CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation.
CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation.
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DOI：
10.1371/journal.pone.0147489
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Elble RC
中科院分区：
文献类型：
--
作者：
Ramena G;Yin Y;Yu Y;Walia V;Elble RC
CLCA 2是一种p53、p63诱导的跨膜蛋白，在乳腺癌中经常下调。它在人乳腺上皮细胞的分化过程中被诱导，并且其敲低引起上皮向间充质转化（EMT）。为了确定CLCA 2如何促进上皮分化，我们使用膜双杂交筛选来寻找相互作用物。我们发现了与细胞连接蛋白EVA 1（上皮细胞V样抗原1）的强相互作用，并通过免疫共沉淀证实了这一点。与CLCA 2一样，EVA 1是一种受p53和p63调控的I型跨膜蛋白。它被认为介导不同上皮组织中的嗜同性细胞-细胞粘附。我们发现EVA 1在乳腺肿瘤和乳腺癌细胞系中经常下调，特别是那些间充质表型。此外，永生化的人乳腺上皮细胞（HMEC）中EVA 1的敲低导致EMT，这意味着EVA 1是上皮分化所必需的。EVA 1和CLCA 2与E-钙粘蛋白在细胞-细胞连接处共定位。通过缺失分析确定相互作用的结构域，揭示相互作用的位点是跨膜段（TMS）。发现CLCA 2 TMS的一级序列在整个哺乳动物的CLCA 2直系同源物中是保守的，表明其与EVA 1的相互作用与乳腺共同进化。对其他连接相互作用物的筛选显示，CLCA 2参与了两种不同的复合物，一种与EVA 1和ZO-1，另一种与β连环蛋白。CLCA 2的过表达导致β连环蛋白和β连环蛋白激活基因的下调。因此，CLCA 2将连接粘附分子连接到调节增殖和分化的胞质信号蛋白。这些结果可以解释CLCA 2的衰减如何导致EMT以及为什么CLCA 2和EVA 1在转移性乳腺癌细胞系中频繁下调。
CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.