Agonists of fibroblast growth factor receptor induce neurite outgrowth and survival of cerebellar granule neurons

Fibroblast growth factor receptor (FGFR) signaling is pivotal in the regulation of neurogenesis, neuronal differentiation and survival, and synaptic plasticity both during development and in adulthood. In order to develop low molecular weight agonists of FGFR, seven peptides, termed hexafins, corresponding to the β6‐β7 loop region of the FGF 1, 2, 3, 8, 9, 10, and 17, were synthesized. This region shares a homologous amino acid sequence with the FG‐loop region of the second fibronectin Type III module of the neural cell adhesion molecule (NCAM) that binds to the FGFR. Hexafins were shown by surface plasmon resonance to bind to FGFR1‐IIIc‐Ig2‐3 and FGFR2‐IIIb‐Ig2‐3. The heparin analog sucrose octasulfate inhibited hexafin binding to FGFR1‐IIIc‐Ig2‐3 indicating overlapping binding sites. Hexafin‐binding to FGFR1‐IIIc resulted in receptor phosphorylation, but inhibited FGF1‐induced FGFR1 phosphorylation, indicating that hexafins act as partial agonists. Hexafin2, 3, 8, 10, and 17 (but not 1 or 9) induced neurite outgrowth from cerebellar granule neurons (CGNs), an effect that was abolished by two inhibitors of FGFR, SU5402 and inositol hexaphosphate (IP6) and a diacylglycerol lipase inhibitor, RHC‐80267. The neuritogenic effects of selected hexafins could also be inhibited by FGF1 which by itself did not induce neurite outgrowth. Moreover, hexafin1, 3, 9, 10, and 17 (but not 2 or 8) promoted survival of CGNs induced to undergo apoptosis. Thus, selected hexafins induced neuronal differentiation and survival, making them promising pharmacological tools for the study of functional FGFR regulation in development of the nervous system. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Soluble N-cadherin stimulates fibroblast growth factor receptor dependent neurite outgrowth and N-cadherin and the fibroblast growth factor receptor co-cluster in cells

A chimeric molecule consisting of the extracellular domain of the adhesion molecule, N-cadherin, fused to the Fc region of human IgG (NCAD-Fc) supports calcium-dependent cell adhesion and promotes neurite outgrowth following affinity-capture to a tissue culture substrate. When presented to cerebellar neurons as a soluble molecule, the NCAD-Fc stimulated neurite outgrowth in a manner equivalent to that seen for N-cadherin expressed as a cell surface glycoprotein. Neurons expressing a dominant-negative version of the fibroblast growth factor (FGF) receptor did not respond to soluble NCAD-Fc. In cells transfected with full-length N-cadherin and the FGF receptor, antibody-clustering of N-cadherin resulted in a co-clustering of the FGF receptor to discrete patches in the cell membrane. The data demonstrate that the ability of N-cadherin to stimulate neurite outgrowth can be dissociated from its ability to function as a substrate associated adhesion molecule. The N-cadherin and the FGF receptor co-clustering in cells provides a basis for the neurite outgrowth response stimulated by N-cadherin being dependent on FGF receptor function.     

In vivo neurogenesis is inhibited by neutralizing antibodies to basic fibroblast growth factor

While extracellular growth factors govern neuronal precursor mitosis in culture, little is known about their roles in regulating neurogenesis in vivo. Previously, we reported that subcutaneously administered basic fibroblast growth factor (bFGF) promoted neuroblast proliferation in P1 rat brain, in regions in which bFGF and FGF receptors are expressed during development. To define the role of endogenous bFGF in neurogenesis, we employed a neutralizing monoclonal antibody to the factor. In culture, bFGF-induced granule precursor proliferation was progressively inhibited by increasing concentrations of antibody. In contrast, heat-inactivated or nonneutralizing anti-bFGF antibodies were ineffective. The inhibition was specific for bFGF, since EGF-induced [³H]dT incorporation was not altered. To study effects in vivo, neutralizing antibody was administered to newborn rats via the cisterna magnum. Four hours after injection, DNA synthesis in cerebellum and hippocampus was decreased by 53% and 63%, respectively, suggesting that endogenous bFGF was involved in brain development. To define effects on neurogenesis specifically, granule cell precursors were isolated after antibody treatment. [³H]dT incorporation in granule precursors was decreased by 50%, indicating that the neutralizing antibody inhibited neuroblast proliferation in vivo. In contrast, no reduction was observed using nonneutralizing or the heat-inactivated antibodies. The inhibition of precursor proliferation following immunoneutralization of bFGF in vivo suggests that the endogenous factor normally regulates brain neurogenesis. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 289–296, 1997     

Inhibition of cell migration by 24-kDa fibroblast growth factor-2 is dependent upon the estrogen receptor

The single-copy gene for fibroblast growth factor-2 (FGF-2) encodes for multiple forms of the protein with molecular masses of 24, 22.5, 22, and 18 kDa. We reported previously that the 24-22-kDa FGF-2 forms inhibit the migration of endothelial and MCF-7 cells by 50% and 70%, respectively. Here we show that this inhibition of migration is mediated by the estrogen receptor (ER). We have found that depletion of the receptor in either cell line abrogates the inhibitory activity of 24-kDa FGF-2 while re-introduction of the ER into deficient cells once again promotes the inhibitory response. To determine whether exposure to 24-kDa FGF-2 resulted in the activation of the estrogen receptor, 3T3 cells were cotransfected with estrogen receptor cDNA and an estrogen regulatory element-luciferase gene reporter construct and treated with 24- and 18-kDa FGF-2. The high molecular weight form stimulated luciferase activity 5-fold while 18-kDa FGF-2 at the same concentration had no effect. Treatment of ER-positive MCF-7 cells transfected with the reporter construct only showed the same results. Inclusion of the pure estrogen antagonist ICI 182,780 blocked the increase in luciferase activity by 24-kDa FGF-2, further indicating that the response was estrogen receptor dependent. Expression of dominant negative FGF receptor 1 inhibited ER activation, indicating that this was the cell surface receptor mediating the effect. Although growth factor-dependent activation of the ER was reported to require mitogen-activated protein kinase-induced phosphorylation at Ser(118) in COS and HeLa cells, this mechanism is not involved with the activation by 24-kDa FGF-2. These results suggest that the addition of 55 amino acids to the amino-terminal end of 18-kDa FGF-2 by alternative translation alters FGF-2 function and allows for the activation of a second signaling pathway involving the estrogen receptor.     

Synergistic activity of fibronectin and fibroblast growth factor receptors on neuronal adhesion and neurite extension through extracellular signal-regulated kinase pathway

Fibroblast growth factor (FGF) is an important modulator of cell growth and differentiation of various cells including neuron. Cells need to adhere specifically to cellular and extracellular components of their environment to carry out diverse physiological functions. Here, we examined whether fibronectin (FN) and FGF can cooperate for neuronal adhesion and neurite outgrowth. Using recombinant FN peptide (FNIII9-10), we found that FNIII9-10-mediated adhesion promotes the effect of FGF1 on neurite outgrowth of PC12 cells, while FGF1 enhances the FNIII9-10-mediated neuronal adhesion of PC12 cells. This collaboration of FNIII9-10 and FGF1 was the result of the sustained activation of extracellular signal-regulated kinase (ERK)-type MAP kinase. Finally, the synergistic activity of FGF1 and FN was inhibited by PD98059, an MEK inhibitor. Taken together, these findings indicate that FN-mediated signaling can collaborate with FGFRs signaling for neurite outgrowth through selective activation of ERK-type MAP kinase in PC12 cells, and suggest that FN and FGF act in concert to regulate cell differentiation in the nervous system.     

