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1.
Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.  相似文献   

2.
Synovial fluid basic calcium phosphate (BCP) crystals are associated with severe destructive arthropathies characterised by synovial proliferation and non-inflammatory degradation of intra-articular collagenous structures. BCP crystals stimulate fibroblast and chondrocyte mitogenesis, metalloprotease secretion and prostaglandin production. As a tissue protective effect of prostaglandins has been suggested, we recently studied the effect of PGE1 on BCP crystal-induced mitogenesis and collagenase mRNA accumulation in human fibroblasts (HF). We demonstrated a dose-dependent inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation. The mechanism of PGE1 inhibition of BCP crystal-induced mitogenesis and collagenase mRNA accumulation was therefore explored. PGE1 (100 ng/ml) increased HF intracellular cAMP 40-fold over control. BCP alone caused no such change but inhibited the PGE1-induced increase in intracellular cAMP by at least 60%. The PGE1-induced increase in intracellular cAMP was also blocked by the adenyl cyclase inhibitor, 2′,5′-dideoxyadenosine (ddA) (10 μM) and ddA reversed the PGE1-mediated inhibition of BCP crystal-induced mitogenesis. Dibutyrul cAMP also inhibited BCP crystal-induced mitogenesis in a concentration-dependent manner. Agents which increase intracellular cAMP levels such as the adenyl cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) mimicked the effect of PGE1 on HF collagenase mRNA levels. PGE1 inhibits the biologic effects of BCP crystals through the cAMP signal transduction pathway and such inhibition may have significant therapeutic implications.  相似文献   

3.
To understand better the molecular nature of the epithelial-mesenchymal interactions that govern folliculogenesis and hair growth, we have studied the behavior of cultured rat dermal papilla cells (rDP), the mesenchymal component of the hair follicle. Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) both potentiated the growth of rDP in culture, and transforming growth factor-beta (TGF-beta) inhibited rDP proliferation. Biosynthetic labeling studies demonstrated that both PDGF and bFGF induced synthesis of a major secreted protein(s) with Mr = 55-60 kD. It was noted that PDGF and bFGF differentially regulated synthesis of this major secreted protein; PDGF-mediated induction was found to be transient, while bFGF allowed prolonged synthesis of the protein. Sodium dodecyl sulfate (SDS)-substrate gel analysis of rDP-conditioned media revealed that this protein is a metalloproteinase with casienolytic activity and Mr approximately 51 kD (unreduced). We have identified the growth factor-regulated rDP protein as the matrix metalloproteinase stromelysin by immunoprecipitation. Northern analysis established that increased secretion of stromelysin was accompanied by an increased expression of stromelysin-specific mRNA. Remarkably, stromelysin antisera interfere with stimulation of dermal papilla cell growth, demonstrating that stromelysin production serves a functional role in mitogen-induced proliferation in these cells. These findings provide insight into the mechanism by which the connective tissue remodeling required for formation of hair embryonically and the postembryonic hair cycle may be regulated.  相似文献   

4.
In culture, nontransformed human diploid fibroblasts divide a limited number of times, resulting in a nonproliferating senescent cell culture which exhibits an altered pattern of gene expression. Previously we reported that an early event in the process of replicative senescence was an increase in the synthesis of two connective tissue degrading metalloproteinases, collagenase and stromelysin, and a decrease in the synthesis of the physiological inhibitor of those enzymes, tissue inhibitor of metalloproteinases-1 (TIMP-1). The cytokine TGF-β1 is known to regulate the expression of each of these three genes and to be synthesized and secreted by cultured human fibroblasts. This suggested the hypothesis that the age-specific modulation of collagenase, stromelysin, and TIMP-1 expression is the result of a change in TGF-β1 activity during replicative senescence. To test this hypothesis, the responses of early, mid, and late passage (presenescent) fibroblast cell cultures to a TGF-β neutralizing antibody were evaluated. In early passage cell cultures, exposure to TGF-β neutralizing antibody resulted in a significant increase in the expression of collagenase and stromelysin and decreased TIMP-1 expression. The antibody did not affect expression of either of those genes by late passage cell cultures, although late passage cultures did respond to added TGF-β1. Quantification of the levels of active TGF-β, using a growth inhibition assay, indicates that the level of active TGF-β1 is decreased during replicative senescence, supporting the conclusion that the modulation of collagenase, stromelysin, and TIMP-1 expression results from diminished TGF-β activity.  相似文献   

5.
In human foreskin fibroblast cultures, two proteins with Mr 60,000 and 55,000 were found to be induced about 3.5-fold by epidermal growth factor (EGF), platelet-derived growth factor, and beta-transforming growth factor. The induced proteins were identified as procollagenases by immunoprecipitation of induced medium with antibodies to purified human fibroblast collagenase. Collagenase enzyme activity in the medium from EGF-treated cultures was also induced at least 3-fold compared to control cultures. Induction of collagenase was dependent upon de novo protein and RNA synthesis and was observed in the medium 10 h after addition of EGF. Although these growth-promoting factors interact with separate membrane receptors, each induced the secretion of a common protein, suggesting that collagenase may be important in some aspect of mitogenesis, cell mobilization, and migration.  相似文献   

