Role of Glutathione Peroxidase 4 in Conjunctival Epithelial Cells

Abstract

Purpose.: The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in conjunctival epithelial cells.

Methods.: An immortalized human conjunctival epithelial cell line was used. Cells were transfected with catalase, GPx1, GPx4, SOD1, SOD2, or control siRNA. Knockdown was confirmed by RT-PCR and immunoblotting. The cytotoxicity induced by knockdown of these antioxidant enzymes was examined by assay of LDH activity. Furthermore, evaluations of lipid peroxidation, cellular levels of reactive oxygen species, cell proliferation, and apoptosis were conducted in cells treated with GPx4 or control siRNA. In oxidative stress study, cells treated with GPx4 or control siRNA were applied with hydrogen peroxide or ferric sulfide, and their cytotoxicity was evaluated by assay of LDH activity.

Results.: Small interfering RNA of catalase, GPx1, GPx4, SOD1, and SOD2 siRNA remarkably inhibited the mRNA and protein expression of each gene. Knockdown of GPx4 and SOD1 but not catalase, GPx1, and SOD2 significantly induced cytotoxicity. Glutathione peroxidase 4 knockdown increased lipid oxidation and reactive oxygen species. The proliferation of GPx4 siRNA-treated cells was reduced compared with control siRNA–treated cells. Moreover, cell death in GPx4 siRNA–treated cells was characterized by positive staining for annexin V. In an oxidation stress study, GPx4 siRNA knockdown enhanced the cytotoxicity induced by hydrogen peroxide or ferric sulfide.

Conclusion.: These results suggest that GPx4 is essential for maintaining oxidative homeostasis and keeping defense against oxidative stress in conjunctival epithelial cells.

Introduction

The ocular surface is constantly endangered to oxidative stress due to exposure to light and oxidative stress is implicated in several ocular surface diseases including pterygium, dry eye, conjunctivochalasis, and atopic keratoconjunctivitis.^1–5 Thus, the ocular surface needs antioxidants to maintain its oxidative homeostasis and redox balance. 

Among various kinds of antioxidative enzymes and small molecules, glutathione peroxidase is one of the major players in the antioxidative defense. The biochemical function of glutathione peroxidase is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water. Eight isozymes of glutathione peroxidase have been identified in humans, which vary in cellular location and substrate specificity.^6–8 Glutathione peroxidase 4 (GPx4) has a high preference for lipid hydroperoxides and directly reduces peroxidized phospholipids in cellular membranes. 

Lipid peroxidation is implicated in a number of pathophysiologic processes of not only systemic diseases such as atopic dermatitis,^9,10 but also ocular surface disorders such as dry eye and conjunctivochalasis.^1–4 Peroxidized lipids are highly reactive and lead to DNA fragmentation and protein modification.⁹ Byproducts of lipid peroxidation such as 4-hydroxynonenal (4-HNE) are known to induce cell damage such as apoptosis and growth inhibition.^11,12 In vivo, knockout mice of GPx4 die at embryonic day 8,¹³ and GPx4 is critically important for neuronal development including photoreceptors.^14,15 Glutathione peroxidase 4 is also important to avoid neurodegeneration,^16,17 β-cell dysfunction,¹⁸ male infertility,¹⁹ and choroidal neovascularization.²⁰ Therefore, GPx4 is thought to be crucial for cell protection from oxidative stress. In our previous study, we generated photoreceptor-specific conditional knockout mice of GPx4, in which photoreceptor cells rapidly underwent drastic degeneration and completely disappeared by P21, indicating GPx4 is a critical antioxidant enzyme for the maturation and survival of photoreceptor cells.¹⁴ 

The purpose of the current study was to examine the role of GPx4 in the conjunctiva, the main component of the ocular surface, using the siRNA knockdown technique. 

