authors: Yang, Lin, Kowalski, J. R., Zhan, X., Thomas, S. M., Luscinskas, F. W.
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.20.5.A863

The underlying mechanisms that regulate leukocyte transendothelial migration (TEM) remain incompletely understood. 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 ICAM‐1 around adherent leukocytes. Here, an in vitro flow model was used to investigate the role of Src‐induced cortactin phosphorylation in TNF‐α‐activated human umbilical vein endothelium (HUVEC) during polymorphonuclear leukocyte (PMN) TEM of HUVEC. Pretreatment of HUVEC with Src kinase inhibitors PP2 or SU6656 reduced PMN transmigration (45± 8% & 36± 6%, respectively). Live cell imaging of GFP‐tagged cortactin in HUVEC revealed re‐distribution of cortactin to transmigrating PMN. Reduction of cortactin protein in HUVEC by small interfering RNA (siRNA) also impaired TEM to a similar degree. Interestingly, Src inhibitors or cortactin siRNA reduced PMN TEM at cell‐cell borders, but did not alter adhesion. The reduced TEM by cortactin siRNA was rescued by reconstitution with wild‐type murine cortactin. Tyrosine phosphorylation of cortactin was important for TEM, because expression of a cortactin mutant, in which the tyrosine phosphorylation sites were mutated to phenylalanine (cortacin3F), failed to rescue TEM. Moreover, expression of cortactin3F alone partially blocked PMN transmigration. These data suggest a model whereby cortactin regulates PMN transmigration through its tyrosine phosphorylation by Src family kinases. Supported by NIH grants HL53393 and HL 36028.