Effects of (-)-epigallocatechin gallate on RPE cell migration and adhesion

Materials

Protease inhibitors, BSA (BSA), and fibronectin were purchased from Sigma Chemical Co. (St Louis, MO). Antibodies (Ab) raised against phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phosphor-phosphoinositide 3-kinases (PI3K), PDGF receptor β (PDGFR-β), and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA). An Ab raised against phospho-PDGFR-β at Tyr⁷¹⁶ was from Upstate Biotech Inc. (Lake Placid, NY). Antibodies raised against phospho-c-Jun N-terminal kinase (JNK) were from New England Biolabs, Inc. (Beverly, MA). Antibodies for phospho-p38 were from R&D systems, Inc. (Minneapolis, MN). A secondary antibody of antirabbit-HRP was purchased from Santa Cruz Biotechnology. EGCG was purchased from Sigma Chemical Co.

Cell culture

Adult human retinal pigment epithelial (ARPE19) cells were purchased from Food Industry Research and Development Institute (Hsinchu, Taiwan) and were maintained in DMEM/F12 supplemented with 10% fetal calf serum (GibcoBRL, Invitrogen Life Technologies, Carlsbad, CA), 100 units/ml penicillin, and 100 mg/ml streptomycin (Sigma Chemical Co.). The cells were cultured in a humidified incubator at 37 °C and 5% CO₂. For most of the experiments, cells reaching a 90%–95% of confluence were starved and synchronized in serum-free DMEM for 24 h before being subjected to further analysis.

Electric cell-substrate impedance sensing migration assays

Eight-well array culture ware (ECIS 8W1E) consisting of one active electrode (250 μm diameter) and one large area counter electrode (100 μm²) per well were purchased from Applied Biophysics (Troy, NY). To study cell behavior with this instrument, cells are grown in culture wells containing gold film surface electrodes, with ordinary culture media serving as the electrolyte. In its normal mode, an approximately constant current source applies an AC signal of 1 μA, usually at 4 kHz, between a small measuring electrode (250 μm diameter) and a large counter electrode. The instrument monitors both the voltage across the electrodes and its phase relative to the applied current. In addition to reporting the total impedance, these data are converted to resistance and capacitance. As the cells attach and spread on the small electrode, their membranes constrict the current and force it to flow beneath and between the cells, resulting in large increases in impedance. The microampere current and the resulting voltage drop of a few millivolts have no measurable effect on the cells and, hence, the monitoring of cell behavior is noninvasive.

The electrode array was placed in an incubator and a medium (200 μl/well) was added to cover the electrodes. The ARPE19 cells were then inoculated at a concentration of 70,000 cells/well in the arrays and incubated for 24 h, while attachment and spreading were followed by means of impedance measurements. The experiments were conducted on wells where the impedance had achieved a steady-state. Then the wells were submitted to an elevated voltage pulse of 40 kHz frequency, 4 V amplitude, and 10 s duration, which led to death and detachment of the cells present on the small active electrode. The medium were then changed to a serum-free cell culture medium with or without PDGF-BB (20 ng/ml) and 10 μM of EGCG. The cells surrounding the small active electrode that have not been subjected to the elevated voltage pulse then migrated inward to replace the killed cells, and the migration was assessed by continuous impedance measurements for 25 h.

Transwell migration assay

Migration assays with RPE cells were performed using a modified Boyden chamber model (Transwell apparatus; 8.0 μm pore size; Costar,, Cambridge, MA) [36]. The lower face of the polycarbonate filter (Transwell insert) was coated with 0.3 mg of fibronectin for 30 min in the laminar flow hood. The Transwell insert was placed back onto the 24-well plate and the lower chamber was filled with 0.6 ml of DMEM/F12 serum-free medium, with or without 20 ng/ml of PDGF-BB, and 1, 3, and 10 µM of EGCG. The EGCG was dissolved in phosphate buffered saline (PBS). Human RPE cells (5×10⁴ cells/well) in 200 µl medium were plated to the upper chamber. After 5 h of incubation at 37 °C, all non-migrated cells were removed from the upper face of the Transwell membrane with a cotton swab, and migrated cells were fixed and stained with 0.5% toluidine blue in 4% permanganate aldehyde-fuchsin (PAF). Migration was quantified by counting the number of stained cells per 100× field (high power field, HPF) with a phase-contrast microscope (DMIL^® ; Leica, Wetzlar, Germany), and 20 HPFs were photographed and counted in each migration.

