Resolvin D1, but not resolvin E1, transactivates the epidermal growth factor receptor to increase intracellular calcium and glycoconjugate secretion in rat and human conjunctival goblet cells

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Purpose:To identify interactions of the epidermal growth factor receptor (EGFR) with the pro-resolving mediator receptors for RvD1 and RvE1 to stimulate an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion from cultured human and rat conjunctival goblet cells.Methods:Goblet cells from human and rat conjunctiva were grown in culture using RPMI media. Cultured goblet cells were pre-incubated with inhibitors, and then stimulated with RvD1, RvE1, EGF or the cholinergic agonist carbachol (Cch). Increase in [Ca2+]i was measured using fura-2/AM. Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-1. Western blot analysis was performed with antibodies against AKT and ERK 1/2.Results:In cultured human conjunctival goblet cells RvE1 -stimulated an increase in [Ca2+]i. RvD1-, but not the RvE1-, stimulated increase in [Ca2+]i and mucin secretion was blocked by the EGFR inhibitor AG1478 and siRNA for the EGFR. RvD1-, but not RvE1-stimulated an increase in [Ca2+]i that was also inhibited by TAPI-1, an inhibitor of the matrix metalloprotease ADAM 17. Inhibition of the EGFR also blocked RvD1-stimulated increase in AKT activity and both RvD1-and RvE1-stimulated increase in ERK 1/2 activity. Pretreatment with either RvD1 or RvE1 did not block the EGFR-stimulated increase in [Ca2+]i.Conclusions:We conclude that in cultured rat and human conjunctival goblet cells, RvD1 activates the EGFR, increases [Ca2+]i, activates AKT and ERK1/2 to stimulate mucin secretion. RvE1 does not transactivate the EGFR to increase [Ca2+]I and stimulate mucin secretion, but does interact with the receptor to increase ERK 1/2 activity.HighlightsRvE1 increases [Ca2+]i in human conjunctival goblet cells and is potent stimulator in rat conjunctival goblet cells.Inhibition of EGFR blocks RvD1-, but not RvE1-stimulated, increase in [Ca2+]i and glycoconjugate secretion.Inhibition of MMP blocks RvD1-, but not RvE1-stimulated, increase in [Ca2+]i.Inhibition of EGFR blocks RvD1, but not RvE1, activation of AKT in rat cultured conjunctival goblet cells.Neither RvD1 nor RvE1 affect EGF-stimulated increase in [Ca2+]i in cultured rat conjunctival goblet cells.

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