Shiomi Takayuki, Inoki Isao, Kataoka Fumio, Ohtsuka Takashi, Hashimoto Gakuji, Nemori Ryoichi, Okada Yasunori
Department of Pathology, School of Medicine, Keio University, Tokyo, Japan.
Lab Invest. 2005 Dec;85(12):1489-506. doi: 10.1038/labinvest.3700351.
Matrix metalloproteinase-7 (MMP-7) (also known as matrilysin-1) is secreted as a proenzyme (proMMP-7) and plays a key role in the degradation of various extracellular matrix (ECM) and non-ECM molecules after activation. To identify the binding proteins related to proMMP-7 activation, a human lung cDNA library was screened by yeast two-hybrid system using proMMP-7 as bait. We identified a candidate molecule CD151, which is a member of the transmembrane 4 superfamily. Complex formation of proMMP-7 with CD151 was demonstrated by immunoprecipitation of the molecules from CaR-1 cells, a human rectal carcinoma cell line, expressing both proMMP-7 and CD151, and CD151 stable transfectants incubated with proMMP-7. Yeast two-hybrid assays using deletion mutants of proMMP-7 and CD151 suggested an interaction between the propeptide of proMMP-7 and the COOH-terminal extracellular loop of CD151. The binding activity of (125)I-labeled proMMP-7 to CD151 on the cell membranes was shown with CD151 stable transfectants. Laser-scanning confocal microscopy demonstrated that proMMP-7 colocalizes with CD151 on the cell membranes of CD151 stable transfectants and CaR-1 cells. In situ zymography using crosslinked carboxymethylated transferrin, a substrate of MMP-7, demonstrated proteinase activity on and around CD151 stable transfectants and CaR-1 cells, while the activity was abolished by their treatment with MMP inhibitors, anti-MMP-7 antibody or anti-CD151 antibody. In human lung adenocarcinoma tissues, colocalization of MMP-7 and CD151 was demonstrated on the carcinoma cells. Metalloproteinase activity was present in these tissues and could be inhibited by antibodies to MMP-7 or CD151. These data demonstrate for the first time that proMMP-7 is captured and activated on the cell membranes through interaction with CD151, and suggest the possibility that similar to the MT1-MMP/MMP-2 system, MMP-7 is involved in the pericellular activation mechanism mediated by CD151, a crucial step in proteolysis on the cell membranes under various pathophysiological conditions including cancer invasion and metastasis.
基质金属蛋白酶-7（MMP-7）（也称为基质溶素-1）以酶原形式（proMMP-7）分泌，激活后在多种细胞外基质（ECM）和非ECM分子的降解中起关键作用。为了鉴定与proMMP-7激活相关的结合蛋白，使用proMMP-7作为诱饵，通过酵母双杂交系统筛选人肺cDNA文库。我们鉴定出一个候选分子CD151，它是跨膜4超家族的成员。通过从表达proMMP-7和CD151的人直肠癌细胞系CaR-1细胞以及与proMMP-7孵育的CD151稳定转染子中免疫沉淀分子，证明了proMMP-7与CD151形成复合物。使用proMMP-7和CD151的缺失突变体进行的酵母双杂交分析表明，proMMP-7的前肽与CD151的COOH末端细胞外环之间存在相互作用。用CD151稳定转染子显示了细胞膜上（125）I标记的proMMP-7与CD151的结合活性。激光扫描共聚焦显微镜显示，proMMP-7与CD151在CD151稳定转染子和CaR-1细胞的细胞膜上共定位。使用交联羧甲基化转铁蛋白（一种MMP-7的底物）进行原位酶谱分析，证明了CD151稳定转染子和CaR-1细胞及其周围存在蛋白酶活性，而用MMP抑制剂、抗MMP-7抗体或抗CD151抗体处理后，该活性被消除。在人肺腺癌组织中，癌细胞上显示出MMP-7和CD151的共定位。这些组织中存在金属蛋白酶活性，并且可以被抗MMP-7或抗CD151抗体抑制。这些数据首次证明proMMP-7通过与CD151相互作用在细胞膜上被捕获并激活，并提示了一种可能性，即类似于MT1-MMP/MMP-2系统，MMP-7参与由CD151介导的细胞周缘激活机制，这是在包括癌症侵袭和转移在内的各种病理生理条件下细胞膜蛋白水解的关键步骤。
