Diquafosol ophthalmic solution enhances mucin expression via ERK activation in human conjunctival epithelial cells with hyperosmotic stress.

PURPOSE

To evaluate the effect of diquafosol tetrasodium on the expression of secretory and membrane-associated mucins in multi-layered cultures of primary human conjunctival epithelial cells (HCEC) using intracellular extracellular signal regulated kinase (ERK) signaling.

METHODS

HCECs were treated with hyperosmotic stress (400 mOsm/l) for 24 h after air-liquid interface cell culture followed by treatment with diquafosol. HCECs were stimulated for 1 h with or without PD98059, an ERK inhibitor, then treated with diquafosol for 6 h and 24 h. Mucin 1 (MUC1), mucin 16 (MUC16), and MUC5AC mRNA and protein expression levels were analyzed, and cell viability was detected using an MTT assay. Western blot analysis was used to examine p44/42 MAPK (Erk1/2) and phosphorylated p44/42 MAPK (Erk1/2) expression.

RESULTS

Hyperosmotic stressed HCECs demonstrated increased MUC5AC secretion and gene expression when treated with diquafosol. MUC1 mRNA levels increased significantly at 24 h (p<0.01), and expression of MUC16 mRNA levels increased at 6 h and were maintained until 24 h (p<0.05).There was no significant difference in cell viability compared to the control group. Immunostaining results for MUC1, MUC16, and MUC5AC in diquafosol tetrasodium-treated HCECs at 24 h showed more positive cells than in the control group. Phosphorylation of p44/42 MAPK (Erk1/2) signaling molecules significantly increased from 5 min to 60 min (p<0.05). The effects of diquafosol on mucin expressions in hyperosmotic stressed HCECs were significantly inhibited by PD98059, an ERK inhibitor, at 6 h and 24 h.

CONCLUSIONS

ERK signaling may regulate the expression levels of MUC1, MUC16, and MUC5AC induced by diquafosol in hyperosmotic stressed HCECs.