PubMed Health⌕ Search

Biomedical subjects

P Mignatti

Publications and source records attributed to P Mignatti.

9 recordsLinked to original sources
PubMed1992

Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex.

Basic fibroblast growth factor (bFGF) modulates functions of a variety of cell types. Whereas bFGF is known to act extracellularly, the protein lacks a transient signal peptide. No defined mechanism for bFGF secretion has been characterized besides release from dead or injured cells. To study this problem we devised an experimental system to examine bFGF-mediated migration of isolated single cells. Under these conditions individual cells are not affected by bFGF derived from other cells. By this method we have previously shown that bFGF released by NIH 3T3 cells transfected with bFGF cDNA modulates migration in an autocrine manner. We have now examined the effects on cell motility of drugs or treatments known to affect various pathways of protein secretion. Drugs that block secretion via the endoplasmic reticulum (ER)-Golgi complex or via multidrug resistance proteins did not inhibit cell motility. Migration was enhanced by the calcium ionophore A23187, which stimulates exocytosis, and was inhibited by methylamine, serum-free, and low temperature (18 degrees C) conditions, which block endo- and exocytosis. The reversal of these effects by the concomitant addition of affinity-purified anti-bFGF IgG or recombinant bFGF showed that the alterations in cell migration were mediated by changes in bFGF externalization. Thus bFGF can be released via a mechanism of exocytosis independent of the ER-Golgi pathway.

Animals↗
PubMed1992

Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity.

Exposure of bovine aortic or capillary endothelial cells to basic FGF (bFGF) for 1 h resulted in an approximately sixfold increase in plasminogen activator (PA) activity by 18 h that returned nearly to basal levels by 36 h. We hypothesized that the decrease in PA activity following bFGF stimulation was mediated by transforming growth factor beta (TGF-beta) formed from its inactive precursor. Conditioned medium collected from endothelial cells 36 h after a 1-h exposure to bFGF, but not control medium, inhibited basal levels of PA activity when transferred to confluent monolayers of bovine aortic endothelial cells. Antibody to TGF-beta neutralized the inhibitory activity of this conditioned medium, indicating that the medium contained active TGF-beta. Northern blot analysis and quantitation of acid activatable latent TGF-beta in conditioned medium demonstrated that bFGF exposure did not increase the amount of transcription or secretion of latent TGF-beta by the endothelial cells. Both aprotinin, an inhibitor of plasmin, and anti-urokinase type PA IgG blocked the generation of active TGF-beta in cultures exposed to bFGF. These results demonstrated that plasmin generated by uPA activity is required for the activation of latent TGF-beta in endothelial cell cultures treated with bFGF. Activation of TGF-beta by endothelial cells exposed to bFGF appears to limit both the degree and duration of PA stimulation. Thus, in bFGF-stimulated endothelial cell cultures, PA levels are controlled by a negative feedback loop: PA, whose expression is stimulated by bFGF, contributes to the formation of TGF-beta, which in turn opposes the effects of bFGF by limiting PA synthesis and activity. These studies suggest a role for TGF-beta in reversing the invasive stage of angiogenesis and contributing to the formation of quiescent capillaries.

Animals↗
PubMed1991

Basic fibroblast growth factor released by single, isolated cells stimulates their migration in an autocrine manner.

Basic fibroblast growth factor (bFGF), a protein with angiogenic, mitogenic, and chemotactic properties, lacks a signal sequence and is not secreted via the classical secretory pathway. However, the growth factor is known to act extracellularly. Since no defined mechanism for bFGF release has been described, it has been suggested that this growth factor is released from dead or damaged cells. To test this hypothesis we characterized the effect of exogenously added bFGF and neutralizing antibody on the migration of single, isolated NIH 3T3 cells transfected with bFGF cDNA. Under these conditions the observed cell cannot be affected by bFGF derived from other cells. Cells were seeded onto colloidal gold-coated coverslips at a density of one cell per coverslip. A cell migrating on this substrate produces a track free of refringent gold particles that is measured by an image analyzer. The results showed that cell motility directly correlated with the amount of bFGF released from the migrating cells. Affinity-purified anti-bFGF antibody, but not irrelevant IgG, reduced the level of migration of the bFGF transfectants to that of the control cells transfected with the vector alone, showing that bFGF stimulates migration of the cell that releases it. Thus, bFGF is secreted by viable cells and mediates cell functions via a "true" autocrine mechanism.

3T3 Cells↗
PubMed1991

Release of basic fibroblast growth factor, an angiogenic factor devoid of secretory signal sequence: a trivial phenomenon or a novel secretion mechanism?

Basic fibroblast growth factor (bFGF), a potent angiogenesis inducer, lacks a signal sequence. Therefore, it has been proposed that bFGF is primarily released from dead or damaged cells. Other proteins devoid of secretion signals, interleukin 1 beta (IL-1 beta) and the muscle lectin L-14, have been shown to be released via exocytosis, a novel secretion pathway independent of the "classic" endoplasmic reticulum-Golgi route. In the light of these findings and of our own recent results, we discuss evidence that bFGF can be released from single, uninjured cells and mediate functions in an autocrine manner. As is the case for IL-1 beta and L-14, externalization of bFGF may occur via exocytosis, a pathway utilized during development and differentiation.

Animals↗
PubMed1991

Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor.

Basic fibroblast growth factor, a potent angiogenesis inducer, stimulates urokinase (uPA) production by vascular endothelial cells. In both basic fibroblast growth factor-stimulated and -nonstimulated bovine capillary endothelial and human umbilical vein endothelial cells single-chain uPA binding is mediated by a membrane protein with a Mr of 42,000. Exposure of bovine capillary or endothelial human umbilical vein endothelial cells to pmolar concentrations of basic fibroblast growth factor results in a dose-dependent, protein synthesis-dependent increase in the number of membrane receptors for uPA (19,500-187,000) and in a parallel decrease in their affinity (KD = 0.144-0.790 nM). With both cells, single-chain uPA binding is competed by synthetic peptides whose sequence corresponds to the receptor-binding sequence in the NH2-terminal domain of uPA. Exposure of bovine capillary endothelial cells to transforming growth factor beta 1, which inhibits uPA production and upregulates type 1 plasminogen activator inhibitor, the major endothelial cell plasminogen activator inhibitor, has no effect on uPA receptor levels. These results show that basic fibroblast growth factor, besides stimulating uPA production by vascular endothelial cells, also increases the production of receptors, which modulates their capacity to focalize this enzyme on the cell surface. This effect may be important in the degradative processes that occur during angiogenesis.

Adrenal Cortex↗
PubMed1990

Growth factor control of extracellular proteolysis.

The involvement of proteases and growth factors in angiogenesis is complex. The angiogenic factor basic fibroblast growth factor (bFGF) induces increased synthesis of both plasminogen activator and collagenase in endothelial cells. In addition, bFGF increases the number of plasminogen activator receptors on the cell surface. Increased production of plasmin may be responsible for the release of soluble complexes of heparan sulfate-bFGF which may be the active form of bFGF. The activity of a negative regulator of angiogenesis, transforming growth factor beta (TGF-beta), is also regulated by proteases since the released latent form of TGF-beta is activated by a surface proteolytic assembly plasminogen activator and plasmin. Since TGF-beta induces an inhibitor of plasminogen activator, the activation reaction is self-regulatory.

Animals↗
PubMedDate not supplied

Properties and kinetics of development of Rous sarcoma virus-infected cells evidenced by methylene blue staining.

Three different kinds of areas of infected cells corresponding to different focus formation stages can be evidenced by methylene blue (MB) staining in cultures of chick embryo (CE) fibroblasts infected at low multiplicity with the temperature-sensitive (ts) mutant of Rous sarcoma virus (RSV), FU19, which transforms these fibroblasts at 37 degrees but not at 41 degrees. These are: (a) areas of MB-stainable cells with transformed phenotype (STP areas=foci); (b) areas of MB-stainable cells with normal phenotype (SNP areas), and (c) areas of MB-unstainable cells with normal phenotype (USNP areas). DNA and RNA synthesis and virus production were followed in these various stages at 37 degrees and at 41 degrees. The results show that when cultures are shifted from 37 degrees to 41 degrees, virus production in the SNP and USNP areas which arise by phenotypic reversion of STP foci remains comparable to that of the latter foci. On the contrary, DNA and RNA synthesis are markedly reduced in SNP and USNP areas, DNA synthesis falling down to the level of uninfected cells, and RNA synthesis remaining somewhat higher. The kinetics of development of SNP and STP areas in cultures infected with FU19 and with the parental virus SR4 were also compared. The results confirm that SNP areas are precursors of STP areas but that this passage occurs at a slower rate in cultures infected with FU19.

Animals↗