Squamosamide Derivative FLZ Protects Retinal Pigment Epithelium Cells from Oxidative Stress through Activation of Epidermal Growth Factor Receptor (EGFR)-AKT Signaling

Reactive oxygen species (ROS)-mediated retinal pigment epithelium (RPE) cell apoptosis is attributed to age-related macular degeneration (AMD) pathogenesis. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, Annona glabra, has displayed significant cyto-protective activity. In the current study, we explored the pro-survival effect of FLZ in oxidative stressed-RPE cells and studied the underlying signaling mechanisms. Our results showed that FLZ attenuated hydrogen peroxide (H2O2)-induced viability decrease and apoptosis in the RPE cell line (ARPE-19 cells) and in primary mouse RPE cells. Western blotting results showed that FLZ activated AKT signaling in RPE cells. The AKT-specific inhibitor, MK-2206, the phosphoinositide 3-kinase (PI3K)/AKT pan inhibitor, wortmannin, and AKT1-shRNA (short hairpin RNA) depletion almost abolished FLZ-mediated pro-survival/anti-apoptosis activity. We discovered that epidermal growth factor receptor (EGFR) trans-activation mediated FLZ-induced AKT activation and the pro-survival effect in RPE cells, and the anti-apoptosis effect of FLZ against H2O2 was inhibited by the EGFR inhibitor, PD153035, or by EGFR shRNA-knockdown. In conclusion, FLZ protects RPE cells from oxidative stress through activation of EGFR-AKT signaling, and our results suggest that FLZ might have therapeutic values for AMD.


Introduction
Age-related macular degeneration (AMD) is a progressive retinal degeneration disease, which causes blindness among elderly people [1]. Ultraviolet (UV) exposure and reactive oxygen species (ROS) damage are the main pathological causes of AMD [2,3]. Under oxidative stress, reactive free radicals, including superoxide, hydroxyl radical, singlet oxygen and hydrogen peroxide (H2O2), induce damage to retinal pigment epithelium (RPE) cells by excessively oxidizing key cellular components [2,3]. Anti-oxidants or zinc-containing supplements could reduce AMD progression in human [4,5]. Thus, oxidative stress prevention is an effective strategy to slow down or even reverse AMD progression. Groups including ours have been adding H2O2 to cultured RPE cells to create a cellular model of AMD and to explore the potential interfering strategies [6][7][8][9][10].
In light of this information, we proposed that FLZ might exert a protective effect against oxidative stress in RPE cells. We thus explored the potential role of FLZ on H2O2-treated RPE cells. We identified a new FLZ-mediated pro-survival pathway that attenuated H2O2-induced RPE cell damage and that may minimize the risk of developing AMD.

FLZ Activates AKT in RPE Cells
Next, we explored the mechanisms underlying the pro-survival effect of FLZ by focusing on AKT signaling. FLZ is shown to activate AKT in other cells [11], and AKT signaling is essential for the survival of RPE cells [18]. Western blotting results in Figure 3A,B demonstrated that FLZ induced AKT activation in both time-and dose-dependent manners. AKT activation was seen as early as two hours after FLZ treatment, and it lasted as least for six hours ( Figure 3A). Further, as shown in Figure 3C, AKT was also activated by FLZ in primary RPE cells. Together, these results confirm AKT activation by FLZ in RPE cells.

Figure 3. FLZ activates AKT in RPE cells. APRE-19 cells (A,B) and primary mouse RPE cells (C)
were treated with FLZ for the indicated time, and phospho-(p-) AKT1 (Ser 473), regular AKT1 and β-actin were tested by western blotting. AKT1 phosphorylation was quantified. Experiments were repeated three times to insure consistency of results. "C" stands for the untreated control group. * p < 0.05 vs. Group "C" (ANOVA).

