Delayed disruption of barrier function in cultured human corneal epithelial cells induced by tumor necrosis factor-alpha in a manner dependent on NF-kappaB.
OBJECTIVE
The corneal epithelium provides a barrier that is both important for corneal homeostasis and dependent on tight junctions (TJs) between adjacent epithelial cells. The authors examined the effects of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, on barrier function and the expression of TJ proteins in simian virus 40-transformed human corneal epithelial (HCE) cells.


METHODS
The barrier function of cultured HCE cells was evaluated by measurement of transepithelial electrical resistance (TER). The subcellular distribution of the TJ proteins zonula occludens-1 (ZO-1) and occludin and that of the p65 subunit of nuclear factor-kappaB (NF-kappaB) were determined by immunofluorescence staining. The expression of ZO-1 and occludin and the phosphorylation and degradation of the NF-kappaB inhibitory protein IkappaB-alpha were examined by immunoblot analysis.


RESULTS
TNF-alpha induced a decrease in the TER of HCE cells in a concentration- and time-dependent manner. It also induced the disappearance of ZO-1 from the interfaces of neighboring HCE cells without affecting the localization of occludin. The abundance of neither ZO-1 nor occludin was affected by TNF-alpha. TNF-alpha induced the phosphorylation and downregulation of IkappaB-alpha and the translocation of the p65 subunit of NF-kappaB to the nucleus. The NF-kappaB inhibitor curcumin blocked the effects of TNF-alpha on TER and the subcellular localization of ZO-1 at late phase.


CONCLUSIONS
TNF-alpha disrupted the barrier function of HCE cells, apparently by affecting the localization of ZO-1 at TJs in a manner dependent on NF-kappaB at late phase. This action of TNF-alpha may contribute to the loss of corneal epithelial barrier function associated with ocular inflammation.
Kimura
Kazuhiro
K
Department of Ocular Pathophysiology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan. k.kimura@yamaguchi-u.ac.jp
Teranishi
Shinichiro
S
Fukuda
Ken
K
Kawamoto
Koji
K
Nishida
Teruo
T
D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003474:Curcumin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D017097:Electric Impedance; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D062826:Zonula Occludens-1 Protein
D020672:I-kappa B Proteins; D008565:Membrane Proteins; C000606204:NFKBIA protein, human; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; C569042:TJP1 protein, human; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D062826:Zonula Occludens-1 Protein; D000072000:NF-KappaB Inhibitor alpha; D003474:Curcumin