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.     

Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.     

Fibroblast growth factor receptor 3 induces gene expression primarily through Ras-independent signal transduction pathways

Fibroblast growth factor receptors (FGFR) are widely expressed in many tissues and cell types, and the temporal expression of these receptors and their ligands play important roles in the control of development. There are four FGFR family members, FGFR-1-4, and understanding the ability of these receptors to transduce signals is central to understanding how they function in controlling differentiation and development. We have utilized signal transduction by FGF-1 in PC12 cells to compare the ability of FGFR-1 and FGFR-3 to elicit the neuronal phenotype. In PC12 cells FGFR-1 is much more potent in the induction of neurite outgrowth than FGFR-3. This correlated with the ability of FGFR-1 to induce robust and sustained activation of the Ras-dependent mitogen-activated protein kinase pathways. In contrast, FGFR-3 could not induce strong sustained Ras-dependent signals. In this study, we analyzed the ability of FGFR-3 to induce the expression of sodium channels, peripherin, and Thy-1 in PC12 cells because all three of these proteins are known to be induced via Ras-independent pathways. We determined that FGFR-3 was capable of inducing several Ras-independent gene expression pathways important to the neuronal phenotype to a level equivalent of that induced by FGFR-1. Thus, FGFR-3 elicits phenotypic changes primarily though activation of Ras-independent pathways in the absence of robust Ras-dependent signals.     

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.     

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.     

FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates

Senescent cells do not proliferate in response to exogenous growth factors, yet the number and affinity of growth factor receptors on the cell surface appear to be similar to presenescent cell populations. To determine whether a defect in receptor signaling exists, we analyzed human umbilical vein endothelial cells (HUVEC) since HUVEC growth is absolutely dependent upon the presence of FGF. We report that in both presenescent and senescent HUVEC populations, FGF-1 induces the expression of cell cycle-specific genes, suggesting that functional FGF receptor (FGFR) may exist on the surface of these cells. However, the tyrosine phosphorylation of FGFR-1 substrates, Src and cortactin, is impaired in senescent HUVEC, and only the presenescent cell populations exhibit a FGF-1-dependent Src tyrosine kinase activity. Moreover, we demonstrate that senescent HUVEC are unable to migrate in response to FGF-1, and these data correlate with an altered organization of focal adhesion sites. These data suggest that the induction of gene expression is insufficient to promote a proliferative or migratory phenotype in senescent HUVEC and that the attenuation of the FGFR-1 signal transduction pathway may be involved in the inability of senescent HUVEC to proliferate and/or migrate.     

The phosphoprotein stathmin is essential for nerve growth factor- stimulated differentiation

Stathmin is a ubiquitous cytosolic protein which undergoes extensive phosphorylation in response to a variety of external signals. It is highly abundant in developing neurons. The use of antisense oligonucleotides which selectively block stathmin expression has allowed us to study directly its role in rat PC12 cells. We show that stathmin depletion prevents nerve growth factor (NGF)-stimulated differentiation of PC12 cells into sympathetic-like neurons although the expression of several NGF-inducible genes was not affected. Furthermore, we found that stathmin phosphorylation in PC12 cells which is induced by NGF depends on mitogen-activated protein kinase (MAPK) activity. We conclude that stathmin is an essential component of the NGF-induced MAPK signaling pathway and performs a key role during differentiation of developing neurons.     

Identification of the Cytoplasmic Regions of Fibroblast Growth Factor (FGF) Receptor 1 Which Play Important Roles in Induction of Neurite Outgrowth in PC12 Cells by FGF-1

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction revealed that the JM region of FGFR-1 conferred strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involved the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functioned primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis revealed that the JM region did not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR.     

Mapping of atypical protein kinase C within the nerve growth factor signaling cascade: relationship to differentiation and survival of PC12 cells

The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-iota was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-iota with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-iota by NGF. At the level of Raf-1, neither PKC-iota nor PI3 kinase was required for activation; however, PKC-iota could weakly activate MEK. Inhibitors of PKC-iota activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-iota were likewise required for NGF-induced NF-kappaB activation and cell survival, whereas Ras was not required for either survival or NF-kappaB activation but was required for differentiation. IKK existed as a complex with PKC-iota, Src and IkappaB. Consistent with a role for Src in regulating NF-kappaB activation, an absence of Src activity impaired recruitment of PKC-iota into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-kappaB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-kappaB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-iota and the NF-kappaB pathway.     

Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction.

Fibroblast growth factor (FGF) receptor (FGFR) gene family consists of at least four receptor tyrosine kinases that transduce signals important in a variety of developmental and physiological processes related to cell growth and differentiation. Here we have characterized the binding of different FGFs to FGFR-4. Our results establish an FGF binding profile for FGFR-4 with aFGF having the highest affinity, followed by K-FGF/hst-1 and bFGF. In addition, FGF-6 was found to bind to FGFR-4 in ligand competition experiments. Interestingly, the FGFR-4 gene was found to encode only the prototype receptor in a region where both FGFR-1 and FGFR-2 show alternative splicing leading to differences in their ligand binding specificities and to secreted forms of these receptors. Ligands binding to FGFR-4 induced receptor autophosphorylation and phosphorylation of a set of cellular polypeptides, which differed from those phosphorylated in FGFR-1-expressing cells. Specifically, the FGFR-1-expressing cells showed a considerably more extensive tyrosine phosphorylation of PLC-gamma than the FGFR-4-expressing cells. Structural and functional specificity within the FGFR family exemplified by FGFR-4 may help to explain how FGFs perform their diverse functions.     

p38 mitogen-activated protein kinase activation is required for fibroblast growth factor-2-stimulated cell proliferation but not differentiation.

Basic fibroblast growth factor (FGF-2) is a member of a family of polypeptides that have roles in a wide range of biological processes. To determine why different cell types show distinct responses to treatment with FGF-2, the array of FGF receptors present on the surface of a cell which differentiates in response to FGF-2 (PC12 cells) was compared with that present on the surface of a cell that proliferates in response to FGF-2 (Swiss 3T3 fibroblasts). Both cell types express exclusively FGFR1, suggesting that there are cell type-specific FGFR1 signaling pathways. Since mitogen-activated protein kinases function as mediators of cellular responses to a variety of stimuli, the roles of these proteins in FGF-mediated responses were examined. FGF-2 activates extracellular signal-regulated kinases with similar kinetics in both fibroblasts and PC12 cells, and a specific inhibitor of extracellular signal-regulated kinase activation blocks differentiation but has little effect on proliferation. In contrast, while p38 mitogen-activated protein kinase is activated weakly and transiently in PC12 cells treated with FGF-2, a much stronger and sustained activation of this kinase is seen in FGF-2-treated fibroblasts. Furthermore, specific inhibitors of this kinase block proliferation but have no effect on differentiation. This effect on proliferation is specific for FGF-2 since the same concentrations of inhibitors have little or no effect on proliferation induced by serum.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.     

Characterization of FGF receptor expression in human neutrophils and their contribution to chemotaxis

Several members of the fibroblast growth factor (FGF) family are potent endothelial cell (EC) mitogens and angiogenic factors, and their activities can be mediated by four tyrosine kinase receptors (FGFR1-4). In addition, FGFs can induce the release of inflammatory mediators by ECs and the expression of adhesion molecules at their surface, thereby favoring the recruitment and transvascular migration of inflammatory cells such as neutrophils. Neither the expression nor the biological activities that could be mediated by FGFRs have been investigated in human neutrophils. By biochemical and cytological analyses, we observed that purified circulating human neutrophils from healthy individuals expressed varying levels of FGFRs in their cytosol and at their cytoplasmic membrane. FGFR-2 was identified as the sole cell surface receptor, with FGFR-1 and -4 localizing in the cytosol and FGFR-3 being undetectable. We assessed the capacity of FGF-1 and FGF-2 to induce neutrophil chemotaxis in a modified Boyden microchamber and observed that they increase neutrophil transmigration at 10(-10) and 10(-9) M and by 1.77- and 2.34-fold, respectively, as compared with PBS-treated cells. Treatment with a selective anti-FGFR-2 antibody reduced FGF-1-mediated chemotaxis by 75% and abrogated the effect of FGF-2, while the blockade of FGFR-1 and -4 partially inhibited (15-40%) FGF-chemotactic activities. In summary, our data are the first to report the expression of FGF receptors in human neutrophils, with FGF-1 and FGF-2 promoting neutrophil chemotaxis mainly through FGFR-2 activation.     

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.     

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.