Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.

Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.     

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.     

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.     

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.     

Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures.

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.     

Collagenase expression and endogenous activation in rabbit synovial fibroblasts stimulated by the calcium ionophore A23187

Collagenase is synthesized and secreted by stimulated rabbit fibroblasts as a proenzyme that must be proteolytically cleaved to yield catalytically active species. The calcium ionophore A23187 has provided new insights into the regulation of collagenase activation cascade by living cells. A23187, at concentrations of 10-40 ng/ml, induced expression of collagenase and stromelysin mRNA and the secretion of procollagenase of 57 and 53 kDa and prostromelysin of 51 kDa. Interestingly, it also stimulated activation of procollagenase to active forms of 47 and 43 kDa. The concentrations and treatment times required for induction of gene expression and activation indicated that they were independent events. Active collagenase constituted up to 16% of the total collagenase present in medium conditioned by A23187-treated cells. When grown on a collagen substrate, A23187-treated cells degraded collagen in a spatially localized manner. In cells treated with agents that induce procollagenase only, collagenase was localized in the perinuclear Golgi area; however, in A23187-treated cells, collagenase was located in widely dispersed granules, suggesting different intracellular pathways for collagenase before, during, and after activation. Addition of serine, thiol-, and metalloproteinase inhibitors with A23187 to rabbit fibroblasts inhibited conversion of procollagenase to its active form to varying degrees, suggesting that enzymes in these classes are involved in a cascade of proteolytic events leading to collagenase activation.     

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.     

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.     

Changes in cell shape correlate with collagenase gene expression in rabbit synovial fibroblasts

Induction of the neutral proteinase, collagenase, is a marker for a specific switch in gene expression observed in rabbit synovial fibroblasts. A variety of agents, including 12-O-tetradecanoylphorbol-13-acetate, cytochalasins B and D, trypsin, chymotrypsin, poly(2-hydroxyethylmethacrylate), and trifluoperazine induced this change in gene expression. Induction of collagenase by these agents was always correlated with a marked alteration in cell morphology, although the cells remained adherent to the culture dishes. The amount of collagenase induced was positively correlated with the degree of shape change produced by a given concentration and, to some extent, with the duration of treatment. Altered cell morphology was required only during the first few hours of treatment with inducing agents; after this time collagenase synthesis continued for up to 6 d even when agents were removed and normal flattened cell morphology was regained. All agents that altered cell morphology also produced a characteristic switch in protein secretion phenotype, characterized by the induction of procollagenase (Mr 53,000 and 57,000) and a neutral metalloproteinase (Mr 51,000), which accounted for approximately 25% and 15% of the protein secreted, respectively. Secretion of another neutral proteinase, plasminogen activator, did not correlate with increased collagenase secretion. In contrast, synthesis and secretion of a number of other polypeptides, including the extracellular matrix proteins, collagen and fibronectin, were concomitantly decreased. That changes in cell shape correlated with a program of gene expression manifested by both degradation and synthesis of extracellular macromolecules may have broad implications in development, repair, and pathologic conditions.     

Mononuclear cell-mediated modulation of synovial cell metabolism. I. Collagen synthesis

Human PHA-stimulated mononuclear cells produce a factor which inhibits synovial cell collagen and non-collagen protein synthesis, whereas it enhances hyaluronic acid (HA) production. Indomethacin (10(-4)-10(-6) M), a cyclo-oxygenase inhibitor, suppresses this effect, suggesting that the mechanism is prostaglandin-mediated. The active material, of apparent molecular weight 12 000-20 000, also displays the properties of the mononuclear cell factor (MCF) previously described by others, since its stimulates collagenase and PGE2 release by the cultured synovial cells. Furthermore, it co-purifies with interleukin 1 (IL 1) as shown by lymphocyte-activating factor activity. This strongly suggests that IL 1 could be responsible for some (or all) the effects observed on MCF-exposed synovial cells. From these data, we deduce the possibility that mononuclear cells may participate in limiting synovial collagen deposition in rheumatoid arthritis.     

