Conjunctival goblet cells play a major role in maintaining the mucous layer of the tear film under physiological conditions as well as in inflammatory diseases like dry eye and allergic conjunctivitis.. Resolution of inflammation is mediated by pro-resolution agonists such as lipoxin A4 (LXA4) that can also function under physiological conditions. The purpose of this study was to determine the actions of LXA4 on cultured rat conjunctival goblet cell mucin secretion, intracellular [Ca2+] ([Ca2+]i) and identify signaling pathways activated by LXA4. ALX/FPR was localized to goblet cells in rat conjunctiva and in cultured goblet cells. LXA4 significantly increased mucin secretion, [Ca2+]i, and ERK 1/2 activation. These functions were inhibited by ALX/FPR2 inhibitors. Stable analogs of LXA4 increased [Ca2+]i to the same extent as LXA4. Sequential addition of either LXA4 or resolvin D1 followed by the second compound decreased [Ca2+]i of the second compound compared to its initial response. LXA4 activated phospholipase C, -D, and A2 and downstream molecules protein kinase C, ERK 1/2, and Ca2+/calmodulin dependent kinase to increase mucin secretion and [Ca2+]i. We conclude that conjunctival goblet cells respond to LXA4 to maintain the homeostasis of the ocular surface and could be a novel treatment for dry eye diseases.

The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M1AchR, M2AchR, and M3AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca2+] ([Ca2+]i) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca2+]i in a concentration-dependent manner. EGF-stimulated increase in [Ca2+]i was blocked by inhibitors of the EGF receptor and removal of extracellular Ca2+. Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M1AchR, M2AchR, and M3AchRs. Cch-stimulated increase in [Ca2+]i was blocked by inhibitors for the M1AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca2+]i and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca2+. This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca2+]i, and activate ERK 1/2 leading to an increase in mucin secretion.