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PubMed1991

Secretion of latent type IV procollagenase and active type IV collagenase by testicular cells in culture.

Testicular peritubular myoid cells, which have properties similar to those of vascular smooth-muscle cells, secrete a variety of metalloproteinases when maintained in culture in a chemically defined medium. The predominant metalloproteinases secreted were identified as latent type IV procollagenases having molecular masses of 72 kDa and 75 kDa, as detected in Western immunoblots with specific antibodies against type IV procollagenase. When peritubular cells were stimulated by dibutyryl cyclic AMP, forskolin or cholera toxin, they secreted increased amounts of type IV procollagenase. However, little if any of the active type IV collagenase, having a lower molecular mass of 66 kDa, could be detected under these conditions. Addition of low concentrations of cytochalasin D to peritubular cells in monoculture resulted in conversion of the latent type IV collagenase into its active form, assessed with antibody-specificity studies and by the appearance of the 66 kDa protein. In contrast, Sertoli cells in culture did not manifest an increased conversion of type IV procollagenase into type IV collagenase in the presence of cytochalasin D, even though cytochalasin D addition invariably resulted in a disruption of the microfilament assembly in each of these gonadal somatic cell populations. When peritubular cells were co-cultured with Sertoli cells, addition of cytochalasin D no longer resulted in formation of increased amounts of the active form of type IV collagenase. Sertoli cells and peritubular cells each secreted a tissue inhibitor of metalloproteinase type 2, detected with a specific antibody in a Western immunoblot to have a molecular mass of 21 kDa. We conclude that cytochalasin D acts on mesenchymal-type peritubular cells, but not on epithelial-type Sertoli cells, to enhance the conversion of latent type IV procollagenase into active type IV collagenase. This conversion of type IV procollagenase into type IV collagenase by peritubular cells was inhibited by factor(s) secreted by Sertoli cells. Interactions between Sertoli cells and peritubular cells are postulated to modulate net proteinase activities in discrete regions of the testis.

Animals↗
PubMed1984

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.

Animals↗
PubMed1991

Biochemical evidence of collagenase-mediated collagenolysis as a mechanism of cervical dilatation at parturition in the guinea pig.

Dilatation of the uterine cervix at parturition is accompanied by a remarkable alteration in its biomechanical characteristics leading to a significant reduction in its strength and stiffness. Our previous studies and those of others have suggested the involvement of collagenolysis leading to cervical dilatation. This study provides further evidence for the occurrence of collagenolysis in the dilated guinea pig cervix at birth. The changes in collagenase and collagenase inhibitory activity in vivo in cervices of nonpregnant, pregnant, and postpartum guinea pigs were determined. There were no significant changes in procollagenase, collagenase inhibitory activity, and net procollagenase in animals at 25 and 50 days of gestation compared with nonpregnant animals. At parturition (68 +/- 2 days), there was a 6-fold increase in procollagenase, a 26-fold increase in collagenase inhibitory activity, and a 2-fold increase in net procollagenase activity. Cervices in organ culture obtained at birth produced 2.9-fold more procollagenase, 1.6-fold more collagenase inhibitory activity, and a 10-fold increase in net procollagenase activity when compared to nonpregnant or 25-day pregnant animals. These studies indicate that dilatation of the guinea pig cervix at parturition involves collagenase-mediated degradation of collagen in the cervix.

Animals↗
PubMed1987

Behavior of epidermolysis bullosa fibroblasts in a hydrated collagen lattice.

To determine if an altered ability to contract a hydrated collagen lattice is characteristic of fibroblasts from patients with recessive dystrophic epidermolysis bullosa (RDEB), we examined contraction by fibroblasts from normal subjects and patients with RDEB, dominant dystrophic epidermolysis bullosa (DDEB), and dominant epidermolysis bullosa simplex (DEBS). An extremely broad range of contractility (normal, poor, and hypercontraction) was observed in all types of epidermolysis bullosa (EB). When contraction in control fibroblasts was defined as the mean +/- 2 SD, (all control values were within this range) and the data were analyzed by the chi-square test, only 32% of EB cells fell within this range, with 47% poorly contractile and 21% hypercontractile. These data, derived from 34 patients, indicate that no single genetic defect resulting in altered contractility in the 3 distinct types of EB is likely. Neither cell viability, collagenase expression, nor PGE2 synthesis as correlated with gel contraction in any group. Indomethacin had no effect on contraction in RDEB. It is possible that the genetic defects in EB cause blister formation in vivo and may lead in some way to an abnormal interaction of fibroblasts with the extracellular matrix resulting in an altered collagen lattice contraction in vitro.

Cells, Cultured↗
PubMed1992

Changes in gene expression by 193- and 248-nm excimer laser radiation in cultured human fibroblasts.

Tissue ablation by ultraviolet excimer lasers results in exposure of viable cells to subablative doses of radiation. To understand the potential biological consequences better, we have studied changes in gene expression in cultured human skin fibroblasts exposed to either 193- or 248-nm laser light. Northern blot analyses revealed that both treatments up-regulate a common set of genes, including interstitial collagenase, tissue inhibitor of metalloprotease, metallothionein, and the proto-oncogene c-fos. Dose-response and kinetic studies of collagenase induction by 193-nm radiation showed a maximal effect with 60 J/m2 and at approximately 24 h. The induction was still persistent 96 h later. In addition to the commonly affected genes, known to be activated also by conventional UV light (254 nm) and tumor-promoting phorbol esters, other genes were found to be selectively induced by the 193-nm radiation. The heat-shock hsp70 mRNA, undetectable in controls and in cultures irradiated at 248 nm, was transiently induced 8 h after exposure to 193-nm radiation. Furthermore, a selective up-regulation of collagen type I expression was observed. The results indicate that the 193- and 248-nm radiations by excimer lasers elicit specific and different cellular responses, in addition to an overlapping pathway of gene activation common also to UV radiation by germicidal lamps. The laser-induced genes could serve as molecular markers in evaluating cell injury in situ.

Collagenases↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1987

Epiphyseal growth plate cartilage and chondrocytes in mineralising cultures produce a low molecular mass angiogenic procollagenase activator.

We have investigated one aspect of the biochemical control of angiogenesis in the mammalian growth plate. This is the first report of an angiogenic compound from normal growing skeletal tissue. The active agent was indistinguishable from ESAF (endothelial cell-stimulating angiogenic factor), previously identified from tumour, retina and synovial fluid. 1. Using a procollagenase activation assay, ESAF was detected in low molecular weight extracts of growth plate cartilage from two species (foetal calf and young rabbit). ESAF content declined with increasing age of the foetal calf growth plate cartilage. 2. Rabbit growth plate chondrocytes in high density culture also produced ESAF 24-48 h after calcification of the culture. Inhibition of alkaline phosphatase in these cultures reduced the stimulation of ESAF production. These findings suggest that ESAF production occurs as a stage in chondrocyte differentiation which is linked with the onset of matrix calcification. A model is proposed for the control of angiogenesis in the growth plate.

Angiogenesis Inducing Agents↗
PubMed1987

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.

Amino Acid Sequence↗