A serum factor promotes collagenase synthesis by an osteoblastic cell line.

Regulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106-01. The cells were stained by the avidin-biotin-complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone-seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secretagogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106-01 cells.     

Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells

Angiogenesis is associated with the fragmentation of blood vessel basement membranes. Since collagen is a major constituent of basement membranes, cultured human endothelial cells derived from umbilical cord veins were assayed for their ability to produce collagenase. Unstimulated cultured human endothelial cells did not secrete detectable levels of active collagenase into the culture medium. However, if the post-culture medium was treated with trypsin or plasmin, low levels of collagenolytic activity were detected, indicating that endothelial cells secrete small amounts of latent collagenase. Addition of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the culture medium stimulated the secretion of collagenase by endothelial cells 5–30 fold. More than 90% of the collagenase was secreted in the latent form. Stimulation of collagenase production was detected at 10^?9 M TPA and was maximal at 10^?8 M TPA. An increase in the rate of collagenase production could be detected within 3 hr after the addition of TPA, and full induction occurred by 12 hr. Cycloheximide (3 μg/ml) or actinomycin D (0.1 μg/ml) inhibited both basal levels of collagenase production and the stimulation of collagenase production by TPA. Phorbol-12,13-didecanoate (PDD), a tumor-promoting analog of TPA, also stimulated collagenase production when administered at the same concentrations that were effective for TPA. However, 4-O-methyl TPA and 4-αPDD, two analogs of TPA which are not tumor promoters, did not stimulate collagenase production at concentrations up to 10^?7 M. The collagenase produced by endothelial cells was a typical vertebrate collagenase as judged by the following criteria: it cleaved collagen into only two fragments which were three quarters and one quarter of the length of the intact molecule; it was inhibited by EDTA and human serum; it was not inhibited by inhibitors of serine, thiol or aspartate proteases. Thus TPA causes an increase in the production of latent collagenase by cultured human endothelial cells.     

Serotonin: an inducer of collagenase in myometrial smooth muscle cells.

Rat myometrial smooth muscle cells in culture actively produce collagenase in medium containing fetal bovine serum, but not in medium containing newborn bovine serum or containing fetal serum adsorbed with dextran-coated charcoal. A dialyzable molecule has been isolated from fetal bovine serum, which restores the ability of the smooth muscle cells to produce collagenase. The molecule has been purified and identified as serotonin (5-hydroxytryptamine). Cells cultured in medium depleted of serotonin for 3 days fail to produce collagenase, as assessed both enzymatically and immunologically. Addition of serotonin promptly restores the ability of the cells to produce the enzyme. The EC50 for serotonin is approximately 2 microM; maximum stimulation of collagenase production is observed at 5 microM. The response is specific for serotonin: a wide variety of compounds tested, either related to serotonin or of potential reproductive significance, were without effect in the induction of collagenase production by the cells. No changes in DNA content, general protein synthesis, or cellular collagen production were observed as a consequence of serotonin depletion or restoration, suggesting a selective effect of the compound on collagenase production. The effect of serotonin was also selective to myometrial smooth muscle cells; collagenase-producing fibroblasts from skin and cervix displayed no serotonin requirement for enzyme production. Studies using specific agonists or antagonists for a variety of serotonin receptor subtypes suggest that the 5-HT-2 receptor mediates the serotonin induction of collagenase in these cells. Preliminary evidence indicates that cultured human myometrial smooth muscle cells are also dependent upon serotonin for collagenase production. The evidence in this study suggests the possibility that serotonin serves as a signal to begin the massive collagen degradation that occurs in the postpartum uterus.     

Collagenase production by human skin fibroblasts.

Normal human skin fibroblasts, when cultured in serum free medium, produce collagenase in an inactive form. The enzyme in the crude medium can be activated by a brief preincubation with trypsin or by autoactivation. Once activated, the fibroblast collagenase is identical in its mechanism of action to human skin collagenase obtained from organ cultures. In addition, an inhibitor of collagenase is also present in the medium of fibroblast cultures. The inhibitor appears to be produced by the cells and its molecular weight is slightly higher than that of the enzyme. The presence of this inhibitor may account for previous inability to detect collagenase in human skin fibroblast cultures. It is also possible that some of the inactive enzyme exists in the medium in the form of a proenzyme.     

Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid

In order to gain insight into the biological significance of a collagenase inhibitor secreted by human skin fibroblasts, we examined various human connective tissues and body fluids for such a protein. The inhibitors found in these tissues were compared immunologically to skin fibroblast inhibitor by Ouchterlony analysis and by the development of a highly specific enzyme-linked immunosorbent assay (ELISA). Using this ELISA, cell cultures of human skin fibroblasts, corneal fibroblasts, gingival fibroblasts, and adult and fetal lung fibroblasts secreted similar amounts of immunoreactive inhibitor protein. Each culture medium displayed a reaction of immunologic identity with skin fibroblast inhibitor when examined in Ouchterlony gel diffusion. In testing for functional inhibitory activity, the same 1:1 stoichiometry of collagenase inhibition was observed in each culture medium that characterizes the human skin inhibitor. Other mesodermally derived human cell types, including human fetal osteoblasts, uterine smooth muscle cells, fibrosarcoma cells, and explants of tendon and articular cartilage behaved in the same manner as the fibroblast cultures. Because collagenase inhibitors with biochemical similarities to skin fibroblast inhibitor have been described in serum and in amniotic fluid, we also examined these sources of inhibitory proteins. The data indicate that both serum and amniotic fluid contain collagenase inhibitors which are immunologically and functionally identical with the skin fibroblast inhibitor. The concentration of inhibitor in serum, as measured by ELISA assay, is 1.03 +/- 0.27 micrograms/ml. The results suggest that collagenase inhibitors which are functionally equivalent and immunologically identical with human skin fibroblast collagenase inhibitor are synthesized by many, if not all, fetal and adult mesodermal tissues in the human organism. The inhibitor apparently gains access to certain body fluids such as serum and amniotic fluid. This inhibitor protein may, therefore, function in the regulation of collagen degradation in most human connective tissues.     

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.     

Fetal bovine serum inhibits chondrocyte collagenase production: interleukin 1 reverses this effect

Bovine articular chondrocytes incubated in medium which was serum free or contained low levels of fetal bovine serum (less than 5%) constitutively produced collagenase. Increasing the concentration of serum in the culture medium inhibited the production of collagenase. Addition of interleukin 1 and lipopolysaccharide reversed the inhibitory effect of serum. Phorbol esters only stimulated collagenase production when the serum concentration was at least 10%. These data suggest that there is a factor(s) in fetal bovine serum that inhibits collagenase production by chondrocytes and this can be reversed by agents such as interleukin 1.     

Collagenase activity in cultures of rat prostate carcinoma.

A specific collagenase (EC 3.4.24.3) has been found and purified from serum-free culture medium of 11095 epidermoid carcinoma of rat prostate. The molecular weight of this collagenase was estimated at 71 000 and the pH optimum was approx. 7. At 26 degrees C, the collagenase cleaved collagen at a site 3/4 the length from the N-terminus. At 37 degrees C, this collagenase degraded collagen to smaller peptides. The enzyme activity was inhibited by serum, cysteine and EDTA, but not by protease inhibitors. The presence of collagenase in rat tumor tissue suggests that this enzyme might play a significant role in tissue invasion by cancer cells.     

Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos

The zebrafish has become a popular model for studies of vertebrate development and toxicology. However,in vitro approaches utilizing this organism have not been fully exploited due to the absence of suitable cell culture systems. Previously, we developed methods for the culture of cells derived from zebrafish blastula-stage embryos. One of these cultures, ZEM-2, was derived in a complex medium containing trout embryo extract, trout serum and medium conditioned by buffalo rat liver cells. In this study we describe a zebrafish embryo cell line, ZEM-2A, derived from ZEM-2 following selection for growth in a simplified medium. Optimal growth of ZEM-2A cells is attained in nutrient medium supplemented with 5% fetal bovine serum.     

A method for the continuous cultivation of mammary epithelium

Summary Established cell lines of mammary epithelium have been obtained from mice, rats, and hamsters. Maintenance and replication of the epithelium in serial subcultures were dependent on their periodic treatment with collagenase. Because collagenase is not cytotoxic and has maximum efficiency at neutral pH in isotonic saline solutions containing calcium and magnesium, this enzyme can be introduced directly into the culture medium; cells have been maintained for 3 days in such a medium with serous enrichment at no detriment to them. Up to 10% concentrations of serum have not interfered with enzymatic activity. Supported by United States Public Health Service Grant CA-08515 and CA-08740 from the National Cancer Institute. General Research Support Grant FR-5582 from the National Institutes of Health, and Grant-in-Aid Contract M-43 from the State of New Jersey.     