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The homologous proteinase inhibitors, human alpha 2-macroglobulin (alpha 2M) and chicken ovostatin, have been compared with respect to their "bait" region sequences and interactions with two human matrix metalloproteinases, collagenase and stromelysin. A stretch of 34 amino acid residues of the ovostatin bait region sequence was determined and the matrix metalloproteinase cleavage sites identified. Collagenase cleaved a X-Leu bond where X was unidentified, whereas the major cleavage site by stromelysin was at the Gly-Phe bond, 4 residues on the COOH-terminal side of the collagenase cleavage site. Collagenase cleaved the alpha 2M bait region at the Gly679-Leu680 bond, and stromelysin at Gly679-Leu680 and Phe684-Tyr685 bonds. Sequence similarity in the bait region of members of the alpha-macroglobulin family is strikingly low. The kinetic studies indicate that alpha 2M is a 150-fold better substrate for collagenase than type I collagen. Structural predictions based on the bait region sequences suggest that a collagen-like triple helical structure is not a prerequisite for the efficient binding of tissue collagenase to a substrate. The binding of stromelysin to alpha 2M is slower than that of collagenase. Stromelysin reacts with ovostatin even more slowly. Despite the preference of chicken ovostatin for metalloproteinases, human alpha 2M, a far less selective inhibitor, reacts more rapidly with collagenase and stromelysin. These results suggest that alpha 2M may play an important role in regulating the activities of matrix metalloproteinases in the extracellular space.  相似文献   

9.
On purification, human fibroblast collagenase breaks down into two major forms (Mr22,000 and Mr 27,000) and one minor form (Mr 25,000). The most likely mechanism is autolysis, although the presence of contaminating enzymes cannot be excluded. From N-terminal sequencing studies, the 22,000-Mr fragment contains the active site; differential binding to concanavalin A shows the 25,000-Mr fragment is a glycosylated form of the 22,000-Mr fragment. These low-Mr forms can be separated by Zn2+-chelate chromatography. An activity profile of this column, combined with data from substrate gels, indicates no activity against collagen in the 22,000-Mr and 25,000-Mr forms, but rather, activity casein and gelatin. The 27,000-Mr form has no activity. The 22,000/25,000-Mr form can act as an activator for collagenase in a similar way to that reported for stromelysin. The activity of the 22,000/25,000-Mr form is not inhibited by the tissue inhibitor of metalloproteinases (TIMP). The 27,000-Mr C-terminal part of the collagenase molecule therefore appears to be important in maintaining the substrate-specificity of the enzyme, and also plays a role in the binding of TIMP.  相似文献   

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The expression of collagenase and stromelysin is believed to be coordinately regulated. In this report however, we provide evidence that suggests subtle differences may exist in the early events of the induction of these enzymes. Rabbit articular chondrocytes treated with interleukin-1, either alone or in combination with fibroblast growth factor, accumulated steady-state mRNA levels for both the enzymes, with the latter treatment more effective in inducing greater levels and within a shorter time. Further, the induction of the enzymes by either protocol was blocked by cycloheximide co-treatment. Cycloheximide added 1 h post-stimulation with interleukin-1 + fibroblast growth factor failed to block stromelysin mRNA expression, but was able to block collagenase steady-state mRNA levels. Transforming growth factor-beta, another inhibitor of metallprotease induction, showed no such differential activity. The results suggest that collagenase and stromelysin may have subtle variations in their induction pathways. Our studies further show that the enzyme induction by interleukin-1 alone or in combination with fibroblast growth factor occurs through different, but related mechanisms.  相似文献   

