Different Anti-Oxidant Effects of Thioredoxin 1 and Thioredoxin 2 in Retinal Epithelial Cells

Abstract

Age-related macular degeneration (AMD) affects the retina and is the most common cause of blindness in elderly persons in developed countries. The retina is constantly subjected to oxidative stress; to avoid the effects of oxidative stress, retinal pigment epithelial (RPE) cells possess potent anti-oxidant systems. Disruption of these systems leads to dysfunction of RPE cells, which then accelerates the development of AMD. Here, we investigated the role of thioredoxins (TRXs), scavengers of intracellular reactive oxygen species, by assessing the effect of TRX overexpression on cell viability, morphology, NF-κB expression, and mitochondrial membrane potential, in RPE cells. TRX-overexpressing cell lines were generated by infection of an established human RPE cell line (ARPE) with adeno-associated virus vectors encoding either TRX1 or TRX2. We showed that overexpression of TRXs reduced cell death caused by 4-hydroxynonenal (4-HNE)–induced oxidative stress; TRX2 was more effective than TRX1 in promoting cell survival. 4-HNE caused perinuclear NF-κB accumulation, which was absent in TRX-overexpressing cells. Moreover, overexpression of TRXs prevented depolarization of mitochondrial membranes; again, TRX2 was more effective than TRX1 in maintaining the membrane potential. The difference in the protective effects of these TRXs against oxidative stress may be due to their expression profile. TRX2 was expressed in the mitochondria, while TRX1 was expressed in the cytoplasm. Thus, TRX2 may directly protect mitochondria by preventing depolarization. These results demonstrate that TRXs are potent antioxidant proteins in RPE cells and their direct effect on mitochondria may be a key to prevent oxidative stress.

Introduction

Age-related macular degeneration (AMD) is a major cause of blindness worldwide, and which, at present, affects 1.75 million people in the United States (Friedman et al., 2004). The number of patients with AMD is also increasing in Japan, as the population ages. AMD is characterized by functional and morphological abnormalities of retinal epithelial (RPE) cells (Sparrow et al., 2012). The accumulation of drusen, the focal extracellular protein deposits formed below RPE cells, in Bruch’s membrane leads to dysfunction of the RPE cells (Sparrow et al., 2012), which, in turn, leads to AMD.

RPE cells have multiple functions related to the maintenance of the retina; these include phagocytosis of shed photoreceptors (Sugano et al., 2003, 2006), formation of the outer blood-retinal barrier, and sustenance of retinal neurons by release of growth factors (Faktorovich et al., 1992; LaVail et al., 1998). RPE cells also underlie photoreceptors, which endure a high level of oxidative stress because of their high oxygen consumption, high levels of polyunsaturated lipids, and their long-term exposure to light (Beatty et al., 2000). Oxidative stress has been thought to be a key factor in the pathogenesis of AMD (Cai et al., 2000; Liang and Godley, 2003; Nelson et al., 2002).

Under normal physiological conditions, the metabolism of oxygen generates reactive oxygen species (ROS), which can cause cellular and mitochondrial damage. The retina possesses various detoxification systems, involving endogenous antioxidant compounds and enzymes that function against oxidative stress. In RPE cells, antioxidants such as the superoxide dismutases and glutathione peroxidases (Lu et al., 2009) are generated to protect the RPE cells, as well as photoreceptors and other retinal cells. RPE cells also possess the thioredoxin (TRX) system for detoxification of ROS (Ohira et al., 1994; Tanito et al., 2005). However, little is known about the role of TRX in RPE cells.

The TRX system is a ubiquitous thiol-reducing system that includes TRX proteins, TRX-interacting protein (TXNIP), TRX reductase (TRXR), and NADPH. TRX proteins, TRX1 and TRX2, protect cells by scavenging intracellular ROS. The oxidized TRX proteins can then be reduced by TRXR in the presence of NADPH. TRX activity and expression are negatively regulated by TXNIP (Perrone et al., 2009), which directly interacts with the catalytically active center of TRXs. This inhibits the interaction of TRXs with other proteins, including the apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAPKKK5), causing cells to be more sensitive to oxidative stress (Nishiyama et al., 1999).

The aim of this study was to analyze the effect of TRX1 and TRX2 in RPE cells. We established TRX-overexpression models in an established human RPE cell line (ARPE) and evaluated the protective effect of overexpression of these proteins individually against oxidative stress caused by 4-hydroxynonenal (HNE) in vitro.

