GRP78 Suppresses Lipid Peroxidation and Promotes Cellular Antioxidant Levels in Glial Cells following Hydrogen Peroxide Exposure

Oxidative stress, caused by the over production of reactive oxygen species (ROS), has been shown to contribute to cell damage associated with neurotrauma and neurodegenerative diseases. ROS mediates cell damage either through direct oxidation of lipids, proteins and DNA or by acting as signaling molecules to trigger cellular apoptotic pathways. The 78 kDa glucose-regulated protein (GRP78) is an ER chaperone that has been suggested to protect cells against ROS-induced damage. However, the protective mechanism of GRP78 remains unclear. In this study, we used C6 glioma cells transiently overexpressing GRP78 to investigate the protective effect of GRP78 against oxidative stress (hydrogen peroxide)-induced injury. Our results showed that the overexpression of GRP78 significantly protected cells from ROS-induced cell damage when compared to non-GRP78 overexpressing cells, which was most likely due to GRP78-overexpressing cells having higher levels of glutathione (GSH) and NAD(P)H:quinone oxidoreductase 1 (NQO1), two antioxidants that protect cells against oxidative stress. Although hydrogen peroxide treatment increased lipid peroxidation in non-GRP78 overexpressing cells, this increase was significantly reduced in GRP78-overexpressing cells. Overall, these results indicate that GRP78 plays an important role in protecting glial cells against oxidative stress via regulating the expression of GSH and NQO1.


Introduction
Reactive oxygen species (ROS) are one of the cytotoxic factors produced from damaged cells that cause oxidative stress and tissue damage during neurotrauma [1]. Hydrogen peroxide (H 2 O 2 ), a ROS, is released from dying cells during neurotrauma and neurodegenerative disease and causes tissue destruction [2,3]. H 2 O 2 can produce hydroxyl radicals (OH?) and mediate cell damage either through direct oxidation of lipids, proteins and DNA or act as a signaling molecule to trigger cellular apoptotic pathways [4][5][6]. Therefore, it is important to protect cells from H 2 O 2 -induced cell damage as a therapeutic strategy against neurotrauma and neurodegenerative diseases [7,8]. Endoplasmic reticulum (ER) stress has been reported to be one of the pathways via which cells are damaged and die following ROS exposure [9,10].
The mechanism by which ER stress promotes apoptosis in cells hinges on driving the accumulation of structurally abnormal proteins that are usually repaired by ER chaperones to prevent cell death [11]. The 78 kDa glucose-regulated protein (GRP78) is one example of an ER chaperone that regulates protein folding in the ER and controls the ER-Ca 2+ balance via trans membrane ER stress sensors, which contribute to cell survival [11][12][13]. GRP78 has been suggested to not only protect cells against highconcentrations of glutamate or tunicamycin, which induce ER stress directly [14], but also protect cells from ROS damage [15][16][17].
Many studies have focused on various antioxidant factors, such as glutathione and NAD(P)H:quinone oxidoreductase 1 (NQO1). A previous study reported that induction of NQO1 and GSH by dimethyl fumarate, 3H-1,2-dithiole-3-thione or tert-butylhydroquinone (tBHQ) protected against neurocytotoxicity caused by dopamine, 6-hydroxydopamine, 4-hydroxy-2-nonenal,or H 2 O 2 [18]. As this study described, these antioxidants have recently been demonstrated to play an important role in protecting cells against oxidative stress [19][20][21]. Glutathione is the most abundant low molecular weight thiol in most organisms [20,22]. There are two types of glutathione, reduced glutathione (GSH) and oxidized glutathione (GSSG), depending on the environment. Reduced glutathione (GSH) is the main non-protein antioxidant and plays a critical role in the detoxification of H 2 O 2 and lipid hydroperoxide, and is involved in the protection against oxidative stress [18]. Similarly, NQO1, one of the most extensively investigated phase 2 enzymes, is an effective antioxidant that protects membrane phospholipids from oxidative damage and plays an important protective role in oxidative stress [19].
Few studies have investigated the influence of GRP78 on NQO1. Some studies have suggested that H 2 O 2 may not be involved in ER stress-dependent cell damage because the response of GRP78 is different following H 2 O 2 exposure and other cytotoxic factors [23,24]. Similarly, a report on PKE-like ER kinase (PERK), a ER-stress sensing protein that resides in the ER, suggested that the PERK pathway is activated after dissociation of GRP78 from PERK monomers and leads to intracellular GSH production [25]. As these studies showed, the role of GRP78 during oxidative stress remains unclear. Therefore, we used cells transiently overexpressing GRP78 to investigate the protective effect of GRP78 against high extracellular concentrations of H 2 O 2 and evaluated the response of glutathione and NQO1.

