Nuclear Factor (Erythroid-Derived)-Related Factor 2-Associated Retinal Pigment Epithelial Cell Protection under Blue Light-Induced Oxidative Stress

Purpose. It is a matter of increasing concern that exposure to light-emitting diodes (LED), particularly blue light (BL), damages retinal cells. This study aimed to investigate the retinal pigment epithelium (RPE) damage caused by BL and to elucidate the role of nuclear factor (erythroid-derived)-related factor 2 (Nrf2) in the pathogenesis of BL-induced RPE damage. Methods. ARPE-19, a human RPE cell line, and mouse primary RPE cells from wild-type and Nrf2 knockout (Nrf2 −/−) mice were cultured under blue LED exposure (intermediate wavelength, 450 nm). Cell death rate and reactive oxygen species (ROS) generation were measured. TUNEL staining was performed to detect apoptosis. Real-time polymerase chain reaction was performed on NRF2 mRNA, and western blotting was performed to detect Nrf2 proteins in the nucleus or cytoplasm of RPE cells. Results. BL exposure increased cell death rate and ROS generation in ARPE-19 cells in a time-dependent manner; cell death was caused by apoptosis. Moreover, BL exposure induced NRF2 mRNA upregulation and Nrf2 nuclear translocation in RPE. Cell death rate was significantly higher in RPE cells from Nrf2 −/− mice than from wild-type mice. Conclusions. The Nrf2 pathway plays an important role in protecting RPE cells against BL-induced oxidative stress.


Introduction
Age-related macular degeneration (AMD) leads to blindness, accounting for approximately 9% of blindness cases worldwide. There are approximately 30 million patients with AMD, of whom > 0.5 million have become blind [1]. AMD can be divided into two categories: wet and dry AMD [2]. In wet AMD, choroidal neovascularization breaks the retinal pigment epithelium (RPE) through to the neural retina, causing the leakage of fluid, lipids, and blood; these changes lead to fibrous scarring and antivascular endothelial growth factor drugs are approved for wet AMD treatment [3][4][5]. In dry AMD, progressive geographic atrophy of RPE occurs, followed by severe damage of the photoreceptors. Severe irreversible blindness from AMD is caused by these advanced forms [6,7].
AMD may have a multifactorial pathogenesis [8] and is characterized by photoreceptor cell death [9][10][11][12][13][14]. Several factors, such as smoking, obesity, eating habits, and light exposure, particularly blue light (BL) exposure, play important roles in the progression of AMD [15][16][17][18]. BL exposure causes an increase in the reactive oxygen species (ROS), which may result in structural damage and decreased viability of retinal cells. It also causes RPE apoptosis via oxidative stress and mitochondrial damage [19][20][21]. 2 Oxidative Medicine and Cellular Longevity One of the most important antioxidation pathways is the nuclear factor (erythroid-derived)-like 2 (Nrf2) pathway. Nrf2 is a 65 kDa molecule with a basic leucine zipper structure [22]. Without oxidative stress, Nrf2 in its inactive state is bound to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm [23]. When cells are exposed to oxidative stress, the active site cysteine residues of Keap1 are oxidized, preventing Keap1 from interacting with NRF2. With Nrf2 accumulation in the cytoplasm, Nrf2 moves to the nucleus and binds to the antioxidant response element [24]. Nrf2 also serves as the master regulator of a highly coordinated antioxidant response in RPE cells [25]. Several studies demonstrated that antioxidative factors prevent RPE cells from being damaged by oxidative stress [26][27][28][29][30][31][32][33][34]. Some antioxidative factors also upregulate Nrf2 signaling. RPE damage may be prevented by these antioxidative factors via the upregulation of Nrf2 signaling. However, little is known regarding whether Nrf2 signaling activation is directly involved in RPE protection. Moreover, to the best of our knowledge, the direct relationship between BL exposure and Nrf2 signaling in RPE cells has not been well elucidated.