Materials and Methods

Cell Culture and Transfection of siRNA

Human conjunctival epithelial cells (Wong-Kilbourne derivative of Chang conjunctiva, American Type Culture Collection) were cultured under 5% CO₂ at 37°C in Medium 199 (Invitrogen, Carlsbad, CA, USA) containing 10% FBS and 100 U penicillin plus 100 μg/mL streptomycin. 

Cells with 30% to 40% confluence were transfected with catalase, GPx1, GPx4, SOD1, SOD2, or control siRNA (Ambion) of 25 nM using lipofectamine²¹ (RNA iMAX; Invitrogen) following manufacturer's instruction. Morphology of transfected cells was assessed with an inverted phase-contrast microscope. 

Real-Time RT-PCR

Cells treated with catalase, GPx1, GPx4, SOD1, SOD2, and control siRNA were used at 2 days after transfection for detecting knockdown efficiency by RT-PCR. Cellular total RNA was isolated with Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. Subsequently, RNA was reverse-transcribed into cDNA by master mix with genomic DNA remover (ReverTra Ace qPCR RT with gDNA Remover; Toyobo, Osaka, Japan). Quantitative real-time PCR was carried out with thermal cycler dice (Takara Bio, Inc., Otsu, Shiga, Japan) using a qPCR kit (Platinum SYBR Green qPCR SuperMix-UDG; Invitrogen). Values for each gene were normalized to expression levels of GAPDH. The sequences of the primers used in the real-time RT-PCR were as follows: human GAPDH (Fwd, 5-TTGATTTTGGAGGGATCTCG-3- and Rev, 5-AACTTTGGCATTGTGGAAGG-3); human catalase (Fwd, 5-GCCTGGGACCCAATTATCTT-3, Rev, 5-GAATCTCCGCACTTCTCCAG-3); human GPx1 (Fwd, 5-CTCTTCGAGAAGTGCGAGGT-3, Rev, 5-TCGATGTCAATGGTCTGGAA-3); GPx4 (Fwd, 5-GCACATGGTTAACCTGGACA-3, Rev, 5-CTGCTTCCCGAACTGGTTAC-3); human SOD1 (Fwd, 5-TGGCCGATGTGTCTATTGAA-3, Rev, 5-GGGCCTCAGACTACATCCAA-3); and SOD2 (Fwd, 5-TTGGCCAAGGGAGATGTTAC-3, Rev, AGTCACGTTTGATGGCTTCC-3). 

Immunoblotting

Cells treated with catalase, GPx1, GPx4, SOD1, SOD2, and control siRNA were used at 2 days after transfection for detecting knockdown efficiency by immunoblotting. SDS-PAGE of cellular proteins was performed on gel (Mini-PROTEAN TGX Any kD; Bio-Rad Laboratories, Hercules, CA) with tris-glycine-SDS running buffer (Bio-Rad Laboratories). Immunoblot analysis was performed by electrotransferring proteins from the gels onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA) at 100 V for 60 minutes at ice-cold temperature using tris-glycine buffer. The membranes were probed with antibodies to β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); catalase (Santa Cruz Biotechnology, Inc.); GPx1 (Cell Signaling Technology, Beverly, MA, USA); GPx4 (Santa Cruz Biotechnology, Inc.); SOD1 (Santa Cruz Biotechnology, Inc.); and SOD2 (GeneTex, Inc., Irvine, CA, USA). Binding of secondary antibodies, conjugated to alkaline phosphatase or to horseradish peroxidase, was visualized with BCIP/NBT substrate (Bio-Rad Laboratories) or chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, USA). 

Cytotoxicity Assay

Membrane breakage and cell death were quantified using release of LDH into the culture medium.²² Four days after transfection, a cytotoxicity assay for SOD1, SOD2 catalase, GPx1, and GPx4 knockdown cells was performed using the lactate dehydrogenase (LDH) cytotoxicity detection kit (Takara Bio, Inc.).²³ Lactate dehydrogenase activity was measured in the extracellular medium and in the cell lysate according to the manufacturer's instructions, and then extracellular LDH activity was calculated as percentage of the total LDH activity. 

In oxidative study, cells treated with GPx4 or control siRNA were then stimulated with hydrogen peroxide (0, 0.1, and 1 μM; Wako, Osaka, Japan) or ferric sulfide (0, 100 and 1000 μM; Wako) at 2 days after transfection. Two days later, we evaluated LDH activity of cells treated with hydrogen peroxide or ferric sulfide. 

Determination of Lipid Peroxidation

The biomarker 4-HNE is known to be useful for lipid peroxidation.^11,12 Determination of lipid peroxidation was assessed by immunohistochemical detection 4-HNE.²⁴ Cells treated with GPx4 or control siRNA at 4 days after transfection were fixed with 4% paraformaldehyde for 15 minutes washed three times with PBS, and permeabilized with 0.1% of Triton X-100 solution containing 5% goat serum in PBS. Permeabilized cells were washed three times with PBS containing 5% goat serum, incubated with anti-4-HNE antibodies (Japan Institute for the Control of Aging, Shizuoka, Japan) for 1 day at 4°C. Then, cells were washed again three times with PBS. AlexaFluor 488–conjugated anti-mouse IgG secondary antibodies (Invitrogen) were applied, the sample left at room temperature for 1 hour, and excess antibodies were removed by washing cells three times with PBS. Fluorescent images were observed with a fluorescence microscope (Keyence Corp., Osaka, Japan). The fluorescence intensities of the dots stained with 4-HNE were quantified using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health [NIH], Bethesda, MD, USA). 

Fluorescent probe C11-BODIPY581/591 is used for indexing lipid peroxidation, and has been used as the indication of lipid peroxidation in mammalian cells.²⁵ The evaluation of lipid peroxidation was conducted by using not only 4-HNE immunostaining but also C11-BODIPY581/591. Cells treated with GPx4 or control siRNA at 4 days after transfection were incubated with 10 μM C11-BODIPY 581/591 (Invitrogen) for 30 minutes, and rinsed with proliferation medium. Then, the fluorescence was analyzed at 485-nm excitation/535-nm excitation.²⁵ 

Determination of Reactive Oxygen Species (ROS)

Chloromethyl derivative (CM)-H2DCFDA is a cell-permeant indicator for ROS,²⁶ and the cellular levels of ROS were determined using CM-H2DCFDA. Cells treated with GPx4 or control siRNA at 4 days after transfection were incubated with 5 μM CM-H2DCFDA (Invitrogen) for 30 minutes, and rinsed with proliferation medium. Then, the fluorescence was analyzed at 485-/535-nm excitation. 

Detection of Apoptosis

Apoptosis was evaluated by annexin V stain.^27,28 At 4 days after transfection, cells treated with GPx4 or control siRNA were stained by AlexaFluor 488 annexin V (Invitrogen) for 15 minutes at room temperature, and rinsed with PBS. Then cells were fixed using 2% paraformaldehyde, and mounted in mounting medium (Vectashield; Vector Laboratories, Burlingame, CA, USA) containing DAPI. Fluorescent images were observed with a fluorescence microscope (Keyence Corp.). The percentages of annexin V-positive, apoptotic cells relative to the total number of DAPI-positive cells were calculated. 

Assay of Proliferation

Proliferation of cells treated with GPx4 or control siRNA was assessed using WST-8 assay²⁹ (Dojindo Molecular Tech, Inc., Rockville, MD, USA) at 0, 1, 3, 5, and 7 days after transfection. We performed the WST-8 assay according to the manufacturer's instructions. 

Statistical Analysis

Data are expressed as mean + SEM. Statistical analysis was performed with 2-tail Student's t-test or Dennett's test. Values of P < 0.05 were considered statistically significant. 

