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Biomedical subjects

Z Werb

Publications and source records attributed to Z Werb.

At least 145 records · Page 8Linked to original sources
PubMed1989

Genes for extracellular-matrix-degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian 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 secrete functional ECM-degrading metalloproteinases, including collagenase and stromelysin, that are inhibitable by the tissue inhibitor of metalloproteinases (TIMP) and that are regulated during peri-implantation development and endoderm differentiation. mRNA transcripts for collagenase, stromelysin, and TIMP were detected as maternal transcripts in the unfertilized egg, were present at the zygote and cleavage stages, and increased at the blastocyst stage and with endoderm differentiation. These data suggest that metalloproteinases function in cell-ECM interactions during growth, development, and implantation of mammalian embryos.

Amino Acid Sequence↗
PubMed1989

Rapid fragmentation and reorganization of Golgi membranes during frustrated phagocytosis of immobile immune complexes by macrophages.

The authors have observed the rapid reorganization of the cellular membranes of macrophages during Fc receptor-mediated frustrated phagocytosis of immune complex-coated surfaces. As the macrophages spread, large, clear basal vacuoles and anastamosing tubules were formed, occasionally contiguous with the adherent surface. Coated vesicles also were observed. This process was accompanied by a rapid reorganization of the Golgi complex region of the macrophages, which was observed using trimetaphosphatase histochemistry and an antibody to a Golgi membrane antigen as markers. On contact of the macrophages with the immune complexes, the Golgi complexes, which were tightly clustered around the centrioles, dispersed into vesicles and reorganized near the basal surface. The Golgi cisternae swelled, fragmented, and decreased in number. Golgi membrane antigen was found in the large basal vacuoles and also associated with the adherent basal surface of the macrophages. This indicates that the Golgi complexes were reorganized, in part, by a direct recruitment of their membranes to the increasing basal surface area of the spreading macrophages. The changes in the structure of the Golgi complexes were reversible; by 2 hours, the complexes had recovered their normal organization, with an accompanying decrease in the number of large basal vacuoles. These data suggest that the dynamic interrelationship among the Golgi membranes, intracellular vacuoles, and the plasma membrane can be perturbed by membrane spreading on a nonphagocytosable surface.

Animals↗
PubMed1989

Directed exocytosis of secretory granules containing apolipoprotein E to the adherent surface and basal vacuoles of macrophages spreading on immobile immune complexes.

As macrophages spread on an immune complex-coated surface, large, clear basal vacuoles and numerous coated vesicles and tubules form rapidly and are occasionally contiguous with the adherent surface, creating a microcompartment between the immune complex-coated surface and the cell membrane. The present study explored the nature of this basal compartment by examining the distribution of a major secretory product of macrophages, apolipoprotein E (ApoE), and of a lysosomal enzyme, acid phosphatase, by enzyme and immunocytochemistry. Upon contact of the macrophages with the immune complexes, intracellular stores of ApoE were secreted rapidly in the first 10 to 20 minutes to the area of ligand-receptor interaction. ApoE filled the large basal vacuoles and was also found in invaginations on the adherent surface that were sealed to the influx of proteins and peptides from the bulk medium. In contrast, the contents of lysosomes were not redistributed to the basal compartment. By 2 hours most of the ApoE had appeared in the bulk medium, suggesting that the protein could move out of the basal compartment. These data suggest that specific ligand-Fc receptor interactions serve to target secretion by macrophages to selective focal areas of contact, and that there are also mechanisms for retrieval of material from these sites.

Animals↗
PubMed1988

Collagenase expression and endogenous activation in rabbit synovial fibroblasts stimulated by the calcium ionophore A23187.

Collagenase is synthesized and secreted by stimulated rabbit fibroblasts as a proenzyme that must be proteolytically cleaved to yield catalytically active species. The calcium ionophore A23187 has provided new insights into the regulation of collagenase activation cascade by living cells. A23187, at concentrations of 10-40 ng/ml, induced expression of collagenase and stromelysin mRNA and the secretion of procollagenase of 57 and 53 kDa and prostromelysin of 51 kDa. Interestingly, it also stimulated activation of procollagenase to active forms of 47 and 43 kDa. The concentrations and treatment times required for induction of gene expression and activation indicated that they were independent events. Active collagenase constituted up to 16% of the total collagenase present in medium conditioned by A23187-treated cells. When grown on a collagen substrate, A23187-treated cells degraded collagen in a spatially localized manner. In cells treated with agents that induce procollagenase only, collagenase was localized in the perinuclear Golgi area; however, in A23187-treated cells, collagenase was located in widely dispersed granules, suggesting different intracellular pathways for collagenase before, during, and after activation. Addition of serine, thiol-, and metalloproteinase inhibitors with A23187 to rabbit fibroblasts inhibited conversion of procollagenase to its active form to varying degrees, suggesting that enzymes in these classes are involved in a cascade of proteolytic events leading to collagenase activation.