Cell Culture, Primary Cell Preparation, and BL Exposure.
ARPE-19, a human RPE cell line, was purchased from the American Type Culture Collection (Rockville, MD, USA), and primary human RPE (hRPE) cell line was purchased from Lonza (Walkersville, MD, USA). Cells were grown in colorless Dulbecco's modified Eagle's medium (DMEM) premixed with Ham's F-12 (1 : 1 ratio, Sigma-Aldrich) and supplemented with 10% fetal bovine serum and the antibiotics streptomycin/penicillin G (Sigma-Aldrich) [13,14]. Primary mouse RPE cells were collected from the wild-type and Nrf2 −/− mice, as previously described [41,42]. In brief, mouse eyecups were washed with sterile PBS, and flatmounts were created. The retina was gently removed to allow RPE layer to be on the surface of the flatmount. The RPE eyecups were rinsed in a chelating agent (Versene, Invitrogen), and RPE cells were enzymatically dislodged by 2% Dispase (Roche Diagnostics). Dislodged RPE cells were collected and cultured in DMEM containing antibiotics at 37 ∘ C containing 5% CO 2 . Animal studies were approved by the Institutional Animal Care and Use Committee of the Nagoya University Graduate School of Medicine. All procedures involving animals were conducted according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Nrf2 −/− mice were provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan [43]. The cells were cultured in the dark or under BL (Zensui LED Lamp Blue6; Zensui Inc., Japan; peak wavelength: 450 m, 1,200 lux).

LDH Assay and ROS Measurement.
The cell death rate was evaluated by measuring the lactate dehydrogenase (LDH) activities using the Cytotoxicity Detection Kit PLUS (Roche Diagnostics, Mannheim, Germany). The supernatant of the culture medium, which contained LDH secreted from dead cells, was collected, followed by the addition of Triton X-100 in the medium to release intracellular LDH from the surviving cells. After measuring the LDH activities in the culture supernatant and medium, the proportions of dead cells among the total cells were calculated. Oxidative stress on BL exposure was evaluated with respect to the amount of ROS, measured using the OxiSelect6 ROS assay kit (Cell Biolabs. Co., Japan). In brief, after BL exposure, the assay was terminated by adding cell lysis buffer, and fluorescence intensity was measured at 493 nm (ex)/523 nm (em) using a fluorescent plate reader at each time point.

Cell Morphology and TUNEL Staining.
Morphological changes of ARPE-19 cells exposed to BL were visualized using a phase-contrast microscope (FSX-100; Olympus, Tokyo, Japan). TUNEL-positive apoptotic cells were detected, as previously described [14,44]. In brief, after 24 h of BL exposure, the cells were fixed with 2% PFA for 20 min at room temperature on the chambered cell culture slides. The cells were stained with the In Situ Cell Death Detection kit (Roche Diagnostics, Mannheim, Germany) and 0.3 mg/mL 4 ,6diamidino-2-phenylindole (Invitrogen, Carlsbad, CA, USA) for 1 h. The stained cells were then observed using a Bio Imaging Navigator fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). The number of TUNEL-positive cells was calculated from images obtained with a 20x lens (537 × 710 m). The average number of TUNEL-positive cells observed in three independent areas was calculated per well (n = number of wells) [14].

Protein and RNA Isolation.
For total protein collection, the cultured human and mouse cells were lysed in RIPA buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). The lysate was centrifuged at 15,000 ×g for 15 min at 4 ∘ C, and the supernatant was collected. Protein concentrations were determined using the Bradford Assay Kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard. To measure Nrf2 abundance in the nucleus, ARPE-19 cells were treated with NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL, USA) as previously described [41]. For realtime polymerase chain reaction (RT-PCR) analyses, total RNA was purified using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol; the RNA concentration and quality were assessed using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA) [13].

