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Retinal Cell Biology  |   May 2015
Glutaredoxin 1 (Grx1) Protects Human Retinal Pigment Epithelial Cells From Oxidative Damage by Preventing AKT Glutathionylation
Author Affiliations & Notes
  • Xiaobin Liu
    Pharmaceutical Sciences University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Jamieson Jann
    Pharmaceutical Sciences University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Christy Xavier
    Pharmaceutical Sciences University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Hongli Wu
    Pharmaceutical Sciences University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, United States
    North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, United States
    Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, Texas, United States
  • Correspondence: Hongli Wu, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA; hongli.wu@unthsc.edu
Investigative Ophthalmology & Visual Science May 2015, Vol.56, 2821-2832. doi:10.1167/iovs.14-15876
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      Xiaobin Liu, Jamieson Jann, Christy Xavier, Hongli Wu; Glutaredoxin 1 (Grx1) Protects Human Retinal Pigment Epithelial Cells From Oxidative Damage by Preventing AKT Glutathionylation. Invest. Ophthalmol. Vis. Sci. 2015;56(5):2821-2832. doi: 10.1167/iovs.14-15876.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Glutaredoxin 1 (Grx1) belongs to the oxidoreductase family and is a component of the endogenous antioxidant defense system. However, its physiological function remains largely unknown. In this study, we investigated whether and how Grx1 overexpression protects the retinal pigment epithelial (RPE) cells against H2O2-induced apoptosis.

Methods.: Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx1-containing plasmid or an empty vector. Primary human RPE cells were transfected with Grx1 small interfering RNA (siRNA) or scrambled siRNA. Cell viability was measured with the WST8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Protein kinase B (AKT) activation was examined by Western blot. Protein kinase B glutathionylation was detected by immunoprecipitation followed by immunoblotting with anti-PSSG antibody.

Results.: Glutaredoxin 1 overexpression protected ARPE-19 cells from H2O2-induced cell viability loss. Conversely, Grx1 gene knockdown sensitized primary human RPE cells to H2O2. Assessment of apoptosis indicated that cells transfected with the Grx1-containing plasmid were more resistant to H2O2 with fewer cells undergoing apoptosis as compared to empty vector-transfected cells. Hydrogen peroxide–induced PSSG accumulation was also attenuated by Grx1 enrichment. Furthermore, Grx1 overexpression prevented H2O2-induced AKT glutathionylation, resulting in a sustained phospho-AKT elevation in RPE cells.

Conclusions.: Glutaredoxin 1 can protect RPE cells against oxidative stress–induced apoptosis. The mechanism of this protection is associated with its ability to stimulate the phosphorylation of AKT by preventing AKT glutathionylation. Considering Grx1's protective abilities in RPE cells, Grx1 could be a potential pharmacological target for retinal degenerative diseases.

