INTRODUCTION
Age-related macular degeneration (AMD) causes irreversible vision loss in elderly people as a consequence of the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaries (1). Early-stage AMD is characterized by the formation of extracellular deposits, called drusen, between Bruch’s membrane and the RPE and/or abnormalities in the RPE (2). The later stages of AMD are classified into two categories: exudative AMD (wet form) and geographic atrophy (dry form). In exudative AMD, angiogenic factors, such as vascular endothelial growth factor (VEGF), are secreted from retinal and RPE cells, promoting the formation of new vessels from the choroid and thereby inducing subretinal fluid accumulation, hemorrhage, and scarring (3). Geographic atrophy (GA) involves gradual degeneration of RPE and photoreceptor cells within the macular area, resulting in vision loss (4). While AMD pathogenesis remains to be elucidated, retinal oxidative stress and inflammation have been shown to play pathogenic roles in AMD (5, 6).
The Wnt/β-catenin pathway controls diverse developmental processes and homeostasis (7, 8). A key regulator of this pathway is the amount of intracellular β-catenin, a transcription coactivator. In the absence of Wnt ligand, β-catenin is phosphorylated by the destruction complex, consisting of casein kinase 1 (CK1), glycogen synthase kinase-3β(GSK-3β), adenomatous polyposis coli (APC), and the scaffold protein axin, at residues Ser45, Thr41, Ser37, and Ser33 (9). These phosphorylation sites are then recognized by F-box β-transducin repeat-containing protein (β-TrCP), a component of the ubiquitin ligase complex, which results in β-catenin degradation (10, 11). Upon association of Wnt ligands (Wnt1, Wnt3a, and Wnt8) with the receptor Frizzled (Fz) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors, the destruction complex is negatively regulated, leading to the accumulation of intracellular β-catenin (12). Then, β-catenin translocates into the nucleus, where it forms a complex with the T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcription factor family, thereby activating the expression of its target genes, including c-myc, cyclin D1 (CCND1), and matrix metalloproteinase-7 (MMP-7) (13-15).
Aberrant upregulation of the Wnt/β-catenin pathway has been observed in the retina and RPE of AMD animal models (16). Levels of LRP5/6 and intracellular β-catenin are elevated in laser-induced choroidal neovascularization (CNV) models (17, 18). Intravitreal injection of a monoclonal anti-LRP6 antibody (Mab2F1) efficiently suppresses the Wnt/β-catenin pathway, thereby ameliorating neovascularization (17). In addition, ectopic expression of a constitutively active mutant β-catenin (S37A) induce the expression of angiogenic and proinflammatory actors such as vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), and nuclear factor-κB (NF-κB) in human RPE cells and normal rat retinas (16). Furthermore, Wnt signaling promotes epithelial-mesenchymal transition, which impairs RPE barrier function and leads to the degenerative disease of the RPE such as AMD (16, 19). Therefore, attenuating the Wnt/β-catenin pathway could be used as a potential strategy for treating AMD. In this study, we found that Wnt5a inhibited the Wnt/β-catenin pathway by promoting β-catenin phosphorylation/degradation, thereby preventing the pathogenic role of the Wnt/β-catenin pathway in RPE cells.
RESULTS
Wnt5a inhibits the Wnt/β-catenin pathway in human RPE cells
he effect of Wnt5a on the canonical Wnt pathway varies in different cells, tissues, and organisms (23, 24). Thus, we investigated whether Wnt5a could affect the Wnt/β-catenin pathway in human RPE cells. To do this, ARPE-19 and hTERT-RPE-1 cells were transiently transfected with the TOPFlash, a synthetic β-catenin/Tcf-dependent firefly luciferase (FL) reporter, followed by incubation with Wnt3a-conditioned medium (Wnt3a-CM). Wnt3a-CM up-regulated TOPFlash activity in both ARPE-19 and hTERT-RPE-1 cells (Fig. 1A). The addition of Wnt5a-conditioned medium (Wnt5a-CM) decreased the Wnt3a-CM-stimulated β-catenin response transcription (CRT) (Fig. 1A). In contrast, Wnt3a-CM and Wnt5a-CM did not affect the activity of FOPFlash, a negative control reporter with mutated β-catenin/ Tcf binding elements (Fig. 1A). Next, we determined the amount of intracellular β-catenin regulating CRT in the Wnt/β-catenin pathway by western blot analysis with an anti-β-catenin antibody. Treatment of ARPE-19 and hTERT-RPE-1 cells with Wnt5a-CM reduced the cytoplasmic level of β-catenin accumulated as a response to Wnt3a-CM (Fig. 1B). These results suggest that Wnt5a attenuates the Wnt/β-catenin pathway by down-regulating the level of intracellular β-catenin in human RPE cells.
