[Interchangeability of growth factors in early G(0)-G(1) period of cell cycle].
Explore the source record for details and available documents.
3T3 Cells↗
Biomedical subjects
T Maciag
Publications and source records attributed to T Maciag.
Intracellular precursor interleukin (IL)-1alpha, but not mature IL-1alpha, is able to regulate human endothelial cell migration in vitro.
The human umbilical vein endothelial cell (HUVEC) has a finite lifespan in vitro, and senescent HUVEC contain elevated levels of the negative growth regulator interleukin (IL)-1alpha. IL-1alpha is translated as a signal peptide sequence-less cytosolic 31-kDa precursor (IL-1alpha p), which undergoes proteolytic activation to release the mature carboxyl terminus 17-kDa protein (IL-1alpha m). Both the IL-1alpha p and IL-1alpha m proteins are biologically active as exogenous cytokines. Interestingly, only IL-1alpha p contains a nuclear localization sequence between residues 79 and 85. To further study the role of intracellular IL-1alpha in the regulation of human endothelial cell function, a spontaneous HUVEC transformant was stably transfected with IL-1alpha p, IL-1alpha m, and the IL-1alpha p K82N mutant, which attenuates the nuclear traffic of IL-1alpha p. Interestingly, the IL-1alpha p transfectants were found to have a lower migratory potential than either IL-1alpha m or IL-1alpha p K82N transfectants, and the addition of the IL-1 receptor antagonist did not alter the migration of these cells. Immunofluorescence microscopy demonstrated that only the IL-1alpha p transfectants exhibited prominent staining for beta-catenin-associated cell-to-cell contacts, as well as pronounced vimentin intermediate filaments and actin cytoskeleton staining. These data suggest that IL-1alpha p, and not IL-1alpha m, may function as an intracellular regulator of the migratory capacity of the human endothelial cell and that the nuclear localization sequence present within IL-1alpha p may be involved in regulating this function.
Cell Movement↗
The immediate-early gene product MAD-3/EDG-3/IkappaB alpha is an endogenous modulator of fibroblast growth factor-1 (FGF-1) dependent human endothelial cell growth.
The tumor promoter phorbol 12-myristic 13-acetate inhibits the growth of human endothelial cells and induces the formation of capillary-like, tubular structures. We report the novel growth regulatory function of the immediate-early gene, edg-3, which is identical to the IkappaB alpha/MAD-3 gene. We employed phosphothioate oligonucleotides (PTO) directed against the translation initiation site of IkappaB alpha to inhibit its expression. The antisense IkappaB alpha PTO-treated cells exhibited an exaggerated growth response to fibroblast growth factor-1 (FGF-1). In contrast, IL-1-induced growth arrest response was not modulated. These data suggest that the early response gene IkappaB alpha is an endogenous regulator of endothelial cell growth in vitro.
Cell Division↗
A carboxyl-terminal domain in fibroblast growth factor (FGF)-2 inhibits FGF-1 release in response to heat shock in vitro.
The fibroblast growth factor (FGF) prototypes, FGF-1 and FGF-2, lack a signal sequence, but both contain a nuclear localization sequence. We prepared a series of FGF-1 deletion mutants fused to the reporter gene, beta-galactosidase (beta-gal) and determined that a domain between residues 83 and 154 is responsible for FGF-1 cytosol retention in NIH 3T3 cells. Using a series of FGF-beta-gal chimeric proteins prepared by the shuffling of cassette-formatted synthetic FGF prototype genes, we were able to demonstrate that the nuclear localization sequence from the 5'-CUG region of FGF-2 is not able to direct the nuclear association of FGF-1 due to its inability to repress the function of the FGF-1 cytosol retention domain. We also observed that while the FGF-1:beta-gal chimera was released in response to heat shock, the FGF-2:beta-gal protein was not. Further, replacement of the FGF-1 cytosol retention domain with the corresponding domain from FGF-2 repressed the release of the chimeric protein. These data suggest that the specificity of the stress-induced secretion pathway for FGF-1 involves a carboxyl-terminal domain that is absent in FGF-2 and that the FGF-1 secretion pathway does not restrict the release of high molecular weight forms of FGF-1.
3T3 Cells↗
Human immunodeficiency virus-associated vasculopathy in transgenic mice.
