Low expression of FGF1 (fibroblast growth factor-1) in rat parasympathetic preganglionic neurons

Fibroblast growth factor-1 (FGF1), a member of the FGF family of growth factors, is localized in cholinergic neurons where it has trophic activity. We recently reported that cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) contain little FGF1, raising the possibility that FGF1 is not localized to parasympathetic preganglionic cholinergic neurons. To clarify this issue, we investigated the co-localization of FGF1 with cholinergic neuron markers in the Edinger-Westphal nucleus (EWN), salivatory nucleus, DMNV, and sacral parasympathetic nucleus by double immunofluorescence using antibodies to FGF1 and choline acetyltransferase (ChAT). The neurons in the EWN were devoid of FGF1. In the salivatory nucleus, 13% of ChAT-positive neurons were also positive for FGF1. In the DMNV, only 8% of ChAT-positive neurons contained FGF1, and in the sacral parasympathetic nucleus, 18% of ChAT-positive neurons were FGF1-positive. We also confirmed that a large number of ChAT-positive motor neurons in the oculomotor nucleus, facial nucleus, hypoglossal nucleus, and spinal motor neurons contained FGF1. The results confirmed that parasympathetic preganglionic neurons are largely devoid of FGF1, which is a unique feature among cholinergic neurons.     

Localization of basic fibroblast growth factor in a subpopulation of rat sensory neurons

Summary The distribution of basic fibroblast growth factor (bFGF)-immunoreactivity (IR) was studied in rat sensory and autonomic ganglia. In postnatal and adult sympathetic superior cervical ganglia and in adult parasympathetic otic ganglia no bFGF-staining was found. Postnatal and adult neural crest-and placode-derived sensory ganglia displayed intensive bFGF-IR in a neuronal subpopulation. This subpopulation was characterized by use of consecutive sections of adult dorsal root ganglia stained with antibodies against substance P, somatostatin, bombesin, and bFGF. Basic FGF was colocalized with the somatostatin/bombesin subpopulation but not with substance P.     

Localization of neurons innervating the columellar muscles of the snail

The topographic arrangement of large and small neurons participating in the mechanism of the defensive reflex was studied in the circumesophageal nerve ring ofHelix pomatia by a modified retrograde cobalt ion transport method. Comparison of the results with those of previous electrophysiological investigations of the mechanism of the defensive relfex leads to the conclusion that this reflex is effected by a system of neurons consisting of nine large and 60–80 small nerve cells.Research Institute of Neurocybernetics, State University, Rostov-on-Don. Translated from Neirofiziologiya, Vol. 12, No. 6, pp. 637–641, November–December, 1980.     

Regulation of rat proximal tubule epithelial cell growth by fibroblast growth factors, insulin-like growth factor-1 and transforming growth factor-beta, and analysis of fibroblast growth factors in rat kidney.

Growth factors may play an important role in regulating the growth of the proximal tubule epithelium. To determine which growth factors could be involved, we have investigated the mitogenicity of various purified factors in rat kidney proximal tubule epithelial (RPTE) cells cultured in defined medium. Fibroblast growth factors, aFGF (acidic FGF) and bFGF (basic FGF), stimulate DNA synthesis in a dose-dependent manner, with ED50 values of 4.5 and 3.2 ng/ml, respectively; their effects are not additive. With cholera toxin in the medium, both aFGF and bFGF can replace insulin or epidermal growth factor (EGF) to attain the maximum level of cell growth, but they cannot replace cholera toxin. Cholera toxin specifically potentiates the effects of FGFs on DNA synthesis. At high cell density, both insulin and insulin-like growth factor 1 (IGF-1) induce DNA synthesis more effectively than EGF, FGFs and cholera toxin. The high concentration (0.2-1.0 microgram/ml) of insulin required for cell growth can be replaced by a low concentration of IGF-1 (10-20 ng/ml), indicating that insulin probably acts through a low affinity interaction with the IGF-1 receptor. Transforming growth factor-beta 1 (TGF-beta 1) inhibits DNA synthesis induced by individual factors and combinations of factors in a concentration-dependent manner. Northern blot analysis shows that mRNA for TGF-beta 1, IGF-1, and aFGF, but not bFGF are present in rat kidney. Western blot analysis and bioassay data confirmed that the majority of FGF-like protein in rat kidney is aFGF. The data suggest that in addition to EGF, IGFs, and TGF-beta, FGFs may also be important kidney-derived regulators of proximal tubule epithelial cell growth in vivo and in vitro.     

