Shedding of membrane type 1 matrix metalloproteinase in a human breast carcinoma cell line.
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 MTI-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.
Harayama
T
T
Department of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University.
Ohuchi
E
E
Aoki
T
T
Sato
H
H
Seiki
M
M
Okada
Y
Y
D017209:Apoptosis; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000201:enzymology* / Q000473:pathology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003208:Concanavalin A / Q000494:pharmacology; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D011480:Protease Inhibitors / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011480:Protease Inhibitors; D003208:Concanavalin A; D019716:Tissue Inhibitor of Metalloproteinase-2; D020782:Matrix Metalloproteinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2