Regulation of fibroblast growth factor-23 signaling by klotho

The aging suppressor gene Klotho encodes a single-pass transmembrane protein. Klotho-deficient mice exhibit a variety of aging-like phenotypes, many of which are similar to those observed in fibroblast growth factor-23 (FGF23)-deficient mice. To test the possibility that Klotho and FGF23 may function in a common signal transduction pathway(s), we investigated whether Klotho is involved in FGF signaling. Here we show that Klotho protein directly binds to multiple FGF receptors (FGFRs). The Klotho-FGFR complex binds to FGF23 with higher affinity than FGFR or Klotho alone. In addition, Klotho significantly enhanced the ability of FGF23 to induce phosphorylation of FGF receptor substrate and ERK in various types of cells. Thus, Klotho functions as a cofactor essential for activation of FGF signaling by FGF23.     

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.     

Maternally-derived antibody to fibroblast growth factor-23 reduced dietary phosphate requirements in growing chicks

Phosphate in manure of monogastric animals pollutes the environment if improperly applied to soil. Strategies that reduce phosphate inputs into animal production systems reduce environmental pollution. Using a novel vaccine to fibroblast growth factor-23 (FGF-23), we induced neutralizing antibodies that reduced the phosphate requirement of growing chickens. Breeding hens were injected with a FGF-23 peptide (AFLPGMNP) conjugate. Antibody was passively transferred from hen to chick and chick response to deficient dietary phosphate intake was determined. Chicks without passive anti-FGF-23 antibody had a 43% and 21% reduction in blood phosphate and bone ash, respectively, when fed a phosphate deficient diet and compared to chicks fed a phosphate replete diet (P<0.05). Chicks with circulating anti-FGF-23 antibodies fed the phosphate deficient diet had plasma phosphate and bone ash that did not differ from chicks fed the phosphate replete diet (P>0.05). Neutralization of FGF-23 offers a new approach to reduce phosphate requirements of farmed animals and may provide a new means to reduce phosphate pollution related to animal farming.     

The anti-angiogenic activity of rPAI-1(23) inhibits fibroblast growth factor-2 functions

Many angiogenesis inhibitors are breakdown products of endogenous extracellular matrix proteins. Plasmin and matrix metalloproteinase-3 generate breakdown products of matrix-bound plasminogen activator inhibitor-1 (PAI-1). We produced a truncated form of PAI-1, rPAI-1(23), that possesses significant anti-angiogenic activity and stimulates high levels of apoptosis in quiescent arterial endothelial cells. Quiescent endothelial cells are less susceptible to apoptosis than angiogenic endothelial cells. The present study was designed to determine the mechanism of the rPAI-1(23) effects in bovine aortic endothelial cells. Apoptosis was measured in annexin V and caspase 3 assays. Expression of death and survival signaling molecules were examined by Western blot and kinase activity. Fibroblast growth factor 2 (FGF2) functions were analyzed in angiogenesis assays. The early response to rPAI-1(23) was an increase in annexin V-positive cells and phosphorylated (p) JNK isoform expression followed by an increase in p-Akt and p-c-Jun expression. Caspase 3 was activated at 4 h, whereas p-Akt was reduced to control levels. By 6 h of rPAI-1(23) treatment cell number was reduced by 35%, and p-c-Jun and p-JNK were degraded by proteasomes. Confocal microscopic images showed increased amounts of FGF2 in the extracellular matrix. However, rPAI-1(23) blocked FGF2 signaling through FGF receptor 1 and syndecan-4, inhibiting cell migration, tubulogenesis, and proliferation. Exogenous FGF2 stimulation could not reverse these effects. We conclude that rPAI-1(23) stimulation of apoptosis in BAEC triggers a cascade of death versus survival events that includes release of FGF2. The rPAI-1(23) anti-angiogenic activity inhibits FGF2 pro-angiogenic functions by blocking FGF2 signaling through FGF receptor 1 and syndecan-4 and downstream effectors p-Akt, p-JNK, and p-c-Jun.     

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.     