Biosynthesis of tissue inhibitor of metalloproteinases by human fibroblasts in culture. Stimulation by 12-O-tetradecanoylphorbol 13-acetate and interleukin 1 in parallel with collagenase

Biosynthesis of the glycoprotein tissue inhibitor of metalloproteinases (TIMP) by human fibroblasts in culture has been characterized by functional assays, immunoprecipitation, and immunocytochemistry with a monospecific antiserum. As determined by radiolabeling with [35S]methionine, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the secreted form of TIMP had an Mr of 29,000, whereas the form associated with the cell layer had an Mr of 24,000. Unstimulated human lung fibroblasts (HFL-1) secreted TIMP at the rate of approximately 2 micrograms/10(6) cells/24 h, and normal foreskin fibroblasts (HS 27) and skin fibroblasts from a patient with Hurler's disease (GM 1391) secreted TIMP at 0.3 and 0.2 micrograms/10(6) cells/24 h, respectively. Secretion of TIMP was stimulated up to 10-fold by treating the cells with 20-100 ng/ml of 12-O-tetradecanoylphorbol 13-acetate or 10 units/ml of human interleukin 1. In the stimulated HFL-1 cells, TIMP accounted for 0.03-0.09% of the total [35S]methionine incorporated into protein, and 0.3-0.8% of the [35S]methionine in secreted protein. Although TIMP accounted for a relatively small proportion of total protein synthesis of the fibroblasts, greater than 80% of untreated and greater than 95% of stimulated fibroblasts synthesized TIMP, as determined by indirect immunofluorescence. The treatments of the human fibroblasts that increased TIMP secretion also induced synthesis and secretion of proenzyme forms of collagenase, indicating that degradative enzymes and their controlling inhibitors may be synthesized in parallel under certain conditions.     

Stromelysin is an activator of procollagenase. A study with natural and recombinant enzymes.

The latent forms of stromelysin and collagenase from human gingival fibroblasts were purified to homogeneity. These latent proenzymes underwent serial small reductions in Mr upon activation by treatment with either 4-aminophenylmercuric acetate or trypsin. Similar shifts in Mr and activation kinetics were observed upon identical treatments of either recombinant prostromelysin or procollagenase. Prostromelysin showed a lag between activation and Mr fall, suggesting an initial activation by conformational change. Collagenase activity was enhanced up to 12-fold by either natural or recombinant stromelysin in the presence of trypsin or 4-aminophenylmercuric acetate. Stromelysin caused a further apparent decrease in the Mr of procollagenase. Since these important connective-tissue-degrading enzymes are usually co-ordinately produced by cells, a cascade mechanism is proposed in which collagenase is activated by stromelysin.     

Interleukin-1-like factor from human B-lymphocytes transformed by the Epstein-Barr virus

The NC-37, EBV-transformed human B-lymphoblastoid cell line spontaneously produces interleukin (IL)-1-like factor, which is able to stimulate proliferation of mouse thymocytes and human fibroblasts. The IL-1-like factor production can be enhanced by prodigiozan and by the conditioned medium of human peripheral blood mononuclear cells. The NC-37 cells have a membrane-associated form of IL-1-like factor also. The molecular mass of the intracellular precursor of this factor determined by gel-filtration is 35-40 kDa, and that of the secretory form--18-20 kDa. The secretory form has the following isoelectric points: 4.5; 6.8; 7.2-8.4.     

