Collagenase is a major gene product of induced rabbit synovial fibroblasts

We have investigated the effects of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on rabbit synovial fibroblasts, and found that this agent induced a major switch in gene expression in these cells that was marked by the specific induction of the neutral proteinase, collagenase, and was always accompanied by alterations in cell morphology. Procollagenase synthesis and secretion was first observed 6-12 h after the addition of TPA. The rate of collagenase production (1-5 U, or approximately 0.2-1 micrograms secreted procollagenase protein per 10(5) cells per 24 h) depended on the TPA concentration (1-400 ng/ml) and time of exposure (1-72 h). Procollagenase was the most prominent protein visible by direct silver staining or by autoradiography after SDS PAGE of [35S]methionine-labeled proteins. The two procollagenase bands of Mr 53,000 and 57,000, which migrated as a family of spots on two-dimensional gels and were immunoprecipitated by antibodies to purified rabbit collagenase, accounted for 23% of the newly synthesized, secreted protein in TPA-treated cells. Cell-free translation of mRNA from TPA-treated cells in rabbit reticulocyte lysate produced a single band of immunoprecipitable preprocollagenase (Mr 55,000) as a major product (5% of total) that migrated as a single spot on two-dimensional gels. Secreted procollagenase, preprocollagenase , and active collagenase (purified to homogeneity; specific activity 1.2 X 10(4) U/mg protein) had related peptide maps. Two other major secreted proteins, a neutral metalloproteinase of Mr 51,000 and a polypeptide of Mr 47,000, were also induced by TPA. In contrast to the induction of these four polypeptides, TPA decreased synthesis and secretion of a number of proteins, including collagen and fibronectin. Thus, collagenase is a convenient marker for major alterations in the pattern of protein synthesis and secretion by rabbit synovial fibroblasts treated with TPA.     

Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization, and substrates

Rabbit synovial fibroblasts induced to undergo a specific switch in gene expression by agents that alter cell morphology secreted the neutral proteinase precursor procollagenase (apparent Mr of 53,000 and 57,000). A major Mr = 51,000 polypeptide that was always induced coordinately with procollagenase has now been identified as the proenzyme form of a metal-dependent proteinase active at neutral pH. We have named this proteinase stromelysin. Prostromelysin and procollagenase were the most prominent [35S]methionine-labeled secreted proteins of the induced fibroblasts. By the use of casein degradation as an assay for enzyme activity, stromelysin was isolated with high yield from the conditioned culture medium of 12-O-tetradecanoylphorbol 13-acetate-treated fibroblasts and migrated as an active form of Mr = 21,000 that was immunologically identical to the proteoglycan-degrading proteinase purified from rabbit bone. Immunoglobulin G from antiserum raised to purified rabbit bone proteoglycanase immunoprecipitated the Mr = 51,000 proenzyme form from conditioned medium of induced rabbit cells and also immunoprecipitated an Mr = 55,000 polypeptide from induced human fibroblasts. When rabbit prostromelysin was activated by trypsin or 4-aminophenylmercuric acetate, the proenzyme was converted to an active form of Mr = 41,000. During the course of the purification, prostromelysin was converted to an additional activatable form of Mr = 35,000 and additional active forms of Mr = 21,000-25,000, which had related peptide maps distinct from collagenase. All of these forms were immunologically cross-reactive. Purified stromelysin degraded casein, cartilage proteoglycans, fibronectin, alpha 1-proteinase inhibitor, and immunoglobulin G2a and had limited activity on laminin, elastin, type IV collagen, and gelatin, but did not degrade type I collagen. Stromelysin was inhibited by EDTA, 1,10-phenanthroline, and the specific glycoprotein tissue inhibitor of metalloproteinases isolated from human amniotic fluid and was therefore classified as a metalloproteinase.     

