Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity

https://doi.org/10.1016/j.bbrc.2016.06.066Get rights and content

Highlights

  • Glutathione depletion induces denitrosylation of protein disulfide isomerase.

  • Protein disulfide isomerase catalyzes dimerization of NO synthase, resulting in increased NO production.

  • Protein disulfide isomerase is constitutively inactivated by S-nitrosylation, and activated by denitrosylation.

Abstract

Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI’s isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity.

Introduction

Glutathione (GSH) is a major endogenous reducing agent involved in phase II metabolic detoxification of various reactive chemical species [1]. GSH depletion is a common phenomenon underlying many forms of oxidative stress and cytotoxicity [2]. Acetaminophen overdose-induced fatal liver damage in humans is probably the best-known clinical example resulting from chemically-induced GSH depletion [3]. It is also widely believed that GSH depletion precedes the development of certain forms of neurodegeneration, such as Parkinson’s disease [4]. Studies have shown that artificial depletion of GSH induces dopaminergic neuronal death in vitro [5] and potentiates neuronal toxicity induced by treating the animals with 6-hydroxydopamine or 1-methyl-4-phenylpyridinium (MPP+) in vivo [6], [7]. Together, these data suggest that GSH depletion contributes to cell death under certain pathogenic conditions [8].

The immortalized HT22 mouse hippocampal neuronal cells lack glutamate receptor, and in recent years, this cell line has become a selective model for studying GSH depletion-induced oxidative cytotoxicity [9]. In this model system, the presence of high concentrations of extracellular glutamate selectively blocks the uptake of cystine (a precursor for GSH synthesis) via the glutamate/cystine antiporter system, and thereby depletes intracellular GSH in a time- and dose-dependent manner [10]. Earlier studies showed that glutamate-induced GSH depletion in HT22 cells leads to reactive oxygen species (ROS) accumulation [11]. Moreover, glutamate induces nitric oxide (NO) formation in cerebellar slices [12] and primary cortical neurons [8], thus leading to the suggestion that NO may be involved in glutamate-induced cytotoxicity. However, the precise mechanism by which glutamate-induced GSH depletion leads to ROS accumulation and cell death is not clear.

In the present study, we sought to use the glutamate-treated HT22 cells as a model system to investigate further the biochemical mechanism as to how GSH depletion leads to ROS formation and ultimately cell death.

Section snippets

Materials

DAF-FM-DA and anti-PDI antibody was purchased from SIGMA-Aldrich (St. Louis, MO). H2-DCF-DA was purchased from Molecular Probe (Eugene, OR). Anti-nNOS and anti-eNOS antibodies were obtained from BD Biosciences (San Jose, CA). Anti-iNOS antibody was from Cell Signaling Technology (Beverly, MA). l-[3H]arginine (Arginine monohydrochloride, [2,3,4-3H]-) was purchased from Perkin Elmer (Boston, MA). siRNA duplexes targeting the mouse PDI and scrambled non-targeting siRNA were purchased from Santa

NO accumulation precedes ROS accumulation and cytotoxicity

To probe whether NO accumulation is involved during GSH depletion-induced oxidative toxicity in HT22 cells, we determined changes in NO levels following glutamate treatment. NO was found to be accumulated by glutamate treatment in a dose- and time-dependent manner (Fig. 1a,b), and its accumulation occurs a little earlier than ROS accumulation (Fig. 1b,c).

To determine whether NO accumulation is involved in ROS accumulation and cell death, we tested the effect of

PDI catalyzes NOS dimerization

The formation of dimer NOS is stabilized by an intermolecular disulfide bond and the dimer NOS is apt for NO production [23]. We postulated that protein disulfide isomerase (PDI) may regulate nNOS dimerization in HT22 cells during glutamate-induced GSH depletion because PDI is a ubiquitous dithiol/disulfide oxidoreductase of the thioredoxin superfamily and is involved in protein processing and translocation by catalyzing intra- and inter-molecular disulfide bridges in proteins [24]. To test

Acknowledgements

The study described here is supported by an Endowment Fund from the University of Kansas Medical Center to Bao-Ting Zhu. We thank Dr. David Schubert (Salk Institute, La Jolla, CA) for providing the HT22 cells as a gift.

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    Present address: Aomori University, Department of Pharmacy, Aomori, 030-0943, Japan.

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