Potassium-chloride cotransporter mediates cell cycle progression and proliferation of human corneal epithelial cells.

Epidermal growth factor (EGF)-induced proliferation of corneal epithelial cells contributes to its renewal, which maintains the protective and refractive properties of the cornea. This study characterized in human corneal epithelial cells (HCEC) the role of the potassium-chloride cotransporter (KCC) in mediating (1) EGF-induced mitogen-activated protein kinase (MAPK) pathway activation; (2) increases in cell cycle progression; and (3) proliferation. The KCC inhibitor [(dihydroindenyl)oxy] alkanoic acid (DIOA) and KCC activator N-ethylmaleimide (NEM), suppressed and enhanced EGF-induced p44/42MAPK activation, respectively. Such selective modulation was mirrored by corresponding changes in cell proliferation and shifts in cell cycle distribution. DIOA induced a 20% increase in G(0)/G(1)-phase cell population, whereas NEM induced a 22% increase in the proportion of cells in the G(2)/M-phase and accelerated the transition from G(0)/G(1)-phase to the S-phase. Associated with these changes, KCC1 content in a plasma membrane enriched fraction increased by 300%. Alterations in regulatory volume capacity were associated with corresponding changes in both KCC1 membrane content and activity. These results indicate that EGF-induced increases in KCC1 activity and content modulate cell volume changes required for (1) activation of the p44/42MAPK signaling pathway, (2) cell cycle progression, and (3) increases in cell proliferation.

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

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, N-ethylmaleimide (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 correlated 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 KCC1 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 hypotonicity-induced activation of the p44/42MAPK signaling pathway required for cell proliferation.

Differential dependence of regulatory volume decrease behavior in rabbit corneal epithelial cells on MAPK superfamily activation.

We characterized the dependence of hypotonicity-induced regulatory volume decrease (RVD) responses on mitogen-activated protein kinase (MAPK) pathway signaling in SV40-immortalized rabbit corneal epithelial cells (RCEC). Following calcein-AM loading, RVD was monitored using a microplate fluorescence reader. Western blot analysis determined MAPK activation. After 30 min, the RVD response restored the relative cell volume to nearly isotonic values, whereas it was inhibited when cells were bathed either in a Cl- -free solution or with the Cl- -channel inhibitors: 5-nitro-2-(3-phenylpropylamino)benzoic acid or niflumic acid. Similar declines occurred with either a high-K+ (20 mM) supplemented solution or the K+ channel inhibitor 4-aminopyridine. Activation of extracellular signal-regulated kinase (ERK), p38, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was time and tonicity-dependent. Stimulation of ERK and SAPK/JNK was maximized earlier than that of p38. Activation of ERK and SAPK/JNK was insensitive to Cl- and K+ channel inhibitors, whereas inhibition with either PD98059 or SP600125, respectively, blocked RVD. However, inhibition of p38 with SB203580had no effect on RVD. Suppression of RVD instead blocked p38 activation. Differences in the dependence of RVD activation on Erk1/2 and p38 signaling were validated in dominant negative (d/n)-Erk1 and d/n-p38 cells. Volume-sensitive Cl- and K+ channel activation contributes, in concert, to RVD in RCEC. Therefore, swelling-induced ERK and SAPK/JNK stimulation precedes Cl- and K+ channel activation, whereas p38 activation occurs as a consequence of RVD.

Fate of hypertonicity-stressed corneal epithelial cells depends on differential MAPK activation and p38MAPK/Na-K-2Cl cotransporter1 interaction.

The capacity of the corneal epithelium to adapt to hypertonic challenge is dependent on the ability of the cells to upregulate the expression and activity of cell membrane-associated Na-K-2Cl cotransporter1 (NKCC1). Yet, the signaling pathways that control this response during hypertonic stress are still unclear. We studied stress-induced changes in proliferation and survival capacity of SV40-immortalized human (HCEC) and rabbit (RCEC) corneal epithelial cells as a function of (i) the magnitude of the hypertonic challenge, (ii) differential changes in activation of mitogen-activated protein kinase (MAPK), and (iii) the extent of p38MAPK interaction with NKCC1. Cells were incubated in hypertonic (up to 600 mOsm) media for varying time periods up to 24 h. Phosphorylated forms of p44/42, p38, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) MAPK were immunoprecipitated from cell lysates, and the amount of each activated NKCC1-associated MAPK was evaluated by Western blot/ECL assay. DNA integrity was assessed by electrophoresis in a 2% agarose gel. Cell survival and proliferation were evaluated based on three criteria: protein content, cell count, and the MTT assay. Exposure to media of 325-350 mOsm increased proliferation of HCEC up to 75%, whereas this response was limited to <16% in RCEC. At higher osmolarities, cell proliferation decreased in both species. SAPK/JNK activity increased 150-fold in HCEC and <10-fold in RCEC, while DNA fragmentation occurred only in HCEC. Compared to HCEC, the better RCEC survival rate was associated with higher p38MAPK activity and near complete restoration of p44/42MAPK activity after the first 30 min. In both cell lines, the amount of phospho-NKCC1 that coimmunoprecipitated with phospho-p38MAPK was proportional to the magnitudes of their respective activation levels. However, no such associations occurred between amounts of phosphorylated p44/42MAPK or SAPK/JNK and phospho-NKCC1. Under isotonic conditions, with bumetanide-induced inhibition of RCEC and HCEC NKCC1 activities, p44/42MAPK activity declined by 40 and 60%, respectively. Such declines led to proportional decreases in cell proliferation. Survival of hypertonicity-stressed corneal epithelial cells depends both on p38MAPK activation capacity and the ability of p38MAPK to stimulate NKCC1 activity through protein-protein interaction. The level of NKCC1 activation affects the extent of cell volume recovery and, in turn, epithelial survival capacity.