Differential Roles of Very Low-density Lipoprotein Receptor Splice Variants in Regulating Wnt Signaling

Abstract

Purpose: Very low-density lipoprotein receptor (VLDLR) is a multi-ligand receptor and has diverse roles beyond lipoprotein metabolism. Two alternative splice variants of VLDLR, variant I (VLDLRI) and variant II (VLDLRII) are differentially expressed in various tissues. Our previous studies have shown that VLDLR negatively regulates Wnt signaling in the retina and plays important roles in the regulation of retinal microvasculature. The present study was undertaken to investigate the differential roles of VLDLR splice variants in the regulation of Wnt signaling.

Methods: The expression of VLDLRI and VLDLRII in various tissues was analyzed by RT-PCR. VLDLRI and VLDLRII adenovirus were separately delivered into the vitreous space of VLDLR^-/- mice, and Wnt signaling activity was evaluated by Western blot analysis. Levels of the shed VLDLR extracellular domain (sVLDLR-N) and full-length VLDLR were measured by Western blot analysis, and the ratio of sVLDLR-N to full-length VLDLR was determined by densitometry. The impact of shed/full-length VLDLRI/II on Wnt signaling activity was evaluated by TOP-FLASH assay. Hypoxia was induced in human retinal epithelial cells by 100nM CoCl₂ for 24h.

Results: Most of the examined murine tissues expressed both VLDLRI and VLDLRII, while the retina expressed only VLDLRII. The exclusive expression of VLDLRII was also verified in the human retina. VLDLRII showed a more potent inhibitory effect on Wnt signaling in the retina of VLDLR^-/- mice compared to VLDLRI. sVLDLR-N was detected in the interphotoreceptor matrix of bovine eyes and conditioned medium (CM) from cells expressing VLDLR. The ratio of shed/full-length VLDLR was dramatically higher in cells expressing VLDLRII than that of cells expressing VLDLRI. Moreover, sVLDLR-N was capable of inhibiting Wnt signaling and exhibited a more potent Wnt inhibitory effect than full-length VLDLR. CM from cells expressing VLDLRII demonstrated a more potent Wnt inhibitory effect than those from cells expressing VLDLRI. Further, levels of sVLDLR-N were significantly decreased in human retinal epithelial cells under CoCl₂-induced hypoxia with/without overexpression of VLDLR.

Conclusions: VLDLR splice variants have differential regulatory effects on Wnt signaling due to the differential extracellular domain shedding, and the shed VLDLR extracellular domain may represent a novel mechanism for Wnt signaling regulation in the retina.