Animals↗
PubMed1988

Developmental expression of PDGF, TGF-alpha, and TGF-beta genes in preimplantation mouse embryos.

Control of growth and differentiation during mammalian embryogenesis may be regulated by growth factors from embryonic or maternal sources. With the use of single-cell messenger RNA phenotyping, the simultaneous expression of growth factor transcripts in single or small numbers of preimplantation mouse embryos was examined. Transcripts for platelet-derived growth factor A chain (PDGF-A), transforming growth factor (TGF)-alpha, and TGF-beta 1, but not for four other growth factors, were found in whole blastocysts. TGF-alpha, TGF-beta 1, and PDGF antigens were detected in blastocysts by immunocytochemistry. Both PDGF-A and TGF-alpha were detected as maternal transcripts in the unfertilized ovulated oocyte, and again in blastocysts. TGF-beta 1 transcripts appeared only after fertilization. The expression of a subset of growth factors in mouse blastocysts suggests a role for these factors in the growth and differentiation of early mammalian embryos.

Animals↗
PubMed1988

Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping.

The presence of macrophages is required for the regeneration of many cell types during wound healing. Macrophages have been reported to express a wide range of mitogenic factors and cytokines, but none of these factors has been shown in vivo to sustain all the wound-healing processes. It has been suggested that transforming growth factor-alpha (TGF-alpha) may mediate angiogenesis, epidermal regrowth, and formation of granulation tissue in vivo. Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, TGF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1. The expression of these transcripts was determined by a novel method for RNA analysis in which low numbers of mouse macrophages were isolated from wound cylinders, their RNA was purified and reverse-transcribed, and the complementary DNA was amplified in a polymerase chain reaction primed with growth factor sequence-specific primers. This single-cell RNA phenotyping procedure is rapid and has the potential for quantification, and mRNA transcripts from a single cell or a few cells can be unambiguously demonstrated, with the simultaneous analysis of several mRNA species. Macrophages from wounds expressed TGF-alpha antigen, and wound fluids contained TGF-alpha. Elicited macrophages in culture also expressed TGF-alpha transcripts and polypeptide in a time-dependent manner after stimulation with modified low-density lipoproteins and lipopolysaccharide endotoxin, which are characteristic of the activators found in injured tissues.

Animals↗
PubMed1988

Pitfalls in ecto-5'-nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages.

We have used both the enzyme cytochemical method with lead nitrate as a capture agent and an immunological method at the electron microscope level to localize plasma membrane 5'-nucleotidase in rat peritoneal resident macrophages during the initial interactions of latex beads or heat-killed Escherichia coli with the cell during phagocytosis. In macrophages at rest, cytochemical reaction product was evenly distributed along the external surface of the plasma membrane. However, when the cells were phagocytosing latex beads or bacteria, reaction product covered the entire surface of the adhering particles. To determine whether the apparent redistribution of 5'-nucleotidase onto the adhering particle was fact or artifact, we localized 5'-nucleotidase using a monoclonal antibody and an immunogold labelling technique. In macrophages binding or beginning to ingest bacteria, gold particles were distributed along the plasma membrane, except at the sites of cell-bacterium internalization. More significantly, the adhering bacteria were free of gold particles and therefore had no 5'-nucleotidase on their surfaces. Latex beads proved to be unsuitable as a test particle because the gold particles stuck to them non-specifically. We conclude that the artifactual redistribution of lead-phosphate reaction product is a major drawback of enzyme cytochemical methods when used on cell surfaces and that the immunogold labelling technique is more reliable.

5'-Nucleotidase↗
PubMed1987

Expression of apolipoprotein E by mouse brain astrocytes and its modulation by interferon-gamma.

We have found that mouse brain astrocytes in culture synthesize and secrete apolipoprotein E (ApoE) and that its expression is modulated by interferon-gamma, which influences the functional state of astrocytes. Interferon-gamma, which induces expression of Ia determinants on the surface of the astrocyte, reduced secretion of ApoE while enhancing intracellular expression of ApoE. Our data suggest that the synthesis and secretion phases of the ApoE pathway are regulated independently. It is possible that, by the production of ApoE, astrocytes may perform functions that are usually ascribed to macrophages and that may be important in immune reactivity in the brain.

Animals↗
PubMed1987

Collagen-fibronectin interactions in normal and Rous sarcoma virus-transformed avian tendon cells: possible mechanisms for increased extracellular matrix turnover after transformation.

