Regulation of human corneal epithelial mucins by rebamipide.
OBJECTIVE
Membrane-associated mucins (MAMs) play important roles in barrier function and tear stability, and their expression on the ocular surface is altered in dry eye disease. Rebamipide is a mucin secretagogue that promotes the production of mucin-like glycoproteins in human corneal epithelial (HCE) cells. However, the expression of MAMs on the corneal epithelia (MUC1, MUC4, MUC16), which is induced by rebamipide, is poorly understood. In this study, we investigated the effect of rebamipide on the regulation of MAM expression in HCE cells.


METHODS
MUC16, Ki67 and PCNA expression levels in HCE cells isolated at confluence and at 24 hours after confluence were examined by Western blotting to assess cell proliferation. HCE cells isolated at 24 hours after confluence were cultured in medium supplemented with 1-10 µM rebamipide or 0.3-30 nM of epidermal growth factor (EGF). Real-time PCR (RT-PCR) and Western blot analysis of MAMs were performed to evaluate the effect of rebamipide. Western blot analysis of cells treated with an EGF receptor inhibitor (AG1478) or MEK1/2 inhibitor (U0126) was performed to reveal the relationship between EGF receptor activation and rebamipide-induced MAM expression.


RESULTS
HCE cells isolated at 24 hours after confluence had lower cell proliferation activity and increased MUC16 expression compared with cells isolated at confluence. RT-PCR and Western blot analysis revealed that rebamipide increased MAM gene expression for 2 hours and protein expression for 24 hours in HCE cells. EGF inhibitor treatment led to reduced levels of all three MAMs that are normally induced by rebamipide, whereas EGF induced the expression of all three MAMs.


CONCLUSIONS
We suggested that rebamipide increased MUC1, MUC4 and MUC16 expression levels through signals involved in EGF receptor activation in the human corneal epithelia. These data suggest that rebamipide may improve subjective symptoms of dry eye disease by upregulating MAM expression.
Ako Research Institute, Otsuka Pharmaceutical Co, Ltd. , Hyogo , Japan.
Shinsaku
S
Itoh
Kuni
K
Itoh
Hisashi
H
Shinohara
D000409:Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000897:Anti-Ulcer Agents / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism; D018396:Mucin-1 / Q000378:metabolism*; D055264:Mucin-4 / Q000378:metabolism*; D055272:Mucin-6 / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D015363:Quinolones / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction
D000897:Anti-Ulcer Agents; D015415:Biomarkers; D019394:Ki-67 Antigen; C060192:MUC1 protein, human; C524658:MUC4 protein, human; C524734:MUC6 protein, human; D018396:Mucin-1; D055264:Mucin-4; D055272:Mucin-6; D018809:Proliferating Cell Nuclear Antigen; D015363:Quinolones; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; C052785:rebamipide; D000409:Alanine