Autocrine growth stimulation by secreted Kaposi fibroblast growth factor but not by endogenous basic fibroblast growth factor

We studied the different potentials of a secreted and a nonsecreted member of the fibroblast growth factor (FGF) family to induce autocrine growth stimulation in human adrenal cortex carcinoma cells (SW-13). These epithelial cells express basic FGF (bFGF) cell surface receptors, and picomolar concentrations of bFGF suffice to induce anchorage-independent growth. The requirement for exogenously added bFGF contrasts with the intracellular storage of biologically active bFGF in SW-13 cells greater than 10,000-fold in excess of the concentration needed to stimulate anchorage independent growth. To study whether the expression of a secreted FGF would alter the growth phenotype of these cells, we transfected them with an expression vector coding for the Kaposi-fgf (K-fgf) oncogene. In contrast to controls, K-fgf-transfected cells secrete significant amounts of biologically active K-fgf protein into the growth media, show up to 50-fold increased colony formation in soft agar, and grow into rapidly progressing, highly vascularized tumors in athymic nude mice. A reversible inhibition of the autocrine growth stimulation in vitro is brought about by the polyanionic compound suramin. We conclude that FGF has to be released from SW-13 cells to function fully as a growth stimulator in vitro and in vivo.     

BMP inhibits neurite growth by a mechanism dependent on LIM-kinase

Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor-beta superfamily. BMPs regulate several crucial aspects of embryonic development and organogenesis. Here, we demonstrate that BMP-2 inhibits the neurite outgrowth of postnatal cerebellar neurons in vitro. Although receptor-regulated Smad proteins are activated by BMP-2, this signal transduction is not necessary for the inhibitory effect of BMP-2. Interestingly, BMP-2 activates LIM-kinase 1 in the neurons, and the dominant negative form of LIM-kinase 1 abolishes the effect of BMP-2. Thus, BMP-2 inhibits neurite outgrowth by a LIM-kinase 1-dependent mechanism, and our findings add a new member to the group of neurite growth inhibitors.     

Focal delivery of fibroblast growth factor-1 by transfected cells induces spiral ganglion neurite targeting in vitro

Sensory cells in the cochlea of the rat transiently express acidic fibroblast growth factor (FGF-1) during the developmental period of terminal innervation in the sensory epithelium. To explore the potential role of FGF-1 in terminal innervation events, the response of cochlear ganglion neurons to FGF-1 was evaluated in culture. Explants from the spiral ganglion of postnatal day 5 rats were cultured in the presence of exogenous FGF-1, with or without heparin. FGF-1 in the culture medium produced a dose-dependent increase in the number and length of neurites produced by spiral ganglion neurons, a response that was enhanced by heparin. To assess the effects of FGF-1 produced by a focal, cellular source, additional explants were cocultured with 3T3 cell transfectants that secrete FGF-1. Neurites that came into contact with FGF-1 secreting cells branched, formed bouton-like terminal swellings on the surface of the transfectants, and stopped extending. The results suggest that FGF-1 may stimulate neurite extension into the sensory epithelium of the cochlea and that focal production of FGF-1 may contribute to the formation of contacts on sensory cells by developing neurites. J. Cell. Physiol. 177:123–129, 1998. © 1998 Wiley-Liss, Inc.     

In vitro evolution of cisplatin/DNA monoadducts into diadducts is dependent upon superhelical density.

DNA binding of antitumor platinum(II) compounds accounts for cellular toxicity. Binding of cis-dichlorodiammineplatinum(II) (cis-DDP) to DNA involves the transient presence of monoadducts which evolve in a second phase into difunctional lesions which are far more toxic than the monoadducts. Temporal control of the monoadducts half-live is at least dependent upon the chemical nature of the cis-platinum derivative and the secondary structure of DNA. The effect of the degree of DNA superhelicity on the binding of cis-platinum derivatives as well as on the evolution of monofunctional adducts has been addressed on plasmid DNA. The rate of platination was not affected by the degree of DNA superhelicity. Similarly, when the evolution of the lesions was complete, no variation of toxicity was found with different populations of topoisomers, as determined by bacterial transformation efficiency. In contrast, when the kinetic of difunctional lesions formation was controlled in vitro, we observed a higher rate of formation on a supercoiled plasmid by comparison with a relaxed one. This result suggests that platinum-DNA adduct toxicity could be modulated by the topology of the chromosome.     

