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At least 937 records · Page 52Linked to original sources
PubMed2001

Density-dependent expression of FGF-2 in response to oxidative stress in RPE cells in vitro.

PURPOSE: The purpose of this study is to demonstrate the effect of culture density on the steady state mRNA levels of fibroblast growth factor-2 (FGF-2) when retinal pigment epithelial (RPE) cells are subjected to oxidative stress in vitro. METHODS: Subconfluent and confluent cultures of the established RPE cell line ARPE-19, were treated with increasing concentrations of tert-butyl hydroperoxide (tBH) or hydrogen peroxide (H(2)O(2)). Cell viability was measured using the WST-1 assay, and intracellular reactive oxygen intermediate (ROI) production was quantified by dichlorofluoroscein (DCF) fluorescence. Steady state changes in heme oxygenase-1 (HO-1) and FGF-2 mRNAs were measured by Northern blot analysis. RESULTS: Confluent cultures of ARPE-19 cells were less susceptible than subconfluent cultures to the toxic effects of the chemical oxidants. The intracellular reactive oxygen intermediate production was higher in subconfluent than confluent cultures with increasing tBH concentration. At nontoxic concentrations of tBH and H(2)O(2), a dose dependent increase in FGF-2 expression was seen as a function of culture density. FGF-2 mRNA expression was induced after tBH treatment in subconfluent, but not confluent cells. On the other hand, FGF-2 mRNA induction was observed after H(2)O( 2) treatment in confluent, but not subconfluent cultures. In contrast, no density dependent induction of HO-1 mRNA was seen after treatment with either tBH or H(2)O(2). CONCLUSIONS: These results suggest that care should be taken to control for cell density in similar types of in vitro experiments.

Blotting, Northern↗
PubMed1996

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↗
PubMed1998

Activation of the MAP kinase pathway by FGF-1 correlates with cell proliferation induction while activation of the Src pathway correlates with migration.

FGF regulates both cell migration and proliferation by receptor-dependent induction of immediate-early gene expression and tyrosine phosphorylation of intracellular polypeptides. Because little is known about the disparate nature of intracellular signaling pathways, which are able to discriminate between cell migration and proliferation, we used a washout strategy to examine the relationship between immediate-early gene expression and tyrosine phosphorylation with respect to the potential of cells either to migrate or to initiate DNA synthesis in response to FGF-1. We demonstrate that transient exposure to FGF-1 results in a significant decrease in Fos transcript expression and a decrease in tyrosine phosphorylation of the FGFR-1, p42(mapk), and p44(mapk). Consistent with these biochemical effects, we demonstrate that attenuation in the level of DNA synthesis such that a 1.5-h withdrawal is sufficient to return the population to a state similar to quiescence. In contrast, the level of Myc mRNA, the activity of Src, the tyrosine phosphorylation of cortactin, and the FGF-1-induced redistribution of cortactin and F-actin were unaffected by transient FGF-1 stimulation. These biochemical responses are consistent with an implied uncompromised migratory potential of the cells in response to growth factor withdrawal. These results suggest a correlation between Fos expression and the mitogen-activated protein kinase pathway with initiation of DNA synthesis and a correlation between high levels of Myc mRNA and Src kinase activity with the regulation of cell migration.

3T3 Cells↗
PubMed2003

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation.

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.

Animals↗
PubMed1998

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain.

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation.

3T3 Cells↗
PubMed2002

Vesicle transmembrane potential is required for translocation to the cytosol of externally added FGF-1.

Externally added fibroblast growth factor-1 (FGF-1) is capable of crossing cellular membranes to reach the cytosol and the nucleus in a number of cell types. We have monitored the translocation of the growth factor by two methods: phosphorylation of FGF-1, and prenylation of an FGF-1 mutant that contains a C-terminal prenylation signal. Inhibition of endosomal acidification by ammonium chloride or monensin did not block the translocation of FGF-1, whereas bafilomycin A1, a specific inhibitor of vacuolar proton pumps, blocked translocation completely. A combination of ionophores expected to dissipate the vesicular membrane potential (valinomycin plus monensin) also fully inhibited the translocation. The inhibition of translocation by bafilomycin A1 was overcome in the presence of monensin or nigericin, while ouabain blocked translocation under these conditions. The data indicate that translocation of FGF-1 to cytosol occurs from the lumen of intracellular vesicles possessing vacuolar proton pumps, and that a vesicular membrane potential is required. Apparently, activation of vesicular Na+/K+-ATPase by monensin or nigericin generates a membrane potential that can support translocation when the proton pump is blocked.

3T3 Cells↗
PubMed1998

Apert syndrome mutations in fibroblast growth factor receptor 2 exhibit increased affinity for FGF ligand.

Dominantly acting mutations of the fibroblast growth factor (FGF) receptor 2 (FGFR2) gene have been implicated in various craniosynostosis syndromes. Apert syndrome, characterized in addition by syndactyly of the limbs, involves specific mutations at two adjacent residues, Ser252Trp and Pro253Arg, predicted to lie in the linker region between IgII and IgIII of the FGFR2 ligand-binding domain. We have analysed the interaction of FGF ligands with wild-type and Apert-type mutant FGFR2 ectodomains in solution. Wild-type and Apert-type receptors form a complex with FGF ligands with a stoichiometry of 2:2 (ligand:receptor). The kinetics and specificity of ligand binding to wild-type and Apert mutant receptors have been analysed using surface plasmon resonance techniques. This reveals that Apert mutations, compared with wild-type, exhibit a selective decrease in the dissociation kinetics of FGF2, but not of other FGF ligands examined. In contrast, the substitution Ser252Leu in FGFR2, previously observed in several asymptomatic individuals, exhibited wild-type kinetics. These findings indicate that Apert syndrome arises as a result of increased affinity of mutant receptors for specific FGF ligands which leads to activation of signalling under conditions where availability of ligand is limiting.

Acrocephalosyndactylia↗
PubMed1998

FGF-2 stimulates migration of Kaposi's sarcoma-like vascular cells by HGF-dependent relocalization of the urokinase receptor.

The spindle-shaped cell line TTB was recently isolated from highly vascularized skin lesions of BKV/HIV-1 tat transgenic mice and shown to possess an autocrine loop for hepatocyte growth factor (HGF). We show that fibroblast growth factor-2 (FGF-2) stimulates TTB cell migration and promotes polarization of uPAR at the leading edge of migrating cells. FGF-stimulated TTB cells presented the typical migratory phenotype, with a triangular cell shape and concomitant breakdown of actin stress fibers and smooth muscle-specific actin isoform. FGF-2-stimulated migration was blocked by antibodies against urokinase-type plasminogen activator (uPA) or uPA receptor (uPAR) and by neutralizing anti-HGF antibodies. The latter also inhibited uPAR relocalization at the cell surface of FGF-2-treated TTB cells. This points to a crosstalk between FGF-2 and HGF that might mediate TTB cell migration by modulating the localization of cell surface uPAR.

Cell Line↗
PubMed2002

Expression of tenascin-C long isoforms is induced in the hypothalamus by FGF-1.

Fibroblast growth factor (FGF)-1 modulates various brain functions, such as the hypothalamic control of feeding. In the rat, we examined the effect of intracerebroventricularly infused FGF-1 on the hypothalamic expression of tenascin-C, a selective mediator of neuron-glial interaction. In situ hybridization revealed little tenascin-C mRNA expression in control brains, but greatly increased expression in ependymal cells around the third ventricle in the FGF-1-infused rats. Reverse transcription-linked PCR analysis of hypothalamic mRNA revealed an FGF-1-induced expression not of the shortest isoform of tenascin-C, but of the long isoforms (with additional fibronectin type III-domain insertions). Quantitative analysis by real time PCR revealed that this induction was transient and dose-dependent. Specific modulation of hypothalamic neuron-glial interactions by tenascin-C may mediate FGF-1-induced feeding suppression.

