Citation: Liu Y, Ren Y, Wang X, Liu X, Xu Y, He Y. Down regulation
of UCP2 expression in retinal pigment epithelium cells under oxidative stress:
an in vitro study. Int J Ophthalmol 2019;12(7):1089-1094
DOI:10.18240/ijo.2019.07.06
·Basic Research·
Down regulation of UCP2 expression in retinal pigment epithelium cells under oxidative stress: an in vitro study
Ying Liu, Yuan Ren, Xia Wang, Xu Liu, Yun Xu, Yuan He
Department of Ophthalmology, the Second Affiliated Hospital of Xi’an Medical University; Ocular Immunology and Inflammation Institute, Shaanxi Provincial Clinical Research Center for Ophthalmology, Xi’an 710038, Shaanxi Province, China
Correspondence to: Yuan He. Department of Ophthalmology, the Second Affiliated Hospital of Xi’an Medical University, Xi’an 710038, Shaanxi Province, China. openji7127@hotmail.com
Received:
Abstract
AIM: To evaluate the expression of uncoupling protein 2 (UCP2) in a retinal pigment epithelium cell line (ARPE-19), under oxidative stress (OS).
METHODS: ARPE-19 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2O2; 0, 150, 300, 500, 700, and 900 µmol/L) for 24h, to induce oxidative damage and cell viability was assessed by MTT assay. UCP2 mRNA expression in cells treated with H2O2 was investigated by reverse transcription-polymerase chain reaction (RT-PCR). UCP2 protein expression was assessed by Western blotting and ROS levels analyzed by flow cytometry (FCM). Further, UCP2-siRNA treated cultures were exposed to H2O2 (0, 75, 150, and 300 µmol/L) for 2h and cell viability determined by MTT assay.
RESULTS: Cells treated with higher concentrations of H2O2 appeared shrunken; their adhesion to adjacent cells was disrupted, and the number of dead cells increased. The results of cell viability assays demonstrated that the numbers of cells were decreased in a dose-dependent manner following treatment with H2O2. Compared with untreated controls, cell viability was significantly reduced after treatment with >300 µmol/L H2O2 (P<0.05). Cell metabolic activity was decreased with increased concentrations of H2O2 as detected by MTT assay. Levels of OS were further decreased in cells treated with UCP2-siRNA compared with those treated with H2O2 alone (P<0.05). The results of RT-PCR and Western blotting demonstrated that UCP2 expression was reduced in H2O2-treated groups compared with controls (P<0.05). FCM analysis showed that cell reactive oxygen species (ROS) levels were increased in H2O2-treated groups and further upregulated by UCP2-siRNA treatment (P<0.05).
CONCLUSION: Expression levels of UCP2 are decreased in ARPE-19 cells treated with H2O2. ROS levels are further increased in cells treated with UCP2-siRNA relative to those treated with H2O2 alone. UCP2 may have a protective role in ARPE-19 cells during oxidative injury.
KEYWORDS: uncoupling protein 2; retinal pigment epithelium cells; oxidative stress
DOI:10.18240/ijo.2019.07.06
Citation: Liu Y, Ren Y, Wang X, Liu X, Xu Y, He Y. Down regulation of UCP2 expression in retinal pigment epithelium cells under oxidative stress: an in vitro study. Int J Ophthalmol 2019;12(7):1089-1094
INTRODUCTION
Age-related macular degeneration (AMD) is the leading cause of blindness among people over 55y in western countries[1]. The number of patients with AMD continues to increase, and it is estimated approximately 198 million people currently suffered from the disease[2]. AMD is generally considered to be caused by various biochemical, immunogenic, and environmental factors[3]. A number of retinal pathologies, including AMD, are associated with mitochondrial dysfunction[4], which induces increased levels of reactive oxygen species (ROS), mitochondrial DNA damage, and defective metabolic activity[5]. The most recent studies point to the key role of oxidative stress (OS) in the pathogenesis of AMD[6, 7].
Uncoupling protein 2 (UCP2), a member of the anion carrier family that localizes to the inner mitochondrial membrane, can decrease ROS production[8], and is widely expressed in rodents and adult human tissues. UCP2 has protective effects on numerous cells, through its function in controlling the production of superoxide and other downstream ROS. We have shown that mitochondrial function declines in aged retinal pigment epithelium (RPE) cells and that OS aggravates this condition, supporting a potential role for mitochondrial dysfunction in RPE cell damage and AMD onset[9, 10]. Therefore, we hypothesized that UCP2 may have a protective role in RPE cells during H2O2 injury.
