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D. A. Antonetti, T. Murakami; Vascular Endothelial Growth Factor (VEGF) Activation of Protein Kinase C Beta (PKCB) Phosphorylates Occludin and Regulates Vascular Permeability in vivo. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5628.
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PKCB activation contributes to VEGF-induced vascular permeability in diabetic retinas. However, the obligatory downstream targets remain unclear. Previously, we demonstrated phosphorylation and subsequent ubiquitination of occludin was required for VEGF-induced permeabilty in endothelial cell culture. Here, we demonstrate that occludin is a downstream target of PKCB required for VEGF-induced permeabilty in cell culture and in vivo.
Primary bovine retinal endothelial cells were transfected with dominant negative PKCB (dnPKCB), wild-type PKCB (wtPKCB), or occludin constructs using Amaxa nucleofection. Cells and retina were treated with specific PKCB inhibitors or proteosome inhibitors. Endothelial permeability to 70kDa RITC dextran was measured across cell monolayers. Evan’s blue dye accumulation was used to measure retinal vascular permeability in vivo after intravitreal injection of VEGF. Immunoprecipitation and Western blotting were used to quantify occludin phosphorylation and ubiquitination. Confocal microscopy was used to detect tight junction protein distribution.
A broad PKC inhibitor and two specific chemical inhibitors of PKCB all blocked VEGF-induced Ser490 phosphorylation and occludin ubiquitination in cell culture and in vivo. Overexpression of wtPKCB stimulated, while dnPKCB blocked VEGF-induced occludin phosphorylation/ubiquitination and subsequent permeability. The effects of PKCB expression on permeability could be overcome completely by expressing a S490A mutant of occludin to block permeabilty or an occludin-ubiquitin chimera to promote permeability. Proteosome inhibition augmented VEGF-induced occludin phosphorylation/ubiquitination, occludin and claudin 5 redistribution, and permeability in both cell culture and in vivo.
These data demonstrate a novel mechanism of PKCB-mediated occludin phosphorylation and subsequent ubiquitination in vivo. Occludin ubiquitination is required for VEGF-induced tight junction redisruption and vascular permeability.
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