Abstract
Purpose: :
Frizzled4 (Fzd4) mutations are associated with Familial Exudative Vitreoretinopathy (FEVR) and have more recently been reported in ROP patients. We previously reported a missense Fzd4 variant, (P33S;P168S) in both types of patients. Fzd4 is a receptor that can activate three intracellular Wnt signaling pathways; Wnt/β-catenin, planar cell polarity and Wnt-Ca++. We do not know which of these pathways are affected by various Fzd4 mutations; information that is essential for designing therapeutic strategies. A cell-culture Luciferase Reporter assay was required to assess the effect of the (P33S;P168S) variant upon the activation of the Wnt/β-catenin signaling pathway.
Methods: :
HEK293 cells were transfected with Fzd4 or mutant plasmids, LRP5 (co-receptor) and a TCF/LEF Firefly-luciferase reporter plasmid (SuperArray) to monitor activation of the Wnt B-Catenin pathway. A mutation previously reported to reduce activation of this pathway (C117R) was compared. Norrin was added to cells 24-hours post-transfection or a Wnt3a vector was co-transfected with other DNAs. Cells were lysed 48-hours post transfection for assay of Firefly and Renilla luciferase activities. Firefly luciiferase activity was normalized to Renilla luciferase activity to correct for transfection efficiency and cell number.
Results: :
There was no significant loss in reporter activity when activating the (P33S;P168S) Fzd4 variant with either Wnt3a or Norrin. However there was a 43% reduction of Wnt3a-induced activity with FZD4 (C117R). Our first trial of Norrin induced activity also resulted in a decrease by 70% when using the FZD4 (C117R) mutant. In the case of Norrin, additional experiments are underway to confirm the later result with the (C117R) mutant.
Conclusions: :
The (P33S;P168S) Fzd4 variant does not appear to block activation of Wnt/β-catenin signaling by Wnt3a or Norrin. Thus therapeutic strategies to increase receptor activation could be corrective or harmful depending on the specific FZD4 variant. Wnt3a has been shown to activate the Wnt-Ca++ pathway, but it is not known whether Norrin can also activate non- Wnt/β-catenin signaling. Further investigations are exploring effects of the (P33S;P168S) variant on the other pathways linked to the FZD4 receptor.
Keywords: retina • retinal development • retinal degenerations: hereditary