Dendrimer-based tumor cell targeting of fibroblast growth factor-1

Fibroblast Growth Factor Receptor (FGFR) is overexpressed in a wide variety of tumors, and therefore is an attractive target for drug delivery. Recombinant FGF-1 was purified and attached to a fifth-generation (G5) polyamidoamine dendrimer. The specific binding and internalization of this conjugate labeled with FITC was demonstrated by flow cytometry as well as by confocal microscopic analysis in cell lines expressing FGFR. The binding and uptake of FGF-conjugated dendrimers was completely blocked by excess nonconjugated FGF-1. Confocal microscopic analysis showed cytosolic as well as nuclear localization. Multivalent G5-FGF nanoparticles may serve as a platform for cytosolic as well as nuclear drug delivery in tumor cells, and as an FGF delivery agent for angiogenesis and wound healing. Our study shows for the first time the applicability of a dendrimer nanodevice for tumor cell targeting through FGFR.     

Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1

Fibroblast growth factor-1 (FGF-1), which stimulates cell growth, differentiation, and migration, is capable of crossing cellular membranes to reach the cytosol and the nucleus in cells containing specific FGF receptors. The cell entry process can be monitored by phosphorylation of the translocated FGF-1. We present evidence that phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC)delta. The phosphorylated FGF-1 is subsequently exported to the cytosol. A mutant growth factor where serine at the phosphorylation site is exchanged with glutamic acid, to mimic phosphorylated FGF-1, is constitutively transported to the cytosol, whereas a mutant containing alanine at this site remains in the nucleus. The export can be blocked by leptomycin B, indicating active and receptor-mediated nuclear export of FGF-1. Thapsigargin, but not leptomycin B, prevents the appearance of active PKCdelta in the nucleus, and FGF-1 is in this case phosphorylated in the cytosol. Leptomycin B increases the amount of phosphorylated FGF-1 in the cells by preventing dephosphorylation of the growth factor, which seems to occur more rapidly in the cytoplasm than in the nucleus. The nucleocytoplasmic trafficking of the phosphorylated growth factor is likely to play a role in the activity of internalized FGF-1.     

Biosynthesis of human fibroblast growth factor-5.

We have analyzed the biosynthesis of human fibroblast growth factor-5 (FGF-5) at the translational and posttranslational levels. FGF-5 RNA synthesized in vitro can be translated in rabbit reticulocyte lysates to yield a 29,500-Da protein, which is consistent with the molecular weight predicted from the coding sequence. The efficiency of FGF-5 translation is dramatically enhanced if an upstream open reading frame (ORF-1) in the RNA is deleted or if both AUG codons in ORF-1 are destroyed by point mutations, while partial enhancement is achieved by individual mutation of either ORF-1 AUG codon. These data suggest that FGF-5 synthesis requires the scanning of ribosomes past the two ORF-1 AUG codons. The introduction of these ORF-1 mutations into a eukaryotic FGF-5 expression vector increases its capacity to transform mouse NIH 3T3 cells up to 50-fold upon transfection. FGF-5 is secreted from transfected 3T3 cells and from human tumor cells as glycoproteins containing heterogeneous amounts of sialic acid. Glycosidase treatments suggest that the growth factor bears both N-linked and O-linked sugars.     

