Tight Junction Transmembrane Protein Claudin Subtypes Expression and Distribution in hTERT Immortalized Corneal Epithelium and in vivo Human Corneal Epithelium

Abstract

Purpose:: To investigate the expression and cellular distribution of the tight junction transmembrane protein claudin subtypes in hTERT immortalized corneal epithelium and in vivo human corneal epithelium.

Methods:: Corneal epithelial cells immortalized by preventing telomere shortening by transduction with hTERT (the generous gift of Dr. I. K. Gipson, Schepens Eye Research Institute, Boston, MA) were cultured on 12 mm Transwell filters (Corning Co., Acton, MA) at a density of 4 x 10⁵ cells/cm². Human corneal tissue (Eye Bank Association of America, Washington, DC) was also examined.Reverse transcription-polymerase chain reaction was used to reveal the claudin mRNA expression. Immunohistochemistry was used to determine the tissue distribution of claudins.

Results:: The transcripts for claudin-1, -2, -3, -4, -7, -9, -10, -14, and -15 were identified in hTERT immortalized corneal epithelium. The transcripts for claudin-1, -2, -3, -4, -7, -9, and -14 were identified in in vivo human corneal epithelium. By light immunohistochemistry, claudin-1, -4, and -7 were localized at the membrane of epithelial cells, but claudin-2 staining was limited in the nucleus in hTERT immortalized corneal epithelium. In in vivo human corneal epithelium, only claudin-1 and -7 were localized at the membrane.

Conclusions:: Corneal epithelium forms a barrier that isolates the eye from the outside environment and regulates passive movement of fluid, electrolytes, macromolecules, and cells through the paracellular pathway. Tight junctions create this barrier. Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Thus far, 24 claudins have been identified and they have tissue specificity. Variations in the tightness of individual paired tight junction strands are determined by the combination and mixing ratios of claudin species. hTERT immortalized corneal epithelium expressed all and more claudins than in vivo human corneal epithelium. This newly developed cell line will be useful for experiments, like the knock down of endogenous claudin gene expression, to resolve the mechanism of barrier regulation.

Keywords: cell adhesions/cell junctions • cornea: epithelium