Presence Of FGF-2 Research Articles

Generation of purified oligodendrocyte progenitors from embryonic stem cells.

Demyelination is a key component in the pathogenesis of many neurological disorders. Transplantation of myelinating cells may offer a therapeutic approach to restore neurological function in these diseases. Recent findings suggest that pluripotent embryonic stem (ES) cells can serve as an unlimited donor source for neural transplantation. The clinical application of ES cells for myelin repair will depend critically on the ability to enrich oligodendroglial progenitors in high purity. Combining controlled differentiation in the presence of growth factors and genetic lineage selection, we devised a cell culture protocol yielding highly purified oligodendrocyte progenitors. Murine ES cell clones stably transfected with a construct encoding the beta-galactosidase-neomycine phosphotransferase fusion protein (beta(geo)) under control of the 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter were differentiated into bipotential glial precursors. Subsequent induction of a CNP-positive stage and selection in neomycine resulted in a homogenous cell population with a pre-oligodendrocyte phenotype. The selected cells continued to proliferate in the presence of FGF-2 and PDGF and, upon growth factor withdrawal, differentiated into mature galactocerebroside (GalC)-positive oligodendrocytes. Transplantation studies in myelin-deficient (md) rats indicate that ES cell-derived oligodendrocyte progenitors generated with this method may serve as an attractive donor source for myelin repair.

Expression pattern of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) system members in bovine corpus luteum endothelial cells during treatment with FGF-2, VEGF or oestradiol.

The development of the corpus luteum (CL) is accompanied by very active angiogenesis. We hypothesize that during this process endothelial cells (EC) are under the control of several angiogenic factors and steroids. The aim of this study was to examine the expression of the angiogenic growth factor systems - fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) - in EC derived from the bovine CL. Endothelial cells were cultured in serum-free medium and treated for 24 h with different concentrations of oestradiol (range from 10(-13) to 10(-5) mol/l), VEGF or FGF-2 (1, 10 and 100 ng/ml, respectively) and compared with untreated controls. Cells were harvested, total RNA extracted and subjected to semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Treatment with oestradiol or FGF-2 stimulated the expression of FGF-2, but VEGF treatment showed no effect on the FGF-2 expression. FGF-2 or VEGF treatment resulted in an up-regulation of the FGF receptor (FGFR) mRNA. However, no FGF-1 expression was detected in EC. For the VEGF system, treatment with FGF-2, VEGF or oestradiol did not affect VEGF expression. However, the presence of FGF-2 in the medium up-regulated the expression of both VEGF receptors (VEGFR-1 and VEGFR-2), whereas oestradiol or VEGF treatment showed no effect on the expression of these receptors. Our results reveal that functional angiogenic growth factor systems were expressed in vitro in bovine EC derived from the CL. This suggests that the angiogenic FGF and VEGF system members were regulated by FGF or VEGF, but not by oestradiol-17beta.

Synthesis of disaccharides derived from heparin and evaluation of effects on endothelial cell growth and on binding of heparin to FGF-2

The disaccharide β- d-GlcA-(1→4)-α- d-GlcNAc-1→OMe and other small nonsulfated oligosaccharides related to heparin/heparan sulfate have been shown to bind to FGF and activated the fibroblast growth factor (FGF) signalling pathway in (F32) cells expressing the FGF receptor. Synthetic routes to β- d-GlcA-(1→4)-α- d-GlcNAc-1→OMe and a glucose analogue β- d-Glc-(1→4)-α- d-GlcNAc-1→OMe are described. The effects of these disaccharides on endothelial cell growth, which is relevant to angiogenesis, were evaluated and it was found they did not mimic the inhibitory effects that were observed for heparin albumin (HA) and that have also been observed by monosaccharide conjugates. They did not alter bovine aortic endothelial cell (BAEC) proliferation, in the presence of FGF-2 in serum free medium or in absence of FGF-2 in serum free and complete medium. Disaccharides (10 μg/mL) reduced by 25–31% the inhibition caused by HA (10 μg/mL) on BAEC growth in serum-free medium but had no effect in complete medium. There was no evidence obtained for the binding of these oligosaccharides to FGF-2 in competition with HA by ELISA.

The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells.

Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Here, we show that Wnt signaling inhibits the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of beta-catenin in mouse cortical NPC cultures induced neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling led to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the beta-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized beta-catenin did not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development.

