- 學位論文
探討嵌膜蛋白酶在人類大腸上皮細胞間緊密連接形成過程中扮演的角色
Delineation of membrane-anchored serine proteases in tight junction formation of colorectal epithelial cells
摘要
細胞連接在維持體內組織的滲透壓平衡中扮演著重要的角色。在近期研究中發現，第二型嵌膜絲胺酸蛋白酶 (TTSP)中的成員，間質蛋白酶 (matriptase) 與攝護腺蛋白酶(prostasin)在大腸腺癌上皮細胞 (Caco-2)的緊密連接的形成過程中扮演正向調控的角色。由先前本實驗的研究結果顯示，另一個TTSP的成員，第二型嵌膜蛋白酶 (TMPRSS2)會活化matriptase, 並參與細胞緊密連接的形成。 然而，TMPRSS2, matriptase及prostasin三者在腸道上皮細胞緊密連接形成過程中，所扮演的角色，仍有許多未知。在本研究中，我們假設TMPRSS2, matriptase及prostasin會形成一個嵌膜蛋白酶疊層，進而影響腸道上皮細胞的細胞緊密連接的形成及通透性。結果顯示TMPRSS2是一個位於matriptase及prostasin上游的調控者。利用TEER測量Caco-2及MDCK細胞的離子通透性可發現，過量表達的TMPRSS2及prostasin都會增加細胞間緊密連接的形成並使離子通透性降低。抑制prostasin的表現量則會降低緊密連接形成以及增加細胞通透性。當在過量表達TMPRSS2的Caco-2細胞中降低matriptase的表現量，將會降低由TMPRSS2所促進的細胞緊密連接並增高其通透性。第一型肝細胞生長因子活化抑制蛋白 (HAI-1)被報導為可抑制matriptase 和prostasin的活性。因此，想進一步探討是否HAI-1的表達藉由抑制matriptase 和prostasin，進而壓抑由TMPRSS2所促進的細胞緊密連接。由細胞離子通透性實驗發現，過量表達的HAI-1，明顯地將TMPRSS2促進細胞提早分化的情形壓抑下來。綜合以上結果，TMPRSS2與prostasin及matriptase在細胞緊密連接過程中均扮演角色，且TMPRSS2是matriptase及prostasin上游的調節蛋白酶，調控細胞緊密連接的形成及通透性。
並列摘要
Junction barriers play a protective role in the tissue homeostasis. Recent studies have shown that type II transmembrane serine proteases (TTSP) matriptase and prostasin have a positive role in tight junction formation. Our preliminary data further showed that another TTSP TMPRSS2 could induce matriptase activation and be involved in junction barrier formation. How the pericellular proteolysis among TMPRSS2, matriptase and prostasin plays a role in junction barrier formation and permeability remain largely unknown. In this study, we hypothesized that a pericellular proteolytic cascade among TMPRSS2, matriptase and prostasin exists and can affect the tight junction formation and permeability of colorectal epithelial cells. The results showed that TMPRSS2 acted as an upstream regulator for matriptase and prostasin in human epithelial cells. Moreover, the results from the TransEpithelial Electric Resistance (TEER) analysis showed that TMPRSS2 and prostasin overexpression increased the formation of tight junction in colorectal cancer Caco-2 cells and MDCK cells. Prostasin knockdown in Caco-2 cells reduced the tight junction formation and increased the permeability of the polarized cells. Furthermore, matriptase knockdown in TMPRSS2-overexpressing Caco-2 cells inhibited TMPRSS2 function on the barrier formation and increase the ionic paracellular permeability. Overexpression of hepatocyte growth factor activator inhibitor (HAI-1) could decrease the barrier formation induced by TMPRSS2. The results together indicate that TMPRSS2 is an upstream regulator of matriptase and prostasin to affect the barrier formation and permeability in epithelial cells.