Cell Signaling Pathways Mediating Epidermal Growth Factor Stimulation of Na:K:2Cl Cotransport Activity in Rabbit Corneal Epithelial Cells

Abstract

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na+]i in these cells loaded with the Na+ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na+]i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 microm RG-13022) and cPLA2 activity (10 microm AACOCF3) obviated EGF-induced increases in [Na+]i. In contrast, PGE2 (10 microg/ml) and cAMP (2 mm) increased [Na+]i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 microm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na+]i. Similarly, EGF failed to increase [Na+]i following inhibition of: 1) PKA activity (10 microm H-89); 2) Erk1/2 (15 microm PD98059) or 3) p38 (15 microm SB203580) activity. Stimulation protein kinase C activity (0.1 microm PMA) transiently increased [Na+]i followed by a decline towards its baseline value. EGF-induced increases in [Na+]i were unaltered by inhibition of K+ conductance (100 microm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA2 activity. Their stimulation increases PGE2 and cAMP levels resulting in PKA and NKCC activation.

Potassium-Chloride Cotransporter Mediates Cell Cycle Progression and Proliferation of Human Corneal Epithelial Cells

“…Specifically, EGF stimulation of p38MAPK induces an increase in NKCC1 function, whereas EGF-and ET-1-induced increases in proliferation are dependent on increases in K + efflux. 6,7 Furthermore, the present study provides evidence that KCC1 is additionally involved in mediating the mitogenic response induced by EGF. This is apparent because stimulation of KCC1 leads to p44/42MAPK activation and increases in cell proliferation ( Figs.…”

“…The present study, in conjunction with our previous work, demonstrates that, both, KCC1 and NKCC1 are essential components of the EGF-induced mitogenic signal in HCEC. 7 We previously demonstrated that a hypertonic challenge activates p38MAPK signaling, resulting in increases NKCC1 activity and cell swelling, 5,43 whereas EGF stimulation induces instead similar activation of NKCC1 and, consequently, enhanced cell proliferation. 7 In contrast, a hypotonic challenge induces KCC activation, leading to cell shrinkage and p44/42MAPK stimulation.…”

“…7 We previously demonstrated that a hypertonic challenge activates p38MAPK signaling, resulting in increases NKCC1 activity and cell swelling, 5,43 whereas EGF stimulation induces instead similar activation of NKCC1 and, consequently, enhanced cell proliferation. 7 In contrast, a hypotonic challenge induces KCC activation, leading to cell shrinkage and p44/42MAPK stimulation. 24 Moreover, the current study demonstrates that KCC1 mediates EGF-induced p44/42MAPK activation and proliferation.…”

“…[4][5][6][7] These signaling pathways have several features in common to pathways linked to the cytokine-receptors that mediate control of the corneal epithelial responses-proliferation and migration-required for corneal epithelial renewal. In many cell types, cell volume/size is linked to cell cycle progression.…”

“…[10][11][12][13][14] One of the well-studied pathways in CEC is the epidermal growth factor (EGF) receptor-linked p44/42MAPK and p38MAPK signaling cascades. 6,7,9,12,13,[15][16][17][18] In these cells, EGF activates NKCC1 and K + channels and the inhibition of either of these mechanisms blocks the mitogenic response to this growth factor. These effects suggest that cell volume modulation is a component of the cell signaling pathway mediating EGF stimulation of cell proliferation.…”