Expression of EGF receptor and FGF receptor isoforms during neuroepithelial stem cell differentiation

To characterize the role of epidermal growth factor (EGF) and fibroblast growth factor (FGF) in regulating neuroepithelial stem cells differentiation, we have examined the expression of FGF, EGF, and their receptors by neuroepithelial (NEP) cells and their derivatives. Our results indicate that undifferentiated NEP cells express a subset of FGF receptor (FGFR) isoforms, but do not express platelet-derived growth factor receptors (PDGFRs) or epidermal growth factor receptor (EGFR). The FGFR pattern of expression by differentiated neuron and glial cells differs from that found on NEP stem cells. FGFR-4 is uniquely expressed on NEP cells, while FGFR-1 is expressed by both NEP cells and neurons, and FGFR-2 is down-regulated during neuronal differentiation. FGFRs present on astrocytes and oligodendrocytes also represent a subset of those present on NEP cells. Expression of FGF and EGF by NEP cells and their progeny was also examined. NEP cells synthesize detectable levels of both FGF-1 and FGF-2, and EGF. FGF-1 and FGF-2 synthesis is likely to be biologically relevant, as cells grown at high density do not require exogenous FGF for their survival and cells grown in the presence of neutralizing antibodies to FGF show a reduction in cell survival and division. Thus, neuroepithelial cells synthesize and respond to FGF, but not to EGF, and are therefore distinct from other neural stem cells (neurospheres). The unique pattern of expression of FGF isoforms may serve to distinguish NEP cells from their more differentiated progeny.     

FGF-8 stimulates neuronal differentiation through FGFR-4a and interferes with mesoderm induction in Xenopus embryos

The role of fibroblast growth factors (FGFs) in neural induction is controversial [1,2]. Although FGF signalling has been implicated in early neural induction [3-5], a late role for FGFs in neural development is not well established. Indeed, it is thought that FGFs induce a precursor cell fate but are not able to induce neuronal differentiation or late neural markers [6-8]. It is also not known whether the same or distinct FGFs and FGF receptors (FGFRs) mediate the effects on mesoderm and neural development. We report that Xenopus embryos expressing ectopic FGF-8 develop an abundance of ectopic neurons that extend to the ventral, non-neural, ectoderm, but show no ectopic or enhanced notochord or somitic markers. FGF-8 inhibited the expression of an early mesoderm marker, Xbra, in contrast to eFGF, which induced ectopic Xbra robustly and neuronal differentiation weakly. The effect of FGF-8 on neurogenesis was blocked by dominant-negative FGFR-4a (DeltaXFGFR-4a). Endogenous neurogenesis was also blocked by DeltaXFGFR-4a and less efficiently by dominant-negative FGFR-1 (XFD), suggesting that it depends preferentially on signalling through FGFR-4a. The results suggest that FGF-8 and FGFR-4a signalling promotes neurogenesis and, unlike other FGFs, FGF-8 interferes with mesoderm induction. Thus, different FGFs show specificity for mesoderm induction versus neurogenesis and this may be mediated, at least in part, by the use of distinct receptors.     

Evolutionarily conserved and divergent expression of members of the FGF receptor family among vertebrate embryos, as revealed by FGFR expression patterns in Xenopus

Fibroblast growth factors (FGFs) mediate many cell-cell signaling events during early development. While the actions of FGFs have been well-studied, the roles played by specific members of the FGF receptor (FGFR) family are poorly understood. To characterize the roles played by individual FGFRs we compared the regulation and expression of the three Xenopus FGFRs described to date (XFGFR-1, XFGFR-2, and XFGFR-4). First, we describe the expression of Xenopus FGFR-4; XFGFR-4 is present as a maternal mRNA and is found in the embryo through at least the tadpole stage. XFGFR-4 and XFGFR-1 mRNAs are present at comparable levels, arguing that both mediate FGF signaling during early development. Second, the expression of XFGFR-4 in animal caps differs from the expression of XFGFR-1 and XFGFR-2, suggesting that the FGFRs are independently regulated in ectoderm. Third, using whole-mount in situ hybridization, we show that XFGFR-1, XFGFR-2, and XFGFR-4 are expressed in dramatically different patterns, arguing that specific FGF signaling events are mediated by different members of the FGFR family. Among these, FGF signaling during the induction of neural crest cells is likely to be mediated by XFGFR-4. Comparison of our results with previously reported FGFR expression patterns reveals that FGFR-1 expression is highly conserved among vertebrate embryos, and FGFR-2 expression shows many features that are conserved and some that are divergent. In contrast, the expression pattern of FGFR-4 is highly divergent among vertebrate embryos. Received: 5 August 1999 / Accepted: 18 January 2000     