Animals↗
PubMed2003

Increased serum and cerebrospinal fluid FGF-2 levels in amyotrophic lateral sclerosis.

Lack of growth factors and hypoxia are two recent hypotheses about mechanisms underlying motor neuron death in amyotrophic lateral sclerosis. With this background, serum from 15 patients with amyotrophic lateral sclerosis and 15 controls, and CSF from 15 patients and 10 controls were analysed for fibroblast growth factor 2 (FGF-2) using an immunoassay. Serum FGF-2 levels were higher in the patient group than in the control group. FGF-2 was detected in CSF in 11/15 patients, but in none of the 10 control subjects. There were no correlations between age, duration of disease or clinical rating and FGF-2 levels. The findings indicate that FGF-2 is involved in the pathophysiological chain of events in this disorder.

Adult↗
PubMed2003

FGF-2 stimulation affects calvarial osteoblast biology: quantitative analysis of nine genes important for cranial suture biology by real-time reverse transcription polymerase chain reaction.

Appropriately timed closure of the cranial sutures is a critical factor in normal postnatal morphogenesis of the cranial vault. Suture patency is necessary to permit rapid neonatal expansion of the cerebral hemispheres, and later ossification is important for bony protection of the cerebrum. Premature suture ossification (craniosynostosis) leads to myriad adverse functional and developmental consequences. Several murine studies have implicated dura-derived fibroblast growth factor-2 (FGF-2) paracrine signaling as a critical factor promoting physiologic posterior frontal suture fusion. In this study, the authors used real-time reverse transcription polymerase chain reaction (RT-PCR) to study an in vitro system that models the in vivo stimulation of suture calvarial osteoblasts by dura-derived FGF-2. The authors advocate real-time RT-PCR as a powerful and rapid technique that offers advantages in the highly sensitive, specific, and reproducible analyses of nine genes known to be important in cranial suture biology. The genes studied were growth factors [FGF-2, transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 3], growth factor receptors (FGF-R1, FGF-R2, TGF-beta RI, and TGF-beta RII), and a marker of osteoblast differentiation (Co1-I alpha I). These analyses provide a "snapshot" of several important genes involved in suture fusion that is more inclusive and quantitative than that which has been previously reported.

Animals↗
PubMed2005

Differentiation-dependent up-regulation of BMP-2, TGF-beta1, and VEGF expression by FGF-2 in human bone marrow stromal cells.

BACKGROUND: Bone tissue formation by bone marrow stromal cells may be supported and enhanced by multiple growth factors, particularly in cases of a compromised local microenvironment. In this study, the authors hypothesized that fibroblast growth factor (FGF)-2 can stimulate the production by human bone marrow stromal cells of osteogenic [i.e., bone morphogenetic protein (BMP)-2 and transforming growth factor (TGF)-beta1] and angiogenic [i.e., vascular endothelial growth factor (VEGF)] factors. METHODS: Human bone marrow stromal cells from six donors were expanded for two passages (expansion phase) and subsequently cultivated in osteogenic medium containing ascorbic acid, beta-glycerophosphate, and dexamethasone (differentiation phase). After each phase, cells were transferred into serum-free medium with or without FGF-2 at different concentrations and for different times, and the expression of BMP-2, TGF-beta1, and VEGF was quantified at the mRNA level by real-time quantitative reverse-transcriptase polymerase chain reaction. The amounts of TGF-beta1 and VEGF released in the culture medium were assessed using enzyme-linked immunosorbent assay kits and normalized to the DNA content. RESULTS: In response to 5 ng/ml FGF-2 for 24 hours, the mRNA expression of VEGF increased at both culture phases (up to 6.1 fold), whereas that of BMP-2 and TGF-beta1 significantly increased only after the expansion (3.1-fold) or differentiation phase (2.1-fold), respectively. Similar trends were observed in the amounts of proteins measured in the culture medium. CONCLUSIONS: The authors' results indicate that FGF-2 up-regulates the expression of BMP-2, TGF-beta1, and VEGF in human bone marrow stromal cells, in a pattern dependent on the cell-differentiation stage. These findings prompt for in vivo investigations on the delivery of FGF-2 for the temporally/functionally regulated enhancement of bone marrow stromal cell-based bone induction.

