Alternative splicing affecting a novel domain in the C. elegans EGL-15 FGF receptor confers functional specificity

Fibroblast growth factor (FGF) receptors trigger a wide variety of cellular responses as diverse as cell migration, cell proliferation and cell differentiation. However, the molecular basis of the specificity of these responses is not well understood. The C. elegans FGF receptor EGL-15 similarly mediates a number of different responses, including transducing a chemoattractive signal and mediating an essential function. Analysis of the migration-specific alleles of egl-15 has identified a novel EGL-15 isoform that provides a molecular explanation for the different phenotypic effects of lesions at this locus. Alternative splicing yields two EGL-15 proteins containing different forms of a domain located within the extracellular region of the receptors immediately after the first IG domain. Neither of these two domain forms is found in any other FGF receptor. We have tested the roles of these EGL-15 receptor isoforms and their two FGF ligands for their signaling specificity. Our analyses demonstrate different physiological functions for the two receptor variants. EGL-15(5A) is required for the response to the FGF chemoattractant that guides the migrating sex myoblasts to their final positions. By contrast, EGL-15(5B) is both necessary and sufficient to elicit the essential function mediated by this receptor.     

Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerization-dependent manner

Adiponectin, an adipocyte-specific secretory protein, is present in serum as three oligomeric complexes. Apart from its roles as an anti-diabetic and anti-atherogenic hormone, adiponectin has been implicated as an important regulator of cell growth and tissue remodeling. Here we show that some of these functions might be mediated by the specific interactions of adiponectin with several important growth factors. Among six different growth factors examined, adiponectin was found to bind with platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (FGF), and heparin-binding epidermal growth factor-like growth factor (HB EGF) with distinct affinities. The bindings of adiponectin with these growth factors are oligomerization-dependent. PDGF-BB bound to the high molecular weight (HMW) and middle molecular weight (MMW) complexes, but not to the low molecular weight (LMW) complex of adiponectin. Basic FGF preferentially interacted with the HMW form, whereas HB EGF bound to all three forms with comparable affinities. These three growth factors did not compete with each other for their bindings to adiponectin, suggesting the involvement of distinct binding sites. The interactions of adiponectin with PDGF-BB, basic FGF, and HB EGF precluded the bindings to their respective membrane receptors and attenuated the DNA synthesis and cell proliferation induced by these growth factors. Small interfering RNA-mediated down-regulation of adiponectin receptors did not affect the suppressive effects of adiponectin on cell proliferation stimulated by these growth factors. These data collectively suggest that the oligomeric complexes of adiponectin can modulate the biological actions of several growth factors by controlling their bioavailability at a pre-receptor level and that this effect might partly account for the anti-atherogenic, anti-angiogenic, and anti-proliferative functions of adiponectin.     

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120ctn, also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.     

Pivotal Role of Translokin/CEP57 in the Unconventional Secretion Versus Nuclear Translocation of FGF2

Intracellular trafficking of fibroblast growth factor 2 (FGF2) exhibits two unusual features: (i) it is secreted despite the lack of signal peptide and (ii) it can translocate to the nucleus after interaction with high- and low-affinity receptors on the cell surface, although it does not possess any classical nuclear localization signal. This nuclear translocation constitutes an important part of the response to the growth factor. Previously, we identified Translokin/CEP57, an FGF2 binding partner, as an intracellular mediator of FGF2 trafficking, which is essential for the nuclear translocation of the growth factor. Here, we report the identification of four Translokin partners: sorting nexin 6, Ran-binding protein M and the kinesins KIF3A and KIF3B. These proteins, through their interaction with Translokin, are involved in two exclusive complexes allowing the bidirectional trafficking of FGF2. Thus, Translokin plays a pivotal role in this original mechanism. In addition, we show that FGF2 secretion is regulated by a negative loop, retro-controlled by FGF receptor and involving FGF2 itself.     

Fibroblast growth factor 3, a protein with a dual subcellular fate, is interacting with human ribosomal protein S2

The secreted isoform of fibroblast growth factor 3 (FGF3) induces a mitogenic cell response, while the nuclear form inhibits cell proliferation. Recently, we identified a nucleolar FGF3-binding protein which is implicated in processing of pre-rRNA as a possible target of nuclear FGF3 signalling. Here, we report a second candidate protein identified by a yeast two-hybrid screen for nuclear FGF3 action, ribosomal protein S2, rpS2. Recombinant rpS2 binds to in vitro translated FGF3 and to nuclear FGF3 extracted from transfected COS-1 cells. Characterization of the FGF3 binding domain of rpS2 showed that both the Arg-Gly-rich N-terminal region and a short carboxyl-terminal sequence of rpS2 are necessary for FGF3 binding. Mapping the S2 binding domains of FGF3 revealed that these domains are important for both NoBP and rpS2 interaction. Transient co-expression of rpS2 and nuclear FGF3 resulted in a reduced nucleolar localization of the FGF. These findings suggest that the nuclear form of FGF3 inhibits cell proliferation by interfering with ribosomal biogenesis.     

