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Abstract
Vascular endothelial growth factor (VEGF) alters tight junctions (TJs) and promotes vascular permeability in many retinal and brain diseases. However, the molecular mechanisms of barrier regulation are poorly understood. Here we demonstrate that occludin phosphorylation and ubiquitination regulate VEGF-induced TJ protein trafficking and concomitant vascular permeability. VEGF treatment induced TJ fragmentation and occludin trafficking from the cell border to early and late endosomes, concomitant with increased occludin phosphorylation on Ser-490 and ubiquitination. Furthermore, both co-immunoprecipitation and immunocytochemistry demonstrated that VEGF treatment increased the interaction between occludin and modulators of intracellular trafficking that contain the ubiquitin interacting motif, including Epsin-1, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Inhibiting occludin phosphorylation by mutating Ser-490 to Ala suppressed VEGF-induced ubiquitination, inhibited trafficking of TJ proteins, and prevented the increase in endothelial permeability. In addition, an occludin-ubiquitin chimera disrupted TJs and increased permeability without VEGF. These data demonstrate a novel mechanism of VEGF-induced occludin phosphorylation and ubiquitination that contributes to TJ trafficking and subsequent vascular permeability.
Highlights
Suggests that a number of pathological eye diseases such as diabetes, retinopathy of prematurity, age-related macular degeneration, inflammation, and infectious diseases disrupt the tight junctions (TJs) altering the blood-retinal barrier
Immunoreactivity for occludin was continuous at the cell borders (Fig. 1A), whereas Vascular endothelial growth factor (VEGF) treatment (50 ng/ml, 15 min) disrupted occludin immunoreactivity at the cell border and increased intracellular punctate labeling, which co-localized with early endosome antigen 1 (Fig. 1, B and E)
bovine retinal endothelial cells (BRECs) were treated with VEGF for the indicated times and immunoprecipitated with an antibody to occludin followed by immunoblotting with an antibody to ubiquitin
Summary
Materials—Recombinant human VEGF165 was purchased from R&D Systems (Minneapolis, MN). Twenty cells transfected with each occludin construct were selected at random, followed by masked grading. Transfection—Transfection of plasmid containing occludin mutants or HA-tagged ubiquitin was achieved using the nucleofection technique (Amaxa Biosystems), according to the manufacturer’s instructions [28]. Co-immunoprecipitation—The protein interactions were examined using co-immunoprecipitation of cell lysates, according to a modified protocol described previously [26]. BREC on 2 ϫ 100-mm culture plates were lysed and homogenized with co-immunoprecipitation buffer (1% Nonidet P-40, 10% glycerol, 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM N-ethylmaleimide, 1 mM NaVO4, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM benzamidine, complete protease inhibitor mixture). Immunoblotting with antibodies against occludin or phosphorylated occludin at Ser490 was performed to evaluate whether the pool of ubiquitinated proteins contain these proteins. A two-tailed t test (two conditions) or analysis of variance (three or more conditions) was performed using software for statistical analysis (InStat 3.05; GraphPad). p Ͻ 0.05 was considered significant
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