Materials and Methods

Cell preparation

All procedures involving rats adhered to the ARVO Resolution on the Use of Animals in Research and the guidelines of the UCSF Committee on Animal Research. Human ARPE-19 (ARPE) cells were kindly supplied by Leonard Hjelmeland (Department of Ophthalmology, Section of Molecular and Cellular Biology, University of California, Davis, CA). The ARPE cells were maintained in a growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen-Gibco, Tokyo, Japan), 10% fetal bovine serum (FBS; Invitrogen-Gibco), and antibiotics (Invitrogen-Gibco), as described previously (Abe et al., 1999). The medium was changed every 3 days, and cultured cells were released by incubation in a 0.125% trypsin/0.01% EDTA solution, and then passaged.

Human embryonic kidney (293T) cells were obtained from the American Type Culture Collection (Rockville, MD). HT1080 cells were supplied by the Cell Resource Center (Tohoku University, Sendai, Japan). The 293T and HT1080 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen-Gibco) supplemented with 10% FBS and were passaged with 0.02% EDTA solution and 0.125% trypsin/0.01% EDTA solutions, respectively.

Cultured cells were incubated in a humidified CO₂ incubator in a mixture of 95% air and 5% CO₂.

Preparation of adeno-associated virus vector carrying the TRX gene constructs

Adeno-associated virus (AAV) vectors carrying the TRX1 or TRX2 genes were constructed as follows. cDNA encoding the individual TRXs (TRX1: Genbank Accession No. AF276919.1, TRX2: Genbank Accession No. AF276920) was fused to a DNA segment encoding a fluorescent protein, pmCherry, in frame with the 3′-end of the TRX cDNA. This TRX-pmCherry encoding DNA was introduced into the EcoRI and HindIII sites of the 6P1 plasmid (Kugler et al., 2003). The synapsin promoter of this plasmid was exchanged for a hybrid cytomegalovirus (CMV) enhancer/chicken β-actin promoter (CAG) (Niwa et al., 1991). The pAAV-RC and pHelper plasmids were obtained from the AAV Helper-Free System (Stratagene, La Jolla, CA).

Recombinant AAV vector production and infection

The AAV vectors were produced according to a previously described method (Sugano et al., 2005). AAV vectors were infected into ARPE cells according to the manufacturer’s instructions (Stratagene). Specifically, AAV-permissive medium was prepared to a final concentration of 40 mM hydroxyurea (Sigma-Aldrich, St. Louis, MO) and 1 mM sodium butyrate (Sigma-Aldrich) in culture medium. Cells were then treated with AAV-permissive medium. After 6 hours of incubation, cells were washed twice with DMEM supplemented with 2% FBS, and were then infected with the AAV constructs, individually, at 37°C for 2 hours. After infection, the medium was replaced with the same volume of DMEM supplemented with 18% FBS to terminate the infection, and the cells were then cultured. Successful transgene expression of the TRX1 or TRX2 was confirmed by assessing the expression of pmCherry fluorescence by microscopy (Axiovert 40; Carl Zeiss, Oberkochen, Germany). The localization of the TRXs in the cells was also further investigated using MitoTracker^® dye (Molecular Probe, Eugene, OR) as a mitochondrial marker.

Recombinant AAV vector titer measurement

To determine the virus titer, the level of AAV2-specific capsid proteins was measured by enzyme-linked immunosorbent assay (ELISA; Progen Biotechnik, Heidelberg, Germany), and the virus titer was designated as capsids per milliliter (Grimm et al., 1999).

Cell viability test

ARPE cells were infected with AAV-TRX1 and AAV-TRX2, individually. After 3 days of infection, the expression of pmCherry fluorescence was confirmed by fluorescence microscopy as before, and plated into a 96-well plate. One day after plating, the medium was exchanged for a medium containing various concentrations of 4-HNE to induce oxidative stress. After 24 hours of incubation, cell viability was assessed using a CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI).

Nuclear morphology

During the assessment of cell viability, cells were stained with Hoechst 33258 (bisbenzimide; Molecular Probes, Japan) at a final concentration of 1 μg/ml for 30 minutes at 37°C, before they were fixed with 4% paraformaldehyde/0.1 M PBS and observed by fluorescence microscopy (Carl Zeiss). This staining also allowed identification of apoptotic cells, which have pyknotic or irregularly shaped nuclei.

Immunocytochemistry

AAV-TRX–infected ARPE cells and non–infected control ARPE cells grown on a culture slide, and which had been exposed to 25 μM of 4-HNE, were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 minutes, and then dried. After blocking with PBS containing 0.05% Triton X-100 and 3% goat serum for 30 minutes, cells were incubated with a rabbit anti-NF-κB p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. After the cells were washed with PBS, they were treated with an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (Molecular Probes) for 1 hour at room temperature. The culture slides were then observed under a fluorescence microscope (Carl Zeiss).