The aims of our experiment were to investigate UCP2 expression in RPE cells exposed to OS in vitro and to explore how UCP2 is involved in the protection of these cells.
MATERIALS AND METHODS
Cell and Chemical Reagents The RPE cell line, ARPE-19, was purchased from ATCC (Manassas, Virginia, USA). Hydrogen peroxide (H2O2) was from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Gibco BRL (Carlsbad, CA, USA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St Louis, MO, USA).
Cell Culture Culture medium consisted of DMEM supplemented with 10%
FBS and 1% penicillin and streptomycin. Cultures were maintained at
Hydrogen Peroxide Treatment ARPE-19 cells were plated in 96-well plates at 1.2×104 cells/well for 12h. H2O2 was added at 0, 150, 300, 500, 700, or 900 µmol/L for 24h.
MTT Assay Cell viability was quantitatively assessed using MTT
assays. In each assay, PBS-treated and untreated cells were used as blank
controls and negative controls, respectively. Cells were washed three times
with PBS, then incubated with 20 µL MTT (5 mg/mL) reaction mixture at
Analysis of UCP2 mRNA
Expression Total mRNA was isolated from cells using TRIzol
extraction according to the manufacturer’s protocol. First-strand cDNA was
synthesized using an iScript cDNA synthesis kit (BioRad), and reverse
transcription-PCR (RT-PCR) was performed using the SuperScript First Strand
Synthesis System for RT-PCR (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) was used as the internal RNA loading control. cDNA aliquots (2 µL) were
subsequently amplified in a total reaction volume of 25 µL, containing
2×TaqPCRMaster Mix (12.5 µL), 1.0 µL each of forward and reverse primers, and
ddH2O (8.5 µL). The following primers were used to amplify human
UCP2: forward,
Western Blot Analysis of UCP2 Expression ARPE-19 cells were lysed using Cytobuster lysis buffer (Novagen, Madison, WI, USA) and protein concentrations estimated using a Dc Protein Assay Kit (BioRad). Protein samples (30 µg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were incubated with primary antibodies (1:1000) for 3h at room temperature after blocking with 5% (wt/vol) nonfat dried milk, followed by washing and incubation with horseradish peroxidase secondary antibodies for 1h at room temperature. Bound antibodies were visualized using the Bio-Rad ECL detection system and protein levels quantified with Image J software.
ROS Measurement ARPE-19 cells were detached by trypsinization, and 2×106 cells/mL incubated with H2-DCF-DA (0.4 µmol/L) at
Treatment of Cells with UCP2-siRNA UCP2-siRNA was purchased from GeneCopoeia and transfected according to the manufacturer’s recommendations. ARPE-19 cells were grown to approximately 80% confluence, then incubated with 1.0 µg scrambled siRNA or UCP2-siRNA in the presence of EndoFectin Lenti transfection reagent (3 µL/mL). Transfection was conducted without serum in the growth medium. After transfection (3h), a one-half volume of DMEM containing 30% FBS was added to the cells, and cultures grown for 48h. UCP2-siRNA efficacy was estimated by confocal microscopy. ARPE-19 cells mock transfected with siRNA were used as negative controls. After transfection (24h) UCP2-siRNA treated cultures were exposed to H2O2 (75, 150, 300 µmol/L) for 2h and cell viability measured by MTT assay.
Statistical Analysis All experiments were conducted in triplicate and each assay was repeated twice. Data are expressed as mean±standard deviation (SD). Differences between groups were analyzed by one-way ANOVA and post-hoc multiple comparisons performed using the Tukey HSD test when ANOVA testing indicated significance (P<0.05) using SPSS version 22.0 for Windows.
RESULTS
Morphological Changes in ARPE-19 Cells Treated With Different H2O2 Concentrations ARPE-19 cells were seeded in 6-well plates at 1.0×105 cells/well and cultured for 24h. Then, H2O2 (0, 150, 300, 500, 700, and 900 µmol/L) was added and cells grown for a further 24h before observation by microscopy to evaluate morphological changes. Cells treated with higher concentrations of H2O2 appeared shrunken cells, with disruption of adhesion between adjacent cells, and the numbers of dead cells was increased (Figure 1).
