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

Abstract

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.

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

“…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.…”

“…5,43 Given that, EGF stimulates both NKCC1 and KCC1-leading to net increases in osmolyte influx and efflux, respectively, and changes in cell volume-coordinated regulation of ion transport pathway activity and their induced transient changes in cell volume are part of the mitogenic response required for corneal epithelial renewal. The involvement of such ion transporter mechanisms in mediating responses to mitogens indicates that their activation is an important component of the second messenger cascade linking receptor stimulation to increases in proliferation.…”

“…Cell proliferation was assessed by direct cell count as previously described in ref. 5. In brief, previously starved HCEC were plated at a density of 10 5 in 60-mm dishes and allowed to attach for 3 h. Cells were pretreated for 30 min, with or without DIOA and NEM prior to supplementing the media with EGF, and allowed to grow for up to 72 h. Cells were harvested at 24, 48 and 72 h by trypsinization followed by counting them with the aid of a hematocytometer, using trypan blue exclusion (0.08%) to monitor viability.…”

“…[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.…”

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

“…Its inhibition suppressed such response. RVI is mediated by p38 protein-protein interaction with the Na:K:2Cl cotransporter 1 (NKCC1), which induces increases in osmolyte uptake followed by rises in NKCC1 gene and protein expression (Bildin et al, 2003;Capo-Aponte et al, 2007b). This dependence of RVI induction on p38 activation also exists in the renal medullary thick ascending limb of Henle's loop (Bustamante et al, 2003;Roger et al, 1999).…”