Autocrine control of collagenase synthesis by synovial fibroblasts

Fibroblasts respond to exogenous stimuli, such as Interleukin 1, phorbol esters, or crystals of monosodium urate monohydrate, by synthesizing and secreting large quantities of collagenase. Here we show that addition of exogenous stimuli results in the production of an autologous protein that is, itself, capable of inducing collagenase. This autocrine has been partially purified. Activity resides in a protein(s) with a pl of 5 or 8 and with Mr of approximately 15K. Conversely, conditioned medium taken from unstimulated cultures contains an inhibitor of collagenase synthesis. This protein, which has a Mr approximately 20-25k by HPLC gel filtration antagonizes collagenase synthesis induced by phorbol esters, exogenous parallel 1, and the autologous inducer. We conclude that synovial fibroblasts regulate collagenase synthesis via an autocrine mechanism that includes the synthesis of both an inducer and inhibitor. Both proteins are active at nanomolar amounts and may function as polypeptide hormones in regulating collagenase synthesis and, hence, connective tissue remodeling.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels.

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Purification and characterization of a collagenase inhibitor produced by bovine vascular smooth muscle cells

A potent polypeptide inhibitor of mammalian collagenases was purified to homogeneity from medium conditioned by bovine aortic smooth muscle cells maintained in culture. This inhibitor was purified by a series of molecular sieve and heparin-Sepharose chromatographic procedures; it had an apparent Mr of 28,500 and was a major protein secreted by the smooth muscle cells. It was found to be active against several mammalian collagenases including those obtained from rabbit and human fibroblasts and a tumor-specific type IV collagenase. In contrast, it had minimal inhibitory activity for bacterial collagenase and was inactive against the serine proteases plasmin and trypsin. The inhibitor shared many characteristics with tissue inhibitor of metalloproteinases including the ability to irreversibly inhibit susceptible proteinases, heat and acid resistance, and sensitivity to trypsin degradation and reduction-alkylation. A polyclonal rabbit antiserum with blocking activity which recognized the Mr 28,500 protein was obtained. This inhibitor, which is likely produced by bovine vascular smooth muscle cells in vivo to protect the collagen matrix of blood vessels, may play an important role in pathological conditions associated with alteration of collagen metabolism in tissues.     

Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells

We have investigated the influence of culture substrata upon glycosaminoglycans produced in primary cultures of mouse mammary epithelial cells isolated from the glands of late pregnant mice. Three substrata have been used for experiments: tissue culture plastic, collagen (type I) gels attached to culture dishes, and collagen (type I) gels that have been floated in the culture medium after cell attachment. These latter gels contract significantly. Cells cultured on all three substrata produce hyaluronic acid, heparan sulfate, chondroitin sulfates and dermatan sulfate but the relative quantities accumulated and their distribution among cellular and extracellular compartments differ according to the nature of the culture substratum. Notably most of the glycosaminoglycans accumulated by cells on plastic are secreted into the culture medium, while cells on floating gels incorporate almost all their glycosaminoglycans into an extracellular matrix fraction. Cells on attached collagen gels secrete approx. 30% of their glycosaminoglycans and assemble most of the remainder into an extracellular matrix. Hyaluronic acid is produced in significant quantities by cells on plastic and attached gels but in relatively reduced quantity by cells on floating gels. In contrast, iduronyl-rich dermatan sulfate is accumulated by cells on floating gels, where it is primarily associated with the extracellular matrix fraction, but is proportionally reduced in cells on plastic and attached gels. The results are discussed in terms of polarized assembly of a morphologically distinct basal lamina, a process that occurs primarily when cells are on floating gels. In addition, as these cultures secrete certain milk proteins only when cultured on floating gels, we discuss the possibility that cell synthesized glycosaminoglycans and proteoglycans may play a role in the maintenance of a differentiated phenotype.     