There is substantial clinical evidence for the development of vascular disorders in human immunodeficiency virus (HIV)-infected individuals, particularly in the form of vasculitis. Transgenic mice carrying a replication-defective HIV-1 provirus with selective deletion of the gag, pol, and env genes developed extensive vasculopathy. Restricted expression of HIV nonstructural genes in smooth muscle cells was accompanied by the migration and proliferation of these cells in blood vessels of all sizes and at different body sites. The frequent infiltration observed in the hypertrophic vessel walls occurred predominantly in the adventitia and was composed of primarily T cells and occasionally plasma cells. The intimal thickening generated significant luminal narrowing in some vessels, and the restricted blood flow led to ischemia in the affected tissues. Interestingly, the endothelium did not appear to support HIV gene expression or be involved in the pathological process. This transgenic model provides an opportunity to dissect the mechanism underlying HIV-associated vasculopathy.
Animals↗
Inhibition of growth factor mitogenicity and growth of tumor cell xenografts by a sulfonated distamycin A derivative.
Interference with growth factor-receptor interactions may have particular relevance in efforts to intervene clinically in both autocrine and paracrine aspects of malignancy. Suramin is a synthetic anticancer agent that works, in part, by blocking the binding of growth factors to their receptors. While initial clinical trials have been encouraging, its use in clinical applications is associated with significant toxicities. Suradista is a novel sulfonated distamycin derivative that is also effective at complexing and inactivating growth factors and cytokines while remaining relatively nontoxic. The goal of this study was to compare the antineoplastic properties of suramin and Suradista. To achieve this, the effects of these compounds on growth factor induced mitogenesis in normal mouse fibroblasts and human umbilical vein endothelial cells were examined, as well as their ability to inhibit the growth of NIH/3T3 cells that had been transformed by the introduction of a fibroblast growth factor (FGF) 1 coding region (residues 1-154) fused to the signal peptide of the hst/KS3 gene (sp-hst/KS3:FGF1-154). In each case, Suradista was more effective than suramin in inhibiting mitogenesis in normal cells, as well as the growth of the transformed cells. Furthermore, Suradista was also shown to be as effective as suramin at inhibiting the growth of sp-hst/KS3:FGF1-154-transformed NIH/3T3 xenografts grown in athymic nude mice when given at only 50% the dosage used for suramin (50 mg/kg for Suradista versus 100 mg/kg for suramin). In summary, these results indicate that novel compounds acting like suramin may be developed as effective antineoplastic agents and may also prove to be of clinical benefit.
3T3 Cells↗
An antisense oligonucleotide to the notch ligand jagged enhances fibroblast growth factor-induced angiogenesis in vitro.
Angiogenesis, or the formation of new blood vessels, plays a central role in a number of physiologic and pathologic conditions, including wound healing, diabetic retinopathy, and solid tumor growth, and endothelial cells can be induced to mimic this process in vitro. Using a modification of the differential display method (Zimrin, A. B., Villeponteau, B., and Maciag, T. (1995) Biochem. Biophys. Res. Commun. 213, 630-638), we isolated the human homolog of the Jagged ligand for the Notch receptor from human endothelial cells exposed to fibrin and demonstrate that the Jagged transcript, but not the Notch 1 or Notch 2 transcripts, are up-regulated by fibrin. Interestingly, the addition of an antisense Jagged oligomer to bovine microvascular endothelial cells grown on a collagen gel resulted in a marked increase in invasion and tube formation in the underlying gel in response to fibroblast growth factor. In contrast, no effect was observed on vascular endothelial growth factor-induced angiogenesis under identical conditions. These data suggest that Jagged-Notch signaling is able to regulate fibroblast growth factor-induced endothelial cell migration in vitro, an early event during angiogenesis in vivo.
Calcium-Binding Proteins↗
The human diploid fibroblast senescence pathway is independent of interleukin-1 alpha mRNA levels and tyrosine phosphorylation of FGFR-1 substrates.
In vitro cellular senescence of human umbilical vein endothelial cells (HUVEC) may involve the intracellular activity of the signal peptide-less cytokine interleukin (IL)-1 alpha. To determine whether senescence of other human diploid cells involves the function of IL-1 alpha, we examined the steady-state expression of IL-1 alpha mRNA in IMR-90 fibroblasts. The IL-1 alpha transcript was not elevated in senescent IMR-90 cells. With the exception of the plasminogen activator inhibitor (PAI)-1 transcript, other IL-1 alpha-response gene mRNAs were not induced in senescent IMR-90, although the mRNA for each gene was induced by exogenous IL-1 alpha. The mRNA expression of cell cycle-specific genes demonstrated that Fos and ornithine decarboxylase (ODC) were induced in young and senescent cells in response to both serum and fibroblast growth factor (FGF)-1. Histone (H)3 mRNA was induced by serum in young cells, but not in senescent cells, and FGF-1 failed to induce H3 mRNA in either young or senescent cells. Further, while young IMR-90 populations were able to respond to serum as an initiator of DNA synthesis and cell growth, they did not exhibit a response to exogenous FGF-1. FGF receptor (R)-1 substrates were not tyrosine phosphorylated in either young or senescent IMR-90 cells. These data demonstrate that IL-1 alpha and FGF-1 may have different functions in HUVEC and IMR-90 fibroblast populations including distinct pathways for the regulation of cellular growth and senescence.