Quantitative Reverse Transcription-PCR (RT-PCR).
The total RNA was reverse transcribed using the Transcriptor Universal cDNA Master Kit (Roche Diagnostics), starting with 2 g of total RNA from each sample [13]. RT-PCR was performed using the Thunderbird Probe qPCR Mix (Toyobo Life Science, Osaka, Japan) and Gene Expression Assay containing primers and an FAM dye-labeled TaqMan probe for detecting human NRF2 (HS00965961-g1; Applied Biosystems, USA) and eukaryotic 18S rRNA (Hs 99999901 s1; Applied Biosystems) that is available for human 18S rRNA [12]. PCR cycles consisted of a predenaturation step at 95 ∘ C for 2 min followed by 40 cycles of denaturing steps at 95 ∘ C for 15 s and annealing and extending steps at 60 ∘ C for 60 s. The relative expressions of the target genes were determined using the 2 −ΔΔCt method.

Outcomes and Statistical
Analysis. Cell death rate, ROS generation, and RT-PCR of Nrf2 mRNA were statistically analyzed using the Mann-Whitney test. P values of <0.05 were considered to be statistically significant.
To investigate the involvement of NRF2 in the effect of BL exposure on ARPE-19 cells, we examined mRNA level and NRF2 protein expression of ARPE-19 cells with and without BL exposure. Compared with the mRNA level of ARPE-19 cells at the onset of treatment (control, 1.00 ± 0.09, = 2), the level at 6 h after BL exposure was significantly higher (Figure 4  expression from total ARPE-19 cells appeared to be decreased by BL exposure (Figure 4(b)). In an active state, Nrf2 showed nuclear translocation. Therefore, we separately obtained Nrf2 protein from the nucleus of ARPE-19 cells. Nrf2 protein was abundantly expressed in the ARPE-19 nucleus (Figure 4(b)).
These results showed that, as a result of BL exposure, Nrf2 in ARPE-19 cells was activated. To shed more light on the importance of Nrf2 activation in BL exposure-induced ARPE-19 cell damage, we collected primary RPE cells from Nrf2 −/− and wild-type (Nrf2 +/+ ) mice and compared their cell In contrast, death rates of RPE cells from wild-type mice were 5.0 ± 0.1 ( = 6) and 25.7 ± 1.4 ( = 6) at 6 and 24 h after exposure, respectively. There were significant increases in the death rate in Nrf2 −/− mouse RPE cells at 6 h ( < 0.001) and 24 h ( < 0.001) compared with those in wild-type mice ( Figure 5). These findings indicated that Nrf2 plays an important role in blocking cell death caused by BL exposureinduced ROS generation.

