Shedding of Membrane Type 1 Matrix Metalloproteinase in a Human Breast Carcinoma Cell Line

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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.