Inhibition of gap junction-mediated intercellular communication by TNF-alpha in cultured human corneal fibroblasts.
OBJECTIVE
Keratocytes are connected to each other by gap junctions, which mediate intercellular communication and contribute to maintenance of corneal homeostasis. The possible effect of tumor necrosis factor (TNF)-alpha, a proinflammatory cytokine, on gap junctional intercellular communication (GJIC) in cultured human corneal fibroblasts was examined.


METHODS
GJIC activity was measured by observing the intercellular diffusion of the fluorescent dye Lucifer yellow. The expression of the gap junction protein connexin43 (Cx43) was evaluated by immunofluorescence and immunoblot analyses with a specific monoclonal antibody. The abundance of Cx43 mRNA was determined by quantitative reverse transcription and polymerase chain reaction analysis.


RESULTS
TNF-alpha induced a time- and concentration-dependent decrease in GJIC activity in human corneal fibroblasts. Immunofluorescence analysis revealed that TNF-alpha reduced the level of specific staining for Cx43 at sites of contact between adjacent cells. Immunoblot analysis detected four specific Cx43 bands, one corresponding to the nonphosphorylated form of the protein and three corresponding to phosphorylated forms. Exposure of cells to TNF-alpha reduced the relative abundance of the three phosphorylated forms of Cx43. The amount of Cx43 mRNA was not affected by TNF-alpha.


CONCLUSIONS
TNF-alpha inhibited GJIC in cultured human corneal fibroblasts, an effect that was possibly mediated by dephosphorylation and consequent degradation of Cx43. The downregulation of GJIC among keratocytes in response to TNF-alpha may contribute to the breakdown of corneal homeostasis during corneal inflammation.
Hao
Ji-Long
JL
Department of Biomolecular Recognition and Ophthalmology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan.
Suzuki
Katsuyoshi
K
Lu
Ying
Y
Hirano
Shinji
S
Fukuda
Ken
K
Kumagai
Naoki
N
Kimura
Kazuhiro
K
Nishida
Teruo
T
D002450:Cell Communication / Q000187:drug effects*; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D003319:Corneal Stroma / Q000166:cytology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007546:Isoquinolines / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D018031:Connexin 43; D007546:Isoquinolines; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C017475:lucifer yellow