Fibronectin fragments, but not intact fibronectin, signalling through the fibronectin receptor induce metalloproteinase gene expression in fibroblasts.
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Amino Acid Sequence↗
Biomedical subjects
Z Werb
Publications and source records attributed to Z Werb.
Tissue inhibitor of metalloproteinases is induced by nerve damage.
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Animals↗
Expression and function of matrix metalloproteinases in development.
Extracellular matrix (ECM) remodeling accompanies cell migration, cell-cell interactions, embryo expansion, uterine implantation and tissue invasion during mammalian embryogenesis. We have found that mouse embryos express mRNA transcripts for collagenase, stromelysin and the tissue inhibitor of metalloproteinases (TIMP) and secrete functional ECM-degrading metalloproteinases, including collagenase and stromelysin. These metalloproteinases are inhibitable by TIMP and are regulated during peri-implantation development and endoderm differentiation. The involvement of a controlled proteolytic reaction, dependent on metalloproteinases, during the implantation of mouse embryos is suggested by the secretion of proteinases by trophoblast during its invasive phase and by the reciprocal expression of TIMP in the maternal deciduum. Exogenous TIMP affects the migration of parietal endoderm cells during blastocyst outgrowth in vitro. Taken together, these data suggest that metalloproteinases function in cell-ECM interactions during mammalian development.
Animals↗
Induction of c-fos transcripts in early postimplantation mouse embryos by TGF-alpha, EGF, PDGF, and FGF.
The activity of growth factor receptors in the early postimplantation mouse embryo was studied by analyzing changes in expression of mRNA transcripts of an early response gene, c-fos, after binding of specific ligands. Reverse transcription of mRNA coupled with the polymerase chain reaction was used to detect gene transcription in single embryos after exposure to growth factors. Postimplantation embryos (at 7.5 days of gestation) had physiologically active receptors for transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), human platelet-derived growth factor (PDGF), recombinant PDGF-AA homodimer, and basic fibroblast growth factor (FGF), as indicated by induced expression of c-fos mRNA, c-fos expression was not induced in untreated embryos or in embryos incubated with active recombinant PDGF-BB homodimer. These results show that growth factor receptors are functional during early mammalian embryogenesis.
Animals↗
92-kD type IV collagenase mediates invasion of human cytotrophoblasts.
The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.
Antibody Specificity↗
Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture.
The extracellular matrix (ECM) is an important regulator of mammary epithelial cell function both in vivo and in culture. Substantial remodeling of ECM accompanies the structural changes in the mammary gland during gestation, lactation and involution. However, little is known about the nature of the enzymes and the processes involved. We have characterized and studied the regulation of cell-associated and secreted mammary gland proteinases active at neutral pH that may be involved in degradation of the ECM during the different stages of mammary development. Mammary tissue extracts from virgin and pregnant CD-1 mice resolved by zymography contained three major proteinases of 60K (K = 10(3) Mr), 68K and 70K that degraded denatured collagen. These three gelatinases were completely inhibited by the tissue inhibitor of metalloproteinases. Proteolytic activity was lowest during lactation especially for the 60K gelatinase which was shown to be the activated form of the 68K gelatinase. The activated 60K form decreased prior to parturition but increased markedly after the first two days of involution. An additional gelatin-degrading proteinase of 130K was expressed during the first three days of involution and differed from the other gelatinases by its lack of inhibition by the tissue inhibitor of metalloproteinases. The activity of the casein-degrading proteinases was lowest during lactation. Three caseinolytic activities were detected in mammary tissue extracts. A novel 26K cell-associated caseinase--a serine arginine-esterase--was modulated at different stages of mammary development. The other caseinases, at 92K and a larger than 100K, were not developmentally regulated. To find out which cell type produced the proteinases in the mammary gland, we isolated and cultured mouse mammary epithelial cells. Cells cultured on different substrata produced the full spectrum of gelatinases and caseinases seen in the whole gland thus implicating the epithelial cells as a major source of these enzymes. Analysis of proteinases secreted by cells grown on a reconstituted basement membrane showed that gelatinases were secreted preferentially in the direction of the basement membrane. The temporal pattern of expression of these proteinases and the basal secretion of gelatinases by epithelial cells suggest their involvement in the remodelling of the extracellular matrix during the different stages of mammary development and thus modulation of mammary cell function.
Animals↗
Regulation of extracellular matrix degradation by cell-extracellular matrix interactions.
