January 2010
Volume 51, Issue 1
Free
Retinal Cell Biology  |   January 2010
Protective Effect of Clusterin from Oxidative Stress–Induced Apoptosis in Human Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • Jeong Hun Kim
    From the Fight against Angiogenesis-Related Blindness Laboratory, Department of Ophthalmology, Seoul National University College of Medicine and Seoul Artificial Eye Center, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea;
  • Jin Hyoung Kim
    From the Fight against Angiogenesis-Related Blindness Laboratory, Department of Ophthalmology, Seoul National University College of Medicine and Seoul Artificial Eye Center, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea;
  • Hyoung Oh Jun
    From the Fight against Angiogenesis-Related Blindness Laboratory, Department of Ophthalmology, Seoul National University College of Medicine and Seoul Artificial Eye Center, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea;
  • Young Suk Yu
    From the Fight against Angiogenesis-Related Blindness Laboratory, Department of Ophthalmology, Seoul National University College of Medicine and Seoul Artificial Eye Center, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea;
  • Bon Hong Min
    the Department of Pharmacology and BK21 Program for Medical Sciences, College of Medicine, Korea University, Seoul, Korea;
  • Kyu Hyung Park
    the Department of Ophthalmology, Seoul National University Bundang Hospital, Seongnam, Korea; and
  • Kyu-Won Kim
    the NeuroVascular Coordination Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea.
  • Corresponding author: Young Suk Yu, Department of Ophthalmology, College of Medicine, Seoul National University and Seoul Artificial Eye Center Clinical Research Institute, Seoul National University Hospital, Seoul, Korea; ysyu@snu.ac.kr
  • Footnotes
    2  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science January 2010, Vol.51, 561-566. doi:10.1167/iovs.09-3774
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      Jeong Hun Kim, Jin Hyoung Kim, Hyoung Oh Jun, Young Suk Yu, Bon Hong Min, Kyu Hyung Park, Kyu-Won Kim; Protective Effect of Clusterin from Oxidative Stress–Induced Apoptosis in Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(1):561-566. doi: 10.1167/iovs.09-3774.

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

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Abstract

Purpose.: Oxidative stress to retinal pigment epithelial (RPE) cells is thought to play a critical role in the pathogenesis of age-related macular degeneration (AMD). This study was conducted to investigate whether clusterin protects human RPE cells from ROS-induced apoptosis through a PI3K/Akt survival pathway.

Methods.: The preventive effect of clusterin on reactive oxygen species (ROS) production and RPE cell death induced by hydrogen peroxide was determined in ARPE-19 cells. The ability of clusterin to protect RPE cells against ROS-mediated apoptosis was assessed by caspase-3 activity and DAPI staining. Furthermore, the protective effect of clusterin via the PI3K/Akt pathway was determined by Western blot analysis.

Results.: Clusterin prevented ARPE-19 cells from H2O2-induced cell death and ROS production. H2O2-induced oxidative stress increased caspase-3 activity, which was significantly inhibited by clusterin, as determined by the abrogation of apoptotic bodies. Interestingly, clusterin induced Akt phosphorylation in human RPE cells under oxidative stress, which contributed to cell viability in ARPE-19 cells. This cell survival by clusterin was blocked by a PI3K inhibitor.

Conclusions.: Clusterin may play a protective role in responding to the local redox environment of human RPE cells, which contributes to the cell survival via the PI3K/Akt pathway. Therefore, clusterin could be considered for the preventive approach to AMD.

