IOVS Clinical Chemistry
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(Investigative Ophthalmology and Visual Science. 2008;49:1827-1835.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-1142
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NFAT5 Induction and Its Role in Hyperosmolar Stressed Human Limbal Epithelial Cells

Joon H. Lee,1 Min Kim,2 Young Sun Im,2 Wungrak Choi,2 Suk Ho Byeon,2 and Hyung Keun Lee2,3

1From the Myung-gok Eye Research Institute, College of Medicine, Konyang University, ChungNam, Korea; the 2Institute of Vision Research, Department of Ophthalmology, and the 3Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, Korea.

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-{kappa}B (nuclear factor-{kappa}B), and MAPK activity. TNF-{alpha} and IL-1β, -6, and -8 levels were determined after inhibition of NFAT5 and/or NF-{kappa}B. 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-1β 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-{kappa}B and NFAT5.

CONCLUSIONS. NFAT5 is induced and translocates to the nucleus in HLECs undergoing hyperosmolar stress through activation of p38. IL-1 β 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.






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