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 microg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 microg/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, gamma-catenin, and p120(Cas). These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.
Goldblum
S E
SE
Division of Infectious Diseases, Department of Medicine, Department of Veterans Affairs Medical Center, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Young
B A
BA
Wang
P
P
Murphy-Ullrich
J E
JE
D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004731:Endotoxins / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000378:metabolism / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects; D007365:Intercellular Junctions / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000276:immunology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011506:Proteins / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D019700:Thrombospondin 1 / Q000378:metabolism* / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D002250:Carbon Radioisotopes; D004731:Endotoxins; D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D019700:Thrombospondin 1; D016212:Transforming Growth Factor beta; D019000:Phosphotyrosine; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases