Transforming growth factor-beta 1 up-regulates type IV collagenase expression in cultured human keratinocytes.

During the wound healing process lysis of basement membranes precedes keratinocyte migration into the wound bed. We studied, in vitro, whether this degradation of basement membranes could be regulated by transforming growth factor-beta 1 (TGF-beta 1), which is known to accelerate wound healing in vivo. Transforming growth factor-beta 1 was found to increase the expression of both 92- and 72-kDa type IV collagenases (gelatinases) in cultured human mucosal and dermal keratinocytes. The 92-kDa enzyme predominated in both unstimulated and stimulated cultures. The 92-kDa form was stimulated over 5-fold, and the other form by a factor of 2-3. This increase in the synthesis of type IV collagenases was associated with a marked increase in the mRNA levels of these enzymes as well. The induction of the 92-kDa enzyme was similar in culture medium containing either 0.15 or 1.2 mM calcium chloride. Rat mucosal keratinocytes secreted only 92-kDa type IV collagenase, the secretion of which was not regulated by TGF-beta 1. Also, TGF-beta 1 did not cause any significant induction (maximum about 1.2-fold) of either type IV collagenase in human gingival fibroblasts. The induction levels of both collagenases in human keratinocytes were independent of the type of the extracellular matrix the cells were grown on. However, the basement membrane matrix (Matrigel) activated about half of the 92-kDa type to its 84-kDa active form. The data suggest that TGF-beta 1 has a specific function in up-regulating the expression of type IV collagenases in human keratinocytes, offering a possible explanation of how keratinocytes detach from basement membranes prior to the migration over the wound bed.     

Monoclonal antibodies to type IV collagenase recognize a protein with limited sequence homology to interstitial collagenase and stromelysin

Type IV collagenase is a metalloproteinase associated with metastatic tumor cells. It specifically cleaves the triple helical basement membrane (type IV) collagen molecule at a single site. Monoclonal antibodies which block the activity of the human type IV collagenase were developed and used to purify this antigen. The purified type IV collagenase was partially sequenced following cyanogen bromide and trypsin cleavage. The amino acid sequence of the human type IV collagenase fragments revealed a region homologous to the human interstitial collagenase and stromelysin. However, several sequences in type IV collagenase were identified which are distinct from the latter. Polyclonal antibodies were raised against a synthetic peptide derived from such a sequence. Following affinity purification, the antibodies recognized the denatured human type IV collagenase in Western immunoblotting. These data indicate that type IV collagenase is a distinct member of a general family of metalloproteinases.     

92-kD type IV collagenase mediates invasion of human cytotrophoblasts

The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.     

Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development.

We report the isolation of a cDNA clone providing the first and complete sequence of mouse 72-kDa type IV collagenase. The clone contains 2800 nucleotides with a 1986-nucleotide open reading frame coding for 662 amino acids. The amino acid sequence includes a 29-residue signal peptide, an 80-residue propeptide, and a 553-residue enzyme proper. The sequence identity between the mouse and human enzymes is 96% with all cysteine residues conserved. The carboxyl-terminal domain of the mouse enzyme contains two more residues than the human enzyme. Northern hybridization analysis revealed considerable expression of the enzyme gene in newborn mouse lung, heart, kidney, and psoas muscle tissues, whereas only weak or no signals were observed in liver, spleen, and brain. Expression of the gene was substantially reduced in the same tissues of 3-month-old mice. In situ hybridization analysis of 72-kDa type IV collagenase expression in 10-15-day-old mouse embryos showed that the gene was intensely expressed in mesenchymal cells. Brain and surface ectoderm were completely negative. The epithelial tissue component of developing organs was negative with the exception of salivary gland. Although the expression varied somewhat between different mesenchymal tissues, no temporal or spatial changes could be associated with the advancement of epithelial branching morphogenesis. These findings together with our previous data on the expression of 72-kDa type IV collagenase in human tumors indicate that this enzyme has some very specific roles both in the physiological and pathological degradation of extracellular matrix. Furthermore, it has become clear that the closely related 92-kDa type IV collagenase differs completely with respect to expression pattern as well as gene regulation. The mouse cDNA clones reported in this study may provide important tools unraveling the actual roles of these enzymes in vivo.     