Age-related macular degeneration (AMD) is a progressive eye disease that manifests as loss of central vision due to the degeneration of photoreceptors and adjacent retinal pigment epithelial (RPE) cells in the macula, the central portion of the retina.1 Between 20 and 25 million people are affected by AMD worldwide, a figure that will triple with the increase in the aging population in the next 30 to 40 years.2 Age-related macular degeneration is a multifactorial late-onset disease occurring due to oxidative damage, chronic inflammation, and alterations in the complement system. Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) and antioxidative defenses. The excessive level of ROS can damage various cell components, disrupt cellular physiological functions, accelerate aging, and lead to various oxidative stress-related diseases, such as neurodegenerative diseases, cancers,3 cardiovascular disorders,4 cataracts,5 and AMD.2 
It is widely accepted that oxidative stress–induced RPE cell dysfunction contributes to various retinal degenerative diseases, including AMD.2 Due to the high ambient oxygen tension required to maintain the high metabolic rate that is essential to maintain the normal function of the photoreceptors and the constant exposure to photo-oxidative stress, the RPE cells are subjected to long-term oxidative stress.6 In addition, the daily phagocytosis of shed photoreceptor outer segments by RPE cells also results in the generation of free radicals, including H2O2.7 Once these free radicals are produced, they attack nearby proteins, lipids, and nucleic acids, causing oxidative damage to these essential macromolecules. Among the macromolecules being attacked, proteins are the main targets of free radicals due to their high abundance and their high reactivity with ROS. Under oxidative stress, the thiols in cysteine residues within proteins (PSH) are among the most oxidant-sensitive targets and can undergo a variety of reversible and irreversible redox modifications.8 Protein glutathionylation, defined as the reversible formation of a mixed disulfide (PSSG) between protein thiols and glutathione (GSH), appears to be the most important mode of thiol oxidation.5 Protein glutathionylation formation can potentially modify protein/enzyme structure, function, and activity, or even disrupt cell signaling, depending on the importance of the cysteine residue in carrying out protein/enzyme function. To prevent protein function loss in the RPE cells, the thiol groups of the proteins must be maintained at their normal, reduced state by effectively cleaving the protein-thiol mixed disulfides, which is catalyzed by thiol-disulfide oxidoreductases. 
Glutaredoxin (Grx), also known as thioltransferase (TTase), is a subfamily of the thiol-disulfide oxidoreductase family, containing a conserved Cys-X-X-Cys domain in its active site. So far, two Grx genes have been identified: cytosolic glutaredoxin 1 (Grx1)9,10 and the recently discovered mitochondrial glutaredoxin 2 (Grx2).11,12 The first evidence of the presence of Grx1 in the neural retina was provided by Lou et al.13 in 1997. Sears et al.14 further proved that Grx1 is also highly expressed in the RPE cells where it modulates protein deglutathionylation. However, physiological roles of endogenous Grx1 have not been well elucidated in the RPE cells. 
The purpose of the present study was to explore the possible protective role of Grx1 against oxidative stress in the human RPE cells. We also examined the crosstalk between PSSG and phosphorylation and revealed protein kinase B (AKT) as a new target for redox regulation in RPE cells. 
Materials and Methods
Materials
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), G418 (Life Technologies, Grand Island, NY, USA), penicillin/streptomycin, 0.25% trypsin, and other cell culture reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Hydrogen peroxide and other chemicals were obtained from Sigma-Aldrich Corp. unless otherwise stated. Anti-Grx1 antibody was purchased from Abcam (#ab45953; Cambridge, MA, USA). Anti-PSSG was from Viogen (#101-A-100; Watertown, MA, USA). Anti-AKT (#2920) and p-AKT (#9271) antibodies were from Cell Signaling (Boston, MA, USA). Anti-β-actin antibody was from Sigma-Aldrich Corp. (#A2228). Horseradish peroxidase–conjugated secondary antibodies (sc2061, sc2060, sc2030) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 
Human RPE Cell Culture
Human fetal RPE cells were purchased at passage 1 from ScienCell Research Laboratories (Carlsbad, CA, USA), and all experiments were performed with cells between passages 2 and 8. A cell line derived from adult human RPE cells, the ARPE-19 cell, was kindly gifted by Fu Shang (Tufts University, Medford, MA, USA). For H2O2-induced apoptotic studies, cells were synchronized by gradual serum deprivation with the following procedure: Cells were cultured overnight in DMEM with 2% FBS followed by incubation in serum-free medium for 30 minutes before exposure to a bolus of 200 μM H2O2 for 24 hours. 
Overexpression of Human Grx1 in ARPE-19 Cells
Plasmid containing human Grx1 was a generous gift from Marjorie F. Lou and Rodrigo Franco (University of Nebraska-Lincoln, Lincoln, NE, USA). Human Grx1 gene was cloned into the multicloning site of Geneticin-resistant mammalian expression vector pCR3.1 (+) to construct sense plasmids. PCR3.1 (+) empty vector was used as a negative control. The plasmids were introduced into ARPE-19 cells by electroporation using GenePulser X Cell (Bio-Rad, Richmond, CA, USA) at 160 V, 500 μF. After electroporation, the cells were transferred into new dishes with fresh culture medium containing 1 mg/mL Geneticin for selection. Thereafter, the cells were fed every 3 days. After 4 weeks of selection, the surviving cells were passaged into new dishes with culture medium supplemented with 400 μg/mL Geneticin. 
SiRNA Transfection
Control and Grx1 small interfering RNA (siRNA) used in this study were chemically synthesized by Santa Cruz Biotechnology. Small interfering RNA duplexes (scrambled siRNA, sc-37007; Grx1 siRNA, sc-72089) were transfected into primary human RPE cells with siRNA transfection reagent (sc-29528; Santa Cruz Biotechnology) according to the manufacturer's instructions. 
Cell Viability Assay
Cell viability was measured by a colorimetric cell viability kit (Promokine, Heidelberg, Germany) with the tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt). Cells were seeded at a density of 5000 cells/well (100 μL total volume/well) in a 96-well assay plate and were treated with 200 μM H2O2 for 24 hours. After treatment, 10 μL WST-8 solution was added to each well of the culture plate and incubated for 2 hours in the incubator. The absorption was evaluated at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). 
Grx1 Enzyme Activity Assay
Glutaredoxin 1 was assayed following the method described by Gladyshev et al.11 Briefly, the reaction mixture contained 0.2 mM β-nicotinamide adenine dinucleotide phosphate, reduced tetra(cyclohexylammonium) salt (NADPH), 0.5 mM GSH, 0.1 M potassium phosphate buffer (pH 7.4), 0.4 units of glutathione reductase (GR), and an aliquot of whole cell lysate in a total volume of 1 mL. The reaction was carried out at 30°C and initiated after a 5-minute preincubation with 2 mM hydroxyethyl disulfide (HEDS). The decrease in absorbance of NADPH at 340 nm was monitored for a kinetic read sequence of 30-second intervals for a total 5 minutes using a spectrophotometer (SpectronicTM 200; Thermo Scientific, Waltham, MA, USA). 
Flow Cytometry Analysis of Cell Apoptosis
Cells were seeded in 100-mm culture plates and incubated overnight at 37°C. After exposure to 200 μM H2O2 for 24 hours, the cells were trypsinized and stained with annexin V and propidium iodide (PI) using annexin V apoptosis kit (Invitrogen, Grand Island, NY, USA) according to the manufacturer's protocol. The stained cells were then analyzed by flow cytometry (FC500; Beckman Coulter, Indianapolis, IN, USA) to differentiate viable (annexin V/PI), early apoptotic (annexin V+/PI), and late apoptotic (annexin V+/PI+) cells. 
Hoechst 33342 Fluorescent Staining
Cells were plated on a 12-well plate at a density of 1 × 105 cells/well on poly-L-lysine–coated glass coverslips and treated with or without 200 μM H2O2 for 24 hours. The cells were washed twice with ice-cold PBS and then fixed with cold 4% paraformaldehyde for 15 minutes followed by PBS wash. The fixed cells were then stained with 0.1 μg/mL Hoechst 33342 (Invitrogen) for 10 minutes and rinsed with PBS. The images were taken using a fluorescence microscope (Olympus, Center Valley, PA, USA). 
Western Blot Analysis
Cells were collected using cell scrapers on ice and were lysed with radioimmunoprecipitation (RIPA) lysis buffer (R0278; Sigma-Aldrich Corp.). 
An equal amount of proteins was applied on 10% SDS-PAGE, and resolved protein bands were transferred to enhanced chemiluminescence membrane (Hybond; GE Healthcare, Piscataway, NJ, USA) and probed with specific antibodies. Corresponding protein bands were detected and visualized using a SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA). 
GSH and Glutathione Disulfide Assays
For GSH measurement, an aliquot of fresh cell lysate was treated with an equal volume of 20% trichloroacetic acid followed by centrifugation. The supernatant was used immediately for GSH analysis using 5,5′-dithiobis(2-nitrobenzic acid) (DTNB) reagent, following the method of Lou et al.15 The glutathione disulfide (GSSG) level was measured by using a GSSG assay kit (ab156681; Abcam) according to the manufacturer's instructions. 
Protein Glutathionylation Detection
Vector and Grx1 overexpressed (OE) cells were treated with 1 mM H2O2 for 30 minutes and lysed with RIPA buffer. Equal amounts of protein (80 μg) in each group were loaded onto an SDS-PAGE gel under nonreducing (no β-mercaptoethanol) conditions and probed with anti-PSSG antibody. For AKT glutathionylation detection, cells were treated with 200 μM H2O2 for 30 minutes, and cell lysates were incubated with anti-AKT antibody magnetic beads conjugate (Thermo Scientific) overnight at 4°C. The immunoprecipitated complexes were analyzed on 12% nonreducing SDS-PAGE followed by detection of glutathionylation by immunoblotting with anti-PSSG antibody. 
Statistics
Each experiment was performed at least three times, and statistical analyses were performed using Student's t-test when comparing between two groups and one-way ANOVA followed by Bonferroni's test as a post hoc test when comparing among three or more groups with the Prism software (GraphPad, La Jolla, CA, USA). The number of experimental samples used in each group is presented in the figure legends. All data are expressed as means ± SD, and differences were considered significant at P < 0.05. 
Results
Effects of H2O2 on Cell Viability and Morphology Changes in RPE Cells
To identify the appropriate concentration of H2O2, nontransfected ARPE-19 cells were treated with 100 μM to 1 mM H2O2 for 24 hours, and WST-8 assay was used to measure the dose-dependent cell toxicity of H2O2 in ARPE-19 cells. As shown in Figure 1A, H2O2 at low concentration of 100 μM had no obvious effect on the viability of RPE cells (control: 100% vs. 100 μM H2O2: 95.3% ± 7.2%). Hydrogen peroxide at higher concentrations of 150, 200, 300, 400, and 500 μM and 1 mM produced a progressive cytotoxic effect in ARPE-19 cells. Therefore, 200 μM H2O2, which induced approximately 60% of cell viability loss, was used in the following experiments. We also examined the morphologic changes of ARPE-19 cells in response to different concentrations of H2O2. As shown in Figure 1B, no obvious morphologic changes were seen in the 100 μM H2O2-treated group. In contrast, 200 and 500 μM H2O2 significantly decreased the cell number of ARPE-19 cells in a dose-dependent manner. 
Figure 1
 
Cytotoxicity of H2O2 in ARPE-19 cells. (A) Dose-dependent effect of H2O2 on the viability of ARPE-19 cells. Cells were exposed to 0, 100, 150, 200, 300, 400, or 500 μM or 1 mM H2O2 for 24 hours. Viable cells were quantified by WST-8 assay. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the 0 μM H2O2 group. (B) Morphologic changes of ARPE-19 cells. Cells were exposed to 0, 100, 200, or 500 μM H2O2 for 24 hours. Cell morphology was examined with phase-contrast microscopy. Scale bars: 50 μm.
Figure 1
 
Cytotoxicity of H2O2 in ARPE-19 cells. (A) Dose-dependent effect of H2O2 on the viability of ARPE-19 cells. Cells were exposed to 0, 100, 150, 200, 300, 400, or 500 μM or 1 mM H2O2 for 24 hours. Viable cells were quantified by WST-8 assay. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the 0 μM H2O2 group. (B) Morphologic changes of ARPE-19 cells. Cells were exposed to 0, 100, 200, or 500 μM H2O2 for 24 hours. Cell morphology was examined with phase-contrast microscopy. Scale bars: 50 μm.
Overexpression of Grx1 in Human ARPE-19 Cells
We first examined the effects of H2O2 on Grx1 expression in ARPE-19 cells. As shown in Supplementary Figure S1, H2O2 activated Grx1 by increasing its expression at both the mRNA and protein levels, which may be an adaptive protective response of the RPE cells to restore the altered redox state. To assess the role of Grx1 in protection against oxidative stress, Grx1 was stably transfected in ARPE-19 cells. As shown in Figure 2A, cells transfected with Grx1-containing plasmid (Grx1 OE) exhibited a 3-fold increase in Grx1 protein expression compared to that of empty vector (vector) and nontransfected control wild-type (WT) cells, suggesting successful Grx1 transfection and expression. Fluorescence microscopic examination was also performed to detect the immunostaining of Grx1. As shown in Figure 2B, Grx1 was evenly distributed throughout the cytoplasm of Grx1 OE cells. On the other hand, in vector cells, only a very low level of florescence signal could be detected. Glutaredoxin 1 immunofluoresence in Grx1 OE cells exhibited a 2-fold increase compared with that of vector cells. With respect to Grx1 enzyme activity, Grx1-containing plasmid transfection stimulated Grx1 activity to 16.3 ± 1.96 mU/mg protein, which was ∼3-fold that in vector cells (Fig. 2C). 
Figure 2
 