The involvement of proteases and protease inhibitors in neovascularization

Bovine capillary endothelial cells have been found to respond to several stimuli by producing increased amounts of plasminogen activator and latent collagenase. These stimulators include the tumor promoter tetradecanoyl phorbol-13-acetate as well as crude preparations from a human hepatoma, bovine retinae, and mouse adipocytes, all of which are known to contain angiogenic factors. Endothelial cells and skin fibroblasts do not respond to these stimuli in the same way, indicating a specificity of the response. In addition, inhibitors of plasmin and vertebrate collagenase have been isolated from cartilage, a tissue resistant to neovascularization. We have proposed that these specific protease inhibitors confer on cartilage its antiangiogenic properties.     

Replacement of vertebrate serum with lipids and other factors in the culture of invertebrate cells,tissues, parasites,and pathogens

Summary Culture medium supplementation with vertebrate serum results in the selection of fibroblastoid insect cell lines and a general decline during continuous subculturing of both morphologic and functional differentiation of the surviving cells. Essential lipid mixtures can substitute for vertebrate serum in the culture of insect and some vertebrate cells, tissues, parasites, and pathogens. The provision of sterols and essential (with nonessential) polyunsaturated fatty acids as phospholipids in oxidation-protected peptoliposomes or proteoliposomes allows cells in culture to duplicate in vivo specific membranes more accurately. Such lipid-corrected membranes allow cultured cells to communicate with neighboring cells through the extracellular matrix, effectively transmit hormonal signals directly and via receptor control, and respond with various tissue-specific functions and differentiation states as directed.     

Regulation of connective tissue collagenase production: stimulators from adult and fetal epidermal cells

We have examined the ability of primary adult rabbit skin cells to regulate collagenase production in vitro. Dermal cells constitutively produce collagenase in culture, and enzyme production by these cells can be influenced by epithelial cells. Co-culture with skin epidermal cells resulted in more enzyme production by dermal cells, whereas co- culture with corneal epithelial cells yielded less enzyme activity. Connective tissue cells from a different source, cornea, also produced collagenase when co-cultured with skin epidermal cells, although the stromal cells alone made no enzyme. The drug cytochalasin B had very little influence on collagenase production by dermal cells, either alone or in co-culture with epidermal cells, but did significantly potentiate enzyme production by corneal stromal cells responding to epidermal effector molecules. Epidermal-cell-conditioned medium from both fetal and adult rabbit skin was a potent source of stimulators (apparent mol wt 20,500 and 55,000) of connective-tissue-cell collagenase production. Stimulator production by epidermal cultures was cell density dependent. Optimal production of stimulators occurred in adult cultures containing 10(6) epidermal cells/ml of medium, and in fetal cultures containing 10(5) cells/ml. Inhibitors of connective tissue cell enzyme production were not detected in conditioned medium from either adult or fetal epidermal cells.     

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.     

Collagenases from human and rat skin fibroblasts purified on a zinc chelating column reveal marked differences in latency as a result of serum culture conditions.

1. Fibroblasts from both human and rat skin were grown in the presence or absence of serum and the collagenase activity in the medium was partially purified on zinc-Sepharose. 2. During chromatography, using a discontinuous elution gradient, the rat collagenase elutes at different pH and ionic strength than the human collagenase. Both latent and active collagenases of both species are retarded by the affinity matrix. 3. Latency of collagenase in media obtained from fibroblast cultures appears to be influenced by the presence of a serum component in the culture medium. 4. The results demonstrate that collagenases secreted by fibroblast cultures established from the same tissue but obtained from different species are biochemically diverse and that, within one species, the amount of active enzyme depends on the presence of a serum factor.     

A component of normal human serum which enhances the activity of vertebrate collagenases.

The activity of vertebrate collagenase is increased by approximately 3-fold in the presence of saturating amounts of a macromolecule found in normal human serum. The activities of collagenases from human skin, rat skin, and tadpole tailfin are all markedly enhanced in the presence of this molecule, but activities of bacterial collagenase, trypsin, chymotrypsin, thermolysin, and a gelatin-specific neutral protease from human skin are unchanged. The enhancer itself has no proteolytic activity and does not change the normal cleavage products of human skin collagenase. The collagenase enhancer is an extremely stable molecule. It is resistant to heat, to extremes of pH at physiological temperature, and appears to be protein in nature. Of particular interest is the requirement that the collagen substrate be in fibrillar form in order for the enhancer to be effective.     

A specific collagenase from rabbit fibroblasts in monolayer culture

1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24 degrees C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH(2)Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.     

