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J. Yang, R. B. Caldwell, A. M. Behzadian; A Dominant Negative Mutant of p38 MAP Kinase Blocks VEGF-Induced uPAR Expression and Barrier Breakdown in Retinal Microvascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):38.
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To define the role of MAP kinases in VEGF-induced signaling pathway that lead to uPAR expression and vascular permeability increase. Activation of MAP kinases has been shown to correlate with increased permeability in endothelial cells treated with VEGF or high glucose and in retinas of diabetic animal models. We have shown previously that p38 MAP kinase inhibitors block VEGF-induced GSK / beta-catenin signaling and uPAR expression. Here, we present data showing that adenoviral vector delivery of dominant negative p38 mutants into retinal endothelial cells blocks VEGF-induced activation of the GSK / beta-catenin signaling pathway, prevents uPAR expression and preserves paracellular barrier function.
Two dominant negative mutants of p38 which were originally obtained from Dr. J. Han’s lab, one was mutated on the Thr180 and Tyr182 phosphorylation sites; the other on its ATP-binding site, thus loosing its kinase activity. The mutants of p38 gene were cloned into pShuttle-IRES-hrGFP and then into pAdEasy vector. Bovine retinal endothelial (BRE) cells were infected with recombinant adenovirus of those p38 dominant negative mutants or without insertions. Fluorescence microscopy was used to monitor expression of recombinant genes in infected BRE cells and Western blotting to analyze phosphorylation of GSK, p38 and the p38 substrates. uPAR expression was measured by quantitative real-time PCR and barrier function was monitored by measuring transcellular electrical resistance.
Both p38 mutants blocked VEGF-induced phosphorylation of p38 substrates MAPKAP-2 and ATF2, suggesting that VEGF-induced p38 activation was blocked. Moreover, VEGF-induced GSK phosphorylation was also reduced by both mutants. However, the VEGF-induced increase in uPAR expression was more potently inhibited by the kinase mutant of p38 as compared with either the phosphorylation mutant or empty vector. This same kinase mutant also strongly prevented VEGF-induced barrier breakdown.
Expression of a dominant negative p38 Map kinase mutant blocks the activation of p38 by VEGF, prevents phosphorylation of GSK, expression of uPAR and preserves endothelial cell barrier function. This indicates that VEGF-induced permeability depends on p38 kinase activity and offers a new strategy for developing potent and specific therapies for treatment of retinal diseases associated with vascular barrier dysfunction.
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