Abstract
Purpose :
Purpose: Previous studies demonstrated that norrin restores blood-retinal barrier (BRB) properties after vascular endothelial growth factor-induced permeability providing a potentially important new therapeutic approach to treat VEGF-driven macular edema. The norrin-induced barrier restoration requires disheveled 1 (Dvl1) as knock-down of Dvl1 prevents the norrin response. Here, we explore the contribution of Dvl1 to bovine retinal endothelial cell (BREC) barrier properties induced by norrin. We hypothesize that norrin signals throughDvl1 promoting claudin-5 interaction and stabilizing tight junctions (TJs).
Methods :
Methods: The localization of Dvl1 at the TJ was analyzed by confocal microscopy. The interaction between Dvl1 with claudin-5 was determined by co-immunoprecipitation in BREC and capture assays in HEK293 cells transfected with Dvl1 mutants. In BREC cells transfected with the C-terminal region of Dvl1 the expression of TJ proteins was evaluated by western blot and barrier properties were analyzed by measurement of the solute flux of 70kDa RITC-dextran.
Results :
Results: Dvl1 immunofluorescence showed co-localization with ZO-1 and claudin-5 at the TJ complex. The co-immunoprecipitation assays demonstrated that Dvl co-precipitates with both ZO-1 and claudin-5 and the interaction with claudin-5 appeared specific for Dvl1. Further, the interaction of Dvl1 with claudin-5 is stronger after the co-stimulation with VEGF/norrin. Dvl1 has previously been shown to form intramolecular binding between the C-terminal PDZ-binding domain (PDZ-bd) with an internal PDZ domain. Co-transfection of HEK293 cells with Dvl1 mutants and claudin-5 revealed that Dvl1 interacts with claudin-5 through the Dvl PDZ domain and claudin-5 PDZ-bd since this interaction increases with deletion of the Dvl1 PDZ-bd and is blocked by expressing the C-terminal fragment of Dvl1. In BREC cells, transfection of the C-terminal fragment of Dvl1 downregulates the expression of claudin-5 but has no effect on expression of other proteins of the TJs including: ZO-1, occludin, claudin-1, or VE-cadherin. Blocking Dvl1/claudin-5 interaction increased basal permeability and prevents norrin induction of barrier properties after VEGF.
Conclusions :
Conclusions: Altogether, these results demonstrate that norrin promotes the interaction between Dvl1 and claudin-5, stabilizing claudin-5 and inducing barrier properties.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.