Tumor necrosis factor-alpha causes barrier dysfunction mediated by tyrosine198 and tyrosine218 in beta-actin

We tested the hypothesis that tumor necrosis factor-alpha (TNF) induces barrier dysfunction of pulmonary microvessel endothelial monolayers (PMEM) mediated by specific tyrosine residues in beta-actin. PMEM were transfected with a wild-type, mutant [tyrosine(198) to phenylalanine(198) (Y198F)], mutant Y218F, or mutant Y306F beta-actin construct tagged with enhanced yellow fluorescent protein (EYFP-beta-actin). The cellular compartmentalization of wild-type and mutant EYFP-beta-actin was displayed using EYFP fluorescence of the tagged beta-actin. beta-Actin was quantified for the EYFP-tagged and native beta-actin using Western blot assay. The effect of the EYFP-beta-actin on a cell junction protein was assessed by association of EYFP-beta-actin with beta-catenin using confocal microscopy and coimmunoprecipitation. The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The cellular compartmentalization of wild-type and mutant EYFP-beta-actin was similar to the native beta-actin. Incubation of PMEM with TNF (100 ng/ml) for 0.5 h resulted in increases in permeability to albumin and a decrease in association of the EYFP-beta-actin with beta-catenin. However, the expression of the EYFP-Y198F beta-actin and EYFP-Y218F beta-actin prevented the effect of TNF on beta-catenin and barrier function. The vehicle, wild-type EYFP-beta-actin, and mutant Y306F beta-actin had no affect on the response to TNF. The data indicate that TNF induces an increase in endothelial permeability that is dependent on tyrosine(198) and tyrosine(218) in beta-actin.