Heterodimerization of VLDLR and LRP6: A novel mechanism for the inhibition of the Wnt/β-catenin pathway.

Abstract

Purpose: We have previously shown that abnormal angiogenesis in the retinas of very low-density lipoprotein receptor knock-out (Vldlr^-/-) mice, which closely resembles that in some forms of wet age-related macular degeneration in humans, is mediated via activation of Wnt/β-catenin signaling. The purpose of this study was to investigate a novel function of VLDLR as an inhibitory regulator of the Wnt pathway, via binding with the Wnt co-receptor, low-density lipoprotein-receptor-related protein 6 (LRP6).

Methods: To assess the impact of VLDLR knock-down on Wnt activity (TCF/β-catenin activity), a luciferase-based promoter assay (TOPFLASH assay) was performed in hTERT-RPE-1 cells transfected with TOPFLASH, control pRL-TK reporter plasmids and siRNA (either Vldlr-specific or scrambled control). Conditioned media with or without Wnt3A were added to the transfected cells, to induce TCF/β-catenin activity. Following the incubation, the cells were harvested, and luciferase activity was measured. Western blotting was performed to investigate the knock-down efficiency of VLDLR. Since Vegf is a downstream target of TCF/β-catenin, we also measured levels of secreted VEGF in cultured medium by ELISA. Furthermore, interactions between endogenous VLDLR and LRP6 in RPE cells were examined with immunoprecipitation.

Results: VLDLR levels were decreased as a result of siRNA transfection. Phosphorylated LRP6 levels were increased by knock-down of VLDLR. Furthermore, the siRNA knock-down of VLDLR led to elevation of transcriptional activity of TCF/β-catenin in the presence or absence of exogenous Wnt3A, verifying the activation of Wnt signaling. Consistently, levels of secreted VEGF were also significantly elevated by VLDLR knock-down. In addition, endogenous VLDLR co-immunoprecipitated with LRP6 in RPE cells, suggesting physical interactions between VLDLR and LRP6.

Conclusions: VLDLR functions as a negative regulator of Wnt signaling through binding with LRP6.