Effect of single wrist exercise on fibroblast growth factor-2, insulin-like growth factor, and growth hormone

Anabolic effects of exercise are mediated, in part, by fibroblast growth factor-2 (FGF-2), insulin-like growth factor-I (IGF-I), and growth hormone (GH). To identify local vs. systemic modification of these mediators, 10 male subjects performed 10 min of unilateral wrist-flexion exercise. Blood was sampled from catheters placed in basilic veins of both arms. Lactate was significantly increased only in the exercising arm. FGF-2 decreased dramatically (P < 0.01) in both the resting (from 1.49 +/- 0.32 to nadir at 0.11 +/- 0.11 pg/ml) and exercising arm (1.80 +/- 0.60 to 0.29 +/- 0.14 pg/ml). Small but significant increases were found in both the resting and exercising arm for IGF-I and IGF binding protein-3 (IGFBP-3). GH was elevated in blood sampled from both the resting (from 1.04 +/- 0.68 to a peak of 2.57 +/- 0.53 ng/ml) and exercising arm (1.04 +/- 0.66 to 2.43 +/- 0.42 ng/ml, P < 0.05). Unilateral wrist exercise was not sufficiently intense to increase circulating lactate or heart rate, but it led to systemic changes in GH, IGF-I, IGFBP-3, and FGF-2. Low-intensity exercise involving small muscle groups can influence the circulating levels of growth factors.     

The discovery of basic fibroblast growth factor/fibroblast growth factor-2 and its role in haematological malignancies

Basic fibroblast growth factor/fibroblast growth factor-2 is one of the best characterized of the pro-angiogenic cytokines. This review describes its history, as well as its role in tumor angiogenesis associated with haematological malignancies, as traced by the main contributions to the international medical literature.     

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.     

A novel role of fibroblast growth factor-2 and pentosan polysulfate in the pathogenesis of intestinal bleeding in mice

Pentosan polysulfate (PPS) is a heparin-like polysaccharide that can affect the binding interactions of fibroblast growth factor (FGF-2) with its high-affinity receptors. Patients with angiogenic tumors frequently show high levels of FGF-2 in the circulation. Since FGF-2 is a heparin-binding angiogenic growth factor, PPS has been used successfully to block its activity in patients with angiogenic tumors. However, because of its heparin-like activity, the major toxic effect of PPS is the development of bleeding disorders. The role that circulating FGF-2 plays in the pathogenesis of bleeding disorders in patients treated with PPS is currently unknown. Here we hypothesized that FGF-2 might play a physiological role in the pathogenesis of intestinal bleeding induced by PPS. This hypothesis is supported by previous studies showing that PPS is accumulated in the intestine and that circulating FGF-2 specifically binds to and modulates the angiogenic activity of intestinal submucosal endothelial cells. We used recombinant adenoviral vectors carrying a secreted form of FGF-2 and LacZ control vectors to determine whether high levels of circulating FGF-2 facilitate the development of intestinal bleeding disorders in FVB/N and C57BL/6J mice treated with PPS. We found that PPS, acting together with FGF-2, induced structural changes in intestinal vessels leading to the development of lethal intestinal hemorrhages. These findings might have wider clinical implications for the systemic use of PPS and other heparinoids in the treatment of patients with angiogenic diseases associated with high levels of circulating FGF-2.     

Human fibroblast growth factor-23 mutants suppress Na+-dependent phosphate co-transport activity and 1alpha,25-dihydroxyvitamin D3 production

The human fibroblast growth factor 23 (hFGF23) and its autosomal dominant hypophosphatemic rickets (ADHR) mutant genes were incorporated into animals by naked DNA injection to investigate the action on phosphate homeostasis in vivo. The hFGF23 mutants (R176Q, R179Q, and R179W) markedly reduced serum phosphorus (6.2-6.9 mg/dl) compared with the plasmid MOCK (8.5 mg/dl). However, native hFGF23 did not affect serum phosphorus (8.6 mg/dl). Both hFGF23 and hFGF23R179Q mRNAs were expressed more than 100-fold in the liver 4 days after injection, however, the C-terminal portion of hFGF23 was detected only in the serum from hFGF23R179Q-injected animals (1109 pg/ml). hFGF23R179Q mutant was secreted as a 32-kDa protein, whereas, native hFGF23 was detected as a 20-kDa protein in the cell-conditioned media. These results suggest the hFGF23R179Q protein is resistant to intracellular proteolytic processing. The hFGF23R179Q suppressed Na/P(i) co-transport activities both in kidney and in small intestine by 45 and 30%, respectively, as well as serum 1alpha,25-dihydroxyvitamin D(3) to less than 15 pg/ml. However, it had little effect on serum parathyroid hormone (PTH). Infusion of hFGF23R179Q protein normalized serum phosphorus in thyroparathyroidectomized rats without affecting serum calcium. Taken together, the FGF23 mutants reduce both phosphate uptake in intestine and phosphate reabsorption in kidney, independent of PTH action.     

Localization of fibroblast growth factor-1 in cholinergic neurons innervating the rat larynx.

Cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) are particularly vulnerable to laryngeal nerve damage, possibly because they lack fibroblast growth factor-1 (FGF1). To test this hypothesis, we investigated the localization of FGF1 in cholinergic neurons innervating the rat larynx by immunohistochemistry using central-type antibodies to choline acetyltransferase (cChAT) and peripheral type (pChAT) antibodies, as well as tracer experiments. In the DMNV, only 9% of cChAT-positive neurons contained FGF1, and 71% of FGF1-positive neurons colocalized with cChAT. In the nucleus ambiguus, 100% of cChAT-positive neurons were FGF1 positive. In the intralaryngeal ganglia, all ganglionic neurons contained both pChAT and FGF1. In the nodose ganglia, 66% of pChAT-positive neurons were also positive for FGF1, and 90% of FGF1-positive ganglionic cells displayed pChAT immunoreactivity. Neuronal tracing using cholera toxin B subunit (CTb) demonstrated that cholinergic neurons sending their axons from the DMNV and nucleus ambiguus to the superior laryngeal nerve were FGF1 negative and FGF1 positive, respectively. In the nodose ganglia, some FGF1-positive cells were labeled with CTb. The results indicate that for innervation of the rat larynx, FGF1 is localized to motor neurons, postganglionic parasympathetic neurons, and sensory neurons, but expression is very low in preganglionic parasympathetic cholinergic neurons.     

Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie-infected neuroblastoma cells.

A growing body of evidence suggests that an altered level or function of the neurotrophic insulin-like growth factor-1 receptor (IGF-1R), which supports neuronal survival, may underlie neurodegeneration. This study has focused on the expression and function of the IGF-1R in scrapie-infected neuroblastoma cell lines. Our results show that scrapie infection induces a 4-fold increase in the level of IGF-1R in two independently scrapie-infected neuroblastomas, ScN2a and ScN1E-115 cells, and that the increased IGF-1R level was accompanied by increased IGF-1R mRNA levels. In contrast to the elevated IGF-1R expression in ScN2a, receptor binding studies revealed an 80% decrease in specific (125)I-IGF-1-binding sites compared with N2a cells. This decrease in IGF-1R-binding sites was shown to be caused by a 7-fold decrease in IGF-1R affinity. Furthermore, ScN2a showed no significant difference in IGF-1 induced proliferative response, despite the noticeable elevated IGF-1R expression, putatively explained by the reduced IGF-1R binding affinity. Additionally, IGF-1 stimulated IGF-1Rbeta tyrosine phosphorylation showed no major change in the dose-response between the cell types, possibly due to altered tyrosine kinase signaling in scrapie-infected neuroblastoma cells. Altogether these data indicate that scrapie infection affects the expression, binding affinity, and signal transduction mediated by the IGF-1R in neuroblastoma cells. Altered IGF-1R expression and function may weaken the trophic support in scrapie-infected neurons and thereby contribute to neurodegeneration in prion diseases.     

Regulation of matrilysin expression in endothelium by fibroblast growth factor-2

Matrilysin (MMP7) is a secreted matrix metalloproteinase, which contributes to angiogenesis by breaking down basement membranes. We show that the angiogenic factor FGF-2 induces MMP7 expression in human endothelial cells. The promoter contains a Lef/Tcf consensus sequence, but using wildtype or Lef/Tcf-mutated promoter constructs, FGF-2-induced MMP7 reporter activity is independent from Lef/Tcf sites. Instead, we show that overexpression of a dominant negative Stat3 mutant reduces FGF-2-mediated MMP7 promoter activity. However, Stat3 does not bind to the MMP7 promoter, but activates MMP7 gene expression indirectly via AP-1. This is confirmed by MMP7 promoter constructs with mutated AP-1 sites which did not respond to FGF-2 and by siRNAs against Stat1 and Stat3, which repressed FGF-2-induced MMP7 protein expression. In conclusion, we show that FGF-2-induced MMP7 expression in endothelium depends on AP-1 and FGF-2 signaling to AP-1 involves a Stat1/3-dependent pathway.     

Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity

Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.     

Conditional expression of fibroblast growth factor-7 in the developing and mature lung

