Mechanism of RSL3-induced ferroptotic cell death in HT22 cells: crucial 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 the 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 HT22 mouse neuronal cells as an in vitro model, we show that treatment of these cells with RSL3 strongly increases NOS protein levels and that PDI-mediated NOS dimerization is activated by RSL3, resulting in NO accumulation. Mechanistically, we find that PDI is activated in cells treated with RSL3 because of its inhibition of TrxR1, and the activated PDI then catalyzes NOS dimerization, which is followed by the 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 resulted from RSL3 inhibition of TrxR1 activity contributes crucially to RSL3-induced ferroptosis in a cell culture model through the PDI→NOS→NO→ROS/lipid-ROS pathway, in addition to its known inhibition of GPX4 activity.

1. Introduction

Ferroptosis is a form of oxidative cell death [1] and is morphologically distinct from apoptosis-associated characteristics [1,2]. Mechanistically, ferroptosis can be selectively induced through depletion of cellular glutathione (GSH) and/or through suppression of glutathione peroxidase 4 (GPX4), an enzyme involved in the reduction of lipid peroxides. RSL3 is a prototypical inducer of ferroptosis [3], and over the past few years, it has been widely thought that RSL3 induces ferroptosis primarily through its suppression of GPX4 function, which then leads to the accumulation of cellular lipid reactive oxygen species (lipid-ROS) [4]. However, a recent study reported that RSL3 can strongly inhibit the catalytic activity of thioredoxin reductase 1 (TrxR1) [5]. Because TrxR1 uses NADPH as a cofactor to reduce the active-site disulfide bonds in thioredoxins, when TrxR1 is inhibited by an inhibitor (such as RSL3), the active site of the thioredoxins would remain in the oxidized state (i.e., containing a disulfide bond in their active sites) [6].

Protein disulfide isomerase (PDI or PDIA1) is the prototype of the PDI family of proteins, which are ubiquitous dithiol/disulfide oxidoreductases of the thioredoxin superfamily [7–9]. PDI is primarily localized in the endoplasmic reticulum of mammalian cells, although a small fraction of this protein is also found in the nucleus, cytosol, mitochondria, plasma membrane and extracellular space [10]. PDI is involved in protein processing by catalyzing the formation of intra- and intermolecular disulfide bridges in proteins [11]. Our recent studies showed that PDI plays an important role in mediating glutamate- and erastin-induced, GSH depletion-associated oxidative cytotoxicity through PDI-mediated NOS activation (i.e., homodimer formation through disulfide bond linkages), which is followed by the accumulation of cellular NO, ROS and lipid-ROS and ultimately the induction of ferroptotic cell death [12].

In the present study, we sought to examine the role of PDI in mediating RSL3-induced ferroptosis in HT22 neuronal cells and the mechanism of PDI activation. We found that PDI plays a crucial role in mediating RSL3-induced ferroptotic cell death in these cells through its ability to catalyze NOS dimerization, which is followed by the accumulation of cellular NO, ROS and lipid-ROS, and ultimately ferroptotic cell death. Genetic or pharmacological inhibition of PDI function could abrogate RSL3-induced ferroptosis. The mechanism by which PDI is activated in RSL3-treated cells is caused by TrxR1 inhibition by RSL3, and this inhibition leaves more PDI proteins in the oxidized state, which is the active form that catalyzes the dimerization of NOS (including both nNOS and iNOS) in HT22 cells.