title: 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 (M(1)AchR, M(2)AchR, and M(3)AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca(2+)] ([Ca(2+)](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 [Ca(2+)](i) in a concentration-dependent manner. EGF-stimulated increase in [Ca(2+)](i) was blocked by inhibitors of the EGF receptor and removal of extracellular Ca(2+). Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M(1)AchR, M(2)AchR, and M(3)AchRs. Cch-stimulated increase in [Ca(2+)](i) was blocked by inhibitors for the M(1)AchRs, 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 [Ca(2+)](i) and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca(2+). This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca(2+)](i), and activate ERK 1/2 leading to an increase in mucin secretion.
authors.affiliation: Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hodges
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Bair
authors.forename: Richard B
authors.initials: RB
authors.lastname: Carozza
authors.forename: Dayu
authors.initials: D
authors.lastname: Li
authors.forename: Marie A
authors.initials: MA
authors.lastname: Shatos
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D003228:Conjunctiva / Q000166:cytology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D020397:Goblet Cells / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D055271:Mucin 5AC / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; C524680:Muc5ac protein, rat; D055271:Mucin 5AC; D034741:RNA, Small Interfering; C049925:fura-2-am; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium; D016257:Fura-2