Bone Marrow Cells↗
PubMed1993

Structural and functional specificity of FGF receptors.

Fibroblast growth factors (FGFs) represent a group of polypeptide mitogens eliciting a wide variety of responses depending on the target cell type. The knowledge of the cell surface receptors mediating the effects of FGFs has recently expanded remarkably. Perhaps not surprisingly, the complexity of the FGF family and FGF induced responses is reflected in the diversity and redundancy of the FGF receptors. The molecular cloning of the signal transducing receptors for fibroblast growth factors has revealed a tyrosine kinase gene family with at least four members. Differential splicing and polyadenylation creates further diversity in the FGF receptor system. These numerous receptor forms have both distinct and redundant properties. We are only now beginning to understand how the different receptors are activated by the various FGFs and how they are expressed by various cells and tissues. FGF binding to the tyrosine kinase receptors needs the assistance of heparan sulphate side chains of proteoglycans present at the cell surface and in the extracellular matrix. As several other growth factors share the heparin binding property of FGFs, the dual receptor system for FGFs might be an example of a more widely used principle.

Animals↗
PubMed2005

Zebrafish hairy/enhancer of split protein links FGF signaling to cyclic gene expression in the periodic segmentation of somites.

Notch and fibroblast growth factor (FGF) signaling pathways have been implicated in the establishment of proper periodicity of vertebrate somites. Here, we show evidence that a Hes6-related hairy/Enhancer of split-related gene, her13.2, links FGF signaling to the Notch-regulated oscillation machinery in zebrafish. Expression of her13.2 is induced by FGF-soaked beads and decreased by an FGF signaling inhibitor. her13.2 is required for periodic repression of the Notch-regulated genes her1 and her7, and for proper somite segmentation. Furthermore, Her13.2 augments autorepression of her1 in association with Her1 protein. Therefore, FGF signaling appears to maintain the oscillation machinery by supplying a binding partner, Her13.2, for Her1.

Animals↗
PubMed2001

Xenopus Sprouty2 inhibits FGF-mediated gastrulation movements but does not affect mesoderm induction and patterning.

Signal transduction through the FGF receptor is essential for the specification of the vertebrate body plan. Blocking the FGF pathway in early Xenopus embryos inhibits mesoderm induction and results in truncation of the anterior-posterior axis. The Drosophila gene sprouty encodes an antagonist of FGF signaling, which is transcriptionally induced by the pathway, but whose molecular functions are poorly characterized. We have cloned Xenopus sprouty2 and show that it is expressed in a similar pattern to known FGFs and is dependent on the FGF/Ras/MAPK pathway for its expression. Overexpression of Xsprouty2 in both embryos and explant assays results in the inhibition of the cell movements of convergent extension. Although blocking FGF/Ras/MAPK signaling leads to an inhibition of mesodermal gene expression, these markers are unaffected by Xsprouty2, indicating that mesoderm induction and patterning occurs normally in these embryos. Finally, using Xenopus oocytes we show that Xsprouty2 is an intracellular antagonist of FGF-dependent calcium signaling. These results provide evidence for at least two distinct FGF-dependent signal transduction pathways: a Sprouty-insensitive Ras/MAPK pathway required for the transcription of most mesodermal genes, and a Sprouty-sensitive pathway required for coordination of cellular morphogenesis.

Amino Acid Sequence↗
PubMed2005

Redesigning symmetry-related "mini-core" regions of FGF-1 to increase primary structure symmetry: thermodynamic and functional consequences of structural symmetry.

