Inhibition of gap junction-mediated intercellular communication by TNF-α in cultured human corneal fibroblasts
Inhibition of gap junction-mediated intercellular communication by TNF-α in cultured human corneal fibroblasts
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DOI：
10.1167/iovs.04-0840
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发表时间：
2005-04-01
影响因子：
4.4
通讯作者：
Nishida, T
中科院分区：
文献类型：
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作者：
Hao, JL;Suzuki, K;Nishida, T
目的.角膜细胞通过缝隙连接相互连接，缝隙连接介导细胞间的通讯并有助于维持角膜的稳态。本研究探讨了促炎细胞因子肿瘤坏死因子（TNF）-α对培养的人角膜成纤维细胞间隙连接细胞间通讯（GJIC）的可能影响。通过观察荧光染料荧光黄的细胞间扩散来测量GJIC活性。通过免疫荧光和免疫印迹分析，用特异性单克隆抗体评估差距连接蛋白连接蛋白43（Cx43）的表达。通过定量逆转录和聚合酶链反应分析确定Cx43 mRNA的丰度。TNF-α诱导人角膜成纤维细胞中GJIC活性的时间和浓度依赖性降低。免疫荧光分析显示，TNF-α降低了相邻细胞之间接触部位的Cx43特异性染色水平。免疫印迹分析检测到四个特定的Cx43带，一个对应于非磷酸化形式的蛋白质和三个对应于磷酸化形式。细胞暴露于TNF-α降低了Cx43的三种磷酸化形式的相对丰度。结论TNF-α对Cx43 mRNA表达无影响。TNF-α抑制培养的人角膜成纤维细胞中的GJIC，这种作用可能是由Cx43的去磷酸化和随后的降解介导的。在角膜炎症过程中，角膜细胞中GJIC对TNF-α的下调可能导致角膜稳态的破坏。
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)-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.