Wnt signaling induces epithelial-mesenchymal transition with proliferation in ARPE-19 cells upon loss of contact inhibition - PubMed (original) (raw)
Wnt signaling induces epithelial-mesenchymal transition with proliferation in ARPE-19 cells upon loss of contact inhibition
Hung-Chi Chen et al. Lab Invest. 2012 May.
Abstract
Proliferation and epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) are hallmarks of proliferative vitreoretinopathy. This study aims at clarifying the role of growth factors, such as epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), and transforming growth factor-β1 (TGF-β1), in controlling how RPE proliferates while undergoing EMT. When contact inhibition of post-confluent ARPE-19 cells was disrupted by EGTA, an increase of BrdU labeling was noted only in the presence of EGF and/or FGF-2, and was accompanied by EMT as evidenced by the loss of a normal RPE phenotype (altered cytolocalization of RPE65, N-cadherin, ZO-1, and Na,K-ATPase) and the gain of a mesenchymal phenotype (increased expression of vimentin, S100A4, and α-smooth muscle actin). EMT with proliferation by EGTA+EGF+FGF-2 was accompanied by activation of canonical Wnt signaling (judged by the TCF/LEF promoter activity, increased nuclear levels of and interaction between β-catenin and LEF1 proteins, and the replication by overexpression of β-catenin), abolished by concomitant addition of XAV939, a Wnt inhibitor, but not associated with suppression of Hippo signaling (negative expression of nuclear TAZ or YAP and cytoplasmic p-TAZ or p-YAP). The causative role of Wnt signaling on EMT with proliferation was confirmed by overexpression of stable S33Y β-catenin with EGTA treatment. In addition, contact inhibition disrupted by EGTA in the presence of TGF-β1 also led to EMT, but suppressed proliferation and Wnt signaling. The Wnt signaling triggered by EGF+FGF-2 was sufficient and synergized with TGF-β1 in activating the Smad/ZEB1/2 signaling responsible for EMT. These findings establish a framework for further dissecting how RPE might partake in a number of proliferative vitreoretinopathies characterized by EMT.
Conflict of interest statement
Duality of Interests: None declared.
Figures
Figure 1
Maturation of cell junctions coincides with contact-inhibition in post-confluent ARPE-19 cells. (a) Proliferation assessed by BrdU labeling was still positive on day 4 post-confluence, but became abruptly negative from day 7 post-confluence (* P <0.05). (b) Immunostaining of adherent junction components such as N-cadherin, α-catenin, β-catenin, and, p120 catenin and tight junction component such as ZO-1 was performed in cells at 25 % confluence (pre-confluence) and 10 days post-confluence. All components moved from the cytoplasm to the intercellular membrane. Scale bar, 100 μm.
Figure 2
Contact inhibition is unlocked by EGTA only in the presence of EGF and/or FGF-2, but not TGF-β1. (a) Immunostaining of BrdU was performed in ARPE-19 cells cultured to day 7 post-confluence after being treated without or with 1mg/mL EGTA immediately followed by PBS, 10 ng/mL EGF, 20 ng/mL FGF-2, 10 ng/mL TGF-β1, 10 ng/mL EGF + 20 ng/mL FGF-2, or 10 ng/mL EGF + 20 ng/mL FGF-2 + 10 ng/mL TGF-β1 for 1 day. Without EGTA, no BrdU labeling was found. When EGTA was added, BrdU labeling was promoted by EGF, FGF-2, or EGF+FGF-2, but not by PBS, TGF-β1 or EGF+FGF-2+TGF-β1. Scale bar, 100 μm. (b) The BrdU labeling index was significantly increased by EGF, FGF-2, or in combination (highest) when EGTA was added (*, †, and ‡, P <0.05).
Figure 3
EMT occurs only when junction is disrupted by EGTA in the presence of EGF+FGF-2 with or without TGF-β1. (a) Immunostaining showed marked dissolution of junctional staining of RPE65, N-cadherin, ZO-1, and, Na,K-ATPase by EGF+FGF-2 with or without TGF-β1 and to a lesser extent by EGF, FGF-2, or TGF-β1 alone. No effect was noted in EGTA alone. (b) The immunostaining to vimentin (upper panel) was positive in all but most obvious in cells treated with and TGF-β1, EGF+FGF-2 or EGF+FGF-2+TGF-β1. The immunostaining to S100A4 (middle panel) was only obviously positive in cells treated with EGF+FGF-2. The immunostaining to α-SMA (lower panel) was most pronounced in cells treated with TGF-β1, EGF+FGF-2, and EGF+FGF-2+TGF-β1. EGTA alone had no effect. Scale bar, 100 μm.
