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Yusuke Takahashi, Harminder Singh, Jian-Xing (Jay) Ma; Distinct Roles of Wnt Co-receptors in the Regulation of Wnt Signaling in Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5371. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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). 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 diabetes-induced Wnt signaling activation. 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 in DR.
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 was analyzed by a PCR products mismatch assay. The protein levels of LRP5 and LRP6 in the WT and KO cells were examined by Western blot analysis. Following treatment of Wnt3a (or control L-cell) conditioned media, Wnt signaling activation in the WT and KO cells were measured by luciferase-based TOP-flash assay as well as Western blot analysis of phosphorylated LRP5/6. Furthermore, to examine the distinct roles of Wnt co-receptors, Wnt ligand expression plasmids (Wnt1 and Wnt3a) were separately transfected into WT and KO cells and Wnt signaling activation was evaluated.
A plasmid expressing Cas9 nuclease and human LRP5 (or LRP6) specific guide-RNA were transfected into ARPE19 cells, and transfected cells were selected by puromycin. Each puromycin resistant cell line showed desired genomic DNA modification in LRP5 or LRP6 gene. In addition, Western blot analyses showed near complete KO of LRP5 and LRP6 in respective KO cell line. We further demonstrated that LRP6 mediates Wnt signaling induced by Wnt1 and Wnt3a, whereas LRP5 mediates Wnt signaling by Wnt1 but not by Wnt3a, suggesting that LRP5 and LRP6 have differential roles in Wnt signaling regulation.
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. This study will also identify whether LRP5 or LRP6 should be used as a drug target for the treatment of DR.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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