Reorganization of polymerized actin: a possible trigger for induction of procollagenase in fibroblasts cultured in and on collagen gels

Changes in cell shape are postulated to modulate gene expression during differentiation of a number of cell types, including rabbit synovial fibroblasts, which are inducible for expression of the zymogen form of the metalloendopeptidase, collagenase. In the work presented here, fibroblasts cultured on and within hydrated collagen gels were allowed to contract by release of the gels from the sides of the culture dish. Within 24 h of cell release, synthesis and secretion of procollagenase was initiated in the absence of any chemical manipulation. Fibroblasts grown in and on collagen also responded to 12-O-tetradecanoylphorbol-13-acetate and cytochalasin B with morphologic change and induced procollagenase. However, colchicine, which altered morphology to varying degrees in cells on plastic, on collagen, and within collagen gels, did not induce procollagenase expression. In all cases, the enzyme was induced only after reorganization of polymerized actin, rather than after a change in cellular morphology per se. As a first approach to identifying other aspects of the stimulated phenotype that could affect collagen turnover, the expression of collagen and endogenous metalloproteinase inhibitors in relation to procollagenase secretion was investigated. Collagen secretion by fibroblasts decreased when procollagenase secretion was induced by the pharmacologic agents, but not when cells were stimulated by contraction on or within collagen gels. The expression of two endogenous inhibitors was not coordinately regulated with induction of procollagenase. Therefore, the extracellular matrix and the cellular actin cytoskeleton may transduce signals that modulate the tissue remodeling phenotype of fibroblasts.     

Cytoskeletal dynamics in rabbit synovial fibroblasts: II. Reformation of stress fibers in cells rounded by treatment with collagenase-inducing agents

Modulation of the synthesis and secretion of extracellular matrix proteins and matrix-degrading metalloproteases by rabbit synovial fibroblasts is an important model system for studying the control of tissue-specific gene expression. Induction of collagenase expression is correlated with changes in cell shape and actin filament distribution, but the role of the cellular cytoskeleton in the sustained synthesis and secretion of metalloproteases has not been closely examined. When cells were allowed to respread after rounding by trypsin or cytochalasin, two known metalloprotease inducers, reformation of stress fibers was observed within 2 h in the presence of serum. In the absence of serum, trypsin-treated cells did not respread substantially, even after 24 h in culture. In contrast, cytochalasin-treated cells recovered almost as rapidly in the absence as in the presence of serum, showing reformation of well-formed microfilament bundles within 30 min of drug removal, especially at the spreading cell edges. High resolution electron-microscopic views of detergent-extracted cytoskeletons confirmed the rapid rebundling of peripheral microfilaments. Acrylamide-treated cells fell between these two extremes, spreading slowly in the absence of serum, but almost as rapidly as cytochalasin-treated cells in its presence. Reestablishment of normal intermediate filament distribution generally lagged slightly behind actin for all treatments, and intermediate filaments always appeared to spread back into the cellular cytoplasm within the confines of the reforming peripheral microfilament bundles. No obvious interaction between these two cytoskeletal elements was observed after any treatment, and no specific role for intermediate filaments in modulating gene expression in these cells is suggested by these results. The serum dependence displayed after trypsin or acrylamide treatment may be due to the disturbances in fibronectin synthesis observed in these cells and is consistent with evidence that both induction and sustained expression of matrix-degrading metalloprotease may involve signals transduced through plasma membrane matrix receptors (integrins).     

Role of Serum Amyloid A as an Intermediate in the IL-1 and PMA-Stimulated Signaling Pathways Regulating Expression of Rabbit Fibroblast Collagenase

The matrix metalloproteinase collagenase is expressed by resident tissue cells only when needed for biological remodeling. Exogenous addition of inflammatory and growth-promoting cytokines stimulates collagenase expression in early passage fibroblast cultures. In addition, the signal for collagenase expression in response to phorbol-12 myristate-13 acetate (PMA) or to agents which alter cell shape in early passage fibroblast cultures is routed extracellularly to an autocrine cytokine intermediate, IL-1α. Importantly, fibroblasts, when freshly isolated from the tissue, are not competent for IL-1α gene expression and, therefore, cannot produce collagenase in response to shape change agents. However, they do make a small amount of collagenase in response to PMA via an IL-1-independent pathway that has not been further characterized. In this paper, we investigate the role of a second autocrine, serum amyloid A3 (SAA3), in IL-1-dependent and -independent collagenase gene expression. We demonstrate that SAA3 is required for effective stimulation of collagenase expression by either exogenous or endogenous IL-1. Furthermore, while freshly isolated fibroblasts cannot express IL-1α they can express SAA3, and this autocrine mediator acts independently of IL-1α to control the low level of collagenase expression that can be stimulated by PMA. These results provide further evidence for a newly emerging paradigm of collagenase regulation which emphasizes the requirement for extracellular routing of signals. They also suggest that SAA3 might be utilized independently of IL-1α to control tissue remodelingin vivo.     

