title: Wnt signaling induces epithelial-mesenchymal transition with proliferation in ARPE-19 cells upon loss of contact inhibition.
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.
authors.affiliation: Tissue Tech, R&D Ocular Surface Center, Ocular Surface Research and Education Foundation, Miami, FL 33173, USA.
authors.forename: Hung-Chi
authors.initials: HC
authors.lastname: Chen
authors.forename: Ying-Ting
authors.initials: YT
authors.lastname: Zhu
authors.forename: Szu-Yu
authors.initials: SY
authors.lastname: Chen
authors.forename: Scheffer C G
authors.initials: SC
authors.lastname: Tseng
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003260:Contact Inhibition; D004533:Egtazic Acid / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D058750:Epithelial-Mesenchymal Transition; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism / Q000494:pharmacology; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D012097:Repressor Proteins / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D018630:Vitreoretinopathy, Proliferative / Q000473:pathology; D051153:Wnt Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000074165:Zinc Finger E-box Binding Homeobox 2; D000071799:Zinc Finger E-box-Binding Homeobox 1; D051176:beta Catenin / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D006575:Heterocyclic Compounds, 3-Ring; D018398:Homeodomain Proteins; D012097:Repressor Proteins; D051785:Smad Proteins; D014157:Transcription Factors; D053773:Transforming Growth Factor beta1; D051153:Wnt Proteins; C544261:XAV939; C486307:ZEB1 protein, human; C492363:ZEB2 protein, human; D000074165:Zinc Finger E-box Binding Homeobox 2; D000071799:Zinc Finger E-box-Binding Homeobox 1; D051176:beta Catenin; D016222:Fibroblast Growth Factor 2; D004533:Egtazic Acid; D004815:Epidermal Growth Factor