12.
We have investigated the effects of ligation of the fibronectin receptor (FnR) on gene expression in rabbit synovial fibroblasts. Monoclonal antibodies to the FnR that block initial adhesion of fibroblasts to fibronectin induced the expression of genes encoding the secreted extracellular matrix-degrading metalloproteinases collagenase and stromelysin. That induction was a direct consequence of interaction with the FnR was shown by the accumulation of mRNA for stromelysin and collagenase. Monoclonal antibodies to several other membrane glycoprotein receptors had no effect on metalloproteinase gene expression. Less than 2 h of treatment of the fibroblasts with anti-FnR in solution was sufficient to trigger the change in gene expression, and induction was blocked by dexamethasone. Unlike other inducers of metalloproteinase expression, including phorbol diesters and growth factors, addition of the anti-FnR in solution to cells adherent to serum-derived adhesion proteins or collagen produced no detectable change in cell shape or actin microfilament organization. Inductive effects were potentiated by cross-linking of the ligand. Fab fragments of anti-FnR were ineffective unless cross-linked or immobilized on the substrate. Adhesion of fibroblasts to native fibronectin did not induce metallo-proteinases. However, adhesion to covalently immobilized peptides containing the arg-gly-asp sequence that were derived from fibronectin, varying in size from hexapeptides up to 120 kD, induced collagenase and stromelysin gene expression. This suggests that degradation products of fibronectin are the natural inductive ligands for the FnR. These data demonstrate that signals leading to changes in gene expression are transduced by the FnR, a member of the integrin family of extracellular matrix receptors. The signaling of changes in gene expression by the FnR is distinct from signaling involving cell shape and actin cytoarchitecture. At least two distinct signals are generated: the binding of fibronectin-derived fragments and adhesion-blocking antibodies to the FnR triggers events different from those triggered by binding of the native fibronectin ligand. Because the genes regulated by this integrin are for enzymes that degrade the extracellular matrix, these results suggest that information transduced by the binding of various ligands to integrins may orchestrate the expression of genes regulating cell behavior in the extracellular environment.  相似文献   

13.
Sparsely cultured chondrocytes from the sternae of 14-day chick embryos grew vigorously with a liquid medium using Falcon tissue culture dishes, when the conditioned medium (CM) prepared from the high-density cultures of neural retinae was used as a growth medium. In CM most cells adhered to the culture substrate, while in the fresh medium, in which cell growth was very slow, many viable cells remained unattached to the substrate. Enhancement of cell growth by CM was much less marked in cultures of chondrocytes suspended in agarose. The main effect of CM in enhancing cell growth is thus considered to be related to the increase of cell adhesiveness to the substrate. The main active factor in CM is non-dialyzable and heat-stable.  相似文献   

14.
Poly A RNA has been isolated from a murine T cell hybridoma ( T2D4 ) that spontaneously secretes suppressive immunoglobulin G-binding factor ( IgGBF ). Translation products, obtained from a rabbit reticulocyte lysate translation system and after injection into Xenopus laevis oocytes, contain material with the biologic activity, the affinity, and the m.w. of murine IgGBF ; it suppresses secondary in vitro IgG antibody production in a dose-dependent fashion. The suppressive factor binds to IgG but not to IgM immunoadsorbents and, after mild NaDodSO4 treatment, dissociates in NaDodSO4 polyacrylamide gels into two peaks at 78 and 40 kD. Translation products from two non- IgGBF -secreting cell lines (BW-5147, a T lymphoma line, and A9, a fibroblast cell line) fail to exert any suppressive activity. On sucrose gradients, the RNA responsible for the biologic activity was found in one major peak located at 11S. IgGBF synthesized in a cellfree translation system by using poly A RNA and sucrose gradient fractions was also characterized by immunoprecipitation with Fc fragments of [35S]methionine-labeled proteins. On NaDodSO4 polyacrylamide gels, it migrates in one peak located at 37 kD. We conclude that IgGBF is coded for by 11S poly A RNA and that no post-translational modifications (other than proteolytic cleavage) are necessary to obtain a biologically active factor with Ig-binding properties.  相似文献   

15.
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-μm 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-μm 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.  相似文献   

16.
Phosphorylation of the insulin receptor by casein kinase I   总被引:1,自引:0,他引:1  
Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.  相似文献   

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A dual enzyme disaggregation method using collagenase and then trypsin was developed that allowed the reproducible initiation of primary cultures from Atlantic salmon Salmo salar gills. Cultures had both epithelial and fibroblast morphology and persisted for an average of 20 passages. Growth was dependent upon a minimum concentration of 5% foetal calf serum (FCS) for fibroblasts and 10% FCS for epithelial cells. Growth was mostly independent of substrate, although epithelial cells showed increased growth on type I collagen gels. Matrigel? cell culture substrate produced reduced growth of fibroblasts and did not benefit epithelial cell growth. Epithelial cells reacted with monoclonal antibodies (MAbs) against mammalian cytokeratins, and fibroblast cells reacted with MAbs against mammalian fibronectin and type I collagen. The method also produced two long‐term cultures: one epithelial and one fibroblast that have been designated RGE‐2 and RGF respectively.  相似文献   

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The protein of molecular weight about 160 kD (designated LMG160) was isolated from purified low mobility group chromatin proteins. Polyclonal antibody directed against the LMG160 protein in mouse was raised. The specificity of the antibody was determined with the use of ELISA. Using chemical cross-linking procedure followed by immunoprecipitation with the antiLMG160 antibody complex formation with chromatin proteins was demonstrated. Among the proteins that form complexes with LMG160, histones H3, H2A, and H4 were identified (Western blotting technique).  相似文献   

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