Effects of retinol and dexamethasone on cytokine-mediated control of metalloproteinases and their inhibitors by human articular chondrocytes and synovial cells in culture

Human articular chondrocytes in culture produced large amounts of specific mammalian collagenase, gelatinase and proteoglycanase when exposed to dialysed supernatant medium derived from cultured human blood mononuclear cells (mononuclear cell factor) or to conditioned medium, partially purified by fractionation with ammonium sulphate (60–90% fraction), from cultures of human synovial tissue (synovial factor). Human chondrocytes and synovial cells also released into culture medium an inhibitor of collagenase of apparent molecular weight about 30 000, which appeared to be similar to the tissue inhibitor of metalloproteinases synthesised by tissues in culture. The amounts of free collagenase inhibitor were reduced in culture media from chondrocytes or synovial cells exposed to mononuclear cell factor or synovial factor. While retinol inhibited the production of collagenase brought about by mononuclear cell factor or synovial factor, it restored the levels of inhibitor, which were reduced in the presence of mononuclear cell factor or synovial factor. Dexamethasone markedly reduced the production of collagenase by synovial cells, while only partially inhibiting factor-stimulated collagenase production by chondrocytes. Addition of puromycin as an inhibitor of protein synthesis reduced the amounts of both collagenase and inhibitor to control or undetectable levels.     

Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen

A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.     

A vertebrate interstitial collagenase inhibitor from bovine scapular cartilage: purification and characterization

A collagenase inhibitor was purified from bovine cartilage by a combination of gel filtration, ion exchange, concanavalin A-Sepharose affinity chromatography, and elution from preparative sodium dodecyl sulfate-polyacrylamide gels. The inhibitor was purified 370-fold and migrated as a single polypeptide with an Mr of 19,000 on SDS-polyacrylamide gels. It stained positively for carbohydrate with periodic acid-Schiff's reagent and bound to lectins, indicating that it is a glycoprotein. The inhibitory activity was stable to heating up to 60 degrees C and between pH 4 and 10. The inhibition of collagenase by the cartilage inhibitor could not be reversed by trypsin or mersalyl. The inhibitory activity did not require the presence of free sulfhydryl groups, and it could be removed from the cartilage extract by incubating with native collagen, suggesting that the inhibitor binds to collagen. The cartilage inhibitor was effective against human and mouse interstitial collagenases, but it did not inhibit trypsin or bacterial collagenase.     

Bradykinin augments fibroblast-mediated contraction of released collagen gels

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10(-9) and 10(-8) M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+ mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.     

Collagenase inhibitor stimulates cleft formation during early morphogenesis of mouse salivary gland

A collagenase inhibitor obtained from the culture medium of bovine dental pulp markedly enhanced the cleft formation of mouse embryonic salivary gland epithelium when the inhibitor was included in the culture medium for 12-day and 13-day salivary glands. Determination of collagenase activity using [3H]collagen as substrate indicated that there was a latent collagenase activity in 12-day glands. In addition, a highly purified Clostridial collagenase freed from protease and hyaluronidase activities, strongly inhibited initiation of the cleft formation of the 12-day epithelium. Scanning electron microscopic observation showed that abundant collagen-like fibrils were seen on the epithelium in the collagenase-inhibitor-treated glands compared to those in the control. These findings suggest that during early morphogenesis tissue collagenase may regulate the cleft formation in the epithelium.     

Insulin stimulates secretion of a collagenase inhibitor by Swarm rat chondrosarcoma chondrocytes

Swarm rat chondrosarcoma chondrocytes produce an inhibitor of collagenase similar to that found in bovine articular chondrocytes and extracts of bovine scapular cartilage. These cells synthesize normal levels of cartilage type proteoglycans when cultured in serum free medium with insulin. Collagen synthesis is also increased when insulin is added to chondrosarcoma chondrocytes. We have demonstrated that insulin stimulates collagenase inhibitor production by these chondrocytes. Enhancement of inhibitory activity occurs over the range of 10 to 1000 ng/ml. A 3.2 fold stimulation was observed at a concentration of 1 microgram/ml. There was a lag period of 24 to 48 hours before the insulin effect became evident. Latent or active collagenase was not detectable under these conditions. These results suggest that the hormone insulin controls the levels of collagen in this tumor by stimulating synthesis of collagen and inhibitors of collagenase.     

