Diminished response of Werner's syndrome fibroblasts to growth factors PDGF and FGF.

Patients with Werner's syndrome, an autosomal recessive disorder, undergo an accelerated aging process that leads to premature death. Fibroblasts from such patients typically grow poorly in culture. Here it is shown that fibroblasts from a patient with Werner's syndrome have a markedly attenuated mitogenic response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). In contrast, they have a full mitogenic response to fetal bovine serum. Both PDGF binding and receptor numbers per cell are unaltered. The Werner's syndrome cells express high constitutive levels of collagenase in vitro. Although PDGF enhances collagenase expression through increased levels of hybridizable collagenase messenger RNA in normal skin fibroblasts, no induction of collagenase occurs in the Werner's syndrome fibroblasts. Moreover, the failure to respond to this agonist effect of PDGF is not restored by fetal bovine serum. The data suggest that failure of one or more PDGF-mediated pathways in Werner's syndrome cells may contribute to the phenotypic expression of the disorder.

Glucocorticoid modulation of collagenase expression in human skin fibroblast cultures. Evidence for pre-translational inhibition.

Glucocorticoids inhibit collagenase accumulation in the medium of human skin explant cultures. To examine the mechanism for this process, skin fibroblasts were placed in serum-free medium containing various steroids. Dexamethasone produced a dose-dependent inhibition of trypsin-activatable collagenase in the culture medium with maximal inhibition of approx. 85% at 10(-6) M. Dexamethasone failed to inhibit collagenase activity directly. The decrease in activity in the medium was paralleled by a decrease in immunoreactive protein, suggesting inhibition of enzyme synthesis. The specificity of the effect was shown in two ways. At 10(-6) M steroid, only dexamethasone and hydrocortisone were inhibitory; estradiol, progesterone and testosterone produced less than 10% inhibition. In biosynthetic studies, exposure to 10(-7) M dexamethasone for 24 h produced approx. 50% inhibition of collagenase synthesis but caused no greater than 10% inhibition of total protein synthesis. The T1/2 for achieving the effect was approx. 16 h after initial exposure to dexamethasone. These kinetics were parallel to the inhibition caused by actinomycin D and cordycepin, two inhibitors of transcription, but were longer than that caused by cycloheximide (T 1/2 less than 3 h). To examine this process, cells were cultured in the presence or absence of 10(-6) M dexamethasone prior to harvesting mRNA for cell-free translation. In each case the inhibition or enzyme activity in the intact cells was paralleled by a reduction in translatable collagenase mRNA from the same cells. At the same time, there was no significant inhibition of total protein translation by the steroid. These data suggest that glucocorticoids regulate collagenase synthesis at a pre-translational level, possibly through inhibition of transcription.

Enhanced cell-free translation of human skin collagenase in recessive dystrophic epidermolysis bullosa.

The regulatory mechanisms for collagenase synthesis in recessive dystrophic epidermolysis bullosa (RDEB) have been studied using messenger RNA (mRNA) harvested from normal and RDEB skin fibroblasts to direct protein synthesis in a rabbit reticulocyte lysate translation system. Fibroblast mRNA encoded the synthesis of approximately 60,000 and approximately 55,000 dalton forms of procollagenase in cell-free translation. In contrast to biosynthesis by intact cells, there was preferential translation of the approximately 60,000 dalton specie. For quantitative comparisons with mRNA from normal cells, mRNA was harvested from fibroblasts of 2 RDEB patents whose intact cells have been documented to have increased synthesis of collagenase. Although total translational activity was equal in normal and RDEB mRNA preparations, translatable collagenase mRNA was increased 3.5- to 10-fold, suggesting that the enhanced collagenase synthesis characteristic of RDEB is due to increased concentrations or preferential translation of collagenase mRNA.

Constitutive synthesis of a 92-kDa keratinocyte-derived type IV collagenase is enhanced by type I collagen and decreased by type IV collagen matrices.