Validation of Grx1 overexpression in ARPE-19 cells. (A) Western blot analysis of Grx1 expression in wild-type (WT, nontransfected), empty vector-transfected (vector), and Grx1 overexpressed (Grx1 OE) cells. Bottom: The relative pixel density of Grx1 over β-actin. (B) Immunostaining of GRX1 in cells transfected with empty vector (vector) of Grx1-containing plasmid (Grx1 OE). Scale bars: 50 μm. Bottom: The average fluorescence intensity of Grx1. (C) Grx1 enzyme activity in vector and Grx1 OE cell lysates. Data presented are a typical representation of triplicate experiments. ***P < 0.001 compared with vector group.
Figure 2
 
Validation of Grx1 overexpression in ARPE-19 cells. (A) Western blot analysis of Grx1 expression in wild-type (WT, nontransfected), empty vector-transfected (vector), and Grx1 overexpressed (Grx1 OE) cells. Bottom: The relative pixel density of Grx1 over β-actin. (B) Immunostaining of GRX1 in cells transfected with empty vector (vector) of Grx1-containing plasmid (Grx1 OE). Scale bars: 50 μm. Bottom: The average fluorescence intensity of Grx1. (C) Grx1 enzyme activity in vector and Grx1 OE cell lysates. Data presented are a typical representation of triplicate experiments. ***P < 0.001 compared with vector group.
Overexpression of Grx1 Protects ARPE-19 Cells Against H2O2-Mediated Cell Death
Next, we evaluated the effects of Grx1 overexpression against H2O2-induced cell death. As summarized in Figure 3A, after 24 hours of 200 μM H2O2 treatment, the percentage of surviving cells in the vector group decreased dramatically to 40%. However, in the Grx1 OE group, more than 80% of cells were still viable after H2O2 treatment. The protective effect of Grx1 overexpression was also assessed by morphologic observation (Fig. 3B). Live cell phase-contrast microscopy showed that 24-hour treatment with 200 μM H2O2 led to cell shrinkage and complete loss of the cell body in vector cells. However, the H2O2-treated Grx1 OE cells remained relatively dense with clear cell body, indicating that Grx1 overexpression protected ARPE-19 cells from oxidative stress. Furthermore, we also found that Grx1 overexpression significantly decreased H2O2-induced ROS production, indicating that Grx1 can effectively detoxify H2O2 and probably all ROS (Supplementary Fig. S2). 
Figure 3
 
Effects of Grx1 overexpression on H2O2-induced cytotoxicity in ARPE-19 cells. (A) Vector-transfected cells (vector) and Grx1 overexpressed cells (Grx1 OE) were exposed to medium with or without H2O2 (200 μM) for 24 hours. After treatment, cell viability was determined by WST-8 assay. The data are expressed as means ± SD of three experiments, n = 8. ***P < 0.001. (B) Cell density and morphology were examined with phase-contrast microscopy. Scale bars: 50 μm. (C) Apoptosis was then determined by detection of cells with apoptotic nuclear morphology using fluorescence microscopy upon Hoechst 33342 staining. Cells with nuclei showing strong chromatin condensation and nuclear fragmentation (labeled with white arrow) were considered apoptotic. Scale bars: 20 μm.
Figure 3
 
Effects of Grx1 overexpression on H2O2-induced cytotoxicity in ARPE-19 cells. (A) Vector-transfected cells (vector) and Grx1 overexpressed cells (Grx1 OE) were exposed to medium with or without H2O2 (200 μM) for 24 hours. After treatment, cell viability was determined by WST-8 assay. The data are expressed as means ± SD of three experiments, n = 8. ***P < 0.001. (B) Cell density and morphology were examined with phase-contrast microscopy. Scale bars: 50 μm. (C) Apoptosis was then determined by detection of cells with apoptotic nuclear morphology using fluorescence microscopy upon Hoechst 33342 staining. Cells with nuclei showing strong chromatin condensation and nuclear fragmentation (labeled with white arrow) were considered apoptotic. Scale bars: 20 μm.
Overexpression of Grx1 Protects ARPE-19 Cells Against H2O2-Induced Cell Apoptosis
To better understand the antiapoptotic effect of Grx1, cell apoptosis was evaluated by two different assays: Hoechst 33342 nuclei staining and annexinV/PI double staining. As shown in Figure 3C, unstressed vector and Grx1 OE cells both showed normal nuclear morphology with relative low Hoechst 33342 fluorescence intensity. After treatment with 200 μM H2O2 for 24 hours, vector cells showed reduced nuclear size, increased Hoechst 33342 fluorescence intensity, and pyknotic nuclei (labeled with arrows). In contrast, Grx1 overexpression effectively prevented H2O2-induced nuclear changes as indicated by fewer cells with nuclear fragmentation and condensation. Next, annexin-V/PI staining and fluorescence-activated cell sorting (FACS) was used to quantify early (annexin V+/PI) or late apoptotic (annexin V+/PI+) cells. As shown in Figures 4A and 4B, cell apoptosis levels were similarly low as in nonstressed vector and Grx1 OE cells. However, after H2O2 treatment, the rate of early apoptosis increased to 24.1% ± 2.8% in the vector group but remained very low (2.2% ± 0.6%) in the Grx1 OE group (P < 0.05). Consistently, the number of late apoptotic cells increased to 45.8% ± 6.5% in the vector group, but only 6.7% ± 2.1% in the Grx1 OE group (P < 0.05), indicating that Grx1 has a strong antiapoptotic effect. 
Figure 4
 
Flow cytometry quantification of apoptosis in H2O2-treated vector and Grx1 OE cells. (A) Flow cytometry analysis of vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) cells treated with or without H2O2 (200 μM) for 24 hours. Representative figures showing population of viable (annexin V/PI), early apoptotic (annexin V+/PI), late apoptotic (annexin V+/PI+), and necrotic (annexin V/PI+) cells. Bar graphs show the quantification of early (B) and late apoptotic (C) cells. Data are mean ± SD of three independent experiments. ***P < 0.001 compared with vector group.
Figure 4
 
Flow cytometry quantification of apoptosis in H2O2-treated vector and Grx1 OE cells. (A) Flow cytometry analysis of vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) cells treated with or without H2O2 (200 μM) for 24 hours. Representative figures showing population of viable (annexin V/PI), early apoptotic (annexin V+/PI), late apoptotic (annexin V+/PI+), and necrotic (annexin V/PI+) cells. Bar graphs show the quantification of early (B) and late apoptotic (C) cells. Data are mean ± SD of three independent experiments. ***P < 0.001 compared with vector group.
Grx1 Overexpression Stimulated Antiapoptotic Bcl-2 Protein Expression and Decreased Proapoptotic Bax Protein Expression During Oxidative Stress
To investigate the possible mechanism of the antiapoptotic effect of Grx1, we examined the involvement of Bcl-2 family proteins in the initiation and amplification of RPE apoptosis cascade. As shown in Figure 5A, the level of Bax, the proapoptotic molecule, rose extensively after oxidative stress in vector cells but much less in cells with enriched Grx1. In contrast, the protein level of antiapoptotic factor Bcl-2 was suppressed by 80% in the vector group after H2O2 treatment. However, cells with Grx1 overexpression could drastically prevent oxidative stress–induced Bcl-2 loss (Fig. 5B). 
Figure 5
 