Cellular Senescence↗
Molecular modeling and deletion mutagenesis implicate the nuclear translocation sequence in structural integrity of fibroblast growth factor-1.
The sequence NYKKPKL in the NH2 terminus of fibroblast growth factor (FGF)-1 has been proposed to affect the long term activities of FGF-1 through its function as a nuclear translocation signal or its role in stabilization of the structure required to sustain binding and activation of the transmembrane receptor kinase. A dynamic molecular model of FGF-1 docked into a duplex of the FGF receptor ectodomain and a hexadecameric heparin chain suggests that the NYKKPKL sequence does not directly interact with heparin or the receptor, but rather the lysine-leucine residues within the sequence indirectly stabilize a major receptor-binding domain. Concurrent with a marked increase in dependence on exogenous heparin for optimal activity, sequential deletion of residues in the NYKKPKL sequence in FGF-1 resulted in a progressive loss of thermal stability, resistance to protease, mitogenic activity, and affinity for the transmembrane receptor. The largest change resulted from deletion of the entire sequence through the lysine-leucine residues. In the presence of sufficiently high concentrations of heparin, the deletion mutants exhibited mitogenic activity equal to wild-type FGF-1. The results confirm that a primary role of the NYKKPKL sequence domain is to maintain the structural integrity of FGF-1 required for optimal binding to and activation of the heparan sulfate-transmembrane receptor complex.
3T3 Cells↗
The nuclear trafficking of extracellular fibroblast growth factor (FGF)-1 correlates with the perinuclear association of the FGF receptor-1alpha isoforms but not the FGF receptor-1beta isoforms.
The alternatively spliced fibroblast growth factor receptor (FGFR)-1 isoforms, FGFR-1alpha and FGFR-1beta, are characterized by the presence of either three or two Ig-like loops in the extracellular domain and are differentially expressed during embryonic development and tumor progression. We have previously shown that in cells irreversibly committed to DNA synthesis by FGF-1, approximately 15% of cell surface FGFR-1 traffics to a perinuclear locale as a structurally intact and functional tyrosine kinase (Prudovsky, I., Savion, N., Zhan, X., Friesel, R., Xu, J., Hou, J., McKeehan, W. L., and Maciag, T. (1994) J. Biol. Chem. 269, 31720-31724). In order to define the structural requirement for association of FGFR-1 with the nucleus, the expression and trafficking of FGFR-1 in FGFR-1alpha and FGFR-1beta L6 myoblast transfectants was studied. Although FGFR-1alpha was expressed as p145 and p125 forms, FGFR-1beta was expressed as p120 and p100 forms in the L6 myoblast transfectants. Tunicamycin and N-glyconase experiments suggest that these forms of FGFR-1alpha and FGFR-1beta are the result of differential glycosylation. However, only the p145 form of FGFR-1alpha and the p120 form of FGFR-1beta were able to bind FGF-1 and activate tyrosine phosphorylation. Pulse-chase analysis of FGFR-1 biosynthesis suggests that the p125 and p100 proteins are the precursor forms of p145 FGFR-1alpha and p120 FGFR-1beta, respectively. Because ligand-chase analysis demonstrated that FGFR-1beta L6 myoblast transfectants exhibited a reduced efficiency of nuclear translocation of exogenous FGF-1 when compared with FGFR-1alpha transfectants, the intracellular trafficking of the FGFR-1alpha and FGFR-1beta isoforms was studied using an in vitro kinase assay to amplify immunoprecipitated FGFR-1. Indeed, the appearance of the FGFR-1alpha but not FGFR-1beta isoform in the nuclear fraction of L6 myoblast transfectants suggests that the distal Ig-like loop in FGFR-1alpha mediates the differential nuclear association of FGFR-1alpha as a structurally intact and functional tyrosine kinase. Further, the FGFR-1beta L6 myoblast transfectants but not the FGFR-1alpha myoblast transfectants exhibited a pronounced morphologic change in response to exogenous FGF-1. Because this phenotype change involves the induction of a rounded cellular shape, it is possible that the FGFR-1alpha and FGFR-1beta may ultimately exhibit differential trafficking to adhesion sites.