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly persons. 1 Taking into account the progression spectrum, AMD may be classified into three forms: early, intermediate, and advanced. More than 80% of all cases of blindness attributable to AMD are the advanced form, 2 which could be grouped as two types, dry form and wet form. Interestingly, pathologic features common to both types, including drusen and pigmentary changes in retinal pigment epithelial (RPE) cells, are limited in RPE cells and adjacent structures. Accordingly, AMD pathophysiologically results from pathologic processes of RPE cells and their interactions with photoreceptors and choriocapillaris. 3 As a result, an approach to rescue RPE cells would be helpful for preventing the occurrence or progression of AMD. 
RPE cells are basically prone to oxidative stress, high oxygen tension, lifelong light illumination, and phagocytosis. 3 Therefore, in accordance with the decrease of antioxidative enzymes in RPE cells with age, oxidative stress is thought to play a critical role in the pathogenesis of AMD. 4 Multiple signaling pathways to coordinate cellular responses and to determine cell fate could be activated by reactive oxygen species (ROS). 5 Transient fluctuations of ROS could serve some regulatory functions, whereas high and sustained levels of ROS cause mitochondrial DNA damage and ultimately lead to the apoptosis of RPE cells. 6  
Clusterin is a major secretory glycoprotein composed of two disulfide linked 35- to 40-kDa subunits (α and β) encoded by a single gene, 7 which is an extracellular chaperone that acts as a functional homologue to the small heat shock proteins that stabilize stressed proteins in a folding-competent state. 8 During increased oxidative stress, clusterin is induced as a physiological defense to maintain cell viability. It is involved in the suppression of pro-death signals after increased oxidative injury through scavenging extracellular abnormal molecules. 9 In addition, clusterin may localize to mitochondria and inhibit apoptosis by interacting with activated Bax. 10 Recently, we demonstrated that clusterin is upregulated in the developing retina and protects retinal cells from H2O2-induced apoptotic cell death, 11 which is mediated by Akt activation. 12 Moreover, we showed that clusterin expression increases in retinal endothelial cells in an in vitro ischemia condition to play a protective role in ischemia-induced retinal endothelial cell apoptosis. 12  
In the present study, we showed for the first time that clusterin protects human RPE cells from ROS-induced apoptosis through the PI3K/Akt survival pathway. Clusterin significantly inhibits ROS generation and caspase-3 activity in human RPE cells under H2O2-induced oxidative stress. Interestingly, the protective activity of clusterin to H2O2-induced oxidative stress is mediated by the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Taken together, clusterin may act as an external survival signal through the PI3K/Akt pathway in human RPE cells. 
Materials and Methods
Cell Culture
ARPE-19 cells (American Type Culture Collection, Manassas, VA) were used for human RPE cells. The cells were routinely maintained in Dulbecco's modified Eagle's medium (Invitrogen, Gibco, Carlsbad, CA) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 100 U/mL penicillin (Sigma Aldrich, St. Louis, MO), 100 μg/mL streptomycin (Invitrogen, Gibco), and 1 mM sodium pyruvate (Sigma Aldrich). ARPE-19 cells used in this study were taken from passages 4 to 6. Clusterin, H2O2 (Sigma Aldrich), N-acetylcysteine (NAC; Sigma Aldrich), and LY294002 (LY; Sigma Aldrich) treatment was carried out in cells cultured in serum-free condition. 
Affinity Purification of Clusterin from Human Serum
Clusterin was purified from fresh normal human plasma, as previously described. 11,12 Human plasma supplemented with 0.5 mM phenylmethylsulfonyl fluoride (PMSF) was precipitated using 12% polyethylene glycol (PEG; MWt 3350; Sigma) overnight at 4°C, and, after centrifugation, the supernatant was reprecipitated with 23% PEG. This precipitate was dissolved in 10 mM Tris buffer (pH 7.4), 0.5 mM PMSF, subjected to DEAE column chromatography (GE Healthcare Life Sciences, Buckinghamshire, UK), and equilibrated with 10 mM Tris buffer (pH 7.4). Fractions were obtained by elution with a linear gradient from 0 to 0.5 M NaCl, and the pool of positive fractions containing clusterin (validated by immunoblotting using clusterin antibody [M18; Santa Cruz Biotechnology, Santa Cruz, CA]) was subjected to heparin sepharose column chromatography (GE Healthcare Life Sciences) equipped with a 20-mM potassium phosphate buffer (pH 6.0) pre-equilibrated column. Proteins bound to heparin sepharose were fractionated using a linear gradient of NaCl (0–2 M), and fractions positive for clusterin were sorted by immunoblotting using anti-clusterin M18. The serum clusterin obtained was finally purified by affinity chromatography using cyanogen bromide-activated agarose (Sigma) covalently conjugated with anti-clusterin monoclonal antibody (1G8) generated in our laboratory using recombinant human full-length clusterin expressed in Escherichia coli as an antigen. The positive pool of clusterin obtained by heparin sepharose column chromatography was then applied to a 1G8 affinity column at 4°C. The column was initially washed with 10 mM potassium phosphate buffer (pH 7.4) containing 0.5 M NaCl and 1% Triton X-100 and then was rewashed with 10 mM potassium phosphate buffer (pH 7.4) containing 0.5 M NaCl. Pure clusterin remaining on the column was collected by eluting with 2 M guanidine-HCl in 0.5 M NaCl. The eluted protein was dialyzed against 5 mM potassium phosphate (pH 6.5) and lyophilized before it was stored at −80°C. 
