Acidic fibroblast growth factor (FGF) potentiates glial-mediated neurotoxicity by activating FGFR2 IIIb protein

Previous studies indicate that astrocytes are the brain cells that express acidic fibroblast growth factor (aFGF) and that the expression is increased upon activation. However, there has been no study investigating the significance of this phenomenon. Here we report that aFGF treatment of IFNγ-stimulated human astrocytes, and LPS/IFNγ-stimulated human microglia, enhances their secretion of inflammatory cytokines and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of aFGF enhancement involves stimulation of the receptor FGFR2 IIIb. We show by RT-PCR that this receptor, but not other FGF receptors, is robustly expressed by astrocytes and microglia. We establish by Western blotting, and immunohistochemistry on postmortem human brain tissue that the FGFR2 IIIb protein is expressed by both of these glial cell types. We blocked the inflammatory stimulant action of aFGF by transfecting microglia and astrocytes with a small inhibitory RNA (siRNA) to FGFR2 IIIb as well as by removal of aFGF using an anti-aFGF antibody. Treatment with bFGF in combination with the stimulants was without effect, but together with aFGF, it partially counteracted the action of aFGF, indicating that it may be a weak antagonist of FGFR2 IIIb. The inflammatory effect was also attenuated by treatment with inhibitors of protein kinase C, Src tyrosine kinase, and MEK-1/2 indicating the involvement of these intracellular pathways. Our data suggest that inhibition of expression or release of aFGF could have therapeutic potential by inhibiting inflammation in neurodegenerative diseases such as Alzheimer disease where many neuroinflammatory molecules are prominently expressed.     

Plasticity in interactions of fibroblast growth factor 1 (FGF1) N terminus with FGF receptors underlies promiscuity of FGF1

Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of fibroblast growth factor receptors 1–3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived fibroblast growth factors (FGFs), respectively. FGF1 is termed the “universal FGFR ligand” because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the “b” and “c” splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 Å resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-19, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-16, Asn-22, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs.     

Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.     

Fibroblast growth factor 21 (FGF21) inhibits chondrocyte function and growth hormone action directly at the growth plate

Fibroblast growth factor 21 (FGF21) modulates glucose and lipid metabolism during fasting. In addition, previous evidence indicates that increased expression of FGF21 during chronic food restriction is associated with reduced bone growth and growth hormone (GH) insensitivity. In light of the inhibitory effects on growth plate chondrogenesis mediated by other FGFs, we hypothesized that FGF21 causes growth inhibition by acting directly at the long bones' growth plate. We first demonstrated the expression of FGF21, FGFR1 and FGFR3 (two receptors known to be activated by FGF21) and β-klotho (a co-receptor required for the FGF21-mediated receptor binding and activation) in fetal and 3-week-old mouse growth plate chondrocytes. We then cultured mouse growth plate chondrocytes in the presence of graded concentrations of rhFGF21 (0.01-10 μg/ml). Higher concentrations of FGF21 (5 and 10 μg/ml) inhibited chondrocyte thymidine incorporation and collagen X mRNA expression. 10 ng/ml GH stimulated chondrocyte thymidine incorporation and collagen X mRNA expression, with both effects prevented by the addition in the culture medium of FGF21 in a concentration-dependent manner. In addition, FGF21 reduced GH binding in cultured chondrocytes. In cells transfected with FGFR1 siRNA or ERK 1 siRNA, the antagonistic effects of FGF21 on GH action were all prevented, supporting a specific effect of this growth factor in chondrocytes. Our findings suggest that increased expression of FGF21 during food restriction causes growth attenuation by antagonizing the GH stimulatory effects on chondrogenesis directly at the growth plate. In addition, high concentrations of FGF21 may directly suppress growth plate chondrocyte proliferation and differentiation.     

Rapid Trimming of Cell Surface Polysialic Acid (PolySia) by Exovesicular Sialidase Triggers Release of Preexisting Surface Neurotrophin

As acidic glycocalyx on primary mouse microglial cells and a mouse microglial cell line Ra2, expression of polysialic acid (polySia/PSA), a polymer of the sialic acid Neu5Ac (N-acetylneuraminic acid), was demonstrated. PolySia is known to modulate cell adhesion, migration, and localization of neurotrophins mainly on neural cells. PolySia on Ra2 cells disappeared very rapidly after an inflammatory stimulus. Results of knockdown and inhibitor studies indicated that rapid surface clearance of polySia was achieved by secretion of endogenous sialidase Neu1 as an exovesicular component. Neu1-mediated polySia turnover was accompanied by the release of brain-derived neurotrophic factor normally retained by polySia molecules. Introduction of a single oxygen atom change into polySia by exogenous feeding of the non-neural sialic acid Neu5Gc (N-glycolylneuraminic acid) caused resistance to Neu1-induced polySia turnover and also inhibited the associated release of brain-derived neurotrophic factor. These results indicate the importance of rapid turnover of the polySia glycocalyx by exovesicular sialidases in neurotrophin regulation.     

Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.     

The expression profile of de-N-acetyl polysialic acid (NeuPSA) in normal and diseased human tissue

Although sialic acids have a key role in many aspects of human biology, the expression of polysialic acid (PSA) in human tissues is thought to be relatively rare. We identified a derivative of PSA called neuraminic acid-containing PSA or NeuPSA that was highly expressed in primary human melanoma tumors and in several cancer cell lines. Moreover, anti-NeuPSA antibodies could induce apoptosis of cancer cells. However, little was known about NeuPSA expression in normal or diseased tissues. In this study we investigated the complete expression profile of NeuPSA in human tissues and a few primary tumors using the anti-NeuPSA monoclonal antibody, SEAM 3. Almost every human tissue tested spanning a representative sample of all organ types was positive for SEAM 3 binding. Specificity of SEAM 3 binding was established by inhibition with NeuPSA but not closely related meningococcal C polysaccharide and loss of SEAM 3 binding when specimens were treated with periodate at high pH, which specifically destroys NeuPSA. Only subsets of cells in each specimen stained positive, and the relative staining between tissues was variable. The distribution and amount of NeuPSA antigen in tissues was correlated with known levels of polysialyltransferase PST or STX expression. The majority of anti-NeuPSA binding occurred intracellularly in the cytoplasm of cells. Tumors generally exhibited considerably increased staining compared with corresponding normal tissues. Identifying the diverse tissue distribution and intracellular location of NeuPSA provides a foundation for investigating the functional role of NeuPSA in human health and disease.     

Immunoreactive fibroblast growth factor (FGF) in a transplantable chondrosarcoma: inhibition of tumor growth by antibodies to FGF

Using a radioimmunoassay for bovine pituitary fibroblast growth factor (FGF), we have established the presence of the immunoreactive mitogen in extracts of a transplantable mouse chondrosarcoma. Both neutral and acidic extracts of the tumor contain an immunoreactive FGF (ir-FGF) that cross-reacts in a parallel and dose-dependent fashion in the radioimmunoassay. The ir-FGF is retained on heparin-Sepharose affinity columns and can be detected in the same molecular weight forms as rat pituitary FGF. Mice (C57/Bl) inoculated with the tumor (10 mg, im) show a decreased rate of tumor growth when passively immunized with the antiserum to FGF. The results establish the presence of FGF in this tumor and implicate its role in the etiology of its development.     

Structural and functional impairments of polysialic acid by a mutated polysialyltransferase found in schizophrenia

Polysialic acid (polySia), a unique acidic glycan modifying neural cell adhesion molecule (NCAM), is known to regulate embryonic neural development and adult brain functions. Polysialyltransferase STX is responsible for the synthesis of polySia, and two single nucleotide polymorphisms (SNPs) of the coding region of STX are reported from schizophrenic patients: SNP7 and SNP9, respectively, giving STX(G421A) with E141K and STX(C621G) with silent mutations. In this study, we focused on these mutations and a binding activity of polySia to neural materials, such as brain-derived neurotrophic factor (BDNF). Here we describe three new findings. First, STX(G421A) shows a dramatic decrease in polySia synthetic activity on NCAM, whereas STX(C621G) does not. The STX(G421A)-derived polySia-NCAM contains a lower amount of polySia with a shorter chain length. Second, polySia shows a dopamine (DA) binding activity, which is a new function of polySia as revealed by frontal affinity chromatography for measuring the polySia-neurotransmitter interactions. Interestingly, the STX(G421A)-derived polySia-NCAM completely loses the DA binding activity, whereas it greatly diminishes but does not lose the BDNF binding activity. Third, an impairment of the polySia structure with an endosialidase modulates the DA-mediated Akt signaling. Taken together, impairment of the amount and quality of polySia may be involved in psychiatric disorders through impaired binding to BDNF and DA, which are deeply involved in schizophrenia and other psychiatric disorders, such as depression and bipolar disorder.     