Alternative Splicing↗
Serum-starvation induces the extracellular appearance of FGF-1.
Autocrine/paracrine stimulation of cell growth by members of the fibroblast growth factor (FGF) family of polypeptides is dependent upon extracellular interactions with specific high affinity receptors at the cell surface. Acidic FGF (FGF-1) lacks a classical signal sequence for secretion, suggesting that intrinsic levels of this mitogen may not stimulate cell growth and utilizes a non-classical pathway to gain access to the extracellular compartment. To evaluate the biological potential of intracellular FGF-1 more rigorously, human cDNA sequences for the growth factor were introduced into primary murine embryonic fibroblasts using retrovirally mediated gene transfer. Heparin affinity, Western analysis, mitogenic assays, in situ immunohistochemical techniques, induction of tyrosine phosphorylation and antibody inhibition studies were used to demonstrate functionality of the FGF-1 transgene in this experimental model. Under normal culture conditions, cells constitutively expressing intracellular FGF-1 exhibited a slight growth advantage. In contrast, when maintained in reduced serum, these cells adopted a transformed phenotype and demonstrated an enhanced growth potential, induction of FGF-specific phosphotyrosyl proteins and the nuclear association of the growth factor. Analysis of the conditioned media from these stressed cells indicated that serum starvation induces the secretion of FGF-1 as latent high molecular mass complexes requiring reducing agents to activate its full biological potential.
Amino Acid Sequence↗
Progress towards a unifying hypothesis for angiogenesis.
Explore the source record for details and available documents.
Animals↗
FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates.
Senescent cells do not proliferate in response to exogenous growth factors, yet the number and affinity of growth factor receptors on the cell surface appear to be similar to presenescent cell populations. To determine whether a defect in receptor signaling exists, we analyzed human umbilical vein endothelial cells (HUVEC) since HUVEC growth is absolutely dependent upon the presence of FGF. We report that in both presenescent and senescent HUVEC populations, FGF-1 induces the expression of cell cycle-specific genes, suggesting that functional FGF receptor (FGFR) may exist on the surface of these cells. However, the tyrosine phosphorylation of FGFR-1 substrates, Src and cortactin, is impaired in senescent HUVEC, and only the presenescent cell populations exhibit a FGF-1-dependent Src tyrosine kinase activity. Moreover, we demonstrate that senescent HUVEC are unable to migrate in response to FGF-1, and these data correlate with an altered organization of focal adhesion sites. These data suggest that the induction of gene expression is insufficient to promote a proliferative or migratory phenotype in senescent HUVEC and that the attenuation of the FGFR-1 signal transduction pathway may be involved in the inability of senescent HUVEC to proliferate and/or migrate.
Base Sequence↗
The cysteine residue responsible for the release of fibroblast growth factor-1 residues in a domain independent of the domain for phosphatidylserine binding.
Fibroblast growth factor (FGF)-1 lacks a classical signal sequence to direct its secretion yet utilizes high affinity cell surface receptors to signal its heparin-dependent angiogenic and neurotrophic activities. We have previously reported that FGF-1 is released in response to temperature stress as a latent homodimer through a pathway that is potentiated by the Golgi inhibitor, brefeldin A (Jackson, A., Tarantini, F., Gamble, S., Friedman, S., and Maciag, T. (1995) J. Biol. Chem. 270, 33-36). In an attempt to further characterize this unconventional secretion mechanism, we sought to define the Cys residue(s) critical for FGF-1 dimer formation and release and to determine whether FGF-1 can associate with known phospholipid components of organelle or plasma membranes, which may be disturbed by brefeldin A. Utilizing FGF-1 Cys mutants, we were able to demonstrate that residue Cys30 is critical for FGF-1 release in response to heat shock. In addition, using solid phase phospholipid binding assays we demonstrate that FGF-1 is able to specifically associate with phosphatidylserine (PS). Heparin inhibits the association between FGF-1 and PS, and synthetic peptide competition assays suggest that the PS-binding domain of FGF-1 lies between residues 114 and 137. These observations indicate that FGF-1 may be able to associate with the PS component of organelle and/or plasma membranes and that the domains responsible for FGF-1 homodimer formation and PS binding are structurally distinct.
3T3 Cells↗
Models of in vitro angiogenesis: endothelial cell differentiation on fibrin but not matrigel is transcriptionally dependent.