A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

Basic fibroblast growth factor (FGF-2) induces cell proliferation and urokinase-type plasminogen activator (uPA) production in fetal bovine aortic endothelial GM 7373 cells. In the present paper we investigated the role of the interaction of FGF-2 with tyrosine-kinase (TK) FGF receptors (FGFRs) in mediating uPA up-regulation in these cells. The results show that FGF-2 antagonists suramin, protamine, heparin, the synthetic peptide FGF-2(112-155), and a soluble form of FGFR-1 do not inhibit FGF-2-mediated uPA up-regulation at concentrations that affect growth factor binding to cell surface receptors and mitogenic activity. In contrast, tyrosine phosphorylation inhibitors and overexpression of a dominant negative TK- mutant of FGFR-1 abolish the uPA-inducing activity of FGF-2, indicating that FGFR and its TK activity are essential in mediating uPA induction. Accordingly, FGF-2 induces uPA up-regulation in Chinese hamster ovary cells transfected with wild-type FGFR-1, -2, -3, or -4 but not with TK- FGFR-1 mutant. Small unilamellar phosphatidyl choline:cholesterol vesicles loaded with FGF-2 increased uPA production in GM 7373 cells in the absence of a mitogenic response. Liposome-encapsulated FGF-2 showed a limited but significant capacity, relative to free FGF-2, to interact with FGFR both at 4 degrees C and 37 degrees C and to be internalized within the cell. uPA up-regulation by liposome-encapsulated FGF-2 was quenched by neutralizing anti-FGF-2 antibodies, indicating that the activity of liposome-delivered FGF-2 is mediated by an extracellular action of the growth factor. Taken together, the data indicate that a distinct interaction of FGF-2 with FGFR, quantitatively and/or qualitatively different from the one that leads to mitogenicity, is responsible for the uPA-inducing activity of the growth factor.     

Role of FGFs in skeletal muscle and limb development

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridgederived factor that participates in limb outgrowth and patterning. © 1994 Wiley-Liss, Inc.     

Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells.

The current paradigm for the role of nerve growth factor (NGF) or FGF-2 in the differentiation of neuronal cells implies their binding to specific receptors and activation of kinase cascades leading to the expression of differentiation specific genes. We examined herein the hypothesis that FGF receptors (FGFRs) are involved in NGF-induced neuritogenesis of pheochromocytoma-derived PC12 cells. We demonstrate that in PC12 cells, FGFR expression and activity are modulated upon NGF treatment and that a dominant negative FGFR-2 reduces NGF-induced neuritogenesis. Moreover, FGF-2 expression is modulated by NGF, and FGF-2 is detected at the cell surface. Oligonucleotides that specifically inhibit FGF-2 binding to its receptors are able to significantly reduce NGF-induced neurite outgrowth. Finally, the duration of mitogen-activated protein kinase (MAPK) activity upon FGF or NGF stimulation is shortened in FGFR-2 dominant negative cells through inactivation of signaling from the receptor to the Ras/MAPK pathway. In conclusion, these results demonstrate that FGFR activation is involved in neuritogenesis induced by NGF where it contributes to a sustained MAPK activity in response to NGF.     

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.     

Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2

Members of the FGF family of growth factors localize to the nuclei in a variety of different cell types. To determine whether FGF receptors are also present within nuclei and if this localization is regulated by FGFs, nuclei were prepared from quiescent and FGF-2-treated Swiss 3T3 fibroblasts and examined for the presence of FGF receptors by immunoblotting with an antibody produced against the extracellular domain of FGF receptor-1 (FGFR-1). Little or no FGFR-1 is detected in nuclei prepared from quiescent cells. When cells are treated with FGF- 2, however, there is a time- and dose-dependent increase in the association of FGFR-1 immunoreactivity with the nucleus. In contrast, treatment with either EGF or 10% serum does not increase the association of FGFR-1 with the nucleus. When cell surface proteins are labeled with biotin, a biotinylated FGFR-1 is detected in the nuclear fraction prepared from FGF-2-treated, but not untreated, cells indicating that the nuclear-associated FGFR-1 immunoreactivity derives from the cell surface. The presence of FGFR-1 in the nuclei of FGF-2- treated cells was confirmed by immunostaining with a panel of different FGFR-1 antibodies, including one directed against the COOH-terminal domain of the protein. Fractionation of nuclei from FGF-2-treated cells indicates that nuclear FGFR-1 is localized to the nuclear matrix, suggesting that the receptor may play a role in regulating gene activity.     

Selectively desulfated heparin inhibits fibroblast growth factor-induced mitogenicity and angiogenesis

Fibroblast growth factors (FGFs) are known to induce formation of new blood vessels, angiogenesis. We show that FGF-induced angiogenesis can be modulated using selectively desulfated heparin. Chinese hamster ovary cells (CHO677) deficient in heparan sulfate biosynthesis were employed to assess the function of heparin/heparan sulfate in FGF receptor-1 (FGFR-1) signal transduction and biological responses. In the presence of FGF-2, FGFR-1 kinase and subsequent mitogen-activated protein kinase Erk2 activities were augmented in a dose-dependent manner, whereas high concentrations of heparin resulted in decreased activity. The length of the heparin oligomer, minimally an 8/10-mer, was critical for the ability to enhance FGFR-1 kinase activity. The N- and 2-O-sulfate groups of heparin were essential for binding to FGF-2, whereas stimulation of FGFR-1 and Erk2 kinases by FGF-2 also required the presence of 6-O-sulfate groups. Sulfation at 2-O- and 6-O-positions was moreover a prerequisite for binding of heparin to a lysine-rich peptide corresponding to amino acids 160-177 in the extracellular domain of FGFR-1. Selectively 6-O-desulfated heparin, which binds to FGF-2 but fails to bind the receptor, decreased FGF-2-induced proliferation of CHO677 cells, presumably by displacing intact heparin. Furthermore, FGF-2-induced angiogenesis in chick embryos was inhibited by 6-O-desulfated heparin. Thus, formation of a ternary complex of FGF-2, heparin, and FGFR-1 appears critical for the activation of FGFR-1 kinase and downstream signal transduction. Preventing complex formation by modified heparin preparations may allow regulation of FGF-2 functions, such as induction of angiogenesis.     

Molecular cloning and developmental expression of rat fibroblast growth factor receptor 3

Fibroblast growth factors (FGFs) are involved in the control of a variety of biological functions including regulation and differentiation of various cell types. Furthermore, they play important roles in the processes of regeneration, angiogenesis, and chemotaxis. The family of FGF receptors (FGFRs) comprises four members, FGFR-1 to -4, which exist in several differentially expressed splice variants. Except for FGFR-3, primary structures and expression of the three other FGFRs have been described in the rat system. Although expression studies with heterologous probes of FGFR-3 from mice have been performed in the rat system, these analyses were limited and the complete set of receptors has not yet been revealed. To understand the developmental functions of FGFR-3, it is important to elucidate the expression pattern in embryos of different stages. In this study, we have isolated a cDNA of FGFR-3 from rat brain. Expression analyses by RT-PCR of adult rat revealed expression in several tissues, however, expression levels were highest in lung and brain. During embryonic development, FGFR-3 displays a diffuse expression in most tissues at embryonic day 14 (E14), as observed by in situ hybridization experiments. In E18 the expression pattern is more restricted, showing strong signals in spinal cord, dorsal root ganglia, cortex, chondrocytes, and endothelial cells. The temporal and spatial pattern of FGFR-3 expression suggests specific functions in several tissues during development.     

