Mechanism of RSL3-Induced Ferroptotic Cell Death in HT22 Cells: Contributing Role of Protein Disulfide Isomerase

Abstract

Protein disulfide isomerase (PDI) was recently shown to be an upstream mediator of erastin-induced, glutathione depletion-associated ferroptosis through its catalysis of nitric oxide synthase (NOS) dimerization and nitric oxide (NO) accumulation. A recent study reported that RSL3, a known ferroptosis inducer and glutathione peroxidase 4 (GPX4) inhibitor, can inhibit thioredoxin reductase 1 (TrxR1). The present study seeks to test a hypothesis that RSL3 may, through its inhibition of TrxR1, facilitate PDI activation (i.e., in a catalytically-active, oxidized state), thereby enhancing RSL3-induced ferroptosis through NOS dimerization and NO accumulation. Using the HT22 mouse neuronal cells as an in-vitro model, we show that treatment of these cells with RSL3 can strongly increase NOS protein level, and the PDI-mediated NOS dimerization is activated by RSL3, resulting in NO accumulation. Mechanistically, we find that PDI is activated in cells treated with RSL3 resulting from its inhibition of TrxR1, and the activated PDI then catalyzes NOS dimerization, which is followed by accumulation of cellular NO, ROS and lipid-ROS, and ultimately ferroptotic cell death. Genetic or pharmacological inhibition of PDI or TrxR1 partially abrogates RSL3-induced NOS activation and the subsequent accumulation of cellular NO, ROS/lipid-ROS, and ultimately ferroptosis in HT22 cells. The results of this study clearly show that PDI activation resulting from RSL3 inhibition of the TrxR1 activity contributes crucially to RSL3-induced ferroptosis in a cell culture model through the PDIl7NOSl7NOl7ROS/lipid-ROS pathway, in addition to its known inhibition of the GPX4 activity.