Elovanoids (ELV) modulate erastin -mediated phosphorylation of the scaffold phosphatidyl ethanolamine binding protein-1 (PEBP-1) and ferroptosis in human Retinal Pigment Epithelial cells (hRPEC).

Abstract

Purpose : Purpose: Ferroptosis is a type of programmed cell death characterized by the accumulation of lipid peroxides, distinct from other forms of regulated cell death such as apoptosis. Erastin is a small molecule capable of initiating ferroptotic cell death.Thus cells treated with erastin are deprived of cysteine and are unable to synthesize the antioxidant glutathione. Depletion of glutathione leads to excessive lipid peroxidation and cell death. Dephosphorylating of PEBP-1 protects against ferroptosis induced by erastin. The novel lipid mediator ELV protects hRPEC from uncompensated oxidative-stress. In the present study, we uncovered that ELV regulate phosphorylation of PEBP-1 in h-RPEC treated with erastin.

Methods : Methods: Overnight grown h-RPE cells (3x10⁵) in 60mM plates were serum starved for 16h at 37°C, exposed to erastin (1-50uM) and treated with 32:6 and 34:6 elovanoids (Me-forms, 200nM) for 6h followed by Western blot analysis and 16h for ferroptosis assessment. PEBP protein phosphorylation and total PEBP protein were detected by Western blot analysis using either anti- PEBP phospho antibody or anti-total PEBP antibody. Ferroptosis assessment was conducted by using Nikon deconvolution microscope.

Results : Results: Our data demonstrates that ELV 32:6-Me and 34:6-Me downregulated phosphorylation of PEBP-1 in h-RPE induced by erastin. On the contrary, there was no change in total PEBP-1 protein observed in identical experimental condition. The time and concentration kinetics showed that 10nM erastin with 200nM ELV for 6h treatment was the ideal condition for the optimum inhibition of PEBP-1 phosphorylation in h-RPEC. Moreover, the ELV mediated downregulation of PEBP-1 phosphorylation in erastin treated cells is specific for erastin as erastin specific inhibitors Ferrostatin-1, and Liproxstatin-1, treated cells were unable to phosphorylate PEBP by erastin whereas non erastin specific inhibitor B1-6C9 was in effective.

Conclusions : Conclusions: Erastin induced ferroptosis, inhibited by ELV required PEBP-1 phosphorylation. These findings provide a new mechanism of RPEC survival induced by erastin, mediated by a unique class of lipid mediator, ELV. The unraveling of this molecular target of ELV on PEBP-1 phosphorylation contribute to explain how ELV sustain PREC survival.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.