Cell Viability Assay
Cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ARPE-19 cells (1 × 105 cells) were incubated with different concentrations of H2O2 (50–500 μM), clusterin (0.5–5 μg/mL), or LY (10 μM) for 24 hours. The medium was then replaced with fresh medium containing 0.5 mg/mL MTT for 4 hours After incubation, the medium was carefully removed from the plate, and dimethyl sulfoxide was added to solubilize formazan produced from MTT by the viable cells. Absorbance was measured at 540 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). 
Intracellular ROS Measurement
ARPE-19 cells (1 × 105 cells) were incubated with different concentrations of H2O2 (50–500 μM), clusterin (0.5–5 μg/mL), or NAC (10 mM) and were then labeled with 20 μM of 2,′7′-dichlorofluorescein-diacetate (2,′7′-DCFH-DA; Sigma-Aldrich) for 30 minutes at 37°C. DCF fluorescence was measured with excitation and emission settings of 495 and 525 nm, respectively. Nonspecific fluorescence values without cells were subtracted from the fluorescence values with cells. 
Caspase-3 Activity Measurement
Caspase-3 activity was measured with an assay kit (Caspase3/CPP32 Fluorometric Protease; Bio Vision, Mountain View, CA). ARPE-19 cells (1 × 105 cells) were incubated with 200 μM H2O2 or 1 μg/mL clusterin for set periods ranging from 0 to 12 hours. The cells were harvested and then suspended in the cell lysis buffer to obtain cell lysate. According to the manufacturer's instruction, after 1 hour of incubation with a 7-amino-4-trifluoromethylcoumarin-derived caspase substrate, 7-amino-4-trifluoromethyl coumarin released by caspase-3 was determined by a fluorometric plate reader (CytoFluor Series 4000; Applied Biosystems, Framingham, MA) with excitation and emission settings of 400 and 505 nm, respectively. 
4,6-Diamidino-2-Phenolindole Staining
We conducted 4,6-diamidino-2-phenolindole (DAPI; Sigma-Aldrich) staining for the identification of apoptotic nuclei. ARPE-19 cells (1 × 105 cells) were treated with 200 μM H2O2, 1 μg/mL clusterin, or 10 mM NAC. Cells were fixed and stained with 10 μg/mL of DAPI (Sigma-Aldrich). After incubation for 5 minutes in the dark, the cells were washed. Slides were mounted and observed under a fluorescence microscope (BX50; Olympus, Tokyo, Japan). 
Reverse Transcriptase-Polymerase Chain Reaction Analysis
Total RNA from cells was isolated using reagent (Trizol; Invitrogen, Rockville, MD) according to the manufacturer's instructions. First-strand cDNA was synthesized with 3 μg each DNA-free total RNA and oligo-(dT) 16 primer by Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). Equal amounts of cDNA were subsequently amplified by PCR in a 50-μL reaction volume containing 1× PCR buffer, 200 μM dNTP, 10 μM specific primer for clusterin (5′-CGAGAAGGCGACGATGAC-3′ and 5′-GGTGGAACAGTCCACAGACA-3′) and GAPDH (5′-TCCCTCAAGATTGTCAGCAA-3′ and 5′-AGATCCACAACGGATACATT-3′), and 1.25 U Taq DNA polymerase (TaKaRa, Tokyo, Japan). PCR was performed with an initial denaturation step followed by denaturation, annealing, and extension. PCR products were separated on agarose gels and visualized using ethidium bromide staining under UV transillumination. 
Western Blot Analysis
Western blot analysis was performed using standard Western blotting methods. The protein concentration was measured with a BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were separated by electrophoresis on 5% to 10% SDS-PAGE and were transferred electrophoretically on nitrocellulose membrane (Amersham, Little Chalfont, UK). The membranes were blocked for 30 minutes in 5% nonfat milk and then were incubated overnight with antibodies against Akt, phospho-Akt (pAkt), pro-caspase-3, or cleaved caspase-3 (Cell Signaling, Danvers, MA) at 4°C. To ensure the equal loading of protein in each lane, the blots were stripped and reprobed with an antibody against β-actin. Intensity values were normalized relative to control values. The blots were scanned using a flatbed scanner, and the band intensity was analyzed with a software program (TINA; Raytest, Staubenhardt, Germany). 
Statistical Analysis
Statistical differences between groups were evaluated using Student's paired t-test. All statistical tests were completed (SPSS for Windows, version 12.0; SPSS, Chicago, IL). Figures are depicted as mean ± SD. P ≤ 0.05 was considered statistically significant. 
Results
Protection of Clusterin from H2O2-Induced Cell Death in RPE Cells
To investigate the cytotoxicity of clusterin on human RPE cells, MTT assay was carried out with different concentrations of clusterin (0.1–20 μg/mL). As shown in Figure 1A, up to 20 μg/mL (20 times the effective therapeutic concentration in the retina 11,12 ) clusterin did not affect the cell viability of ARPE-19 cells. Based on our previous report that clusterin inhibits retinal cells from H2O2-induced cell death, 11 we next investigated whether H2O2-treated cell viability of human RPE cells could be prevented by clusterin treatment. When ARPE-19 cells were treated with higher H2O2 doses (greater than 200 μM), cell viability was significantly decreased (Fig. 1B). However, as demonstrated in Figure 1C, clusterin treatment effectively prevented ARPE-19 cells from H2O2-induced cell death. 
Figure 1.
 