Figure 1 Morphological changes in ARPE-19 cells following treatment with various concentrations of H2O2 H2O2 [0 (A), 150 (B), 300 (C), 500 (D), 700 (E), and 900 µmol/L (F)] was added and cells cultured for 24h. The number of ARPE-19 cells decreased with increased H2O2 concentration and their morphology became irregular and shrunken, with increased numbers of dead cells. Scale bar: 200 µm. Magnification, ×100.
Effect of H2O2 on ARPE-19 Cell Metabolic Activity MTT assay demonstrated that ARPE-19 cell metabolic activity clearly decreased after H2O2 treatment. As concentrations of H2O2 increased, the number of viable cells decreased. Following post-hoc correction for multiple comparisons, there was a significant difference in the viability of cells treated with 300 µmol/L H2O2 compared with untreated controls (n=5; P=0.000); however, no significant differences were found on comparison of 300 vs 500 µmol/L, 500 vs 700 µmol/L, or 700 vs 900 µmol/L H2O2 treatment groups (all P>0.05; Figure 2). Nevertheless, increased concentration of H2O2 was associated with reduced cell metabolic activity, as detected by decreased absorbance (OD) values, indicating that H2O2 could induce apoptosis of ARPE-19 cells via OS (Figure 2).
Figure 2 Analysis of cell metabolic activity Cell metabolic activity gradually decreased with increasing H2O2 concentration. Compared with the untreated control group, cell viability was significantly reduced by treatment with 300 µmol/L H2O2 (n=5, aP<0.05).
UCP2 mRNA Expression in ARPE-19 Cells Exposed to Various H2O2 Concentrations RT-PCR results revealed that the
expression of UCP2 was decreased in a dose-dependent manner on treatment
with H2O2. Following, post-hoc multiple comparisons, mRNA
levels of UCP2 were significantly reduced compared with the untreated control
group (n=5, P<0.05; Figure
Figure 3 UCP2 mRNA levels in ARPE-19 cells under OS A: RT-PCR showing that down regulation of UCP2 expression in ARPE-19 cells under H2O2-induced OS; B: The expression of UCP2 was decreased in a dose-dependent manner in response to H2O2 compared with the untreated control group (aP<0.05) and no significant differences were detected between cell groups treated with different concentrations of H2O2 (P>0.05).
UCP2 Protein Expression in ARPE-19 Cells Treated with H2O2 Western blotting showing that the expression of UCP2 was significantly decreased after treatment with 75, 150 and 300 µmol/L H2O2 compared with untreated controls (P<0.05); however, there were no significant differences in UCP2 expression levels between the different treatment groups (P>0.05; Figure 4).
Figure 4 Western blot showing that the expression of UCP2 was significantly decreased after treatment with 75, 150, 300 µmol/L H2O2 relative to the untreated control group (aP<0.05).
Effect of UCP2-siRNA on ROS Levels in H2O2-treated ARPE-19 Cells Measurement of ROS by FCM, according to H2-DCF-DA fluorescence, in ARPE-19 cells treated with H2O2 (0, 75, 150, 300 µmol/L) demonstrated that ROS levels were significantly increased in groups treated with H2O2 compared with untreated controls (P<0.05; Figure 5). The ROS levels of cells treated with UCP2-siRNA exhibited an additional increase compared with those treated with 300 µmol/L H2O2 alone (P<0.05; Figure 6).
Figure 5 Flow cytometry measurement of ROS levels by detection of H2-DCF-DA fluorescence Data are shown as fold-change in fluorescence levels compared with controls. ROS levels in ARPE-19 cells were significantly increased in cells treated with H2O2 compared with untreated controls (aP<0.05).
Figure 6 Flow cytometry measurement of ROS levels by H2-DCF-DA fluorescence The ROS levels in cells treated with UCP2-siRNA were increased compared with those exposed to 300 µmol/L H2O2 alone (aP<0.05).
Effect of UCP2-siRNA on Viability in H2O2-treated ARPE-19 Cells ARPE-19 cells treated with UCP2-siRNA exhibited reduced UCP2 expression compared with controls (Figure 7). H2O2 (75, 150, 300 µmol/L) was added to UCP2-siRNA treated cultures 24h after transfection for 2h. The control group was not treated with either UCP2-siRNA or H2O2. MTT assay showed that UCP2-siRNA-treated cell metabolic activity clearly decreased after treatment with H2O2. Cells treated with UCP2-siRNA exhibited a further decrease in viability, compared with those treated with H2O2 alone. Cell viability was significantly reduced relative to controls (n=5, P<0.05; Figure 8).
