NFAT5 induction and its role in hyperosmolar stressed human limbal epithelial cells.
OBJECTIVE
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.
Lee
Joon H
JH
Myung-gok Eye Research Institute, College of Medicine, Konyang University, ChungNam, Korea.
Kim
Min
M
Im
Young Sun
YS
Choi
Wungrak
W
Byeon
Suk Ho
SH
Lee
Hyung Keun
HK
D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology*; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D009997:Osmotic Pressure; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012462:Saline Solution, Hypertonic / Q000494:pharmacology; D015398:Signal Transduction; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D004791:Enzyme Inhibitors; D053583:Interleukin-1beta; D016328:NF-kappa B; C118125:NFAT5 protein, human; D050778:NFATC Transcription Factors; D012462:Saline Solution, Hypertonic; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases