Claudin-1 contributes to the epithelial barrier function in MDCK cells
Claudin-1 contributes to the epithelial barrier function in MDCK cells
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DOI：
10.1016/s0171-9335(99)80086-7
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发表时间：
1999-12-01
影响因子：
6.6
通讯作者：
Shibata, Y
中科院分区：
文献类型：
--
作者：
Inai, T;Kobayashi, J;Shibata, Y
紧密连接（TJ）形成细胞旁通透性屏障，并且还充当防止顶端和基底外侧质膜之间蛋白质和脂质混合的栅栏。最近，claudin-1被鉴定为定位于TJ的整合膜蛋白，并且引入claudin-1可以在成纤维细胞中形成类似TJ的网络。为了研究claudin-1的功能，用含有myc标记的小鼠claudin-1的哺乳动物表达载体转染MDCK细胞，并获得了4个稳定的克隆。 myc 标记的claudin-1 与occludin 和ZO-1 在细胞-细胞接触位点精确共定位，表明外源claudin-1 正确靶向TJ。免疫印迹分析显示claudin-1 的过表达增加了ZO-1 的表达，但不增加occludin 或ZO-2 的表达。通过跨上皮电阻（TER）和细胞旁细胞评估这些细胞的屏障功能。表达 Claudin-1 的细胞表现出比野生型 MDCK 细胞高约四倍的 TER。与 TER 的增加一致，过表达claudin-1的细胞表现出细胞旁通量减少，估计为 4 和 40 kD FITC-葡聚糖。这些结果表明claudin-1 参与TJ 的屏障功能。
Tight junctions (TJs) create a paracellular permeability barrier and also act as a fence preventing intermixing of proteins and lipids between the apical and basolateral plasma membranes. Recently, claudin-1 has been identified as an integral membrane protein localizing at TJs, and introduced claudin-1 can form TJ-like networks in fibroblasts. To investigate the function of claudin-1, MDCK cells were transfected with a mammalian expression vector containing myc-tagged mouse claudin-1, and four stable clones were obtained. The myc-tagged claudin-1 precisely colocalized with both occludin and ZO-1 at cell-cell contact sites, indicating that exogenous claudin-1 was properly targeted to the TJs, Immunoblot analysis revealed that overexpression of claudin-1 increased expression of ZO-1 but not of occludin or ZO-2. The barrier functions of these cells were evaluated by transepithelial electrical resistance (TER) and paracellular nux. Claudin-1-expressing cells exhibited about four times higher TER than wild-type MDCK cells, Consistent with the increase of TER, the cells overexpressing claudin-1 showed reduced paracellular flux, estimated at 4 and 40 kD FITC-dextrans. These results suggest that claudin-1 is involved in the barrier function at TJs.