The role of proteases in matrix breakdown during cellular invasion.
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Amnion↗
Biomedical subjects
D B Rifkin
Publications and source records attributed to D B Rifkin.
Transformation by basic fibroblast growth factor requires high levels of expression: comparison with transformation by hst/K-fgf.
Basic fibroblast growth factor is a potent mitogen for a variety of cell types and has been suggested to have transforming activity. To test this hypothesis, we have introduced a human bFGF cDNA into NIH 3T3 cells either by DNA transfection or by retrovirus infection. We have compared the properties of cell lines obtained with cells prepared similarly but expressing the hst/K-fgf growth factor. While bFGF does not contain an amino terminal signal sequence and is not secreted from cells in which it is synthesized, hst/K-fgf does contain a signal sequence and is secreted from cells. Our results show that the transformed phenotype correlates directly with the level of bFGF expression, since all transformed clones expressed high levels of bFGF, while nontransformed clones expressed comparatively low levels of bFGF. In contrast, even low levels of hst/K-fgf expression resulted in a transformed phenotype. These results suggest that bFGF is an inefficient transforming protein and that this may relate to its lack of secretion.
Animals↗
Monospecific antibodies implicate basic fibroblast growth factor in normal wound repair.
Exogenous polypeptide growth factors influence the rate of wound healing and other biological processes, but there is no direct evidence that these peptides have an intrinsic role. To test whether basic fibroblast growth factor is involved in wound repair, rats were implanted with subcutaneous polyvinyl alcohol sponges containing slow-release pellets releasing either a polyclonal neutralizing antiserum directed against basic fibroblast growth factor, preimmune IgG, or nothing. Histological and biochemical evaluation of the granulation tissue that infiltrated the sponges showed anti-basic fibroblast growth factor to cause significant reductions in DNA, protein, and collagen content when compared with either preimmune IgG or placebo at the early stages of wound repair.
Animals↗
Membrane and matrix localization of proteinases: a common theme in tumor cell invasion and angiogenesis.
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Angiogenesis Inducing Agents↗
The development of a quantitative RIA for basic fibroblast growth factor using polyclonal antibodies against the 157 amino acid form of human bFGF. The identification of bFGF in adherent elicited murine peritoneal macrophages.
Polyclonal antibodies which have the capacity to neutralize the biological activity of basic fibriblast growth factor (bFGF) in vitro, have been raised in rabbits against the 157 amino acid form of bFGF purified from human placenta. In a dot blot assay the anti-bFGF antibodies do not recognize the acidic form of FGF (aFGF) with which the basic form shares significant amino acid sequence homology. As determined by immunoblotting, bFGF antibodies recognized only bFGF in a mixture of placentally derived heparin-binding proteins, demonstrating the specificity of these antibodies. Using the anti-human bFGF antibodies, we have developed a solid-phase competitive radioimmunoassay sensitive to 7.8 ng/ml (0.4 pmol/ml) for bFGF. aFGF does not compete with bFGF for binding to the antibodies in the radioimmunoassay even at 2.04 micrograms/ml. The specificity of the assay was further demonstrated by a lack of competition of cytochrome C, myoglobin, epidermal growth factor or bovine serum albumin with bFGF for binding to the antibodies. We have identified bFGF in extracts of adherent thioglycollate-stimulated mouse peritoneal macrophages by immunological criteria including the ability of the extract to compete with 125I-bFGF for binding to affinity-purified anti-human bFGF antibodies in the RIA and the ability of these antibodies in inhibit the bFGF-like biological activity of the macrophage extract.
Amino Acids↗
In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor.
When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.
Animals↗
Multiple forms of an angiogenesis factor: basic fibroblast growth factor.
An angiogenesis factor has been isolated from human placenta and human hepatoma cells on the basis of its ability to stimulate protease production in cultured capillary endothelial cells. The purified angiogenesis factor also stimulated DNA synthesis and motility in capillary endothelial cells and induced angiogenesis in vivo. Amino acid sequence data revealed that the angiogenesis factor was human basic fibroblast growth factor (bFGF). Other angiogenesis factors isolated on the basis of their ability to stimulate endothelial cell proliferation have also been identified as bFGFs. The bFGFs that have been sequenced show variability in their N-termini. These different forms of bFGF may be naturally occurring processed forms or may be generated by proteases released during the isolation procedure. Recently a bFGF with a large N-terminal extension has been identified. This Mr 25,000 bFGF has the same biological activity and the same affinity for the bFGF receptor as the typical Mr 18,000 bFGFs. The Mr 25,000 bFGF can be converted into an Mr 18,000 form by treatment with low concentrations of trypsin, suggesting that it may be a precursor to the Mr 18,000 bFGF.
Amino Acid Sequence↗
Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation.
Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.
Animals↗
Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis.
We have found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic fibroblast growth factor (bFGF). Cell movement is blocked by purified polyclonal rabbit IgG to bFGF as well as affinity purified anti-bFGF IgG and anti-bFGF F(ab')2 fragments. The inhibitory effect of the immunoglobulins is dependent upon antibody concentration, is reversible, is overcome by the addition of recombinant bFGF, and is removed by affinity chromatography of the antiserum through a column of bFGF-Sepharose. Cell movement is also reversibly inhibited by the addition of protamine sulfate and suramin; two agents reported to block bFGF binding to its receptor. The addition of recombinant bFGF to wounded monolayers accelerates the movement of cells into the denuded area. Transforming growth factor beta which has been shown to antagonize several other effects of bFGF also inhibits cell movement. The anti-bFGF IgG prevents the movement of bovine capillary endothelial cells, BHK-21, NIH 3T3, and human skin fibroblasts into a denuded area. Antibodies to bFGF, as well as suramin and protamine sulfate also suppress the basal levels of plasminogen activator and DNA synthesis in bovine aortic endothelial cells.