Cell Culture and Culture Conditions
The rat C6 glioma cell line was obtained from American Type Culture Collections (ATCC, Manassas, VA, USA). Cells were plated onto 6 cm tissue culture dishes and maintained at 37uC with 5% (v/v) CO 2

Intracellular GRP78 and NQO1 Detection
The IntraStain H reagent kit (DAKO) has previously been used successfully for the detection of intracellular GRP78 or NQO1 protein expression under conditions of tunicamycin treatment and bactin [14]. Following exposure to treatment, cells were fixed and permeabilized using IntraStain according to the manufacturer's instructions. For the detection of cytoplasmic GRP78 or NQO1, cells were stained with rabbit polyclonal anti-GRP78 or mouse monoclonal anti-NQO1, and then stained with the Alexa 488conjugated anti-rabbit secondary antibody or PE-conjugated antimouse secondary antibody. GRP78 expression and b-actin expression were measured with a BD LSRFortessa TM apparatus (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo TM Software (Tree Star Inc. Ashland, OR, USA).

Construction of Chimeric Proteins and Transfection
The pIRES2-AcGFP1 plasmid was purchased from Clontech Laboratories. The plasmid contains the internal ribosome entry site (IRES) of the encephalomycarditits virus between the multiple cloning site (MCS) and the Aequorea coerulescens green florescent protein (Ac GFP) cording region. This permits both the gene of interest and the Ac GFP1 gene to be translated to a single bicistronic mRNA. A 1.9-kb cDNA of rat GRP78 (GenBank M14059), covering a whole open reading frame, was composed by Takara Bio. Inc. (Otsu, Japan) carrying XhoI/PstI cut sites and coned into pIRES2-AcGFP1 plasmid (Fig. 1). The pIRES-AcGFP1 vector was designed so that cells transfected transiently express GFP and the protein. Therefore, cells transfected with the GRP78 gene and expressing the protein express GFP. C6 cells were transfected with the indicated plasmids using the Neon TM transfection system (Invitrogen/Life Technologies). After transfec-

Western Blot Analysis
After 36 h of transfection, GFP-positive and -negative cells were sorted using the FACSAria TM apparatus (Becton Dickinson, San Jose, CA, USA). Total extracted proteins from cultured cells were quantified using the Lowry method. Equal amounts of total protein from each sample were loaded onto a 12.5% (w/v) SDSpolyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were then incubated with the primary antibodies at 4uC overnight, followed by incubation with the HRP-linked anti-rabbit IgG (DAKO), anti-goat IgG (DAKO), or anti-mouse IgG (DAKO) antibodies for 1 h. Signals were detected using Immobilon Western Chemiluminescent HRP (Millipore). Intensity of specifically amplified products was quantified by densitometric scans of the films using computer software for a Macintosh ''CS analyzer'' (ATTO, Tokyo, Japan).

Annexin V/Propidium Iodide (PI) Double Staining
Cells undergoing apoptosis or necrosis can be stained and quantified using Annexin V and propidium iodide (PI). Cells treated with H 2 O 2 , were washed once with phosphate buffered saline (PBS), and stained for 15 min with allophycocyaninconjugated (APC)-Annexin V and PI (Becton Dickinson), according to the manufacturer's instructions. Quantification of apoptotic/necrotic cell-death staining with APC-annexin V and PI under each condition were measured with a BD LSRFortessa TM apparatus (Becton Dickinson) and analyzed using FlowJo TM Software (Tree Star Inc. Ashland, OR, USA). Annexin Vnegative/PI-negative cells were considered living cells.

Evaluation of Lipid Peroxidation
The lipid peroxidation of the cell membrane after H 2 O 2 exposure was examined using cis-parinaric acid as a probe. Oxidation of this probe is accompanied by decreased fluorescence and absorption [26,27]. cis-Parinaric AcidH was purchased from Molecular Probes/Life Technologies and the method was performed with slight modification as described by Hedley et al. [26]. Briefly, at 5 h after incubation with H 2 O 2 , 10 mM of cis-  Parinaric Acid was added and the incubation was continued for 1 h (total incubation time was 6 h). Cells were collected with PBS and the fluorescence was measured using the BD LSRFortessa TM apparatus (Becton Dickinson) and analyzed using FlowJo TM Software (Tree Star Inc.) The excitation and emission wavelengths were 320 and 420 nm, respectively.    Dulbecco's PBS and incubated in Dulbecco's PBS containing 10 mM (final concentration) ThiolTracker Violet for 30 min at 37uC. Cells were measured using the BD LSRFortessa TM apparatus (Becton Dickinson) and analyzed using FlowJo TM Software (Tree Star Inc.). The excitation and emission wavelengths were 404 and 526 nm, respectively.

