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Ji-Long Hao, Katsuyoshi Suzuki, Ying Lu, Shinji Hirano, Ken Fukuda, Naoki Kumagai, Kazuhiro Kimura, Teruo Nishida; Inhibition of Gap Junction–Mediated Intercellular Communication by TNF-α in Cultured Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2005;46(4):1195-1200. doi: 10.1167/iovs.04-0840.
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purpose. 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)-α, 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-α induced a time- and concentration-dependent decrease in GJIC activity in human corneal fibroblasts. Immunofluorescence analysis revealed that TNF-α 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-α reduced the relative abundance of the three phosphorylated forms of Cx43. The amount of Cx43 mRNA was not affected by TNF-α.
conclusions. TNF-α 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-α may contribute to the breakdown of corneal homeostasis during corneal inflammation.
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