Wnt/beta-Catenin Signaling Mediates Regeneration of Retinal Pigment Epithelium After Laser Photocoagulation in Mouse Eye

Abstract

PURPOSE. Laser photocoagulation of retinal pigment epithelium (RPE) is used to stimulate the regenerative processes of the RPE. However, the molecular mechanisms that control RPE proliferation and the epithelial-mesenchymal transition (EMT) during regeneration remain poorly understood. We investigated the role of Wnt/beta-catenin signaling in the regeneration of mouse RPE after laser photocoagulation. METHODS. C57BL/6J mice were photocoagulated unilaterally. To determine the beta-catenin-dependent Wnt signal transduction in the photocoagulated RPE, the expression levels of Wnts, beta-catenin, and their target genes were analyzed using real time-PCR and Western blotting. Retinal pigment epithelium proliferation and EMT were determined by 5-ethynyl-2'--deoxyuridine (EdU) incorporation assay and by profiling expression of EMT markers, respectively, in eyes injected intravitreally with a Wnt/beta-catenin signaling antagonist, Dkk-1, after laser photocoagulation. RESULTS. Expression of several of the 19 Wnt genes was significantly increased in laser-treated RPE. The expression levels of beta-catenin signaling target genes cyclin D1, Otx2, and Mitf were higher in laser-treated RPE than in control RPE. The number of EdU-positive cells in the laser-treated area was significantly lower in the Dkk-1-injected group than in the laser-treated group or laser-treated + vehicle-injected group. There were more Otx2- and Mitf-positive cells after laser photocoagulation and markedly fewer in the Dkk-1-injected group. Otx2- and Mitf-positive cells were colocalized with EdU-positive cells. The EMT markers vimentin and alpha-smooth muscle actin (alpha-SMA) were upregulated in the laser-treated area and significantly downregulated in the Dkk-1-injected group. CONCLUSIONS. Laser photocoagulation activates a Wnt/beta-catenin signal transduction pathway, promoting both RPE proliferation and EMT. Wnt/beta-catenin signaling also upregulates the expression of Otx2 and Mitf, key transcription factors in RPE formation. Our results demonstrate an important role for Wnt/beta-catenin signaling in RPE proliferation and EMT, and suggest that manipulating Wnt/beta-catenin signaling may have therapeutic potential for RPE regeneration.