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Published on: March 15, 2024
DNA-Dependent Protein Kinase Catalytic Subunit Prevents Ferroptosis in Retinal Pigment Epithelial Cells
Xueying Wang1, Xi Wang1, Zhenzhen Zhao1
1Department of Ophthalmology of Tongji Hospital and Laboratory of Clinical and Visual Sciences of Tongji Eye Institute, School of Medicine, Tongji University, Shanghai, China.
Purpose:
The purpose of this study was to investigate the activated core kinases involved in the DNA damage responses (DDR) during ferroptosis of retinal pigment epithelial (RPE) cells in vitro and their regulatory effects on ferroptosis.
Methods:
Ferroptosis was induced by erastin in induced RPE (iRPE) cells derived from human umbilical cord mesenchymal stem cells (hUCMSCs), hUCMSCs, and induced pluripotent stem cell-derived RPE (iPSC-RPE) cells. CCK8 was employed to measure the cell viability. Calcein/PI staining was used to detect the ferroptotic cells. The γ-H2AX, 8-oxoG, and phosphorylated DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were determined through immunostaining. The phosphorylation of DNA-PKcs and ERK1/2 was determined by Western blotting. Lipid peroxides were detected by BODIPY581/591-C11 staining.
Results:
The iRPE cells exhibited a stronger ability to resist ferroptosis compared to hUCMSCs. Ferroptosis induced DNA damage in cells, and DNA-PKcs was rapidly phosphorylated in iRPE cells on the treatment of erastin. In addition, inhibition of DNA-PKcs phosphorylation promoted ferroptosis in iRPE cells, suggesting that DNA-PKcs prevents ferroptosis. Meanwhile, DNA-PKcs inhibited ERK1/2 phosphorylation only at the early stage of ferroptosis induction, whereas ERK1/2 phosphorylation played a protective role in iRPE cells. Furthermore, erastin inducing DNA-PKcs phosphorylation and inhibition of its phosphorylation promoting ferroptosis were also verified in iPSC-RPE cells.
Conclusions:
The present study elucidates that the key DDR kinase DNA-PKcs is activated and plays protective role during ferroptosis in RPE cells in vitro, which will provide new research targets and strategies for inhibiting ferroptosis in RPE cells.
Insights
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activation protects retinal pigment epithelial cells from ferroptosis by preventing DNA damage. This kinase is a potential therapeutic target for RPE cell ferroptosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Ophthalmology
Background:
- Ferroptosis is a regulated form of cell death involving iron-dependent lipid peroxidation.
- Retinal pigment epithelial (RPE) cells are crucial for retinal health and susceptible to ferroptosis.
- Understanding the molecular mechanisms of ferroptosis in RPE cells is vital for treating retinal diseases.
Purpose of the Study:
- To investigate the role of DNA damage response (DDR) kinases in ferroptosis of RPE cells.
- To identify the regulatory effects of activated kinases on ferroptosis in RPE cells.
- To explore potential therapeutic targets for inhibiting RPE cell ferroptosis.
Main Methods:
- Ferroptosis was induced in various RPE cell models (iRPE, hUCMSCs, iPSC-RPE) using erastin.
- Cell viability was assessed using CCK8 assay, and ferroptosis was detected via Calcein/PI staining.
- DNA damage markers (γ-H2AX, 8-oxoG) and kinase phosphorylation (DNA-PKcs, ERK1/2) were analyzed by immunostaining and Western blotting.
- Lipid peroxidation was quantified using BODIPY581/591-C11 staining.
Main Results:
- Induced RPE (iRPE) cells showed greater ferroptosis resistance than human umbilical cord mesenchymal stem cells (hUCMSCs).
- Erastin-induced ferroptosis triggered rapid DNA-PKcs phosphorylation in iRPE cells, indicating a protective role.
- Inhibition of DNA-PKcs phosphorylation exacerbated ferroptosis, while DNA-PKcs suppressed ERK1/2 phosphorylation early in ferroptosis.
- These findings were consistent in induced pluripotent stem cell-derived RPE (iPSC-RPE) cells.
Conclusions:
- The DNA damage response kinase DNA-PKcs is activated during RPE cell ferroptosis and exerts a protective effect.
- DNA-PKcs represents a novel therapeutic target for modulating ferroptosis in RPE cells.
- This study provides new strategies for inhibiting ferroptosis in RPE cells, relevant to retinal disease treatment.
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