Poly(ADP-Ribose) Polymerase-1 inhibition potentiates cell death and phosphorylation of DNA damage response proteins in oxidative stressed retinal cells

• Published in: Experimental eye research Vol. 188; p. 107790
• Main Authors: Martín-Guerrero, Sandra M., Casado, Pedro, Muñoz-Gámez, José A., Carrasco, María-Carmen, Navascués, Julio, Cuadros, Miguel A., López-Giménez, Juan F., Cutillas, Pedro R., Martín-Oliva, David
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-11-2019
• Subjects: Animals; Ataxia Telangiectasia Mutated Proteins - metabolism; Blotting, Western; Caspase 3 - metabolism; Cell Death; Cell Line; DNA Damage; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Eye Proteins - metabolism; Histones - metabolism; Hydrogen Peroxide - toxicity; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Oxidants - toxicity; Oxidative Stress; Phenanthrenes - pharmacology; Phosphoproteins - metabolism; Phosphoproteomic; Phosphorylation; Photoreceptor; Poly (ADP-Ribose) Polymerase-1 - antagonists & inhibitors; Poly (ADP-Ribose) Polymerase-1 - metabolism; Poly(ADP-ribose) Polymerase Inhibitors - pharmacology; Poly(ADP-Ribose) Polymerase-1; Retina - metabolism; Retina - pathology; Poly(ADP-Ribose) Polymerase-1; DSBs; PAR; Oxidative stress; MDC1; DNA damage; INL; Photoreceptor; BRCA1; OxS; Phosphoproteomic; ONL; PARP-1; GCL; LC-MS/MS; SDS-PAGE; DDR; TUNEL; Cell death; ROS; SSBs; ATM; ATR
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2019.107790

Oxidative stress (OxS) is involved in the development of cell injures occurring in retinal diseases while Poly(ADP-ribose) Polymerase-1 (PARP-1) is a key protein involved in the repair of the DNA damage caused by OxS. Inhibition of PARP-1 activity with the pharmacological inhibitor PJ34 in mouse retinal explants subjected to H2O2-induced oxidative damage resulted in an increase of apoptotic cells. Reduction of cell growth was also observed in the mouse cone like cell line 661 W in the presence of PJ34 under OxS conditions. Mass spectrometry-based phosphoproteomics analysis performed in 661 W cells determined that OxS induced significant changes in the phosphorylation in 1807 of the 8131 peptides initially detected. Blockade of PARP-1 activity after the oxidative treatment additionally increased the phosphorylation of multiple proteins, many of them at SQ motifs and related to the DNA-damage response (DDR). These motifs are substrates of the kinases ATM/ATR, which play a central role in DDR. Western blot analysis confirmed that the ATM/ATR activity measured and the phosphorylation at SQ motifs of ATM/ATR substrates was augmented when PARP-1 activity was inhibited under OxS conditions, in 661 W cells. Phosphorylation of ATM/ATR substrates, including the phosphorylation of the histone H2AX were also induced in organotypic cultures of retinal explants subjected to PARP-1 inhibition during exposure to OxS. In conclusion, inhibition of PARP-1 increased the phosphorylation and hence the activation of several proteins involved in the response to DNA damage, like the ATM protein kinase. This finally resulted in an augmented injury in mouse retinal cells suffering from OxS. Therefore, the inhibition of PARP-1 activity may have a negative outcome in the treatment of retinal diseases in which OxS is involved.
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•Oxidative insult by H2O2 induces the activation of PARP-1 in retinal cells.•PARP-1 inhibition potentiates retinal explants degeneration after H2O2 treatment.•PARP-1 inhibition increases protein phosphorylation after the oxidative insult.•Proteins involved in the DNA damage response are affected by PARP-1 inhibition.•Blocking PARP-1 activity diminishes the survival of oxidative stressed-retinal cells.