Matrix metalloproteases are secreted by mammalian cells as zymogens and, upon activation, initiate tissue remodeling by proteolytic degradation of collagens and proteoglycans. Activation of the secreted proenzymes and interaction with their specific inhibitors determine the net enzymatic activity in the extracellular space. We have previously demonstrated that 72T4Cl can be activated by a plasma membrane-dependent mechanism specific for this enzyme. Here, we report purification of the membrane activator of 72T4Cl, which is a new metalloprotease identical to a recently cloned membrane-type matrix metalloprotease (MT-MMP). We demonstrate that activated MT-MMP acts as a cell surface tissue inhibitor of metalloprotease 2 (TIMP-2) receptor with Kd = 2.54 x 10(-9) M. The activator.TIMP-2 complex in turn acts as a receptor for 72T4Cl (Kd = 0.56 x 10(-9) M, binding to the carboxyl-end domain of the enzyme. Activation of 72T4Cl on the cell membrane provides a basic mechanism for spatially regulated extracellular proteolysis and presents a new target for prognosis and treatment of metastatic disease. The activation, purified as a tri-molecular complex of MT-MMP.TIMP2.carboxyl-end domain of 72T4Cl, is itself an activated form of MT-MMP, posing the following question: what is the mechanism of the activator's activation?

Simian virus 40 (SV40)-transformed human lung fibroblasts secrete both 72-kDa type IV collagenase and a closely related 92-kDa type IV collagenase that was not detected in the parental cell line. The 92-kDa type IV procollagenase purified from these cells exists in a noncovalent complex with the tissue inhibitor of metalloproteases, TIMP. Here we report that the 72-kDa type IV procollagenase purified from HRAS-transformed human bronchial epithelial cells, SV40-transformed lung fibroblasts, and normal skin fibroblasts exists in a stable but noncovalent stoichiometric complex with a 24-kDa inhibitor referred to here as "TIMP-2." TIMP-2 is closely related to TIMP, as demonstrated by comparison of the partial amino acid sequence of this protein to that of TIMP, although it does not cross-react with TIMP-specific antibody. The TIMP-2 inhibitor interacts with the 72-kDa type IV collagenase in preference to the 92-kDa type IV collagenase that forms a complex exclusively with TIMP. The 72-kDa type IV collagenase-TIMP-2 complex can be activated with organomercurials to yield a catalytically competent enzyme. Activation occurs concomitantly with autoproteolytic cleavage of the amino terminus of the protein and does not require dissociation of the complex. Both activity and activation of the complex can be completely inhibited by further addition of stoichiometric quantities of purified TIMP-2 or recombinant TIMP.Tissue inhibitor of metalloproteases (TIMP) is a ubiquitous (1) glycoprotein (Mr 30,000) that originally was purified from serum-free medium conditioned by normal human skin fibroblasts (2). Inhibition of interstitial collagenase is stoichiometric, with a 1:1 molar ratio of inhibitor to enzyme being required for complete inhibition of enzyme activity. Interstitial procollagenase can neither interact with TIMP nor bind to collagen; both require extracellular activation of the collagenase zymogen (3, 4). Active enzyme binds with nearly equal affinity to both monomeric collagen and aggregated fibrils. The K, of enzyme-inhibitor binding was determined to be less than 10-9 M (4).The gene encoding TIMP has been cloned as a collagenase inhibitor (5, 6) and as a factor with erythroid-potentiating activity (7) that stimulates colony formation by relatively mature erythroid precursors (CFU-E). The TIMP gene has been localized to the X chromosome (8, 9). The secreted protein consists of 184 amino acid residues and contains six disulfide bonds and two glycosylation sites containing Nlinked oligosaccharides. The sequence of the inhibitor has no substantial homology to previously sequenced protease inhibitors with the exception of the metalloprotease inhibitor isolated from bovine scapular cartilage (10) and from normal murine fibroblasts (11, 12). The inhibitor extracted from cartilage has a molecular mass of 27,400 with an NH2-terminal sequence of 45 residues that shows 65% homology to TIMP.Recent observations suggest that expression of TIMP may be involved in suppression of tumorigenicity of immortal murine 3T3 cells...

