Abstract
Purpose :
Fuchs Endothelial Corneal Dystrophy (FECD) is an oxidative stress disorder leading to accelerated loss of corneal endothelial cells (CEnCs) and exhibits greater accumulation of DNA damage compared to normal aging CEnCs. Ataxia Telangiectasia Mutated (ATM) is a key DNA damage response (DDR) kinase in post-mitotically arrested cells, which is activated and recruited to the damage site by Mre11-Nbs1-Rad50 (MRN) complex, leading to downstream activation of cell cycle checkpoints, DNA repair and/or apoptosis. In this study, we investigate the role of altered DDR mediated by ATM in FECD pathogenesis.
Methods :
Immortalized normal CEnC line HCEnC-SV-67F-16 and FECD patient-derived cell line FECD-SV-73F-74 (with TCF4 CTG repeat expansion) were treated with or without 25 µM menadione (MN) previously shown to induce intracellular ROS and rosette formation in vitro. Cells were lysed at 30, 60, 90- or 120 min post treatment and 60 min time-point was chosen to compare early DDR signaling between cell lines. The phosphorylation status and levels of γH2AX (Ser139) and pATM (Ser1981) and its downstream effectors pCHK2 (Thr68) and pp53 (Ser15) were evaluated by western blotting. The binding partners of pATM were determined by immunoprecipitation (IP) assay using pATM antibody.
Results :
In HCEnC-SV-67F-16 cells, MN induced a time-dependent increase in DNA damage marker γH2AX. FECD-SV-73F-74 cells exhibited increased activation of pATM (6.9-fold), which was significantly higher compared to HCEnC-SV-67F-16 (4.3 fold). At a similar time-point, pp53 levels were also significantly higher in FECD (4.1-fold) compared to normal CEnCs (2.1-fold). Further, increased co-IP of pp53 by pATM in FECD shows that ATM directly binds and activates p53 to initiate DDR signaling cascade. Whereas, there was only a marginal increase in pCHK2 levels in FECD-SV-73F-74 cells compared to a significant increase in normal CEnCs (2.7-fold) indicating that ATM possibly activates p53 preferentially by direct phosphorylation over activation via CHK2 in FECD.
Conclusions :
We demonstrate increased activation of ATM-mediated DDR upon ROS induction in FECD. This activation is via ATM-p53 signaling axis through increased pATM-mediated interaction and thus activation of its direct substrate p53 in FECD compared to normal CEnCs. This study provides insights into understanding the role of DDR signaling cascade in determining the apoptotic cell fate of CEnCs in FECD.
This is a 2020 ARVO Annual Meeting abstract.