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

W Shen, S Li, SH Chung, L Zhu, J Stayt, T Su… - European journal of cell …, 2011 - Elsevier
W Shen, S Li, SH Chung, L Zhu, J Stayt, T Su, PO Couraud, IA Romero, B Weksler…
European journal of cell biology, 2011Elsevier
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 …
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 14C-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 14C-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.
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