Effects of Grx1 overexpression on Bax, Bcl-2, and caspase 3 levels. Vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) ARPE-19 cells were treated with or without 200 μM H2O2 for 24 hours. Whole cell lysates (40 μg proteins) from vector and Grx1 OE groups were separated by 12% SDS–PAGE and hybridized with indicated specific antibodies. (A) Effects of Grx1 overexpression on the Bax protein expression in ARPE-19 cells (left). Right: The relative pixel density of Bax over β-actin. (B) Effects of Grx1 overexpression on the Bcl-2 protein expression in ARPE-19 cells. Right: The relative pixel density of Bcl-2 over β-actin. (C) Effects of Grx1 overexpression on caspase 3 cleavage in ARPE-19 cells. Samples were immunoblotted against antibodies specific for pro-caspase 3 and cleaved caspase 3, respectively. Beta-actin was also used as a loading control. The relative pixel density of the cleaved caspase 3 at 17 kDa over pro-caspase 3 at 35 kDa is shown at right. The data presented above are a typical representation of triplicate experiments. ***P < 0.001 compared with vector group.
Figure 5
 
Effects of Grx1 overexpression on Bax, Bcl-2, and caspase 3 levels. Vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) ARPE-19 cells were treated with or without 200 μM H2O2 for 24 hours. Whole cell lysates (40 μg proteins) from vector and Grx1 OE groups were separated by 12% SDS–PAGE and hybridized with indicated specific antibodies. (A) Effects of Grx1 overexpression on the Bax protein expression in ARPE-19 cells (left). Right: The relative pixel density of Bax over β-actin. (B) Effects of Grx1 overexpression on the Bcl-2 protein expression in ARPE-19 cells. Right: The relative pixel density of Bcl-2 over β-actin. (C) Effects of Grx1 overexpression on caspase 3 cleavage in ARPE-19 cells. Samples were immunoblotted against antibodies specific for pro-caspase 3 and cleaved caspase 3, respectively. Beta-actin was also used as a loading control. The relative pixel density of the cleaved caspase 3 at 17 kDa over pro-caspase 3 at 35 kDa is shown at right. The data presented above are a typical representation of triplicate experiments. ***P < 0.001 compared with vector group.
Caspase 3 activation is another marker of cell apoptosis. Caspase 3 is normally present as an inactive form of procaspase 3 (35 kDa) until stress-induced apoptotic signaling cleaves procaspase 3 to the active form (17 kDa). As shown in Figure 5C, cleaved caspase 3 was not seen in nontreated vector and Grx1 OE cells. After H2O2 treatment, cleaved caspase 3 was increased in vector cells but barely detectable in Grx1 OE cells (Fig. 5C). 
GSH Level and GSSG Level
The GSH-GSSG redox pair is considered the most important cellular redox buffer system. As shown in Figure 6A, under H2O2 treatment, intracellular GSH level in vector cells was significantly decreased from 22.5 ± 2.5 to 5.1 ± 2.5 nmol/mg protein. However, only less than a 10% decrease in GSH level was observed in Grx1 OE cells after H2O2 treatment. On the other hand, as shown in Figure 6B, the level of GSSG (oxidized GSH) was significantly increased in vector cells in response to H2O2 but remained relatively low in Grx1 OE cells (vector: 1.2 ± 0.3 versus Grx1 OE: 0.6 ± 0.1 nmol/mg protein, P < 0.05). 
Figure 6
 
Effects of Grx1 overexpression on levels of GSH, GSSG, and protein-GSH mixed disulfide (PSSP) levels. Vector-transfected cells (vector) and Grx1 overexpressed cells (Grx1 OE) were exposed to medium with and without H2O2 (200 μM) for 24 hours. Glutathione level (A) and GSSG level (B) were measured as described in the Materials and Methods. (C) Comparison of protein glutathionylation (PSSG) level in H2O2-treated vector and Grx1 OE cells. The cells were treated with 1 mM H2O2 for 30 minutes, and total proteins were separated on a 12% SDS gel under nonreducing condition (no β-mercaptoethanol) for blot analysis using an anti-PSSG antibody. The graph on the right depicts the relative pixel density of all the PSSG bands over β-actin. Data are mean ± SD of three independent experiments. ***P < 0.001 compared with vector group.
Figure 6
 
Effects of Grx1 overexpression on levels of GSH, GSSG, and protein-GSH mixed disulfide (PSSP) levels. Vector-transfected cells (vector) and Grx1 overexpressed cells (Grx1 OE) were exposed to medium with and without H2O2 (200 μM) for 24 hours. Glutathione level (A) and GSSG level (B) were measured as described in the Materials and Methods. (C) Comparison of protein glutathionylation (PSSG) level in H2O2-treated vector and Grx1 OE cells. The cells were treated with 1 mM H2O2 for 30 minutes, and total proteins were separated on a 12% SDS gel under nonreducing condition (no β-mercaptoethanol) for blot analysis using an anti-PSSG antibody. The graph on the right depicts the relative pixel density of all the PSSG bands over β-actin. Data are mean ± SD of three independent experiments. ***P < 0.001 compared with vector group.
Grx1 Overexpression Diminishes H2O2-Induced Protein Glutathionylation
Since the major function of Grx1 is to catalyze the reduction of PSSG, we next investigated whether Grx1 overexpression decreases the accumulation of glutathionylated proteins in ARPE-19 cells in response to treatment with H2O2. Since PSSG is a relatively fast and dynamic process, in order to capture the most obvious PSSG level difference between tested groups, we used a higher concentration of H2O2 (1 mM) and a shorter incubation time (30 minutes) to treat the cells to compare the oxidation-induced PSSG formation. As shown in Figure 6C, without H2O2 treatment, the basal levels of PSSG were equally low in both vector and Grx1 OE cells. Hydrogen peroxide treatment significantly increased PSSG formation in vector cells, whereas Grx1 enrichment effectively prevented oxidative stress–induced PSSG accumulation, indicating that high levels of Grx1 may accelerate the deglutathionylation process to repair oxidative protein damage within a short period of time. 
Grx1 Overexpression Enhances Oxidant-Mediated AKT Activation Through Preventing AKT Glutathionylation
It has been previously reported that oxidant-mediated AKT activation plays a central role in protecting RPE cells against oxidative stress–induced apoptosis.16 To better understand the involvement of AKT activation in the cytoprotective effects of Grx1, we first tested if Grx1 overexpression enhances AKT phosphorylation at its serine residue 473 (Ser473), which fully activates the enzymatic function of AKT kinases. As shown in Figure 7A, the acute application of H2O2 triggered a robust but transient increase in AKT phosphorylation; the level of phosphor-AKT (p-AKT) reached its peak at approximately 30 minutes, but declined rapidly and returned to basal level 120 minutes after H2O2 challenge. In contrast, in Grx1 OE cells, H2O2 treatment resulted in a long-lasting activation of AKT. When Grx1 OE cells were exposed to 200 μM H2O2, p-AKT quickly increased and reached a maximum level within 30 minutes, and the level persisted for up to 240 minutes with no obvious sign of decline. 
Figure 7
 
Glutaredoxin 1 overexpression enhances AKT phosphorylation through preventing AKT-SSG formation. (A) Time-dependent AKT activation in vector and Grx1 OE cells under oxidative stress. Vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) cells were treated with 200 μM H2O2 for different times (0–240 minutes). Total AKT and phospo-AKT (p-AKT) were determined by using total AKT and p-AKT-specific antibodies. Bottom: The relative pixel density of p-AKT over total AKT. (B) Detection of glutathionylated AKT (AKT-SSG) in vector and Grx1 OE cells. Vector and Grx1 OE cells were treated with or without 200 μM H2O2 for 30 minutes. Protein kinase B was then immunoprecipitated from cell lysates by using AKT-antibody, followed by Western blot detection of PSSG. Normal rabbit IgG was used as a negative control. The graph at the bottom depicts the relative pixel density of AKT-SSG bands over total AKT. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vector group.
Figure 7
 
