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T. Murakami, E. A. Felinski, D. A. Antonetti; Occludin Phosphorylation and Ubiquitination Regulate VEGF-induced Vascular Permeability. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4978.
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Alterations to the tight junctions promote vascular permeability in retinal diseases. However, the molecular mechanisms of barrier regulation are poorly understood. Here we investigated how VEGF regulates the phosphorylation and ubiquitination of occludin and how these posttranslational modifications contribute to VEGF-induced tight junction (TJ) protein trafficking and concomitant vascular permeability.
Primary bovine retinal endothelial cells (BRECs) were grown with MCDB-131 media supplemented with 10% FBS. Plasmid expressing human occludin and occludin mutants were transfected with Amaxa Nucleofection. Western blot was performed for the evaluation of protein contents, immunoprecipitation (IP) for detecting ubiquitination, and co-immunoprecipitation (co-IP) for protein-protein interaction. Immunostaining of cells on coverslip was performed with fluorescent secondary antibodies and analyzed by confocal microscopy (Leica). BREC permeability was evaluated by the flux of 70kDa RITC labeled dextran or transendothelial electrical resistance (TER).
VEGF treatment induced TJ fragmentation and occludin trafficking from the cell border to early and late endosomes, concomitant with increased occludin phosphorylation on Ser490 and ubiquitination. Further, both co-IP 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, Eps15 and Hrs. Mutating occludin Ser490 to Ala suppressed VEGF-induced interaction between occludin and these modulators of intracellular trafficking, inhibited the redistribution of TJ proteins and prevented the increase in endothelial permeability. In addition, an occludin-ubiquitin chimera disrupted TJs and increased endothelial permeability in the absence of VEGF.
These data demonstrate a novel mechanism of VEGF-induced occludin phosphorylation and ubiquitination that contributes to the trafficking of tight junctions and subsequent vascular permeability.
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