, but residues 418 -474 were not important. A similar pattern was seen using cell membrane-associated MT1 MMP; residues 568 -631 were required for binding and activation of progelatinase A, whereas residues 418 -474 were not. Neither region was required for activation in solution. The addition of TIMP2 to HT1080 membrane preparations expressing MT1 MMP, but depleted of endogenous TIMP2, resulted in potentiation of progelatinase A activation. This effect was dependent upon TIMP2 binding to MT1 MMP rather than at an independent membrane site. Together, the data suggest that TIMP2 forms a receptor with MT1 MMP that regulates the concentration and efficient generation of functionally active gelatinase A.

M r 72,000 gelatinase) has been implicated in extracellular matrix remodelling in relation to developmental processes (1), inflammation (2), and tumor invasion and metastasis (3, 4). Like other MMPs, gelatinase A is secreted as an inactive proenzyme, and it has been postulated that the proteolytic cascades associated with the activation process are a significant regulatory feature of these enzymes. Progelatinase A is unique in that the plasmin generation cascade implicated for other pro-MMPs does not effect activation (5, 6). Recently the potential for activation via matrilysin (7) and collagenase (8) has been demonstrated, and there is also evidence that activation by selfcleavage can occur at high progelatinase A concentrations (9). More strikingly, a cell-mediated mechanism for progelatinase A activation has been the focus of much recent work (10 -15). We reported that human skin fibroblasts stimulated by concanavalin A can bind, proteolytically process, and activate progelatinase A. The importance of the C-terminal domain of the enzyme for both the binding to cell membranes and subsequent activation was demonstrated (12)(13)(14). Cell membranemediated cleavage of progelatinase A to the M r 66,000 active form (N terminus Tyr 81 ) occurs via a M r 68,000 intermediate (N terminus Leu 38 (14)), and exogenous TIMP-2 specifically inhibits the activation process. Sato et al. (16) reported the cloning of a novel transmembrane member of the MMP family, membrane type 1 MMP (MT1-MMP), and showed that cells transfected with MT1-MMP cDNA can activate progelatinase A. Using an inactive mutant of progelatinase A, we have shown that cell membrane-mediated activation is, by analogy with other MMPs, likely to be a complex activation cascade involving MT1-MMP which can be induced in fibroblasts by treatment with concanavalin A, followed by bimolecular autolysis (17). Strongin et al. (15) also purified a fibroblast plasma membrane metalloproteinase, either related to or identical with MT1-MMP, and demonstrated that it bound TIMP-2. It was proposed that the resulting MT1-MMP⅐TIMP-2 complex acted as a receptor for progelatinase A, potentiating cleavage by active MT1-MMP at adjacent sites.In this article, we look more closely at the biochemical properties of MMT1-MMP particularly in relation to its ability to process progelatinase A and the effects of tissue inhibitors of metalloproteinases as regulators. A soluble recombinant form of pro-MT1-MMP consisting of the catalytic domain alone has been expressed into the periplasm of Escherichia coli and purified. The active form of this enzyme, which is a weak general proteinase, exhibits limited cleavage of the progelatinase A propeptide, leading to autoproteolytic activation of gelatinase

Soluble proenzyme forms of the catalytic domains of membrane-type matrix metalloproteinases 1 and 2 (MTI-MMP and MT2-MMP) and a form of MTI-MMP containing the catalytic and hemopexin domains were expressed as soluble recombinant proteins. Purified, activated forms of the MT-MMP were shown to degrade fibronectin, tenascin, nidogen, aggrecan and perlecan. Only MT2-MMP showed activity against laminin. MT1 -MMP retaining the hemopexin domain was able to specifically cleave native type-I and type-I11 collagens into the 3/4-1/4 fragments typical of the specific collagenases. The catalytic domain alone did not retain this activity. The MT-MMP did not degrade interleukin-lp, but, similarly to many other MMP, could process a pro [tumor necrosis factor (TNF) a] fusion protein to release mature TNF. However, the latter was subsequently degraded into smaller fragments. These results demonstrate that, in addition to their ability to activate other MMP, such as progelatinase A/proMMP2 and procollagenase-3/proMMPI 3, MT-MMP degrade a number of extracellular matrix macromolecules. Their location at the surface of cells implies that they could play a significant role in the modulation of cell-matrix interactions.Keywords: tissue inhibitor of matrix metalloproteinase; extracellular matrix ; cytokine; wound healing ; invasion.Extracellular matrices (ECM) play a key role in cell biology. Not only do they contribute to tissue architecture, but they also provide immobilized ligands for cellular receptors, sequester growth factors, and in basement membrane form selectively permeable barriers between tissue compartments. Hence, ECM components influence and regulate cell growth and differentiation, cell adhesion, mobility and spreading. Therefore their turnover is an important process in physiological and pathophysiological situations.Matrix metalloproteinases (MMP) have a prominent role in the degradation of ECM, and have a wide spectrum of other substrates, cleaving for example interleukin 1p (ILIP) [I] MATERIALS AND METHODS Expression and purification of MT-MMP. RecombinantMT-MMP were expressed as soluble proteins with a C-terminal His tag in the periplasm of Escherichia coli as described previously [lo]. The three constructs used were the proform of the catalytic domain of human MT1-MMP, des-(269-559)-MTI-