Immunoblot analysis

Cells were exposed to 25 μM of 4-HNE for 3 or 6 hours. No-treated cells were used as a control. After the exposure, cells were lysed in RIPA buffer and kept on ice for 20 minutes. The lysates were centrifuged and the supernatant was used for western blot analysis. Thirty μg of the lysates was separated by 4–15% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA). The blots were first probed with antibodies to the phosphorylated form of protein (anti-p-IκB-α; Santa Cruz biotechnology). The blots were then stripped of the first antibody and reprobed with antibodies that detect the total protein (anti-β-Actin; Santa Cruz biotechnology). For the detection of these antibodies, alkaline-phosphatase–conjugated anti -rabbit IgG or -mouse IgG (Promega) was used as the secondary antibody. Protein bands were detected by Chemiluminescent imager (Flour-S MAX, BIORAD).

Analysis of mitochondrial membrane potentials

Changes in the mitochondrial membrane potential were visualized using a mitochondrial membrane potential-sensitive dye, JC-1 (Molecular Probes). Six hours after exposure to 4-HNE, the medium containing JC-1 (10 μg/ml final concentration) was added to each well, and cells incubated at 37°C for 10 minutes. After the cells were washed with PBS, they were photographed under a fluorescence microscope. The changes in the ratio of green/red fluorescence were analyzed using AxioVision 4.3 software (Carl Zeiss).

Statistical analysis

Statistical analysis was performed using GraphPad Prism (Graph-Pad Software, San Diego, CA). The un-paired t-test or Bonferroni’s multiple comparison test was used for the cell viability test or analysis of mitochondrial membrane potential, respectively. The criterion for statistical significance was P<0.05.

Results

Expression of TRX1 and TRX2 in ARPE cells

The expression of the pmCherry and TRX1-pmCherry was localized to the cytoplasm. while TRX2-pmCherry was observed in the mitochondria (Fig. 1). To refine the localization of TRX1 and TRX2, we also stained the mitochondria using MitoTracker^® dye (Molecular Probe); TRX2-pmCherry expression overlapped with the fluorescence of this mitochondrial dye.

Fig. 1

Expression profile of TRX1 and TRX2 in ARPE Cells. ARPE cells were transfected with AAV-TRX1 or AAV-TRX2, and overexpressions of TRX1 or TRX2 were established (ARPE-TRX1, ARPE-TRX2). The expression of TRX was confirmed by a fluorescent protein, pmCherry which was fused with TRX. To reveal the expression profiles of TRXs, mitochondrial marker (Mitotraker) was used. As the control of pmCherry expression, ARPE cells were transfected with AAV-pmcherry and examined.

Cell viability

We used 4-HNE to induce oxidative stress; after exposure to 4-HNE, ARPE cells died in a dose-dependent manner (Fig. 2). Only 83.1% and 58.9% of the ARPE cells survived in the presence of 12.5 μM and 25 μM of 4-HNE, respectively. However, the level of cell death induced by 12.5 μM of 4-HNE was reduced in cells overexpressing TRX1 and TRX2 (cell survival was 88.5%±0.8% and 102.3%±3.9%, respectively). Similarly, when the cells were exposed to 25 μM of 4-HNE, overexpression of TRX1 and TRX2 rescued cell viability, yielding cell survival rates of 67.8%±2.3% and 78.9%±5.7%, respectively. There was no effect on the cell survival by only expressing pmCherry (data not shown).

Fig. 2

Cell viability test in ARPE cells exposed to 4-HNE. ARPE-TRX1, ARPE-TRX2, and native ARPE cells were exposed to 4-HNE to induce oxidative stress. After 24 h of incubation, cell viability was assessed by CellTiter^® 96 Aqueous Non-Radioactive Cell Proliferation Assay. Data are shown as mean (SD) of N=5. *: P<0.05; **: P<0.01; unpaired t-test.

Nuclear morphology

To study the influence of 4-HNE on the cells, morphological changes of the nuclei were analyzed. After exposure to 4-HNE, ARPE cells showed apoptosis-like features, such as chromatin condensation and fragmented nuclei (Fig. 3). These morphological changes were more severe in untransformed ARPE cells than in TRX-transformed cells. With regard to untransformed ARPE cells, many of the cells loosened from the plate, resulting in low cell density, as shown in Fig. 3A. There were no differences in cell number between TRX1- and TRX2-transformed cells (Data not shown).

