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VN Bildin, Z Wang, PS Reinach; Epidermal Growth Factor and Endothelin-1 Regulation of Mitogen-Activated Protein Kinase Activity, Na-K-2Cl Cotransporter and Na/K-ATPase Expression and Proliferation in Bovine Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1651.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine if there is an interaction between epidermal growth factor (EGF) and endothelin-1 (ET-1) in their stimulation of mitogen-activated protein kinase (MAPK) activity, Na-K-2Cl cotransporter (NKCC) and Na/K-ATPase protein expression as well as proliferation in cultured bovine corneal epithelial cells (BCEC). Methods: After 48 h of serum starvation, the BCEC were incubated with or without EGF and/or ET-1 at different concentrations: 1.) for 10 min to analyze MAPK activation; 2.) 24 h to determine subsequent changes in NKCC and Na/K-ATPase protein expression and cell proliferation. Western blot/ECL assay was used to evaluate phosphorylated MAPK, NKCC and Na/K-ATPase protein amounts in the cytosolic fraction and membrane-containing pellet of cell lysates, respectively. Proliferation was quantified based on measurements of 3H-thymidine uptake. Results: Both mitogens applied alone dose dependently stimulated MAPK activity with a maximal increase of about 10-fold for 1-10 nM ET-1 and more than 20-fold for 5-10 ng/ml EGF. EGF and ET-1 at those concentrations similarly upregulated NKCC and Na/K-ATPase proteins expression levels, (i.e. 6-7- and 3-4-fold, respectively). Cell proliferation was also proportionally stimulated. On the other hand, the combined effects of EGF and ET-1 at all concentrations on MAPK activity, NKCC and Na/K-ATPase proteins levels and cell proliferation were similar to those with 5-10 ng/ml EGF alone. Conclusion: The dose dependent stimulation of NKCC and Na/K-ATPase protein expression and proliferation induced by EGF and ET-1 in BCEC closely correlate with increases in MAPK activity produced by these cytokines. This suggests that EGF and ET-1 receptor control of proliferation occurs through changes in cell volume. EGF and ET-1 receptor regulation of NKCC and Na/K-ATPase protein expression and cell proliferation both occur through common signaling pathways and involve MAPK activation.
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