title: Inhibition of gap junction-mediated intercellular communication by TNF-alpha in cultured human corneal fibroblasts.
abstract: 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.
authors.affiliation: Department of Biomolecular Recognition and Ophthalmology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan.
authors.forename: Ji-Long
authors.initials: JL
authors.lastname: Hao
authors.forename: Katsuyoshi
authors.initials: K
authors.lastname: Suzuki
authors.forename: Ying
authors.initials: Y
authors.lastname: Lu
authors.forename: Shinji
authors.initials: S
authors.lastname: Hirano
authors.forename: Ken
authors.initials: K
authors.lastname: Fukuda
authors.forename: Naoki
authors.initials: N
authors.lastname: Kumagai
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Kimura
authors.forename: Teruo
authors.initials: T
authors.lastname: Nishida
mesh_terms: 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*
chemical_list: D018031:Connexin 43; D007546:Isoquinolines; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C017475:lucifer yellow