AKT Activation Mediates the FLZ-Induced Pro-Survival Effect against H2O2
Our group has shown that a number of agents, including NGF [6], ginsenoside Rg-1 [21], salvianolic acid A [8] and α-MSH [9], induce the pro-survival effect in RPE cells, which is mediated, at least in part, by AKT activation. The results above have shown that FLZ activated AKT in RPE cells. Next, we explored the role of AKT activation in the pro-survival effect. As shown in Figure 4A,B, in APRE-19 cells, the AKT-specific inhibitor, MK-2206 (MK) [22,23], and the phosphoinositide 3-kinase (PI3K)/AKT pan inhibitor, wortmannin (WT) [24], largely inhibited the pro-survival and anti-apoptosis activities of FLZ against H2O2, indicating that AKT activation is required for FLZ's effect. To further support this hypothesis, we utilized targeted shRNA to selectively knockdown AKT1 in ARPE-19 cells ( Figure 4C), and the results showed that FLZ-induced pro-survival and anti-apoptosis activities against H2O2 were diminished when AKT was depleted by shRNA ( Figure 4D,E). Thus, AKT activation is important for the FLZ-mediated survival effect in RPE cells. ; and cell apoptosis was tested by the TUNEL staining assay (B). The control APRE-19 cells (no shRNA infection) or stable APRE-19 cells infected with scramble-shRNA (sc shRNA) or AKT1-shRNA were treated with H2O2 (400 μM) or plus FLZ (1 μM) for 24 h, cell viability was tested by the MTT assay (D), and apoptosis was tested by the TUNEL staining assay (E); FLZ (1 μM)-induced AKT1 phosphorylation was tested (4 h) and AKT1 phosphorylation and expression (vs. β-actin) were quantified (C). Experiments were repeated three times to ensure the consistency of the results. "C" stands for the untreated control group. Vehicle stands for 0.1% DMSO. * p < 0.05 (ANOVA).

EGFR (Epidermal Growth Factor Receptor) Trans-Activation Mediates FLZ-Induced AKT Activation and Pro-Survival Effect in RPE Cells
Next, we studied the potential upstream signaling for AKT activation by FLZ in RPE cells. The EGF-EGFR signaling network is among one of the best-characterized signaling systems [25]. Besides being activated by its ligands, EGFR could also be activated indirectly by a number of agents, a process termed EGFR "trans-activation" [26][27][28][29][30]. Western blotting results in Figure 5A demonstrated that FLZ induced EGFR Tyr 1068 phosphorylation, the EGFR activation indicator, in cultured ARPE-19 cells, which was blocked by the EGFR inhibitor, PD153035 (PD) [31,32]. Meanwhile, PD153035 prevented FLZ-induced AKT activation ( Figure 5A), indicating that EGFR trans-activation is required for FLZ-induced AKT activation. This is further supported by the fact that EGFR depletion by shRNA dramatically inhibited AKT activation by FLZ in APRE-19 cells ( Figure 5C). H2O2 by itself could also slightly induce phosphorylation of EGFR and AKT in ARPE-19 cells, which was further increased by co-administration of FLZ ( Figure 5B). Significantly, the EGFR inhibitor, PD153035, blocked EGFR-AKT phosphorylation by FLZ plus H2O2 ( Figure 5A,B). These results suggest that trans-activation of EGFR and activation of its downstream signaling, AKT, are early events in H2O2-treated RPE cells (see the related reports [33][34][35]). FLZ increases AKT phosphorylation in H2O2-treated APRE-19 cells through enhancing EGFR activation. In APRE-19 cells, the pro-survival and anti-apoptosis activities of FLZ against H2O2 were alleviated by PD153035 or EGFR shRNA knockdown ( Figure 5D,E). As shown in Figure 5F, in primary RPE cells, FLZ-induced pro-survival activity was similarly inhibited by PD153035 and wortmannin (WT). Together, we concluded that EGFR mediated FLZ-induced AKT activation and pro-survival activity in RPE cells ( Figure 6).

Discussion
The vision loss among elderly AMD patients starts from abnormalities in the RPE, along with photoreceptor damage, Bruch's membrane thickening and choriocapillary hypo-perfusion [36]. These are considered as the main characteristics of AMD [37]. UV radiation and subsequent oxidative stress damage are known as the main contributors to AMD [38][39][40][41][42][43]. In the current study, we showed that FLZ activated EGFR-AKT signaling and attenuated H2O2-induced RPE cell death and apoptosis. These effects by FLZ were inhibited by AKT/EGFR inhibition or depletion. Thus, we suggest that FLZ prevents H2O2-induced RPE cell damage through activating EGFR-AKT signaling ( Figure 6).
EGFR is functionally expressed in RPE cells [44][45][46]. EGFR and their ligands modulate cellular functions in a variety of ways, including proliferation, survival, adhesion, migration and apoptosis resistance [47]. The EGFR ligands, including EGF, amphiregulin and transforming growth factor alpha (TGFα), as well as and heparin binding EGF (HB-EGF) and epiregulin directly bind to and activate EGFR [47]. Activated EGFR recruits adaptor proteins to activate downstream signaling molecules (i.e., AKT signaling) [48] and to promote cell survival [47]. EGFR could also be trans-activated indirectly by various stimuli [26][27][28][29][30]. In the current study, we found that FLZ-induced AKT activation was also mediated through EGFR trans-activation. The conclusion was supported by the fact that EGFR inhibitor or silencing inhibited AKT activation by FLZ. Meanwhile, the pro-survival activity of FLZ against H2O2 was also attenuated by EGFR inhibitor or depletion. Thus, EGFR trans-activation is the upstream signal for FLZ-induced AKT activation, which promotes RPE cell survival ( Figure 6).

