Pigment epithelium-derived factor inhibits oxidative stress-induced cell death by activation of extracellular signal-regulated kinases in cultured retinal pigment epithelial cells

Life Sciences 79 (2006) 545 – 550 www.elsevier.com/locate/lifescie

Pigment epithelium-derived factor inhibits oxidative stress-induced cell death by activation of extracellular signal-regulated kinases in cultured retinal pigment epithelial cells Yeou-Ping Tsao a,b,⁎,1 , Tsung-Chuan Ho a,1 , Show-Li Chen b,c , Huey-Chuan Cheng a,d a

b

Department of Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan c Department of Microbiology, School of Medicine, National Taiwan University, Taipei, Taiwan d Mackay Medicine, Nursing and Management College, Taipei, Taiwan Received 28 October 2005; accepted 26 January 2006

Abstract Oxidative stress-induced retinal pigment epithelial (RPE) cell death is involved in the pathogenesis of age-related macular degeneration (AMD). Pigment epithelium-derived factor (PEDF) is an anti-angiogenic/neurotropic dual functional factor, and recently it was also shown to mediate anti-oxidative action. In the present study, the influence of PEDF in hydrogen peroxide (H2O2)-induced RPE cell death was investigated using nontransformed human RPE cell line (ARPE-19). The recombinant PEDF was purified from E. coli. The MTT cell viability assay showed that PEDF rescued ARPE-19 from H2O2-induced cell death in a dose- and time-dependent manner. Western blot analysis revealed that PEDF stimulated the extracellular signal-regulated kinases (ERK1/2) phosphorylation. The PEDF cytoprotective effect was significantly attenuated by the ERK1/2 inhibitor PD98059. In this study, we demonstrate that PEDF induces ERK1/2 phosphorylation and we further suggest that the ERK signal cascade contributes to RPE cell's cytoprotection against oxidative stress. © 2006 Elsevier Inc. All rights reserved. Keywords: PEDF; Oxidative stress; RPE; ARPE-19; ERK1/2

Introduction Age-related macular degeneration (AMD) is the most common cause of vision loss in patients. Degeneration, dysfunction or loss of retinal pigment epithelial (RPE) cells by oxidative injury is highly related to the development of AMD (Winkler et al., 1999; Cai et al., 2000). RPE cells are responsible for the clearance of lipid peroxidants derived from photoreceptors by phagocytosis. This process is associated with a frequent exposure to a number of reactive oxygen species (ROS) including H2O2 (Miceli et al., 1994). Cytotoxic levels of H2O2 cause programmed cell death (Barak et al., 2001; Jin et al., 2001) in ⁎ Corresponding author. Department of Ophthalmology, Mackay Memorial Hospital, Number 92, Sec. 2, Chung Shan North Road, Taipei, Taiwan. Tel.: +886 2 28094661x3076; fax: +886 2 28085952. E-mail address: [email protected] (Y.-P. Tsao). 1 These authors contributed equally to this work. 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.01.041

RPE cells and are associated with significant mitochondrial (Ballinger et al., 1999) and genomic (Verna et al., 2000) DNA damage. It is conceivable that RPE cells may possess selfprotective mechanisms to survive oxidative damage. The overloading of these mechanisms may lead to RPE cell death and AMD. Pigment epithelium-derived factor (PEDF) was first identified from the conditioned medium of fetal human RPE cell cultures (Tombran-Tink and Johnson, 1989; Tombran-Tink et al., 1991) and is expressed throughout life (Tombran-Tink et al., 1996). Interestingly, PEDF was shown to protect cells such as pericytes, endothelial cells and neuronal cells against oxidative damage (Cao et al., 1999; Yamagishi et al., 2004, 2005; Amano et al., 2005). However, whether PEDF also exerts a protective effect on RPE cells has not been documented yet. Several different mechanisms have been reported to play a role in the protective effect of PEDF in different cell systems. For instance, PEDF increases glutathione peroxidase expression which in turn protects cultured retinal pericytes against high

