Migratory cells including invasive tumor cells frequently express CD44, a major receptor for hyaluronan and membrane-type 1 matrix metalloproteinase (MT1-MMP) that degrades extracellular matrix at the pericellular region. In this study, we demonstrate that MT1-MMP acts as a processing enzyme for CD44H, releasing it into the medium as a soluble 70-kD fragment. Furthermore, this processing event stimulates cell motility; however, expression of either CD44H or MT1-MMP alone did not stimulate cell motility. Coexpression of MT1-MMP and mutant CD44H lacking the MT1-MMP–processing site did not result in shedding and did not promote cell migration, suggesting that the processing of CD44H by MT1-MMP is critical in the migratory stimulation. Moreover, expression of the mutant CD44H inhibited the cell migration promoted by CD44H and MT1-MMP in a dominant-negative manner. The pancreatic tumor cell line, MIA PaCa-2, was found to shed the 70-kD CD44H fragment in a MT1-MMP–dependent manner. Expression of the mutant CD44H in the cells as well as MMP inhibitor treatment effectively inhibited the migration, suggesting that MIA PaCa-2 cells indeed use the CD44H and MT1-MMP as migratory devices. These findings revealed a novel interaction of the two molecules that have each been implicated in tumor cell migration and invasion.

ABSTRACT

Membrane-type 1 matrix metalloproteinase (MT1-MMP)/MMP-14 is the activator of progelatinase A (proGelA)/proMMP-2 on the cell surface. However, it was a paradox that a tissue inhibitor of metalloproteinase-2 (TIMP-2), which is an inhibitor of MT1-MMP, is required for proGelA activation by the cells expressing MT1-MMP. In this study, a truncated MT1-MMP having a FLAG-tag sequence at the C terminus (MT1-F) was immobilized onto agarose beads (MT1-F/B) and used to analyze the role of TIMP-2. The proteolytic activity of MT1-F/B against a synthetic peptide substrate was inhibited by TIMP-2 in a dose-dependent manner. In contrast, TIMP-2 promoted the processing of proGelA by MT1-F/B at low concentrations and inhibited it at higher concentrations. TIMP-2 promoted the binding of proGelA to the MT1-F on the beads by forming a trimolecular complex, which was followed by processing of proGelA. A stimulatory effect of TIMP-2 was observed under conditions in which unoccupied MT1-F was still available. Thus, the ternary complex is thought to act as a means to concentrate the substrate to the bead surface and to present it to the neighboring free MT1-F. Matrix metalloproteinases (MMPs)1 are zinc-dependent endopeptidases that play critical roles in the physiological and pathological turnover of extracellular matrix (ECM) by degrading the macromolecules (1-6). MMPs are produced as a zymogen (proMMP) that needs proteolytic activation by eliminating the N-terminal propeptide for the enzymes to function (7). Serine proteases such as plasmin, neutrophil elastase, and trypsin are well known activators for proMMPs. These activators digest the propeptide sequences at the basic amino acid motifs and eventually induce autocatalytic activation (8).However, proGelA lacks such a basic motif and therefore cannot be activated by serine proteinases (9). ProGelA had been reported to be activated by an unknown MMP-like activity on the surface of cancer and fibroblastic cells (10 -15), and we identified MT1-MMP as such an activator on the cell surface (16,17). Three other genes encoding similar enzymes that have a transmembrane domain and a short cytoplasmic tail were identified (18 -20); at least two of them (MT2-MMP and MT3-MMP) activated proGelA in vitro (21).Upon cell-mediated activation, proGelA binds to the cells through its hemopexin-like domain (HLD) (22). Using the HLD of GelA, Strongin et al. (23) isolated TIMP-2 complexed with the activated form of MT1-MMP from the cell membrane extract. We also purified a shaded fragment of MT1-MMP from the culture medium of the human breast carcinoma cell line MDA-MB-231 as a form inhibited by TIMP-2 (24). The Cterminal domain of the TIMP-2 in the complex was available for further complex formation with proGelA through its HLD (trimolecular complex). Strongin et al. also demonstrated that a small amount of TIMP-2 is an essential component for the activation of proGelA on the surface, in contrast to evidence that TIMP-2 is a well established inhibitor for all of the known MMPs. It would thus be of inter...

Skin wound healing depends on cell migration and extracellular matrix remodeling. Both processes, which are necessary for reepithelization and restoration of the underlying connective tissue, are believed to involve the action of extracellular proteinases. We screened cDNA libraries and we found that six matrix metalloproteinase genes were highly expressed during rat skin wound healing. They were namely those of stromelysin 1, stromelysin 3, collagenase 3, gelatinase A (GelA), gelatinase B, and membrane type-1 matrix metalloproteinase (MT1-MMP). The expression kinetics of these MMP genes, the tissue distribution of their transcripts, the results of cotransfection experiments in COS-1 cells, and zymographic analyses performed using microdissected rat wound tissues support the possibility that during cutaneous wound healing pro-GelA and pro-gelatinase B are activated by MT1-MMP and stromelysin 1, respectively. Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki. 1994. Nature (Lond.). 370: 61–65), our finding that GelA and MT1-MMP transcripts were expressed in stromal cells exhibiting a similar tissue distribution suggests that MT1-MMP activates pro-GelA at the stromal cell surface. This possibility is further supported by our observation that the processing of proGelA to its mature form correlated to the detection of MT1-MMP in cell membranes of rat fibroblasts expressing the MT1-MMP and GelA genes. These observations, together with the detection of high levels of the mature GelA form in the granulation tissue but not in the regenerating epidermis, suggest that MT1-MMP and GelA contribute to the restoration of connective tissue during rat skin wound healing.