Human keratinocytes synthesize interstitial collagenase, a 72-kDa gelatinase, and a recently described 92-kDa gelatinase/type IV collagenase. We examined the synthesis of this novel enzyme by basal keratinocytes apposed to plastic, basement membrane collagen (type IV), and interstitial dermal collagen (type I). Samples of conditioned medium were electrophoresed on a 10% polyacrylamide, gelatin-ladened zymogram. Protein bands with gelatin-cleaving properties were identified by clarification of the gel and quantified by densitometry. A 92-kDa band had marked gelatinolytic activity and increased in culture over 72 h. The identification of this 92-kDa band as type IV collagenase was demonstrated by Western immunoblotting using monospecific antibody to the 92-kDa type IV collagenase. Keratinocytes apposed to type I collagen exhibited a threefold increase in the synthesis of the 92-kDa enzyme compared to cultures apposed to type IV collagen and a 1.5-times increase compared to plastic. The specificity of this enhancement was shown by constant levels of other proteins (e.g., the 72-kDa gelatinase). This study demonstrates that cell-matrix interactions modulate the synthesis of a recently described, keratinocyte-derived, 92-kDa gelatinase and that specific collagen types (I versus IV) have opposite effects upon the synthesis of this enzyme.

Epithelial organoids and mononuclear phagocytes from DMBA-induced mammary tumors of the rat secrete collagenase in vitro.

The production of collagenase has been examined in primary cultures of multicellular epithelial organoids and of stromal cells isolated from DMBA-induced mammary tumors of the rat. Plastic culture dishes and dishes coated with collagen fibrils were used to study the effect of such a substrate on collagenase release. Cultures of 51-micron epithelial organoids consisted of cuboidal cells and a myoepithelial-like cell type which formed a continuous layer under the cuboidal cells. A transient low production of collagenase with an apparent molecular weight (MW) of 72 kD was detected on both substrates. Upon separation by trypsin only cuboidal cells released collagenase. Cultures of 27-micron organoids contained only few myoepithelial-like cells. On plastic, they formed dense monolayers of cuboidal cells and released more collagenase than the greater aggregates. On collagen fibrils, these organoids formed cords and ridges and collagenase production was about 4- to 6-fold higher. These results indicate that collagenase release is influenced by the nature of the interaction of cuboidal cells with the substrate on which they grow. Similar organoids prepared from virgin mammary glands failed to secrete collagenase on either substrate. Primary cultures of stromal cells derived from tumor tissues comprised one basic cell type that expressed a series of properties characteristic for monocytes/macrophages. These cultures were capable of producing collagenase with an apparent MW of 56 kD. Collagenase with a similar size was detected in the extracts of 51 from 65 mammary tumors.

Age-related changes in collagenase expression in cultured embryonic and fetal human skin fibroblasts.

Since skin collagenase is required for initiation of the degradation of types I and III collagens, the major collagens of the human dermis, we examined its expression during embryonic and fetal development. When using skin fibroblasts cultured from human embryos and fetuses, immunoreactive collagenase concentrations were strongly correlated with estimated gestational age (p less than 0.001), with levels at 7-8 weeks of gestation that were about one-twentieth of those in the 29-week cell cultures. In crude culture medium, the apparent catalytic efficiency (activity per unit immunoreactive protein) was variable, an observation attributable in part to variable expression of a collagenase-inhibitory protein. Following chromatographic purification, four of ten fetal collagenases were found to have greater than or equal to 4-fold decrease in specific activity, suggesting that these particular fetal collagenases may be structurally and/or catalytically altered. Since the decreased levels of immunoreactive protein suggested that decreased enzyme synthesis was the major mechanism, we examined collagenase synthesis in a cell-free translation system. Here, we quantitated collagenase expression in the culture medium of intact cells prior to harvesting mRNA. Compared with the intact adult cells, the fetal cells had 3-17 times less collagenase activity in the medium, while in cell-free translation there was a 2- to 3-fold decrease in collagenase synthesis. These data suggest that decreased in vitro expression is correlated with decreased levels of translatable collagenase mRNA but that other factors, such as the collagenase inhibitor and altered specific activity of the enzyme, may be important in modulating collagenase activity.

Human fibroblast collagenase: glycosylation and tissue-specific levels of enzyme synthesis.