Clusterin protects human RPE cells from H2O2-induced cell death. (A) Cell viability was measured in ARPE-19 cells treated with different concentrations of clusterin (0.1–20 μg/mL) by MTT assay. Each value represents the mean ± SE of three independent experiments. (B) Cell viability was measured in ARPE-19 cells treated with different concentrations of H2O2 (50–500 μM) by MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) Cell viability was measured in ARPE-19 cells treated with 200 μM H2O2 or 1 μg/mL clusterin by MTT assay. Figures were selected as representative data from three independent experiments. Scale bar, 100 μm. Each value represents the mean ± SE of three independent experiments (*P < 0.05).
Figure 1.
 
Clusterin protects human RPE cells from H2O2-induced cell death. (A) Cell viability was measured in ARPE-19 cells treated with different concentrations of clusterin (0.1–20 μg/mL) by MTT assay. Each value represents the mean ± SE of three independent experiments. (B) Cell viability was measured in ARPE-19 cells treated with different concentrations of H2O2 (50–500 μM) by MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) Cell viability was measured in ARPE-19 cells treated with 200 μM H2O2 or 1 μg/mL clusterin by MTT assay. Figures were selected as representative data from three independent experiments. Scale bar, 100 μm. Each value represents the mean ± SE of three independent experiments (*P < 0.05).
Inhibition of H2O2-Induced ROS Production by Clusterin in Human RPE Cells
To evaluate the inhibitory effect of clusterin on ROS production in human RPE cells, we investigated whether clusterin could inhibit H2O2-induced ROS production using the cell-permeable fluorescence dye. As shown in Figure 2A, the intensity of the mean oxidized DCF peak was increased by 4.9-fold compared with controls after 200 μM H2O2 treatment in ARPE-19 cells, which was significantly suppressed by cotreatment with 1 μg/mL clusterin. The inhibitory effect of clusterin on ROS production was similar to that of 10 mM NAC, a ROS inhibitor (Fig. 2A). 
Figure 2.
 
Clusterin inhibits H2O2-induced ROS production in human RPE cells. (A) ARPE-19 cells were treated with 200 μM H2O2, 1 μg/mL clusterin, or 10 mM NAC. For measuring H2O2 production, the cells were then labeled with DCFH-DA. Figures were selected as representative data from three independent experiments. Scale bar, 100 μm. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) ARPE-19 cells were treated with different concentrations of H2O2 (50–500 μM) and clusterin (0.1–5 μg/mL). For measuring H2O2 production, the cells were then labeled with DCFH-DA. Quantitative analysis was performed by measuring the fluorescence intensity. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) Cell viability was measured in ARPE-19 cells treated with 0.2 μM rotenone and 1 μg/mL clusterin by MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05).
Figure 2.
 
