Klotho attenuates epithelial-mesenchymal transition of retinal pigment epithelial cells in subretinal fibrosis by suppressing the ERK1/2 and Wnt/β-catenin signaling pathways

Retinal pigment epithelial (RPE) cells undergoing epithelial-mesenchymal transition (EMT) are a key factor in promoting the progression of subretinal fibrosis. The klotho protein and gene exert anti-fibrotic effects in multiple fibrotic diseases. However, the mechanisms involved in the role of klotho are unclear in subretinal fibrosis. The aim of the present study was to explore the effects of klotho on subretinal fibrosis induced by laser photocoagulation in mice and EMT induced by TGF-beta 1 in RPE cells and the underlying molecular mechanisms. In vitro, klotho overexpression or knockdown was performed in ARPE-19 cells (adult retinal Pigment Epithelial-19), then TGF-beta 1 treatment was applied. Using western blotting, expression of epithelial markers (zonula occludens-1), mesenchymal signs (alpha-smooth muscle actin, alpha-SMA, N-cadherin, N-cad and collagen I), and the ERK1/2 and Wnt/beta-catenin signaling pathways were assessed. The proliferative ability of ARPE-19 cells was examined by CCK-8 and EdU test, and the migratory ability was examined by wound healing and Transwell assays. Furthermore, to explore the underlying molecular pathway of klotho overexpression, RNA-sequencing (seq) was performed. In vivo, photocoagulation was used to induce subretinal fibrosis in mice, which occurs as a result of choroidal neovascularization (CNV), then recombinant mouse klotho protein was administered intravitreally. Upregulation of epithelial and downregulation of mesenchymal markers demonstrated that klotho overexpression prevented TGF-beta 1-induced EMT; klotho knockdown resulted in the opposite effects. Additionally, klotho overexpression suppressed cell proliferation and migration and attenuated ERK1/2 and Wnt/beta-catenin signaling activated by TGF-beta 1. RNA-seq results demonstrated that several signaling pathways, including cellular senescence and the TNF signaling pathway, were associated with anti-fibrotic effects of klotho overexpression. In vivo, subretinal fibrotic areas were attenuated following klotho treatment in laser-induced CNV lesions, as illustrated by immunofluorescence and Masson staining of the mouse eyes. Western blotting results that the protein levels of mesenchymal markers were significantly downregulated and those of epithelial markers were upregulated. In summary, the present study suggested that klotho may have therapeutic value in management of fibrotic vitreoretinal disorders such as subretinal fibrosis.