In vitro maturation of collagen fibrils modulates spreading, DNA synthesis, and collagenolysis of epidermal cells and fibroblasts

Collagen fibrils were maturated in vitro by incubating them in a serum-containing culture medium at 37 degrees C for varied lengths of time. Epidermal cells and fibroblasts were cultured on these maturated collagen gels to see the effects of maturation on cellular morphology and physiology. The spreading and DNA synthesis of both types of cells on the maturated collagen gels were significantly enhanced compared to those on fresh gels. The maturation did not affect the cellular adhesiveness to the substrate. The secretion of collagenase by epidermal cells was suppressed on the maturated collagen gels, the extent of the suppression being related to the length of maturation of the gels. These maturation-related effects of collagen were also observed when collagen was incubated in the medium without serum, indicating that the effects are not due to deposition of serum proteins to collagen gels during maturation. Physical and chemical characterizations of the maturated collagen were performed: the mechanical strength of collagen gels increased in maturated collagen gels, the amounts of insoluble collagen increased with the maturation. These changes in the chemical and physical nature of the maturated collagen gel strongly suggested that there was an increase in intermolecular crosslinks during the process of maturation. These maturation-induced changes in collagen were marked when collagen gels were incubated in the presence of glucose, indicating that a glucose-protein reaction such as the Maillard reaction is involved in this phenomenon.     

Collagen-induced proMMP-2 activation by MT1-MMP in human dermal fibroblasts and the possible role of alpha2beta1 integrins

Culture of human dermal fibroblasts within a three-dimensional matrix composed of native type I collagen fibrils is widely used to study the cellular responses to the extracellular matrix. Upon contact with native type I collagen fibrils human skin fibroblasts activate latent 72-kDa type IV collagenase/ gelatinase (MMP-2) to its active 59- and 62-kDa forms. This activation did not occur when cells were cultured on plastic dishes coated with monomeric type I collagen or its denatured form, gelatin. Activation could be inhibited by antibodies against MT1-MMP, by the addition of TIMP-2 and by prevention of MT1-MMP processing. MT1-MMP protein was detected at low levels as active protein in fibroblasts cultured as monolayers. In collagen gel cultures, an increase of the active, 60-kDa MT1-MMP and an additional 63-kDa protein corresponding to inactive MT1-MMP was detected. Incubation of medium containing latent MMP-2 with cell membranes isolated from fibroblasts grown in collagen gels caused activation of the enzyme. Furthermore, regulation of MT1-MMP expression in collagen cultures seems to be mediated by alpha2beta1 integrins. These studies suggest that activation of the proMMP-2 is regulated at the cell surface by a mechanism which is sensitive to cell culture in contact with physiologically relevant matrices and which depends on the ratio of proenzyme and the specific inhibitor TIMP-2.     

Comparison of collagen gels and mammary extracellular matrix as substrata for study of terminal differentiation in rabbit mammary epithelial cells

Mammary epithelial cells were prepared by collagenase digestion of tissue from mid-pregnant rabbits and cultured for up to 6 days on either collagen gels or an extracellular matrix prepared from the same tissue. The behaviour of the cells in serum-supplemented medium containing combinations of insulin, prolactin, hydrocortisone, estradiol and progesterone were monitored by measuring rates of casein synthesis, lactose synthesis, DNA synthesis and protein degradation. After 6 days, epithelial cells on floating collagen gels showed substantial increases in casein synthesis and DNA synthesis over freshly-prepared cells, following a decline during the first 3 days when the collagen gels are contracting. The optimum hormone combination for casein synthesis was insulin + prolactin + hydrocortisone, whereas for optimum DNA synthesis the additional presence of estradiol and progesterone was required. Cells on extracellular matrix showed increased rates of both casein synthesis and DNA synthesis by day 6 in the presence of insulin + prolactin + hydrocortisone, with additional estradiol + progesterone having an inhibitory effect. Whereas on day 2 rates of intracellular protein degradation were generally lower in cells on extracellular matrix, by day 6 rates of protein degradation were lowest in cells cultured on collagen gels with insulin + prolactin + hydrocortisone. In all cases, rates of lactose synthesis fell to low levels as the culture proceeded. Pulse-chase labelling of freshly-prepared cells with [32P]orthophosphate in medium containing serum and insulin + prolactin + hydrocortisone demonstrated that newly-synthesized casein was degraded during its passage through the epithelial cell. The influences of the collagen gels and extracellular matrix and of the hormone combinations on epithelial cell differentiation and secretory activity are discussed.     