Fig. 3

Nuclear morphology of RPE cells exposed to 4-HNE. Native RPE cells, AAV-TRX1- and AAV-TRX2-transformed ARPE cells were exposed to 25μM 4-HNE; after 24 h, nuclear morphology was observed using Hoechst 33258 under fluorescence microscopy. Arrows indicate nuclear fragmentation. (A) Native ARPE cells, (B) AAV-TRX1-transformed ARPE cells, (C) AAV-TRX2-transformed ARPE cells.

Immunocytochemistry

The NF-κB expression was also studied in ARPE cells before and after exposure to 4-HNE. The fluorescence indicating NF-κB p65 in cultured ARPE cells was observed uniformly throughout the cytoplasm (Fig. 4A, D, G). Six hours after the exposure to 4-HNE, this fluorescence was observed to translocate to the nuclear and perinuclear region in native ARPE cells (Fig. 4C). On the other hand, the p65-associated fluorescence in the TRX1- and TRX2-transformed cells remained in the cytoplasm (Fig. 4F, I).

Fig. 4

NF-κB expression profiles in ARPE cells exposed to 4-HNE. ARPE cells, with or without transformation by AAV-TRX1 and AAV-TRX2, were exposed to 4-HNE. Pre- and post-exposure, cells were fixed and immunocytochemically stained for NF-κB expression. (A–C) Native ARPE cells, (D–F) AAV-TRX1-transformed ARPE cells, (G–I) AAV-TRX2-transformed ARPE cells. Arrows indicate the NF-κB translocation to the nuclear and perinuclear region.

Immunoblot analysis

To demonstrate the translocation of NF-κB to the perinuclear region, we analyzed the IκB signaling. Phosphorylated form of IκB (p-IκB) was increased at 6 hours of 4-HNE exposure (Fig 5). But this elevation was inhibited by the expression of Trx1 or Trx2. Trx2 expression suppressed the phosphorylation more effectively.

Fig. 5

Effect of Trx1 and Trx2 on the phosphorylation of IκB. Cells were exposed to 4-HNE for 3 or 6 hours. As the control, no-treated with 4-HNE cells were analyzed. Protein levels of phosphorylated IκB-α were detected by the chemiluminescence method. The blots were then stripped and reprobed with antibodies to β-actin for the loading control.

Analysis of mitochondrial membrane potentials

We used JC-1 as an indicator of mitochondrial membrane potential to assess whether overexpression of TRXs could preserve the mitochondrial function in cells exposed to 4-HNE-induced oxidative stress. As shown in Fig 6, the ratio of the intensity of green fluorescence to red fluorescence, a marker of depolarization, increased after exposure to 4-HNE (100±24.11). However, depolarization was suppressed by the overexpression of either TRX1 (26.33±10.81) or TRX2 (9.21±9.43).

Fig. 6

Analysis of mitochondrial membrane potential. Changes in mitochondrial membrane potential were visualized using JC-1 dye. After 6-h exposure of ARPE cells to 4-HNE, JC-1 was added to the medium; after washing with PBS, cells were photographed under fluorescence microscopy. Changes in the ratio of green/red fluorescence were analyzed using AxioVision 4.3 software. Data are shown as mean±SD of N=5. **: P<0.01; Bonferroni’s multiple comparison test.

Discussion

Healthy RPE cells are indispensable for the maintenance of the retina; abnormalities in RPE cells lead to photoreceptor degeneration and to AMD. To protect retinal cells from oxidative stress, RPE cells have multiple detoxification systems, including TRXs, which are important regulators of the cellular redox state (Ohira et al., 1994). Here, we established RPE cell lines overexpressing TRX1 and TRX2 by using AAV vectors (Fig. 1). Expression of TRX1 could be observed in the cytoplasm of APRE cells, while that of TRX2 was localized to the mitochondria. The expression of the TRXs was not punctate, and confirmed results from a previous localization study (Caprioli et al., 2009). These results indicate that the TRX-overexpression systems established in this study were stable and functional.

The cytoprotective effects of TRX were analyzed in native ARPE, and in TRX1- or TRX2-overexpressing ARPE cells, by exposing the cells to 4-HNE, a major lipid peroxidation product in retinal and RPE cells (Toyokuni et al., 1994). 4-HNE induced RPE cell death in a dose-dependent manner. Although overexpression of both TRX1 and TRX2 had a protective effect against 4-HNE-induced oxidative stress, the effect of TRX2 was more potent than that of TRX1 (Fig. 2). We also observed apoptosis-like morphological changes in the nuclei of ARPE cells that had been exposed to 4-HNE (Fig. 3). This nuclear fragmentation was more severe in native ARPE cells than in cells overexpressing TRXs; in this respect, there was no difference between cells overexpressing TRX1 or TRX2.

