The counteradhesive proteins SPARC and TSP regulate an endothelial paracellular pathway through protein tyrosine phosphorylation

Abstract

SPARC (Secreted Protein Acidic and Rich in Cysteine) and thrombospondin-1 (TSP) are secreted glycoproteins that influence multiple endothelial cell (EC) functions including cell attachment to and spreading on substrates, cell motility and angiogenesis. These two counteradhesive proteins have been grouped together solely on a functional basis. By definition, both SPARC and TSP influence EC-extracellular matrix interactions and promote focal adhesion disassembly. We studied whether SPARC and TSP influence homophilic EC-EC adhesion and vascular endothelial barrier function. Actin-dependent homophilic EC-EC adhesion is vital to the formation and maintenance of an endothelial barrier. Although the signal transduction pathways responsible for regulating endothelial barrier function and opening of the paracellular pathway are unclear, tyrosine phosphorylation of actin-dependent cell-cell adherens junction proteins diminishes homophilic cell-cell adhesion. We have now demonstrated that SPARC and TSP regulate movement of a 14C-bovine serum albumin (BSA) tracer molecule across postconfluent EC monolayers in a tyrosine phosphorylation-dependent manner, in vitro. Pretreatment of EC monolayers with the protein tyrosine kinase (PTK) inhibitors, herbimycin A or genistein, protected against both SPARC- and TSP-induced barrier dysfunction. In contrast, pretreatment of EC monolayers with the protein tyrosine phosphatase (PTP) inhibitors, sodium orthovanadate (vanadate) or phenylarsine oxide enhanced the SPARC- and TSP-induced increments in 14C-BSA flux. SPARC- and TSP-exposed EC monolayers also exhibited actin reorganization and intercellular gap formation using F-actin epifluorescence microscopy. Further, PTK inhibition protected against intercellular gap formation. In the presence of PTP inhibition, both SPARC- and TSP-exposed EC exhibited dose-and-time dependent increases in protein tyrosine phosphorylation by immunoblotting. Several tyrosine phosphorylated proteins were identified as cell-cell adherens junction proteins. Further, these increases in protein tyrosine phosphorylation were localized to the EC-EC boundaries using epifluorescence microscopy. These findings indicate that SPARC and TSP regulate an endothelial paracellular pathway through protein tyrosine phosphorylation. In addition, our studies with the PTP inhibitor, vanadate, corroborate our findings with SPARC and TSP, and suggest that vascular endothelial barrier function is regulated through tyrosine phosphorylation of cell-cell adherens junction proteins.