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.
Gertzberg
Nancy
N
Department of Pharmaceutical Science, Albany College of Pharmacy, Albany, NY 12208, USA.
Gurnani
Tina
T
Neumann
Paul
P
Forbes
Anne-Kay
AK
Jean-Louis
Natacha
N
Johnson
Arnold
A
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000378:metabolism; D008168:Lung / Q000098:blood supply*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism*
D000199:Actins; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C054562:yellow fluorescent protein, Bacteria; D014443:Tyrosine