Upregulation of EphA2 is associated with apoptosis in response to H2O2 and UV radiation-induced cataracts

Wei Zhao; Shuying Chen; Bing Lu; Di Wu; Yuzhou Gu; Shengjie Hao; Feiyin Sheng; Yili Xu; Yu Han; Rongrong Chen; Lei Zhou; Qiuli Fu; Ke Yao

doi:10.1016/j.abb.2023.109756

Upregulation of EphA2 is associated with apoptosis in response to H₂O₂ and UV radiation-induced cataracts

Arch Biochem Biophys. 2023 Oct 1:747:109756. doi: 10.1016/j.abb.2023.109756. Epub 2023 Sep 14.

Authors

Wei Zhao¹, Shuying Chen¹, Bing Lu¹, Di Wu¹, Yuzhou Gu¹, Shengjie Hao¹, Feiyin Sheng¹, Yili Xu¹, Yu Han¹, Rongrong Chen¹, Lei Zhou², Qiuli Fu³, Ke Yao⁴

Affiliations

¹ Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China.
² School of Optometry, Department of Applied Biology and Chemical Technology, Research Centre for SHARP Vision (RCSV), The Hong Kong Polytechnic University, Hong Kong, China; Centre for Eye and Vision Research (CEVR), 17W Hong Kong Science Park, Hong Kong, China.
³ Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China. Electronic address: 2313009@zju.edu.cn.
⁴ Eye Center, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Laboratory of Ophthalmology, Zhejiang Provincial Clinical Research Center for Eye Diseases, Zhejiang Provincial Engineering Institute on Eye Diseases, Hangzhou, Zhejiang, China. Electronic address: xlren@zju.edu.cn.

PMID: 37714253
DOI: 10.1016/j.abb.2023.109756

Abstract

In this article, we examine the role of erythropoietin-producing hepatocellular receptor A2 (EphA2) in the apoptosis of lens epithelial cells (LECs) in H₂O₂ and UV radiation-induced cataracts. We treated SRA01/04 cells with H₂O₂ or ultraviolet (UV) radiation to create a cataract cell model. We constructed a cataract lens model by exposing mice to UV radiation. We used CCK8 assays, Annexin V-FITC analysis, and immunohistochemical staining to explore proliferation and apoptosis of the cataract model. Thereafter, we used quantitative real-time PCR (qPCR) analysis, Western blot assays, and immunofluorescence to determine gene and protein expression levels. We also employed Crispr/Cas9 gene editing to create an EphA2 knockout in SRA01/04 cells. Results: H₂O₂ or UV radiation induced SRA01/04 cells showed EphA2 gene upregulation. CCK8 and apoptosis assays showed that EphA2 over-expression (OE) reduced epithelial cell apoptosis, but knockout of EphA2 induced it in response to H₂O₂ and UV radiation, respectively. Mutation of the EphA2 protein kinase domain (c.2003G > A, p. G668D) had a limited effect on cell apoptosis. In vivo, the EphA2 protein level increased in the lenses of UV-treated mice. Our results showed that EphA2 was upregulated in SRA01/04 cells in response to H₂O₂ and UV radiation. Mutation of the EphA2 protein kinase domain (c.2003G > A, p. G668D) had a limited effect on H₂O₂ and UV radiation-induced cell apoptosis. We confirmed this change with an experiment on UV-treated mice. The present study established a novel association between EphA2 and LEC apoptosis.

Keywords: Apoptosis; Cataract; Crispr/Cas9 genome editing; EphA2; Oxidative stress.