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Monica Diaz-Coranguez, Cheng-mao Lin, David A Antonetti; Disheveled localizes at tight junction sites and interacts with ZO1 in BREC. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4617.
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© ARVO (1962-2015); The Authors (2016-present)
Norrin signaling through FZD4, LRP5/6, and TSPAN12 receptor complex is required for developmental angiogenesis and formation of the blood-retinal barrier (BRB). We demonstrated previously that VEGF allows Norrin to signal by promoting TSPAN12 localization at the membrane and Norrin signaling after VEGF results in BRB restoration in a βCatenin-dependent manner. Here, we examined the contribution of disheveled (Dvl) in Norrin-induced BRB restoration. Our overall hypothesis is that Norrin signals through Dvl to promote tight junction (TJ) stabilization.
Dvl3-WT, -ΔDIX or -ΔPDZ mutants were transfected in primary bovine retinal endothelial cells (BREC) for measurements of solute flux of 70kDa RITC-dextran. Dvl expression was determined by Western blot and qRT-PCR, and its localization was observed by immunofluorescence (IF) confocal microscopy. Dvl/ZO1 interaction was analyzed by co-immunoprecipitation (CoIP) assays in BREC or in HEK293 cells co-transfected with Dvl mutants and ZO1. Data was analyzed with one-way ANOVA.
Permeability to 70 kDa RITC-dextran flux across BREC monolayers was increased after 24h of VEGF stimulation, and this was significantly reduced by ~50% by the transfection of Dvl3-ΔDIX suggesting a non-canonical signaling pathway contributes to BRB restoration. Dvl3-ΔPDZ mutant but not Dvl3-WT, recapitulated this pro-barrier effect suggesting a potential requirement for Dvl activation that is circumvented by the deletion mutants. All Dvl isoforms are expressed in BREC but only Dvl3 content increased with VEGF stimulation. Moreover, Norrin reduced Dvl3 content to control levels and induced changes in Dvl gel migration, suggesting that Norrin promotes post-translational modifications of Dvl. IF staining demonstrated co-localization of Dvl with ZO1 and claudin-5 at the TJ complex and CoIP experiments demonstrated that Dvl formed a complex with ZO1. Importantly, Dvl3/ZO1 interaction was most abundant in the presence of VEGF and Norrin together. These results were corroborated by co-transfection of Dvl3 or Dvl3 mutants and ZO1 in HEK293, where Dvl3 and ZO1 CoIP was readily observed. Consistent with permeability assays, mutations in DIX or PDZ domains did not prevent Dvl3/ZO1 interaction.
These results demonstrate that Norrin signaling promotes Dvl3 interaction with ZO1 and suggests Dvl may directly contribute to TJ complex formation and Norrin-induced BRB restoration after VEGF.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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