To investigate the protective effect of TRX overexpression against 4-HNE-induced apoptosis-like cell death, we then analyzed the expression of NF-κB by immunocytochemistry. NF-κB has been reported to generate a proinflammatory signal (Salminen et al., 2008) involved in histone H3 chromatin remodeling (Perrone et al., 2009) and oxidative stress (Wu et al., 2005). As RPE cells age, they are subject to continued oxidative stress, which induces inflammation. In response to these reactions, chronic neovascularization is found to develop in 15–20% of AMD cases (Kaarniranta and Salminen, 2009). Moreover, Kaarniranta et al. revealed that prevention of NF-κB signaling in inflammation can be considered a potential treatment for AMD (Kaarniranta and Salminen, 2009). The NF-κB protein complex is retained in an inactive state in the cytoplasm by binding to inhibitory proteins, such as IκBa, IκBb, IκBc, IκBe, and Bcl3, also collectively known as IκB family. Once a stress stimulus, such as oxidative stress, is encountered, IκB kinase (IKK) is activated, which in turn phosphorylates IκB proteins, making them susceptible to degradation by the ubiquitin-proteasome system (Barnes and Karin, 1997). Subsequently, the NF-κB complex undergoes nuclear translocation where it can bind to various promoter areas of its target genes and induce gene transcription of the corresponding genes, most of which are implicated in the regulation of inflammation. Our results showed that 4-HNE caused NF-κB accumulation in the perinuclear region at 6 hours after commencement of exposure to 4-HNE (Fig. 4C). This indicated that 4-HNE caused oxidative stress in ARPE cells; however, this accumulation was not present in ARPE cells overexpressing either of the TRXs (Fig. 4F and I). Western blotting also revealed that increment of phosphorylation of IκB at 6 hours of 4-HNE exposure was diminished in Trx1- or Trx2-expressed cells (Fig. 5). Moreover, Trx2 expressed cells did not cause any elevation of p-IκB. Our data represented 4-HNE exposure caused phosphorylation of IκB in native APRE cells, however NFκB p65 located at the nuclear and perinuclear region. Emdad et al., reported that astrocyte elevated gene-1 (AEG-1) activated NFκB via IκB-α degradation and p65 translocation and also showed the NFκB localization at the perinuclear (Emdad et al., 2006).

To analyze the protective effect caused by overexpression of TRXs, cells were subjected to 4-HNE stress and stained with JC-1; JC-1 is a cationic dye that exhibits potential-dependent fluorescence accumulation in mitochondria. In mitochondria, the potential difference is maintained by a hydrogen ion concentration gradient across the inner membrane, and is related to ATP production. Typically, mitochondrial membranes are hyperpolarized, and depolarization followed by disappearance of the membrane potential causes fragmentation of the membrane structure. Mitochondrial depolarization has been reported to be a sign of the early stage of apoptosis (Mancini et al., 1997; Wadia et al., 1998). We found that overexpression of either of the TRXs significantly prevented the loss of mitochondrial potential induced by 4-HNE (Fig. 6); although TRX2-overexpression in ARPE cells more effectively maintained the membrane potential than did TRX1 overexpression. These results were also comparable with our data that indicate that TRX2 is more effective in improving survival of cells exposed to 4-HNE than TRX1 is (Fig. 2). It is also congruent with our finding that TRX2, rather than TRX1, is localized particularly in the mitochondrion.

In conclusion, oxidative stress leads to the generation of inflammatory signals in RPE cells, while TXNIP, the endogenous inhibitor of TRXs, is increased, as described by Sreekumar et al. (2009). The inhibition of TRXs by TXNIP leads to an increase in oxidative stress and also to an increase in NF-κB expression (Nishiyama et al., 1999). However, when TRXs are overexpressed, their levels override TXNIP inhibition. This may reduce the inflammatory signals, by decreasing perinuclear NF-κB accumulation. In our study, TRX2 was a more potent antioxidant than TRX1 during oxidative stress; this may be due to the difference in subcellular localization of these proteins. Given that the primary effect of oxidative stress is degeneration of the mitochondria, and that TRX2 is localized to the mitochondria, it, rather than TRX1, may function in preventing depolarization of the mitochondrial membrane. These results demonstrate that TRXs are potent antioxidant proteins in RPE cells and that their direct effect on mitochondria may be a key to prevention of oxidative stress.

Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (nos. 23791960, 24390393, and 23659804); Program for Promotion of Fundamental studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO).

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