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.     

Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression

We have investigated the effects of ligation of the fibronectin receptor (FnR) on gene expression in rabbit synovial fibroblasts. Monoclonal antibodies to the FnR that block initial adhesion of fibroblasts to fibronectin induced the expression of genes encoding the secreted extracellular matrix-degrading metalloproteinases collagenase and stromelysin. That induction was a direct consequence of interaction with the FnR was shown by the accumulation of mRNA for stromelysin and collagenase. Monoclonal antibodies to several other membrane glycoprotein receptors had no effect on metalloproteinase gene expression. Less than 2 h of treatment of the fibroblasts with anti-FnR in solution was sufficient to trigger the change in gene expression, and induction was blocked by dexamethasone. Unlike other inducers of metalloproteinase expression, including phorbol diesters and growth factors, addition of the anti-FnR in solution to cells adherent to serum-derived adhesion proteins or collagen produced no detectable change in cell shape or actin microfilament organization. Inductive effects were potentiated by cross-linking of the ligand. Fab fragments of anti-FnR were ineffective unless cross-linked or immobilized on the substrate. Adhesion of fibroblasts to native fibronectin did not induce metallo-proteinases. However, adhesion to covalently immobilized peptides containing the arg-gly-asp sequence that were derived from fibronectin, varying in size from hexapeptides up to 120 kD, induced collagenase and stromelysin gene expression. This suggests that degradation products of fibronectin are the natural inductive ligands for the FnR. These data demonstrate that signals leading to changes in gene expression are transduced by the FnR, a member of the integrin family of extracellular matrix receptors. The signaling of changes in gene expression by the FnR is distinct from signaling involving cell shape and actin cytoarchitecture. At least two distinct signals are generated: the binding of fibronectin-derived fragments and adhesion-blocking antibodies to the FnR triggers events different from those triggered by binding of the native fibronectin ligand. Because the genes regulated by this integrin are for enzymes that degrade the extracellular matrix, these results suggest that information transduced by the binding of various ligands to integrins may orchestrate the expression of genes regulating cell behavior in the extracellular environment.     

Effects of products from macrophages, blood mononuclear cells and or retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied on cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of 3H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of alpha 1 chains; the amount of alpha 2 chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. alpha 2 chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen, and therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Procollagenase activator produced by rabbit uterine cervical fibroblasts.

Culture medium from rabbit uterine cervical fibroblasts contained a procollagenase and a neutral proproteinase which acts as a procollagenase activator. These two proenzymes have been purified by a combination of ion-exchange, affinity and gel chromatographies. The purified neutral proproteinase showed Mr 60,000 with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This neutral proproteinase was activated by trypsin, 4-aminophenylmercuric acetate (APMA) and plasmin, and the active species of the proteinase had Mr 53,000 when activated by APMA; kallikrein and urokinase did not activate this proproteinase. The purified neutral proteinase was inhibited by EDTA, 1,10-phenanthroline and rabbit plasma, but not by serine proteinase inhibitors, suggesting that this proteinase is a metal-dependent proteinase. The purified enzyme could also degrade gelatin, casein, proteoglycan and type IV procollagen. The purified procollagenase had Mr 55,000 and was activated by trypsin, APMA and the active neutral proteinase. These activations were accompanied by decrease in Mr, and the activated species had an Mr which was approx. 10,000 less than that of the procollagenase. In particular, procollagenase activation with neutral proteinase depended on incubation time and proteolytic activity of proteinase. These results indicate that activation of procollagenase by the rabbit uterine neutral proteinase is related to limited proteolysis in the procollagenase molecule.     

