Peroxynitrite-dependent activation of protein phosphatase type 2A mediates microvascular endothelial barrier dysfunction.
OBJECTIVE
We investigated the mechanism by which proinflammatory stimulation induces microvascular endothelial barrier dysfunction. Since protein phosphatase type 2A (PP2A) can mediate paracellular leak and can be inactivated by tyrosine phosphorylation in its catalytic subunit (PP2Ac), we hypothesized that microvascular endothelial cells exposed to proinflammatory stimulation produce peroxynitrite that nitrates PP2Ac, and this nitration inhibits tyrosine phosphorylation of PP2Ac and thereby increases PP2A activity to mediate endothelial barrier dysfunction.


RESULTS
Exposure of mouse skeletal muscle microvascular endothelial cell monolayers to a proinflammatory stimulus [lipopolysaccharide (LPS) + interferon (IFN)gamma] increased permeability to albumin, and this barrier dysfunction was attenuated by PP2A inhibitor okadaic acid or by siRNA (small interfering ribonucleic acid) against PP2Ac. LPS + IFN gamma increased synthesis of peroxynitrite precursors nitric oxide (NO) and superoxide by inducible NO synthase (iNOS) and NADPH oxidase, respectively. PP2Ac immunoprecipitates isolated from LPS + IFN gamma- or peroxynitrite-treated cells showed increased tyrosine nitration, decreased tyrosine phosphorylation and increased phosphatase activity. 3-Nitrotyrosine immunoprecipitates from LPS + IFN gamma-stimulated cells also exhibited increased PP2A activity. Further, iNOS inhibitor 1400W, iNOS deficiency, NADPH oxidase inhibitor apocynin, or p47phox deficiency prevented the increase in PP2A activity and preserved barrier function.


CONCLUSIONS
LPS + IFN gamma stimulates endothelial cells to produce iNOS-derived NO and NADPH oxidase-derived superoxide, which form peroxynitrite that nitrates tyrosine residues in PP2Ac and inhibits their phosphorylation. This nitration in PP2Ac is correlated with PP2A activation that mediates endothelial barrier dysfunction.
Wu
Feng
F
Department of Exercise and Nutrition Sciences, University at Buffalo, 3435 Main Street, G10 Farber Hall, Buffalo, NY 14214-8028, USA. fengwu@buffalo.edu
Wilson
John X
JX
D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology*; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000098:blood supply; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
D008070:Lipopolysaccharides; D013481:Superoxides; D030421:Peroxynitrous Acid; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D054648:Protein Phosphatase 2
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