Fig. 1.Wnt5a downregulates the levels of β-catenin in human retinal pigment cells. (A) ARPE-19 and hTERT-RPE-1 cells were cotransfected with TOPFlash and pCMV-RL plasmids and incubated with Wnt3a-CM and/or Wnt5a-CM for 24 h. Luciferase activities were measured at 48 h post transfection. TOPFlash activity was reported as relative light units (RLU) normalized to Renilla luciferase activity. Results were the average of three experiments, and the bars indicate standard deviations. *P < 0.05, compared to the Wnt3aCM treated group. (B) Cytosolic proteins were prepared from ARPE-19 and hTERT-RPE-1 cells treated with Wnt3a-CM and/ or Wnt5a-CM for 24 h and were subjected to western blotting with an anti-β-catenin antibody. Blots were re-probed with an anti-actin antibody as loading control. *P < 0.05 compared to the Wnt3a-CM treated group.
Wnt5a promotes β-catenin phosphorylation and degradation in human RPE cells
To examine whether the reduction of cytoplasmic β-catenin protein by Wnt5a was due to the repression of β-catenin gene expression in human RPE cells, we performed semi-quantitative RT-PCR to determine the expression level of β-catenin mRNA. In contrast to β-catenin protein expression, mRNA expression of β-catenin was unchanged by treatment of Wnt3a-CM and/or Wnt5a-CM (Fig. 2A). Next, we used MG-132 proteasome inhibitor to explore whether Wnt5a-induced β-catenin down-regulation was mediated by the proteasomal degradation pathway. As shown in Fig. 2B, down-regulation of β-catenin in response to Wnt5a-CM was substantially abrogated when ARPE-19 cells were incubated with MG-132, suggesting that Wnt5a inhibits Wnt/β-catenin signaling via proteasome-dependent β-catenin degradation rather than by repressing β-catenin gene expression in human RPE cells. Phosphorylation at the N-terminal Ser33/Ser37 residues plays an essential role in proteasome-dependent degradation (25). Therefore, we determined whether Wnt5a could induce β-catenin Ser33/37/Thr41 phosphorylation by western blot analysis with a phospho-specific β-catenin antibody. In consistent with previous reports (11), incubation of ARPE-19 cells with Wnt3a-CM suppressed β-catenin Ser33/Ser37 phosphorylation (Fig. 2C). Moreover, Wnt5a-CM restored β-catenin Ser33/ Ser37 phosphorylation in the presence of Wnt3a-CM (Fig. 2C).
Fig. 2.Wnt5a inhibits the canonical Wnt pathway by promoting β-catenin phosphorylation and degradation in human retinal pigment epithelial cells. (A) Semi-quantitative RT-PCR of β-catenin and GAPDH using cDNA of total RNA extracted from ARPE-19 cells treated with Wnt3a-CM and/or Wnt5a-CM for 24 h. (B) Cytosolic proteins were prepared from ARPE-19 cells that were incubated with Wnt3a-CM and/or Wnt5a-CM and exposed to MG-132 (20 μM) for 8 h. Proteins were subjected to western blotting with an anti-β-catenin antibody. Protein blot was re-probed with an anti-actin antibody as loading control. (C) Cytosolic proteins were prepared from ARPE-19 cells that were incubated with Wnt3a-CM and/or Wnt5a-CM and then subjected to western blotting with anti-phospho-Ser33/37/Thr41-β-catenin or β-catenin antibody. The same amount of β-catenin was loaded into each lane. In (A-C), the histogram shows the average volume density corrected for the loading control (n = 3) and bars indicate standard deviations group. *P < 0.05 compared to the Wnt3a-CM treated group.