Figure 4
EGTA plus EGF+FGF-2 activates Wnt/β-catenin signaling. (a) Immunostaining revealed nuclear β-catenin and LEF1 only when cells were treated with EGTA plus EGF+FGF-2 but not EGTA alone. Scale bar, 100 μm (b) Western blot analysis confirmed increased nuclear contents of β-catenin and LEF1 in the nuclear extract, but decreased β-catenin in the membranous extract when cells were treated by EGTA with EGF+FGF-2. Levels of connexin 43, α-tubulin, and histone were used as loading controls for membranous, cytosolic and nuclear extracts, respectively. (c) Overexpression of S33Y β-catenin in post-confluent ARPE treated with EGTA without growth factors increased BrdU labeling, nuclear β-catenin and S100A4, and cytoplasmic α-SMA. (d) Immunoprecipitation by anti-β-catenin antibody followed by Western blotting with anti-LEF-1 antibody confirmed tight association between β-catenin and LEF-1 in the nuclear extract from EGTA plus EGF+FGF-2 but not from PBS or EGTA alone.
Figure 5
EMT with proliferation induced by EGTA with EGF+FGF-2 is caused by Wnt/β-catenin signaling by rescued by SS3Y β-catenin. (a) The TCF/LEF reporter activity was silent in cells treated by PBS, EGTA, EGF, FGF-2, TGF-β1, or EGF+FGF-2+TGF-β1 but elevated 15-fold by EGTA with EGF+FGF-2. The latter was abolished by XAV939 (*, P <0.05). (b) Immunostaining revealed that XAV939 abolished nuclear staining of BrdU, enhanced cytoplasmic staining of RPE65, and reverted the cytoplasmic staining pattern of N-cadherin, ZO-1, and Na,K-ATPase to the membranous staining pattern. (c) Immunostaining also revealed that XAV939 diminished the cytoplasmic staining of vimentin, reverted nuclear staining of β-catenin to the membranous staining, and abolished nuclear staining of S100A4 and LEF1, as well as cytoplasmic staining of α-SMA. (d) Inhibition of proliferation and EMT by XAV 939 was rescued by overexression of stable SS3Y β-catenin, resulting in an increase of BrdU labeling, nuclear staining of β-catenin and S100A4, and cytoplasmic staining of α-SMA. Scale bar, 100 μm.
Figure 6
Wnt signaling by EGTA plus EGF+FGF-2 is sufficient and additive with TGF-β1 to trigger Smad/ZEB1/2 signaling. (a) Immunostaining showed nuclear staining of p-Smad2/3 (upper panel), ZEB1 (middle panel), and ZEB2 (lower panel) in cells only treated by EGTA with TGF-β1, EGF+FGF-2 or EGF+FGF-2+TGF-β1, but not EGTA alone. Scale bar, 100 μm. (b) Western blot analysis confirmed that the nuclear levels of p-Smad2/3, ZEB1, and ZEB2 were not activated by EGTA alone until TGF-β1, EGF+FGF-2, or EGF+FGF-2+TGF-β1 was added with an additive action between the former two. (c) Western blot analysis also confirmed that the blocking of the Wnt signaling by XAV939 decreased the nuclear level of p-Smad2/3 only in cells treated wtih EGF+FGF-2 but not TGF-β1. Histone was used as a loading control.
Figure 7
Scheme of canonical Wnt signaling and Smad/ZEB1/2 signaling in regulating EMT with or without proliferation by growth factors. Upon disruption of contact inhibition, EGF+FGF-2 promotes EMT with proliferation via activating Wnt/β-catenin signaling, while TGF-β1 promotes EMT without proliferation via Smad/ZEB1/2 signaling. Wnt/β-catenin signaling promoted by EGF+FGF-2 is sufficient and synergized with TGF-β1 in activating Smad/ZEB1/2 signaling responsible for EMT.
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