Figure 7 UCP2-siRNA transfected ARPE-19 cells Cells transfected with UCP2-siRNA showed reduced UCP2 expression compared with controls.
Figure 8 H2O2 (75, 150, 300 µmol/L) was added to UCP2-siRNA treated cultures 24h after transfection and incubated for 2h MTT assay showing that metabolic activity in UCP2-siRNA treated cells was clearly decreased following H2O2 exposure. Cells treated with UCP2-siRNA exhibited a further decrease compared with those treated with H2O2 alone. Cell viability was significantly reduced, relative to controls (n=5, P<0.05). aP<0.05 vs H2O2 treatment, bP<0.05 vs control.
DISCUSSION
The molecular mechanisms underlying AMD remain poorly understood. Multiple influences contribute to AMD, including biochemical, immunogenic, and environmental factors, as well as OS. OS is a major contributor to the pathologies associated with AMD. The degeneration of RPE cells in the sub retinal pigment epithelial space and choroid is an initial pathological characteristic of AMD and the leading cause of severe vision loss in older people. Moreover, OS is implicated as a major inducer of RPE cell death.
Mitochondria are the main source of ROS production and excess ROS can induce mitochondrial damage and lead to disease[11, 12]. High ROS levels result in cell membrane damage via lipid peroxidation, which enhances oxidative injury, causing energy depletion and increasing ROS production. Uncoupling proteins (UCPs) localize to the mitochondrial inner membrane and belong to a superfamily of mitochondrial anion transporters that uncouple ATP synthesis from oxidative phosphorylation, by controlling the leakage of protons across the inner mitochondrial membrane[13, 14]. UCPs are important natural antioxidants involved in the maintenance of ROS homeostasis, alongside scavenging enzymes (e.g. superoxide dismutase and catalase) and low molecular weight antioxidants (e.g. ascorbic acid and glutathione)[15]. To date, five UCP subunits (UCP1-5) have been discovered in mammals, among which, UCP2 is widely distributed in various tissues and has been extensively studied[16]. UCP2 is an important mediator of oxidative damage. It is established that increased UCP2 expression can attenuate oxidant tissue damage[17, 18].
Available animal models fall short of accurately representing the
characteristics of AMD, as they lack human genetic polymorphisms and long-term
exposure to OS and environmental factors[19];
however, some cell models have been described. Culture of RPE cells with H2O2 is an established model for the study of OS. Our previous research demonstrated
that UCP2-deficient RPE cells had increased ROS levels and that increasing UCP2
expression using pigment epithelium-derived factor dampened ROS production in
aging and deficient cells exposed to OS[20]. In
the present study, we found that UCP2 mRNA levels were reduced in
ARPE-19 cells under OS, relative to controls. UCP2 decreases ROS production by
regulating ATP generation in mitochondria. In this study, UCP2 expression was
decreased in ARPE-19 cells exposed to OS. Further, we analyzed the metabolic
activity of cells treated with UCP2-siRNA and H2O2,
demonstrating that it was further decreased in UCP2-siRNA-treated cells exposed
to OS, compared with those treated with H2O2 alone,
according to increased ROS levels detected in UCP2-siRNA treated cells. These
results clarify that UCP2 functions to partially protect ARPE-19 cells under
OS. Our previous study also showed that UCP2 could reduce ROS generation;
therefore, we speculated that reduced expression of UCP2 was associated with
elevated ROS, resulting in decreased cellular anti-oxidation function. This
finding is consistent with those of several previous studies, where over expression
of UCP2 was reported to attenuate oxidant tissue damage[17, 18]; however, we did not detect significant
differences among cell groups treated with different concentrations of H2O2,
implying that its function in regulating ATP generation or maintaining energy
balance may not be the primary role of UCP
The present study demonstrates that UCP2 gene expression is decreased in human ARPE-19 cells in response to H2O2-induced OS in vitro. ROS levels in these cells were further increased in UCP2-siRNA treated cells. Hence, the UCP2 gene may have a protective role in RPE cells under OS.
ACKNOWLEDGEMENTS
Foundations: Supported by the National Natural Science Foundation of China (No.81100665; No.81770929).
Conflicts of Interest: Liu Y, None; Ren Y, None; Wang X, None; Liu X, None; Xu Y, None; He Y, None.
REFERENCES