Regulation of Wnt/beta-catenin signaling by very low-density lipoprotein receptor and recombinant monoclonal antibodies

Purpose: Our overall goal is to illustrate how very low-density lipoprotein receptor (VLDLR), a member of the low-density lipoprotein receptor (LDLR) family, modulates Wnt/β-catenin signaling. Molecular mechanisms underlying VLDLR-mediated regulation of Wnt/β-catenin signaling allow us to evaluate anti-VLDLR monoclonal antibody as a potential drug intervention in Wnt-related diseases such as melanoma and diabetic retinopathy (DR). In addition, we generated a monoclonal antibody that blocks lowdensity lipoprotein receptor-related protein (LRP) 6 and tested its efficacy to attenuate Wnt signaling activation in two models of DR.;Methods: We utilized multiple approaches to understand the regulatory role of VLDLR in Wnt/β-catenin signaling. For loss- or gain-of-function studies, we either transfected small interfering RNA or expression plasmids of VLDLR (including full- length (FL), Cterminus deletion (VLDLRΔC), N-terminus deletion (VLDLRΔN), and soluble extracellular domain of VLDLR (sVLDLR-N)), respectively, in hTERT-RPE-1 cells. To measure Wnt/β-catenin signaling in RPE cells, a vector expressing firefly luciferase under the control of 8xTCF/LEF binding elements in the proximal promoter region was cotransfected with a control renilla luciferase vector. We used co-immunoprecipitation assays, surface biotinylation, and protein half-life measurement to validate our putative mechanism, in which VLDLR destabilizes LRP6 through a physical interaction at the plasma membrane. Moreover, monoclonal antibodies specific to the extracellular domain of VLDLR (α-VLDLR) were utilized to prevent heterodimerization between VLDLR and LRP6 and activate Wnt signaling. As a proof of concept study, we tested the antiproliferative effect of monoclonal anti-VLDLR antibody (3D10C9) in murine B16-F1 melanoma cells using a cell proliferation assay and cell cycle analysis. Moreover, efficacy of anti-LRP6 antibody (Mab2F1) was tested to evaluate inhibitory effect on Wnt signaling, vascular leakage, and inflammation in retinal cells and in animal models of DR.;Results: Knockdown of Vldlr resulted in elevation of LRP6 levels and activation of Wnt/β-catenin signaling, while over-expression of VLDLR suppressed it. Overexpression of VLDLR deletion mutants revealed that the extracellular domain of VLDLR is responsible Wnt/β-catenin signaling inhibition. Further, the soluble VLDLR ectodomain is essential and sufficient to inhibit Wnt/β-catenin signaling. The VLDLR ectodomain accelerated degradation of LRP6 and reduced LRP6 levels on the cell surface, suggesting accelerated internalization and degradation of LRP6. The accelerated degradation of LRP6 may be through heterodimerization between LRP6 and VLDLR through their extracellular domains, as shown by co-immunoprecipitation. Monoclonal antibodies specific for the VLDLR ectodomain blocked the VLDLR:LRP6 heterodimerization, resulting in enhanced Wnt/β-catenin signaling in vitro. Intravitreal injection of one clone of the monoclonal antibodies enhanced Wnt/β-catenin signaling in the retina. Further, intraperitoneal injection of the monoclonal antibody in BAT-gal mice developed positive β-galactosidase staining in the kidney and small intestine, validating activation of Wnt/β-catenin signaling by dissociation of the heterodimer in vivo. Mab2F1 showed robust inhibition of Wnt signaling and downregulation of angiogenic/inflammatory factors. In addition, Mab2F1 ameliorated retinal vascular leakage in oxygen-induced retinopathy and in diabetic rats. Moreover, Mab2F1 suppressed diabetes-induced inflammation in the retinae of STZ-diabetic rats.;Conclusion: Our results demonstrate that heterodimerization of VLDLR and LRP6 through their ectodomains mediates accelerated turnover of LRP6 and represents a new regulatory mechanism of Wnt/β-catenin signaling. These results suggest that dynamic interactions between LDLR family members serve as an endogenous mechanism for modulating Wnt/β-catenin signaling. This novel mechanism qualifies the VLDLR ectodomain as a means to suppress Wnt/β-catenin signaling. Furthermore, the α-VLDLR antibody could provide clinical benefits to activate Wnt/β-catenin signaling in diseases associated with diminished Wnt signaling, such as melanoma. On the other hand, anti- LRP6 antibody, Mab2F1, clearly demonstrated the importance of LRP6 in Wnt signaling activation, and had beneficial effects in the pathogenesis of DR.