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/β-catenin signaling in the regeneration of mouse RPE after laser photocoagulation.
Methods: C57BL/6J mice were photocoagulated unilaterally. To determine the β-catenin-dependent Wnt signal transduction in the photocoagulated RPE, the expression levels of Wnts, β-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/β-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 β-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 α-smooth muscle actin (α-SMA) were upregulated in the laser-treated area and significantly downregulated in the Dkk-1-injected group.
Conclusions: Laser photocoagulation activates a Wnt/β-catenin signal transduction pathway, promoting both RPE proliferation and EMT. Wnt/β-catenin signaling also upregulates the expression of Otx2 and Mitf, key transcription factors in RPE formation. Our results demonstrate an important role for Wnt/β-catenin signaling in RPE proliferation and EMT, and suggest that manipulating Wnt/β-catenin signaling may have therapeutic potential for RPE regeneration.