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glucose- or H2O2-induced cell death (Amano et al., 2005). PEDF also suppresses NADPH oxidase activity in endothelial cells (Yamagishi et al., 2004, 2005). Moreover, the mechanism of PEDF-mediated neuroprotection has been reported to depend on an activation of the cyclic AMP-responsive element binding protein (CREB) and the subsequent induction of anti-apoptotic genes (Yabe et al., 2001, 2005). CREB activation occurs via phosphorylation at serine 133 (Ser133) by various kinases, including mitogen-activated protein kinases (MAPKs) (Shaywitz and Greenberg, 1999; Mayr and Montminy, 2001). ERK phosphorylates CREB through the p90rsk (Xing et al., 1996; Bonni et al., 1999). Furthermore, the ERK1/2 pathway generates survival signals during harmful conditions in several cell systems (Xia et al., 1995; Guyton et al., 1996; Hecquet et al., 2003). Whether PEDF can activate any of these signaling mechanisms in RPE cells is unknown. Since RPE is frequently exposed to high oxidative stress, we sought to document the possible autocrine anti-oxidative effect of PEDF on RPE. We utilized a human RPE cell line, known as ARPE-19, in our studies. This is a nontransformed cell line with properties similar to those of primary human RPE cells (Dunn et al., 1996). We are also interested in developing the ARPE-19 cell line into a model system to address the signaling pathway activated after PEDF exposure. Materials and methods Materials Hydrogen peroxide and other pharmacological and general biochemical reagents were purchased from Sigma (St. Louis, MO). PD98059 was purchased from Calbiochem (San Diego, CA).

Fig. 2. Effect of H2O2 treatment on the viability of ARPE-19 cells. (A) The cells were incubated with different H2O2 concentrations as indicated for 2h. After treatment, cells were incubated in 10% FBS-DMEM and allowed to recover for 16h. The viability was determined by the MTT assay. Four independent assays were performed, and data are represented as the mean ± S.D. (n = 4). (B) Phasecontrast photomicrographs of ARPE-19 cells with H2O2 treatment for 2h (left panel) or after a further 16h of recovery in 10% FBS-DMEM (right panel). Original magnification ×100.

fetal bovine serum (FBS), 50units/ml penicillin/streptomycin and 2.5 mM glutamine at 37°C incubator with 5% CO2.

Cell culture

Expression and purification of recombinant PEDF

The human RPE cell line ARPE-19 obtained from American Type Culture Collections (Manassas, VA) was cultured in Dulbecco's modified essential medium supplemented with 10%

In this study, full length PEDF cDNA was cloned from total RNA of hepatoma SK-Hep-1 by reverse transcriptase-PCR. The cloning of the N-terminal truncated PEDF form (Asp44– Pro418) was as previously described (Becerra et al., 1993) except that the DNA sequence was inserted into the pET-15b expression vector (Novagen, Madison, WI). The PEDF cDNA inserted in the vector obtained a 6 × histidine (His) tag at the 5′ end. Protein extraction was performed using BugBuster Extraction Reagent (Novagen, Madison, WI) according to the manufacturer's instructions. The inclusion body was resolved by 4M urea at room temperature for 2h as described (Becerra et al., 1993) and then purified by His Bind Kits (Novagen, Madison, WI) according to the manufacturer's instructions. PEDF was dissolved in the elusion buffer and kept at − 30 °C. •

Fig. 1. Protein analysis of the different cellular fractions of E. coli expressing recombinant PEDF protein. Bacterial cell lysates were resolved using 10% SDSPAGE and stained with Coomassie blue; (1) protein markers; (2) extract from nontransformed BL21-CodonPlus (DE3)-RP; (3, 4) transformants before and after IPTG induction, respectively; (5) soluble cellular fraction; (6) insoluble cellular fraction; (7) insoluble fraction dissolved in 4M urea; (8) N-terminaltruncated-PEDF (42.8kDa) eluted from an Ni-NTA-agarose column.