Cell adhesion assay

Ninety-six-well plates (Costar) were coated with 50 μl of fibronectin (15 μg/ml) in PBS at 4 °C overnight. After a brief wash with PBS, the plates were blocked with 10% BSA at 37 °C for 1 h. After trypsinization, suspended RPE cells were labeled with BCECF/AM (10 μg/ml) for 30 min at 37 °C. Until it is cleaved by the intracellular esterase to become green fluorescent BCECF, BCECF/AM is intrinsically nonfluorescent. The labeled cells were washed and resuspended in DMEM to a density of 1×10⁵ cells/ml. The suspended cells (95 μl) were incubated with 5 μl of PBS or various concentrations of EGCG for 1 h at 37 °C. After washing twice with PBS and after the addition of a radioimmunoprecipitationassay buffer, the non-adherent cells were removed by aspiration and the 96-well plates were subjected to measurement with a Wallac Victor 3 1420 multilabel counter (Perkin Elmer, Turku, Finland) using excitation and emission wavelengths of 485 and 535 nm, respectively. Eight plates were quantified for each assay.

Dot binding assay

A nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) was soaked in a buffer (25 mM Tris, 192 mM glycine and 20% methanol) for 30 s. Recombinant PDGF-BB (2 μg/ml in 50 μl) was applied to the membrane with a Bio-Dot microfiltration apparatus (Bio-Rad Laboratories, Hercules, CA) by suction. 2.5 μl of PBS, 3 mM and 10 mM of EGCG, and 5 mM of lycopene were directly spotted on the same membrane. The membrane was then blocked with BSA (5% in PBS) for 0.5 h. After washing with PBS, the membrane was incubated with PDGF-BB (0.5 μg/ml) in PBS for 1 h at room temperature (RT). A brief wash was followed, and the membrane was then incubated with anti-PDGF-BB Ab (2 μg/ml in 1% BSA-containing PBS) for 1 h at RT. After another brief wash, the membrane was incubated with horseradish peroxidase-conjugated Ab before being developed by enhanced chemiluminescence (ECL; NEN, Boston, MA).

Immunofluorescence microscopy

The procedure for staining actin cytoskeleton has previously been described [37]. Briefly, the trypsinized RPE cells were suspended at 37 °C for 30 min, and then pretreated with or without EGCG for an additional 1 h. The cells were then allowed to adhere on glass coverslips that were precoated with fibronectin (15 μg/ml) for 1 h. Afterwards, they were washed, fixed with 1% PAF for 20 min, and permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 3% BSA, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated phalloidin (1:200; Sigma). The coverslips were mounted under an Olympus BX51 microscope and the immunofluorescence images were taken using a Spot RT Camera System (Diagnostics Instruments, Inc., Sterling Height, MI).

Cell lysate preparation and western blot analysis

Platelet-derived growth factor-BB (10 ng/ml) was preincubated with a vehicle or EGCG for 30 min and was then added to human RPE cells. The effect of EGCG on PDGFR-β, PI3K, and MAPKs phosphorylation was analyzed by western blotting. Retinal pigment epithelial cells were washed with prechilled PBS and lysed in a radioimmunoprecipitation assay buffer (20 mM Tris–HCl, pH 7.4, 137 mM NaCl, 2 mM EDTA. One mM sodium fluoride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 1 mM sodium orthovanadate, 1 mM PMSF, and 1 μg/ml aprotinin and leupeptin [freshly prepared]). After sonication, the lysate was centrifuged (14,000× g for 15 min at 4 °C) and the supernatant was transferred to a tube. The protein content was quantified with the Pierce protein assay kit (Pierce, Rockford, IL). Total protein was separated by electrophoresis on 10% SDS polyacrylamide gels and the proteins were electroblotted onto polyvinylidene fluoride (PVDF) membranes and probed using the specific antibodies mentioned. Immunoblots were detected by enhanced chemiluminescence (Chemiluminescence Reagent Plus; NEN, Boston, MA). For some of the experiments, the PVDF membrane was stripped at 60 °C for 30 min with a stripping buffer containing 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM β-mercaptoethanol.

Statistical analysis

Data are expressed as mean±standard error (SE), unless otherwise indicated. Comparison of the means of two groups of data are made using an unpaired, two-tailed Student's t test. We consider p values <0.05 as statistically significant. The data are analyzed using SigmaPlot for Windows Version 10.0.