Wnt5a down-regulates the levels of angiogenic/inflammatory factors in human RPE cells
Activation of the Wnt/β-catenin pathway is known to significantly up-regulate the expression of angiogenic and inflammatory factors in human RPE cells (16). Given the fact that Wnt5a suppresses the Wnt/β-catenin pathway, we examined whether Wnt5a could represses the expression of angiogenic and inflammatory factors. In consistent with previous results (16), western blot analysis showed that the levels of VEGF, NF-κB, and TNF-α, which are known pathogenic factors in age-related macular degeneration and diabetic retinopathy, were increased in ARPE-19 and hTERT-RPE-1 cells treated with Wnt3a-CM (Fig. 3A, B). However, Wnt5a-CM suppressed the Wnt3a-induced up-regulation of these factors in ARPE-19 and hTERT-RPE-1 cells (Fig. 3A, B).
Fig. 3.Wnt5a downregulates the levels of angiogenic/inflammatory factors in human RPE cells. Total proteins were prepared from ARPE-19 (A) and hTERT-PRE-1 (B) cells treated with Wnt3a-CM and/or Wnt5a-CM and subjected to western blot analysis with antibody against NF-κB, TNF-α, or VEGF. The blots were re-probed with anti-actin antibody as loading control. The histogram shows the average volume density corrected for the loading control (n = 3) and bars indicate standard deviations group. *P < 0.05 compared to the Wnt3a-CM treated group.
Wnt5a suppresses epithelial-mesenchymal transition in human RPE cells
Epithelial-mesenchymal transition (EMT) is characterized by the loss of epithelial cell-cell contacts with a decrease in the epithelial adhesion molecule E-cadherin (26). EMT impairs RPE structural integrity, leading to retinal degenerative diseases such as AMD (27). As shown in Fig. 4A and B, incubation of ARPE-19 cells with Wnt3a-CM resulted in significant down-regulation of E-cadherin at both protein and mRNA levels. However, the addition of Wnt5a-CM nullified the effect of Wnt3a-CM on E-cadherin expression (Fig. 4A, B). Next, we analyzed ARPE-19 cell migration in response to Wnt3a-CM and/or Wnt5a-CM. Wnt3aCM consistently increased ARPE-19 cell migration. However, co-incubation of Wnt3a-CM and Wnt5a-CM inhibited cell migration (Fig. 4C), indicating that Wnt5a suppresses Wnt/β-catenin-induced EMT in RPE cells.
Fig. 4.Wnt5a suppresses epithelial-mesenchymal transition in human RPE cells. (A) Cytosolic proteins were prepared from ARPE-19 cells and subjected to western blotting with antibody against E-cadherin or β-catenin. The blot was re-probed with anti-actin antibody as loading control. (B) Semi-quantitative RT-PCR for E-cadherin and GAPDH was performed using cDNA of total RNA extracted from ARPE-19 cells. (C) ARPE-19 cells were grown to confluence, scratched using a razor blade, and treated with Wnt3a-CM and/or Wnt5a-CM. Representative images of cell migration at 0 h and 24 h post treatment. (D) Total proteins were prepared from ARPE-19 cells and subjected to western blotting with antibody against Snail. The blot was re-probed with anti-β-actin antibody as loading control. (E) Total proteins were prepared from ARPE-19 cells and subjected to western blotting with anti-phospho-Snail or Snail antibody. The same amount of Snail was loaded into each lane. In (A, B, D and E), the histogram shows the average volume density corrected for the loading control (n = 3) and bars indicate standard deviations group. *P < 0.05 compared to the Wnt3a-CM treated group.
