Shedding of Membrane Type 1 Matrix Metalloproteinase in a Human Breast Carcinoma Cell Line |
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| Authors: | Takeshi Harayama Eiko Ohuchi Takanori Aoki Hiroshi Sato Motoharu Seiki Yasunori Okada |
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| Affiliation: | Departments of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934;Department of Biology, Faculty of Science, Kanazawa University, Kakumamachi, Kanazawa 920-1192;Biopharmaceutical Department, Fuji Chemical Industries, Ltd., 530 Chokeiji, Takaoka 933-0951;Departments of Virology and Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934;Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Tokyo 108-0071;Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016 |
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| Abstract: | Membrane type 1 matrix metalloproteinase (MT1-MMP) with a transmembrane domain is a new member of the MMP gene family and is expressed on the cell surfaces of many carcinoma cells to activate the zymogen of MMP-2 (gelatinase A). We have previously reported that MT1-MMP is released into culture media in a complex form with tissue inhibitor of metalloproteinases 2 (TIMP-2) from a human breast carcinoma cell line, MDA-MB-231, treated with concanavalin A (Con A). In the present study, we further studied the release mechanism of MT1-MMP. Immunoblot analysis indicated that the amounts of MT1-MMP in culture media increase with the time of exposure and the concentration of Con A, and those in cell lysates conversely decrease in a similar way. Time- and dose-dependent release of MT1-MMP into the media was confirmed by a sandwich enzyme immunoassay specific to MT1-MMP. The molecular weight of the immunoreactive MT1-MMP in the media was Mr 56,000, which was 4,000-Mr smaller than that in the cell lysates. Northern blot analysis demonstrated that the mRNA expression level of MT1-MMP is about 3-fold enhanced after a 24 h-exposure to Con A and this is maintained up to 72-h exposure. The release of MT1-MMP from the Con A-treated cells was inhibited by metalloproteinase inhibitors such as EDTA and o-phenanthroline, but not by MMP inhibitors including TIMP-1, TIMP-2 and BB94 or other proteinase inhibitors of serine, cysteine and aspartic proteinases. During the Con A treatment of the cells, cell viability decreased time- and dose-dependently and dead cells reacted positively in the TdT-mediated dUTP Nick-End Labeling (TUNEL) method. Con A-treated MDA cells showed apoptotic morphology when stained with Hoechst dye and hematoxylin and eosin. DNA ladder formation was detected by electrophoresis of the DNA from Con A-treated MDA cells. These results suggest that MT1-MMP release from Con A-treated cells is due to shedding mediated by metalloproteinase(s) other than MMPs, and is associated with apoptosis. |
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| Keywords: | Membrane type matrix metalloproteinase Shedding Invasion and metastasis Apoptosis Concanavalin A |
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