Horizontal cells invest retinal capillaries in the tree shrew Tupaia belangeri

Fibroblast growth factor (FGF) receptors constitute a family of four membrane-spanning tyrosine kinases (FGFR1-4) which serve as high-affinity receptors for 17 growth factors (FGF1-17). To study functions of FGF/ FGFR signals in development, mice that carry mutations in each receptor have been created by gene targeting. Analysis of these mutant mice revealed essential functions of FGF receptors in multiple biological processes, including mesoderm induction and patterning, cell growth and migration, organ formation and bone growth. In this review we discuss recent work with FGF receptors to illustrate mechanisms, through which the FGF/FGFR signals specify vertebrate limb initiation, outgrowth and patterning.     

Intracellular partners of fibroblast growth factors 1 and 2 - implications for functions

Fibroblast growth factors 1 and 2 (FGF1 and FGF2) are mainly considered as ligands of surface receptors through which they regulate a broad spectrum of biological processes. They are secreted in non-canonical way and, unlike other growth factors, they are able to translocate from the endosome to the cell interior. These unique features, as well as the role of the intracellular pool of FGF1 and FGF2, are far from being fully understood. An increasing number of reports address this problem, focusing on the intracellular interactions of FGF1 and 2. Here, we summarize the current state of knowledge of the FGF1 and FGF2 binding partners inside the cell and the possible role of these interactions. The partner proteins are grouped according to their function, including proteins involved in secretion, cell signaling, nucleocytoplasmic transport, binding and processing of nucleic acids, ATP binding, and cytoskeleton assembly. An in-depth analysis of the network of these binding partners could indicate novel, non-classical functions of FGF1 and FGF2 and uncover an additional level of a fine control of the well-known FGF-regulated cellular processes.     

Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells

Previous studies have established that embryonal carcinoma (EC) cells produce several different growth factors, but express few, if any, receptors for epidermal growth factor, platelet-derived growth factor, or transforming growth factor type-beta. In this study, the production and utilization of fibroblast growth factor (FGF) by EC cells and their differentiated cells were investigated. We have determined that EC cells produce a heat-labile, heparin-binding factor that competes with FGF for binding to membrane receptors and appears to be immunologically related to FGF. The same or a similar factor is produced by three different EC cell lines, including a multipotent human EC cell line. However, production of this factor is apparently reduced when each EC cell line differentiates. Unlike the parental EC cells, the differentiated cells respond to FGF by growth stimulation and the growth responses to FGF correlate with increased binding of FGF. Although the binding data indicate that both the EC cells and their differentiated cells exhibit high affinity receptors for FGF, the differentiated cells express these receptors at levels approximately 10-fold higher. These findings suggest that the FGF-related growth factor could influence the growth of EC cells or their differentiated cells.     

Growth factor-induced shedding of syndecan-1 confers glypican-1 dependence on mitogenic responses of cancer cells

The cell surface heparan sulfate proteoglycan (HSPG) glypican-1 is up-regulated by pancreatic and breast cancer cells, and its removal renders such cells insensitive to many growth factors. We sought to explain why the cell surface HSPG syndecan-1, which is also up-regulated by these cells and is a known growth factor coreceptor, does not compensate for glypican-1 loss. We show that the initial responses of these cells to the growth factor FGF2 are not glypican dependent, but they become so over time as FGF2 induces shedding of syndecan-1. Manipulations that retain syndecan-1 on the cell surface make long-term FGF2 responses glypican independent, whereas those that trigger syndecan-1 shedding make initial FGF2 responses glypican dependent. We further show that syndecan-1 shedding is mediated by matrix metalloproteinase-7 (MMP7), which, being anchored to cells by HSPGs, also causes its own release in a complex with syndecan-1 ectodomains. These results support a specific role for shed syndecan-1 or MMP7-syndecan-1 complexes in tumor progression and add to accumulating evidence that syndecans and glypicans have nonequivalent functions in vivo.     