Wnt5a down-regulates Snail protein by promoting Snail phosphorylation
Snail is a zinc finger transcription factor that mediates EMT by binding to E-box motifs located in the E-cadherin promoter region to repress E-cadherin expression (28). To investigate the mechanism underlying Wnt5a-mediated E-cadherin repression, we analyzed Snail protein expression by western blot analysis with an anti-Snail antibody. As shown in Fig. 4D, incubation of ARPE-19 cells with Wnt3a-CM induced accumulation of Snail protein. However, Wnt5a-CM nullified Wnt3a-mediated Snail stabilization (Fig. 4D). Since CK1/GSK-3β-mediated Snail phosphorylation is a prerequisite for β-TrCP-dependent Snail degradation (29), we determined the status of Snail Ser96 phosphorylation after Wnt3a-CM and/or Wnt5a-CM treatment in ARPE-19 cells. When GSK-3β activity was inhibited by Wnt3aCM, Snail Ser96 phosphorylation was decreased (Fig. 4E). The addition of Wnt5a-CM rescued Snail phosphorylation at this residue in the presence of Wnt3a-CM (Fig. 4E). These results indicate that Wnt5a promotes Snail degradation via Ser96 phosphorylation in human RPE cells.
DISCUSSION
The Wnt/β-catenin pathway is known to regulate various biological and pathological processes including AMD. Recently, Mab2F1 that specifically interacts with the extracellular domain of LRP6 has been shown to inhibit the Wnt/β-catenin pathway, thereby attenuating retinal vascular leakage and retinal inflammation in a laser-induced CNV model (17). In the present study, we demonstrated for the first time that Wnt5a, which antagonizes the Wnt/β-catenin pathway, down-regulated the expression of proinflammatory and angiogenic factors and suppressed epithelial-mesenchymal transition (EMT) in human RPE cells.
Several studies have reported that Wnt5a antagonizes the Wnt/β-catenin pathway (23, 24), but its precise mechanism remains debatable. Wnt5a stimulates intracellular Ca2+ release to activate nuclear factor of activated T cells (NFAT), calmodulin-dependent kinase II (CaMKII), and protein kinase C (PKC) (30-32). In Xenopus embryos, XWnt5a induces translocation of XNF-AT and then mediates ventral signals by suppressing the Wnt/β-catenin pathway. The Wnt5a-mediated Ca2+ signaling activates the TAK1-NLK pathway via CaMKII and then active NLK phosphorylates T cell factor (TCF), thereby preventing the TCF/β-catenin complex from binding to DNA. Recently, PKCα has been shown to negatively regulate the Wnt/β-catenin pathway through β-catenin phosphorylation and degradation. However, Topol et al. (33) have reported that Wnt5a induces β-catenin degradation through induction of Siah2, which interacts with adenomatous polyposis coli (APC) and promotes β-catenin ubiquitination. In this study, we found that Wnt5a destabilized β-catenin protein in part by increasing β-catenin phosphorylation at Ser33/Ser37 residues. Further studies are needed to determine the mechanism underlying Wnt5a-mediated β-catenin degradation in RPE cells.
The Wnt/β-catenin pathway has been reported to up-regulate vascular endothelial growth factor (VEGF) gene expression (34). In addition, the Wnt/β-catenin pathway stimulates the generation of reactive oxygen species (ROS), which can accumulate in the form of oxidized macromolecules in RPE cells, leading to retinal degeneration (35). ROS subsequently activates the nuclear factor-κB (NF-κB) pathway, which up-regulates the expression of proinflammatory factors, such as tumor necrosis factor-a (TNF-α), in RPE cells (36). The current study demonstrated that Wnt5a repressed the expression levels of VEGF, NF-κB and TNF-α, which were up-regulated by activation of the Wnt/β-catenin pathway.