Results

Knockdown of the Antioxidant Enzymes Using siRNA

Human conjunctival epithelial cells were transfected with catalase, GPx1, GPx4, SOD1, or SOD2 siRNA to cause the knockdown of each antioxidant enzyme. The messenger RNA expression was evaluated by quantitative RT-PCR. The messenger RNA expression of all antioxidant enzymes was downregulated by more than 85% (Fig. 1A). Moreover, protein expression level was determined by immunoblotting analysis. The treatment of siRNA prominently reduced the protein expression of each gene in all antioxidant enzymes (Fig. 1B). We confirmed remarkable gene knockdown of all antioxidant enzymes in the expression of mRNA and protein. 

Figure 1

 

Knockdown of antioxidative enzymes, catalase, GPx1, GPx4, SOD1, and SOD, by siRNA in conjunctival epithelial cells. (A) mRNA expression of each antioxidative enzyme was quantified by real-time RT-PCR, and normalized to GAPDH mRNA level. Data were means + SEM. (n = 3–4). (B) Protein expression of each antioxidative enzyme was determined by immunoblot analysis. Reproducibility was confirmed in triplicate.

Effects of Antioxidant Enzymes Knockdown on Morphologic Changes of Conjunctival Epithelial Cells

The morphologic characteristics of conjunctival epithelial cells treated with each targeted siRNA were investigated at 4 days after transfection. Cells treated with control siRNA appeared to be compact, uniform and cobblestone pavement in shape (Fig. 2). Shape of cells treated with catalase, GPx1, or SOD2 was similar to that of cells treated with control siRNA (Fig. 2). On the other hand, cells treated with GPx4, and SOD1 siRNA exhibited signs of cell damage such as spheroids. 

Figure 2

 

Phase contrast morphology of conjunctival epithelial cells treated with control, catalase, GPx1, GPx4, SOD1, or SOD2 siRNA at 4 days after transfection. Scale bar: 50 μm.

Effects of Antioxidant Enzymes Knockdown on LDH Activity

Cytotoxicity was evaluated by measuring LDH activity. Knockdown of GPx4 and SOD1 significantly increased the activity of LDH (Fig. 3). On the other hand, knockdown of catalase, GPx1, and SOD2 did not affect LDH activity (Fig. 3). These results suggest that GPx4 and SOD1 play important roles in maintaining oxidative homeostasis under physiological condition in conjunctival cells. 

Figure 3

 

Lactate dehydrogenase release from conjunctival epithelial cells treated with control, catalase, GPx1, GPx4, SOD1, or SOD2 siRNA 4 days after transfection. Data are means + SEM (n = 4). **P < 0.01 relative to control siRNA group (Dunnett's test).

Effects of GPx4 Knockdown on Lipid Peroxidation and ROS

A common byproduct of lipid peroxidation, 4-HNE also causes cellular damage.^11,12 To assess lipid hydroperoxide generation in the GPx4 knockdown cells, we performed immunostaining for 4-HNE. We found 4-HNE was significantly elevated in GPx4 knockdown cells as demonstrated by immunofluorescence microscopic detection (Figs. 4A, 4B). In addition, GPx4 knockdown significantly increased the fluorescence intensity of oxidized BODIPY-C11 (Figs. 4C). These results suggest that GPx4 is implicated in controlling the lipid hydroperoxides production of conjunctival epithelial cells. 

Figure 4

 

Determination of lipid peroxidation and ROS. (A) Detection of 4-HNE by fluorescence microscopy using 4-HNE antibodies. (B) The fluorescence intensities of 4-HNE were quantified using ImageJ (NIH). Data are means + SEM (n = 5). (C) The fluorescence intensities of oxidized lipid marker Bodipy C-11. Data are means + SEM (n = 12). (D) The fluorescence intensities of CM-H2DCFDA, the indicator of ROS. Data are means + SEM (n = 11–12). **P < 0.01 and *P < 0.05 relative to control siRNA group (Student's t-test). Scale bar: 10 μm.

Moreover, GPx4 knockdown significantly increased fluorescence intensity of CM-H2DCFDA (Figs. 4D). Thus, GPx4 is thought to play a role in regulating also ROS production of conjunctival epithelial cells. 