Proteoglycans: many forms and many functions.

Proteoglycans are produced by most eukaryotic cells and are versatile components of pericellular and extracellular matrices. They belong to many different protein families. Their functions vary from the physical effects of the proteoglycan aggrecan, which binds with link protein to hyaluronan to form multimolecular aggregates in cartilage; to the intercalated membrane protein CD44 that has a proteoglycan form and is a receptor and a cell-binding site for hyaluronan; to heparan sulfate proteoglycans of the syndecan and other families that provide matrix binding sites and cell-surface receptors for growth factors such as fibroblast growth factor (FGF). One feature that recurs in proteoglycan biology is that their structure is open to extensive modulation during cellular expression. Examples of protein changes are known, but a major source of structural variation is in the glycosaminoglycan chains. The number of chains and their length can vary, as well as their pattern of sulfation. This may result in the switching of different chain types with different properties, e.g., chondroitin sulfate and heparan sulfate, and it may also result in the selective expression of sulfated chain sequences that have specific functions. The control of glycosaminoglycan structure is not well understood, but it does appear to be used to change the properties of proteoglycans to suit different biological needs. Proteoglycan forms of proteins are thus important modifiers of the organization of the pericellular and extracellular matrices and modulators of the processes that occur there.     

The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis.

During development of the Drosophila tracheal (respiratory) system, the cell bodies and apical and basal surfaces of the tracheal epithelium normally move in concert as new branches bud and grow out to form tubes. We show that mutations in the Drosophila ribbon (rib) gene disrupt this coupling: the basal surface continues to extend towards its normal targets, but movement and morphogenesis of the tracheal cell bodies and apical surface is severely impaired, resulting in long basal membrane protrusions but little net movement or branch formation. rib mutant tracheal cells are still responsive to the Branchless fibroblast growth factor (FGF) that guides branch outgrowth, and they express apical membrane markers normally. This suggests that the defect lies either in transmission of the FGF signal from the basal surface to the rest of the cell or in the apical cell migration and tubulogenesis machinery. rib encodes a nuclear protein with a BTB/POZ domain and Pipsqueak DNA-binding motif. It is expressed in the developing tracheal system and other morphogenetically active epithelia, many of which are also affected in rib mutants. We propose that Rib is a key regulator of epithelial morphogenesis that promotes migration and morphogenesis of the tracheal cell bodies and apical surface and other morphogenetic movements.     

Basic fibroblast growth factor and its receptors in human embryonic stem cells

Human embryonic stem cells (hESCs) are pluripotent stem cells with long-lasting capacity to self-renew and differentiate into various cell types of endodermal, ectodermal or mesodermal origin. Unlike mouse ESCs (mESCs), which can be maintained in an undifferentiated state simply by adding leukemia inhibitory factor (LIF) into the culture medium, hESCs are notorious for the sustained willingness to differentiate and not yet clearly defined signaling pathways that are crucial for their "stemness". Presently, our knowledge involves only limited number of growth factor signaling pathways that appear to be biologically relevant for stem cell functions in vitro. These include BMP, TGFbeta, Wnt, and FGF signaling pathway. The purpose of this review is to summarize recent data on the expression of FGFs and their receptors in hESCs, and critically evaluate the potential effects of FGF signals for their undifferentiated growth and/or differentiation in context with our current understanding of FGF/FGFR biology.     

Localization of basic fibroblast growth factor to the developing capillaries of the bovine retina

The basic fibroblast growth factor (FGF) is a potent mitogen that has vascular endothelium as one of its principle target cells. Recent work has provided both the complete amino acid sequence of basic FGF and the nucleotide sequence of the genes for both human and bovine basic FGF. Although capillary endothelial cells have been shown to produce basic FGF in vitro and to deposit basic FGF in their extracellular matrix in vitro as well, no direct evidence yet exists for the distribution of basic FGF in vivo. Antipeptide antibodies were prepared against a 15-amino-acid sequence from the amino terminus of basic FGF in order to avoid cross-reactivity with acidic FGF, a protein with 55% overall homology to basic FGF. After affinity purification, these antisera were used to localize the basic fibroblast growth factor in the fetal and adult bovine retina. Immunoreactive material was found in capillaries of the inner nuclear layer, a capillary network undergoing development during the third trimester in the fetal bovine eye. Although the resolution of the technique does not permit a unique assignment of cellular localization, the presence of stain immediately adjacent to the lumen of capillaries suggests that capillary endothelial cells may produce the basic fibroblast growth factor in vivo during vascular development.     