In vitro regulation of pig Sertoli cell growth and function: effects of fibroblast growth factor and somatomedin-C

The effects of insulin, somatomedin-C (Sm-C), epidermal growth factor (EGF), fibroblast growth factor (FGF), vitamin E, and retinoic acid on growth and function of immature cultured pig Sertoli cells were investigated. All these factors, except vitamin E, stimulated Sertoli cell DNA synthesis and proliferation. The mitogenic effects of insulin observed only at micromolar concentrations were similar to those induced by nanomolar concentrations of Sm-C or EGF, but significantly less than those induced by FGF. The effects of EGF and Sm-C were almost additive, whereas those of Sm-C and FGF were synergistic. After a 6-day treatment, FGF and retinoic acid induced a significant increase in the number of follicle-stimulating hormone (FSH) receptors per cell, and in FSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Sm-C, which alone had no effect on these two parameters, potentiated FGF action. Basal plasminogen activator activity was enhanced after the 6-day treatment with EGF plus insulin and, particularly, with FGF plus insulin. Similarly, the response of the latter group to FSH was significantly higher than in any other group of cells. FGF was also able to stimulate cell multiplication and enhanced the FSH receptor number of Sertoli cells isolated from 15- and 26-day-old rats. Thus, FGF is the most potent known mitogenic factor for cultured Sertoli cells, and it stimulates the phenotypic expression of these cells.     

Signaling via fibroblast growth factor receptor-1 is dependent on extracellular matrix in capillary endothelial cell differentiation

Differentiation of endothelial cells, i.e., formation of a vessel lumen, is a prerequisite for angiogenesis. The underlying molecular mechanisms are ill defined. We have studied a brain capillary endothelial cell line (IBEC) established from H-2Kb-tsA58 transgenic mice. These cells form hollow tubes in three-dimensional type I collagen gels in response to fibroblast growth factor-2 (FGF-2). Culture of IBEC on collagen gels in the presence of FGF-2 protected cells from apoptosis and allowed tube formation (i.e., differentiation) but not growth of the cells. FGF-induced differentiation, but not cell survival, was inhibited by treatment of the cells with an anti-beta1-integrin IgG. Changes in integrin expression in the collagen-gel cultures could not be detected. Rather, cell-matrix interactions critical for endothelial cell differentiation were created during the culture, as indicated by the gradual increase in tyrosine phosphorylation of focal adhesion kinase in the collagen-gel cultures. Inclusion of laminin in the collagen gels led to FGF-2-independent formation of tube structures, but cells were not protected from apoptosis. These data indicate that FGF receptor-1 signal transduction in this cell model results in cell survival. Through mechanisms dependent on cell-matrix interactions, possibly involving the alpha3beta1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells.     

Skeletal development is regulated by fibroblast growth factor receptor 1 signalling dynamics

Ligand-dependent signalling pathways have been characterised as having morphogen properties where there is a quantitative relationship between receptor activation and response, or threshold characteristics in which there is a binary switch in response at a fixed level of receptor activation. Here we report the use of a bacterial artificial chromosome (BAC)-based transgenic system in which a hypermorphic mutation has been introduced into the murine Fgfr1 gene. These mice exhibit cranial suture and sternal fusions that are exacerbated when the BAC copy number is increased. Surprisingly, increasing mutant BAC copy number also leads to the de novo appearance of digit I polydactyly in the hind limb and transformations of the vertebrae. Polydactyly is accompanied by a reduction of programmed cell death in the developing hind limb. Candidate gene analysis reveals downregulation of Dkk1 in the digit I field and upregulation of Wnt5a and Hoxd13. These findings show that Fgfr1-mediated developmental pathways exhibit differing signalling dynamics, whereby development of the cranial sutures and sternum follows a morphogen mode, whereas development of the vertebral column and the hind limbs has threshold signalling properties.     

Rac activation upon cell-cell contact formation is dependent on signaling from the epidermal growth factor receptor

