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...

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Mononuclear phagocytes are developmentally and functionally complex cells that play critical roles in extracellular matrix remodeling. We hypothesized that differentiated mononuclear phagocytes, typified by alveolar macrophages, use a spectrum of metalloproteinases to degrade various matrix macromolecules. To test this hypothesis, we have evaluated synthesis and secretion of four metalloproteinases (interstitial collagenase, stromelysin, 72-kD type IV collagenase, and 92-kD type IV collagenase) by human mononuclear phagocytes with regard to (a) the effect of cellular differentiation, (b) regulation of secretion, and (c) comparisons/contrasts with a prototype metalloproteinase-secretory cell, the human fibroblast. We found that regulated secretion of greater quantities and a wider spectrum of metalloenzymes correlated with a more differentiated cellular phenotype. As extreme examples, the 92-kD type IV collagenase was released by peripheral blood monocytes and uninduced U937 monocyte-like cells, whereas stromelysin was secreted only by lipopolysaccharide-stimulated alveolar macrophages. Macrophage production of interstitial collagenase, stromelysin, and 72-kD type IV collagenase was approximately 20%, 10%, and 1-2%, respectively, of that from equal numbers of fibroblasts; secretion of the 92-kD type IV collagenase was not shared by fibroblasts. This work confirms the potential of macrophages to directly degrade extracellular matrix via secreted metalloproteinases in a manner that differs both qualitatively and quantitatively from that of fibroblasts.Moreover, varying regulation of metalloenzyme synthesis, evidenced by distinct patterns of basal and stimulated secretion during differentiation, can be studied at a molecular level in this model system. (J. Clin. Invest. 1990Invest. . 86:1496Invest. -1502

Human skin procollagenase has been isolated, in pure form, from the medium of fibroblasts cultured in the presence or absence of added serum. Purification was achieved using a combination of cation-exchange (phosphocellulose or carboxymethylcellulose) and gel-filtration chromatography. Two forms (60 000 and 55 000 daltons) of the procollagenase were detected by electrophoresis in sodium dodecyl sulfatepolyacrylamide gels and could be separated by chromatography on Ultrogel AcA-44. Each form was converted to active enzyme by trypsin, producing species of 50 000 and 45 000 daltons, respectively. An autoactivation process also occurred, which yielded active enzyme without a detectable change in molecular weight. Procollagenase also was found in organ cultures of human skin but only when serum was added to the medium. This suggests that a serum-inhibitable proteolytic system is present in these cultures which, like trypsin, converts procollagenase to the active enzyme forms that can be isolated from serum-free organ culture medium. The collagenase species obtained from either fibroblast or organ culture medium were chromatographically and electrophoretically identical.