Tyrosine phosphorylation of VE-cadherin and claudin-5 is associated with TGF-β1-induced permeability of centrally derived vascular endothelium

• DOI: 10.1016/j.ejcb.2010.10.013
• Publication Date: 2010-12-19T09:06:29Z
• Authors: Weiyong Shen; Shiying Li; Sook Hyun Chung; Ling Zhu; Jason Stayt; Tao Su; Pierre-Olivier Co...; Ignacio A. Romero; Babette Weksler; Mark C. Gillies
• Keywords: 0303 health sciences; Membrane Proteins; Cadherins; 3. Good health; Capillary Permeability; Transforming Growth Factor beta1; 03 medical and health sciences; Antigens, CD; Animals; Humans; Tyrosine; Cattle; Claudin-5; Endothelium, Vascular; Phosphorylation; Protein Tyrosine Phosphatases; Vanadates; Cells, Cultured

Breakdown of the inner blood-retinal barrier and the blood-brain barrier is associated with changes in tight and adherens junction-associated proteins that link vascular endothelial cells. This study aimed to test the hypothesis that transforming growth factor (TGF)-β1 increases the paracellular permeability of vascular endothelial monolayers through tyrosine phosphorylation of VE-cadherin and claudin-5. Bovine retinal and human brain capillary endothelial cells were grown as monolayers on coated polycarbonate membranes. Paracellular permeability was studied by measuring the equilibration of (14)C-inulin or fluorescence-labelled dextran. Changes in VE-cadherin and claudin-5 expression were studied by immunocytochemistry (ICC) and quantified by cell-based enzyme linked immunosorbent assays (ELISA). Tyrosine phosphorylation of VE-cadherin and claudin-5 was studied by ICC, immunoprecipitation and Western blotting. We found that exposure of endothelial cells to TGF-β1 caused a dose-dependent increase in paracellular permeability as reflected by increases in the equilibration of (14)C-inulin. This effect was enhanced by the tyrosine phosphatase inhibitor orthovanadate and attenuated by the tyrosine kinase inhibitor lavendustin A. ICC and cell-based ELISA revealed that TGF-β1 induced both dose- and time-dependent decreases in VE-cadherin and claudin-5 expression. Assessment of cell viability indicated that changes in these junction-associated proteins were not due to endothelial death or injury. ICC revealed that tyrosine phosphorylation of endothelial monolayers was greatly enhanced by TGF-β1 treatment, and immunoprecipitation of cell lysates showed increased tyrosine phosphorylation of VE-cadherin and claudin-5. Our results suggest that tyrosine phosphorylation of VE-cadherin and claudin-5 is involved in the increased paracellular permeability of central nervous system-derived vascular endothelium induced by TGF-β1.