The distribution of the growth factors FGF-2 and VEGF, and their receptors, in growing red deer antler

The cellular distributions of the growth factors FGF-2 and VEGF, and their receptors FGFR1, FGFR2 and FGFR3, and VEGFR-2 respectively, were visualized by immunohistochemistry and light microscopy in sections of growing red deer antler. Both of these signalling systems were widely expressed in the integument and osteocartilaginous compartments. FGF-2 was found in the same cells as all three FGFRs, indicating that FGF signalling may be principally autocrine. The patterns of labelling for VEGF and its receptor were similar to those seen for FGF-2 and FGFR-3, in both compartments. Our data are consistent with the findings of others in suggesting that FGF-2 induces expression of VEGF, to stimulate and maintain high rates of neovascularisation and angiogenesis, thereby providing nutrients to both velvet and bone as they rapidly grow and develop. The presence of FGF and VEGF and their receptors in epithelial cells suggests that these signalling systems play a role in skin development, raising the possibility that one or both may be involved in the close coupling of the coordinated growth of the integument and osteocartilage of antler, a process which is poorly understood at present.     

Thrombin-induced chemotaxis and aggregation of neutrophils

Thrombin-induced neutrophil chemotaxis and aggregation were studied using cells isolated from either human or sheep blood. Sheep neutrophils (10(8) cells/ml) exhibited maximum chemotactic migration towards 10(-8)M human alpha-thrombin, 10(-8)M gamma-thrombin (which lacks the fibrinogen site), and 10(-12)MD-Phe-Pro-Arg-CH2-alpha-thrombin (catalytically inactive thrombin). Chemotactic responses of the same magnitude were obtained with human neutrophils (10(8) cells/ml). The chemotactic responses to thrombin were comparable to those obtained with diluted (1:200 v/v) zymosan activated serum (ZAS) and 10(-11)M FMLP. Premixing of the thrombin forms with hirudin in 1:1 stoichiometric amounts abolished the chemotaxis but not chemokinesis Aggregatory responses of human and sheep neutrophils were comparable for ZAS, alpha-thrombin, and gamma-thrombin. The responses of both human and sheep neutrophils to D-Phe-Pro-Arg-CH2-alpha-thrombin were attenuated, indicating that the proteolytic site may be involved in the aggregatory response. The results suggest that thrombin-induced neutrophil chemotaxis and aggregation are mediated by different mechanisms, since chemotaxis is a catalytically independent response whereas aggregation is an active site independent response.     

Normal Human Fibroblasts Produce Membrane-Bound and Soluble Isoforms of FGFR-1

Fibroblast growth factors (FGFs) are polypeptide mitogens for a wide variety of cell types and are involved in other processes such as angiogenesis and cell differentiation. FGFs mediate their biological responses by activating high-affinity tyrosine kinase receptors. Currently, there are four human fibroblast growth factor receptor (FGFR) genes. To investigate the mechanisms by which αFGF and βFGF may mediate mitogenic signal transduction in human skin-derived fibroblasts, we analyzed these cells for the presence of high-affinity FGFRs. We show that normal human dermal fibroblasts express a single high-affinity FGFR gene, FGFR-1. Cloning and sequencing of two distinct FGFR-1 cDNAs suggested that normal human dermal fibroblasts express a membrane-bound and a putatively secreted form of FGFR-1. We show that normal human dermal fibroblasts produce two FGFR-1 proteins, one of which exists in conditioned media. The mRNA for the putatively secreted form of FGFR-1 appears to be down-regulated by serum treatment of the cells.     

Fibroblast growth factor receptors have different signaling and mitogenic potentials.