Cell viability ARPE-19 cells were seeded in 96-well microculture plates (12,000 cells/well) for 24h, then the culture medium was replaced by 0.5% FBS-DMEM for 16h prior to H2O2 or PEDF treatment, followed by a 16 h recovery period in 10%

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media. The cell viability was determined by the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described (Garg and Chang, 2003). The results from MTT assay were also confirmed by trypan blue exclusion assay as previously described (Strunnikova et al., 2004). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay ARPE-19 cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with 1 × TBS, permeabilized with 0.1% Triton X-100 for 30min. Fixed monolayer cell cultures were stained with TUNEL (In Situ Cell Death Detection kit, FITC; Roche Molecular Biochemicals, Indianapolis, IN, USA). Cells were counterstained with the DNA specific dye DAPI. TUNEL-positive nuclei were expressed as a percent of the total number of cells per individual field. Western blot analysis Cells were cultured in 6-well plates. The sample preparation, protein concentration determination, electrophoretic analysis and Western blotting were performed as previously described (Garg and Chang, 2003). Statistical analysis Data are expressed as the mean ± S.D. The Mann–Whitney U test was used to determine the statistically significant differences and P b 0.05 was considered significant. Results Cloning and expression of PEDF recombinant protein For the establishment of an in vitro model to study the potential effects of PEDF in RPE cellular anti-oxidative mechanisms, the complete coding sequence of PEDF was

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cloned and the protein was expressed in E. coli. The PEDF is an N-terminal truncated PEDF form (Asp44–Pro418) as previously described (Becerra et al., 1993). This form was used owing to its higher expression in E. coli than the full length form (Met1– Pro418). The steps of protein purification are shown in Fig. 1; most of the PEDF existed in inclusion body of E. coli after IPTG induction (lane 6) but it was dissolved in 4M urea (lane 7). Importantly, high purity PEDF was obtained after nickel column purification (lane 8) and the concentration was 1.2mg/ml. PEDF attenuates H2O2-induced cytotoxicity in human retinal pigment epithelial cells We first tested the cytotoxic effect of H2O2 on ARPE-19 cells. As shown in Fig. 2A, the exposure of cells for 2 h, at various concentrations of H2O2, revealed that a concentration at 0.2mM did not affect cell viability. Higher concentrations (0.8∼1.2 mM) caused a dramatic decrease of viability in ARPE19 cells. At 1 mM, H2O2 lowered the viability to 32% of controls. The results indicated that treatment of cells with H2O2 for 2h caused a dose-dependent decrease of viability with an LD50 of ∼0.87mM. When cell morphology was observed, no obvious changes were detected immediately after the 2 h of H2O2 treatment (Fig. 2B). However, a change in the cell integrity and the detachment of cells was obvious at 16 h after H2O2 treatment (Fig. 2C). Many nuclei formed highly condensed masses and became surrounded by cell debris (Fig. 2C). Next, the protective effect of PEDF against H2O2 cytotoxicity was investigated by the MTT method. Since treating the cells with PEDF and H2O2 simultaneously did not provide protection against H2O2-induced cell death (Fig. 3A, time 0min), we tested whether PEDF preconditioning was required. Treating cells with PEDF (50 ng/ml) for various periods of time prior to exposure to H2O2 at 1 mM revealed that protection was evident after 30 min of PEDF preconditioning. PEDF protection was significantly more effective after 2h of preconditioning

Fig. 3. PEDF protection against H2O2-induced ARPE-19 cell death is dose- and time-dependent. (A) Time-dependent protective effect of PEDF on ARPE-19 cells against H2O2-induced cell death. Cell treatments and MTT assay for estimation of cell viability are described in “Materials and methods”. Values are expressed as percent control value. Variations shown represent S.D. from three independent experiments (n = 4 dishes). **P b 0.02 compared with the H2O2-treated cells; #P b 0.005 vs. 60min PEDF-preconditioned cells. (B) Dose-dependent protective effect of PEDF on ARPE-19 cells against H2O2-induced cell death. *P b 0.05 vs. H2O2 only.

