探討第二型嵌膜蛋白酶參與大腸腺癌細胞間緊密連結的形成

Abstract

嵌膜絲胺酸蛋白酶 (Transmembrane serine protease) 對於組織恆定性以及緊密連結 (Tight junction) 的形成扮演著重要的角色。近年來，第二型嵌膜絲胺酸蛋白酶家族 (TTPSs) 中的成員，第二型嵌膜蛋白酶 (TMPRSS2) 受到越來越多的關注，因其表達量和攝護腺癌之進程有高度相關性。在一些組織例如：攝護腺及結腸都會選擇性地表達TMPRSS2。然而，TMPRSS2的生理功能及其受質至今仍待研究。實驗室過去的研究指出TMPRSS2會誘發TTSP中的另一個成員，間質蛋白酶 (Matriptase) ，在攝護腺癌細胞中的活化。由於最近的報導指出Matriptase可以調控腸道上皮細胞之Tight junction的形成及其屏障通透性。為了進一步探討TMPRSS2是否在Tight junction的形成中扮演角色？我使用結腸癌細胞 (Caco-2) 作為細胞分化模式，並觀察到此細胞在其分化過程中，TMPRSS2及Matriptase之蛋白表現量，都會隨之增加。當過量表達TMPRSS2，發現此蛋白酶可降低Caco-2細胞的離子通透性，促進屏障功能，且不會影響細胞生長。進一步的研究結果顯示，減弱TMPRSS2之表現量，會降低Matriptase的活化程度但不影響其基因表達量。在過量表達TMPRSS2的細胞中，從共軛焦顯微鏡影像顯示出TMPRSS2，ZO-1及Matriptase共同坐落在Tight junction的區域。進一步的研究發現，當降低TMPRSS2之表現量會削弱ZO-1, occludin, 及claudin-1等Tight junction蛋白質分佈在分化的細胞膜上的量。綜合以上結果，TMPRSS2可促進Tight junction之形成並影響離子通透性，此作用可能是藉由活化Matriptase而達成。因此，本研究成果建議TMPRSS2扮演角色參與調控表皮細胞緊密連結及分化細胞的通透性。

Pericellular serine proteases have an important role in tissue homeostasis and tight junction formation. Recently, type II transmembrane serine protease (TTSP) TMPRSS2 has received increasing attention because its expression level is correlated with prostate cancer progression. Several tissues such as prostate and colon also selectively express this protease. However, the biological function and substrate(s) of TMPRSS2 are still unclear. Our preliminary data show that TMPRSS2 can induce the activation of matriptase, another member of TTSP, in prostate cancer cells. Since recent studies have shown that matriptase can regulate intestinal tight junction formation and permeability, we then hypothesized that TMPRSS2 exhibited a role in the tight junction formation of colorectal cells and was crucial for barrier function and permeability. To further investigate the role of TMPRSS2 in tight junction formation, the protein levels of TMPRSS2 and matriptase were examined during the Caco-2 differentiation, and both proteins were increased during the period of cell differentiation. It was observed that TMPRSS2 overexpression in Caco-2 cells decreased iron permeability and increased barrier function during cell differentiation, which were independent of cell growth. Moreover, knockdown of TMPRSS2 reduced the activated level of matriptase in Caco-2 cells. In TMPRSS2-overexpressing Caco-2 cells, the confocal images indicated that TMPRSS2 may co-localize with ZO-1 and matriptase in the TJ region. In addition, TMPRSS2 silencing resulted in the down-regulation of ZO-1, occludin, and claudin-1 in the insoluble membrane fraction of polarized Caco-2 cells. The data together indicate that TMPRSS2 can promote TJ formation and may be via the regulation of matriptase, and suggest that TMPRSS2 plays roles in modulating the TJ formation and permeability of epithelial cells.