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.
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