Signaling Mechanism of Blood-Retinal Barrier Regulation: Role of Mitogen-Activated

Abstract

Breakdown of the blood-retinal barrier (BRB) is an early hallmark of diabetic retinopathy. A critical component in retinal vascular hyper-permeability is increased production of vascular endothelial growth factor (VEGF). VEGF is a potent permeability factor that activates mitogen-activated protein (MAP) kinases. Pigment epithelium-derived factor (PEDF), an endogenous anti-permeability factor, blocks VEGF-induced vascular permeability increase. However, the mechanisms underlying the actions of VEGF and PEDF in regulating endothelial permeability are not yet clear. Previous studies in our laboratory have shown that VEGF induces paracellular permeability via beta-catenin nuclear translocation/transcriptional activation and subsequent upregulation of urokinase plasminogen activator receptor (uPAR). This current study tests the role of two MAP kinases, p38 and extracellular-signal regulated kinase (ERK), in regulating VEGFinduced beta-catenin signaling, uPAR expression and BRB breakdown. We also evaluate the effects of PEDF on this VEGF permeability inducing pathway. The role of MAP kinase in this VEGF permeability inducing pathway was first evaluated using inhibitors of p38 and ERK. These inhibitors preserve the endothelial barrier function upon VEGF treatment. In confluent endothelial cells, cytosolic beta-catenin is phosphorylated by glycogen synthase kinase (GSK) then ubiquitinated and degraded. With VEGF treatment, GSK is phosphorylated/inactive followed by beta-catenin cytosolic accumulation, nuclear translocation and subsequent uPAR expression. These effects were blocked by MAP kinases inhibitors. This indicates p38 and ERK as mediators of VEGF-induced beta-catenin signaling, uPAR expression and endothelial barrier breakdown. Next, it was found that PEDF not only blocks VEGF-induced endothelial permeability increase and MAP kinase activation but also prevents the activation of GSK/beta-catenin signaling as well as uPAR expression. However, PEDF did not block VEGF receptor-2 (VEGFR-2) phosphorylation suggesting that PEDF acts downstream of VEGFR-2 and upstream of MAP kinase level. To further evaluate the role of p38 in regulating VEGF-induced permeability, adenovirusmediated delivery of p38alpha mutants was used. One p38alpha mutant has an altered ATP-binding site thus looses its activity. It is more efficient in blocking VEGF-induced GSK/beta-catenin signaling, uPAR expression and paracellular permeability increase. This study identifies p38alpha and ERK as mediators of VEGF permeability-inducing signaling. They could also serve as potential therapeutic targets for diseases featured by blood-retinal barrier dysfunction.