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M. S. Chang, S.-H. Presley, J.-L. Yang, P. K. Russ, F. R. Haselton; Convergence of Adherens and Tight Junction Signaling in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5200.
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© ARVO (1962-2015); The Authors (2016-present)
Bves (blood vessel epicardial substance) is a novel adhesion molecule that regulates tight junction (TJ) formation in human corneal epithelial (HCE) cells. In addition, loss of Bves is associated with epithelial-mesenchymal transformation (EMT) in HCE cells. Recently, we also observed decreased levels of E-cadherins, an adherens junction (AJ) protein, associated with loss of Bves function. These observations led to the hypothesis that EMT in HCE cells associated with loss of Bves is through both TJ and AJ signaling pathways.
HCE cells were stably transfected to overexpress dominate-negative Bves (HCE-dnBves). Cells were evaluated for epithelial vs. mesenchymal characteristic (morphology, motility, soft agar assay) and for formation of TJ and AJ (immunofluorescent staining, Western blotting). Since TJ directly regulates GEF-H1, an activator of RhoA and ZONAB/DbpA, a Y-box transcription factor, cells were evaluated for changes in RhoA activation by FRET and for ZONAB/DbpA activity with a luciferase reporter. For AJ associated signaling, changes for Beta-catenin localization were evaluated by immuno-staining. Since AJ is associated with Wnt signaling, Top Flash assay was used to asses changes in Wnt signaling tone.
Cells overexpessing dnBves exhibit fibroblast morphology, increased motility, and increased anchorage independent proliferation (soft agar assay), indicating EMT. The levels of AJ and TJ protein were decreased in HCE-dnBves cells compared to parental HCE cells. HCE-dnBves cells exhibited increased levels of RhoA activation and ZONAB/DbpA transcriptional activity. Furthermore, HCE-dnBves cells exhibited nuclear localization of Beta-catenin and, the Top Flash assay is consistent with increased Wnt signaling.
Loss of Bves function through overexpression of dnBves induces EMT in HCE cells. In addition, overexpression of dnBves leads to alteration in TJ and AJ associated signaling. These alterations include increased ZONAB/DbpA transcriptional activity and increased Wnt tone. Our findings suggest that through Bves there is convergence of TJ and AJ signaling, and convergence may be a novel a regulatory mechanism in modulating epithelial-mesenchymal phenotype in HCE cells.
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