Endothelial cells can be induced to form a branching network of tubular structures using a variety of cell culture conditions. We have examined this differentiation process using several sets of conditions: plating human umbilical vein endothelial cells (HUVEC) on Matrigel, adding collagen to the apical surface of HUVEC grown on fibronectin, and plating HUVEC on fibrin in the presence of FGF-1. We determined that although the first two conditions produce dramatic morphologic changes in the HUVEC population, gene transcription and translation are not required for the regulation of the process. Rather, post-translational events are involved since the Matrigel-dependent process could be inhibited by the addition of nocodazole, suramin or H7, a protein kinase inhibitor. In contrast, the fibrin matrix-dependent differentiation pathway involved transcriptional and translational events since the addition of either actinomycin D or cycloheximide inhibited this pathway. A modified differential display of RNA extracted from HUVEC after 0, 2, 5, and 24 hours on fibrin revealed expression of a novel cDNA.
Base Sequence↗
Angiogenesis-directed implantation of genetically modified endothelial cells in mice.
By virtue of their location within blood vessels and their ability to express foreign genes, endothelial cells are attractive vehicles for the delivery of therapeutic molecules in vivo. We wished to determine whether i.v.-injected, genetically modified endothelial cells can become incorporated into sites of active angiogenesis in vivo. To do so, we studied the fate of i.v.-injected, lacZ-expressing human umbilical vein endothelial cells in athymic nude mice bearing lethally irradiated NIH 3T3 murine fibroblast cells transfected with a sp-hst/KS3:fibroblast growth factor-1 chimera that forces the secretion of the angiogenic protein, fibroblast growth factor-1. Following i.v. injection, lacZ-labeled human umbilical vein endothelial cells accumulated at sites of fibroblast growth factor-1-induced angiogenesis, persisting for at least 4 weeks. These results suggest that i.v.-administered, genetically modified endothelial cells can migrate into and survive within an angiogenic site. This strategy may be useful for delivery of therapeutic molecules to sites of pathological angiogenesis during tumor metastasis.
3T3 Cells↗
Identification of the domain within fibroblast growth factor-1 responsible for heparin-dependence.
While the prototype members of the fibroblast growth factor (FGF) family, FGF-1 and FGF-2 are structurally related, the structural differences between these polypeptides predict that they will ultimately exhibit different biological roles. Indeed, a significant difference between these proteins is the dependence of FGF-1 on heparin for the generation of maximal mitogenic activity. In order to gain structural insight into the issue of FGF-1 heparin-dependence, a synthetic gene encoding FGF-2 was constructed with oligonucleotides in a four-cassette format similar to a synthetic gene previously constructed for FGF-1 (Forough et al. 1992, Biochem. Biophys. Acta 1090 293-298). This strategy permitted the molecular shuffling of corresponding cassette(s) between FGF-1 and FGF-2 to yield FGF-1:FGF-2 chimeras. Three amino acid changes (Lys86-->Glu, Tyr120-->His, and Thr121-->Ala) were introduced into the synthetic FGF-2 gene by the cassette format to generate convenient FGF-1 restriction sites, but these alterations did not significantly affect the mitogenic activity or the heparin-binding affinity of the recombinant FGF-2 protein when compared with native FGF-2. Among the various FGF-1:FGF-2 chimeric constructs, one designated FGF-C(1(1/2)1 1), which represents FGF-1 containing FGF-2 amino acid residues 65 to 81, displayed FGF-1-like heparin-binding affinity but it did not require the addition of exogenous heparin to manifest its mitogenic activity. These data suggest that the sequence within residues 65 and 81 from FGF-2 significantly contributes to the heparin-dependent character of FGF-1.
Amino Acid Sequence↗
Characterization of edg-2, a human homologue of the Xenopus maternal transcript G10 from endothelial cells.
We have isolated by polymerase chain reaction-amplified subtractive hybridization technique, several cDNA clones that are induced by phorbol myristic acetate in human umbilical vein endothelial cells (HUVEC). One such clone, termed edg-2, was sequenced and was found to encode a human homologue of a Xenopus maternal transcript G10. The deduced amino acid sequence of edg-2 contains a putative nuclear translocation sequence, an N-terminal acidic domain and a cysteine-rich C-terminal domain containing a putative Zinc-finger structure. The structure of edg-2 polypeptide suggests that it may be a nuclear regulator of transcription. The edg-2 mRNA was expressed ubiquitously in cell lines of epithelial and mesenchymal lineages. In addition, the edg-2 polypeptide sequence is highly conserved in evolution and is expressed by lower organisms such as yeast and C. elegans, suggesting that it may be an important regulator of general nuclear function.
Amino Acid Sequence↗