Martínez-Estrada Ofelia M, Cullerés Albert, Soriano Francesc X, Peinado Hector, Bolós Victoria, Martínez Fernando O, Reina Manuel, Cano Amparo, Fabre Myriam, Vilaró Senén
Departament de Biologia Cellular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.
Biochem J. 2006 Mar 1;394(Pt 2):449-57. doi: 10.1042/BJ20050591.
Claudin-1 is an integral membrane protein component of tight junctions. The Snail family of transcription factors are repressors that play a central role in the epithelial-mesenchymal transition, a process that occurs during cancer progression. Snail and Slug members are direct repressors of E-cadherin and act by binding to the specific E-boxes of its proximal promoter. In the present study, we demonstrate that overexpression of Slug or Snail causes a decrease in transepithelial electrical resistance. Overexpression of Slug and Snail in MDCK (Madin-Darby canine kidney) cells down-regulated Claudin-1 at protein and mRNA levels. In addition, Snail and Slug are able to effectively repress human Claudin-1-driven reporter gene constructs containing the wild-type promoter sequence, but not those with mutations in two proximal E-box elements. We also demonstrate by band-shift assay that Snail and Slug bind to the E-box motifs present in the human Claudin-1 promoter. Moreover, an inverse correlation in the levels of Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of Claudin-1 expression. Taken together, these results support the hypothesis that Claudin-1 is a direct downstream target gene of Snail family factors in epithelial cells.
闭合蛋白-1是紧密连接的一种整合膜蛋白成分。蜗牛家族转录因子是阻遏物，在上皮-间质转化过程中起核心作用，该过程发生于癌症进展期间。蜗牛蛋白和蛞蝓蛋白成员是E-钙黏蛋白的直接阻遏物，通过与E-钙黏蛋白近端启动子的特定E盒结合发挥作用。在本研究中，我们证明蛞蝓蛋白或蜗牛蛋白的过表达会导致跨上皮电阻降低。在MDCK（麦迪逊-达比犬肾）细胞中过表达蛞蝓蛋白和蜗牛蛋白会在蛋白质和mRNA水平下调闭合蛋白-1。此外，蜗牛蛋白和蛞蝓蛋白能够有效抑制含有野生型启动子序列的人闭合蛋白-1驱动的报告基因构建体，但不能抑制两个近端E盒元件发生突变的构建体。我们还通过凝胶迁移试验证明蜗牛蛋白和蛞蝓蛋白与人闭合蛋白-1启动子中存在的E盒基序结合。此外，在乳腺癌细胞系中观察到闭合蛋白-1和蛞蝓蛋白转录物水平呈负相关。在表达低水平闭合蛋白-1转录物的乳腺癌细胞系中，闭合蛋白-1启动子中的E盒元件发挥关键的负调控作用。值得注意的是，在侵袭性人类乳腺肿瘤中，高水平的蜗牛蛋白和蛞蝓蛋白与低水平的闭合蛋白-1表达相关。综上所述，这些结果支持闭合蛋白-1是上皮细胞中蜗牛家族因子的直接下游靶基因这一假说。