Clusterin inhibits H2O2-induced ROS production in human RPE cells. (A) ARPE-19 cells were treated with 200 μM H2O2, 1 μg/mL clusterin, or 10 mM NAC. For measuring H2O2 production, the cells were then labeled with DCFH-DA. Figures were selected as representative data from three independent experiments. Scale bar, 100 μm. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) ARPE-19 cells were treated with different concentrations of H2O2 (50–500 μM) and clusterin (0.1–5 μg/mL). For measuring H2O2 production, the cells were then labeled with DCFH-DA. Quantitative analysis was performed by measuring the fluorescence intensity. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) Cell viability was measured in ARPE-19 cells treated with 0.2 μM rotenone and 1 μg/mL clusterin by MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05).
Next, we measured intracellular ROS induced by variable concentrations of H2O2 (50–500 μM) with different concentrations of clusterin (0.5–5 μg/mL) in ARPE-19 cells. As shown in Figure 2B, intracellular ROS was significantly increased by H2O2 in all concentrations (2.5- to 4.2-fold), which was, however, effectively reduced by clusterin treatment. Interestingly, in less than 1 μg/mL clusterin, intracellular ROS was dramatically decreased, whereas it was sustained at similar levels from 1 μg/mL clusterin (Fig. 2B). It seems that the antioxidant activity of clusterin in ARPE-19 cells is maximized at 1 μg/mL, similar to the effective therapeutic concentration in the retina. 11,12  
In addition, using rotenone, a widely used inhibitor of mitochondrial complex I, we determined whether clusterin could inhibit ROS generation induced by an inducer of endogenous ROS production in RPE cells. At a low concentration of rotenone (<0.25 μM), the respiratory activity of the mitochondria was not substantially inhibited, but ROS were generated that could damage mitochondrial DNA 13 ; 0.2 μM rotenone was treated on RPE cells, and ROS production was measured by DCF fluorescence. As shown in Figure 2C, 0.2 μM rotenone significantly induced ROS production in RPE cells 1.56 (± 0.24)-fold compared with the control (P < 0.05), which was significantly inhibited by treatment with 1 μg/mL clusterin (P < 0.05). These data suggest that clusterin may inhibit ROS generation induced by an inducer of endogenous ROS production in RPE cells. 
H2O2-Induced Apoptosis Inhibition by Clusterin through Suppression of Caspase-3 Activity
To validate the mechanism by which clusterin protects human RPE cells from oxidative stress, the inhibitory effect of clusterin on caspase-3 activity related to apoptotic processes was assessed using caspase-3/CPP fluorometric protease assay. As demonstrated in Figure 3A, caspase-3 activity increased to threefold over control at 6 hours after 200 μM H2O2 treatment and was sustained to 18 hours after 200 μM H2O2 treatment. The increased activity of caspase-3 was, however, significantly inhibited by cotreatment with 1 μg/mL clusterin (Fig. 3A). In addition, compared with no fragmented DNA in control, strong fluorescent spots, indicating apoptotic bodies, were detected by DAPI staining of 200 μM H2O2-treated ARPE-19 cells that were almost completely abrogated by 1 μg/mL clusterin (Fig. 3B). 
Figure 3.
 
Clusterin protects human RPE cells from H2O2-induced apoptosis via inhibition of caspase-3 activity. ARPE-19 cells were treated with 200 μM H2O2 or 1 μg/mL clusterin. (A) Caspase-3 activation was determined during 24-hour culture using caspase-3/CPP32 fluorometric protease assay kit. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) Cell viability was measured using DAPI staining. Strong fluorescent spots show apoptotic nuclei. Figures were selected as representative data from three independent experiments. Scale bar, 10 μm.
Figure 3.
 
Clusterin protects human RPE cells from H2O2-induced apoptosis via inhibition of caspase-3 activity. ARPE-19 cells were treated with 200 μM H2O2 or 1 μg/mL clusterin. (A) Caspase-3 activation was determined during 24-hour culture using caspase-3/CPP32 fluorometric protease assay kit. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) Cell viability was measured using DAPI staining. Strong fluorescent spots show apoptotic nuclei. Figures were selected as representative data from three independent experiments. Scale bar, 10 μm.
Activation of PI3K/Akt Pathway by Clusterin to Protect Human RPE Cells from H2O2-Induced Cell Death
Given that Akt activation protects human RPE cells from oxidant-induced cell death, 14 we first determined whether clusterin could induce Akt phosphorylation in human RPE cells under oxidative stress. When ARPE-19 cells were exposed to 200 μM H2O2 for 2 hours, Akt phosphorylation did not significantly increase compared with control, whereas it was significantly increased with 1 μg/mL clusterin treatment (Fig. 4A). However, the cotreatment of LY294002, an inhibitor of upstream of Akt, PI3K, effectively inhibited Akt phosphorylation (Fig. 4A), indicating that cell survival by clusterin in human RPE cells may be mediated by the PI3K/Akt pathway. 
Figure 4.
 
