Protection of human corneal epithelial cells from hypoxia-induced disruption of barrier function by keratinocyte growth factor

PURPOSE: The possible detrimental effect of hypoxia on the barrier function of corneal epithelial cells and whether keratinocyte growth factor (KGF) might protect against such an effect were investigated. METHODS: Simian virus 40-transformed human corneal epithelial (HCE) cells were cultured for 4 days to allow the establishment of barrier function. They were then deprived of serum for 24 hours before exposure to 1% (hypoxia) or 21% (normoxia) oxygen for 24 hours. Barrier function was evaluated by measurement of transepithelial electrical resistance (TER). The localization of ZO-1 and occludin was determined by immunofluorescence microscopy, and the expression of these tight junctional proteins as well as the phosphorylation of the mitogen-activated protein kinases ERK, p38, and JNK were examined by immunoblot analysis. RESULTS: Hypoxia induced a decrease in the TER of HCE cells compared with that of cells maintained under normoxia. The localization of ZO-1 at cell-cell borders was disrupted by hypoxia, whereas the distribution of occludin was not affected. Hypoxia also induced the downregulation of ZO-1 and a decrease in the phosphorylation of ERK without affecting the phosphorylation of p38 or JNK. All these effects of hypoxia were inhibited by KGF. The effects of KGF on TER and ZO-1 localization in cells exposed to hypoxia were inhibited by PD98059, an inhibitor of ERK signaling. Neither hypoxia nor KGF exhibited mitogenic or cytotoxic effects in HCE cells. CONCLUSIONS: Hypoxia induces disruption of the barrier function of HCE cells by eliciting the redistribution and degradation of ZO-1, and this effect is inhibited by KGF in a manner dependent on ERK activation.