Effect of FGF-binding protein 3 on vascular permeability.
ABSTRACT: Fibroblast growth factor-binding protein 1 (FGF-BP1 is BP1) is involved in the regulation of embryonic development, tumor growth, and angiogenesis by mobilizing endogenous FGFs from their extracellular matrix storage. Here we describe a new member of the FGF-BP family, human BP3. We show that the hBP3 protein is secreted from cells, binds to FGF2 in vitro and in intact cells, and inhibits FGF2 binding to heparin. To determine the function of hBP3 in vivo, hBP3 was transiently expressed in chicken embryos and resulted in > 50% lethality within 24 h because of vascular leakage. The onset of vascular permeability was monitored by recording the extravasation kinetics of FITC-labeled 40-kDa dextran microperfused into the vitelline vein of 3-day-old embryos. Vascular permeability increased as early as 8 h after expression of hBP3. The increased vascular permeability caused by hBP3 was prevented by treatment of embryos with PD173074, a selective FGFR kinase inhibitor. Interestingly, a C-terminal 66-amino acid fragment (C66) of hBP3, which contains the predicted FGF binding domain, still inhibited binding of FGF2 to heparin similar to full-length hBP3. However, expression of the C66 fragment did not increase vascular permeability on its own, but required the administration of exogenous FGF2 protein. We conclude that the FGF binding domain and the heparin binding domain are necessary for the hBP3 interaction with endogenous FGF and the activation of FGFR signaling in vivo.
Project description:Fibroblast growth factor 2 (FGF2) plays important roles in tissue development and repair. Using heparan sulfates (HS)/heparin as a cofactor, FGF2 binds to FGF receptor (FGFR) and induces downstream signaling pathways, such as ERK pathway, that regulate cellular behavior. In most cell lines, FGF2 signaling displays biphasic dose-response profile, reaching maximal response to intermediate concentrations, but weak response to high levels of FGF2. Recent reports demonstrated that the biphasic cellular response results from competition between binding of FGF2 to HS and FGFR that impinge upon ERK signaling dynamics. However, the role of HS/heparin in FGF signaling has been controversial. Several studies suggested that heparin is not required for FGF-FGFR complex formation and that the main role of heparin is to protect FGF from degradation. In this study, we investigated the relationship between FGF2 stability, heparin dependence and ERK signaling dynamics using FGF2 variants with increased thermal stability (FGF2-STABs). FGF2-STABs showed higher efficiency in induction of FGFR-mediated proliferation, lower affinity to heparin and were less dependent on heparin than wild-type FGF2 (FGF2-wt) for induction of FGFR-mediated mitogenic response. Interestingly, in primary mammary fibroblasts, FGF2-wt displayed a sigmoidal dose-response profile, while FGF2-STABs showed a biphasic response. Moreover, at low concentrations, FGF2-STABs induced ERK signaling more potently and displayed a faster dynamics of full ERK activation and higher amplitudes of ERK signaling than FGF2-wt. Our results suggest that FGF2 stability and heparin dependence are important factors in FGF-FGFR signaling complex assembly and ERK signaling dynamics.
Project description:Sucrose octasulfate (SOS) is believed to stimulate fibroblast growth factor (FGF) signaling by binding and stabilizing FGFs. In this report, we show that SOS induces FGF-dependent dimerization of FGF receptors (FGFRs). The crystal structure of the dimeric FGF2-FGFR1-SOS complex at 2.6-A resolution reveals a symmetric assemblage of two 1:1:1 FGF2-FGFR1-SOS ternary complexes. Within each ternary complex SOS binds to FGF and FGFR and thereby increases FGF-FGFR affinity. SOS also interacts with the adjoining FGFR and thereby promotes protein-protein interactions that stabilize dimerization. This structural finding is supported by the inability of selectively desulfated SOS molecules to promote receptor dimerization. Thus, we propose that SOS potentiates FGF signaling by imitating the dual role of heparin in increasing FGF-FGFR affinity and promoting receptor dimerization. Hence, the dimeric FGF-FGFR-SOS structure substantiates the recently proposed "two-end" model, by which heparin induces FGF-FGFR dimerization. Moreover, the FGF-FGFR-SOS structure provides an attractive template for the development of easily synthesized SOS-related heparin agonists and antagonists that may hold therapeutic potential.