Clusterin activates the PI3K/Akt pathway to protect human RPE cells from H2O2-induced cell death. (A, B) ARPE-19 cells were treated with 200 μM H2O2, 1 μg/mL clusterin, or 10 μM LY. (A) After 2-hour exposure to H2O2, Western blot analysis using p-Akt and Akt antibodies was performed. β-Actin served as the loading control. Figures were selected as representative data from three independent experiments. Quantitative analysis was performed by measuring the intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) After 24-hour exposure to H2O2, cell viability was measured with the MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) ARPE-19 cells were treated with 200 μM H2O2 or 1 μg/mL clusterin. RT-PCR was performed with specific primers for clusterin. GAPDH served as an internal control. Western blot analysis using clusterin, pro-caspase-3, and cleaved caspase-3 antibodies was performed. β-Actin served as the loading control. Figures were selected as representative data from three independent experiments.
Figure 4.
 
Clusterin activates the PI3K/Akt pathway to protect human RPE cells from H2O2-induced cell death. (A, B) ARPE-19 cells were treated with 200 μM H2O2, 1 μg/mL clusterin, or 10 μM LY. (A) After 2-hour exposure to H2O2, Western blot analysis using p-Akt and Akt antibodies was performed. β-Actin served as the loading control. Figures were selected as representative data from three independent experiments. Quantitative analysis was performed by measuring the intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) After 24-hour exposure to H2O2, cell viability was measured with the MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) ARPE-19 cells were treated with 200 μM H2O2 or 1 μg/mL clusterin. RT-PCR was performed with specific primers for clusterin. GAPDH served as an internal control. Western blot analysis using clusterin, pro-caspase-3, and cleaved caspase-3 antibodies was performed. β-Actin served as the loading control. Figures were selected as representative data from three independent experiments.
We next addressed whether the cell viability of human RPE cells under oxidative stress is closely related to the Akt phosphorylation by clusterin. As shown in Figure 4B, H2O2-treated cell viability was significantly reduced and was, interestingly, more reduced when Akt activation was blocked by LY, a PI3K inhibitor. These data were supported by a previous report that Akt activation plays a crucial role in cell survival against oxidative stress in human RPE cells. 14 Clusterin effectively prevented ARPE-19 cells from H2O2-induced cell death, that was, however, completely inhibited by LY, a PI3K inhibitor (Fig. 4B). 
Furthermore, we determined whether clusterin is taken up by the RPE cells through RT-PCR and Western blot analysis after clusterin treatment. As shown in the Figure 4C, 1 μg/mL clusterin treatment increased the protein levels without a change in mRNA levels. With 200 μM H2O2 treatment, clusterin expression was decreased because of decreasing mRNA expression and increasing apoptotic cell death. However, with 1 μg/mL clusterin treatment, clusterin expression was recovered, and apoptotic cell death was prevented. Therefore, our results suggest that clusterin is taken up by RPE cells as protection against oxidative stress-induced apoptosis. 
Discussion
In the present study, we have demonstrated that clusterin effectively protects human RPE cells from oxidative stress-induced cell death via the suppression of caspase-3 activity and the activation of Akt. Taken together, our data confirm the protective effect of clusterin on oxidative stress in human RPE cells as determined in other retinal cells in our previous studies. 11,12,15  
Clusterin has been known to be upregulated in response to diverse pathophysiological stresses to exert as an extracellular chaperone, which stabilizes stressed proteins in a folding-competent state. 8 In addition to extracellular chaperone activity to keep the extracellular environment free of abnormal proteins, clusterin may have an antiapoptotic function interfering with Bax proapoptotic activity, 10 which consequently would be linked to the inhibition of the caspase pathway. Moreover, clusterin was recently reported to upregulate Akt phosphorylation and Bad phosphorylation and to cause a decrease of cytochrome c release, which implicates the antiapoptotic function of clusterin. 16 Thus, accumulating evidence suggests that clusterin could be a survival factor directly or indirectly associated with the apoptotic pathway. Actually, clusterin effectively rescued human RPE cells from H2O2-induced cell death. H2O2 strongly enhanced intracellular ROS production in human RPE cells that was significantly inhibited by clusterin treatment. Clusterin also effectively reduced H2O2-induced apoptotic cell death in human RPE cells, as determined by caspase-3 activity and staining for apoptotic nuclei. Given that clusterin inhibits apoptotic processes in mitochondria, 10,16 the primary target of oxidative stress in RPE cells, 17 it is possible that the inhibition of intracellular ROS production by clusterin is causally related to the prevention of H2O2-induced cell death. 
The PI3K/Akt pathway links extracellular survival signals to the apoptosis-related pathway and plays a critical role in keeping cells alive by blocking apoptotic pathways. 18 Akt activation is especially dependent on PI3K to promote cell survival. 19 Activation of the PI3K/Akt pathway results in the phosphorylation of key survival molecules, including Bad, caspase-9, and glycogen synthase kinase-3β, which inhibits apoptotic processes. 19 ROS result predominantly from increased metabolic activity, which directly exerts the cell survival pathways. 5 Interestingly, ROS could lead to transient, rapid activation of Akt, 20 which was reproducible in human RPE. 14 However, temporary Akt activation by ROS was too transient to maintain for 1 hour in human RPE cells. 14 Our choice for exposure of human RPE cells to H2O2 for 2 hours was to avoid this transient effect of ROS on Akt activation. Moreover, given that cumulative oxidative stress leads to AMD pathogenesis, 24 high and sustained doses of ROS may contribute to oxidant-induced cell death in RPE cells. Actually, Akt phosphorylation was not elevated in 2-hour stimulation of H2O2, whereas clusterin treatment significantly enhanced Akt activation, which was completely inhibited by the PI3K inhibitor. These data suggest that clusterin activates survival signals through the PI3K/Akt signaling pathway, as determined by cell viability assay. Furthermore, the PI3K inhibitor enhanced H2O2-induced RPE cell death, which supports that activation of the PI3K/Akt signaling pathway protects human RPE cells from oxidant-induced cell death. 6,14  
Interestingly, clusterin taken up by the RPE cells to protect from oxidative stress-induced apoptosis showed no influence to cell viability and morphology of human RPE cells up to 20 μg/mL, equivalent to 20 times the effective therapeutic dose in other retinal cells 11,12 and in ARPE-19 cells. Therefore, considering the absence of clusterin-induced retinal and systemic toxicity in the mice, 11,12 clusterin could be safely applied to the in vivo model without toxicity. 
In summary, clusterin protects human RPE cells from H2O2-induced cell death, which is closely related to the inhibition of H2O2-induced ROS generation and to the suppression of caspase-3 activity. This survival effect of clusterin was regulated through the transduction mechanism involving the PI3K/Akt pathway, though the details require further investigation. Moreover, clusterin never demonstrated cytotoxicity to human RPE cells 20-fold over the effective therapeutic concentration. Based on this available evidence, we suggest that clusterin may protect human RPE cells from oxidative stress-induced cell death. Therefore, clusterin may play a special role in responding to the local redox environment of human RPE cells and may be considered as a therapeutic approach to AMD. 
Footnotes
 Supported by Bundang Seoul National University Hospital Research Fund Grant 02–2007-007; Bio-signal Analysis Technology Innovation Program Grant M1064501001–06n4501–00110 of the Ministry of Science and Technology; and the Korea Science and Engineering Foundation.
Footnotes
 Disclosure: J. Hun Kim, None; J. Hyoung Kim, None; H.O. Jun, None; Y.S. Yu, None; B.H. Min, None; K.H. Park, None; K.-W. Kim, None
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Figure 1.
 