Using gelatin, casein, and fibronectin as substrates and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we have identified protein-degrading enzymes in both normal and Rous sarcoma virus-transformed primary avian tendon cells. Although there are some consistent differences in the profile of the gelatinolytic activities (mainly metalloproteinases) between normal and transformed cells, the amounts of fibronectin-degrading activities seem to be comparable. In vitro studies reported here demonstrate that the degradation of fibronectin is partially and specifically inhibited by gelatin and collagen. We therefore propose that the abundant collagen present in normal tendon cells protects fibronectin against degradation. Conversely, in transformed cells, where collagen levels are drastically reduced, fibronectin may be more accessible to degradation. Thus differences in the steady-state levels of fibronectin on normal and transformed cells may be, at least in part, a consequence of changes in collagen levels.

Animals↗
PubMed1987

Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes.

Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. We studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggests coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis.

Animals↗
PubMed1986

Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity.

We have characterized the biosynthesis of two metalloproteinases, procollagenase and prostromelysin, by rabbit brain capillary endothelial cells (RBCE) by means of immunochemical, biosynthetic, and functional assays. Unstimulated RBCE secreted no detectable metalloproteinases. Secretion of both procollagenase and prostromelysin was induced within 6 h by treating the cells with 50 ng/ml 12-O-tetradecanoylphorbol-13-acetate. In treated cells, the two proenzymes accounted for up to 20% of the [35S]methionine-labeled secreted proteins; about 15 micrograms of each protein was secreted in 48 h by 10(6) RBCE. Although RBCE secreted approximately as much procollagenase and prostromelysin as did rabbit fibroblasts, virtually no enzyme activity could be measured in RBCE-conditioned medium, even after activation of the proenzymes by trypsin or an organomercurial agent.

Animals↗
PubMed1986

Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors.

Rabbit brain capillary endothelial cells treated with 12-O-tetradecanoylphorbol-13-acetate produce the metalloproteinases, procollagenase and prostromelysin, as up to 20% of their total secreted protein. However, little or no catalytic activity of these enzymes can be found after treatment with either trypsin or an organomercurial agent, which are able to activate the proenzymes in the medium from stimulated rabbit fibroblasts. We now have shown that enzyme activities of procollagenase and prostromelysin are revealed after conditioned medium is analyzed by gel filtration chromatography or by electrophoresis on sodium dodecyl sulfate-substrate gels. In both systems, the metalloproteinases were separated from metalloproteinase inhibitors. The major inhibitor of Mr = 30,000 from capillary endothelial cells was immunologically identical with the rabbit tissue inhibitor of metalloproteinases. Two additional inhibitors of metalloproteinases at Mr = 22,000 and 19,000 were also observed. Inhibitors were present in the conditioned medium from rabbit fibroblasts in much lower quantities and were also qualitatively different. When gel filtration chromatography was used to remove the tissue inhibitor of metalloproteinases from medium conditioned by stimulated capillary endothelial cells, both activatable procollagenase and prostromelysin were readily demonstrable. These data suggest that endogenous inhibitors regulate the expression of metalloproteinases secreted by endothelial cells.

Animals↗
PubMed1986

The cell and molecular biology of apolipoprotein E synthesis by macrophages.

Mononuclear phagocytes secrete over 50 different proteins that are regulated during differentiation and that are under the influence of various materials and factors in their extracellular milieu as part of the inflammatory response. The complex nature of the regulation of the expression of these molecules is displayed by apolipoprotein E (ApoE). ApoE mRNA first appears as monocytes differentiate into macrophages, and this expression is paralleled by the secretion of ApoE by the cells. In mature macrophages ApoE synthesis and secretion are decreased by activation of macrophages with endotoxin and interferon-gamma. Although these macrophages contain abundant translatable ApoE mRNA, little ApoE is synthesized, suggesting that this decrease occurs largely at the translational level. ApoE is also controlled at the level of secretion. ApoE is concentrated in the Golgi complex of macrophages and is also found in endoplasmic reticulum, secretion vesicles and coated vesicles. When macrophages come in contact with immune complexes the intracellular ApoE compartment degranulates rapidly. Therefore, ApoE is regulated at the levels of secretion, translation and transcription.

Animals↗
PubMed1986

Rapid redistribution of clathrin onto macrophage plasma membranes in response to Fc receptor-ligand interaction during frustrated phagocytosis.