Fibroblast Growth Factor Enriches the Embryonic Liver Cultures for Hepatic Progenitors

Fibroblast growth factors (FGFs) play an important role in hepatic induction during development. The aim of our study was to investigate the effect of exogenous FGFs on ex vivo liver development. We begin our analysis by examining FGF signaling during early mouse liver development. Phospho-FGF receptor (Tyr653/654) was detected in embryonic day 10 (E10) to E12 livers only. Next, E10 livers were cultured in the presence of FGF1, FGF4, or FGF8 for 72 hours and examined for histology, proliferation, apoptosis, and differentiation. FGFs especially FGF8 promoted sheet-like architecture, cell proliferation, and survival as compared to the control. All FGFs induced a striking increase in the number of c-kit and alpha-fetoprotein-positive progenitors, without altering albumin staining. However these progenitors were CK-19-positive (biliary and bipotential progenitor marker) only in the presence of FGF1 or FGF4 and not FGF8. FGFs also induced beta-catenin, a stem cell renewal factor in these cultures. In conclusion, the presence of activated FGFR indicates a physiological role of FGF during early liver development. FGF1 and FGF4 enrich the embryonic liver cultures for bipotential hepatic progenitors. FGF8 promotes such enrichment and induces a one-step differentiation toward a unipotential hepatocyte progenitor. Thus, FGFs might be useful for enrichment and propagation of developmental hepatic progenitors.

FGF2-mediated upregulation of urokinase-type plasminogen activator expression requires a MAP-kinase dependent activation of poly(ADP-ribose) polymerase.

Poly(ADP-ribosyl)ation is a post-translational modification of protein occurring in the nucleus by poly(ADP-ribose) polymerase enzyme activity. The main role of poly(ADP-ribose) polymerase system as "nick sensor" and DNA breaks repair is based on its activation via DNA strand breaks. Furthermore, poly(ADP-ribose) polymerase modifies the binding to DNA of several transcriptional factors by poly(ADP-ribosyl)ation, thereby regulating also transcriptional gene expression. We have analyzed whether poly(ADP-ribose) polymerase activity is involved in basic fibroblast growth factor (FGF2)-mediated upregulation of urokinase-type plasminogen activator (uPA) mRNA. We demonstrated that specific inhibition of poly(ADP-ribose) polymerase activity via 3-aminobenzamide (3ABA) or NAD+ deprivation prevents FGF2-mediated uPA mRNA over-expression and cell-associated plasminogen activator (PA) production in GM7373 endothelial cell line. We verified that FGF2 stimulates poly(ADP-ribose) polymerase activity by a DNA strand breaks-independent manner which involves a mitogen-activated protein kinases (MAPK)-dependent pathway, as confirmed by using PD98059 inhibitor and anisomycin stimulation. Poly(ADP-ribose) polymerase involved in this mechanism is mainly the 60 kDa molecular mass isoform, that presents an increase in serine phosphorylation in the presence of FGF2.

The presence of FGF2 signaling determines whether β-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells

Neural stem cells proliferate and maintain multipotency when cultured in the presence of FGF2, but subsequent lineage commitment by the cells is nevertheless influenced by the exposure to FGF2. Here we show that FGF2 effects on neural stem cells are mediated, in part, by β-catenin. Conversely, the effects of β-catenin in neural stem cells depend in part upon whether there is concurrent fibroblast growth factor (FGF) signaling. FGF2 increases β-catenin signaling through several different mechanisms including increased expression of β-catenin mRNA, increased nuclear translocation of β-catenin, increased phosphorylation of GSK-3β, and tyrosine phosphorylation of β-catenin. Overexpression of β-catenin in the presence of FGF2 helps to maintain neural progenitor cells in a proliferative state. However, overexpression of β-catenin in the absence of FGF2 enhances neuronal differentiation. Further, chromatin immunoprecipitation (ChIP) assays demonstrate that both β-catenin and Lef1 bind directly to the neurogenin promoter, and luciferase reporter assays demonstrate that β-catenin is directly involved in the regulation of neurogenin 1 and possibly other proneural genes when neural stem cells are cultured in the presence of FGF2. We suggest that the balance between the mitogenic effects and the proneural effects of β-catenin is determined by the presence of FGF signaling.

Basic fibroblast and epidermal growth factors stimulate survival in adult porcine photoreceptor cell cultures.

To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas. Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium. Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF. Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways. Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase. Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1. Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter. In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments. Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF. Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone. There was no detectable selective loss of rods or cones in the experimental model. Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands. The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.

Differential activation of phosphatidylinositol 3-kinase signaling during proliferation and differentiation of lens epithelial cells.