Clusterin protects human RPE cells from H2O2-induced cell death. (A) Cell viability was measured in ARPE-19 cells treated with different concentrations of clusterin (0.1–20 μg/mL) by MTT assay. Each value represents the mean ± SE of three independent experiments. (B) Cell viability was measured in ARPE-19 cells treated with different concentrations of H2O2 (50–500 μM) by MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) Cell viability was measured in ARPE-19 cells treated with 200 μM H2O2 or 1 μg/mL clusterin by MTT assay. Figures were selected as representative data from three independent experiments. Scale bar, 100 μm. Each value represents the mean ± SE of three independent experiments (*P < 0.05).
Figure 1.
 
Clusterin protects human RPE cells from H2O2-induced cell death. (A) Cell viability was measured in ARPE-19 cells treated with different concentrations of clusterin (0.1–20 μg/mL) by MTT assay. Each value represents the mean ± SE of three independent experiments. (B) Cell viability was measured in ARPE-19 cells treated with different concentrations of H2O2 (50–500 μM) by MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) Cell viability was measured in ARPE-19 cells treated with 200 μM H2O2 or 1 μg/mL clusterin by MTT assay. Figures were selected as representative data from three independent experiments. Scale bar, 100 μm. Each value represents the mean ± SE of three independent experiments (*P < 0.05).
Figure 2.
 
