We tested the hypothesis that tumor necrosis factor-α (TNF) induces barrier dysfunction of pulmonary microvessel endothelial monolayers (PMEM) mediated by specific tyrosine residues in β-actin. PMEM were transfected with a wild-type, mutant [tyrosine198 to phenylalanine198 (Y198F)], mutant Y218F, or mutant Y306F β-actin construct tagged with enhanced yellow fluorescent protein (EYFP-β-actin). The cellular compartmentalization of wild-type and mutant EYFP-β-actin was displayed using EYFP fluorescence of the tagged β-actin. β-Actin was quantified for the EYFP-tagged and native β-actin using Western blot assay. The effect of the EYFP-β-actin on a cell junction protein was assessed by association of EYFP-β-actin with β-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-β-actin was similar to the native β-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-β-actin with β-catenin. However, the expression of the EYFP-Y198F β-actin and EYFP-Y218F β-actin prevented the effect of TNF on β-catenin and barrier function. The vehicle, wild-type EYFP-β-actin, and mutant Y306F β-actin had no affect on the response to TNF. The data indicate that TNF induces an increase in endothelial permeability that is dependent on tyrosine198 and tyrosine218 in β-actin.
Activation of muscarinic cholinoceptor ameliorates tumor necrosis factor-α-induced barrier dysfunction in intestinal epithelial cells