Altered translation initiation of Gja1 limits gap junction formation during epithelial-mesenchymal transition.
Altered translation initiation of Gja1 limits gap junction formation during epithelial-mesenchymal transition.
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DOI：
10.1091/mbc.e17-06-0406
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发表时间：
2018-04-01
影响因子：
3.3
通讯作者：
Smyth JW
中科院分区：
文献类型：
--
作者：
James CC;Zeitz MJ;Calhoun PJ;Lamouille S;Smyth JW
Connexin43 mRNA可以在内部翻译生成n端截断的异构体，包括GJA1-20k。在TGF-β诱导的上皮-间质转化过程中，GJA1-20k的表达受到抑制，稳定高尔基体全长Connexin43，减少通道寡聚化、细胞表面表达和Connexin43间隙连接形成。上皮-间质转化（EMT）在发育、伤口愈合和包括纤维化和癌症转移在内的病理过程中被激活。EMT的特征是细胞间连接和粘附蛋白的重塑，包括间隙连接。编码间隙连接蛋白connexin43 （Cx43）的GJA1 mRNA转录物已被证明可以进行内部翻译起始，产生调节间隙连接的截断同种异构体。PI3K/Akt/mTOR通路是翻译调控的核心，在EMT期间被激活，这使我们假设翻译起始的改变会导致间隙连接的丢失。使用TGF-β诱导的EMT作为模型，我们发现尽管Cx43蛋白的转录和稳定增加，但Cx43间隙连接减少。生化实验显示，内部翻译的Cx43异构体GJA1-20k以Smad3和erk依赖的方式受到抑制。GJA1-20k的异位表达不会阻止EMT，但足以挽救间隙连接的形成。GJA1-20k定位于高尔基体，使用超分辨率定位显微镜我们发现GJA1-43k在缺乏GJA1-20k的间充质细胞中保留在高尔基体上。NativePAGE表明GJA1-20k水平调节GJA1-43k六聚体寡聚，这是Cx43贩运的一个限制步骤。这些发现揭示了翻译起始的改变是细胞在EMT期间调节Cx43间隙连接形成的一种未被探索的机制。
Connexin43 mRNA can be internally translated to generate N-terminally truncated isoforms including GJA1-20k. GJA1-20k expression is suppressed during TGF-β–induced epithelial–mesenchymal transition, stabilizing full-length Connexin43 in the Golgi, reducing channel oligomerization, cell surface expression, and connexin43 gap junction formation. Epithelial–mesenchymal transition (EMT) is activated during development, wound healing, and pathologies including fibrosis and cancer metastasis. Hallmarks of EMT are remodeling of intercellular junctions and adhesion proteins, including gap junctions. The GJA1 mRNA transcript encoding the gap junction protein connexin43 (Cx43) has been demonstrated to undergo internal translation initiation, yielding truncated isoforms that modulate gap junctions. The PI3K/Akt/mTOR pathway is central to translation regulation and is activated during EMT, leading us to hypothesize that altered translation initiation would contribute to gap junction loss. Using TGF-β–induced EMT as a model, we find reductions in Cx43 gap junctions despite increased transcription and stabilization of Cx43 protein. Biochemical experiments reveal suppression of the internally translated Cx43 isoform, GJA1-20k in a Smad3 and ERK-dependent manner. Ectopic expression of GJA1-20k does not halt EMT, but is sufficient to rescue gap junction formation. GJA1-20k localizes to the Golgi apparatus, and using superresolution localization microscopy we find retention of GJA1-43k at the Golgi in mesenchymal cells lacking GJA1-20k. NativePAGE demonstrates that levels of GJA1-20k regulate GJA1-43k hexamer oligomerization, a limiting step in Cx43 trafficking. These findings reveal alterations in translation initiation as an unexplored mechanism by which the cell regulates Cx43 gap junction formation during EMT.