Yi X, Wang Y, Yu F S
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, USA.
Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4093-100.
To investigate the expression and cellular distribution of putative tight junction (TJ) proteins occludin, ZO-1, ZO-2, and claudin-1 in rat corneal epithelium and alterations of TJs in cultured human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge.
Immunohistochemistry was used to determine tissue distribution of occludin, ZO-1, ZO-2, and claudin-1 in the rat cornea. Reverse transcription-polymerase chain reaction was used to reveal the expression of mRNAs for claudins in simian virus (SV)40-immortalized human corneal epithelial (THCE) cells. To assess epithelial response to LPS challenge, THCE cells were cultured on the upper chamber of Transwell filters (Costar, Cambridge, MA), transepithelial electrical resistance (TER) was measured using a voltohmmeter. Immunocytochemistry and immunoblotting were used to assess alteration in the levels and localization of TJ-associated proteins occludin, ZO-1, and ZO-2 in LPS-treated THCE cells.
Occludin, ZO-1, and ZO-2 were found at the cell borders of the superficial layer, whereas claudin-1 was localized mainly in the basal and wing cell layers of rat corneal epithelium. In addition to claudin-1, the transcripts for several other isotypes of claudins-2, -3, -7, -9, -14, and -15 were identified in THCE cells. Treatment of cultured THCE cells with LPS caused a dose- and time-dependent increase in monolayer permeability as assessed by TER measurements. The maximal decrease of TER was observed at approximately 6 to 9 hours after LPS challenge. The TER was then recovered gradually and returned to baseline after 24 hours. Examination of specific proteins associated with TJs by immunoblot analysis and immunomicroscopy revealed changes in the expression levels and localization of some of these proteins after their exposure to LPS. Specifically, LPS challenge resulted in a decrease in the levels of ZO-1 and ZO-2 compared with untreated cells. Reduction of the ZO-2 level was associated with the disappearance of ZO-2 staining from cell borders in 6-hour LPS-treated cells.
Occludin, ZO-1, and ZO-2, but not claudin-1, are components of corneal epithelial TJs. LPS induces breakdown of the epithelial barrier through disruption of TJs, and ZO-1 and ZO-2 are targets for the induction.
研究紧密连接（TJ）相关蛋白闭合蛋白、紧密连接蛋白1（ZO-1）、紧密连接蛋白2（ZO-2）和闭合蛋白1在大鼠角膜上皮中的表达及细胞分布，以及脂多糖（LPS）刺激后培养的人角膜上皮细胞中紧密连接的变化。
采用免疫组织化学法检测闭合蛋白、ZO-1、ZO-2和闭合蛋白1在大鼠角膜中的组织分布。运用逆转录-聚合酶链反应揭示猿猴病毒（SV）40永生化人角膜上皮（THCE）细胞中闭合蛋白的mRNA表达。为评估上皮细胞对LPS刺激的反应，将THCE细胞培养在Transwell小室滤膜（美国马萨诸塞州剑桥市康宁公司）的上室，使用电压电阻计测量跨上皮电阻（TER）。采用免疫细胞化学和免疫印迹法评估LPS处理的THCE细胞中TJ相关蛋白闭合蛋白、ZO-1和ZO-2的水平及定位变化。
闭合蛋白、ZO-1和ZO-2在大鼠角膜上皮表层细胞边界处被发现，而闭合蛋白1主要定位于大鼠角膜上皮的基底细胞层和翼状细胞层。除闭合蛋白1外，在THCE细胞中还鉴定出了几种其他同型闭合蛋白（闭合蛋白2、3、7、9、14和15）的转录本。通过TER测量评估，用LPS处理培养的THCE细胞导致单层通透性呈剂量和时间依赖性增加。LPS刺激后约6至9小时观察到TER的最大下降。然后TER逐渐恢复，24小时后恢复到基线水平。通过免疫印迹分析和免疫显微镜检查与紧密连接相关的特定蛋白，发现这些蛋白在暴露于LPS后其表达水平和定位发生了变化。具体而言，与未处理细胞相比，LPS刺激导致ZO-1和ZO-2水平降低。在LPS处理6小时的细胞中，ZO-2水平的降低与细胞边界处ZO-2染色的消失有关。
闭合蛋白、ZO-1和ZO-2是角膜上皮紧密连接的组成成分，而闭合蛋白1不是。LPS通过破坏紧密连接诱导上皮屏障的破坏，ZO-1和ZO-2是诱导的靶点。
