Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion

Abstract

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.

Polymorphism in THBS1 Gene Is Associated with Post-Refractive Surgery Chronic Ocular Surface Inflammation

“…Likewise, disruption in type 2 repeats may interfere with epidermal growth factor receptor signaling of mucin-secreting goblet cells of the ocular surface. 44,45 A marginally significant increase in genotype frequencies of both SNP2 and 3 in subjects with dry eye suggested a trend that is in line with the functions associated with the corresponding TSP1 regions. Identical frequencies of these 2 SNPs also are consistent with a perfect LD between them.…”

Activation of Epidermal Growth Factor Receptor Mediates Mucin Production Stimulated by p40, a Lactobacillus rhamnosus GG-derived Protein

“…Our study suggests that p40 treatment does not deplete mucin from goblet cells because p40 increases the intracellular mucus level in LS174T cells and in colon epithelial cells in mice, and the number of goblet cells in mice identified by MUC2 immunostaining is not affected by p40 treatment. In addition to increasing mucin synthesis (16), EGF has been shown to regulate mucin secretion (44), indicating that p40 plays a possible role in mucin secretion. p40 promotes mucin production but does not affect the number of goblet cells in the colon in mice, suggesting that p40 may increase mucin synthesis in the existing goblet cells but does not have significant effects on goblet cell differentiation and proliferation under the conditions presented in this study.…”

Conjunctival epithelial and goblet cell function in chronic inflammation and ocular allergic inflammation

“…[27, 28]. Exogenous addition of agonists of muscarinic and VIP receptors also elevate secretion [28-30]. In allergic conjunctivitis both dromic activation of nerves and antidromic release of neurotransmitters could stimulate goblet cell secretion.…”