The contrasting roles of hepcidin and prion protein in phototoxic injury to retinal pigment epithelial cells; an in vitro study

Abstract

Purpose : Recently we reported local synthesis of hepcidin, an iron regulating peptide hormone, in corneal endothelial, trabecular meshwork (TM), and lens epithelial cells in addition to its reported presence in the retina. Though helpful in stringent regulation of iron, cytokine-mediated upregulation of hepcidin in response to phototoxic damage is likely to aggravate the damage by increasing intracellular iron and iron-catalyzed reactive oxygen species (ROS). Here, we explored the response of local hepcidin and prion protein (PrP^C), a mainly neuronal protein known to protect the retina from light-induced damage, in mitigating chemically induced phototoxicity in retinal pigment epithelial (RPE) cells.

Methods : ARPE19 cells were treated with all-trans-retinal to mimic phototoxicity, and expression of IL-6 and hepcidin was quantified by RT-PCR. Expression of ferritin, an iron storage protein, LC3II, a marker of autophagosomal activity, pro-hepcidin, and PrP^C were analyzed by Western blotting.

Results : All-trans-retinal induced time-dependent increase in IL-6 and pro-hepcidin in ARPE19 cells. The increase in hepcidin was accompanied by upregulation of ferritin and LC3II. Interestingly, majority of PrP^C in ARPE-19 cells was cleaved at the β-site, a proteolytic event associated with oxidative stress. Modification of the N-terminal region of PrP^C such that it interfered with β-cleavage induced cell death, indicating that β-cleavage of PrP^C protects against oxidative stress, or is otherwise essential for ARPE-19 cell survival. This contrasts with neuronal cells where majority of PrP^C is cleaved at the α-site, and undergoes β-cleavage only under specific pathological conditions including oxidative stress.

Conclusions : Together, the above results indicate cytokine-mediated upregulation of local hepcidin by phototoxic signal(s) in RPE cells, resulting in the accumulation of intracellular iron and induction of autophagy. PrP^C mitigates oxidative stress by undergoing β-cleavage, an obligatory proteolytic process in RPE cells that is currently being explored. The cornea, TM, and lens epithelium are likely to elicit a similar response to phototoxicity, underscoring the significance of hepcidin and PrP^C, proteins considered to be of little consequence in the eye, as prominent players in ocular conditions associated with oxidative stress.

This is a 2020 ARVO Annual Meeting abstract.