Human skin fibroblasts secrete collagenase as two proenzyme forms (57 and 52 kDa). The minor (57-kDa) proenzyme form is the result of a partial posttranslational modification of the major (52-kDa) proenzyme through the addition of N-linked complex oligosaccharides. Human endothelial cells as well as fibroblasts from human colon, cornea, gingiva, and lung also secrete collagenase in two forms indistinguishable from those of the skin fibroblast enzyme. In vitro tissue culture studies have shown that the level of constitutive synthesis of this fibroblast-type interstitial collagenase is tissue specific, varies widely, and correlates with the steady-state level of a single collagenase-specific mRNA of 2.5 kilobases. The tumor promoter, phorbol 12-myristate 13-acetate, apparently blocks the control of collagenase synthesis resulting in a similarly high level of collagenase expression (approximately equal to 3-7 micrograms of collagenase per 10(6) cells per 24 hr) in all examined cells. The constitutive level of synthesis of a 28-kDa collagenase inhibitor does not correlate with that of the enzyme. Phorbol 12-myristate 13-acetate stimulates the production of this inhibitor that in turn modulates the activity of collagenase in the conditioned media. As a result, the apparent activity of the enzyme present in the medium does not accurately reflect the rate of its synthesis and secretion.

Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein.

We have determined the complete sequence of the cDNA clone representing the full size human skin collagenase mRNA. Collagenase is synthesized in preproenzyme form, Mr 54,092, with a 19 amino acid long signal peptide. The primary secretion products of the enzyme consist of a minor glycosylated form, Mr 57,000, and a major unmodified polypeptide of predicted Mr 51,929. Proteolytic activation of human skin procollagenase results in removal of 81 amino acid residues from the amino-terminal portion of the proenzyme. Both potential N-glycosylation sites are contained within the proteolytically activated form of the enzyme. The primary structure of the coding region of the presented clone is homologous to an oncogene-induced rat protein whose function is still unknown, although preliminary observations suggest that it is not rat skin collagenase.

Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells.

We have purified and determined the complete primary structure of human stromelysin, a secreted metalloprotease with a wide range of substrate specificities. Human stromelysin is synthesized in a preproenzyme form with a calculated size of 53,977 Da and a 17-amino acid long signal peptide. Prostromelysin is secreted in two forms, with apparent molecular masses on NaDodSO4/PAGE of 60 and 57 kDa. The minor 60-kDa polypeptide is a glycosylated form of the major 57-kDa protein containing N-linked complex oligosaccharides. Zymogen activation by trypsin results in the removal of 84 amino acids from the amino terminus of the enzyme generating a 45-kDa active enzyme species. Human stromelysin is capable of degrading proteoglycan, fibronectin, laminin, and type IV collagen but not interstitial type I collagen. The enzyme is not capable of activating purified human fibroblast procollagenase. Analysis of its primary structure shows that stromelysin is in all likelihood the human analog of rat transin, which is an oncogene transformation-induced protease. The pattern of enzyme expression in normal and tumorigenic cells revealed that human skin fibroblasts in vitro secrete stromelysin constitutively (1-2 micrograms per 10(6) cells per 24 hr). Human fetal lung fibroblasts transformed with simian virus 40, human bronchial epithelial cells transformed with the ras oncogene, fibrosarcoma cells (HT-1080), and a melanoma cell strain (A 2058), do not express this protease nor can the enzyme be induced in these cells by treatment with phorbol 12-myristate 13-acetate. Our data indicate that the expression and the possible involvement of secreted metalloproteases in tumorigenesis result from a specific interaction between the transforming factor and the target cell, which may vary in different species.

The structure of the human skin fibroblast collagenase gene.

Genomic clones containing the complete gene encoding human fibroblast interstitial collagenase were isolated from a lambda phage human DNA library. The gene is comprised from 10 exons and spans 8.2 kilobase pairs. We have mapped the relative positions and determined the DNA sequence of all the exon/intron borders of the gene. The organization of the human interstitial collagenase gene is very similar to that of rabbit collagenase and of two other extracellular matrix (ECM) metalloproteases: rat stromelysin (transin) and rat transin 2. All four genes are organized into 10 exons of virtually identical size while the length of the 3' proximal introns is subject to variation. The protein sequence comprising the putative active center is coded for by exon 5 of all four genes and contains a strongly conserved zinc binding site. This observation suggests that the organization of the ECM metalloprotease genes reflect the structure of the functional domains of the enzyme proteins. The structural data accumulated so far provides evidence for the existence of a gene family coding for secreted ECM metalloproteases and suggests that gene duplication played an important role in its formation.

H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen.

H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.