Project description:Fibroblast growth factors (FGFs) participate in organ development and tissue maintenance, as well as the control of vascular function. The paracrine-acting FGFs are stored in the extracellular matrix, and their release is controlled by a secreted FGF-binding protein (FGF-BP, FGFBP1, and BP1) that modulates FGF receptor signaling. A genetic polymorphism in the human FGFBP1 gene was associated with higher gene expression and an increased risk of familial hypertension. Here, we report on the effects of inducible BP1 expression in a transgenic mouse model. Induction of BP1 expression in adult animals leads to a sustained rise in mean arterial pressure by >30 mm?Hg. The hypertensive effect of BP1 expression is prevented by candesartan, an angiotensin II (AngII) receptor antagonist, or by tempol, an inhibitor of reactive oxygen species. In vivo, BP1 expression sensitizes peripheral resistance vessels to AngII constriction by 20-fold but does not alter adrenergic vasoconstriction. FGF receptor kinase inhibition reverses the sensitization to AngII. Also, constriction of isolated renal afferent arterioles by AngII is enhanced after BP1 expression and blocked by FGF receptor kinase inhibition. Furthermore, AngII-mediated constriction of renal afferent arterioles is abolished in FGF2-/- mice but can be restored by add-back of FGF2 plus BP1 proteins. In contrast to AngII, adrenergic constriction is not affected in the FGF2-/- model. Proteomics and gene expression analysis of kidney tissues after BP1 induction show that MAPK (mitogen-activated protein kinase) signaling via MKK4 (MAPK kinase 4), p38, and JNK (c-Jun N-terminal kinase) integrates the crosstalk of the FGF receptor and AngII pathways and thus impact vascular tone and blood pressure.
Project description:Activation of fibroblast growth factor (FGF) signaling is initiated by a multiprotein complex formation between FGF, FGF receptor (FGFR), and heparan sulfate proteoglycan on the cell membrane. Cross-talk with other factors could affect this complex assembly and modulate the biological response of cells to FGF. We have previously demonstrated that anosmin-1, a glycosylated extracellular matrix protein, interacts with the FGFR1 signaling complex and enhances its activity in an IIIc isoform-specific and HS-dependent manner. The molecular mechanism of anosmin-1 action on FGFR1 signaling, however, remains unknown. Here, we show that anosmin-1 directly binds to FGFR1 with high affinity. This interaction involves domains in the N terminus of anosmin-1 (cysteine-rich region, whey acidic protein-like domain and the first fibronectin type III domain) and the D2-D3 extracellular domains of FGFR1. In contrast, anosmin-1 binds to FGFR2IIIc with much lower affinity and displays negligible binding to FGFR3IIIc. We also show that FGFR1-bound anosmin-1, although capable of binding to FGF2 alone, cannot bind to a FGF2.heparin complex, thus preventing FGFR1.FGF2.heparin complex formation. By contrast, heparin-bound anosmin-1 binds to pre-formed FGF2.FGFR1 complex, generating an anosmin-1.FGFR1.FGF2.heparin complex. Furthermore, a functional interaction between anosmin-1 and the FGFR1 signaling complex is demonstrated by immunofluorescence co-localization and Transwell migration assays where anosmin-1 was shown to induce opposing effects during chemotaxis of human neuronal cells. Our study provides molecular and cellular evidence for a modulatory action of anosmin-1 on FGFR1 signaling, whereby binding of anosmin-1 to FGFR1 and heparin can play a dual role in assembly and activity of the ternary FGFR1.FGF2.heparin complex.
Project description:Fibroblast growth factors (FGFs) participate in embryonic development, in maintenance of tissue homeostasis in the adult, and in various diseases. FGF-binding proteins (FGFBP) are secreted proteins that chaperone FGFs stored in the extracellular matrix to their receptor, and can thus modulate FGF signaling. FGFBP1 (alias BP1, FGF-BP1, or HBp17) expression is required for embryonic survival, can modulate FGF-dependent vascular permeability in embryos, and is an angiogenic switch in human cancers. To determine the function of BP1 in vivo, we generated tetracycline-regulated conditional BP1 transgenic mice. BP1-expressing adult mice are viable, fertile, and phenotypically indistinguishable from their littermates. Induction of BP1 expression increased mouse primary fibroblast motility in vitro, increased angiogenic sprouting into subcutaneous matrigel plugs in animals and accelerated the healing of excisional skin wounds. FGF-receptor kinase inhibitors blocked these effects. Healing skin wounds showed increased macrophage invasion as well as cell proliferation after BP1 expression. Also, BP1 expression increased angiogenesis during the healing of skin wounds as well as after ischemic injury to hindlimb skeletal muscles. We conclude that BP1 can enhance FGF effects that are required for the healing and repair of injured tissues in adult animals.
