Cellular Senescence Due to Epithelial Mesenchaymal Transition During Clonal Expansion of Murine Limbal Epithelial Cells

Abstract

Purpose:: 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.

Methods:: 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.

Results:: 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.

Conclusions:: 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.

Keywords: cornea: epithelium • cornea: basic science • differentiation