To investigate whether phosphatidylinositol 3-kinase (PI-3K) signaling is involved in lens epithelial cell proliferation and differentiation promoted by growth factors. Proliferation of rabbit lens epithelial cells grown in culture was measured with a DNA-binding fluorescent dye in a proliferation assay. Primary cultures of embryonic chicken lens epithelial cells that develop lentoids were used for differentiation-related studies, and delta-crystallin synthesis in these cultures was determined by metabolic labeling with [(35)S]methionine. Immunoprecipitation and immunoblot analyses were also used. The PI-3K inhibitors wortmannin and LY294002 blocked the insulin-, insulin-like growth factor (IGF)-1-, and fibroblast growth factor (FGF)-2-promoted cell proliferation in rabbit lens epithelial cells. Inhibition of PI-3K activity by these inhibitors unexpectedly increased the synthesis of early differentiation marker protein delta-crystallin in chicken lens epithelial cells. Insulin and IGF-1 stimulated activation of PI-3K in proliferating and differentiating cultures. FGF-2 showed no direct effect on PI-3K activation. Platelet-derived growth factor (PDGF) did not induce significant proliferation or increased expression of delta-crystallin, but stimulated PI-3K. The presence of FGF-2 in proliferating rabbit lens epithelial cells enhanced the IGF-1-, but not the PDGF-mediated PI-3K activation, suggesting a possible integration of FGF-2 signals with IGF-1. Whereas there was a gradual decrease in insulin/IGF-1-mediated activation of PI-3K and its downstream target Akt, with progression of differentiation in chicken lens epithelial cells, Erk2 phosphorylation induced by these growth factors was not decreased; rather, it remained increased in early stages of differentiation. The results reveal significant differences in the modulation of PI-3K signaling by different growth factors during proliferation in rabbit lens epithelial cells and differentiation in chicken lens epithelial cells and demonstrate that regulation of the PI-3K pathway plays a key role in these processes. A balance between the nonactivation of PI-3K and the activation of Erk2 may be necessary during early stages of epithelial cell transformation.

Fibroblast growth factor receptor-1 mediates the inhibition of endothelial cell proliferation and the promotion of skeletal myoblast differentiation by SPARC: a role for protein kinase A.

The role of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) in modulation of vascular cell proliferation is believed to be mediated, in part, by its ability to regulate the activity of certain growth factors through direct binding. In this study, we demonstrate that SPARC does not bind to basic fibroblast growth factor (bFGF/FGF-2) or interfere with complex formation between FGF-2 and its high-affinity FGF receptor-1 (FGFR1), yet both native SPARC and a peptide derived from the C-terminal high-affinity Ca(2+)-binding region of protein significantly inhibit ligand-induced autophosphorylation of FGFR1 (>80%), activation of mitogen-activated protein kinases (MAPKs) (>75%), and DNA synthesis in human microvascular endothelial cells (HMVEC) stimulated by FGF-2 (>80%). We also report that in the presence of FGF-2, a factor which otherwise stimulates myoblast proliferation and the repression of terminal differentiation, both native SPARC and the Ca(2+)-binding SPARC peptide significantly promote (>60%) the differentiation of the MM14 murine myoblast cell line that expresses FGFR1 almost exclusively. Moreover, using heparan sulfate proteoglycan (HSPG)-deficient myeloid cells and porcine aortic endothelial cells (PAECs) expressing chimeric FGFR1, we show that antagonism of FGFR1-mediated DNA synthesis and MAPK activation by SPARC does not require the presence of cell-surface, low-affinity FGF-2 receptors, but can be mediated by an intracellular mechanism that is independent of an interaction with the extracellular ligand-binding domain of FGFR1. We also report that the inhibitory effect of SPARC on DNA synthesis and MAPK activation in endothelial cells is mediated in part (>50%) by activation of protein kinase A (PKA), a known regulator of Raf-MAPK pathway. SPARC thus modulates the mitogenic effect of FGF-2 downstream from FGFR1 by selective regulation of the MAPK signaling cascade.

The heparan sulfate proteoglycan agrin modulates neurite outgrowth mediated by FGF-2.

Although the role of agrin in the formation of the neuromuscular junction is well established, other functions for agrin have remained elusive. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the heparin-binding growth factor basic fibroblast growth factor (FGF-2), which we have shown previously to bind to agrin with high affinity and that has been shown to mediate neurite outgrowth from a number of neuronal cell types. Using both an established neuronal cell line, PC12 cells, and primary chick retina neuronal cultures, we find that agrin potentiates the ability of FGF-2 to stimulate neurite outgrowth. In PC12 cells and retinal neurons agrin increases the efficacy of FGF-2 stimulation of neurite outgrowth mediated by the FGF receptor, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF-2. We also examined possible mechanisms by which agrin may modulate neurite outgrowth, analyzing ERK phosphorylation and c-fos phosphorylation. These studies indicate that agrin augments a transient early phosphorylation of ERK in the presence of FGF-2, and augments and sustains FGF-2 mediated increases in c-fos phosphorylation. These data are consistent with established mechanisms where heparan sulfate proteoglycans such as agrin may increase the affinity between FGF-2 and the FGF receptor. In summary, our studies suggest that neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF-2.

