TNF-α increases tyrosine phosphorylation of vascular endothelial cadherin and opens the paracellular pathway through fyn activation in human lung endothelia

Tumor necrosis factor (TNF)-α is a key mediator of sepsis-associated multiorgan failure, including the acute respiratory distress syndrome. We examined the role of protein tyrosine phosphorylation in TNF-α-induced pulmonary vascular permeability. Postconfluent human lung microvascular and pulmonary artery endothelial cell (EC) monolayers exposed to human recombinant TNF-α displayed a dose- and time-dependent increase in transendothelial [¹⁴C]albumin flux in the absence of EC injury. TNF-α also increased tyrosine phosphorylation of EC proteins, and several substrates were identified as the zonula adherens proteins vascular endothelial (VE)-cadherin, and β-catenin, γ-catenin, and p120 catenin (p120^ctn). Prior protein tyrosine kinase (PTK) inhibition protected against the TNF-α effect. TNF-α activated multiple PTKs, including src family PTKs. Prior PTK inhibition with the src-selective agents PP1 and PP2 each protected against ∼60% of the TNF-α-induced increment in [¹⁴C]albumin flux. PP2 also blocked TNF-α-induced tyrosine phosphorylation of VE-cadherin, γ-catenin, and p120^ctn. To identify which src family kinase(s) was required for TNF-α-induced vascular permeability, small interfering RNA (siRNA) targeting each of the three src family PTKs expressed in human EC, c-src, fyn, and yes, were introduced into the barrier function assay. Only fyn siRNA protected against the TNF-α effect, whereas the c-src and yes siRNAs did not. These combined data suggest that TNF-α regulates the pulmonary vascular endothelial paracellular pathway, in part, through fyn activation.