Analysis of connexin expression in human conjunctival epithelial cells

Purpose: Dry eye disease (DED) is a common and distressing ocular condition in the over-55s, and a significant cause of visual morbidity. In DED, the conjunctival cells are physically altered, a process known as "squamous metaplasia". The cells become enlarged, flattened-out (lose cell-cell contacts) and adopt characteristics more typical of corneal epithelial cells. The mechanism by which this change occurs is poorly understood, but logically must affect epithelial transport and cell junctions as the cells react to hyperosmotic changes in the overlying tear fluid. Connexins (Cx) are the constituent proteins of Gap Junctions and play a central role in the coordination of cellular activities. These specialised connections are considered to be essential in maintaining the integrity and function of mucous membranes, permitting the exchange of small molecules and ions between neighbouring cells. Cx31.1 and Cx43 are known to have roles in corneal wound healing and apoptosis, however, little is known of Cx expresson in human conjunctiva. The aim of this study is to analyse Cx expression in a human conjunctival cell line to further our understanding of cell-cell communication in this tissue and in DED.

Methods: All experiments were performed on the immortalised Chang human conjunctival epithelial cell line. Morphometric analysis was performed to determine cell size. Reverse-Transcription-PCR (RT-PCR), western blotting (WB) and confocal immunofluorescence (IF) were used to assess Cx gene and protein expression.

Results: RT-PCR of Chang cells showed Cx43 and Cx45 expression at the mRNA level. Cx26 and Cx37 mRNA expression was absent.WB of Chang cell lysates indicated a band at 43kd, suggesting Cx43 protein expression. IF studies demonstrated punctate Cx43 and Cx45 staining, specific to intercellular junctions of confluent Chang cells.

Conclusions: This study shows for the first time that Chang conjunctival epithelial cells express Cx43 and Cx45 at both the mRNA and protein level, especially at cell confluence. This indicates a potential role for these specific Cxs in conjunctival epithelial cell communication at the ocular surface.