Statistical Analysis
Data are expressed as the mean 6 standard deviation (SD). Analysis of variance and the Student's t-test were used to assess differences between the means of test samples and controls. Significance was set at P,0.05.

Cell Damage and GRP78 Expression Following H 2 O 2 Treatment
Flow cytometric (FACS) analysis revealed a significant increase in the number of apoptotic/necrotic cells, namely annexin V/PIpositive cells (AP-positive cells) following 1-6 mM H 2 O 2 treatment at 6,24 h compared to that in the controls (Fig. 2A). The number of GRP78-expressing cells following treatment with H 2 O 2 did not significantly increase compared to that in untreated C6 cells at any concentration ( Fig. 2A). Similarly, western blot analysis revealed that the expression of the GRP78 protein in the groups treated with 1-6 mM H 2 O 2 for 6 and 24 h did not increase obviously when compared to untreated C6 cells (n = 3; Fig. 2B).

Evaluation of cis-Parinaric Acid and GSH Following H 2 O 2 Treatment
FACS analysis revealed that the mean fluorescence intensity of cis-Parinaric acid significantly decreased following treatment at all concentrations of H 2 O 2 when compared to untreated C6 cells (n = 5; P,0.05) (Fig. 3A). This result indicated that H 2 O 2 causes lipid peroxidation of the cell membrane. Similarly, the mean fluorescence intensity of GSH significantly decreased following treatment at all concentrations of H 2 O 2 when compared with untreated C6 cells (n = 4; P,0.05) (Fig. 3B). These results indicated that intracellular GSH expression levels reduced when cells were subjected to excessive oxidative stress.

Expression of GRP78 Protein and NQO1 in GFP-positive Cells after Transfection
At 36 h post-transfection, an average of 43% of cells was GFPpositive (Fig. 4A). Quantitation of immunoblots of GFP-positive cell extracts revealed a significant increase in GRP78 protein levels when compared with GFP-negative cells and non-treated C6 cells (n = 3, P,0.05) (Fig. 4B). This result showed that GFP-positive cells overexpress GRP78 protein before exposure to H 2 O 2 when compared with GFP-negative cells and non-treated C6 cells. Moreover, we observed that NQO1 expression significantly increased in GFP-positive cell extracts when compared with GFP-negative cells and non-treated C6 cells (n = 3, P,0.05) (Fig. 5).

Evaluation of GRP78 Overexpressing Cells Following H 2 O Exposure
After transfection, a mixture of cells consisting of GFP-positive and -negative cells were incubated for 36 h with 10% (v/v) FBS/ DMEM. Cells were then treated with 1-6 mM H 2 O 2 for 6 h. To evaluate the protective effect of GRP78, apoptotic/necrotic cells were labeled with annexin V and PI, and the ratio of AP-positive cells in GFP-positive and -negative cells were analyzed byFACS. FACS analysis revealed that the percentage of GFP-positive cells that were AP-positive significantly decreased when compared with GFP-negative cells following exposure to 1,3 and 6 mM of H 2 O 2 (GFP-positive % vs. GFP-negative % at 0 mM = 3.2 vs. 2.1; at 1 mM = 3.6 vs.9.7; at 3 mM = 13.3 vs. 38.3; at 6 mM = 13.6 vs.49.9; at 9 mM = 28.1 vs. 66.3) (n = 5, P,0.05) (Fig. 6). These findings indicated that GRP78 overexpressing cells could survive longer than non-GRP78 overexpressing cells following H 2 O 2 exposure.
Evaluation of GRP78 Overexpression on Cell Lipid Peroxidation Following H 2 O 2 Exposure As described above, a mixture of cells consisting of GFP-positive and -negative cells were treated with H 2 O 2 1-6 mM for 6 h, and labeled with cis-Pariaric acid to evaluate lipid peroxidation of cell membranes. FACS revealed that the mean fluorescence intensity of cis-Pariaric acid in GFP-positive cells was significantly higher than that in GFP-negative cells following H 2 O 2 exposure at 1, 3 and 6 mM (GFP-positive vs. GFP-negative at 0 mM = 1458 vs. 1287; at 1 mM = 1185 vs.1027; at 3 mM = 1016 vs. 631; at 6 mM = 880 vs. 446) (n = 5, P,0.05) (Fig. 7). These findings indicate that GRP 78 overexpressing cells can suppress lipid peroxidation more than non-GRP78 overexpressing cells following H 2 O 2 exposure.