Differentiation of Engrafted Neuronal-Restricted Precursor Cells Is Inhibited in the Traumatically Injured Spinal Cord

Differentiation of pluripotent neural stem cells engrafted into the adult normal and injured spinal cord is restricted to the glial lineage, suggesting that in vitro induction toward a neuronal lineage prior to transplantation and/or modification of the host environment may be necessary to initiate and increase the differentiation of neurons. In the present study, we investigated the differentiation of neuronal-restricted precursors (NRPs) grafted into the normal and contused adult rat spinal cord. NRPs proliferated through multiple passages in the presence of FGF2 and NT3 and differentiated into only neurons in vitro in the presence of retinoic acid and the absence of FGF2. Differentiated NRPs expressed GABA, glycine, glutamate, and ChAT. Two weeks to 2 months after engraftment of undifferentiated NRPs into adult normal spinal cord, large numbers of surviving cells were seen in all of the animals. The majority differentiated into βIII-tubulin-positive neurons. Some transplanted NRPs expressed GABA and small numbers were glutamate- and ChAT-positive. NRPs were also transplanted into the epicenter of the contused adult rat spinal cord. Two weeks to 2 months after transplantation, some engrafted NRPs remained undifferentiated nestin-positive cells. Small numbers were MAP2- or βIII-tubulin-positive neurons. However, the expression of GABA, glutamate, or ChAT was not observed. These results show that NRPs can differentiate into different types of neurons in the normal adult rat spinal cord, but that such differentiation is inhibited in the injured spinal cord. Manipulation of the microenvironment in the injured spinal cord will likely be necessary to facilitate neuronal replacement.

Effect of fucoidan on fibroblast growth factor-2-induced angiogenesis in vitro

Fucoidans are sulfated polysaccharides extracted from brown marine algae. A purified fucoidan fraction exhibits the same venous antithrombotic activity as heparin in rabbits, but with a lower anticoagulant effect. Because of its heparin-like structure, we postulated that fucoidan might modulate heparin-binding angiogenic growth factor activity. We thus studied its effect, at antithrombotic concentrations, on fibroblast growth factor (FGF)-2-induced proliferation and differentiation of human umbilical vein endothelial cells. The fucoidan effect on endothelial cell differentiation was evaluated by studying the expression of surface proteins (i.e. integrin, adhesion molecule) known to be modulated by FGF-2 and involved in angiogenesis, and by quantifying closed areas delimited by vascular tubes formed on reconstituted basement membrane. Fucoidan had no modulatory effect on the mitogenic activity of FGF-2, but significantly increased tubular structure density induced by FGF-2. Fucoidan alone increased α 6 integrin subunit expression with only partially organized tubular structure. In the presence of FGF-2, fucoidan enhanced α 6, β 1 and PECAM-1 and inhibited α vβ 3 integrin expression. Heparin had no effect in these systems. The most striking effect of fucoidan was observed on α 6 expression and tube formation was abolished by monoclonal anti-α 6 antibodies. Fucoidan plus FGF-2 effect on α 6 expression was markedly decreased by monoclonal anti-FGF-2 antibodies, indicating that fucoidan acts mainly via FGF-2. These results show that, at antithrombotic concentrations, contrary to heparin, fucoidan can enhance vascular tube formation induced by FGF-2 with a modulation of the expression of surface proteins (mainly α 6) involved in angiogenesis.

Vitreous-induced Modulation of Integrins in Retinal Pigment Epithelial Cells: Effects of Fibroblast Growth Factor-2

Growth in the presence of vitreous results in transformation of human RPE cells from an epithelioid to a fibroblast-like appearance and leads to an elevation of the expression of α5 and α2 integrins, while the level of α3 integrin is reduced. These changes are inhibited by the presence of FGF-2. Vitreous treatment increases mobility, as does antibody neutralization of FGF-2 or antibody blockade of FGF receptors. The vitreous-induced rise in mobility depends on an increase in α5 integrin expression since it is inhibited by anti-α5 integrin antibodies. Expression of α5 integrin as a result of infection of RPE cells with an α5 integrin-encoding adenovirus induced morphological transformation and an increase in mobility similar to that seen with vitreous. It is concluded that a decrease in FGF-2 plays an important role in vitreous-induced alterations of RPE cell morphology, integrin expression and mobility. High FGF-2 levels prevent at least some of the increased mobility of RPE cells induced by vitreous. This is mediated via extracellular FGF-2 binding to FGF receptor(s) since antibodies to FGF-2 or to its receptor(s) mimic the effects of vitreous. Changes in mobility and morphology involve altered α5 integrin expression since mobility is blocked by antibodies against these proteins while elevated α5 integrin expression increases mobility and leads to morphological changes.

