Soluble Fibroblast Growth Factor Receptor Research Articles

Juvenile toxicity study of PF-07256472/recifercept, a recombinant human soluble fibroblast growth factor receptor 3, in 2-3-month-old cynomolgus monkeys.

Achondroplasia is an autosomal disorder caused by point mutation in the gene encoding fibroblast growth factor receptor 3 (FGFR3) and resulting in gain of function. Recifercept is a potential disease modifying treatment for achondroplasia and functions as a decoy protein that competes for ligands of the mutated FGFR3. Recifercept is intended to restore normal bone growth by preventing the mutated FGFR3 from negative inhibitory signaling in pediatric patients with achondroplasia. Here we evaluated the potential effects of twice weekly administration of recifercept to juvenile cynomolgus monkeys (approximately 3-months of age at the initiation of dosing) for 6-months. No adverse effects were noted in this study, identifying the high dose as the no-observed-adverse-effect-level and supporting the use of recifercept in pediatric patients from birth. Considering that juvenile toxicity studies in nonhuman primates are not frequently conducted, and when they are conducted they typically utilize animals ≥9months of age, this study demonstrates the feasibility of executing a juvenile toxicity study in very young monkeys prior to weaning.

In vitro and in vivo characterization of Recifercept, a soluble fibroblast growth factor receptor 3, as treatment for achondroplasia.

Achondroplasia is a rare genetic disorder caused by mutations in the Fibroblast Growth Factor receptor 3 (FGFR3). These mutations lead to aberrant increase of inhibitory signaling in proliferating chondrocytes at the growth plate. Recifercept is a potential treatment for this disease using a decoy approach to sequester FGFR3 ligands subsequently normalizing activation of the mutated FGFR3 receptor. Recifercept binds to FGF isoforms in vitro and in cellular model systems and reduces FGFR3 signaling. In addition, in a transgenic mouse model of achondroplasia, Recifercept restores reduced body weight and long bone growth in these mice. These data suggest that Recifercept treatment could lead to clinical benefits in children treated with this molecule.

Postnatal Soluble FGFR3 Therapy Rescues Achondroplasia Symptoms and Restores Bone Growth in Mice

Achondroplasia is a rare genetic disease characterized by abnormal bone development, resulting in short stature. It is caused by a single point mutation in the gene coding for fibroblast growth factor receptor 3 (FGFR3), which leads to prolonged activation upon ligand binding. To prevent excessive intracellular signaling and rescue the symptoms of achondroplasia, we have developed a recombinant protein therapeutic approach using a soluble form of human FGFR3 (sFGFR3), which acts as a decoy receptor and prevents FGF from binding to mutant FGFR3. sFGFR3 was injected subcutaneously to newborn Fgfr3(ach/+) mice-the mouse model of achondroplasia-twice per week throughout the growth period during 3 weeks. Effective maturation of growth plate chondrocytes was restored in bones of treated mice, with a dose-dependent enhancement of skeletal growth in Fgfr3(ach/+) mice. This resulted in normal stature and a significant decrease in mortality and associated complications, without any evidence of toxicity. These results describe a new approach for restoring bone growth and suggest that sFGFR3 could be a potential therapy for children with achondroplasia and related disorders.

FGF Signaling Preserves the Integrity of Endothelial Adherens Junctions

Fibroblast growth factor (FGF)-activated signaling regulates an array of cellular processes ranging from embryonic development to tissue repair. A recent paper by Murakami et al. identifies a potentially important role for FGF signaling in maintenance of endothelial barrier homeostasis through the regulation of adherens junctions.

Adenovirus-mediated expression of a soluble fibroblast growth factor receptor inhibits in vitro growth of prostate DU145 cells.

Fibroblast growth factor (FGF) family plays a key role in prostate cancer. The soluble FGF receptor (sFGFR) has been studied with regards to inhibiting cancer growth and was shown to have a dominant negative effect on cellular signaling and function. Using replication deficient adenovirus-mediated gene transfer, we tested if sFGFR expression may have a suppressive effect on in vitro growth of prostate cancer cells. Western analysis was used to verify expression of sFGFR1 and to examine the effect of sFGFR1 on MAP kinase phosphorylation. The effect on proliferation and invasiveness of DU145 cells was examined using the WST-1 and Matrigel Invasion assay, respectively. Activation of MAP kinase (pERK1 and 2) by exogenous FGF1, 2, and 7 was suppressed to baseline levels by sFGFR, which was not seen with EGF. Proliferation and invasion of DU145 cells were significantly suppressed by sFGFR. A replication deficient adenoviral vector system reproducibly expresses sFGFR in prostate cells. Suppression of in vitro growth in DU145 cells by sFGFR provides the basis of a novel therapeutic approach.

FGF signaling is required for pulmonary homeostasis following hyperoxia.

