NFAT5 Induction and Its Role in Hyperosmolar Stressed Human Limbal Epithelial Cells

Purpose: To introduce a tonicity response gene regulator, NFAT (nuclear factor of activated T-cell)-5 and determine its expression mechanism and specific roles in human limbal epithelial cell (HLECs) subjected to hyperosmolar stress.

Methods: NFAT5 expression was determined in various hyperosmolar conditions in HLECs by RT-PCR and Western immunoblot analyses. NFAT5 translocation during hyperosmolar stress was observed by immunocytochemistry. NFAT5-related signal transduction activity was measured on the basis of inhibition of NF-kappaB (nuclear factor-kappaB), and MAPK activity. TNF-alpha and IL-1beta, -6, and -8 levels were determined after inhibition of NFAT5 and/or NF-kappaB. Hyperosmotic apoptotic cell death, with or without inhibition of NFAT5, was measured by flow cytometry.

Results: NFAT5 was induced and translocated to the nucleus under conditions of hyperosmolar stress. It was inhibited by SB239063, a p38 MAPK inhibitor. Among the inflammatory cytokines induced in hyperosmolar stress conditions, IL-1beta and TNF-alpha levels were significantly reduced after inhibition of NFAT5. Of interest, even after 48 hours of hyperosmolar stress, 45% of HLECs survived. HLEC apoptosis increased markedly as a result of NFAT5 suppression. Moreover, most of the HLECs underwent cell death on dual inhibition of NF-kappaB and NFAT5.

Conclusions: NFAT5 is induced and translocates to the nucleus in HLECs undergoing hyperosmolar stress through activation of p38. IL-1 beta and TNF-alpha are induced via NFAT5 activation. Our data collectively indicate that NFAT5 may be an important gene regulator and survival factor in hyperosmolar stressed HLECs.