Functional and molecular characterization of multiple K–Cl cotransporter isoforms in corneal epithelial cells

Abstract

The dependence of regulatory volume decrease (RVD) activity on potassium-chloride cotransporter (KCC) isoform expression was characterized in corneal epithelial cells (CEC). During exposure to a 50% hypotonic challenge, the RVD response was larger in SV40-immortalized human CEC (HCEC) than in SV40-immortalized rabbit CEC (RCEC). A KCC inhibitor-[(dihydroindenyl)oxy] alkanoic acid (DIOA)-blocked RVD more in HCEC than RCEC. Under isotonic conditions, Nethylmaleimide (NEM) produced KCC activation and transient cell shrinkage. Both of these changes were greater in HCEC than in RCEC. Immunoblot analysis of HCEC, RCEC, primary human CEC (pHCEC), and primary bovine CEC (BCEC) plasma membrane enriched fractions revealed KCC1, KCC3, and KCC4 isoform expression, whereas KCC2 was undetectable. During a hypotonic challenge, KCC1 membrane content increased more rapidly in HCEC than in RCEC. Such a challenge induced a larger increase and more transient p44/42MAPK activation in HCEC than RCEC. On the other hand, HCEC and RCEC p38MAPK phosphorylation reached peak activations at 2.5 and 15 min, respectively. Only in HCEC, pharmacological manipulation of KCC activity modified the hypotonicity-induced activation of p44/42MAPK, whereas p38MAPK phosphorylation was insensitive to such procedures in both cell lines. Larger increases in HCEC KCC1 membrane protein content correlate with their ability to undergo faster and more complete RVD. Furthermore, pharmacological activation of KCC increased p44/42MAPK phosphorylation in HCEC but not in RCEC, presumably a reflection of low KCC membrane expression in RCEC. These findings suggest that KCC1 plays a role in (i) maintaining isotonic steady-state cell volume homeostasis, (ii) recovery of isotonic cell volume after a hypotonic challenge through RVD, and (iii) regulating hypotonicityinduced activation of the p44/42MAPK signaling pathway required for cell proliferation.

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

“…We recently described the role of KCC1 in mediating hypotonicityinduced activation of the p44/42MAPK pathway in HCEC. 24 Since p44/42MAPK and p38MAPK activation play vital roles in the mitogenic and migratory signals induced by EGF in CEC, respectively, we examined the involvement of KCC in mediating EGF-induced MAPK pathway activation. 14,30 Consistent with previous reports on HCEC, 10 ng/ml EGF induced a time-dependent biphasic change in the phosphorylation status of p44/42MAPK and p38MAPK (Fig.…”

“…These results regarding the inability of DIOA and NEM to modulate EGF-induced phosphorylation of p38MAPK are in agreement with the previously reported failure of these reagents to alter hypotonicity-induced activation of this pathway. 24 KCC mediates EGF-induced cell proliferation. Because EGF-induced p44/42MAPK activationrequired to induce a mitogenic responseis actively modulated by KCC, we examined the involvement of KCC in mediating EGF-induced cell proliferation.…”

“…The similarities in time course and extent of activation of the MAPK pathways stimulated by EGF in this study and those previously reported to be induced by hypotonicity suggest that these stimuli have common upstream components in their signaling pathways, which are implicated in inducing volume regulation, as well as in cell proliferation and migration. 24 To evaluate the involvement of KCC in mediating EGF-induced p44/42MAPK and p38MAPK activation, HCEC were pretreated with the KCC inhibitor, DIOA, or the KCC activator, NEM. EGF-induced increases in phospho-p44/42MAPK were completely abolished by DIOA (100 mM), but augmented in the presence of 1 mM NEM (eight-fold) (Fig.…”

“…Cell lysates were prepared as previously described in ref. 24. In brief, HCEC were grown in 100-mm culture dishes (Fisher Scientific, Pittsburgh, PA) to 60-70% confluence and subjected to various indicated experimental conditions.…”

“…24 In that study, KCC1's role was established in maintaining cell volume homeostasis and, most importantly, its involvement in mediating hypotonicity-induced p44/42MAPK, but not p38MAPK, activation in HCEC. However, the role(s) of KCC in modulating EGF-induced p44/42MAPK and p38MAPK activation and proliferation of HCEC has not been evaluated.…”