Cleavage of type VII collagen by interstitial collagenase and type IV collagenase (gelatinase) derived from human skin

Type VII collagen is the major structural protein of anchoring fibrils, which are believed to be critical for epidermal-dermal adhesion in the basement membrane zone of the skin. To elucidate possible mechanisms for the turnover of this protein, we examined the capacities of two proteases, human skin collagenase, which degrades interstitial collagens, and a protease with gelatinolytic and type IV collagenase activities, to cleave type VII collagen. At temperatures below the denaturation temperature, pepsin cleaves type VII collagen into products of approximately 95 and approximately 75 kDa. Human skin collagenase cleaved type VII collagen into two stable fragments of approximately 83 and approximately 80 kDa, and the type IV collagenase (gelatinase) produced a broad band of approximately 80 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleavage of type VII collagen was linear with time and enzyme concentration for both enzymes. Although the Km values were similar for both enzymes, the catalytic rate of cleavage by type IV collagenase is much faster than by interstitial collagenase, and shows a greater rate of increase with increasing temperature. Sequence analysis of the cleavage products from both enzymes showed typical collagenous sequences, indicating a relaxation in the helical part of the type VII collagen molecule at physiological temperature which makes it susceptible to gelatinolytic degradation. Interstitial collagenase from both normal skin cells and cells from patients with recessive dystrophic epidermolysis bullosa, a severe hereditary blistering disease in which both an anchoring fibril defect and excessive production of collagenase can be observed, produced identical cleavage products from type VII collagen. These data suggest a pathophysiological link between increased enzyme levels and the observed decrease or absence of anchoring fibrils.     

The proteolytic activity of the recombinant cryptic human fibronectin type IV collagenase from E. coli expression

Human plasma fibronectin (pFN) contains a cryptic metalloprotease present in the collagen-binding domain. The enzyme could be generated and activated in the presence of Ca²⁺ from the purified 70-kDa pFN fragment produced by cathepsin D digestion. In this work we cloned and expressed the metalloprotease, designated FN type IV collagenase (FnColA), and a truncated variant (FnColB) in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, and subjected to folding and autocatalytic degradation in the presence of Ca²⁺, and yielded an active enzyme capable of digesting gelatin, helical type II and type IV collagen, - and -casein, insulin b-chain, and a synthetic Mca-peptide. In contrast, isolated plasma fibronectin, type I collagen, and the DNP-peptide were no substrates. Both catalytically active recombinant pFN fragments were efficiently inhibited by EDTA, and batimastat, and, in contrast to the glycosylated enzyme isolated from plasma fibronectin, were also inhibited by TIMP-2.     

Activation of the 92 kDa type IV collagenase by tissue kallikrein

Type IV collagenases are secreted as latent 92 and 72 kDa proenzymes which are then activated extracellularly. The mechanisms by which they are activated in vivo are not clear. We have studied the activation of porcine endothelial cell type IV collagenases by tissue and plasma kallikrein, and found that tissue kallikrein was a very efficient activator of the 92 kDa type IV collagenase. Enzyme cleavage was observed at concentrations of tissue kallikrein as low as 0.1 μg/ml. Plasma kallikrein had no effect. By comparison, plasmin, which has been proposed to be the physiological activator of interstitial collagenase and stromelysin, and elastase were much less effective, and high concentrations (plasmin at 100–200 μg/ml and elastase at 20 μg/ml) were required to cause only a limited cleavage which was not associated with an increase in activity, as observed by the gelatin-gel lysis assay. In addition tissue kallikrein was found by immunohistochemistry to be present in the extracellular matrix of the intima of porcine aortic vessel wall. These findings suggest that tissue kallikrein can be a potential activator of the 92 kDa type IV collagenase in vivo. © 1993 Wiley-Liss, Inc.     

Laminin increases the release of type IV collagenase from malignant cells

We have studied the effect of laminin on type IV collagenolytic activity elaborated by malignant cells in culture. Laminin (at concentrations of 4-8 micrograms/ml) added to serum-free culture supernatants of subconfluent A2058 human melanoma cells significantly increased the release of the type IV collagenolytic activity (200-300%). The induction of type IV collagenase was more pronounced (580%) using a fragment of laminin which binds to the cell surface laminin receptor. A monoclonal antibody against the human laminin receptor blocked the effect of laminin on type IV collagenase, suggesting that occupation of the laminin receptor may be necessary for the effect. Increase in the type IV collagenolytic activity mediated by laminin was also demonstrated in two other malignant cell lines, HT fibrosarcoma (168%) and mouse melanoma (B16-F10) (271%). The increase in type IV collagenase was found to be specific for laminin because another cell-binding matrix protein, fibronectin, did not have any effect, and epidermal growth factor and transferrin actually decreased the type IV collagenase in human melanoma culture medium (epidermal growth factor, 50% at 20 ng/ml; and transferrin, 20% at 10 micrograms/ml). These studies suggest that tumor cell binding to laminin, which comprises the first step of basement membrane invasion, will induce the second step, namely the collagenolytic dissolution of the basement membrane.     