Effects of GPx4 Knockdown on Apoptosis and Cell Proliferation

Apoptosis was assessed by annexin V and DAPI staining. The percentage of annexin V–positive cells increased in GPx4 siRNA treated cells (Fig. 5). This result suggests that GPx4 knockdown contributes to apoptosis in conjunctival cells. 

Figure 5

 

Apoptosis caused by knockdown of GPx4 in conjunctival epithelial cells. (A) Cells stained with annexin V and DAPI by fluorescence microscopy. (B) The percentage of cells annexin V–positive cells relative to the total number of DAPI-positive cells. Data are means + SEM (n = 5). **P < 0.01 relative to control siRNA group (Student's t-test). Scale bar: 25 μm.

Next, the effects of GPx4 knockdown on conjunctival epithelial cell growth were examined. The proliferation was evaluated by WST-8 assay. There were no significant differences in cell proliferation among GPx4 and control siRNA treated cells until 3 days after transfection (Fig. 6). However, at 5 and 7 days after transfection, proliferation of GPx4 siRNA–treated cells was significantly lower than that of control siRNA-treated cells, suggesting that GPx4 was essential for growth of conjunctival epithelial cells (Fig. 6). 

Figure 6

 

Proliferation of cells treated with GPx4 siRNA. Proliferation was evaluated by WST-8 assay at 0, 1, 3, 5, and 7 days after transfection. Data are means + SEM (n = 5). **P < 0.01 relative to control siRNA group (Student's t-test).

Effects of GPx4 Knockdown on Cytotoxicity Induced by Oxidative Stress

Hydrogen peroxide and iron are potent generators of oxidative stress, and these agents are reported to induce oxidative damage in many cells.^30–32 We investigated the effects of GPx4 knockdown on cytotoxicity induced by hydrogen peroxide or iron in conjunctival epithelial cells. Lactate dehydrogenase activity of cells treated with control siRNA was not changed by 0.1 and 1 μM hydrogen peroxide or 100 and 1000 μM ferric sulfide (Figs. 7A, 7B). On the other hand, LDH of cells treated with GPx4 siRNA significantly increased by 1 μM hydrogen peroxide or 1000 μM ferric sulfide. Knockdown of GPx4 enhanced cytotoxicity by oxidative stress, and these results suggest that GPx4 is involved in the defense against oxidative stress. 

Figure 7

 

Glutathione peroxidase 4 knockdown enhanced LDH release induced by oxidative stress. Lactate dehydrogenase activity was evaluated at 2 days after application hydrogen peroxide or iron. Data are means + SEM (n = 4). **P < 0.01 relative to control siRNA group of each peroxide or iron dose (Student's t-test). ##P < 0.01 and #P < 0.05 relative to untreated GPx4 siRNA group between the groups of GPx4 siRNA (Dunnett's test).

Discussion

The major findings of the present study were: 

1.  
   GPx4 was an essential antioxidative enzyme for maintaining redox homeostasis in conjunctival epithelial cells. In the other antioxidative enzymes, SOD1 but not catalase, GPx1, or SOD2 seemed also essential for conjunctival cells.
2.  
   Reduction in the expression of GPx4 caused the apoptosis and inhibited the proliferation of conjunctival epithelial cells.
3.  
   Furthermore, GPx4 has an important role in protecting cells from cytotoxicity induced by the stimulation of oxidative stress in conjunctival epithelial cells.