Chemical and Reagents
FLZ is a white powder with 99% purity and is insoluble in water [16]. FLZ was synthesized by Wuxi Ya-tai Bio Company (Wuxi, China) based on the structure described in [16]. FLZ was dissolved in dimethyl sulfoxide (DMSO) and added to the cell culture medium with a maximum DMSO concentration of 0.1%. PD153035, MK-2206 and wortmannin were purchased from Calbiochem (Darmstadt, Germany). The antibody against β-actin was purchased from Sigma (St. Louis, MO, USA). All other antibodies used in this study were obtained from Cell Signaling Tech (Danvers, MA, USA).

Primary Mouse RPE Cell Isolation and Culture
As reported [7,9], C57/B6 mice at the age of 3-5 days were given anesthesia by 75% alcohol, and the eyeballs in asepsis were taken out and diluted several times with D-hank's fluid. After soaking in the DMEM/F-12 for 6 h, the eyeballs were taken out, and the retinas were striped carefully. Parenzyme (0.125%) was added to digest for 20 min at 37 °C before adding culture medium containing blood serum to terminate digestion. Then, the supernatant was centrifuged twice at 1000 r/min in the culture medium (80% DMEM/F-12, 20% FBS) to produce a cell suspension after inoculation into the 75-cm 2 culture flask. Cells were divided and were used for the designed experiments. For all experiments, RPE cells (primary and ARPE-19 cells) were serum-starved overnight using serum-free DMEM medium, and the next day, FLZ and inhibitors were added to the cells.

Cell Viability Assay
Cell viability was assessed by the 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) (Sigma, Shanghai, China) assay. In brief, RPE cells were collected and seeded in 96-well plates at a density of 1 × 10 5 cells/well in 200 mL of culture medium. After treatment, 20 μL of MTT solution (5 mg/mL) were added to each well for 4 h at 37 °C, and cell viability was determined by measuring absorbance at 490 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The OD value was detected as an indicator of RPE cell viability.

Annexin-V/PI FACS (Fluorescence-Activated Cell Sorting) Assay
RPE cell apoptosis was measured by Annexin-V fluorescence-activated cell sorting (FACS) according to the manufacturer's protocol (Sigma). Briefly, after treatment, cells were washed twice with cold PBS (phosphate buffer solution) and incubated in 300 μL binding buffer containing 3 μL of Annexin-V-FITC (fluorescein isothiocyanate) and 3 μL of propidium iodine (PI) in the dark for 15 min at room temperature. The stained samples (containing 200,000 cell/sample) were then analyzed on a FACSCalibur flow cytometer within 1 h following the manufacturer's protocol (Coulter, Hialeah, FL, USA). Annexin-V percentage was recorded as an indicator of apoptosis intensity; while Annexin-V −/− and PI +/+ cells were labeled as necrotic cells. All experiments were performed in triplicate.

TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) Staining
RPE cell apoptosis was detected by the TUNEL. In Situ Cell Death Detection Kit (Roche Molecular Biochemicals, Indianapolis, IN, USA), according to the manufacturer's instructions. RPE cells were also stained with 4',6'-diamino-2-phenylin-dole (DAPI, blue fluorescence; Molecular Probes) to visualize the cell nuclei. The apoptosis rate was determined by TUNEL percentage, which was calculated by the number of TUNEL-positive cells divided by the number of DAPI-stained cells. At least 1000 total cells in 10 views from 10 repeat wells (1 × 100) of each condition were included for counting the TUNEL percentage.

Transit Knockdown of Epidermal Growth Factor Receptor (EGFR) by shRNA
The lentiviral particles containing scramble shRNA (sc-108080) or human EGFR shRNA (sc-29301-V) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). Lentiviral shRNA particles (20 μL/mL) were added to the ARPE-19 cells for 24 h, and the expression level of EGFR and the loading control in infected cells were tested.

Statistical Analysis
All data were presented as the mean ± standard deviation (SD). Statistics were analyzed by one-way ANOVA followed by a Scheffe's f-test by using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Significance was chosen as p < 0.05.

Conclusions
FLZ prevents H2O2-induced RPE cell apoptosis through activating EGFR-AKT signaling. Since AMD is characterized by a progressive decay of RPE cells at the posterior pole of the eye and ROS are the major contributors of RPE damages in AMD, our results suggest that FLZ might have therapeutic value for AMD.