Glutaredoxin 1 overexpression enhances AKT phosphorylation through preventing AKT-SSG formation. (A) Time-dependent AKT activation in vector and Grx1 OE cells under oxidative stress. Vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) cells were treated with 200 μM H2O2 for different times (0–240 minutes). Total AKT and phospo-AKT (p-AKT) were determined by using total AKT and p-AKT-specific antibodies. Bottom: The relative pixel density of p-AKT over total AKT. (B) Detection of glutathionylated AKT (AKT-SSG) in vector and Grx1 OE cells. Vector and Grx1 OE cells were treated with or without 200 μM H2O2 for 30 minutes. Protein kinase B was then immunoprecipitated from cell lysates by using AKT-antibody, followed by Western blot detection of PSSG. Normal rabbit IgG was used as a negative control. The graph at the bottom depicts the relative pixel density of AKT-SSG bands over total AKT. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vector group.
To get a better insight into the contribution of AKT redox regulation to RPE survival, we examined whether Grx1-mediated AKT deglutathionylation is responsible for the observed persistent activation of AKT in H2O2-exposed Grx1 OE cells. Total AKT protein was immunoprecipitated from either vector or Grx1 OE cells treated with or without H2O2, followed by immunoblotting with anti-PSSG antibody to detect AKT glutathionylation. As demonstrated in Figure 7B, there was no detectable level of AKT glutathionylation in both vector and Grx1 OE cells under nonstressed conditions. However, when cells were exposed to 200 μM H2O2 for 30 minutes, the level of AKT glutathionylation was much lower in Grx1 OE cells than in the vector cells, indicating that Grx1 enrichment effectively prevented H2O2-induced AKT glutathionylation. 
Grx1 Knockdown Sensitizes Primary RPE Cells to Oxidation and Prevents H2O2-Induced AKT Activation
We next examined the effect of Grx1 knockdown on cell viability and AKT activation in RPE cells. To provide more biologically relevant results, we used primary human RPE cells for siRNA analysis. As indicated in Figure 8A, Grx1 siRNA transfection successfully suppressed Grx1 expression to 90% of the control (nontransfected cells), whereas scramble siRNA showed no obvious effect. Meanwhile, Grx1 activity was significantly lower in Grx1 knockdown cells (Fig. 8B). As shown in Figure 8C, Grx1 knockdown alone decreased cell viability of RPE cells by 20%, suggesting that Grx1 plays a critical role in cell survival. When exposed to 100 μM H2O2 for 24 hours, scramble siRNA cells showed only a 40% cell viability loss while Grx1 siRNA cells showed a 75% reduction in cell viability. Challenging these cells with 100 μM H2O2 for various times demonstrated the expected robust activation of AKT in scramble siRNA cells but marked inhibition of AKT phosphorylation in Grx1 knockdown cells. These results collectively imply that Grx1 plays an important role in RPE survival through regulating oxidation-dependent AKT activation. 
Figure 8
 
Glutaredoxin 1 inhibition sensitizes primary human RPE cells to oxidative stress. Primary human RPE cells were transfected with Grx1 siRNA, with scramble siRNA, or without transfection (control) for 48 hours. The cells were then treated with or without 100 μM H2O2 for another 24 hours. (A) Validation of Grx1 inhibition with Western blot. Glutaredoxin 1 level in control, scramble siRNA, and Grx1 siRNA groups was analyzed by Western blot using anti-Grx1 antibody. Right: The relative pixel density of Grx1 over β-actin. (B) Glutaredoxin 1 enzyme activity in control, scramble siRNA, and Grx1 siRNA cell lysates. Glutaredoxin 1–specific activity was measured as described in the Materials and Methods. (C) Cell viability was measured by WST-8 in control, scramble siRNA, and Grx1 siRNA groups after H2O2 treatment. The data are expressed as means ± SD; n = 6 from three separate experiments. (D) Time-dependent AKT activation in scramble siRNA and Grx1 siRNA cells under oxidative stress. Scramble siRNA and Grx1 siRNA cells were treated with 100 μM H2O2 for different times (0–240 minutes). Total AKT and phospo-AKT (p-AKT) were determined by using total AKT and p-AKT–specific antibodies. Bottom: The relative pixel density of p-AKT over total AKT. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with scramble siRNA group.
Figure 8
 