Cell adhesion molecule E-cadherin plays important roles in maintaining the epithelial phenotype. Its expression is mainly regulated by transcription factor Snail. Snail binds to E-box consensus sequences in the E-cadherin promoter and repress the expression of E-cadherin at transcriptional level (28). Snail is normally maintained at low levels in cells through CK1/GSK-3β-mediated phosphorylation and subsequent β-TrCP-dependent proteasomal degradation (29). Activation of the Wnt/β-catenin pathway negatively regulated GSK-3β, leading to inhibition of Snail phosphorylation and up-regulation of Snail protein levels (37). In addition, the inflammatory cytokine TNF-α has been shown to stabilize Snail protein through the activation of the NF-κB pathway (38). Our results revealed that Wnt5a reduced the levels of Snail through promoting Snail phosphorylation/ degradation and suppressing the TNFα/NF-κB pathway, thereby increasing the expression of E-cadherin in human RPE cells.
In conclusion, our findings demonstrated that Wnt5a suppressed the pathogenic effects of the Wnt/β-catenin pathway in human RPE cells. Wnt5a promoted β-catenin phosphorylation and degradation, thereby reducing the levels of proinflammatory and angiogenic factors, such as NF-κB, TNF-α, and VEGF. In parallel, Wnt5a induced Snail phosphorylation and degradation, thereby increasing the expression of E-cadherin and inhibiting cell migration. Taken together, our results might provide valuable insights to assist in the development of effective therapeutics for AMD.
MATERIALS AND METHODS
Cell culture
ARPE-19 cells, hTERT-RPE-1 cells, Wnt5a-secreting L cells, and Wnt3a-secreting L cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 120 μg/ml penicillin, and 200 μg/ml streptomycin. HEK293-FL reporter (TOPFlash) and control (FOPFlash) cells were established as described previously (20). Wnt5a- and Wnt3a-conditioned media (Wnt5a-CM and Wnt3aCM) were prepared by culturing Wnt3a-secreting L cells in DMEM with 10% FBS for four days. The media were harvested and sterilized using a 0.22-μm filter. Fresh media was added and cells were cultured for another three days. Culture media was again collected and combined with the previous media.
Plasmids, transfection, and luciferase assay
Reporter plasmid pTOPflash was obtained from Upstate Biotechnology (Lake Placid, NY). Plasmid pCMV-RL was purchased from Promega (Madison, WI). Transfections were performed using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Luciferase assay was performed using Dual Luciferase Assay Kit (Promega).
Western blot analysis
Cytosolic fractions were prepared as described previously (21). Proteins were separated by SDS-PAGE using 4-12% gradient gel (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). These membranes were blocked with 5% nonfat milk and probed with anti-β-catenin (BD Transduction Laboratories), anti-phospho-β-catenin (Cell Signaling), anti-VEGF (Santa Cruz), anti-p65 (Santa Cruz), anti-TNF-α (Santa Cruz), anti-Snail 1 (Santa Cruz), or anti-actin (Cell Signaling) antibody. Membranes were then incubated with horseradish-peroxidaseconjugated anti-mouse IgG (Santa Cruz) or anti-rabbit IgG (Santa Cruz) antibody and visualized using ECL system (Santa Cruz). To determine the levels of phosphorylation, relative amounts of β-catenin and Snail1 protein were quantitated using densitometry (Multi Gauge V2.2). Based on the density of each β-catenin and Snail1 protein band, the same amount of β-catenin and snail was adjusted and loaded into each lane.
RNA extraction and semi-quantitative RT-PCR
Total RNA was isolated using TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions. cDNA synthesis, reverse transcription, and polymerase chain reaction (PCR) were performed as described previously (22). PCR products were separated onto 2% agarose gels and stained with ethidium bromide.
Cell migration assay
ARPE-19 cells grown to confluence on 30-mm cell culture dishes were treated with Wn3a-CM and/or Wnt5a-CM. A scratch was made into the cell layer using a razor blade. After incubating for 24 h, cells were washed with phosphate-buffered saline (PBS) and the scratched areas were photographed.
Statistical analysis
Student’s t-tests were used to compare means between control and experimental groups. All experiments were performed three times. Statistical significance was considered when P value was less than 0.05. Results were presented as the mean ± SD.
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