Abstract
Purpose :
Wnt signaling is a common signaling pathway to regulate multiple physiological processes. Recent studies reported that aberrant activation of Wnt signaling in the eye plays a key role in retinal inflammation, vascular leakage and neovascularization in diabetic retinopathy (DR) and age-related macular degeneration (AMD). Although both low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6 are co-receptors for Wnt ligands and both expressed in the retina, it is not clear which co-receptor plays a key role in over-activation of Wnt signaling in DR and AMD. In the present study, we generated LRP5 knockout (KO) and LRP6 KO cell lines to study the distinct roles of LRP5 and LRP6 in the regulation of Wnt signaling.
Methods :
LRP5 KO and LRP6 KO ARPE19 cells, a cell line derived from human retinal pigment epithelium (RPE) cells, were generated using CRISPR-Cas9 technology. Efficiency of genomic DNA modification in LRP5 and LRP6 genes were analyzed by a PCR products mismatch assay. Protein levels of LRP5 and LRP6 in the WT and KO cells were examined by Western blot analysis. Wnt signaling activation in the WT and KO cells were evaluated by a TOP-flash (Wnt signaling activation) assay. Wnt signaling activation was further confirmed by Western blot analyses of phosphorylated LRP5/6 (pLRP5/6), non-phosphorylated β-catenin and up-regulation of Wnt signal target genes (VEGF, c-Myc, Cyclin-D).
Results :
Generated LRP5 KO and LRP6 KO ARPE-19 cells showed expected genomic DNA modification in the LRP5 or LRP6 genes. Western blot analyses showed near complete KO of LRP5 and LRP6 in each KO cells. Wnt signaling activation assay, Western blot of marker proteins (pLRP6 and nuclear β-catenin) and up-regulation of Wnt signal target genes indicated Wnt signaling activation. We further examined ligand specificity for each co-receptor using expression plasmids of individual canonical Wnt ligands (Wnt1, 2, 2B, 3, 3A, 6, 7B, 8A, 8B, 9A, 10A, 10B and 16). The results suggested that LRP5 and LRP6 have differential roles in mediating Wnt signaling.
Conclusions :
The present study generated a unique experimental system to study the distinct role of the Wnt co-receptors in the RPE cells. The outcome will address an important scientific question, which co-receptor of Wnt signaling plays a key role in DR and AMD. This study will also identify whether LRP5 or LRP6 should be used as a drug target for the treatment of DR and AMD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.