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D.A. Dartt, J. Gu, M.A. Shatos; EGF Activates p42/p44 Mitogen–Activated Protein Kinase to Stimulate Cultured Rat Conjunctival Goblet Cell Proliferation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2328.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the presence and localization of ErbB receptors and to determine if EGF stimulates goblet cell proliferation through activation of p42/p44 mitogen–activated protein kinase (MAPK).
Conjunctiva was removed from male Sprague Dawley rats and pieces were placed in culture in RPMI medium to propagate goblet cells. Cultured cells were identified as goblet cells by the presence of the lectin UEA–1. Goblet cells were homogenized in buffer containing 30 mm Tris–HCl, pH–7.5, 10 mm EGTA, 5mm EDTA, 1 mm DTT and 250 mm sucrose and proteins were separated by SDS–PAGE. Goblet cells were also grown on coverslips and immunofluorescence microscopy experiments (IF) were performed. The presence and localization of ErbB receptors was evaluated by western blot analysis (WB) and IF, respectively. Cultured goblet cells were treated with U0126, an inhibitor specific to MAPK, 2 h prior to addition of EGF for 24 h and cell proliferation measured by the WST–8 assay. Activation of MAPK was measured by IF using an antibody against phosphorylated (active) MAPK and Ki–67, which appears in proliferating cells. Mounting media containing DAPI was used to determine the total number of cells present. Cells positive for both activated MAPK and Ki–67 were counted and the results expressed as a percent of the total number of cells.
ErbB1, 2, 3 and 4 were identified in cultured goblet cells by WB. ErbB1, 2, and 3 were localized to the plasma membrane of cultured goblet cells. ErbB4 could not be detected by IF. EGF significantly stimulated goblet cell proliferation 1.44 ± 0.03 fold over basal that was completely inhibited by U0126 (10–5 M). EGF (10–7 M) caused a biphasic response in the activation of MAPK with 2.5% of the cells expressing both activated MAPK and Ki–67 after 5min. A second wave of proliferation was detected beginning at 16 hours after addition of EGF with 5.0% of the cells expressing MAPK and Ki–67.
We conclude that all four erbB receptors are present in cultured goblet cells and that EGF stimulates their proliferation by activating MAPK in a biphasic manner.
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