TIMP-2 Promotes Activation of Progelatinase A by Membrane-type 1 Matrix Metalloproteinase Immobilized on Agarose Beads

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...

Type IV Collagen Induces Matrix Metalloproteinase 2 Activation in HT1080 Fibrosarcoma Cells

“…7A). These data are in agreement with previous observations that cell-associated TIMP-2 is required for pro-MMP-2 activation [20,28,53].…”

“…It can be hypothesized that type IV collagen mediates its effect by bringing closer cell membrane-associated MT1-MMP/TIMP-2/MMP-2 complexes, therefore promoting the intermolecular autolytic cleavage of intermediate MMP-2 and thus leading to the generation of the fully mature species. Additionally, type IV collagen decreases the level of secreted TIMP-2, thus potentially furthering the processing of pro-MMP-2 which is known to be inhibited by high TIMP-2 concentrations [20,31,53]. It is worth noting that the TIMP-2 degradation process illustrated here potentially represents an additional regulatory mechanism of the proteolytic activity of the cells.…”

“…TIMP-2 has previously been demonstrated to play a dual role in pro-MMP-2 activation: at low concentrations, it promotes activation while, at higher concentrations, it completely inhibits this process [20,28,53]. To detect a potential modification of TIMP-2 concentration induced by pro-MMP-2 activating treatments, TIMP-2 concentrations in conditioned media and total cell extracts were quantified by ELISA (Fig.…”

“…On the basis of the dual role of TIMP-2 during MMP-2 activation, promoting MMP-2 activation when present at low concentrations [20,29,31,54,63,64], but acting as an efficient inhibitor of this process when present at higher doses [19,20,26,31,53,[65][66][67], we hypothesized that modulations of TIMP-2 concentration could play important roles in the regulation of MMP-2 processing. Consequently, we investigated the modifications of both TIMP-2 concentration and distribution between conditioned media and cell extracts induced by MMP-2-activating treatments.…”

The possible role of matrix metalloproteinase (MMP)-2 and MMP-9 in cancer, e.g. acute leukemia

“…Three mechanisms for proMMP-2 activation have been described at this moment; autocatalytical activation, TIMP-2 dependent activation and cell-surface associated urokinase-type plasminogen activator (uPA)/plasmin system dependent activation [34][35][36][37][38][39][40]. The mechanisms differ substantially, but have a characteristic phenomenon.…”