Tricyclic antidepressant amitriptyline activates fibroblast growth factor receptor signaling in glial cells: involvement in glial cell line-derived neurotrophic factor production

Recently, both clinical and animal studies demonstrated neuronal and glial plasticity to be important for the therapeutic action of antidepressants. Antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production through monoamine-independent protein-tyrosine kinase, extracellular signal-regulated kinase (ERK), and cAMP responsive element-binding protein (CREB) activation in glial cells (Hisaoka, K., Takebayashi, M., Tsuchioka, M., Maeda, N., Nakata, Y., and Yamawaki, S. (2007) J. Pharmacol. Exp. Ther. 321, 148-157; Hisaoka, K., Maeda, N., Tsuchioka, M., and Takebayashi, M. (2008) Brain Res. 1196, 53-58). This study clarifies the type of tyrosine kinase and mechanism of antidepressant-induced GDNF production in C6 glioma cells and normal human astrocytes. The amitriptyline (a tricyclic antidepressant)-induced ERK activation was specifically and completely inhibited by fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors and siRNA for FGFR1 and -2. Treatment with amitriptyline or several different classes of antidepressants, but not non-antidepressants, acutely increased the phosphorylation of FGFRs and FGFR substrate 2α (FRS2α). Amitriptyline-induced CREB phosphorylation and GDNF production were blocked by FGFR-tyrosine kinase inhibitors. Therefore, antidepressants activate the FGFR/FRS2α/ERK/CREB signaling cascade, thus resulting in GDNF production. Furthermore, we attempted to elucidate how antidepressants activate FGFR signaling. The effect of amitriptyline was inhibited by heparin, non-permeant FGF-2 neutralizing antibodies, and matrix metalloproteinase (MMP) inhibitors. Serotonin (5-HT) also increased GDNF production through FGFR2 (Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., and Nakata, Y. (2008) J. Neurochem. 106, 244-257); however, the effect of 5-HT was not inhibited by heparin and MMP inhibitors. These results suggest that amitriptyline-induced FGFR activation might occur through an extracellular pathway, in contrast to that of 5-HT. The current data show that amitriptyline-induced FGFR activation might occur by the MMP-dependent shedding of FGFR ligands, such as FGF-2, thus resulting in GDNF production.     

Highly sulfated nonreducing end-derived heparan sulfate domains bind fibroblast growth factor-2 with high affinity and are enriched in biologically active fractions

Human fibroblast growth factor-2 (FGF2) regulates cellular processes including proliferation, adhesion, motility, and angiogenesis. FGF2 exerts its biological function by binding and dimerizing its receptor (FGFR), which activates signal transduction cascades. Effective binding of FGF2 to its receptor requires the presence of heparan sulfate (HS), a linear polysaccharide with N-sulfated domains (NS) localized at the cell surface and extracellular matrix. HS acts as a platform facilitating the formation of a functional FGF-FGFR-HS ternary complex. Crystal structures of the signaling ternary complex revealed two conflicting architectures. In the asymmetrical model, two FGFs and two FGFRs bind a single HS chain. In contrast, the symmetrical model postulates that one FGF and one FGFR bind to the free end of the HS chain and dimerization require these ends to join, bringing the two half-complexes together. In this study, we screened a hexasaccharide HS library for compositions that are able to bind FGF2. The library was composed primarily of NS domains internal to the HS chain with minor presence of non-reducing end (NRE) NS. The binders were categorized into low versus high affinity binders. The low affinity fraction contained primarily hexasaccharides with low degree of sulfation that were internal to the HS chains. In contrast, the high affinity bound fraction was enriched in NRE oligosaccharides that were considerably more sulfated and had the ability to promote FGFR-mediated cell proliferation. The results suggest a role of the NRE of HS in FGF2 signaling and favor the formation of the symmetrical architecture on short NS domains.     

Knockout of Nuclear High Molecular Weight FGF2 Isoforms in Mice Modulates Bone and Phosphate Homeostasis

We previously reported that targeted overexpression of the fibroblast growth factor 2 (FGF2) high molecular weight (HMW) isoforms in osteoblastic lineage cells in mice resulted in phenotypic changes, including dwarfism, rickets, osteomalacia, hypophosphatemia, increased serum parathyroid hormone, and increased levels of the phosphatonin FGF23 in serum and bone. This study examined the effects of genetically knocking out the FGF2HMW isoforms (HMWKO) on bone and phosphate homeostasis. HMWKO mice were not dwarfed and had significantly increased bone mineral density and bone mineral content in femurs and lumbar vertebrae when compared with the wild-type (WT) littermates. Micro-computed tomography analysis of femurs revealed increased trabecular bone volume, thickness, number, and connective tissue density with decreased trabecular spacing compared with WT. In addition, there was significantly decreased cortical porosity and increased cortical thickness and sub-periosteal area in femurs of HMWKO. Histomorphometric analysis demonstrated increased osteoblast activity and diminished osteoclast activity in the HMWKO. In vitro bone marrow stromal cell cultures showed there was a significant increase in alkaline phosphatase-positive colony number at 1 week in HMWKO. At 3 weeks of culture, the mineralized area was also significantly increased. There was increased expression of osteoblast differentiation marker genes and reduced expression of genes associated with impaired mineralization, including a significant reduction in Fgf23 and Sost mRNA. Normal serum phosphate and parathyroid hormone were observed in HMWKO mice. This study demonstrates a significant negative impact of HMWFGF2 on biological functions in bone and phosphate homeostasis in mice.