Delayed disruption of barrier function in cultured human corneal epithelial cells induced by tumor necrosis factor-α in a manner dependent on NF-κB
Delayed disruption of barrier function in cultured human corneal epithelial cells induced by tumor necrosis factor-α in a manner dependent on NF-κB
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DOI：
10.1167/iovs.07-0419
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发表时间：
2008-02-01
影响因子：
4.4
通讯作者：
Nishida, Teruo
中科院分区：
文献类型：
--
作者：
Kimura, Kazuhiro;Teranishi, Shinichiro;Nishida, Teruo
目的.角膜上皮提供了一个屏障，它对角膜稳态很重要，并依赖于相邻上皮细胞之间的紧密连接（TJ）。作者研究了肿瘤坏死因子-α（TNF-α）（一种促炎细胞因子）对猴病毒40转化的人角膜上皮（HCE）细胞屏障功能和TJ蛋白表达的影响。通过测量跨上皮电阻（TER）来评价培养的HCE细胞的屏障功能。用免疫荧光染色法检测TJ蛋白封闭小带-1（ZO-1）和封闭蛋白（occludin）以及核因子-κ B（NF-κ B）p65亚基的亚细胞分布。ZO-1和occludin的表达以及NF-κ B抑制蛋白I κ B-α的磷酸化和降解通过免疫印迹分析进行检测。TNF-α以浓度和时间依赖性方式诱导HCE细胞的TER降低。它还诱导ZO-1从邻近HCE细胞的界面消失，而不影响occludin的定位。ZO-1和occludin的丰度都不受TNF-α的影响。TNF-α诱导I κ B-α的磷酸化和下调，以及NF-κ B的p65亚基向细胞核的移位。NF-κ B B抑制剂姜黄素阻断了TNF-α对TER的影响，并阻断了ZO-1在晚期的亚细胞定位。TNF-α破坏了HCE细胞的屏障功能，显然是通过影响ZO-1在TJ的定位，其方式依赖于晚期的NF-κ B。TNF-α的这种作用可能导致与眼部炎症相关的角膜上皮屏障功能丧失。
PURPOSE. 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-kappa B (NF-kappa B) were determined by immunofluorescence staining. The expression of ZO-1 and occludin and the phosphorylation and degradation of the NF-kappa B inhibitory protein I kappa B-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 I kappa B-alpha and the translocation of the p65 subunit of NF-kappa B to the nucleus. The NF-kappa B 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-kappa B at late phase. This action of TNF-alpha may contribute to the loss of corneal epithelial barrier function associated with ocular inflammation.