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

¹Department of Pharmaceutical Science, Albany College of Pharmacy; ²Research Service of the Stratton Veterans Affairs Medical Center; ³Albany Medical College; and ⁴State University of New York at Albany, Albany, New York

Submitted 6 March 2007 ; accepted in final form 21 August 2007

We tested the hypothesis that tumor necrosis factor- (TNF) induces barrier dysfunction of pulmonary microvessel endothelial monolayers (PMEM) mediated by specific tyrosine residues in -actin. PMEM were transfected with a wild-type, mutant [tyrosine¹⁹⁸ to phenylalanine¹⁹⁸ (Y198F)], mutant Y218F, or mutant Y306F -actin construct tagged with enhanced yellow fluorescent protein (EYFP--actin). The cellular compartmentalization of wild-type and mutant EYFP--actin was displayed using EYFP fluorescence of the tagged -actin. -Actin was quantified for the EYFP-tagged and native -actin using Western blot assay. The effect of the EYFP--actin on a cell junction protein was assessed by association of EYFP--actin with -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--actin was similar to the native -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--actin with -catenin. However, the expression of the EYFP-Y198F -actin and EYFP-Y218F -actin prevented the effect of TNF on -catenin and barrier function. The vehicle, wild-type EYFP--actin, and mutant Y306F -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 -actin.

edema; permeability; nitration