Glutaredoxin 1 inhibition sensitizes primary human RPE cells to oxidative stress. Primary human RPE cells were transfected with Grx1 siRNA, with scramble siRNA, or without transfection (control) for 48 hours. The cells were then treated with or without 100 μM H2O2 for another 24 hours. (A) Validation of Grx1 inhibition with Western blot. Glutaredoxin 1 level in control, scramble siRNA, and Grx1 siRNA groups was analyzed by Western blot using anti-Grx1 antibody. Right: The relative pixel density of Grx1 over β-actin. (B) Glutaredoxin 1 enzyme activity in control, scramble siRNA, and Grx1 siRNA cell lysates. Glutaredoxin 1–specific activity was measured as described in the Materials and Methods. (C) Cell viability was measured by WST-8 in control, scramble siRNA, and Grx1 siRNA groups after H2O2 treatment. The data are expressed as means ± SD; n = 6 from three separate experiments. (D) Time-dependent AKT activation in scramble siRNA and Grx1 siRNA cells under oxidative stress. Scramble siRNA and Grx1 siRNA cells were treated with 100 μM H2O2 for different times (0–240 minutes). Total AKT and phospo-AKT (p-AKT) were determined by using total AKT and p-AKT–specific antibodies. Bottom: The relative pixel density of p-AKT over total AKT. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with scramble siRNA group.
Discussion
Protein glutathionylation, like protein phosphorylation, is an important reversible posttranslational modification that works as a regulatory mechanism of redox signaling pathways for many cellular events, including apoptosis.17 However, the precise molecular mechanism and possible role of S-glutathionylation during oxidative stress, especially in RPE cells, remain largely unexplored. The findings from this study define a new redox-based regulatory system that controls RPE cell survival involving Grx1-catalyzed deglutathionylation of AKT. We propose that the antiapoptotic effect of Grx1 is likely associated with its ability to increase AKT activation by preventing oxidation-induced AKT glutathionylation. The evidence supporting this notion is as follows: (1) Grx1 is important for RPE survival under oxidative stress conditions; (2) H2O2 treatment stimulated PSSG accumulation in RPE cells, indicating that protein S-glutathionylation may play a critical role in H2O2-mediated RPE cell death; (3) Grx1 is able to maintain the redox homeostasis in RPE cells by preserving the intracellular GSH pool; and (4) Grx1 overexpression increased AKTSer473 phosphorylation and protected RPE cells against oxidative stress–induced apoptosis by preventing oxidation-induced AKT-SSG formation. 
Glutaredoxin was originally identified in 1976 as a GSH-dependent electron donor for ribonucleotide reductase, and it was later found to be a ubiquitously expressed and highly conserved thiol-disulfide oxidoreductase, which is involved in many biological processes.9 The major function of Grx1 is to catalyze the reduction of PSSG and repair oxidative damage to cysteine residues. In recent years, the vital roles of Grx1 in cell survival, especially under oxidative stress, have been highlighted. Several lines of evidence suggest that overexpression of Grx1 improves cellular viability under oxidative stress conditions,1820 whereas cells isolated from Grx1 knockout mice exhibited an increased sensitivity to oxidative reagents.21 Moreover, a recent study demonstrated that Grx1 enrichment in dopaminergic cells attenuated the cell toxicity of paraquat, an environmental Parkinsonian toxin that can cause oxidative damage in neuronal cells.22 These studies strongly suggest that Grx1 may play a key role in cellular defense mechanisms against oxidative stress. However, it is worth emphasizing that there is no direct evidence to suggest that Grx1 has similar cytoprotective effects in RPE cells. The present study is the first report of the antiapoptotic effect of Grx1 against oxidative stress in RPE cells. We demonstrated that Grx1 overexpression significantly decreases oxidative stress–induced RPE apoptosis as indicated by higher cell viability, lower apoptotic rate, higher level of antiapoptotic protein Bcl-2, lower level of proapoptotic protein Bax, and less caspase-3 activation in H2O2-treated Grx1 OE cells. 
As the most abundant low-molecular-weight thiol-containing molecule, GSH takes an active part in scavenging ROS, both by direct reaction with ROS and as the cofactor of other antioxidant enzymes, including glutathione peroxidase and Grx1. The resulting product of these GSH-related scavenging reactions is GSSG, the dimeric oxidized form of GSH. Under physiological conditions, the cell contains 5 to 10 mM GSH, and this high GSH/GSSG ratio minimizes PSSG. However, during oxidative stress, ROS molecules oxidize GSH to GSSG, and GSSG accumulation promotes PSSG through thiol-disulfide exchange reactions between a free protein thiol (PSH) and GSSG.5 In our study, when cells were exposed to H2O2, the empty vector group showed enhanced glutathionylation of proteins, and this increase in PSSG levels was accompanied by a dramatic increase in cell death, suggesting that protein-S-glutathionylation may directly contribute to H2O2-induced RPE cell death. In Grx1 OE cells, H2O2-induced PSSG elevation was largely inhibited, suggesting that high levels of Grx1 could prevent the lethal accumulation of PSSG, thus protecting the cells from oxidative damage. This notion is further supported by Sears et al.,14 whose report demonstrated that exogenous Grx1 (purified recombinant protein) could prevent oxidative stress–induced PSSG formation in RPE cells.14 
Protein kinase B is a serine/threonine kinase that plays a key role in cellular survival pathways in a variety of cell types.23 The unphosphorylated form of AKT is virtually inactive, and its activation requires phosphorylation at Thr308 and Ser473. Protein kinase B phosphorylation/activation is involved in multiple cellular processes, such as glucose metabolism,24 gene transcription,25 protein synthesis,26 cell proliferation,27 and migration,28 all working toward the goal of cell survival. Activation of the AKT pathway results in the inhibition of downstream proapoptotic molecules, including Bad, ASK1, Bax, and caspase 9, thus preventing cells from apoptosis.29 At the early stage of oxidative stress, some endogenous protein kinases that have strong cytoprotective effects, including AKT and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), are activated as stress adaption mechanisms.30 In the retina, AKT pathway has been linked to the pathophysiology of AMD-inducing oxidative stress and has been proposed to enhance RPE cell survival.16 In this study, our data clearly illustrated that H2O2 exposure led to transient and rapid activation of AKT in RPE cells. Upon oxidative stress, p-AKT in empty vector cells reached its maximum level within 30 minutes and gradually decreased to the basal level 2 hours after H2O2 treatment. These results are consistent with the findings reported previously by Jaffe et al.16 Interestingly, we found that Grx1-overexpressing cells exhibited substantial increase in p-AKT, and the levels of AKT phosphorylation remained elevated throughout the 4-hour time course of H2O2 treatment. It has been shown that enhanced AKT phosphorylation protects a variety of cells from oxidant-induced cell death.31 On the other hand, impaired AKT phosphorylation resulted in reduced AKT activity, subsequently sensitizing cells to oxidative stress–induced apoptosis.16 Our findings highlight a critical role for the AKT pathway in regulating apoptotic pathway and promoting RPE cell survival following oxidation by H2O2
Although the survival-promoting effects of AKT in H2O2-stimulated cells have been well defined, the redox regulation of this pathway remains largely unknown. Huang et al.32 investigated the crystal structure of the inactive AKT, and an intramolecular disulfide bond was found between two cysteines in the activation loop. More recently, Murata et al.33 also identified an intramolecular disulfide bond formed between Cys-297 and Cys-311 of AKT in cardiac H9C2 cells treated with H2O2, indicating that AKT is a cysteine-rich protein and that the redox status of these thiols may influence AKT activation. However, as far as we know, the correlation between AKT glutathionylation and its phosphorylation has not been reported. In the current study, we found that cell exposure to H2O2 led to the accumulation of glutathionylated AKT and accompanied loss of AKT phosphorylation, whereas Grx1 overexpression increased AKT activation by protecting AKT from oxidation-induced glutathionylation (Fig. 7). Furthermore, phosphorylation of AKT was significantly inhibited in Grx1 siRNA-treated cells (Fig. 8D), further validating the importance of redox homeostasis in maintaining AKT activation. Collectively, we demonstrate for the first time that AKT is a direct target for S-glutathionylation, and Grx1-mediated AKT deglutathionylation serves as a regulatory mechanism for AKT activation. However, the precise mechanism of this crosstalk remains unclear and deserves further investigation. One possible explanation is that addition of GSH, a positively charged residue, can alter the secondary and tertiary structure of AKT. The conformational change induced by GSH attachment could in turn induce a conformational change in the phosphorylation sites, which may decrease the binding of AKT with its activating kinases such as phosphoinositide-dependent kinase 1 (PDPK1) and the mammalian target of rapamycin complex 2 (mTORC2). Another possibility is that AKT glutathionylation may enhance the association of AKT with protein phosphatase 2A (PP2A), the phosphatase that directly dephosphorylates and inhibits AKT. This theory justifies the decreased AKT activation in Grx1-inhibited RPE cells. These hypotheses will be tested in our future studies. 
In summary, we have demonstrated that Grx1 is critical for protecting human RPE cells against oxidative damage. The potential protective effect of Grx1 is associated with its ability to prevent oxidative stress–induced AKT glutathionylation and thus stimulates AKT activation. In the presence of oxidative stress such as H2O2, GSH is oxidized to the disulfide dimer GSSG, thereby increasing glutathionylation of AKT. Glutaredoxin 1 effectively reduces AKT-SSG, thereby promoting AKT phosphorylation. Protein kinase B activation prevents the expression of proapoptotic proteins such as Bax and enhances antiapoptotic proteins such as Bcl-2, leading to decreased caspase 3 activity and diminished apoptosis. A diagram of such a hypothesis is shown in Figure 9
Figure 9
 
Proposed mechanism by which Grx1 regulates thiol/disulfide redox status of AKT upon oxidative stress. In the presence of oxidative stress such as H2O2, GSH is oxidized to the disulfide dimer GSSG, leading to glutathionylation of AKT. Increased AKT glutathionylation diminishes the phosphorylation and activation of AKT. Overexpression of Grx1 protects AKT from H2O2-induced glutathionylation, resulting in a sustained phosphorylation and activation of AKT. Protein kinase B activation decreases the expression of proapoptotic proteins such as Bax and induces antiapoptotic proteins such as Bcl-2, leading to decreased caspase 3 activity and diminished apoptosis. GSH, glutathione; GSSG, glutathione disulfide, oxidized glutathione; AKT-SSG, glutathionylated AKT; p-AKT, phosphorylated AKT (activated).
Figure 9
 
Proposed mechanism by which Grx1 regulates thiol/disulfide redox status of AKT upon oxidative stress. In the presence of oxidative stress such as H2O2, GSH is oxidized to the disulfide dimer GSSG, leading to glutathionylation of AKT. Increased AKT glutathionylation diminishes the phosphorylation and activation of AKT. Overexpression of Grx1 protects AKT from H2O2-induced glutathionylation, resulting in a sustained phosphorylation and activation of AKT. Protein kinase B activation decreases the expression of proapoptotic proteins such as Bax and induces antiapoptotic proteins such as Bcl-2, leading to decreased caspase 3 activity and diminished apoptosis. GSH, glutathione; GSSG, glutathione disulfide, oxidized glutathione; AKT-SSG, glutathionylated AKT; p-AKT, phosphorylated AKT (activated).
Acknowledgments
The authors thank Marjorie Lou, PhD, and Rodrigo Franco, PhD (University of Nebraska-Lincoln), for Grx1-containing plasmid; Fu Shang, PhD (Tufts University), for ARPE-19 cells, Xiangle Sun, PhD (University of North Texas Health Science Center), for flow cytometry assistance; and Marjorie Lou, PhD (University of Nebraska-Lincoln), Iok-Hou Pang, Liangjun Yan, PhD, Michael Jann, PharmD, Katura Bullock, PharmD, and Caitlin Gibson, PhD, (University of North Texas Health Science Center) for their critical reading of the manuscript. 
Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Orlando, Florida, United States, May 2014. 
Supported by a New Faculty Start-Up grant from the University of North Texas Health Science Center. 
Disclosure: X. Liu, None; J. Jann, None; C. Xavier, None; H. Wu, None 
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Figure 1
 
