Cross-Inhibition of Norrin and TGF-ß Signaling Modulates Development of Retinal and Choroidal Vasculature.

ABSTRACT

Purpose: Norrin is essential for the formation of the retinal vasculature during development and promotes its repair after damage via activation of Wnt/ß-catenin signaling. Since retinal TGF-ß signaling has essentially opposite effects on the retinal vasculature we investigated if and how Norrin inhibits TGF-ß signaling, and vice versa. Methods: Eyes from transgenic mice with an overexpression of Norrin (ßB1-Norrin) and/or active TGF-ß (ßB1-TGF-ß1) in the lens were generated and analyzed by light microscopy, immunohistochemistry, and TUNEL. Further on, protein as well as mRNA levels were investigated by Western blot analyses and real-time RT-PCR, respectively. Results: In ßB1-TGF-ß1 mice, the lack of retinal vascular development and choriocapillaris maintenance was rescued when transgenic Norrin was additionally overexpressed in the eye. In addition, retinal Wnt/ß-catenin signaling and the levels of SMAD7, an inhibitor of the canonical TGF-ß pathway, were substantially suppressed in retinae of ßB1-TGF-ß1 mice. In contrast, Norrin normalized Wnt/ß-catenin signaling and SMAD7 levels in double transgenic mice. Moreover, in retinae of ßB1-TGF-ß1 mice, the amounts of phosphorylated SMAD3, a downstream mediator of TGF-ß signaling, were increased compared to those of ßB1-Norrin/ßB1-TGF-ß1 mice. In vitro, Norrin substantially reduced the TGF-ß-mediated induction of target genes, an effect that was blocked by Dickkopf-1, a specific inhibitor of Wnt/ß-catenin signaling. Conclusions: High amounts of TGF-ß in the eye cause a substantial reduction in the activity of Wnt/ß-catenin signaling. This effect is inhibited in the presence of high amounts of Norrin, which further induce the expression of SMAD7 to inhibit TGF-ß signaling.