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Fig. 4. The effect of H2O2 on cell death. Nuclear condensation and fragmentation induced by H2O2 in ARPE-19 cells were visualized by DAPI/TUNEL staining. (A) Serum free control, (B) 1mM H2O2 treatment for 2h, (C, D) cells were preconditioned with solvent or with 50 ng/ml PEDF before the H2O2 treatment. (E) Results of TUNEL assay are presented as percentage of serum free-untreated cell counts (mean ± S.D.) on seven randomly selected regions. ⁎P b 0.05 vs. untreated samples.

when compared to only 1 h of preconditioning (Fig. 3A, P b 0.005). To study whether the concentration of PEDF affects its protective ability, cells were exposed to PEDF at various concentrations from 10 to 200ng/ml for 2 h and then treated with 1 mM H2O2 for 2 h. The results revealed that PEDF concentrations higher than 25ng/ml were clearly cytoprotective (Fig. 3B, P b 0.05 vs. H2O2 only). It seems that the protection provided by PEDF for ARPE-19 cells is both time- and dosedependent. Since H2O2 is reported to induce apoptosis (Garg and Chang, 2003), DNA fragmentation and nuclear condensation were visualized by TUNEL staining and the nuclear morphology was shown by DAPI counterstaining. As shown in Fig. 4B, after 16 h there was an observable increase in TUNEL-positive cells in the samples treated with 1 mM H2O2 but not in the untreated cells (Fig. 4A). In addition, DAPI staining revealed that nuclear shrinkage and DNA condensation occurred in a large portion of H2O2-treated cells (Fig. 4B). The preconditioning with a solvent did not alter the frequency of nuclear changes (Fig. 4C). On the other hand, preconditioning with 50 ng/ml of PEDF reduced the fraction of TUNEL-positive cells and cells with nuclear condensation (Fig. 4D). As shown in Fig. 4E, the TUNEL assay also reflected the statistically significant increase TUNEL-positive cells after exposure to

H2O2 (68 ± 5%). Cells preconditioned with PEDF, prior to the challenge with H2O2, showed reduced TUNEL-positive staining (∼22 ± 8%). However, the result of the TUNEL assay on cells pretreated with the PEDF solvent was similar to the result in H2O2-treated cells (66 ± 5%). Serum free-untreated samples showed only 2% TUNEL-positive cells (Fig. 4E). This indicates that PEDF may protect ARPE-19 cells by preventing apoptosis. PEDF induces Erk1/2 phosphorylation We further tested whether PEDF induces a survival signal while protecting RPE against oxidative damage. The ERKmediated survival signal has been suggested to protect ARPE19 (Garg and Chang, 2003). Therefore, we studied the ERK1/2 activation by examining phosphorylated ERK1/2 (p-ERK1/2) levels by Western blot analysis. In such studies, cells treated with PEDF (50 ng/ml) revealed that the levels of p-ERK1/2 was gradually increased by 3 to 4 fold when compared to the results of untreated cells (Fig. 5A, compare 120min with 0min). The most prominent p-ERK1/2 level appeared in cells treated with PEDF for 2 h. Control experiments revealed that the basal level of ERK1/2 was not affected by the solvent (data not shown).

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p-ERK1 p-ERK2

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Fig. 5. PEDF stimulates phosphorylation of ERK1/2 in ARPE-19 cells. Cells were treated with 50 ng/ml PEDF. Whole cell extracts (20 μg) were examined by Western blot using antibodies against p-ERK1/2 (44/42kDa) (A) or analyzed by anti-ERK1 antibody (B).

