Woo P L, Cha H H, Singer K L, Firestone G L
Department of Molecular and Cell Biology, University of California at Berkeley 94720, USA.
J Biol Chem. 1996 Jan 5;271(1):404-12. doi: 10.1074/jbc.271.1.404.
The synthetic glucocorticoid, dexamethasone, stimulated the transepithelial electrical resistance and suppressed the DNA synthesis of 31EG4 nontransformed mouse mammary epithelial cells. The addition of transforming growth factor-beta 1 (TGF-beta) to mammary cells simultaneously with or up to 24 h after dexamethasone treatment prevented the steroid induction of transepithelial electrical resistance and stimulated the incorporation of [3H]thymidine. However, the TGF-beta inhibition of tight junction formation did not require de novo DNA synthesis. Confocal microscopy revealed that the organized immunostaining pattern of the tight junction protein, ZO-1, and F-actin at the cell periphery was disrupted by TGF-beta, resulting in disorganized and diffuse staining patterns throughout the cell. Western blot analysis demonstrated that TGF-beta did not alter the protein levels of ZO-1. In contrast to cells not treated or pretreated with steroid for up to 24 h, TGF-beta had no effect on cells pretreated with dexamethasone for 48 h. Transfection of chimeric reporter genes containing promoters responsive to either glucocorticoid or TGF-beta demonstrated that the mutual antagonism of tight junction dynamics by dexamethasone and TGF-beta occurs in the presence of intact signaling pathways. Taken together, our results establish for the first time that glucocorticoids and TGF-beta can antagonistically regulate tight junction formation in a nontransformed mammary cell line.
合成糖皮质激素地塞米松可刺激31EG4非转化小鼠乳腺上皮细胞的跨上皮电阻，并抑制其DNA合成。在对地塞米松处理的同时或处理后长达24小时向乳腺细胞中添加转化生长因子β1（TGF-β），可阻止类固醇诱导的跨上皮电阻，并刺激[3H]胸苷的掺入。然而，TGF-β对紧密连接形成的抑制作用并不需要从头合成DNA。共聚焦显微镜显示，紧密连接蛋白ZO-1和细胞周边F-肌动蛋白的有组织免疫染色模式被TGF-β破坏，导致整个细胞中染色模式紊乱且弥散。蛋白质印迹分析表明，TGF-β不会改变ZO-1的蛋白质水平。与未处理或长达24小时未用类固醇预处理的细胞相比，TGF-β对用地塞米松预处理48小时的细胞没有影响。对含有对糖皮质激素或TGF-β有反应的启动子的嵌合报告基因进行转染表明，地塞米松和TGF-β对紧密连接动力学的相互拮抗作用发生在完整信号通路存在的情况下。综上所述，我们的结果首次证实糖皮质激素和TGF-β可在非转化乳腺细胞系中拮抗调节紧密连接的形成。