High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development

cDNA clones for murine 92 kD type IV collagenase (gelatinase B) were generated for the determination of its primary structure and for analysis of temporal and spatial expression in vivo. The mouse enzyme has 72% sequence identity with the human counterpart, the major difference being the presence of a 16-residue segment absent from the human enzyme. In situ hybridization analyses of embryonic and postnatal mouse tissues revealed intense signals in cells of the osteoclast cell lineage. Clear expression above background was not observed in macrophages, polymorphonuclear leukocytes, monocytes, or epithelial cells which have been shown to express the gene in vitro in cell cultures. Expression of the gene was first observed at early stage of cartilage and tooth development at E13, where signals were seen transiently in surrounding mesenchymal cells. At later developmental stages and postnatally strong expression was seen in large cells at the surface of bones. These cells were presumably osteoclasts as their location correlated with that of TRAP positive cells. Signals above background were not observed in a number of other tissues studied. The results represent the first demonstration of a highly osteoclast specific extracellular proteinase. The results suggest that during normal development of embryonic organs the 92-kD type IV collagenase does not have a major role in basement membrane degradation, but is rather mainly used for the turnover of bone matrix, possibly as a gelatinase required for the removal of denatured collagen fragments (gelatin) generated by interstitial collagenase.     

Isolation and characterisation of a chicken gelatinase (type IV collagenase).

The proform of chick gelatinase (type IV collagenase) was isolated and purified to a high specific activity of 12,071 U/mg from cultured embryonic skin fibroblasts stimulated with cytochalasin-B. The enzyme was activated in the presence of 4-aminophenylmercuric acetate with a fall in molecular weight from 66,000-58,000 on non-reducing polyacrylamide gel electrophoresis and was active over the pH range of 6.0-8.9 against a number of substrates. Further biochemical characterisation showed that the organomercurial activated form of the enzyme behaved like a typical mammalian gelatinase, actively degrading gelatin, soluble type I collagen, collagenase generated type I fragments, type IV collagen (producing 3/4 and 1/4 fragments) and type V collagen, whilst having little effect on laminin. The enzyme was inhibited by metal chelators such as EDTA and 1,10-phenanthroline, but not by inhibitors is suggested that this may be TIMP-2. An antiserum was raised to the proenzyme and was found to localise intra- and extra-cellularly in both tissue sections and cell cultures.     

Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer showed that scFv M97 transfection significantly prolonged the survival time of nude mice. The results indicate that intracellular antibody technology represents a novel and efficient way to abrogate selectively the activity of type IV collagenase.     

Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer     

Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator.

Regulation of the activity of proteolytic enzymes is of major importance in the turnover of connective tissues. The search for physiologically relevant activation mechanisms of principal tissue-degrading enzymes, e.g., metalloproteinases, has therefore been of wide interest. We have now studied whether the initiating factor of the fibrinolytic system, urokinase plasminogen activator (u-PA), may also function in the early steps of activation of one of the metalloproteinases, the M(r) 72,000 gelatinase/type IV collagenase produced by cultured fibroblasts. Treatment of the secreted M(r) 72,000 proteinase by u-PA yielded a cleavage product of M(r) 62,000 as revealed by fluorography of radioactively labeled proteins as well as by gelatin zymography SDS-PAGE gels. The u-PA-catalyzed cleavage of the M(r) 72,000 proteinase was blocked by anti-u-PA antibodies, but was unaffected by the plasmin inhibitor aprotinin, thus indicating a specific action for the activator. On the contrary, the tissue activator of plasminogen, t-PA, did not cleave the type IV collagenase in similar assays. u-PA-catalyzed cleavage of recombinant type IV collagenase, produced in a baculovirus expression system, yielded a similar M(r) 62,000 activity in gelatinolysis assay. Zymograms of the isolated pericellular matrices of cultured fibroblasts also revealed M(r) 72,000 gelatinolytic polypeptide that was converted to an M(r) 62,000 form by u-PA. Both polypeptides were recognized in immunoblotting by antibodies against the gelatinase/type IV collagenase, suggesting immunological identity with the secreted enzyme. Thus the M(r) 72,000 gelatinase/type IV collagenase is not only secreted, but also deposited into the pericellular fibroblast matrix, and both forms are substrates for u-PA. The results suggest a new potential role for u-PA as a direct regulator of metalloproteinase-mediated extracellular proteolysis via the cleavage of the M(r) 72,000 gelatinase/type IV collagenase to an M(r) 62,000 form.