DISRUPTION OF CELL—CELL ADHESION IN THE PRESENCE OF SODIUM BUTYRATE ACTIVATES EXPRESSION OF THE 92kDa TYPE IV COLLAGENASE IN MDCK CELLS

We have investigated the effects of altered cell shape on the regulation of the 92kDa type IV collagenase. In MDCK cells, anti-E-cadherin antibodies alter cell shape by disrupting normal cell—cell contacts, while sodium butyrate causes a marked flattening and spreading of cells. The disruption of cell—cell contacts led to a faint expression of the 92kDa collagenase. This effect was enhanced by sodium butyrate, which by itself did not induce collagenase expression. In contrast, stromelysin expression was not regulated in these conditions. Although mRNA expression was enhanced, the secreted collagenase activity was not altered in these conditions in either cell line. Examination of cytoskeletal and extracellular matrix proteins and cell—cell and cell—matrix adhesion proteins by immunofluorescence and Western blot revealed a disruption of the actin network, tight junctions, and fibronectin deposition by anti E-cadherin antibodies, and alterations in actin, cytokeratin 8, cytokeratin 14, laminin and β1 integrin induced by sodium butyrate. Thus, the induction of collagenase expression in epithelial cells by disrupted cell—cell adhesion and sodium butyrate is associated with changes in cell shape and structure.     

Uncoordinate regulation of collagenase,stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: Selective enhancement of collagenase gene expression in human dermal fibroblasts in culture

The degradative effects of interleukin-1 (IL-1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non-steroidal anti-inflammatory drugs. We have examined the role of prostaglandin E₂ (PGE₂) in IL-1-induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01–1.0 μg/ml) of PGE₂ led to a dose-dependent elevation of collagenase mRNA steady-state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE₂ had no additive or synergistic effect with IL-1 on collagenase gene expression. Furthermore, commonly used non-steroidal anti-inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL-1-induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE₂ did not potentiate those of IL-1 on collagenase gene expression in vitro, one could speculate that massive production of PGE₂ by connective tissue cells in vivo in response to inflammatory mediators such as IL-1 or tumor necrosis factor-α, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.     

Mycobacterium tuberculosis up-regulates matrix metalloproteinase-1 secretion from human airway epithelial cells via a p38 MAPK switch

Pulmonary cavitation is vital to the persistence and spread of Mycobacterium tuberculosis (MTb), but mechanisms underlying this lung destruction are poorly understood. Fibrillar type I collagen provides the lung's tensile strength, and only matrix metalloproteinases (MMPs) can degrade it at neutral pH. We investigated MTb-infected lung tissue and found that airway epithelial cells adjacent to tuberculosis (Tb) granulomas expressed a high level of MMP-1 (interstitial collagenase). Conditioned media from MTb-infected monocytes (CoMTb) up-regulated epithelial cell MMP-1 promoter activity, gene expression, and secretion, whereas direct MTb infection did not. CoMTb concurrently suppressed tissue inhibitor of metalloprotease-1 (TIMP-1) secretion, further promoting matrix degradation, and in Tb patients very low TIMP-1 expression was detected. MMP-1 up-regulation required synergy between TNF-alpha and G protein-coupled receptor signaling pathways. CoMTb stimulated p38 MAPK phosphorylation, and this is the point of TNF-alpha synergy with G protein-coupled receptor activation. Furthermore, p38 phosphorylation was the switch up-regulating MMP-1 activity and decreasing TIMP-1 secretion. Activated p38 localized to MMP-1-secreting airway epithelial cells in Tb patients. These data reveal a monocyte-epithelial cell network whereby MTb may drive tissue destruction, and they demonstrate that p38 phosphorylation is a key regulatory point in the generation of a matrix-degrading phenotype.     

Induction of collagenase secretion in human fibroblast cultures by growth promoting factors

In human foreskin fibroblast cultures, two proteins with Mr 60,000 and 55,000 were found to be induced about 3.5-fold by epidermal growth factor (EGF), platelet-derived growth factor, and beta-transforming growth factor. The induced proteins were identified as procollagenases by immunoprecipitation of induced medium with antibodies to purified human fibroblast collagenase. Collagenase enzyme activity in the medium from EGF-treated cultures was also induced at least 3-fold compared to control cultures. Induction of collagenase was dependent upon de novo protein and RNA synthesis and was observed in the medium 10 h after addition of EGF. Although these growth-promoting factors interact with separate membrane receptors, each induced the secretion of a common protein, suggesting that collagenase may be important in some aspect of mitogenesis, cell mobilization, and migration.     

