Tsukamoto T, Nigam S K
Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Jun 27;272(26):16133-9. doi: 10.1074/jbc.272.26.16133.
A key feature of the ischemic epithelial cell phenotype is the disruption of tight junctions (TJ). In a Manin-Darby canine kidney cell model for ischemia-reperfusion/hypoxia-reoxygenation injury which employs inhibitors of glycolysis (2-deoxy-D-glucose) and oxidative phosphorylation (antimycin A), transepithelial electrical resistance, a measure of TJ integrity, dropped rapidly, correlating well with declining ATP levels. Although immunocytochemical studies revealed only subtle changes in the distribution of the TJ proteins, zonula occludens (ZO)-1, ZO-2, and cingulin, examination of the Triton X-100 solubilities of these proteins, an indicator of cytoskeletal association, revealed a striking shift of all three TJ proteins into the insoluble pool, consistent with increased cytoskeletal interaction during ATP depletion. In addition, rate-zonal centrifugation analysis of a detergent-soluble fraction showed an increase in the amount of ZO-1 and ZO-2 in high density fractions following ATP depletion, providing further evidence for association of TJ proteins into a large complex possibly involving the cytoskeleton. Analysis of immunoprecipitation data from [35S]methionine-labeled cells revealed that ATP depletion led to the association of a 240-kDa protein with the ZO-1-containing complex. Western blots of this protein immunoprecipitated with anti-ZO-1 antibodies confirmed its identity as fodrin, a protein believed to link membrane and other proteins to the actin-based cytoskeleton. Together, our data suggest that in the absence of major immunocytochemical changes, ATP depletion leads TJ proteins to form large insoluble complexes and associate with the cytoskeleton. We propose a model in which a key, potentially regulated, step in the generation of the ischemic epithelial cell phenotype is the interaction between TJ proteins and fodrin and/or other cytoskeletal proteins.
缺血性上皮细胞表型的一个关键特征是紧密连接（TJ）的破坏。在一个用于缺血再灌注/缺氧复氧损伤的犬肾细胞模型中，该模型使用糖酵解抑制剂（2-脱氧-D-葡萄糖）和氧化磷酸化抑制剂（抗霉素A），跨上皮电阻（一种衡量TJ完整性的指标）迅速下降，与ATP水平的下降密切相关。尽管免疫细胞化学研究仅揭示了TJ蛋白（闭合蛋白（ZO）-1、ZO-2和cingulin）分布的细微变化，但对这些蛋白的Triton X-100溶解性（一种细胞骨架关联指标）的检测显示，所有三种TJ蛋白都显著转移到不溶性组分中，这与ATP耗竭期间细胞骨架相互作用增加一致。此外，对去污剂可溶部分的速率区带离心分析表明，ATP耗竭后高密度组分中ZO-1和ZO-2的量增加，这为TJ蛋白形成可能涉及细胞骨架的大型复合物提供了进一步证据。对[35S]甲硫氨酸标记细胞的免疫沉淀数据的分析表明，ATP耗竭导致一种240 kDa的蛋白与含ZO-1的复合物结合。用抗ZO-1抗体免疫沉淀该蛋白的蛋白质免疫印迹证实其为血影蛋白，一种被认为将膜和其他蛋白与基于肌动蛋白的细胞骨架连接的蛋白。总之，我们的数据表明，在没有重大免疫细胞化学变化的情况下，ATP耗竭导致TJ蛋白形成大型不溶性复合物并与细胞骨架结合。我们提出了一个模型，其中缺血性上皮细胞表型产生过程中的一个关键且可能受调控的步骤是TJ蛋白与血影蛋白和/或其他细胞骨架蛋白之间的相互作用。