ERK activation mediates the PEDF protective effect Since we observed the induction of ERK phosphorylation by PEDF, while ARPE-19 cells were protected, we were interested in the potential protective role of ERK. In this study, we employed PD98059, a specific inhibitor of MEK, to block ERK1/2 activation. We wanted to determine whether ERK1/2 phosphorylation was important for PEDF protection against H2O2-mediated cytotoxicity. ARPE-19 cells were pretreated with 20 μM PD98059 in the presence or absence of PEDF followed by a H2O2 challenge. The 20μM PD98059 concentration was chosen because it can completely inhibit PEDFinduced ERK1/2 phosphorylation as was revealed by Western blot analysis (data not shown). The MTT assay also showed that exposure to 20 μM PD98059 alone for 4 h was not cytotoxic to ARPE-19 cells (data not shown). As shown in Fig. 6, PD98059 only slightly enhanced the cell death induced by exposure to 1 mM of H2O2 (column 3 compared to column 2). Importantly, PD98059 significantly decreased the PEDF cytoprotection effect (P b 0.05; column 5 compared to column 4). Our results are consistent with the notion that ERK activation has a protective function in the rescuing of RPE cells in an oxidative environment. Also, PEDF can provide protection by stimulating ERK activation. Discussion

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Here we demonstrated that PEDF can protect ARPE-19 cells from H2O2-induced cell death. Because PEDF is expressed and secreted by RPE cells, physiologically this may represent an autocrine protection mechanism that counteracts damage from oxidative stress. Although PEDF has been shown to protect pericytes, endothelial cells and neuronal cells (Cao et al., 1999; Yamagishi et al., 2004, 2005; Amano et al., 2005), our report is the first to show its protection on RPE cells. H2O2 is generated in the RPE during the phagocytosis of the oxidized outer segment of photoreceptors in the physiological process of photoreceptor renewal (Ramachandran et al., 1991; Miceli et al., 1994; Ballinger et al., 1999). Since the real concentration of PEDF in RPE layer is difficult to assess, whether the level of PEDF in RPE is adequate to protect RPE is difficult to prove. However, the concentration of PEDF used in this study

was lower than its physiological concentration (5 μg/ml) in blood (Petersen et al., 2003; Amano et al., 2005), supporting the possibility of protection under physiological conditions. In this study, an N-terminal truncated PEDF was used. A previous report indicated the full function of the full length PEDF (Becerra et al., 1993). It also induces transcription factor NF-κB activation (Yabe et al., 2001) and displays neuroprotective functions (Yabe et al., 2001) which is preserved in the N-terminal of the truncated form. In this study, while trying to identify the mechanism relative to PEDF protection, we found that ERK1/2 phosphorylation is induced by PEDF. Further, the prevention of ERK1/2 phosphorylation reduced the protective effect. These findings strongly suggest that p-ERK1/2 plays a role in the protection of ARPE-19 cells. Previously, PEDF was shown to activate transcription factor NF-κB resulting in further induction of certain pro-survival genes in neuronal cells (Yabe et al., 2001, 2005). PEDF also induces the phosphorylation of CREB that results in CREB activation, and an anti-apoptotic effect was evident (Yabe et al., 2005). Since CREB was found recently to be the target of p-ERK1/2 (Xing et al., 1996; Bonni et al., 1999), it is plausible that PEDF may activate ERK1/2 and then phosphorylate CREB to exert its protection. The protection provided by PEDF is time-dependent in our study (Fig. 3A). Up to 120min of preconditioning is required. This supports a model that a signal cascade may be activated and gene expression may be involved. Further study is required to completely elucidate the mechanism of PEDF protection. In our observation, the protective effect of PEDF against H2O2-induced cytotoxicity was prominent; however, it was not completely inhibited by the addition of the MEK kinase inhibitor PD98059 (Fig. 6). This suggested that mechanisms other than ERK1/2 activation are involved in the protective effect of PEDF. To rule out the possibility of incomplete inhibition of ERK1/2 activation, Western blot analysis was

MTT (% control)

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Fig. 6. Influence of PEDF-mediated cytoprotection with or without the MEK inhibitor PD98059 (PD). ARPE-19 cells were pretreated with either 50ng/ml PEDF or 20μM PD, or also pretreated with PEDF and PD as indicated for 2h prior to the H2O2 challenge for 2h. *P b 0.05 vs. PEDF + H2O2.

