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Ek Kia Tan, Ying-Ting Zhu, Hung-Chi Chen, Szu-Yu Chen, Scheffer C.G. Tseng; Epithelial-mesenchymal transition (EMT) Caused by EGTA with EGF+FGF-2 via Activation of Wnt Signaling. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1149.
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Proliferation and epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a hallmark of proliferative vitreoretinopathy. We aim at clarifying the role of EGF, FGF-2, and TGF-β1 in controlling how RPE proliferates while undergoing EMT.
Post-confluent Day 7 ARPE-19 cells were treated for 24 h with 1 mM EGTA, 10 ng/mL EGF, 20 ng/mL FGF-2, 10 ng/mL TGF-β1, or 10 µM XAV939, individually or in combination. Before termination, cells were further treated with 10 μM BrdU for 4 h. Immunostaining was performed to monitor cytolocalization of RPE65, p120, Kaiso, α-catenin, β-catenin, N-cadherin, ZO-1, S100A4, Smad, ZEB1/2, and BrdU labeling. Western blotting was used to measure their protein levels in the nuclear compartment.
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 S100A4 and α-SMA). EMT with proliferation by EGTA plus EGF+FGF-2 was accompanied by activation of canonical Wnt signaling (judged by the TCF/LEF promoter activity and increased nuclear levels of β-catenin and LEF1 proteins), which was abolished by concomitant addition of Wnt inhibitor XAV939, but not associated with p120-Kaiso signaling. 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 differential role of EGF, FGF-2 and TGF-β and Wnt signaling when cell junction of RPE is disrupted to undergo EMT with proliferation.
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