An appropriate balance of extracellular matrix synthesis and degradation is required for normal morphogenesis and maintenance of tissue architecture. Extracellular matrix molecules and their receptors, as well as proteinases and their inhibitors, are all involved in matrix remodeling. In this report we show that signal transduction through extracellular matrix receptors regulates matrix remodeling.
Animals↗
Ultrastructural localization of a macrophage-restricted sialic acid binding hemagglutinin, SER, in macrophage-hematopoietic cell clusters.
Resident bone marrow macrophages in hematopoietic clusters have previously been shown to express a novel lectin-like sheep erythrocyte receptor, SER, which mediates binding of unopsonized sheep erythrocytes via recognition of sialylated glycoconjugates and may interact with sialylated ligands on murine bone marrow cells. In this study, the distribution of SER on macrophages within hematopoietic clusters was localized by a monoclonal antibody, SER-4, by immunofluorescence and immunoelectron microscopy. SER was found to be diffusely localized at the contact zones between macrophages and erythroblasts, whereas the receptor was highly concentrated at the contacts between macrophages and developing myelomonocytic cells. These data suggest that SER on resident bone marrow macrophages interacts differentially with sialylated ligands on developing myeloid cells and that this may influence their development.
Animals↗
Vectorial secretion of extracellular matrix proteins, matrix-degrading proteinases, and tissue inhibitor of metalloproteinases by endothelial cells.
In a study of the vectorial secretion of proteins by bovine aortic arch endothelial cells, we found that the extracellular matrix macromolecules collagen and fibronectin as well as several matrix-degrading metalloproteinases were secreted selectively in the basal direction. In contrast, the tissue inhibitor of metalloproteinases showed only a weak preference for the basal direction. Three proteins at 18-35 kDa were secreted with preference apically, counter to the basal secretion of approximately 70% of the total secreted protein. As expected, rabbit synovial fibroblasts, which were used as a control, secreted proteins, including collagen, gelatin-degrading proteinases, and casein-degrading proteinases, equally in apical and basal directions. The basal secretion of collagen, fibronectin, gelatinases, and tissue inhibitor of metalloproteinases by bovine aortic arch endothelial cells suggests that the structural and functional polarity of these cells is manifested, in part, at the level of polarized secretion of matrix-related proteins.
Animals↗
Expression of EGF and TGF-alpha genes in early mammalian development.
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Animals↗
mRNA phenotyping for studying gene expression in small numbers of cells: platelet-derived growth factor and other growth factors in wound-derived macrophages.
The understanding of biologic and pathophysiologic processes in lung is aided by a technique for ascertaining the transcriptional phenotype of small numbers of cells examined in vivo. The mRNA phenotyping procedure consists of scaled-down methods for isolating mRNA from small numbers of cells, followed by reverse transcription of the RNA and specifically primed amplification of the cDNA by the polymerase chain reaction. We show an example of the use of the technique in a study of expression of platelet-derived growth factor and other growth factor genes in macrophages isolated from wound cylinders implanted in mice. With this technique, the transcriptional phenotype of purified normal lung epithelial and mesenchymal cells, macrophages, or cells obtained from lavaged lungs or punch biopsy specimens can be examined as a rapid first step in understanding molecular processes in the lung.
Animals↗
Platelet alpha-granule fibrinogen, albumin, and immunoglobulin G are not synthesized by rat and mouse megakaryocytes.
It has been assumed that endogenous synthesis by the platelet precursor cell, the bone marrow megakaryocyte, is the major source of platelet alpha-granule protein. To test this hypothesis, we used mRNA phenotyping to detect in megakaryocytes the presence of mRNA transcripts specific for various proteins. Our results indicate that megakaryocytes synthesize platelet factor 4, a protein relatively specific for platelets, but do not express mRNA transcripts for the fibrinogen, albumin, or IgG found in alpha-granules. We have previously shown that megakaryocytes endocytose circulating proteins, including fibrinogen, albumin, and IgG, and incorporate them into alpha-granules. Thus, platelets appear to contain a unique type of secretory granule whose contents originate by both endogenous synthesis and endocytosis from plasma. Under basal conditions, the source of alpha-granule fibrinogen is plasma.
Animals↗
Expression of extracellular matrix-degrading metalloproteinases and metalloproteinase inhibitors is developmentally regulated during endoderm differentiation of embryonal carcinoma cells.
