Shedding of membrane type 1 matrix metalloproteinase in a human breast carcinoma cell line

Membrane type 1 matrix metalloproteinase (MT1-MMP) with a transmembrane dom ain is a new member of the MMP gene family and is expressed on the cell sur faces 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 farm with tissue inhibitor of metalloproteinases 2 (TIMP-2) f rom a human breast carcinoma cell line, MDA-MB-231, treated with concanaval in A (Con A). In the present study, we further studied the release mechanis m 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 C on 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 sa ndwich enzyme immunoassay specific to MT1-MMP. The molecular weight of the immunoreactive MT1-MMP in the media was M-r 56,000, which was 4000-M-r smal ler than that in the cell lysates, Northern blot analysis demonstrated that the mRNA expression level of MT1-MMP is about S-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 inc luding TIMP-1, TIMP-2 and BB94 or other proteinase inhibitors of serine, cy steine and aspartic proteinases, During the Con A treatment of the cells, c ell viability decreased time- and dose-dependently and dead cells reacted p ositively 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 electropho resis of the DNA from Con A-treated MDA cells, These results suggest that M T1-MMP release from Con A-treated cells is due to shedding mediated by meta lloproteinase(s) other than MMPs, and is associated with apoptosis.