Previous reports detailing mutational effects within the hydrophobic core of human acidic fibroblast growth factor (FGF-1) have shown that a symmetric primary structure constraint is compatible with a stably folded protein. In the present report, we investigate symmetrically related pairs of buried hydrophobic residues in FGF-1 (termed "mini-cores") that are not part of the central core. The effect upon the stability and function of FGF-1 mutations designed to increase primary structure symmetry within these "mini-core" regions was evaluated. At symmetry-related positions 22, 64, and 108, the wild-type protein contains either Tyr or Phe side chains. The results show that either residue can be readily accommodated at these positions. At symmetry-related positions 42, 83, and 130, the wild-type protein contains either Cys or Ile side chains. While positions 42 and 130 can readily accommodate either Cys or Ile side chains, position 83 is substantially destabilized by substitution by Ile. Tertiary structure asymmetry in the vicinity of position 83 appears responsible for the inability to accommodate an Ile side chain at this position, and is known to contribute to functional half-life. A mutant form of FGF-1 with enforced primary structure symmetry at positions 22, 64, and 108 (all Tyr) and 42, 83, and 130 (all Cys) is shown to be more stable than the reference FGF-1 protein. The results support the hypothesis that a symmetric primary structure within a symmetric protein superfold represents a solution to achieving a foldable, stable polypeptide, and highlight the role that function may play in the evolution of asymmetry within symmetric superfolds.

3T3 Cells↗
PubMed2004

Screening of FGF target genes in Xenopus by microarray: temporal dissection of the signalling pathway using a chemical inhibitor.

Microarray is a powerful tool for analysing gene expression patterns in genome-wide view and has greatly contributed to our understanding of spatiotemporal embryonic development at the molecular level. Members of FGF (fibroblast growth factor) family play important roles in embryogenesis, e.g. in organogenesis, proliferation, differentiation, cell migration, angiogenesis, and wound healing. To dissect spatiotemporally the versatile roles of FGF during embryogenesis, we profiled gene expression in Xenopus embryo explants treated with SU5402, a chemical inhibitor specific to FGF receptor 1 (FGFR1), by microarray. We identified 38 genes that were down-regulated and 5 that were up-regulated in response to SU5402 treatment from stage 10.5-11.5 and confirmed their FGF-dependent transcription with RT-PCR analysis and whole-mount in situ hybridization (WISH). Among the 43 genes, we identified 26 as encoding novel proteins and investigated their spatial expression pattern by WISH. Genes whose expression patterns were similar to FGFR1 were further analysed to test whether any of them represented functional FGF target molecules. Here, we report two interesting genes: one is a component of the canonical Ras-MAPK pathway, similar to mammalian mig6 (mitogen-inducible gene 6) acting in muscle differentiation; the other, similar to GPCR4 (G-protein coupled receptor 4), is a promising candidate for a gastrulation movement regulator. These results demonstrate that our approach is a promising strategy for scanning the genes that are essential for the regulation of a diverse array of developmental processes.

Animals↗
PubMed2006

FGF-2 deficiency does not alter vulnerability of the dopaminergic nigrostriatal system towards MPTP intoxication in mice.

Fibroblast growth factor 2 (FGF-2) was the first growth factor discovered that exerted prominent protective and regenerative effects in an animal model of Parkinson's disease, the MPTP-lesioned dopaminergic nigrostriatal system. To address the putative physiological relevance of endogenous FGF-2 for midbrain dopaminergic neurons, we have analysed densities of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and TH-positive fibers in the striatum and amygdala of adult FGF-2-deficient mice. We found that densities of TH-immunoreactive (ir) cells in the SN as well as densities of TH-ir fibers in the striatum and amygdala were unaltered as compared with wild-type littermates. There is evidence to suggest that growth factor deficits do not become apparent unless a system is challenged in a lesioning paradigm. We therefore tested the ability of the nigrostriatal system with respect to its ability to cope with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) intoxication. Treatment with 20 mg/kg MPTP on three consecutive days reduced dopamine levels in the striatum by about 80%. Densities of TH-positive neurons in the SN were reduced by 71%. However, both parameters did not significantly differ between FGF-2(-/-) mice and wild-type littermates. Our results therefore suggest that FGF-2, despite its prominent pharmacological potency as a neurotrophic factor for the dopaminergic nigrostriatal system, is not crucial for maintaining its structural integrity and ability to cope with MPTP intoxication.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