We present a cascade of proteolytic events catalyzed by the proteases secreted by cultured keratinocytes and fibroblasts that results in the activation of interstitial procollagenase. Cultured human skin fibroblasts constitutively secrete interstitial collagenase and stromelysin as proenzymes. In contrast, interstitial collagenase found in serum-free skin organ culture conditioned medium is activated. Cocultivation of the major cellular components of skin organ culture, dermal fibroblasts and epidermal keratinocytes, induces activation of interstitial procollagenase and prostromelysin in the presence of plasminogen. This activation occurs through a urokinasedependent pathway where added keratinocytes secrete the plasminogen activator urokinase, which converts p!asminogen into plasmin. Plasmin is capable of activating purified procollagenase and prostromelysin. Plasmin-dependent activation of procollagenase generates an enzyme species, by amino-terminal processing, identical to those generated by limited proteolysis with trypsin or treatment with organomercurial compounds. Catalytic amounts of activated stromelysin can in turn convert plasmin-or trypsin-activated collagenase into a fully active enzyme by removal of 15 amino acid residues from the carboxyl end of the enzyme. This results in a 5-to 8-fold increase in collagenase specific activity that is due to its proteolytic cleavage and not to the presence of the activator stromelysin. Stromelysin alone in both pro-and activated forms is not capable of efficient activation of human fibroblast interstitial procollagenase.Intensified tissue remodeling during morphogenesis, wound healing, and tumor invasion requires the presence of secreted metalloproteases capable of initiating the degradation of macromolecules ofthe extracellular matrix. Several enzymes of this class have been identified: fibroblast (1-3) and granulocyte (4,5) collagenases, which degrade interstitial collagens, type IV collagenase, which degrades both basement membrane collagen and denatured collagen (gelatin) (6-8), and stromelysin, which degrades proteoglycans (9-13). We have determined the primary structure of fibroblast interstitial collagenase (14), stromelysin (12), and type IV collagenase (7), demonstrated a close structural relationship between these proteins (7), and compared their substrate specificities. Analysis of the genomic organization of the human fibroblast interstitial collagenase gene (15) in comparison with that of rabbit collagenase (16) and rat stromelysin (17) revealed that a close structural relationship on the protein level is reflected in the very similar genomic organization of at least two members of the secreted metalloprotease gene family. Interstitial collagenase (1, 18), stromelysin (12), and type IV collagenase (7) are constitutively secreted by cultured human skin fibroblasts in a proenzyme form and are subject to activation extracellularly. Both procollagenase and prostromelysin are secreted in two forms, one of which contains N-linked complex oligosacc...

We show that activated collagenase (MMP-1) moves processively on the collagen fibril. The mechanism of movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. Inactivation of the enzyme by a single amino acid residue substitution in the active center eliminates the bias without noticeable effect on rate of diffusion. Monte Carlo simulations using a model similar to a ''burnt bridge'' Brownian ratchet accurately describe our experimental results and previous observations on kinetics of collagen digestion. The biological implications of MMP-1 acting as a molecular ratchet tethered to the cell surface suggest new mechanisms for its role in tissue remodeling and cell-matrix interaction.

We have purified and determined the complete primary structure of human stromelysin, a secreted metalloprotease with a wide range of substrate specificities. Human stromelysin is synthesized in a preproenzyme form with a calculated size of 53,977 Da and a 17-amino acid long signal peptide. Prostromelysin is secreted in two forms, with apparent molecular masses on NaDodSO4/PAGE of 60 and 57 kDa. The minor 60-kDa polypeptide is a glycosylated form of the major 57-kDa protein containing N-linked complex oligosaccharides. Zymogen activation by trypsin results in the removal of 84 amino acids from the amino terminus of the enzyme generating a 45-kDa active enzyme species. Human stromelysin is capable of degrading proteoglycan, fibronectin, lamiiin, and type IV collagen but not interstitial type I collagen. The enzyme is not capable of activating purified human fibroblast procollagenase. Analysis of its primary structure shows that stromelysin is in all likelihood the human analog of rat transin, which is an oncogene transformation-induced protease. The pattern of enzyme expression in normal and tumorigenic cells revealed that human skin fibroblasts in vitro secrete stromelysin constitutively (1-2 jug per 106 cells per 24 hr). Human fetal lung fibroblasts transformed with simian virus 40, human bronchial epithelial cells transformed with the ras oncogene, fibrosarcoma cells (HT-1080), and a melanoma cell strain (A 2058), do not express this protease nor can the enzyme be induced in these cells by treatment with phorbol 12-myristate 13-acetate. Our data indicate that the expression and the possible involvement of secreted metalloproteases in tumorigenesis result from a specific interaction between the transforming factor and the target cell, which may vary in different species.The extracellular matrix (ECM) of multicellular organisms plays an active role in the formation and maintenance of tissues. The meshwork of ECM macromolecules is deposited by resident cells and provides a substrate for cell adhesion and migration, as well as a permeability barrier in cell-cell communication. A number of processes in normal tissue maintenance [e.g., wound healing (1, 2), bone resorption (3), and uterine involution (4)] as well as certain pathologic processes [for example, rheumatoid arthritis (5, 6), epidermolysis bullosa (7,8), corneal (9, 10) and gingival disease (11)] require connective tissue remodeling, involving a guided degradation of the preexisting ECM. Secreted proteases, required for initiation of degradation of the proteinaceous components of the ECM, also play an important part in processes requiring cell movement, such as tumor invasion and metastasis (12, 13). These enzymes constitute a family of structurally related metalloendoproteases. Previously, we characterized the expression, properties, and primary structure of human fibroblast collagenase (14-16), an enzyme responsible for the initiation of interstitial collagen degradation. In this report, we present the characterization of human fibroblast stromel...