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performed to demonstrate complete abrogation of ERK1/2 phosphorylation by the 20μM PD98059 that was used in this study (data not shown). Thus, the partial abrogation of PEDFmediated protection by PD98059 suggests that other mechanisms are involved in PEDF-mediated protection. Acknowledgements The authors thank Hong-Kong Chen, Ju-Yun Wu and I-Pin Choung for excellent technical support. This study was supported by grants from National Science Council, Taiwan (NSC 94-3112-B-195-001, NSC 94-2314-B-195-002) and Mackay Memorial Hospital (MMH-E-94006, MMH-9501). References Amano, S., Yamagishi, S., Inagaki, Y., Nakamura, K., Takeuchi, M., Inoue, H., Imaizum, T., 2005. Pigment epithelium-derived factor inhibits oxidative stress-induced apoptosis and dysfunction of cultured retinal pericytes. Microvascular Research 69 (1–2), 45–55. Barak, A., Morse, L.S., Goldkorn, T., 2001. Ceramide: a potential mediator of apoptosis in human retinal pigment epithelial cells. Investigative Ophthalmology and Visual Science 42 (1), 247–254. Ballinger, S.W., Van Houten, B., Jin, G.F., Conklin, C.A., Godley, B.F., 1999. Hydrogen peroxide causes significant mitochondrial DNA damage in human RPE cells. Experimental Eye Research 68 (6), 765–772. Becerra, S.P., Palmer, I., Kumar, A., Steele, F., Shiloach, J., Notario, V., Chader, G.J., 1993. Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli. A functionally active neurotrophic factor. Journal of Biological Chemistry 268 (31), 23148–23156. Bonni, A., Brunet, A., West, A.E., Datta, S.R., Takasu, M.A., Greenberg, M.E., 1999. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286 (5443), 1358–1362. Cai, J., Nelson, K.C., Wu, M., Sternberg Jr., P., Jones, D.P., 2000. Oxidative damage and protection of the RPE. Progress in Retinal and Eye Research 19 (2), 205–221. Cao, W., Tombran-Tink, J., Chen, W., Mrazek, D., Elias, R., McGinnis, J.F., 1999. Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death. Journal of Neuroscience Research 57 (6), 789–800. Dunn, K.C., Aotaki-Keen, A.E., Putkey, F.R., Hjelmeland, L.M., 1996. ARPE19, a human retinal pigment epithelial cell line with differentiated properties. Experimental Eye Research 62 (2), 155–169. Garg, T.K., Chang, J.Y., 2003. Oxidative stress causes ERK phosphorylation and cell death in cultured retinal pigment epithelium: prevention of cell death by AG126 and 15-deoxy-delta 12, 14-PGJ2. BMC Ophthalmology 3, 5, doi:10.1186/1471-2415-3-5. Guyton, K.Z., Liu, Y., Gorospe, M., Xu, Q., Holbrook, N.J., 1996. Activation of mitogen-activated protein kinase by H2O2. Journal of Biological Chemistry 27 (8), 4138–4142. Hecquet, C., Lefevre, G., Valtink, M., Engelmann, K., Mascarelli, F., 2003. Activation and role of MAP kinase-dependent pathways in retinal pigment epithelium cells: JNK1, P38 kinase, and cell death. Investigative Ophthalmology and Visual Science 44 (3), 1320–1329. Jin, G.F., Hurst, J.S., Godley, B.F., 2001. Hydrogen peroxide stimulates apoptosis in cultured human retinal pigment epithelial cells. Current Eye Research 22 (1), 165–173. Mayr, B., Montminy, M., 2001. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nature Reviews. Molecular Cell Biology 2 (8), 599–609.

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