Cytotoxicity of H2O2 in ARPE-19 cells. (A) Dose-dependent effect of H2O2 on the viability of ARPE-19 cells. Cells were exposed to 0, 100, 150, 200, 300, 400, or 500 μM or 1 mM H2O2 for 24 hours. Viable cells were quantified by WST-8 assay. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the 0 μM H2O2 group. (B) Morphologic changes of ARPE-19 cells. Cells were exposed to 0, 100, 200, or 500 μM H2O2 for 24 hours. Cell morphology was examined with phase-contrast microscopy. Scale bars: 50 μm.
Figure 1
 
Cytotoxicity of H2O2 in ARPE-19 cells. (A) Dose-dependent effect of H2O2 on the viability of ARPE-19 cells. Cells were exposed to 0, 100, 150, 200, 300, 400, or 500 μM or 1 mM H2O2 for 24 hours. Viable cells were quantified by WST-8 assay. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the 0 μM H2O2 group. (B) Morphologic changes of ARPE-19 cells. Cells were exposed to 0, 100, 200, or 500 μM H2O2 for 24 hours. Cell morphology was examined with phase-contrast microscopy. Scale bars: 50 μm.
Figure 2
 
Validation of Grx1 overexpression in ARPE-19 cells. (A) Western blot analysis of Grx1 expression in wild-type (WT, nontransfected), empty vector-transfected (vector), and Grx1 overexpressed (Grx1 OE) cells. Bottom: The relative pixel density of Grx1 over β-actin. (B) Immunostaining of GRX1 in cells transfected with empty vector (vector) of Grx1-containing plasmid (Grx1 OE). Scale bars: 50 μm. Bottom: The average fluorescence intensity of Grx1. (C) Grx1 enzyme activity in vector and Grx1 OE cell lysates. Data presented are a typical representation of triplicate experiments. ***P < 0.001 compared with vector group.
Figure 2
 
Validation of Grx1 overexpression in ARPE-19 cells. (A) Western blot analysis of Grx1 expression in wild-type (WT, nontransfected), empty vector-transfected (vector), and Grx1 overexpressed (Grx1 OE) cells. Bottom: The relative pixel density of Grx1 over β-actin. (B) Immunostaining of GRX1 in cells transfected with empty vector (vector) of Grx1-containing plasmid (Grx1 OE). Scale bars: 50 μm. Bottom: The average fluorescence intensity of Grx1. (C) Grx1 enzyme activity in vector and Grx1 OE cell lysates. Data presented are a typical representation of triplicate experiments. ***P < 0.001 compared with vector group.
Figure 3
 
Effects of Grx1 overexpression on H2O2-induced cytotoxicity in ARPE-19 cells. (A) Vector-transfected cells (vector) and Grx1 overexpressed cells (Grx1 OE) were exposed to medium with or without H2O2 (200 μM) for 24 hours. After treatment, cell viability was determined by WST-8 assay. The data are expressed as means ± SD of three experiments, n = 8. ***P < 0.001. (B) Cell density and morphology were examined with phase-contrast microscopy. Scale bars: 50 μm. (C) Apoptosis was then determined by detection of cells with apoptotic nuclear morphology using fluorescence microscopy upon Hoechst 33342 staining. Cells with nuclei showing strong chromatin condensation and nuclear fragmentation (labeled with white arrow) were considered apoptotic. Scale bars: 20 μm.
Figure 3
 
Effects of Grx1 overexpression on H2O2-induced cytotoxicity in ARPE-19 cells. (A) Vector-transfected cells (vector) and Grx1 overexpressed cells (Grx1 OE) were exposed to medium with or without H2O2 (200 μM) for 24 hours. After treatment, cell viability was determined by WST-8 assay. The data are expressed as means ± SD of three experiments, n = 8. ***P < 0.001. (B) Cell density and morphology were examined with phase-contrast microscopy. Scale bars: 50 μm. (C) Apoptosis was then determined by detection of cells with apoptotic nuclear morphology using fluorescence microscopy upon Hoechst 33342 staining. Cells with nuclei showing strong chromatin condensation and nuclear fragmentation (labeled with white arrow) were considered apoptotic. Scale bars: 20 μm.
Figure 4
 
Flow cytometry quantification of apoptosis in H2O2-treated vector and Grx1 OE cells. (A) Flow cytometry analysis of vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) cells treated with or without H2O2 (200 μM) for 24 hours. Representative figures showing population of viable (annexin V/PI), early apoptotic (annexin V+/PI), late apoptotic (annexin V+/PI+), and necrotic (annexin V/PI+) cells. Bar graphs show the quantification of early (B) and late apoptotic (C) cells. Data are mean ± SD of three independent experiments. ***P < 0.001 compared with vector group.
Figure 4
 
Flow cytometry quantification of apoptosis in H2O2-treated vector and Grx1 OE cells. (A) Flow cytometry analysis of vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) cells treated with or without H2O2 (200 μM) for 24 hours. Representative figures showing population of viable (annexin V/PI), early apoptotic (annexin V+/PI), late apoptotic (annexin V+/PI+), and necrotic (annexin V/PI+) cells. Bar graphs show the quantification of early (B) and late apoptotic (C) cells. Data are mean ± SD of three independent experiments. ***P < 0.001 compared with vector group.
Figure 5
 
Effects of Grx1 overexpression on Bax, Bcl-2, and caspase 3 levels. Vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) ARPE-19 cells were treated with or without 200 μM H2O2 for 24 hours. Whole cell lysates (40 μg proteins) from vector and Grx1 OE groups were separated by 12% SDS–PAGE and hybridized with indicated specific antibodies. (A) Effects of Grx1 overexpression on the Bax protein expression in ARPE-19 cells (left). Right: The relative pixel density of Bax over β-actin. (B) Effects of Grx1 overexpression on the Bcl-2 protein expression in ARPE-19 cells. Right: The relative pixel density of Bcl-2 over β-actin. (C) Effects of Grx1 overexpression on caspase 3 cleavage in ARPE-19 cells. Samples were immunoblotted against antibodies specific for pro-caspase 3 and cleaved caspase 3, respectively. Beta-actin was also used as a loading control. The relative pixel density of the cleaved caspase 3 at 17 kDa over pro-caspase 3 at 35 kDa is shown at right. The data presented above are a typical representation of triplicate experiments. ***P < 0.001 compared with vector group.
Figure 5
 
Effects of Grx1 overexpression on Bax, Bcl-2, and caspase 3 levels. Vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) ARPE-19 cells were treated with or without 200 μM H2O2 for 24 hours. Whole cell lysates (40 μg proteins) from vector and Grx1 OE groups were separated by 12% SDS–PAGE and hybridized with indicated specific antibodies. (A) Effects of Grx1 overexpression on the Bax protein expression in ARPE-19 cells (left). Right: The relative pixel density of Bax over β-actin. (B) Effects of Grx1 overexpression on the Bcl-2 protein expression in ARPE-19 cells. Right: The relative pixel density of Bcl-2 over β-actin. (C) Effects of Grx1 overexpression on caspase 3 cleavage in ARPE-19 cells. Samples were immunoblotted against antibodies specific for pro-caspase 3 and cleaved caspase 3, respectively. Beta-actin was also used as a loading control. The relative pixel density of the cleaved caspase 3 at 17 kDa over pro-caspase 3 at 35 kDa is shown at right. The data presented above are a typical representation of triplicate experiments. ***P < 0.001 compared with vector group.
Figure 6
 