Project description:The biological response of cells to fibroblast growth factors (FGFs) depends on heparan sulphate glycosaminoglycans sharing particular structural motifs. Heparin induced FGF dimerization has been suggested to mediate receptor dimerization and activation. Here we demonstrate that heparin-derived oligosaccharides that promote receptor binding and activation specifically induce the dimerization of basic FGF (FGF2). These heparin-induced dimers of FGF2 acquire high affinity for receptor binding and are biologically active. Using biotinylated FGF2 bound to immobilized streptavidin gradually saturated with biotin, enabled a quantitative analysis of heparin-dependent and heparin-independent FGF2 monomers and oligomers. Streptavidin induced FGF2 dimers bind and activate FGF receptors only in the presence of heparin. An excess of streptavidin, forcing biotin-FGF2 into monomers, reduces receptor binding and blocks FGF-dependent cell proliferation. All these suggest predominant receptor binding and activation by heparin associated FGF2 oligomers. Unexpectedly, heparin induced dimers and higher order oligomers lose most of their affinity towards heparin. Direct binding of soluble FGF receptors (FGFRs) to either monomers or dimers of FGF2, immobilized on heparin, confirm the preferred association of FGFRs with dimers of FGF2. Computerized molecular docking predicts a cis-oriented FGF2 dimer, stabilized by heparin, which complies with all the experimental data.
Project description:Fibroblast growth factor (FGF) signalling is involved in a wide range of important biological activities with differential effects in various cell types. The activity of FGF is modulated by heparin/heparan sulphate-like glycosaminoglycans (HSGAGs), found both in the extracellular matrix and on the cell surface. HSGAGs affect FGF signalling by interacting with both the growth factor and the FGF receptor (FGFR). In this study we sought to investigate whether HSGAGs at the cell surface of bovine aortic endothelial cells (BAEC) and smooth muscle cells (SMC) can differentially modulate FGF signalling in these cell types and modulate their differential response to FGF. We find that SMC and BAEC express the same FGFR isoforms and bind FGF2 with equal affinity at the cell surface, yet FGF has a markedly higher proliferative effect on SMC than on BAEC. Isolated HSGAGs from these two cell types were found to elicit distinct patterns of proliferation in chlorate-treated cells. Furthermore, examination of focal sequences reveals that HSGAGs from SMC, but not those from BAEC, retain the sulphation pattern necessary to induce FGF2 activity. As such, the differences in FGF2-mediated proliferation can be explained by the distinct cell surface HSGAGs of the two cell types. We conclude that the focal sequences of cell surface HSGAGs from SMC and BAEC govern, at least in part, the differential activity of FGF2 on these two cell types.
Project description:Fibroblast growth factor (FGF) signaling is essential for normal and cancer biology. Mammalian FGF family members participate in multiple signaling pathways by binding to heparan sulfate and FGF receptors (FGFR) with varying affinities. FGF2 is the prototype member of the FGF family and interacts with its receptor to mediate receptor dimerization, phosphorylation, and activation of signaling pathways, such as Ras-MAPK and PI3K pathways. Excessive mitogenic signaling through the FGF/FGFR axis may induce carcinogenic effects by promoting cancer progression and increasing the angiogenic potential, which can lead to metastatic tumor phenotypes. Dysregulated FGF/FGFR signaling is associated with aggressive cancer phenotypes, enhanced chemotherapy resistance and poor clinical outcomes. In vitro experimental settings have indicated that extracellular FGF2 affects proliferation, drug sensitivity, and apoptosis of cancer cells. Therapeutically targeting FGF2 and FGFR has been extensively assessed in multiple preclinical studies and numerous drugs and treatment options have been tested in clinical trials. Diagnostic assays are used to quantify FGF2, FGFRs, and downstream signaling molecules to better select a target patient population for higher efficacy of cancer therapies. This review focuses on the prognostic significance of FGF2 in cancer with emphasis on therapeutic intervention strategies for solid and hematological malignancies.
Project description:The extracellular part of the fibroblast growth factor (FGF) receptor (FGFR) consists of up to three Ig modules (Ig1-Ig3), in which the Ig2 and Ig3 modules determine affinity and specificity for FGF and heparin. The FGFR isoforms lacking the Ig1 module have higher affinity for FGF and heparin than the triple Ig-module isoforms, suggesting that the Ig1 module is involved in the regulation of the FGFR-ligand interaction. We show here by surface plasmon resonance and NMR analyses that the Ig1 module binds to the Ig2 module, and identify by NMR the binding sites involved in the Ig1-Ig2 interaction. The identified binding site in the Ig2 module was found to be in the area of the FGF-Ig2 and Ig2-heparin contact sites, thus providing direct structural evidence that the Ig1 module functions as a competitive autoinhibitor of the FGFR-ligand interaction. Furthermore, the Ig1 binding site of the Ig2 module overlaps the Ig2-Ig2 contact site. This suggests that the function of the Ig1 module is not only regulation of the FGFR-ligand binding affinity but also prevention of spontaneous FGFR dimerization (through a direct Ig2-Ig2 interaction) in the absence of FGF.
Project description:Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-A resolution. FGF4 adopts a beta-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the betaC'-betaE loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O- and 6-O-sulfates in heparin to exert its optimal biological activity.