Blockade of VEGF-induced GSK/β-catenin signaling, uPAR expression and increased permeability by dominant negative p38α.

Abstract

The goal of this study was to define the role of p38alpha MAP kinase in VEGF-induced vascular permeability increase. Activation of p38 is correlated 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 inhibitors preserve endothelial barrier function and block VEGF-induced GSK/beta-catenin signaling. Here, we present data demonstrating that adenoviral vector delivery of a dominant negative p38alpha mutant blocks this signaling pathway and preserves barrier function. This p38alpha mutant was altered on its ATP-binding site, which eliminates its kinase activity. Bovine retinal endothelial (BRE) cells were transduced with recombinant adenovirus containing the p38alpha mutants or empty vector. Successful transduction was confirmed by expression of GFP and p38 increase. Blockade of p38 activity by p38alpha mutant was demonstrated by inhibition of VEGF-induced phosphorylation of a p38 target, MAP kinase activated protein kinase 2 (MK-2). The mutant also prevented VEGF-induced GSK phosphorylation and beta-catenin cytosolic accumulation and nuclear translocation as shown by cell fractionation and Western blotting. Quantitative real-time PCR demonstrated that this mutant inhibited VEGF-induced uPAR gene expression. Importantly, this same mutant also strongly abrogated VEGF-induced endothelial barrier breakdown as determined by measuring transcellular electrical resistance and tracer flux through endothelial cell monolayer. This study indicates a critical role of p38alpha in VEGF-induced permeability and offers a new strategy for developing potent and specific therapies for treatment of retinal diseases associated with vascular barrier dysfunction.

• Authors: Jinling Yang, Ruth B Caldwell, M Ali Behzadian
• Journal: Experimental eye research (Exp Eye Res) Vol. 100 Pg. 101-8 (Jul 2012) ISSN: 1096-0007 [Electronic] England
• PMID: 22564969 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: Copyright © 2012 Elsevier Ltd. All rights reserved.

Chemical References

• Receptors, Urokinase Plasminogen Activator
• Vascular Endothelial Growth Factor A
• beta Catenin
• Glycogen Synthase Kinase 3 beta
• Mitogen-Activated Protein Kinase 14
• Glycogen Synthase Kinase 3

Topics

• Adenoviridae (genetics)
• Animals
• Capillary Permeability (physiology)
• Cattle
• Electric Impedance
• Endothelial Cells (cytology, metabolism)
• Gene Expression Regulation, Enzymologic
• Genetic Vectors
• Glycogen Synthase Kinase 3 (antagonists & inhibitors, metabolism)
• Glycogen Synthase Kinase 3 beta
• Mitogen-Activated Protein Kinase 14 (physiology)
• Phosphorylation
• Real-Time Polymerase Chain Reaction
• Receptors, Urokinase Plasminogen Activator (metabolism)
• Retinal Vessels (cytology)
• Signal Transduction (drug effects)
• Vascular Endothelial Growth Factor A (pharmacology)
• beta Catenin (antagonists & inhibitors, metabolism)