Discussion
Visible light exposure-induced damage in retinal cells occurs through type I (free radical) and type II (oxygen-dependent) mechanisms. Free radicals induce cells to undergo necrosis, whereas oxygen-dependent mechanisms induce them to undergo apoptosis [47]. The mechanism of AMD was considered to involve oxidative stress caused by several factors driving RPE cells to die via apoptosis. BL exposure induces ROS generation, damaging mitochondrial DNA and cell structure; then, RPE cells are forced to enter an apoptotic state [15][16][17]. In this study, we demonstrated that BL exposure increased ROS generation in RPE cells and induced cell death in a time-dependent manner. In addition, TUNEL staining suggested that apoptotic RPE cell death was caused by BL exposure. To an extent, these findings simulate the pathogenesis of AMD. ARPE-19 cells have some differences from primary hRPE cells in terms of promoter strength [48], proliferation, and cell death [49]. Previous studies have demonstrated that ARPE-19 cells are stronger and tolerable for oxidative stress compared with primary hRPE cells. In the present study, cell death rate and ROS of primary hRPE cells at 6 h of BL exposure (Figures 1(d) and 1(e)) were higher than those of ARPE-19 cells (Figures 1(a) and 1(b)). It has been previously demonstrated that primary hRPE cells are more sensitive than ARPE-19 cells against BL exposure-derived oxidative stress. On the other hand, ROS generation at 24 h was lower than that at 6 h in primary hRPE cells. This is possibly because primary hRPE cells were killed by 24 h BL exposure, and lower ROS generation was measured from the remaining (smaller number of) cells. Although both cells were damaged by BL exposure-derived ROS in a time-dependent manner, we considered that the use of these cells for this BL exposure study design is suitable.
The Nrf2 pathway is one of the most important pathways for protecting cells against oxidative stress [22]. Without oxidative stress, Nrf2, in its inactive state, is kept in the cytoplasm [23]. When cells are exposed to oxidative stress, this Nrf2 is released from Keap1 and moves to the nucleus where it functions in antioxidative protective mechanisms [24]. Other cells, such as epidermoid carcinoma cells [50] and retinal ganglion cells [51], also showed increased Nrf2 protein expression on BL exposure in vitro. These reports suggested that BL exposure increased ROS generation, activated Nrf2 signaling, and reduced cell viability in a time-dependent manner. In our study, BL exposure of RPE cells increased ROS generation and apoptotic cell death rate in a timedependent manner. In addition, Nrf2 mRNA level and Nrf2 protein expression in the nucleus were increased in RPE cells in a time-dependent manner. These findings indicated that BL exposure induces the upregulation of ROS and Nrf2, which are involved in antioxidative protective mechanisms as previously reported.
If the Nrf2 pathway does not properly function, antioxidative protection would be weaker and oxidative stress would cause severe cell damage. Nrf2 −/− mice developed ocular pathology similar to the cardinal features of human AMD; deregulated autophagy is a likely mechanistic link between oxidative injury and inflammation [35]. Nrf2 −/− RPE cells are susceptible to oxidative stress induced by tbutylhydroperoxide [32]. However, these studies did not use light exposure as an oxidative stressor. In dermatology, some studies revealing the relationship between Nrf2 −/− cells and light exposure have been published [52][53][54][55]. Ultraviolet lightirradiated Nrf2 −/− cells exhibited accelerated photoaging, resulting in the necrosis of irradiated cells, inflammatory cell infiltration, TUNEL-positive apoptotic cell formation, and the accumulation of oxidative DNA products, which are caused by oxidative stress. In this study, Nrf2 −/− RPE cells died at rates of 10.5%, 16.4%, and 56.1% at 0, 6, and 24 h of BL exposure, respectively. This rate of cell death at 0 h tended to be higher than that of wild-type RPE cells, and the rates at 6 and 24 h were significantly higher than those of the wildtype RPE cells. These findings suggest that Nrf2 −/− RPE cells are weaker than wild-type RPE cells, and Nrf2 signaling plays a key protective role against BL-induced oxidative stress.
Previous studies demonstrated that some materials, such as polyphenol, salvianolic acid, 4-acetoxyphenol, 17-beta-estradiol triterpenoid RTA-408, pinosylvin, alphamangostin, and coconut water, protect cells against oxidative stress via Nrf2 signaling. They showed that these materials upregulate Nrf2 protein expression and concluded that these materials protect cells against oxidative stress by increasing Nrf2 protein expression. However, it is unknown whether Nrf2 protein expression is increased by these materials to rescue cells from oxidative stress or whether the expression is secondarily increased in a manner independent of these materials [26][27][28][29][30][31][32][33][34]. These materials would be expected to stimulate the Nrf2 pathway and increase Nrf2 protein expression to protect cells against oxidative stress.
A limitation of this study is that we examined only an in vitro biological change. A previous study used Nrf2 −/− mice subjected to direct light exposure in vivo and revealed tissue change caused by oxidative stress [54]. Studying the effect of BL exposure on living Nrf2 −/− mice will provide us with more precise information regarding the adverse effects of BL exposure on the eyes and the importance of the Nrf2 pathway in protecting eyes during BL exposure.
In conclusion, BL exposure induced ROS generation and caused cell death via apoptosis in RPE cells. The Nrf2 pathway plays a protective role against oxidative stress on BL exposure. Our findings in this study are meaningful for demonstrating the direct relationship between BL exposure and Nrf2 signaling, as proved by Nrf2 −/− in RPE cells.