Localization of cholinergic neurons in the cat lower brain stem

The localization of cholinergic neurons in the cat lower brain stem was determined immunocytochemically with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. ChAT-positive neurons were observed in four major cell groups: cranial nerve motor and special visceromotor neurons: parasympathetic preganglionic visceromotor neurons; neurons located in the ponto-mesencephalic tegmentum including area X (or pedunculopontine tegmental nucleus), nucleus laterodorsalis tegmenti (Ldt) of Castaldi, and peri-locus coeruleus alpha (peri-alpha); and neurons located in nucleus reticularis magnocellularis (Mc) and adjacent nucleus reticularis gigantocellularis (Gc) of the medulla.     

Stimulatory role of dopamine on fibroblast growth factor-2 expression in rat striatum

We have previously shown that systemic injection of (-)nicotine produces a selective up-regulation of fibroblast growth factor (FGF)-2 mRNA levels in rat striatum. Because (-)nicotine can increase striatal release of dopamine and glutamate, in the present study we have investigated the contribution of these neurotransmitters in the modulation of FGF-2 expression. We found that coinjection of dopaminergic D1 (SCH23390) or D2 (haloperidol) receptor antagonists prevents nicotine-induced elevation of FGF-2 expression. However, injection of the NMDA receptor antagonist MK-801 produced a significant increment of FGF-2 mRNA and protein levels in rat striatum similar to the effect produced by (-)nicotine alone. Interestingly this effect of MK-801 could also be prevented by D1 or D2 receptor antagonists, suggesting that an elevation of dopamine levels may be required for the regulation of the trophic molecule. Accordingly we found that the non-selective dopaminergic agonist apomorphine can similarly increase striatal FGF-2 mRNA levels. Despite the observation that both D1 and D2 receptors appear to contribute to the modulation of FGF-2 expression, only a direct activation of D2 receptors, through quinpirole administration, was able to mimic the effect of apomorphine. On the basis of FGF-2 neurotrophic activity, these results suggest that direct or indirect activation of dopaminergic system can be neuroprotective and might reduce cell vulnerability in degenerative disorders.     

Expression of the fibroblast growth factor-5 gene in the mouse embryo.

Fibroblast growth factors (FGFs) are structurally related mitogens that can regulate the differentiation of a wide variety of cells. As a step towards elucidating the developmental roles played by one of these factors, we have used in situ hybridization methods to examine expression of the murine F gf-5 gene during embryogenesis. F gf-5 RNA was detected at seven distinct sites in the developing mouse embryo: (1) postimplantation epiblast (embryonic day 5 1/4-7 1/2), (2) lateral splanchnic mesoderm (E9 1/2-10 1/2), (3) lateral somatic mesoderm (E10 1/2-12 1/2), (4) myotomes (E10 1/2-12 1/2), (5) mastication muscle (E11 1/2-14 1/2), (6) limb mesenchyme (E12 1/2-14 1/2), and (7) acoustic ganglion (E12 1/2-14 1/2). At several of these sites, expression is spatially restricted within the tissues. We offer several hypotheses regarding the roles of FGF-5 in murine development.     

Intracellular trafficking in neurones and glia of fibroblast growth factor-2, fibroblast growth factor receptor 1 and heparan sulphate proteoglycans in the injured adult rat cerebral cortex

The potent gliogenic and neurotrophic fibroblast growth factor (FGF)-2 signals through a receptor complex comprising high-affinity FGF receptor (FGFR)1 with heparan sulphate proteoglycans (HSPGs) as co-receptors. We examined the intracellular dynamics of FGF-2, FGFR1 and the HSPGs syndecan-2 and -3, glypican-1 and -2, and perlecan in neurones and glia in and around adult rat cerebral wounds. In the intact cerebral cortex, FGF-2 and FGFR1 mRNA and protein were constitutively expressed in astrocytes and neurones respectively. FGF-2 protein was localized exclusively to astrocyte nuclei. After injury, expression of FGF-2 mRNA was up-regulated only in astrocytes, whereas FGFR1 mRNA expression was increased in both glia and neurones, a disparity indicating that FGF-2 may act as a paracrine and autocrine factor for neurones and glia respectively. FGF-2 protein localized to both cytoplasm and nuclei of injury-responsive neurones and glia. There was weak or no staining of HSPGs in the normal cerebral neuropil and glia nuclei, with a few immunopositive neurones. Specific HSPGs responded to injury by differentially co-localizing with trafficked intracellular FGF-2 and FGFR1. The spatiotemporal dynamics of FGF-2-FGFR1-HSPG complex formation implies a role for individual HSPGs in regulating FGF-2 storage, nuclear trafficking and cell-specific injury responses in CNS wounds.     

