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T. Kawakita, S. Shimmura, K. Tsubota, J. Shimazaki, S. C. G. Tseng; Cellular Senescence Due to Epithelial Mesenchaymal Transition During Clonal Expansion of Murine Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):449.
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© ARVO (1962-2015); The Authors (2016-present)
Epithelial-mesenchymal transition (EMT) is involved in adulttissue fibrosis in the lung and the kidney following wound healing. We reported previously that p63-positive limbal epithelial cells can also undergo EMT in an air-lift rabbit corneal/limbal explant. We have established a murine corneal/limbal epithelial cell line (TKE2) that maintains clonal growth potential. We would like to determine whether EMT could also occur in TKE2 cells in culture.
TKE2 cells were cultured on plastic in a serum-free KSFM medium. EMT was determined by immunostaining to alpha-SMA and S100A4 expression in epithelial cells expressing cytokeratins, p63 and E-cadherin. Cell proliferation was determined by immunostaining to PCNA. Activation of Wnt pathway was determined by immunohistochemical analysis of E-cadherin, beta-catenin, and LEF-1. The inflammatory cytokines concentration of condition medium in three different cell seeding density (500-50000 cells per cm2) was determined by Bioplex®#174. EMT was evaluated following density-dependent endogenous production of TGF-beta or addition of TGF-beta neutralizing antibody.
alpha-SMA positive cells were spontenously observed in the periphery of TKE2 cells during clonal expansion, and their occurrence was promoted by and increased cell seeding density and increased cell-cell contact. Such alpha-SMA positive cells also expressed p63, PCNA or S100A4 in nuclei, confirming their original epithleial phenotype. Endogenous production of TGF-beta dose-dependently correlated with increased cell density, and an increase of alpha-SMA positive cells. Addition of TGF-beta neutralizing antibody inhibited alpha-SMA expression in a high seeding density culture (50000 cells per cm2). The Wnt signaling pathway was activated in the periphery of large colonies after expansion, where alpha-SMA positive cells were noted. At a high cell density, conditioned media contained not only high levels of TGF-beta but also contained high levels of GM-CSF and IL-1 alpha which might explain why TGF-beta neutralizing antibody failed to inhibit EMT completely in a high density culture.
When murine limbal/corneal epithelial cells clonally expanded and aged in culture, they inevitably undergo EMT, which depended not only on cell-cell contact, but also on autocrine and/or paracrine TGF-beta signaling.
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