Effect of retinoic acid on epithelial differentiation and mucin expression in primary human corneal limbal epithelial cells

Purpose: Retinoic acid (RA) is essential for epithelial differentiation and maintenance of the mucous phenotype. This study investigated the effect of RA on corneal epithelial differentiation and mucin expression in a primary human corneal limbal epithelial cell (HCLEC) culture model. Methods: HCLECs were grown in RA-supplemented media at various concentrations (0, 10 ^-9 to 10 ^-6 M). Stratified HCLECs were examined using immunohistochemical or immunofluorescent staining for p63, ABCG2, CK3, CK19, and Western blotting for ABCG2 and CK12 to assess differentiation. Ultrastructural morphology was investigated using scanning and transmission electron microscopy. They were incubated with rose bengal dye to examine barrier function. The effects of RA on the expression of MUC1, -4, and -16 were analyzed by immunohistochemistry, quantitative real-time PCR and Western blot analysis. Results: HCLEC grown without RA showed hyperkeratosis, whereas those grown with 10 ^-8 to 10 ^-7 M RA induced non-keratinized stratified epithelium with a normal appearance. Under these conditions, p63, ABCG2, CK3, CK19, MUC1, -4, and -16 staining patterns were similar to in vivo limbal epithelium. A higher concentration (10 ^-6 M) of RA resulted in abnormal differentiation. HCLECs grown with RA were tightly apposed and maintained intact barrier function against dye penetration. In addition, MUC1, -4, and -16 expressions were highly associated with RA concentrations. Conclusions: This study showed that cultured HCLEC could mimic physiologic and functional phenotypes by controlling RA concentrations in medium. Also, our results suggested modulating effect of RA on differentiation and mucin expression in corneal epithelium.