To assess the role of fibroblast growth factor (FGF) signaling in pulmonary function in the postnatal period, we generated transgenic mice in which a soluble FGF receptor (FGFR-HFc) was conditionally expressed in respiratory epithelial cells of the mouse lung, thereby inhibiting FGF activity. Although FGFR-HFc did not alter postnatal lung morphogenesis, male FGFR-HFc transgenic mice were more susceptible to hyperoxia and failed to recover when ambient oxygen concentrations were normalized. Inflammation, alveolar-capillary leak, and mortality were increased following exposure to 95% Fi(O(2)). Expression of surfactant protein (SP)-A and SP-B were significantly decreased in association with decreased immunostaining for thyroid transcription factor-1. FGF signaling is required for maintenance of surfactant homeostasis and lung function during hyperoxia in vivo, mediated, at least in part, by its role in the maintenance of SP-B expression.

Soluble fibroblast growth factor receptor 1 IIIc suppresses in vitro proliferation and invasion of DU145 cells

Soluble fibroblast growth factor receptor 1 IIIc suppresses in vitro proliferation and invasion of DU145 cells

Prenatal, but Not Postnatal, Inhibition of Fibroblast Growth Factor Receptor Signaling Causes Emphysema

Although fibroblast growth factor (FGF) signaling is required for the formation of the lung in the embryonic period, it is unclear whether FGF receptor activity influences lung morphogenesis later in development. We generated transgenic mice expressing a soluble FGF receptor (FGFR-HFc) under conditional control of the lung-specific surfactant protein C promoter (SP-C-rtTA), to inhibit FGF activity at various times in late gestation and postnatally. Although expression of FGFR-HFc early in development caused severe fetal lung hypoplasia, activation of the transgene in the postnatal period did not alter alveolarization, lung size, or histology. In contrast, expression of the transgene at post-conception day E14.5 decreased lung tubule formation before birth and caused severe emphysema at maturity. FGFR-HFc caused mild focal emphysema when expressed from E16.5 but did not alter alveolarization when expressed after birth. Although FGF signaling was required for branching morphogenesis early in lung development, postnatal alveolarization was not influenced by FGFR-HFc.

Basic fibroblast growth factor: a potential inhibitor of glutamine synthetase expression in injured neural tissue.

Basic fibroblast growth factor (bFGF) was recently shown to promote the survival of neural cells and tissues, raising hopes for its therapeutic potential in degenerative disorders of the CNS. Here we examine the effect of bFGF on the expression of glutamine synthetase, a key enzyme in the detoxification of the neurotransmitter glutamate. Expression of this enzyme is regulated by systemic glucocorticoids and, in chick neural retina tissue, is restricted to Müller glial cells. We report that exogenous supply of bFGF to retinal explants inhibits hormonal induction of glutamine synthetase expression. This inhibition appears to be mediated by the c-Jun protein which accumulated, in response to bFGF, exclusively in Müller glial cells. Ischemic conditions, which reportedly stimulate the release of endogenous bFGF, also led to an increase in c-Jun protein and a decline in glutamine synthetase expression. This decline could be competitively prevented by a soluble fibroblast growth factor receptor but not by a soluble epidermal growth factor receptor. The finding that endogenous release of bFGF or its exogenous supply down-regulates glutamine synthetase expression suggests that in addition to its reported neuroprotective effect, bFGF may exacerbate glutamate mediated neurotoxicity through direct down-regulation of glutamine synthetase.

A Soluble Fibroblast Growth Factor Receptor is Released from HL-60 Promyelocytic Leukemia Cells: Implications for Paracrine Growth Control

The biological activities of fibroblast growth factors (FGF) are mediated by specific cell membrane receptors (FGFR), which have three immunoglobulin-like IgG domains in the extracellular region. The carboxy-terminal segment of the third IgG domain of FGFR1 could be encoded by different exons, designated IIIa, IIIb, or IIIc. While exons IIIb or IIIc encode receptor forms with both intracellular and extracellular domains, the FGF receptor becomes potentially a secreted form lacking the intracellular domain and the transmembrane region when exon IIIa is expressed. Using reverse transcription polymerase chain reaction, we have found that mRNAs encoding the nucleotide sequences of FGFR1-IIIa and FGFR1-IIIc are expressed in HL-60 cells. FGFR1-IIIa fragment was synthesized by a glutathione S-transferase gene fusion system. The purified 33kDa FGFR1-IIIa fragment fusion protein could bind [125I]-labelled FGF-2 in Western ligand blot analysis. Three species of proteins with the molecular weights of 82, 60, and 50 kDa were identified in serum-free, conditioned medium from HL-60 cells by Western blot using an antiserum against purified FGFR1-IIIa fragment fusion protein. Exposure to FGF-2 caused an increase in [3H]-thymidine incorporation into DNA of HL-60 cells and increased cell proliferation, but the addition of FGFR1-IIIa fragment fusion protein inhibited FGF-2-stimulated DNA synthesis and caused a dose-dependent inhibition of FGF-2-stimulated cell proliferation. The effects on DNA synthesis were partly reversed by antibody against the FGFR1-IIIa fragment. These results indicate that both cell membrane spanning and secreted FGF receptors are expressed in HL-60 cells, and that the actions of FGFs as paracrine growth factors could be modulated by secreted FGF receptor forms.