Clusterin inhibits H2O2-induced ROS production in human RPE cells. (A) ARPE-19 cells were treated with 200 μM H2O2, 1 μg/mL clusterin, or 10 mM NAC. For measuring H2O2 production, the cells were then labeled with DCFH-DA. Figures were selected as representative data from three independent experiments. Scale bar, 100 μm. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) ARPE-19 cells were treated with different concentrations of H2O2 (50–500 μM) and clusterin (0.1–5 μg/mL). For measuring H2O2 production, the cells were then labeled with DCFH-DA. Quantitative analysis was performed by measuring the fluorescence intensity. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) Cell viability was measured in ARPE-19 cells treated with 0.2 μM rotenone and 1 μg/mL clusterin by MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05).
Figure 2.
 
Clusterin inhibits H2O2-induced ROS production in human RPE cells. (A) ARPE-19 cells were treated with 200 μM H2O2, 1 μg/mL clusterin, or 10 mM NAC. For measuring H2O2 production, the cells were then labeled with DCFH-DA. Figures were selected as representative data from three independent experiments. Scale bar, 100 μm. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) ARPE-19 cells were treated with different concentrations of H2O2 (50–500 μM) and clusterin (0.1–5 μg/mL). For measuring H2O2 production, the cells were then labeled with DCFH-DA. Quantitative analysis was performed by measuring the fluorescence intensity. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) Cell viability was measured in ARPE-19 cells treated with 0.2 μM rotenone and 1 μg/mL clusterin by MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05).
Figure 3.
 
Clusterin protects human RPE cells from H2O2-induced apoptosis via inhibition of caspase-3 activity. ARPE-19 cells were treated with 200 μM H2O2 or 1 μg/mL clusterin. (A) Caspase-3 activation was determined during 24-hour culture using caspase-3/CPP32 fluorometric protease assay kit. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) Cell viability was measured using DAPI staining. Strong fluorescent spots show apoptotic nuclei. Figures were selected as representative data from three independent experiments. Scale bar, 10 μm.
Figure 3.
 
Clusterin protects human RPE cells from H2O2-induced apoptosis via inhibition of caspase-3 activity. ARPE-19 cells were treated with 200 μM H2O2 or 1 μg/mL clusterin. (A) Caspase-3 activation was determined during 24-hour culture using caspase-3/CPP32 fluorometric protease assay kit. Quantitative analysis was performed by measuring the fluorescence intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) Cell viability was measured using DAPI staining. Strong fluorescent spots show apoptotic nuclei. Figures were selected as representative data from three independent experiments. Scale bar, 10 μm.
Figure 4.
 
Clusterin activates the PI3K/Akt pathway to protect human RPE cells from H2O2-induced cell death. (A, B) ARPE-19 cells were treated with 200 μM H2O2, 1 μg/mL clusterin, or 10 μM LY. (A) After 2-hour exposure to H2O2, Western blot analysis using p-Akt and Akt antibodies was performed. β-Actin served as the loading control. Figures were selected as representative data from three independent experiments. Quantitative analysis was performed by measuring the intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) After 24-hour exposure to H2O2, cell viability was measured with the MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) ARPE-19 cells were treated with 200 μM H2O2 or 1 μg/mL clusterin. RT-PCR was performed with specific primers for clusterin. GAPDH served as an internal control. Western blot analysis using clusterin, pro-caspase-3, and cleaved caspase-3 antibodies was performed. β-Actin served as the loading control. Figures were selected as representative data from three independent experiments.
Figure 4.
 
Clusterin activates the PI3K/Akt pathway to protect human RPE cells from H2O2-induced cell death. (A, B) ARPE-19 cells were treated with 200 μM H2O2, 1 μg/mL clusterin, or 10 μM LY. (A) After 2-hour exposure to H2O2, Western blot analysis using p-Akt and Akt antibodies was performed. β-Actin served as the loading control. Figures were selected as representative data from three independent experiments. Quantitative analysis was performed by measuring the intensity relative to the control. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (B) After 24-hour exposure to H2O2, cell viability was measured with the MTT assay. Each value represents the mean ± SE of three independent experiments (*P < 0.05). (C) ARPE-19 cells were treated with 200 μM H2O2 or 1 μg/mL clusterin. RT-PCR was performed with specific primers for clusterin. GAPDH served as an internal control. Western blot analysis using clusterin, pro-caspase-3, and cleaved caspase-3 antibodies was performed. β-Actin served as the loading control. Figures were selected as representative data from three independent experiments.
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