The differentiation of F9 and PSA-1 embryonal carcinoma cells to embryoid bodies composed of a mixture of parietal and visceral endoderm was accompanied by changes in their secretion of metalloproteinases. Differentiation was induced by retinoic acid and dibutyryl cyclic AMP (for F9 cells) or by removing cells from a substrate of feeder cells to alter cell-cell interaction and adhesion (for PSA-1 cells). The embryoid bodies attached to gelatin-coated dishes, and the parietal endoderm cells spread out over the matrix. The differentiated cells secreted specific gelatin- and casein-degrading proteinases, including enzymes that comigrated with proenzyme forms of collagenase and stromelysin. Total proteinase activity as well as specific collagenase activity increased with the time of differentiation. All of the gelatin- and casein-degrading proteinases detectable by substrate gel zymography were inhibited by inhibitors of metalloproteinases but not by inhibitors of serine or cysteine proteinases, indicating that they were metalloproteinases. Both cell lines showed increased collagenolytic activity, which was activated by treatment with plasmin. In addition, both cell lines showed increased secretion of specific metalloproteinase inhibitors, including tissue inhibitor of metalloproteinases, with differentiation. Analysis of mRNA from undifferentiated and differentiated F9 cells by RNA blot analysis or reverse transcription coupled with the polymerase chain reaction showed that increased expression of genes for collagenase, stromelysin and tissue inhibitor of metalloproteinases is associated with differentiation of these cells. These results suggest that the expression of extracellular matrix-degrading metalloproteinases and their inhibitors is developmentally regulated during the differentiation and spreading of the parietal endoderm.
Animals↗
Two alkaline phosphatase genes are expressed during early development in the mouse embryo.
Alkaline phosphatase (AP) activity is stage specific in mouse embryos and may be associated with compaction and separation of trophectoderm from inner cell mass in preimplantation development. We previously sequenced a cDNA and two mouse AP genes that could contribute to the AP activity in embryos. Oligonucleotide primers were constructed from the three sequences and used in the reverse transcription-polymerase chain reaction technique to establish that two of the three AP isozymes are transcribed during preimplantation development. The predominant transcript (E-AP) is from a gene highly homologous to the human tissue-specific APs, but different from the mouse intestinal AP. Tissue non-specific (TN) AP also is transcribed, but there is approximately 10 times less TN-AP than E-AP transcript. The TN-AP isozyme is the predominant transcript of 7 to 14 day embryos and primordial germ cells. A switch in predominance from E-AP to TN-AP must occur during early postimplantation development. This study establishes a framework for experiments to determine the functions of the two isozymes during preimplantation development.
Alkaline Phosphatase↗
Phorbol diesters regulate expression of the membrane neutral metalloendopeptidase (EC 3.4.24.11) in rabbit synovial fibroblasts and mammary epithelial cells.
The neutral zinc metalloendopeptidase (NEP, EC 3.4.24.11) is an integral membrane protein found in brain tissue, polymorphonuclear leukocytes, and many epithelia. We show here that NEP is expressed on rabbit synovial fibroblasts and on simian virus 40 (SV40) DNA- and H-ras-transformed rabbit mammary epithelial cells. Treatment of these cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h decreased expression of NEP mRNA transcripts and decreased the biosynthetically labeled immunoprecipitable NEP antigen. In contrast to its effects on NEP, TPA treatment induced expression of the secreted metalloproteinase collagenase and the tissue inhibitor of metalloproteinases. TPA induced stromelysin, another secreted metalloproteinase, only in the fibroblasts. These data provide evidence that the expression of the membrane-bound NEP is regulated in several cell types.
Animals↗
Novel method for studying mRNA phenotypes in single or small numbers of cells.
Biological processes, such as growth control, are often governed by biochemical steps involving mRNA transcripts that are short-lived and have a low copy number. Furthermore, the cells involved in these processes are often available in low numbers from in vivo sources. We now report a method that is superior to in situ hybridization, RNA blot analysis, and the nuclease protection assay for the study of short-lived, low-copy-number mRNA transcripts. The method consists of a microprocedure for isolating RNA from one to a few thousand cells and two coupled enzymatic steps: reverse transcription of whole cellular RNA, followed by amplification of the cDNA by a specifically primed polymerase chain reaction to give specific cDNA fragments that can be visualized on agarose gels by ethidium bromide staining. With this method we have detected actin mRNA from a single cell, or less than 100 cRNA molecules, and have quantified differences in RNA concentrations of less than threefold. The reverse transcription reaction products can be divided for the polymerase chain reaction, and several mRNA species can be assayed simultaneously. Therefore, we call the method single-cell mRNA phenotyping. This technique is applicable to the analysis of low-copy-number growth factor transcripts in cells in culture and in vivo.
Actins↗
Proteinases and extracellular matrix remodeling.
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Amino Acid Sequence↗
Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression.
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.
Animals↗