We have observed increases in assembled clathrin on the plasma membrane during "frustrated phagocytosis," the spreading of macrophages on immobilized immune complexes. Resident macrophages freshly harvested from the peritoneal cavity of mice and attached to bovine serum albumin (BSA)-anti-BSA-coated surfaces at 4 degrees C had almost no clathrin basketworks on their adherent plasma membrane (less than 0.01 coated patch/micron 2), as observed by immunofluorescence, immunoperoxidase, and platinum-carbon replica techniques, although abundant assembled clathrin was observed in the perinuclear Golgi region. When the cells were warmed to 37 degrees C they started to spread by 4 min and reached their maximum extent by 20 min. Spreading preceded clathrin assembly at the plasma membrane. Clathrin-coated patches were first observed on the adherent plasma membrane at 6 min. Between 12 and 20 min assembled clathrin coats appeared on both adherent and nonadherent plasma membranes with a concomitant decrease in identifiable clathrin in the perinuclear region. A new steady state emerged by 2 h, as perinuclear clathrin began to reappear. At 20 min at 37 degrees C the adherent plasma membranes of macrophages spreading on BSA alone had 0.9 coated patch/micron 2, whereas in cells spread on immune complex-coated surfaces, the clathrin patches increased, dependent on ligand concentration, to a maximum of 2.1 coated patches/micron 2. Because frustrated phagocytosis of immune complex-coated surfaces at 37 degrees C increased the area of adherent plasma membrane, the total area coated by clathrin basket-works increased 5-fold (28 micron 2/cell) as compared with cells plated on BSA alone (5.6 micron 2/cell) and 200-fold as compared with cells adhering to immune complexes at 4 degrees C. We then determined that macrophages cultured on BSA-coated coverslips for 24 h already have abundant surface clathrin. When immune complexes were formed by the addition of anti-BSA IgG to already spread macrophages cultured on BSA-coated coverslips for 24 h, clathrin assembled at the sites of ligand-receptor interaction even at 4 degrees C, before spreading, and a 2.6-fold increase in assembled clathrin was observed on the adherent plasma membrane of cells on immune complexes as compared with cells on BSA alone. Clathrin was reversibly redistributed to the Golgi region, returning to the steady state by 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Commitment to expression of the metalloendopeptidases, collagenase and stromelysin: relationship of inducing events to changes in cytoskeletal architecture.

Agents that alter the morphology of rabbit synovial fibroblasts induce synthesis of the metalloendopeptidases, collagenase and stromelysin. We studied the relationship of cytoskeletal changes to the commitment to expression of these metalloendopeptidases. Cells treated with cytochalasin B (CB) or 12-O-tetradecanoylphorbol-13-acetate rounded, and only cells that had lost their stress fibers expressed collagenase and stromelysin, as determined by immunofluorescence. We concentrated on the effects of CB because of its rapid reversibility. When CB was added for 1-24 h, then removed, the cells respread within 30-60 min. The minimum period of CB treatment that committed cells to the subsequent synthesis of collagenase and stromelysin was 3 h. After initial treatment with 2 micrograms/ml CB for 3-24 h, or with various concentrations of CB (0-2 micrograms/ml) for 24 h, both enzyme activity and biosynthesis of the proenzymes showed a graded increase when measured at 24 h. Even after treatment with 2 micrograms/ml CB for only 3 h, greater than 85% of all cells were positive for both collagenase and stromelysin when cells were monitored by immunofluorescence. In contrast, when the dependence of collagenase and stromelysin expression on the inducing concentration of CB was examined, there was a dose-dependent increase in the number of cells positive for collagenase and stromelysin, as determined by immunofluorescence. Thus, at low concentrations of CB (less than 0.5 micrograms/ml), a heterogeneous population response was observed. These results suggest that the commitment of fibroblasts to induction of the metalloproteinases is a stochastic process in which a second signal that correlates with the disruption of the actin cytoskeleton may be rate-limiting for collagenase and stromelysin gene expression.

Actins↗
PubMed1986

Reorganization of polymerized actin: a possible trigger for induction of procollagenase in fibroblasts cultured in and on collagen gels.

Changes in cell shape are postulated to modulate gene expression during differentiation of a number of cell types, including rabbit synovial fibroblasts, which are inducible for expression of the zymogen form of the metalloendopeptidase, collagenase. In the work presented here, fibroblasts cultured on and within hydrated collagen gels were allowed to contract by release of the gels from the sides of the culture dish. Within 24 h of cell release, synthesis and secretion of procollagenase was initiated in the absence of any chemical manipulation. Fibroblasts grown in and on collagen also responded to 12-O-tetradecanoylphorbol-13-acetate and cytochalasin B with morphologic change and induced procollagenase. However, colchicine, which altered morphology to varying degrees in cells on plastic, on collagen, and within collagen gels, did not induce procollagenase expression. In all cases, the enzyme was induced only after reorganization of polymerized actin, rather than after a change in cellular morphology per se. As a first approach to identifying other aspects of the stimulated phenotype that could affect collagen turnover, the expression of collagen and endogenous metalloproteinase inhibitors in relation to procollagenase secretion was investigated. Collagen secretion by fibroblasts decreased when procollagenase secretion was induced by the pharmacologic agents, but not when cells were stimulated by contraction on or within collagen gels. The expression of two endogenous inhibitors was not coordinately regulated with induction of procollagenase. Therefore, the extracellular matrix and the cellular actin cytoskeleton may transduce signals that modulate the tissue remodeling phenotype of fibroblasts.

Actins↗