Inhibition, by lanthanides, of neutral proteinases secreted by human, rheumatoid synovium

Fragments of human, rheumatoid synovium were maintained on organ culture for three days under serum-less conditions. Their conditioned media contained collagenolytic, gelatinolytic and caseinolytic activities, which were susceptible to inhibition by lanthanide ions. Of the four lanthanides tested, Sm3+ proved the best inhibitor of gelatinase and caseinase, while La3+ inhibited collagenase the most strongly. Inhibition of collagenase by La3+ was uncompetitive. A direct binding assay confirmed the greater association between collagen fibrils and collagenase in the presence of La3+. Ca2+ was not required for binding of the uninhibited enzyme to collagen, but acted to stabilize collagenase against thermoinactivation.     

Regulation of human skin collagenase activity by hydrocortisone and dexamethasone in organ culture

Hydrocortisone and dexamethasone (9α-fluoro, 16α-methyl prednisolone) prevent the appearance of collagenase in cultures of normal human skin, human rheumatoid synovium and rat uterus. Hydrocortisone is maximally inhibiting at 10^?7M and dexamethasone at 10^?8M in culture medium. Neither steroid is an inhibitor of enzyme activity. The loss of collagenase activity in cultured tissue is not accompanied by detectable inhibition of protein synthesis. Reduction of enzyme activity in culture medium is concomitant with a parallel cessation of tissue collagen degradation, indicating that the tissue fails to produce active collagenase in the presence of physiologic levels of glucocorticoids.     

Suramin inhibits C6 glioma-induced angiogenesis in vitro

Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-μm thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-μm pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently from networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiongenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis.     

Purification and characterization of collagenase activator protein synthesized by articular cartilage

We have isolated an activator of collagenase from medium conditioned with articular cartilage. The activity is contained in an acidic protein appearing as a doublet band of Mr 57,000 and 56,000 on sodium dodecyl sulfate polyacrylamide gels. Both components of the doublet have identical isoelectric points as demonstrated by gel electrophoresis. Purified synovial collagenase has a high dependence on the presence of this factor for activity. Other known activators of latent proteolytic enzymes such as trypsin and mercurials will stimulate collagenase but only if activator protein is present. The activator protein is itself a latent metalloprotease because in the presence of p-aminophenylmercuric acetate and calcium it will digest casein. The caseinase activity and collagenase activation activity have identical heat inactivation profiles, both being stable to a temperature of 60 degrees C and partially inactivated at 80 degrees C. The synthesis of the activator is localized in the superficial zone of articular cartilage.     

A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.     

Gelatinases and metalloproteinase inhibitor secreted by murine colonic carcinoma cells with differing metastatic potential

Gelatinase activity and inhibitory activity against collagenase were measured in serum-free medium conditioned by murine colonic carcinoma cells with different spontaneous metastatic potentials to the lung. The medium conditioned with poorly metastatic NM11 cells gave higher inhibitory activity than that conditioned with highly metastatic LuM1 cells, while the level of secreted gelatinases in the same medium was lower in NM11 medium than in LuM1 case. Northern analysis showed the higher gene expression of both tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in NM11 cells than in LuM1 cells, suggesting that both TIMPs are responsible for the increase of inhibitory activity in NM11 conditioned medium. Examination of the balance of gelatinases and inhibitor revealed that the amount of inhibitor exceeded that of gelatinases in the medium conditioned with NM11 cells. In contrast, the medium conditioned with LuM1 cells contained excess amounts of gelatinases. The results indicated a close correlation between the balance of gelatinases and inhibitors and the metastatic behavior of murine tumor cells.