Effects of fibroblast growth factor-7 (FGF-7) on lung morphogenesis, respiratory epithelial cell differentiation, and proliferation were assessed in transgenic mice in which the human FGF-7 cDNA was controlled by a conditional promoter under the direction of regulatory elements from either the human surfactant protein-C (SP-C) or rat Clara cell secretory protein (ccsp) genes. Expression of FGF-7 was induced in respiratory epithelial cells of the fetal lung by administration of doxycycline to the dam. Prenatally, doxycycline induced FGF-7 mRNA in respiratory epithelial cells in both Sp-c and Ccsp transgenic lines, increasing lung size and causing cystadenomatoid malformation. Postnatally, mice bearing both Ccsp-rtta and (Teto)(7)-cmv-fgf-7 transgenes survived, and lung morphology was normal. Induction of FGF-7 expression by doxycycline in the Ccsp-rtta x (Teto)(7)-cmv-fgf-7 mice caused marked epithelial cell proliferation, adenomatous hyperplasia, and pulmonary infiltration with mononuclear cells. Epithelial cell hyperplasia caused by FGF-7 was largely resolved after removal of doxycycline. Surfactant proteins, TTF-1, and aquaporin 5 expression were conditionally induced by doxycycline. The Sp-c-rtta and Ccsp-rtta activator mice provide models in which expression is conditionally controlled in respiratory epithelial cells in the developing and mature lung, altering lung morphogenesis, differentiation, and proliferation.     

Glycated fibroblast growth factor-2 is quickly produced in vitro upon low-millimolar glucose treatment and detected in vivo in diabetic mice

Angiogenesis impairment in hyperglycemic patients represents a leading cause of severe vascular complications of both type-1 and -2 diabetes mellitus (DM). Angiogenesis dysfunction in DM is related to glycemic control; however, molecular mechanisms involved are still unclear. Fibroblast growth factor-2 (FGF-2) is a potent angiogenic factor and, according to previous evidence, may represent a key target of molecular modifications triggered by high-sugar exposure. Therefore, the purpose of this study was to investigate whether short incubation with hyperglycemic levels of glucose affected FGF-2 and whether glucose-modified FGF-2 was detectable in vivo. Biochemical analyses carried out with SDS-PAGE, fluorescence emission, mass-spectrometry, immunoblot, and competitive ELISA experiments demonstrated that human FGF-2 undergoes a rapid and specific glycation upon 12.5-50 mm glucose exposure. In addition, FGF-2 exposed for 30 min to 12.5 mm glucose lost mitogenic and chemotactic activity in a time- and dose-dependent manner. Under similar conditions, binding affinity to FGF receptor 1 was dramatically reduced by 20-fold, as well as FGF receptor 1 and ERK-1/2 phosphorylation, and FGF-2 lost about 45% of angiogenic activity in two different in vivo angiogenic (Matrigel and chorioallantoic-membrane) assays. Such glucose-induced modification was specific, because other angiogenic growth factors, namely platelet-derived growth factor BB and placental-derived growth factor were not significantly or markedly less modified. Finally, for the first time, glycated-FGF-2 was detected in vivo, in tissues from hyperglycemic nonobese diabetic mice, in significantly higher amounts than in normoglycemic mice. In conclusion, hyperglycemic levels of glucose may strongly affect FGF-2 structure and impair its angiogenic features, and endogenous glycated-FGF-2 is present in diabetic mice, indicating a novel pathogenetic mechanism underlying angiogenesis defects in DM.     

Proliferation of neointimal smooth muscle cells after arterial injury. Dependence on interactions between fibroblast growth factor receptor-2 and fibroblast growth factor-9

The growth factor signaling mechanisms responsible for neointimal smooth muscle cell (SMC) proliferation and accumulation, a characteristic feature of many vascular pathologies that can lead to restenosis after angioplasty, remain to be identified. Here, we examined the contribution of fibroblast growth factor receptors (FGFRs) 2 and 3 as well as novel fibroblast growth factors (FGFs) to such proliferation. Balloon catheter injury to the rat carotid artery stimulated the expression of two distinctly spliced FGFR-2 isoforms, differing only by the presence or absence of the acidic box, and two distinctly spliced FGFR-3 isoforms containing the acidic box and differing only by the presence of either the IIIb or IIIc exon. Post-injury arterial administration of recombinant adenoviruses expressing dominant negative mutant forms of these FGFRs were used to assess the roles of the endogenous FGFR isoforms in neointimal SMC proliferation. Dominant negative FGFR-2 containing the acidic box inhibited such proliferation by 40%, whereas the dominant negative FGFR-3 forms had little effect. Expression of FGF-9, known to be capable of binding to all four neointimal FGFR-2/-3 isoforms, was abundant within the neointima. FGF-9 markedly stimulated both the proliferation of neointimal SMCs and the activation of extracellular signal-related kinases 1/2, effects which were abrogated by the administration of antisense FGF-9 oligonucleotides to injured arteries and the expression of the dominant negative FGFR-2 adenovirus in cultured neointimal SMCs. These studies demonstrate that, although multiple FGFRs are induced in neointimal SMCs following arterial injury, specific interactions between distinctly spliced FGFR-2 isoforms and FGF-9 contribute to the proliferation of these SMCs.     

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.