GSH Expression Levels in GRP78 Overexpressing Cells Following H 2 O 2 Treatment
We observed GSH expression levels in glutathione using FASC analysis. FACS revealed that the mean fluorescence intensity of GSH in GFP-negative cells significantly decreased when compared with GFP-positive cells following exposure to 1, 3 and 6 mM of H 2 O 2 (GFP-positive vs. GFP-negative at 0 mM = 62660 vs. 41590; at 1 mM = 56450 vs. 33050; at 3 mM = 60250 vs. 26850; at 6 mM = 29030 vs. 11360) (n = 5; P,0.05) (Fig. 8). Thus, intracellular GSH expression levels in GRP78 overexpressing cells were significantly higher than that of non-GRP78 overexpressing cells.

Discussion
GRP78 levels have been reported to increase in cells following cytotoxic-induced ER stress, where it contributes to cell survival [11,12,28]. According to reports that metals are implicated in the etiology or pathogenesis of Alzheimer's disease, some metals such as lead (Pb) induce the expression of GRP78, which is often associated with oxidative stress, and Pb impairs GRP78 function following binding [29]. Moreover, it has been reported that GRP78 may play a role in the modulation of the sensitivity of cells to stress after oxidative injury. Increases in the mRNA expression of GRP78 are observed in retinal pigment epithelial cells exposed to oxidative stress [17]. Experiments using cultured neurons reveal that GRP78 may protect cells against oxidative stress via actions involving mainly the maintenance of calcium homeostasis [16]. Meanwhile, it was reported that GRP78 expression did not increase when cells were exposed to H 2 O 2 , which suggested that H 2 O 2 exposure may not induce the ER stress pathway [24]. In our study, an increase in GRP78 expression in C6 cells was not observed after treatment with H 2 O 2 , however, the viability of cells decreased. Considering previous reports, our results suggest that GRP78 itself could play an important role in protecting cells against H 2 O 2 injury regardless of whether the pathways that mediate GRP78 expression respond to their extracellular stimuli.
H 2 O 2 causes cytotoxicity via the formation of more potent oxidants including OH?, which causes lipid peroxidation of the cell membrane [7,30]. Lipid peroxidation disrupts the normal structure of cellular and subcellular membranes. In addition, the process produces byproducts such as 4-hydroxynonenal (4-HNE) or acrolein, both of which bind to proteins and damage their structure and function [30,31]. The present results show that GRP78 overexpressing cells suppress lipid peroxidation and may contribute to cell survival following H 2 O 2 treatment. These data suggest that GRP78 can promote the expression of some antioxidants and may contribute to the protection of cells against While a number of antioxidants are involved in the detoxification of H 2 O 2 , GSH is the primary defense against H 2 O 2 [18]. GSH inhibits lipid peroxidation initiation by scavenging OH? or other ROS. Moreover, GSH also serves as a co-factor for GSH peroxidases that remove H 2 O 2 [20,22]. It was reported that GSH was useful for curtailment of lipid peroxidation damage in acute spinal cord injury [20]. The ratio of GSH reduces when an increase in ROS induces ER stress [31,32]. In our results, when cells were exposed to H 2 O 2 , GSH expression in GRP78 overexpressing cells was high when compared with non-GRP78 overexpressing cells. These results suggest that the increase of GRP78 by gene transfection may contribute to the increase in GSH or inhibit GSH consumption, thus leading to cell survival.
We observed the influence of GRP78 on NQO1 in this study. NQO1 catalyzes the electron reduction of quinone and quinoid compounds to hydroquinones, thus limiting the formation of semiquinone radicals, and the subsequent generation of ROS [19,33]. According to reports regarding the role of NQO1, H 2 O 2dependent formation of reactive oxygen intermediates was shown to be reduced following treatment with neuroprotective agents that induce NQO1 expression [34,35]. In our results, NQO1 expression levels in GRP78 overexpressing cells was higher than in non-GRP78 overexpressing cells. This phenomenon continued following H 2 O 2 exposure. These findings indicate that GRP78 may be advantageous to the expression of NQO1.
NQO1 has the ability to use both NADPH and NADH equally efficiently, thus NQO1 may contribute to the regulation of the redox balance by modulating reduced/oxidized pyridine nucleotide ratios [36]. NADPH is required to reduce oxidized glutathione (GSSG) to GSH [37]. This NQO1 role appears not to contradict our results that GSH expression levels in GRP78 overexpressing cells were higher than non-GRP78 overexpressing cells.
Taken together, this present study suggests that GRP78 plays an important role in protecting glial cells against H 2 O 2 toxicity by regulating GSH and NQO1 expression. However, there are several pathways and factors related to GRP78 expression in cells and further studies are required to understand the mechanisms involved and the direct relationship between GRP78, GSH and NQO1 in order for molecular/pharmacological treatments of neurotrauma or neurodegenerative diseases to be developed.