Cytoskeletal dynamics in rabbit synovial fibroblasts: I. Effects of acrylamide on intermediate filaments and microfilaments

Rabbit synovial fibroblasts respond to changes in cell shape and cytoskeletal architecture by altering specific gene expression. We have tested the ability of acrylamide, a neurotoxin that alters the distribution of intermediate filaments in cultured PtK1 cells, to induce metalloprotease expression in synovial fibroblasts. Cells treated with 2-20 mM acrylamide for 5 to 24 h underwent shape changes similar to cells treated with the tumor promoter phorbol myristate acetate. Intermediate filaments visualized with anti-vimentin antibodies did not collapse into a perinuclear cap in these rounded cells, but were still present in the extended cell processes. Unexpectedly, when actin was visualized in acrylamide-treated cells, extensive dissociation and clumping of microfilaments was observed. Concentrations of acrylamide greater than 10 mM were cytotoxic, but cells recovered completely after 24 h incubation with 5 mM acrylamide. Like other agents that alter cell shape and actin distribution in synovial fibroblasts, acrylamide also induced expression of the secreted metalloprotease collagenase. Although some recent evidence suggests that acrylamide may be able to exert its collagenase-inducing effects extracellularly, perhaps through transmembrane matrix receptors, our observation that this neurotoxin dramatically alters protein synthesis in synovial fibroblasts suggests that direct effects on cell metabolism may also play a role in acute acrylamide intoxication.     

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.     

Purification of a calcium-activated neutral proteinase from human placenta

A calcium-activated neutral proteinase has been purified to homogeneity from human placenta. The purified enzyme is a dimer composed of Mr 73 000 and 30 000 subunits. Half-maximal activity is observed at 250 microM Ca2+. It requires reduced sulfhydryl groups and neutral pH for optimal activity. Leupeptin, antipain, E-64, sulfhydryl-blocking agents and endogenous proteinase inhibitor inhibit the purified enzyme. This paper is the first to describe the purification and characterization of a calcium-activated neutral proteinase from a human non-muscular parenchymatous organ.     

Induction and stimulation of 92-kDa gelatinase/type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumor necrosis factor alpha

Production of a 92-kDa gelatinase/type IV collagenase and tissue inhibitor of metalloproteinases (TIMP) was investigated with human sarcoma cell lines. Among the cytokines and growth factors examined, only human recombinant tumor necrosis factor alpha (TNF alpha) induced and stimulated the proteinase with concomitant increase in TIMP expression, but matrix metalloproteinase 2 (72-kDa gelatinase/type IV collagenase) expression was unchanged. These data suggest that gene expression of the two metalloproteinases is regulated in a different fashion and TNF alpha may be important to allow cancer cells to be more invasive and metastatic.     

Collagen degradation in ischaemic rat hearts.

Myocardial extracellular matrix is organized into a complex arrangement of intercellular and pericellular fibres and fibrils that serves as a supporting framework for contracting cells. Recent evidence suggests that changes in ventricular shape and function occurring after ischaemic injury may be related to alterations of this matrix. In this report we describe the rapid and extensive loss of collagen in myocardial infarction produced by ligating the left anterior descending coronary artery of the rat for 1-3 h. The total collagen content in the myocardial infarct zones after 1, 2 and 3 h of ligation was 75 +/- 8%, 65 +/- 7% and 50 +/- 10% respectively (mean +/- S.D.) of that of either the non-infarcted tissue controls or of the same regions in sex- and age-matched normal left ventricles. A marked decrease also occurred in the residual collagens which were not extractable with 6 M-guanidine hydrochloride, suggesting that rapid degradation of insoluble collagen fibres may also occur. The decreased collagen content in the 3 h myocardial infarct coincided with the appearance of several enzyme activities. Collagenase, other neutral proteinase and presumed lysosomal serine proteinase activities were increased by 3, 3 and 2 times the control values respectively. These results suggest that the increased activities of collagenase and other neutral proteinases may be responsible for the rapid degradation of extracellular matrix collagen in myocardial infarct.