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Xuwen Liu, David Antonetti; Occludin Ser490 phosphorylation regulates endothelial cell growth control and angiogenesis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1186.
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We have previously shown that occludin phosphorylation and ubiquitination regulate VEGF-induced tight junction protein trafficking and concomitant vascular permeability. Our recent studies report a novel function of occludin as a regulator of centrosome separation and mitotic entry in Madin-Darby canine kidney (MDCK) cells. However, it remains unclear if occludin contributes to growth control in endothelial cells. We hypothesize that phosphorylation of occludin Ser490 regulates endothelial cell proliferation and subsequently induces retinal angiogenesis.
Phosphorylation of occludin Ser490 was determined with phosphospecific Ab (P-Ser490Ab) in primary bovine retinal endothelial cells (BREC) through cell cycle. Immunofluorescence confocal microscopy was used to determine the location of phosphorylated occludin in mitotic BREC. Western Blot was performed with occludin P-Ser490Ab during VEGF-induced tube formation in BREC. VEGF-induced tube formation assay was performed in BREC transiently transfected with nonphosphorylatable alanine mutant of occludin Ser490, S471 (S490A, S471A) and aspartic acid phosphomimetic mutant (S490D, S471D) in pmaxFP-Green-C vector. Live-cell imaging of tube formation assay was performed on Confocal Microscope System at every 15 min for 22 h. The effect of occludin S490A mutants on in vitro angiogenesis was further evaluated by Click-iT EdU cell proliferation assay and modified Boyden chamber migration assay.
Analysis of cell cycle revealed that occludin Ser490 phosphorylation increased in mitotic endothelial cells. The increase in occludin Ser490 phosphorylation was consistent with the progression of BREC tube formation. Immunocytochemistry demonstrated phospho-Ser490 occludin at centrosomes in mitotic cells and was supported by live-cell imaging monitoring the dynamic trafficking of GFP-Occludin S490D during BREC tube formation. Transfection with occludin mutant S490A exhibited reduced cell proliferation and migration. Further, VEGF-induced tube formation was nearly completely prevented in BREC transfected by occludin S490A mutant, while there was little effect in occludin S471A mutant.
Occludin Ser490 phosphorylation is important to endothelial proliferation and angiogenesis. Further studies are warranted to identify the occludin interacting protein(s) and determine the molecular mechanisms of occludin-regulated retinal angiogenesis in vivo.
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