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Shedding of membrane type 1 matrix metalloproteinase in a human breast carcinoma cell line
1Department of Molecular Immunology and Pathology, Cancer Research Institute, Kanazawa University.
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 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.
Insights
Concanavalin A induces apoptosis in breast carcinoma cells, leading to the release of membrane type 1 matrix metalloproteinase (MT1-MMP). This shedding is mediated by metalloproteinases other than MMPs.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Membrane type 1 matrix metalloproteinase (MT1-MMP) is crucial for carcinoma cell invasion and matrix remodeling.
- MT1-MMP activates pro-MMP-2 (gelatinase A) and is expressed on the surface of various carcinoma cells.
- Previous studies showed MT1-MMP release from MDA-MB-231 cells treated with concanavalin A (Con A).
Purpose of the Study:
- To elucidate the release mechanism of MT1-MMP from carcinoma cells induced by Con A.
- To investigate the association between MT1-MMP release and apoptosis.
- To identify the enzymes involved in MT1-MMP shedding.
Main Methods:
- Immunoblot analysis to quantify MT1-MMP levels in cell lysates and media.
- Sandwich enzyme immunoassay for time- and dose-dependent MT1-MMP release.
- Northern blot analysis to assess MT1-MMP mRNA expression.
- Treatment with various inhibitors (metalloproteinase, MMP, serine, cysteine, aspartic proteinase inhibitors) to determine involvement in release.
- Cell viability assays, TUNEL assay, Hoechst staining, and DNA ladder formation to assess apoptosis.
Main Results:
- Con A treatment increased MT1-MMP in culture media and decreased it in cell lysates in a time- and dose-dependent manner.
- Released MT1-MMP showed a lower molecular weight compared to cell-associated MT1-MMP.
- MT1-MMP mRNA expression was upregulated by Con A.
- MT1-MMP release was inhibited by metalloproteinase inhibitors like EDTA and o-phenanthroline, but not by specific MMP inhibitors.
- Con A treatment induced apoptosis in MDA-MB-231 cells, evidenced by decreased viability, positive TUNEL staining, apoptotic morphology, and DNA ladder formation.
Conclusions:
- MT1-MMP release from Con A-treated cells is a shedding process.
- The shedding is mediated by metalloproteinases distinct from MMPs.
- MT1-MMP release is closely associated with Con A-induced apoptosis in carcinoma cells.