BALB/C 3T3 CELLS CO-EXPRESSING FGF-2 AND SOLUBLE FGF RECEPTOR ACQUIRE TUMORIGENICITY

The physiological significance of the soluble fibroblast growth factor (FGF) receptors is not clear yet although they are present in blood, vitreous fluid and in the extracellular matrix of vascular endothelial cells. A hypothesis that they might help FGF-2 release from cells is very interesting because FGF-2 does not have a clear secretion signal and the mechanism of the secretion of FGF-2 is still unclear. Single overexpression of FGF-2 is related neither to the secretion potential of the molecule nor to the tumourigenicity of the cells. In this report, BALB/c 3T3 cells transformed with the full length of human FGF-2 cDNA are further transformed with the cDNA coding the extracellular domain of human FGF receptor 1. The obtained transformants co-expressing FGF-2 and soluble FGF receptor are highly tumorigenic in nude mice, while the parental cells do not show any tumorigenicity. In the conditioned medium of the double-transformants, FGF-2 is immunologically detected. These results suggest that naturally produced soluble form of FGF receptor supports the release of FGF-2 from the cells and that over-expression of these two molecules leads to induce the malignant tumours in vivo.

Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1.

Recent studies have demonstrated the existence of a soluble fibroblast growth factor (FGF) receptor type 1 (FGFR1) extracellular domain in the circulation and in vascular basement membranes. However, the process of FGFR1 ectodomain release from the plasma membrane is not known. Here we report that the 72-kDa gelatinase A (matrix metalloproteinase type 2, MMP2) can hydrolyze the Val368-Met369 peptide bond of the FGFR1 ectodomain, eight amino acids upstream of the transmembrane domain, thus releasing the entire extracellular domain. Similar results were obtained regardless of whether FGF was first bound to the receptor or not. The action of MMP2 abolished binding of FGF to an immobilized recombinant FGFR1 ectodomain fusion protein and to Chinese hamster ovary cells overexpressing FGFR1 The released recombinant FGFR1 ectodomain was able to bind FGF after MMP2 cleavage, suggesting that the cleaved soluble receptor maintained its FGF binding capacity. The activity of MMP2 could not be reproduced by the 92-kDa gelatinase B (MMP9) and was inhibited by tissue inhibitor of metalloproteinase type 2. These studies demonstrate that FGFR1 may be a specific target for MMP2 on the cell surface, yielding a soluble FGF receptor that may modulate the mitogenic and angiogenic activities of FGF.

Purification and Characterization of a Functional Soluble Fibroblast Growth Factor Receptor 1

Naturally encoded human soluble fibroblast growth factor receptor 1 was cloned, abundantly expressed by recombinant baculovirus-infected insect cells and purified to apparent homogeneity. Pure soluble receptor bound as monomeric and dimeric complexes and inhibited the mitogenic activity of basic and acidic fibroblast growth factors.

Biochemical characterization of the molecular interaction between recombinant basic fibroblast growth factor and a recombinant soluble fibroblast growth factor receptor

The extracellular domain of human fibroblast growth factor receptor (XC-FGF-R) was expressed in Escherichia coli. The protein was purified to homogeneity and the interaction with basic fibroblast growth factor (bFGF), its physiological ligand, was examined. Using resins on which bFGF was reversibly bound, we analysed the characteristics of the binding between XC-FGF-R and immobilized bFGF. We also investigated the stoichiometry of the binding between XC-FGF-R and recombinant human bFGF (rhbFGF) applying non-denaturing gel electrophoresis, chemical cross-linking followed by SDS/PAGE, and gel-filtration chromatography. In cross-linking and gel-filtration chromatography experiments, a 1:1 complex between rhbFGF and XC-FGF-R was observed. The complex was separated from the non-complexed proteins using non-denaturing PAGE in the presence of 0.1% Triton X-100. The band corresponding to the complex was recognized by specific antibodies directed against bFGF and its receptor, blotted on poly(vinylidene difluoride) membranes and submitted to sequence and amino acid analysis. The data obtained from these determinations confirmed the formation of a 1:1 complex between rhbFGF and XC-FGF-R.

The fibroblast growth factor receptor is not required for herpes simplex virus type 1 infection.

The early events mediating herpes simplex virus type 1 (HSV-1) infection include virion attachment to cell surface heparan sulfates and subsequent penetration. Recent evidence has suggested that the high-affinity fibroblast growth factor (FGF) receptor mediates HSV-1 entry. This report presents three lines of experimental evidence showing that the high-affinity FGF receptor is not required for HSV-1 infection. First, rat L6 myoblasts lacking FGF receptors were as susceptible to HSV-1 infection as L6 cells genetically engineered to express the FGF receptor. Second, a soluble FGF receptor fragment that inhibited FGF binding and receptor activation did not inhibit HSV-1 infection. Finally, basic FGF (but not acidic FGF) inhibited HSV-1 infection in L6 cells lacking FGF receptors, presumably by blocking cell surface heparan sulfates also required for HSV-1 infection. These results show that the high-affinity FGF receptor is not required for HSV-1 infection but instead that specific low-affinity basic FGF binding sites are used for HSV-1 infection.