Animals↗
Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.
We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.
Animals↗
Cell-associated plasminogen activation: regulation and physiological functions.
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Amyloid beta-Protein Precursor↗
New aspects of blood vessel growth: tumor and tissue-derived angiogenesis factors.
Angiogenesis factors have been recently purified from both tumors and nonneoplastic tissues. The biological properties shown by these factors in vivo and on cultured endothelial cells will be discussed.
Angiogenesis Inducing Agents↗
A form of human basic fibroblast growth factor with an extended amino terminus.
The amino acid sequence of a human placental bFGF was determined by a combination of protein and cDNA sequencing. The placental bFGF consists of 157 amino acid residues with a calculated molecular weight of 17,464 and is highly homologous to bovine pituitary bFGF. The human protein contains an amino terminal extension when compared to the sequence established for bovine bFGF (Esch et al., 1985) and to the sequence of the predicted translation product based on human bFGF cDNA clones (Abraham et al., 1986).
Amino Acid Sequence↗
Endothelial cell growth factors and the vessel wall.
The role of endothelial cell growth factors in the maintenance of the blood vessel wall is, as we have described here, much more complex than merely stimulating the mitogenesis of endothelial cells. The FGFs are capable of eliciting an array of responses in endothelial cells, some, or all, of which are important for neovascularization and the control of clot dissolution. These endothelial cell responses include protease elaboration, chemotaxis, and mitogenesis. That these growth factors seem neither to be constitutively released into the medium of cultured cells that synthesize bFGF, nor released into the bloodstream in vivo suggests that the temporal and local control of neovascularization may involve the regulation of growth factor release from cells such as endothelial cells, fibroblasts, and macrophages. Although there is no known example of this for bFGF, it is well known that both thrombin and Factor Xa stimulate the release of a mitogenic protein from endothelial cells and that low oxygen tension stimulates the release of macrophage-derived angiogenesis factor. In addition, both TGF beta and heparin alone appear to play a role in wound healing and vessel wall maintenance. The work of Roberts et al suggests that TGF beta is not only angiogenic, but also stimulates the growth of fibrotic tissue as well. Studies on mast cells demonstrated that released heparin is chemotactic for endothelial cells and can potentiate tumor angiogenesis. An attractive hypothesis is that these molecules not only act as FGF potentiators or inhibitors but that they also may exert their angiogenic effects by inducing FGF release from cells. Perhaps angiogenin, an angiogenic molecule with no mitogenic activity, works in this way. However, no evidence as yet exists concerning this point. A second level of control of neovascularization may involve the interaction of FGF with other molecules released into the same microenvironment. For example, thrombin and TGF beta released from platelets, as well as heparin released from mast cells, have all been demonstrated to affect bFGF activity in vitro and may act as modifiers of FGF activity in vivo. Since bFGF can modulate fibrinolytic activity, one could imagine that its release into a wound region of the vasculature could have detrimental effects on clot formation and subsequent wound healing. Thus, the transient inhibition of bFGF activity by TGF beta would allow clot formation before the induction of neovascularization by bFGF, TGF beta thereby playing a role in the regulation of the sequence in which events occur.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor.
A Mr 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig brain along with the typical Mr 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The Mr 25,000 form, like the Mr 18,000 form, was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The Mr 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentrations of 0.1-10 ng/ml, the same range that was effective for guinea pig and human Mr 18,000 bFGFs. The binding of human 125I-labeled bFGF to baby hamster kidney cells is inhibited equally by the Mr 25,000 guinea pig protein and the Mr 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the Mr 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the Mr 25,000 and Mr 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the Mr 25,000 guinea pig bFGF was converted to a Mr 18,000 protein. These results show that the two molecules are closely related and suggest that the Mr 25,000 protein shares substantial homology with the Mr 18,000 bFGF.
Animals↗
The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells.
Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor.
Animals↗
Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade.
To understand the role of proteinases in tumor invasion, the effects of inhibitors of metallo-, serine-, and cysteine-proteinases on this process were studied using 125I-iododeoxyuridine-labeled B16/BL6 cells grown on human amnion basement membrane. Cellular invasion was quantitated by measuring the radioactivity associated with the amniotic membrane after the B16/BL6 cells on the basement membrane were removed by lysis followed by scraping. The results obtained with proteinase inhibitors showed that inhibitors of collagenase and plasmin prevented invasion of the amnion. Tissue invasion was also blocked by antiurokinase antibodies. On the contrary, cysteine-proteinase inhibitors and anti-tissue plasminogen activator antiserum were ineffective. Mersalyl, a compound known to activate collagenase, stimulated invasion under conditions where plasmin formation or activity were inhibited. Evidence for the role of a plasminogen activator-plasmin-collagenase activation cascade in B16 invasion is provided.
Amnion↗
Both normal and tumor cells produce basic fibroblast growth factor.
We have previously purified from human placenta a basic fibroblast growth factor (FGF)-like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial (BCE) cells, and induces angiogenesis in vivo. The ability of basic FGF to stimulate PA production in BCE cells was used as an assay for the presence of basic FGF-like molecules in extracts of both normal and tumor-derived cultured cells. The identity of the PA-stimulatory activity with basic FGF was confirmed by its high affinity for heparin and by its cross-reactivity with antibodies to human placental basic FGF. Basic FGF-like molecules were identified in eight of ten cell lines tested, and the amount of FGF-like activity present in these cells bore no relation to their origin from normal or tumor tissue. The test cells, BCE cells, had one of the highest levels of FGF-like activity, suggesting that it may have an autocrine role in these cells.
Animals↗