Insulin suppresses collagenase stimulatory effect of stratifin in dermal fibroblasts

A delicate balance between synthesis and degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is an essential feature of tissue remodeling. We have recently demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, plays a critical role in modulating collagenase (MMP-1) mRNA expression in human dermal fibroblasts. In this study, we further characterized the collagenase stimulatory effect of stratifin in dermal fibroblasts and evaluated its effect in the presence and absence of insulin. Our data indicate that stratifin increases the expression of collagenase mRNA more than 20-fold in dermal fibroblasts, grown in either Dulbecco's modified Eagle's medium (DMEM) plus 2% or 10% fetal bovine serum (FBS). Collagenase stimulatory effect of stratifin was completely blocked, when fibroblasts were cultured in test medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% DMEM. The collagenase suppressive effect of test medium was directly proportional to the volume of KSFM used. As this medium contained insulin, we then evaluated the collagenase stimulatory effect of stratifin in dermal fibroblasts in the presence and absence of insulin. The results revealed that stratifin significantly increased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio, while insulin significantly decreased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio. The insulin inhibitory effect on collagenase mRNA expression was time and dose dependent. The maximal inhibitory effect of insulin was seen at 36 h post treatment. In conclusion, stratifin stimulates the expression of collagenase mRNA expression in dermal fibroblasts and this effect is suppressed by insulin treatment.     

Clonal growth of differentiated rabbit cartilage cells

Single cell suspensions have been prepared from rabbit cartilage by collagenase digestion in the presence of fetal bovine serum. When inoculated into medium F12 supplemented with 5% fetal bovine serum and 5% rabbit serum, the cartilage cell suspensions yield large healthy colonies of epithelial-like cells with an average plating efficiency of 47%. At the end of a 14 day incubation period, the centers of many of the colonies are thickened into multilayered aggregates. Such colonies exhibit serveral cartilage-like properties, including metachromatic staining with toluidine blue, dense brown staining with bismark brown, bright green staining with alcian green, and a refractile matrix which can be seen with the phase contrast microscope to surround the living cells. Similar properties have also been observed in second and third passage subcultures.     

Activation of human breast carcinoma collagenase through plasminogen activator

Latent collagenase activity was detected in the media of a well-characterized line of human breast carcinoma cells maintained for over two years in culture. The media also contained sufficient plasminogen activator to convert extrinsically added plasminogen to plasmin which in turn activated the collagenase. During culture of the breast carcinoma in serum-free medium, collagenase activity was maximum on day 12 whereas plasminogen activator activity changed little with time. Using type I collagen as a substrate, the activated breast tumor collagenase produced $\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments consistent with a mammalian collagenase. These findings suggest a pathologic role of plasminogen activator in the activation of latent collagenase during tumor invasion.A number of investigators have postulated that proteases may play a role in tumor invasion (1–5). Collagenase is one such protease which is active at neutral pH and specifically cleaves triple helical collagen into two ($\text{3}\text{4}\text{?}\text{1}\text{4}$ fragments (6). Secretion of collagenase by tumor cells migrating from the primary mass provides an attractive hypothesis for the mechanism of tumor invasion of surrounding host connective tissue—since the local environment would likely be at neutral pH. Consequently, a number of investigators have reported significant levels of collagenase activity in a wide variety of tumors (7–14). Abramson (13) has correlated aggressive $\text{in vivo}$ growth in carcinomas of the head and neck with collagenase activity, and Kuettner et al. (14) have postulated that inhibitors of collagenase may prevent tumors from invading cartilage.Collagenase is produced in both latent and active forms (6). The latent form can be activated with brief protease treatment (15). Since one of the proteases capable of activating collagenase is plasmin (15), the possibility arose that tumor cells could activate collagenase through plasminogen activator. Plasminogen activator secreted by tumor cells (4, 5) could convert plasminogen zymogen to plasmin which would in turn activate latent tumor collagenase. Testing this hypothesis $\text{in vitro}$ was the subject of the present study.Previous studies on collagenase from human carcinoma (7, 13, 14) have suffered from the drawback that contaminating inflammatory cells and fibroblasts may have been the source of the collagenase. Therefore, we have studied collagenase production from cultured human breast carcinoma cells which have been well characterized to be mammary epithelial in origin, malignant in karyotype, and able to grow in nude mice. Production of collagenase from these cells is therefore unequivocally of human carcinoma origin. The time course of latent collagenase and plasminogen activator secretion by these cultured tumor cells was studied following withdrawal of serum. To test whether plasminogen activator was secreted in sufficient amounts to indirectly activate latent collagenase, collagenase activity of the culture media was studied after the extrinsic addition of plasminogen. Finally, to verify that the tumor-secreted collagenase cleaved type I collagen at a single locus, enzyme degradation products were studied by gel electrophoresis.