Mucin Gene Expression in Immortalized Human Corneal–Limbal and Conjunctival Epithelial Cell Lines

Abstract

The corneal and conjunctival epithelia, which cover the ocular surface, play an important role in preventing pathogen penetrance into the eye and maintaining a wet-surface phenotype by producing highly hydrophilic mucin molecules for their apical surfaces. Ocular surface infections, wounding, and pathologies resulting in dry eye threaten sight and can cause blindness. Understanding the ocular surface defense mechanisms that mucins provide has been hampered by the lack of immortalized human corneal and conjunctival epithelial cell lines that retain mucin gene expression patterns of the native tissue. The purpose of this work was to characterize newly developed immortalized corneal and conjunctival cell lines using mucin gene expression as markers of differentiation. The cell lines were derived as described by a previously published process. Primary cultures of corneal-limbal and conjunctival epithelia were sequentially transduced to express a dominant negative p53 protein and a p16(INK4A/Rb)-resistant, mutant cdk4 protein, which enabled the cells to bypass a senescence mechanism recently identified for primary cultures of keratinocytes. These cells were then transduced to express the catalytic subunit of telomerase to permit them to retain their telomeres and divide indefinitely. Cellular morphology and expression of mucin genes in the two cell lines, designated HCLE for the human corneal-limbal line and HCjE for the human conjunctival cell line, were determined after culture on plastic, type I collagen, or Matrigel, in coculture with fibroblasts, and in severe combined immunodeficient (SCID) mice. Expression of the epithelial cell mucins was assayed by reverse transcription, real-time polymerase chain reaction, immunoblot analysis, or immunohistochemistry and compared with expression in native cornea and conjunctiva. When grown in high-calcium medium on plastic and type I collagen, cells of both lines stratified, exhibiting multiple cell layers. In Matrigel, both cell lines formed cell aggregates that contained lumens. In the SCID mice, the conjunctival cell line formed stratified layers under the kidney capsule. The corneal cell line expressed keratins K3 and K12, the keratins that are corneal-epithelial-specific, and both cell lines expressed K19. As in native tissue, the HCLE and HCjE cell lines expressed the membrane-associated mucins, MUC1, -4, and -16, although their levels were generally lower. Levels of MUC4 and -16 mRNA were the most comparable to native tissue, particularly when cultured on plastic. Apical cells of the stratified cultures were the cells that expressed the membrane-associated mucins MUC1 and -16. Goblet-cell-specific MUC5AC mRNA and protein was detected in a small population of HCjE cells only when using type I collagen as a substrate or when cells were cocultured with fibroblasts. Both cell lines produced glycosylated mucins as indicated by binding of H185 antibody, an antibody that recognizes a carbohydrate epitope on mucins. The immortalized corneal (HCLE) and conjunctival (HCjE) cell lines exhibit the mucin gene expression repertoire of their native epithelia. These cell lines will be useful in determining regulation of ocular surface mucin gene expression and, potentially, goblet cell differentiation.