成纤维细胞生长因子18(FGF18)的研究进展

成纤维细胞生长因子18 (fibroblast growth factor 18,FGF18)是成纤维细胞生长因子家族( FGFs)的成员之一.研究发现,FGF 18不仅在骨骼发育和生长期对软骨形成和成骨生成起着重要的作用,其功能也已延伸至其他许多生物过程,尽管对FGF18作为一个有用治疗靶点发挥作用的功能和机制仍有待进一步的发现及研究.现针对FGF18的特点,及其在骨骼发育中的功能,特别其在未来具有潜在应用领域上的研究进展进行综述.     

Sensitized genetic backgrounds reveal a role for C. elegans FGF EGL-17 as a repellent for migrating CAN neurons

Although many molecules are necessary for neuronal cell migrations in C. elegans, no guidance cues are known to be essential for any of these cells to migrate along the anteroposterior (AP) axis. We demonstrate that the fibroblast growth factor (FGF) EGL-17, an attractant for the migrating sex myoblasts (SMs), repels the CANs, a pair of neurons that migrate posteriorly from the head to the center of the embryo. Although mutations in genes encoding EGL-17/FGF and a specific isoform of its receptor EGL-15/FGFR had little effect on CAN migration, they enhanced the CAN migration defects caused by mutations in other genes. Two cells at the anterior end of the embryo express EGL-17/FGF, raising the possibility that EGL-17/FGF functions as a repellent for migrating CANs. Consistent with this hypothesis, ectopic expression of EGL-17/FGF shifted the final CAN cell positions away from these novel sites of expression. Cell-specific rescue experiments demonstrated that EGL-15/FGFR acts in the CANs to promote their migration. We also found that the tyrosine phosphatase receptor CLR-1 regulates CAN migration by inhibiting EGL-15/FGFR signaling, and that the FGFR adaptor protein SEM-5/GRB2 may mediate EGL-15/FGFR signaling in CAN migration. Thus, EGL-17/FGF signaling through an EGL-15/FGFR isoform and possibly SEM-5/GRB2 mediates both attraction of the SMs and repulsion of the CANs. This study also raises the possibility that several guidance cues regulate cell migrations along the C. elegans AP axis, and their role in these migrations may only be revealed in sensitized genetic backgrounds.     

Nucleolar association of fibroblast growth factor 3 via specific sequence motifs has inhibitory effects on cell growth.

The dual subcellular fate of fibroblast growth factor 3 (FGF3) is determined by the competing effects of amino-terminal signals for nuclear localization and secretion (P. Kiefer, P. Acland, D. Pappin, G. Peters, and C. Dickson, EMBO J. 13:4126-4136, 1994). Mutation analysis has implicated additional basic domains in the carboxy-terminal region of the protein as necessary for nuclear uptake and the association of FGF3 with the nucleoli. Immunogold electron microscopy shows that FGF3 is predominantly within the dense fibrillar component of the nucleolus. A form of FGF3 that localizes exclusively in the nucleus and nucleolus was generated by removing signals for secretion, and expression of this nonsecreted FGF3 in a mammary epithelial cell line resulted in slowly growing colonies of enlarged cells. Thus, nuclear import and nucleolar association of FGF3 are determined by the concerted interaction of several distinct motifs, and the exclusive production of the nuclear isoform can inhibit DNA synthesis and cell proliferation.     

Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro

Spermatogonial stem cells (SSCs) support life-long spermatogenesis by self-renewing and producing spermatogonia committed to differentiation. In vitro, SSCs form three-dimensional spermatogonial aggregates (clusters) when cultured with glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2); serial passaging of clusters results in long-term SSC maintenance and expansion. However, the role of these growth factors in controlling patterns of SSC division and fate decision has not been understood thoroughly. We report here that in a short-term culture, GDNF and FGF2 increase the number of dividing SSCs, but not the total SSC number, compared to a no-growth-factor condition. Since the total germ cell number increases with growth factors, these results suggest that GDNF and FGF2 promote a SSC division pattern that sustains the size of the stem cell pool while generating committed progenitors. Our data also show that SSC numbers increase when the cluster structure is disintegrated and cell–cell interaction in clusters is disrupted. Collectively, these results suggest that in this culture system, GDNF and FGF2 stimulate SSC divisions that promote self-renewal and differentiation in the SSC population, and imply that the destruction of the cluster structure, a potential in vitro niche, may contribute to SSC expansion.     

Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line

The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.