Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC–EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations ≥1 μg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 μg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, γ-catenin, and p120(Cas). These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.