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

Abstract

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.

Dependence of Corneal Epithelial Cell Proliferation on Modulation of Interactions Between ERK1/2 and NKCC1

“…Panel B shows that substitution of the control 306 mOsm NaCl Ringers with one diluted by 50% induced after about 1 min a transient increase in fluorescence reflective of swelling since calcein is a self-quenching dye (n=4 coverslips; p<0.005 versus isotonic control). The pattern of this transient was very similar to that induced by EGF, but instead is reflective of hypotonic-induced stimulation of KCC and K + channel activity rather than NKCC1 activation by EGF shown in panel A [7,17].…”

“…In HCEC, some of the downstream signaling events induced by EGFR stimulation include transient activation of the MAPK superfamily as well as the PI3-K/Akt signaling cascade, PKC and time-dependent stimulation of K + channel, KCC along with increases in NKCC1 activity [7][8][9][31][32][33]. All of these changes are linked to NF-B activation whose p50/p65 subunits induce promoter activation leading to the expression of genes underlying proliferation [3].…”

“…Such a stress induces a regulatory volume decrease response leading initially to a decrease in cell volume below the isotonic volume. This decline occurs in HCEC as a consequence of a net loss of KCl mediated by stimulation of K + channel activity and KCC activity [7,8,19]. Isotonic volume is eventually restored subsequent to a volume overshoot below the isotonic volume.…”

“…Swelling is needed to accommodate the increases in chromatin content for preserving its equivalence between the parental and daughter cells. In the corneal epithelium, the aforementioned increases in K + channel, KCC and NKCC1 activity occurring during cell cycle progression can also be induced during exposure to an anisoosmotic challenge resulting in activation of regulatory volume behavior by HCEC and rabbit corneal epithelial cells [7,[15][16][17][18][19]. Exposure to a hypertonic challenge initially induces shrinkage followed by restoration of isotonic volume due to stimulation of NKCC1 activity.…”

“…A number of studies indicate that EGFR activation elicits control of these responses through transient stimulation of a myriad of interacting cell signaling kinase pathways [4][5][6]. One aspect of this signaling process entails EGF stimulation of KCl cotransporters (KCC), K + channels and Na:K:2Cl cotransporter 1 (NKCC1) activity [7][8][9]. However, it is uncertain if ion transport stimulation leads to cell volume swelling and if NKCC1 phosphorylation is reflective of its activation by EGF.…”