Concanavalin A-stimulated fibroblasts expressing MT1-MMP and fibroblast-derived plasma membranes were able to process human procollagenase-3 via a M r 56,000 intermediate form to the final M r 48,000 active enzyme which, by analogy with progelatinase A activation, may represent a model system for in vivo activation. Inhibition experiments using tissue inhibitor of metalloproteinases, plasminogen activator inhibitor-2, or aprotinin demonstrated that activation in the cellular model system was due to MT1-MMP/gelatinase A and excluded the participation of serine proteinases such as plasmin during procollagenase-3 activation. We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by crude plasma membrane preparations from concanavalin Astimulated fibroblasts, thus confirming our results using purified progelatinase A and MT1-MMP. This new activation cascade may be significant in human breast cancer pathology, where all three enzymes have been implicated as playing important roles.Degradation of the extracellular matrix during tumor invasion is thought to result from a combined action of several proteolytic enzyme systems, including the collagenases and other matrix metalloproteinases (MMPs) 1 (1, 2) and serine proteases, such as plasmin generated by the urokinase pathway of plasminogen activation (3).Human collagenase-3 (MMP-13), a new member of the MMP family, is expressed by breast tumors and is likely to play a crucial role in the modulation of extracellular matrix degradation and cell-matrix interactions involved in metastasis (4). Procollagenase-3 comprises three distinct domains which include an 85-amino acid residue propeptide that is lost during activation (5), and in which the conserved sequence PRCGVPD is responsible for the latency of the MMPs (6). This sequence is followed by the catalytic domain containing the active site of the enzyme linked via a short hinge sequence motif to the third, C-terminal domain, that shows homology to vitronectin and which is essential for the collagenolytic activity of collagenase-3.2 Collagenase-3 is a powerful collagenolytic and gelatinolytic enzyme that preferentially cleaves type II collagen, and it can therefore be implied that this enzyme may play a considerable role in connective tissue turnover (5). One of the key events in the regulation of extracellular collagenolytic activity is the activation of procollagenase-3, but there are currently only limited data available on how this may occur in vivo. We have recently shown that procollagenase-3 can be directly activated by stromelysin (5); however, other mechanisms may be of physiological and pathophysiological significance.Increasing evidence is accumulating that the newly discovered membrane associated MMPs (MT-MMPs) act as cell surface activator(s) of progelatinase A (proMMP-2) under physiological or pathophysiological conditions (7-12). This mechanism was thought to be specific for progelatinase A, since other MMPs such as progelatinase B, fibroblast procollage...

The complementary DNA sequence of a novel matrix metalloproteinase was isolated from a human lung cDNA library. It consists of 3530 bp and encodes a polypeptide of 669 amino acids. In comparison to other matrix metalloproteinases, the deduced sequence of the amino acid chain exhibits closest similarity to a recently discovered membrane-type matrix metalloproteinase of 582 amino acids. Likewise, it is composed of a signal peptide, a prodomain, a catalytic domain, a hemopexin-homologous domain and a C-terminal domain. Furthermore, the novel matrix metalloproteinase shares a similar activation site with its 582-amino-acid homologue, an insertion of eight amino acids in the catalytic domain and a tract of more than 20 hydrophobic amino acids near the C-terminus. The hydrophobic structure in the C-terminal domain suggests that the novel matrix metalloproteinase is also membrane bound. When lung cell membrane fractions were probed in immunoblots with polyclonal antibodies against a recombinant fragment of the 669-amino-acid chain, a protein of M(r) 72,000 reacted preferentially with the antibodies. Northern-blot analysis demonstrated quite different tissue distributions of mRNA for the two membrane-type matrix metalloproteinases. While mRNA for the 582-amino-acid enzyme was found predominantly in lung, placenta, kidney, ovary, intestine, prostate and spleen, mRNA for the 669-amino-acid enzyme appeared to be synthesized preferentially in liver, placenta, testis, colon and intestine. Substantial amounts of the latter mRNA were also detected in pancreas, kidney, lung, heart and skeletal muscle.