(-)-Epigallocatechin gallate inhibits platelet-derived growth factor-BB-induced retinal pigment epithelial migration

In the ECIS migration assays, cultured RPE cells were plated in chambers containing gold electrodes, and time-dependent impedance measurements were made (Figure 1). When incubated with PDGF-BB, ARPE19 cell migration was stimulated and the impedance (7933±305 mOhm at 24 h) increased faster and was significantly larger compared to cells without PDGF-BB (6989±491 mOhm at 24 h, p<0.05). (-)-Epigallocatechin gallate inhibited ARPE19 cell migration even in the absence of PDGF-BB (6989±491mOhm without EGCG and without PDGF-BB versus 3600±238mOhm with EGCG at 24 h and without PDGF-BB, n=4, p<0.05). For ARPE 19 cells treated with PDGF-BB, the impedance (7933±305 mOhm at 24 h) was significantly reduced with the addition of 10 μM EGCG (4743±370 mOhm at 24 h, p<0.05), indicating that EGCG inhibits PDGF-BB-induced RPE cell migration.

In the Transwell migration assay, coating with the fibronectin alone was observed to induce only a small amount of ARPE19 cell migration when compared to samples without the coating (data not shown).Without EGCG or PDGF-BB, PBS served as the control. Significant RPE cell migration was observed after PDGF-BB stimulation for 5 h at 37 °C (153±5.6% of control; Figure 2). We found that EGCG suppressed RPE cell migration without PDGF-BB (Figure 2A). Quantitative analysis indicates that in the absence of PDGF-BB, 55% of migration (45±4.2% of control) was inhibited by 10 μM of EGCG (Figure 2C). Moreover, EGCG suppressed PDGF-BB-induced RPE cell migration in a dose-dependent manner (106.4±3.1%, 83.1±3.2% and 55.3±3.7% of control for 1, 3 and 10 μM of EGCG, respectively; Figure 2B). A total of 64% of PDGF-BB-induced RPE cell migration (153±5.6% versus 55.3±3.7% of control, p<0.05) was inhibited by 10 μM of EGCG (Figure 2C). Therefore, the Transwell migration assay demonstrates that EGCG inhibits PDGF-BB-induced RPE cell migration (n=8).

(-)-Epigallocatechin gallate inhibits retinal pigment epithelial cell adhesion to fibronectin

We further studied the inhibitory effects of EGCG on RPE cell adhesion to fibronectin. Figure 3 shows that RPE cell adhesion did not take place without fibronectin and was only activated when fibronectin was added (609.2±86.1% of control). The addition of ECGC significantly inhibited RPE cell adhesion in a dose- dependent manner (426.5±64.1%, 304.8±25.7%, and 187.5±13.6% of control at 1, 3, and 10 μM, respectively, p<0.05, n=5).

(-)-Epigallocatechin gallate does not directly bind to platelet-derived growth factor-BB in dot binding assay

Recombinant human PDGF-BB, EGCG, and lycopene were immobilized on the nitrocellulose (NC) membrane. After incubation with or without PDGF-BB, the membrane was further incubated with antibodies against PDGF-BB and then developed. We observed that immobilized PDGF-BB can be recognized by the anti-PDGF-BB Ab, suggesting the specificity of Ab. The 3 mM and 10 mM of EGCG did not directly bind to PDGF-BB, but a positive binding signal was detected on the lycopene spot incubated with PDGF-BB. The data indicates that EGCG cannot directly bind to PDGF-BB (Figure 4).

(-)-Epigallocatechin gallate changes actin cytoskeleton organization during cell adhesion

We next determined if actin cytoskeleton organization during cell adhesion would be affected by EGCG. Our immunofluorescence microscopy confirmed that fibronectin-adherent RPE cells spread well. Typical long stress fibers running across the cell body were observed (Figure 5A). In the presence of EGCG, the changes in the organization of actin cytoskeleton, indicated by the disappearance of the stress fibers and the generation of protrusions at the cell periphery, were prominent (Figure 5B).

(-)-Epigallocatechin gallate inhibits platelet-derived growth factor-BB-induced platelet-derived growth factor receptor-β, downstream PI3K/Akt and MAPK phosphorylation

It has been reported that PDGF-BB binding to a PDGF receptor (PDGFR) is associated with dimerization, autophosphorylation, and activation of PDGFR-tyrosine kinase activity [38], which subsequently causes RPE cell migration through activation of PI3K/Akt and MAPKs signaling [39]. To determine whether a PDGF-BB-induced signaling pathway is affected by EGCG in human RPE cells, the extent of phosphorylation of PDGFR-β and its downstream components was examined. We observed that stimulation of RPE cells with PDGF-BB results in PDGFR-β phosphorylation, as determined by western blotting with Abs directed against phosphotyrosine and PDGFR-β at Tyr⁷¹⁶ (Figure 6). All concentrations of EGCG at or above 3 μM significantly inhibited PDGF-BB-induced PI3K/Akt, ERK1/2, and p38 phosphorylation in human RPE cells in a concentration-dependent manner. However, JNK phosphorylation was not significantly affected by EGCG.