Upregulation of Tight-Junctional Proteins in Corneal Epithelial Cells by Corneal Fibroblasts in Collagen Vitrigel Cultures

Abstract

To investigate the effects of corneal fibroblasts on the differentiation of corneal epithelial cells in a coculture system based on a collagen vitrigel membrane. Simian virus 40-transformed human corneal epithelial (HCE) cells and human corneal fibroblasts were cultured on opposite sides of a collagen vitrigel membrane. The distribution of HCE cells and corneal fibroblasts on the collagen membrane was determined by immunofluorescence staining and immunoblot analysis of marker proteins. Expression of the tight-junctional proteins ZO-1, occludin, and claudin and of the adherens-junctional proteins E- and N-cadherin in HCE cells was determined at the mRNA and protein levels by reverse transcription-polymerase chain reaction analysis and immunoblot analysis, respectively. The abundance of ZO-1, occludin, and claudin mRNA and proteins in HCE cells was markedly increased by coculture with corneal fibroblasts. The expression of E- or N-cadherin did not differ between HCE cells cultured with corneal fibroblasts and those cultured without them. PD98059, a specific inhibitor of signaling by extracellular signal regulated kinase (ERK), prevented the upregulation of tight-junctional proteins in HCE cells by corneal fibroblasts. Human corneal fibroblasts regulated the expression of tight-junctional proteins in HCE cells, suggesting that corneal fibroblasts may play an important role in the differentiation of corneal epithelial cells.