Effects of Grx1 overexpression on levels of GSH, GSSG, and protein-GSH mixed disulfide (PSSP) levels. Vector-transfected cells (vector) and Grx1 overexpressed cells (Grx1 OE) were exposed to medium with and without H2O2 (200 μM) for 24 hours. Glutathione level (A) and GSSG level (B) were measured as described in the Materials and Methods. (C) Comparison of protein glutathionylation (PSSG) level in H2O2-treated vector and Grx1 OE cells. The cells were treated with 1 mM H2O2 for 30 minutes, and total proteins were separated on a 12% SDS gel under nonreducing condition (no β-mercaptoethanol) for blot analysis using an anti-PSSG antibody. The graph on the right depicts the relative pixel density of all the PSSG bands over β-actin. Data are mean ± SD of three independent experiments. ***P < 0.001 compared with vector group.
Figure 6
 
Effects of Grx1 overexpression on levels of GSH, GSSG, and protein-GSH mixed disulfide (PSSP) levels. Vector-transfected cells (vector) and Grx1 overexpressed cells (Grx1 OE) were exposed to medium with and without H2O2 (200 μM) for 24 hours. Glutathione level (A) and GSSG level (B) were measured as described in the Materials and Methods. (C) Comparison of protein glutathionylation (PSSG) level in H2O2-treated vector and Grx1 OE cells. The cells were treated with 1 mM H2O2 for 30 minutes, and total proteins were separated on a 12% SDS gel under nonreducing condition (no β-mercaptoethanol) for blot analysis using an anti-PSSG antibody. The graph on the right depicts the relative pixel density of all the PSSG bands over β-actin. Data are mean ± SD of three independent experiments. ***P < 0.001 compared with vector group.
Figure 7
 
Glutaredoxin 1 overexpression enhances AKT phosphorylation through preventing AKT-SSG formation. (A) Time-dependent AKT activation in vector and Grx1 OE cells under oxidative stress. Vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) cells were treated with 200 μM H2O2 for different times (0–240 minutes). Total AKT and phospo-AKT (p-AKT) were determined by using total AKT and p-AKT-specific antibodies. Bottom: The relative pixel density of p-AKT over total AKT. (B) Detection of glutathionylated AKT (AKT-SSG) in vector and Grx1 OE cells. Vector and Grx1 OE cells were treated with or without 200 μM H2O2 for 30 minutes. Protein kinase B was then immunoprecipitated from cell lysates by using AKT-antibody, followed by Western blot detection of PSSG. Normal rabbit IgG was used as a negative control. The graph at the bottom depicts the relative pixel density of AKT-SSG bands over total AKT. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vector group.
Figure 7
 
Glutaredoxin 1 overexpression enhances AKT phosphorylation through preventing AKT-SSG formation. (A) Time-dependent AKT activation in vector and Grx1 OE cells under oxidative stress. Vector-transfected (vector) and Grx1 overexpressed (Grx1 OE) cells were treated with 200 μM H2O2 for different times (0–240 minutes). Total AKT and phospo-AKT (p-AKT) were determined by using total AKT and p-AKT-specific antibodies. Bottom: The relative pixel density of p-AKT over total AKT. (B) Detection of glutathionylated AKT (AKT-SSG) in vector and Grx1 OE cells. Vector and Grx1 OE cells were treated with or without 200 μM H2O2 for 30 minutes. Protein kinase B was then immunoprecipitated from cell lysates by using AKT-antibody, followed by Western blot detection of PSSG. Normal rabbit IgG was used as a negative control. The graph at the bottom depicts the relative pixel density of AKT-SSG bands over total AKT. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vector group.
Figure 8
 
Glutaredoxin 1 inhibition sensitizes primary human RPE cells to oxidative stress. Primary human RPE cells were transfected with Grx1 siRNA, with scramble siRNA, or without transfection (control) for 48 hours. The cells were then treated with or without 100 μM H2O2 for another 24 hours. (A) Validation of Grx1 inhibition with Western blot. Glutaredoxin 1 level in control, scramble siRNA, and Grx1 siRNA groups was analyzed by Western blot using anti-Grx1 antibody. Right: The relative pixel density of Grx1 over β-actin. (B) Glutaredoxin 1 enzyme activity in control, scramble siRNA, and Grx1 siRNA cell lysates. Glutaredoxin 1–specific activity was measured as described in the Materials and Methods. (C) Cell viability was measured by WST-8 in control, scramble siRNA, and Grx1 siRNA groups after H2O2 treatment. The data are expressed as means ± SD; n = 6 from three separate experiments. (D) Time-dependent AKT activation in scramble siRNA and Grx1 siRNA cells under oxidative stress. Scramble siRNA and Grx1 siRNA cells were treated with 100 μM H2O2 for different times (0–240 minutes). Total AKT and phospo-AKT (p-AKT) were determined by using total AKT and p-AKT–specific antibodies. Bottom: The relative pixel density of p-AKT over total AKT. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with scramble siRNA group.
Figure 8
 
Glutaredoxin 1 inhibition sensitizes primary human RPE cells to oxidative stress. Primary human RPE cells were transfected with Grx1 siRNA, with scramble siRNA, or without transfection (control) for 48 hours. The cells were then treated with or without 100 μM H2O2 for another 24 hours. (A) Validation of Grx1 inhibition with Western blot. Glutaredoxin 1 level in control, scramble siRNA, and Grx1 siRNA groups was analyzed by Western blot using anti-Grx1 antibody. Right: The relative pixel density of Grx1 over β-actin. (B) Glutaredoxin 1 enzyme activity in control, scramble siRNA, and Grx1 siRNA cell lysates. Glutaredoxin 1–specific activity was measured as described in the Materials and Methods. (C) Cell viability was measured by WST-8 in control, scramble siRNA, and Grx1 siRNA groups after H2O2 treatment. The data are expressed as means ± SD; n = 6 from three separate experiments. (D) Time-dependent AKT activation in scramble siRNA and Grx1 siRNA cells under oxidative stress. Scramble siRNA and Grx1 siRNA cells were treated with 100 μM H2O2 for different times (0–240 minutes). Total AKT and phospo-AKT (p-AKT) were determined by using total AKT and p-AKT–specific antibodies. Bottom: The relative pixel density of p-AKT over total AKT. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with scramble siRNA group.
Figure 9
 
Proposed mechanism by which Grx1 regulates thiol/disulfide redox status of AKT upon oxidative stress. In the presence of oxidative stress such as H2O2, GSH is oxidized to the disulfide dimer GSSG, leading to glutathionylation of AKT. Increased AKT glutathionylation diminishes the phosphorylation and activation of AKT. Overexpression of Grx1 protects AKT from H2O2-induced glutathionylation, resulting in a sustained phosphorylation and activation of AKT. Protein kinase B activation decreases the expression of proapoptotic proteins such as Bax and induces antiapoptotic proteins such as Bcl-2, leading to decreased caspase 3 activity and diminished apoptosis. GSH, glutathione; GSSG, glutathione disulfide, oxidized glutathione; AKT-SSG, glutathionylated AKT; p-AKT, phosphorylated AKT (activated).
Figure 9
 
Proposed mechanism by which Grx1 regulates thiol/disulfide redox status of AKT upon oxidative stress. In the presence of oxidative stress such as H2O2, GSH is oxidized to the disulfide dimer GSSG, leading to glutathionylation of AKT. Increased AKT glutathionylation diminishes the phosphorylation and activation of AKT. Overexpression of Grx1 protects AKT from H2O2-induced glutathionylation, resulting in a sustained phosphorylation and activation of AKT. Protein kinase B activation decreases the expression of proapoptotic proteins such as Bax and induces antiapoptotic proteins such as Bcl-2, leading to decreased caspase 3 activity and diminished apoptosis. GSH, glutathione; GSSG, glutathione disulfide, oxidized glutathione; AKT-SSG, glutathionylated AKT; p-AKT, phosphorylated AKT (activated).
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