Cadherins are transmembrane receptors that mediate cell-cell adhesion. They play an essential role in embryonic development and maintenance of tissue architecture. The Rho family small GTPases regulate actin cytoskeletal dynamics in different cell types. The function of two family members, Rho and Rac, is required for the stability of cadherins at cell-cell contacts. Consistent with the published data we have found that Rac is activated upon induction of intercellular adhesion in epithelial cells. This activation is dependent on functional cadherins (Nakagawa, M., Fukata, M., Yamaga, M., Itoh, N., and Kaibuchi, K. (2001) J. Cell Sci. 114, 1829-1838; Noren, N. K., Niessen, C. M., Gumbiner, B. M., and Burridge, K. (2001) J. Biol. Chem. 276, 3305-3308). Here we show for the first time that clustering of cadherins using antibody-coated beads is sufficient to promote Rac activation. In the presence of Latrunculin B, Rac can be partially activated by antibody-clustered cadherins. These results suggest that actin polymerization is not required for initial Rac activation. Contrary to what has been described before, phosphatidylinositol 3-kinases are not involved in Rac activation following cell-cell adhesion in keratinocytes. Interestingly, inhibition of epidermal growth factor receptor signaling efficiently blocks the increased Rac-GTP levels observed after contact formation. We conclude that cadherin-dependent adhesion can activate Rac via epidermal growth factor receptor signaling.     

Basic fibroblast growth factor is a beta-rich protein.

The conformation of the 153-residue form of human basic fibroblast growth factor (bFGF) was studied with circular dichroism (CD) and sequence prediction methods. The far-UV CD spectrum with a minimum at 202 nm resembled that of an unordered polypeptide/protein or a protein rich in distorted antiparallel -sheets. Analysis of the CD spectrum by the least-squares method of Changet al. (1978) and the CONTIN program of Provencher and Glöckner (1981) suggested that about one half of the molecule consisted of -sheet and there was no -helix. These estimates agreed with the prediction by the sequence method of Garnieret al. (1978) using decision constants based on CD results. bFGF had an unusual CD band at 187 nm, which disappeared upon ionization of Tyr side chains atpH 11.7. It also had another unusual property of irreversibly converting the CD spectrum to a helix-like one with a double minimum at 205 and 215 and a maximum at 189 nm upon heating the solution to above 55°C. The helicity was also enhanced in trifluoroethanol and in sodium dodecyl sulfate. The mutant bFGF in which cysteines 76 and 94 were replaced by serine residues had essentially the same properties as the wild-type.     

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury

To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8±1.5% and 96.5±3.5% of preischemic levels in control and bFGF-treated hearts (10 g/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 g bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease.     

Biotinylated basic fibroblast growth factor is biologically active.

Basic fibroblast growth factor (bFGF) was modified by biotinylation via amino group substitution, using biotin-N-hydroxysuccinimide ester at molar reaction ratios of 20, 200, and 2000 per bFGF molecule (respectively named bio-bFGF.20, bio-bFGF.200, and bio-bFGF.2000). The biotinylated bFGF derivatives, bio-bFGF.20 and bio-bFGF.200, conserved the same affinity for heparin as native bFGF, in contrast to bio-FGF.2000 which lost this property. Bio-bFGF.20 and bio-bFGF.200 were as effective as native bFGF in their capacity to compete with 125I-bFGF for binding to bFGF receptor on bovine brain membranes. The biological activity of these bFGF derivatives was tested on CCL39 cells; bio-bFGF.20 and bio-bFGF.200 were as able as native bFGF to promote growth of CCL39.     

Interaction of platelet factor 4 with fibroblast growth factor 2 is stabilised by heparan sulphate

The CXC chemokine platelet factor 4 (PF4) appears to inhibit tumour growth through its modulation of the activity of angiogenic growth factors. We investigated the heparan sulphate-dependent mechanism of PF4 inhibition of fibroblast growth factor 2 (FGF-2). The ability of PF4 to bind simultaneously to both FGF-2 and HS was assessed using affinity gel chromatography. Thirty-three to forty-two percent more HS bound to the FGF-2 affinity gel in the presence of PF4 than with HS alone. Protection assays showed that PF4 and FGF-2 bound to adjacent or overlapping sites together covering a 12 kDa stretch of HS. This study suggests that the three components may form a ternary complex. PF4 released at sites of angiogenesis may bind to angiogenic growth factors attached to endothelial cell surface HS to disrupt or prevent them from interacting with their signalling receptors. Manipulation of this mechanism may prove useful for clinical intervention of angiogenesis.     

ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation

Homophilic binding in trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen-activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)-mediated neurite outgrowth in the PC12-E2 cell line. We found that both FGFR substrate-2 and Grb2 play important roles in NCAM as well as in FGF2-stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/Fyn signaling pathway.