Structure-function studies of heparin-binding (acidic fibroblast) growth factor-1 using site-directed mutagenesis.

The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF activities. In this report, we summarize evidence that indicates that the heparin-binding and mitogenic activities of HBGF-1 can be dissociated from the receptor-binding activities of the growth factor by site-directed mutagenesis of a single lysine residue. Thus, the mutant HBGF-1 has normal receptor-binding activity and is capable of stimulating tyrosine kinase activity and proto-oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto-oncogene expression from the mitogenic response is observed when the human wild-tupe HBGF-1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild-type or mutant HBGF-1. Production of wild-type induces a transformed phenotype, whereas over-production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site-directed mutagenesis of putative nuclear translocation sequences in the wild-type protein do not affect mitogenic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular trafficking of endogenous fibroblast growth factor-2

We have previously reported how the release of fibroblast growth factor-2 (FGF-2) is mediated by shed vesicles. In the present study, we address the question of how newly synthesized FGF-2 is targeted to the budding vesicles. Considering that in vitro cultured Sk-Hep1 hepatocarcinoma cells release FGF-2 and shed membrane vesicles only when cultured in the presence of serum, we added serum to starved cells and monitored intracellular movements of the growth factor. FGF-2 was targeted both to the cell periphery and to the nucleus and nucleolus. Movements toward the cell periphery were not influenced by drugs affecting microtubules, but were inhibited by cytocalasin B. Involvement of actin in FGF-2 trafficking toward the cell periphery was supported by coimmunoprecipitation and immune localization experiments. Colocalization of FGF-2 granules moving to the cell periphery and FM4-64-labelled intracellular lipids were not observed. Ouabain and methylamine, two inhibitors of FGF-2 release, were analyzed for their effects on FGF-2 intracellular localization and on vesicle shedding. Ouabain inhibited FGF-2 movements toward the cell periphery. The FGF-2 content of shed vesicles was therefore reduced. Methylamine inhibited vesicle shedding; in its presence, FGF-2 clustered at the cell periphery, but the rate of its release decreased. FGF-2 targeting to the nucleus and nucleolus was not affected by cytocalasin B, whereas it was inhibited by drugs that modify microtubule dynamics. Neither ouabain, nor methylamine interfered with FGF-2 translocation to the nucleus and nucleolus. FGF-2 targeting to the budding vesicles and to the nucleus and nucleolus is therefore mediated by fundamentally different mechanisms.     

Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis

The cytokine/extracellular matrix protein osteopontin (OPN/Eta-1) is an important component of cellular immunity and inflammation. It also acts as a survival, cell-adhesive, and chemotactic factor for endothelial cells. Here, subtractive suppression hybridization showed that serum-deprived murine aortic endothelial (MAE) cells transfected with the angiogenic fibroblast growth factor-2 (FGF2) overexpress OPN compared with parental cells. This was confirmed by Northern blotting and Western blot analysis of the conditioned media in different clones of endothelial cells overexpressing FGF2 and in endothelial cells treated with the recombinant growth factor. In vivo, FGF2 caused OPN expression in newly formed endothelium of the chick embryo chorioallantoic membrane (CAM) and of murine s.c. Matrigel plug implants. Recombinant OPN (rOPN), the fusion protein GST-OPN, and the deletion mutant GST-DeltaRGD-OPN were angiogenic in the CAM assay. Angiogenesis was also triggered by OPN-transfected MAE cells grafted onto the CAM. OPN-driven neovascularization was independent from endothelial alpha(v)beta(3) integrin engagement and was always paralleled by the appearance of a massive mononuclear cell infiltrate. Accordingly, rOPN, GST-OPN, GST-DeltaRGD-OPN, and the conditioned medium of OPN-overexpressing MAE cells were chemotactic for isolated human monocytes. Also, rOPN triggered a proangiogenic phenotype in human monocytes by inducing the expression of the angiogenic cytokines TNF-alpha and IL-8. OPN-mediated recruitment of proangiogenic monocytes may represent a mechanism of amplification of FGF2-induced neovascularization during inflammation, wound healing, and tumor growth.     