Fibroblast growth factor (FGF) receptors (FGFRs) are structurally related receptor protein tyrosine kinases encoded by four distinct genes. Activation of FGFR-1, -2, and -3 by FGFs induces mitogenic responses in various cell types, but the mitogenic potential of FGFR-4 has not been previously explored. We have compared the properties of BaF3 murine lymphoid cells and L6 rat myoblast cells engineered to express FGFR-1 or FGFR-4. Acidic FGF binds with high affinity to and elicits tyrosine phosphorylation of FGFR-1 or FGFR-4 receptors displayed on BaF3 cells, but only FGFR-1 activation leads to cell survival and growth. FGFR-4 activation also fails to elicit detectable signals characteristic of the FGFR-1 response: tyrosine phosphorylation of SHC and extracellular signal-related kinase (ERK) proteins and induction of fos and tis11 RNA expression. The only detected response to FGFR-4 activation was weak phosphorylation of phospholipase C gamma. A chimeric receptor containing the extracellular domain of FGFR-4 and the intracellular domain of FGFR-1 confers FGF-dependent growth upon transfected BaF3 cells, demonstrating that the intracellular domains of the receptors dictate their functional capacity. Activation of FGFR-1 in transfected L6 myoblasts induced far stronger phosphorylation of phospholipase C gamma, SHC, and ERK proteins than could activation of FGFR-4 in L6 cells, and only FGFR-1 activation induced tyrosine phosphorylation of a characteristic 80-kD protein. Hence, the signaling and biological responses elicited by different FGF receptors substantially differ.     

Cell transformation by fibroblast growth factors can be suppressed by truncated fibroblast growth factor receptors.

Ligand-induced dimerization and transphosphorylation are thought to be important events by which receptor tyrosine kinases generate cellular signals. We have investigated the ability of signalling-defective, truncated fibroblast growth factor (FGF) receptors (FGFR-1 and FGFR-2) to block the FGF response in cells that express both types of endogenous FGF receptors. When these dominant negative receptors are expressed in NIH 3T3 cells transformed by the secreted FGF-4, the transformed properties of the cells can be reverted to various degrees, with better reversion phenotype correlating with higher levels of truncated receptor expression. Furthermore, truncated FGFR-2 is significantly more efficient at producing reversion than FGFR-1, indicating that FGF-4 preferentially utilizes the FGFR-2 signalling pathway. NIH 3T3 clones expressing these truncated receptors are more resistant to FGF-induced mitogenesis and also exhibit reduced tyrosine phosphorylation upon treatment with FGF. The block in FGF-signalling, however, can be overcome by the addition of excess growth factor. The truncated receptors have binding affinities that are four- to eightfold lower than those of wild-type receptors, as measured by Scatchard analysis. We also observed a partial specificity in the responses of truncated-receptor-expressing clones to FGF-2 or FGF-4. Our results suggest that the block to signal transduction produced by kinase-negative FGF receptors is achieved through a combination of dominant negative effects and competition for growth factor binding with functional receptors.     

Heparan and chondroitin sulfate on growth plate perlecan mediate binding and delivery of FGF-2 to FGF receptors.

Fibroblast growth factor (FGF)-2 regulates chondrocyte proliferation in the growth plate. Heparan sulfate (HS) proteoglycans bind FGF-2. Perlecan, a heparan sulfate proteoglycan (HSPG) in the developing growth plate, however, contains both HS and chondroitin sulfate (CS) chains. The binding of FGF-2 to perlecan isolated from the growth plate was evaluated using cationic filtration (CAF) and immunoprecipitation (IP) assays. FGF-2 bound to perlecan in both the CAF and IP assays primarily via the HS chains on perlecan. A maximum of 123 molecules of FGF-2 was calculated to bind per molecule of perlecan. When digested with chondroitinase ABC to remove its CS chains, perlecan augmented binding of FGF-2 to the FGFR-1 and FGFR-3 receptors and also increased FGF-2 stimulation of [(3)H]-thymidine incorporation in BaF3 cells expressing these FGF receptors. These data show that growth plate perlecan binds to FGF-2 by its HS chains but can only deliver FGF-2 to FGF receptors when its CS chains are removed.