Matriptase Cleaves EpCAM and TROP2 in Keratinocytes, Destabilizing Both Proteins and Associated Claudins
Matriptase Cleaves EpCAM and TROP2 in Keratinocytes, Destabilizing Both Proteins and Associated Claudins
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DOI：
10.3390/cells9041027
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发表时间：
2020-04-01
期刊：
影响因子：
6
通讯作者：
Udey, Mark C.
中科院分区：
文献类型：
--
作者：
Wu, Chuan-Jin;Lu, Michael;Udey, Mark C.
EpCAM和TR 0 P2的同源物都与紧密连接蛋白-1和紧密连接蛋白-7相互作用，它们经常在包括皮肤在内的上皮细胞中共表达。肠道独特地表达高水平的EpCAM，但不表达TROP 2。我们先前将EpCAM鉴定为膜锚定蛋白酶matriptase的底物，并将HAI-2、matriptase、EpCAM和claudin-7连接在对肠上皮细胞（IEC）稳态至关重要的途径中。在此，我们揭示了TR 0 P2也是间质蛋白酶底物。当纯化的重组蛋白在体外混合时，间质蛋白酶切割TR 0 P2。与EpCAM一样，TR 0 P2在293 T细胞中共转染间质蛋白酶后也被切割。EpCAM和TR 0 P2裂解均不被蛋白酶失活的间质蛋白酶或具有鱼鳞病相关G827 R突变的间质蛋白酶促进。我们证实EpCAM和TR 0 P2都在皮肤中表达，并且在通过siRNA敲低解除HAI蛋白对间质蛋白酶的生理抑制后，检测到这些蛋白在人角质形成细胞（HaCaT细胞）中的切割。EpCAM或TR 0 P2的单独敲除仅对紧密连接蛋白-1和紧密连接蛋白-7水平具有小的影响，而两者的消除显著降低了紧密连接蛋白水平。HAI-1敲除促进EpCAM和TR 0 P2切割，伴随着封闭蛋白的减少，而HAI-2敲除几乎没有影响。HAI-1和HAI-2的双敲低诱导EpCAM和TR 0 P2的几乎完全切割和紧密连接蛋白的急剧减少。这些作用通过同时的间质蛋白酶敲低而消除。紧密连接蛋白水平的降低也被溶酶体抑制剂氯喹减少，并且裂解的EpCAM/TR 0 P2片段优先积累。我们证明，TROP 2和EpCAM表现出冗余的调节密封蛋白代谢和HAI，matriptase，EpCAM和密封蛋白途径类似于我们所描述的IEC中存在于角质形成细胞。这项研究可能会提供深入了解间质蛋白酶失调诱导鱼鳞病的机制基础。
The homologs EpCAM and TROP2, which both interact with claudin-1 and claudin-7, are frequently coexpressed in epithelia including skin. Intestine uniquely expresses high levels of EpCAM but not TROP2. We previously identified EpCAM as a substrate of the membrane-anchored protease matriptase and linked HAI-2, matriptase, EpCAM and claudin-7 in a pathway that is pivotal for intestinal epithelial cells (IEC) homeostasis. Herein, we reveal that TROP2 is also a matriptase substrate. Matriptase cleaved TROP2 when purified recombinant proteins were mixed in vitro. TROP2, like EpCAM, was also cleaved after co-transfection of matriptase in 293T cells. Neither EpCAM nor TROP2 cleavage was promoted by protease-disabled matriptase or matriptase that harbored the ichthyosis-associated G827R mutation. We confirmed that EpCAM and TROP2 are both expressed in skin and detected cleavage of these proteins in human keratinocytes (HaCaT cells) after the physiologic inhibition of matriptase by HAI proteins was relieved by siRNA knockdown. Knockdown of EpCAM or TROP2 individually had only small effects on claudin-1 and claudin-7 levels, whereas elimination of both markedly diminished claudin levels. HAI-1 knockdown promoted EpCAM and TROP2 cleavage accompanied by reductions in claudins, whereas HAI-2 knockdown had little impact. Double knockdown of HAI-1 and HAI-2 induced nearly complete cleavage of EpCAM and TROP2 and drastic reductions of claudins. These effects were eliminated by concurrent matriptase knockdown. Decreases in claudin levels were also diminished by the lysosomal inhibitor chloroquine and cleaved EpCAM/TROP2 fragments accumulated preferentially. We demonstrate that TROP2 and EpCAM exhibit redundancies with regard to regulation of claudin metabolism and that an HAI, matriptase, EpCAM and claudin pathway analogous to what we described in IECs exists in keratinocytes. This study may offer insights into the mechanistic basis for matriptase dysregulation-induced ichthyosis.