The activation of human skin fibroblast procollagenase. Sequence identification of the major conversion products

Human skin collagenase is secreted by cultured fibroblasts in a proenzyme form and can be activated to a catalytically competent enzyme by a number of processes. All modes of activation studied lead to conversion of the proenzyme to a stable 42-kDa active enzyme, concomitant with removal of an 81-amino acid peptide from the amino-terminal end of the molecule. The sequence of events leading to the formation of this enzyme form has been determined by analyzing the primary structure of the conversion intermediates. Trypsin-induced activation of procollagenase occurs as a result of the initial cleavage of the peptide bond between Arg-55 and Asn-56, generating a major intermediate of 46 kDa. Treatment of the proenzyme with organomercurials, which have no intrinsic ability to cleave peptide bonds, initially results in activation of the enzyme without loss of molecular weight. This is followed by conversion to two lower molecular weight species of 44 and 42 kDa, the latter corresponding to the stable active enzyme form. The final cleavage producing this form of collagenase is not restricted to a single polypeptide bond but can occur on the amino-terminal side of any one of three contiguous hydrophobic residues, Phe-100, Val-101, Leu-102. The data suggest that both trypsin and organomercurials activate procollagenase by initiating an intramolecular autoproteolytic reaction resulting in the formation of a stable 42-kDa active enzyme species.     

Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity

The atrophy of extracellular matrix is a common event during the aging of connective tissues. In this study, we tested the hypothesis that the altered ability of senescent cells to be modulated by serum growth factors correlated with a loss of regulation of collagenase synthesis. We examined the levels of immunoreactive procollagenase and collagenase inhibitor (the tissue inhibitor of metalloproteinases, TIMP) associated with young and senescent fibroblasts cultured in vitro. Young fibroblasts cultured in low (0.5%) concentrations of fetal bovine serum respond to increased (10%) serum by increasing levels of procollagenase and TIMP beginning 4.0 h after serum stimulation. In contrast, senescent fibroblasts constitutively produce relatively high levels of procollagenase even when cultured in low levels of serum and do not respond to serum stimulation by increasing procollagenase synthesis. In addition, senescent fibroblasts constitutively express a relatively small amount of TIMP which is not induced upon serum stimulation. This altered expression of collagenase and TIMP appears unique to the senescent phenotype and not merely a result of growth inhibition, since young cells growth arrested by density-dependent growth inhibition displayed a temporal pattern of procollagenase and TIMP expression upon serum stimulation similar to that of subconfluent young cultures. An assay of net collagenase activity revealed a greater than 20-fold elevation of activity in trypsin-activated extracts from senescent versus young fibroblasts when cultured in a low concentration of fetal bovine serum. These results demonstrate for the first time a direct correlation between alterations in the molecular pathways regulating connective tissue homeostasis and those of replicative senescence. The increased collagenolytic activity of senescent compared to young fibroblasts cultured in the presence of a low serum concentration suggests that aging fibroblasts may become increasingly fibroclastic causing many of the age-associated alterations in dermal collagen observed during aging in vivo.     

Monoclonal antibodies to human fibroblast procollagenase. Inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH2-terminal end of the activated enzyme

This study describes 11 monoclonal antibodies (Mabs) against human fibroblast collagenase that (i) inhibit the specific catalytic activity of the enzyme and/or (ii) react with one or more forms of the enzyme on Western blots. Each of the Mabs specifically immunoprecipitated the Mr 57,000/52,000 procollagenase from [35S]methionine-labeled culture medium. Five Mabs, designated VI-3, VI-4, 2C5, 4A2, and 7C2, inhibited the activity of fibroblast-type collagenase against soluble monomeric collagen and against reconstituted collagen fibrils but did not inhibit the genetically distinct human PMN leukocyte collagenase. The interstitial collagenase produced by human mucosal keratinocytes (SCC-25) was also inhibited, whereas the corresponding enzyme from rat was not. Assignment of epitopes to structural domains within the molecule based on immunoperoxidase staining of Western blots of collagenase and its autocatalytic fragments revealed that 9 of 11 epitopes, including those recognized by 4 inhibitory Mabs, were clustered in a 169-residue domain, which constitutes the NH2-terminal part of the Mr 46,000/42,000 active enzyme. One Mab (X-2a) specifically recognized the Mr 57,000/52,000 zymogen species and failed to react with the active Mr 46,000/42,000 form. The inhibitory Mab VI-3 was used for immunoaffinity purification of procollagenase from culture media with a recovery better than 80% and a yield of approximately 1.4 mg of enzyme/L of medium.