Establishment of human corneal epithelial cells stably expressing human connexin43

Abstract

Corneal epithelial cells communicate with each other through gap junctions. Whereas this property is retained in corneal epithelial cells in primary culture, it is often lost in immortalized epithelial cells. However, the life span of primary cultured corneal epithelial cells is short and the availability of human tissue for their preparation is limited. To examine the role of the gap-junction protein connexin43 (Cx43) in human corneal epithelial cells, we set out to establish an immortal human corneal epithelial cell line that stably expresses this protein. An expression vector encoding human Cx43 fused to enhanced green fluorescent protein (EGFP) was constructed and introduced by transfection into SV40-immortalized human corneal epithelial (HCE) cells. Stable transfectants were isolated by selection with the antibiotic G418. The expression and localization of the Cx43-EGFP fusion protein were examined by immunoblot analysis and fluorescence microscopy, respectively, and gap-junctional intercellular communication was monitored on the basis of dye coupling. HCE cells stably expressing Cx43-EGFP manifested intercellular dye transfer, whereas those stably expressing EGFP alone did not. Cx43-EGFP localized to the interfaces of neighboring cells. Stable expression of Cx43-EGFP in HCE cells did not affect the expression of keratins 3 and 12, which is a characteristic of corneal epithelial cells, but it did inhibit cell proliferation. We have established an HCE cell line that stably expresses human Cx43 and forms functional gap junctions. These cells may prove useful for studies of the role of gap junctions in the human corneal epithelium.