Abstract
Purpose :
Previous studies reveal that norrin reverses VEGF-induced permeability in a β-catenin dependent pathway. Here, we explored the contribution of disheveled-1 (Dvl1) in norrin-induced blood-retinal barrier (BRB) restoration. We hypothesized that Dvl1 promotes tight junction (TJ) stabilization through both canonical and non-canonical signaling pathways.
Methods :
BRB properties in primary bovine retinal endothelial cells (BREC) were determined by measurements of transendothelial electrical resistence (TEER) or solute flux of 70kDa RITC-dextran. The knockdown of Dvl1 using siRNAs was confirmed by qRT-PCR and Axin2 mRNA content was used as a measure of β-catenin signaling activity. Dvl1 localization at the TJ was observed by immunofluorescence confocal microscopy and the interaction between Dvl1 and claudin-5 or ZO-1, was analyzed by co-immunoprecipitation assays in BREC or in HEK293 cells co-transfected with Dvl1 mutants and claudin-5 or ZO-1.
Results :
Analysis of BRB properties in BREC demonstrated that norrin was able to completely restore TEER after VEGF. The knockdown of Dvl1 using siRNAs specifically reduced basal barrier properties and ablated norrin-induced barrier restoration, despite increased b-catenin signaling in knockdown samples. Similar results were found in flux assays of a 70 kDa RITC-dextran molecule, suggesting that Dvl1 is required for norrin-induced BRB restoration. Dvl immunofluorescence staining showed co-localization of Dvl1 with ZO-1 and claudin-5 at the TJ complex. Dvl1 and TJ proteins interaction analysis by co-immunoprecipitation of endogenous protein in BREC, demonstrated that Dvl1 interacts with both claudin-5 and ZO-1 and this interaction was most abundant in the presence of VEGF/norrin co-stimulation. Studies in HEK293 cells co-transfected with Dvl1 mutants and claudin-5 or ZO-1, reveal a requirement of the Dvl1 PDZ domain for this interaction. Transfection of the C-terminal fragment of Dvl1, containing the PDZ binding motif but not the DIX, PDZ or DEP domains, blocked Dvl1/claudin-5 interaction, increased basal permeability and prevented norrin-induced BRB restoration.
Conclusions :
Together, these results demonstrate that norrin signals though Dvl1 to stimulate barrier properties and suggest a non-canonical signaling role of Dvl1 in regulation of barrier properties through direct binding to claudin-5 and ZO-1.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.