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V.N. Bildin, Z. Wang, P. Reinach; Role of NKCC1 Translocation in Control of Human Corneal Epithelial Cell Proliferation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2199.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In corneal epithelial cells, the mitogenic response to epidermal growth factor (EGF) is elicited through the stimulation of signaling events that include activation of extracellular signal–regulated protein kinases (Erk1/2) and specific isoforms of protein kinase C (PKC). However, it is unclear if this response is dependent on translocation and activation of housekeeping Na–K–2Cl cotransporter (NKCC1). Methods: Serum starved SV40–immortalized human corneal epithelial cells (HCEC) were stimulated by 10 ng/ml EGF and/or incubated with PKC inhibitors (up to 5 µM bisindolylmaleimide 1 or calphostin C) for 15 min or 22 h. Levels of phosphorylated and total Erk1/2 and NKCC1 in a crude cell extract and cell membrane fraction were evaluated by Western blotting/ECL assay. Amounts of total NKCC1 were assessed with T4 antibody and its phosphorylated form using an anti–phospho–NKCC1 antibody R5 raised against a Thr212 and Thr217 human NKCC diphosphopeptide. Cell proliferation was determined based on measurements of 3H–thymidine incorporation. Results: PKC inhibition doubled Erk1/2 activity and produced up to a 25% increase in proliferation of serum starved HCEC, without changing their mitogenic response to EGF. Regardless of whether increases in cell proliferation were elicited by EGF or PKC inhibition, the mitogenic response was proportional to NKCC1 accumulation. Both of them produced fast (5–15 min) and extensive (up to 50–fold increases in amount) translocation of a high molecular weight (∼190 kDa) NKCC1 form to the cell membrane. In the serum starved cells, NKCC1 was predominately present in its low molecular weight form (∼160 kDa). Both isoforms immunostained with T4 whereas only the lighter form interacted with the R5 antibody. EGF or PKC inhibitors caused waning of the lower molecular form. Conclusions: In serum starved HCEC, some PKCs are involved in the control of cell cycle arrest. Entrance into the cell cycle is related with activation (accumulation) of ion transporters involved in regulation of cell volume. Phosphorylation of Thr212 and Thr217 residues of human NKCC1 is required for its trafficking to the cell membrane rather than for mediating net ion transport activity.
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