The present study found that GPx4 plays a critical role in maintaining redox homeostasis and preventing cytotoxicity in conjunctival epithelial cells. Additionally, we found that not many of the antioxidant enzymes could be essential to prevent cytotoxicity because the knockdown of catalase, SOD2, and GPx1 did not increase cytotoxicity in conjunctival epithelial cells. It has been reported that GPx4 is essential in different kinds of cells and loss of GPx4 leads to cell death in vivo and in vitro.^14–16 Our results in the present study is considered to concur with these reports. The unique role of GPx4 that directly reduces the peroxidized lipid in cell membrane is considered important for conjunctival epithelial cells in redox homeostasis, morphological integrity, and the regulation of cell death and proliferation. In the GPx4-deficient conjunctival epithelial cells, an increased accumulation of lipid hydroperoxide and increased cytotoxicity was confirmed. Knockdown GPx4 also caused the increase of ROS production. Glutathione peroxidase 4 cannot directly control the production of ROS. However, the byproducts of lipid hydroperoxide such as 4-HNE are reported to enhance ROS production.^33,34 Thus, it is thought that GPx4 indirectly reduces ROS production. Because the accumulation of lipid hydroperoxide byproducts is associated with conjunctival diseases such as allergic conjunctivitis and dry eye,^1,3 the GPx4 expression may be important for the susceptibility to conjunctival diseases and may be a therapeutic target. In addition, because the cytotoxicity induced by loss of GPx4 can be at least partially rescued by endogenous ³⁵ or exogenous supplementation of vitamin E,¹⁵ it may also be important to evaluate the role of vitamin E for the disorders of conjunctival epithelium. 

In our experiments, we detected the increase in cytotoxicity and 4-HNE at 4 days after transfection. However, we did not detect the similar increase at 2 days when the expression of GPx4 was already suppressed. The results suggest that there was a time lag from the change in the expression of genes to the change in the biological toxicity. 

The superoxide dismutase family is a major antioxidant system.³⁶ A previous study reported that the corneal and conjunctival epithelia are damaged in the elderly SOD1-deficient mice.⁴ In the present study, knockdown of SOD1 caused cell cytotoxicity (Figs. 2, 3), and these results indicate that not only GPx4 but also SOD1 would be irreplaceable for the maintenance of oxidative homeostasis in conjunctival cells. On the other hand, our results indicate that SOD2 is not as important as SOD1 or GPx4 for conjunctival epithelial cells. The result was unexpected considering that SOD2 is a major antioxidant enzyme in mitochondria, SOD2 knockout mice are lethal at neonatal stage,³⁷ and loss of SOD2 in neuronal³⁸ and musculature³⁹ cells leads to significant cytotoxicity and cell death. However, cell type–specific essentiality of antioxidant enzymes can be possible, and we speculate that other antioxidant enzymes in mitochondria can play a complementary role for SOD2. As for catalase and GPx1, the intracellular hydrogen peroxide can be degraded by both catalase or GPx1.⁴⁰ Glutathione peroxidase 1 and catalase can be substitute for each other in the reduction of hydrogen peroxide.⁴⁰ Therefore, our results were considered reasonable that the knockdown of these enzymes did not induce significant cytotoxicity in conjunctival epithelial cells. 

In the present study, we showed that loss of GPx4 led to conjunctival epithelial cell death in vitro, and we found annexin V–positive apoptosis in the cell death. Although the cell death in the loss of GPx4 itself was in line with literature,^14–16 there have currently been a discussion on a novel mechanism of cell death, ferroptosis, where GPx4 is involved in cancer cells.^41,42 Ferroptosis is an iron-dependent cell death and important in cancer cells where iron is highly concentrated. Glutathione peroxidase 4 plays an important role in preventing cancer cell death through inhibition lipid peroxidation.⁴⁰ However, we consider that in conjunctival epithelial cells, noncancer cells, apoptosis is a major cell death pathway induced by loss of GPx4 as indicated in literature.^14,15 

In conclusion, the results in the present study demonstrated that GPx4 is an essential antioxidant enzyme for not only maintaining redox homeostasis, but also keeping defense against oxidative stress in conjunctival epithelial cells. Loss of GPx4 might cause the aggravation of pathology in conjunctiva, and GPx4 might be a new therapeutic target for conjunctival disorders such as dry eye and keratoconjunctivitis. 

Acknowledgments

Supported by Grant-in-Aid for Exploratory Research (25670725) from Japan Society for the Promotion of Science. 

Disclosure: O. Sakai, None; T. Uchida, None; H. Imai, None; T. Ueta, None; S. Amano, None