A nuclear export sequence located on a beta-strand in fibroblast growth factor-1

Receptor-bound and endocytosed fibroblast growth factor-1 (FGF-1) is able to cross the vesicle membrane and translocate to cytosol and nucleus. This suggests an intracellular role of FGF-1, which also signals by activating transmembrane FGF receptors. Phosphorylation of internalized FGF-1 by nuclear protein kinase C delta induces rapid export from the nuclei by a leptomycin B-sensitive pathway. In the present work, we have searched for and identified a Leu-rich nuclear export sequence (NES) at the C terminus of FGF-1 required for its nuclear export and able to confer nuclear export activity to a reporter protein in an in vivo system. Mutants where hydrophobic amino acids within the NES were exchanged for alanine exhibited reduced or abolished nuclear export. As demonstrated in co-immunoprecipitation experiments, a complex containing FGF-1, exportin-1, and its co-factor Ran-GTP, was formed in vitro. Formation of this complex in vivo was demonstrated by a peroxisomal targeting assay. Formation of the FGF-1-exportin-1-Ran-GTP complex in vitro as well as nuclear export of FGF-1 in vivo was dependent on phosphorylation of FGF-1, and it was abolished by leptomycin B. The FGF-1 NES was found to be situated along a beta-strand, which has not been reported before, since NESs usually are alpha-helical.     

Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2.

Fibroblast growth factor-4 (FGF4), like other FGFs, shares a high affinity for the anionic glycosaminoglycans heparin and heparan sulfate (HS), which in turn enhance FGF-receptor (FGFR) binding and activation. Here we demonstrate using a cell free system that, at low concentrations of heparin, FGF4 binds only to FGFR-2, while much higher heparin levels are required for binding to FGFR-1. Chemical crosslinking of radiolabeled FGF4 to the soluble FGF receptors confirms the preferential formation of FGF4-FGFR-2 complexes under restricted heparin availability, with maximal ligand-receptor interactions at almost 20-fold lower heparin concentrations then those required for the affinity labeling of FGFR-1. In accordance, HS-deficient cells expressing FGFR-2 proliferate in response to FGF4 at extremely low exogenous heparin concentrations, while FGFR-1 expressing cells are completely unresponsive under the same conditions. We suggest that FGFR-2 is the preferred receptor for FGF4 under restricted HS conditions and that the bioavailability of structurally distinct HS motifs may differentially control receptor specificity of FGF4 in vivo.     

Development of cholinergic neurons of the rat retina

Biochemical and electrophysiological features of cholinergic neurons from the developing rat retina were analyzed in an attempt to identify important periods in the maturation of these neurons. Acetylcholine synthesis was assayed in intact, isolated retinas; choline acetyltransferase activity was measured in homogenates. Spontaneous and evoked acetylcholine release and certain aspects of synapse formation were examined in a retina-muscle cell culture system. In this system, retinal cells from rats of different ages were dissociated with trypsin and added to previously cultured rat striated muscle cells which served as postsynaptic targets for cholinergic neurons. The results indicate that two developmental periods can be described. The first stage occurs during the final week of gestation well before morphological signs of synapses appear. During this early period, cholinergic neurons acquire the ability to synthesize and release acetylcholine and to form functional synapses. These developing neurons progressively lose their ability to form and to maintain synapses with an inappropriate target. A second stage of maturation begins at the end of the first postnatal week. In this phase, dramatic increases in acetylcholine synthesis, choline acetyltransferase activity, and high-affinity choline uptake are coupled temporally with the morphological differentiation of synapses.