Abstract
Purpose: :
During exposure of corneal epithelial cells to a hypertonic stress, their ability to survive and proliferate is dependent on cell volume regulatory mechanisms and cell signaling pathways. However, the types of interactions that occur between these processes are poorly understood. This study performed to evaluate if the fate of SV40–immortalized human (HCEC) and rabbit (RCEC) corneal epithelial cells is dependent on the: 1) degree of the hypertonic challenge; 2) changes of MAPK superfamily limbs activation; 3) magnitude of Na:K:2Cl cotransporter (NKCC) stimulation.
Methods: :
Cells were incubated in hypertonic media up to 600 mOsm for up to 24 h. Phosphorylated forms of p44/42, p38MAPK and SAPK/JNK were immunoprecipitated from cell lysates. Amounts of activated MAPKs and NKCC associated with each of them were evaluated with Western blot/ECL assay. DNA integrity was assessed by electrophoresis in a 2% agarose gel. Protein content measurement determined cell amount.
Results: :
Incubation of HCEC in 325–350 mOsm media caused increases of up to 75% in proliferation whereas in RCEC such increases were limited to less than 10%. These rises were preceded by corresponding transient activations of p44/42MAPK. In both species, there were osmolarity–dependent decreases in p44/42MAPK activity, cell proliferation and increased rates of apoptosis. The higher RCEC survival rate was associated with more rapid and larger (i.e.10–fold) elevations in their p38MAPK activity, and near complete restoration of p44/42MAPK activity after the initial 30 min. In HCEC, 4 h exposure to 600 mOsm led to more than a 150–fold increase in SAPK/JNK activity and extensive cell death (apoptosis). In RCEC, such a challenge induced instead only moderate increases in SAPK/JNK activity and cell death. In both cell lines, the amount of NKCC that coimmunoprecipitated with phospho–p38MAPK was proportional to the magnitudes of their activation levels. However, no such associations occurred between phosphorylated p44/42MAPK or SAPK/JNK and NKCC amounts. Under isotonic conditions, following inhibition of RCEC and HCEC NKCC activity with 50 µM bumetanide, p44/42MAPK activity declined by 15% and 60%, respectively. Such declines led to proportional decreases in cell survival.
Conclusions: :
Survival of hypertonicity–stressed corneal epithelial cells depends both on p38MAPK activation capacity and the ability of p38MAPK to stimulate NKCC activity through protein–protein interaction. The level of NKCC activation affects the extent of cell volume recovery and in turn epithelial survival capacity.
Keywords: cornea: epithelium • cell survival • signal transduction