Identification of an N-cadherin motif that can interact with the fibroblast growth factor receptor and is required for axonal growth.

In this study, we show that the neurite outgrowth response stimulated by N-cadherin is inhibited by a recently developed and highly specific fibroblast growth factor receptor (FGFR) antagonist. To test whether the N-cadherin response also requires FGF function, we developed peptide mimetics of the receptor binding sites on FGFs. Most mimetics inhibit the neurite outgrowth response stimulated by FGF in the absence of any effect on the N-cadherin response. The exceptions to this result were two mimetics of a short FGF1 sequence, which has been shown to interact with the region of the FGFR containing the histidine-alanine-valine motif. These peptides inhibited FGF and N-cadherin responses with similar efficacy. The histidine-alanine-valine region of the FGFR has previously been implicated in the N-cadherin response, and a candidate interaction site has been identified in extracellular domain 4 of N-cadherin. We now show that antibodies directed to this site on N-cadherin inhibit the neurite outgrowth response stimulated by N-cadherin, and peptide mimetics of the site inhibit N-cadherin and FGF responses. Thus, we can conclude that N-cadherin contains a novel motility motif in extracellular domain 4, and that peptide mimetics of this motif can interact with the FGFR.

The Use of Growth Factors in the Proliferation of Avian Articular Chondrocytes in a Serum-Free Culture System

The purpose of this research was to develop a serum-free culture system for the proliferation of articular chondrocytes. Various growth factors and hormones were tested for their ability to stimulate avian articular chondrocyte proliferation in a defined, serum-free media. Multiple members of the fibroblast growth factor (FGF) family (FGFs: 2, 4, and 9), insulin-like growth factor-1 (IGF-1) and transforming growth factor β (TGF-β) significantly stimulated 3H-thymidine uptake by chondrocytes grown in an adherent serum-free, culture system. Double or triple combinations of these mitogenic growth factors further stimulated cell proliferation to levels that were equivalent to, or surpassed those of cells grown in serum. Although proliferation was maximally stimulated, chondrocytes grown in the presence of FGF-2, IGF-1, and TGF-β, began to exhibit changes in morphology and collagen II expression declined. This culture system could be used to rapidly expand a population of articular chondrocytes prior to transferring these cells to a non-adherent culture system, which could then stabilize the chondrocyte phenotype and maximize matrix synthesis and integrity.

Fibroblast Growth Factor (FGF)-2 Mediates Cell Attachment through Interactions with Two FGF Receptor-1 Isoforms and Extracellular Matrix or Cell-Associated Heparan Sulfate Proteoglycans

In the presence of FGF-2, cells in suspension expressing FGF receptor-1 will attach to monolayers of cells expressing heparan sulfates. This attachment provides physical evidence for the formation of a trimolecular complex between FGF-2, heparan sulfate, and FGF receptors. We have used this system to determine if receptor isoforms containing or lacking the first of three immunoglobulin-like domains are equally able to form complexes with FGF-2 and heparan sulfates. In the presence of FGF-2, cells expressing either isoform of the receptor were able to attach to monolayers of CHO cells expressing heparan sulfates. No attachment was observed in the absence of FGF-2 or if heparin was included in the incubation medium. Attachment of cells expressing the two receptor isoforms occurred at similar concentrations of FGF-2, and similar concentrations of heparin were required to disrupt the interactions. Thus, there appeared to be little difference between these receptor isoforms in their ability to form trimolecular complexes with FGF-2 and cell-associated heparan sulfates. We also found that, in the presence of FGF-2, cells expressing FGF receptor-1 are able to form complexes with both extracellular matrix and cell-surface heparan sulfates.

The Proliferative and Migratory Activities of Breast Cancer Cells Can Be Differentially Regulated by Heparan Sulfates

To explore how heparan sulfate (HS) controls the responsiveness of the breast cancer cell lines MCF-7 and MDA-MB-231 to fibroblast growth factors (FGFs), we have exposed them to HS preparations known to have specificity for FGF-1 (HS glycosaminoglycan (HSGAG A)) or FGF-2 (HSGAGB). Proliferation assays confirmed that MCF-7 cells were highly responsive to FGF-2 complexed with GAGB, whereas migration assays indicated that FGF-1/HSGAGA combinations were stimulatory for the highly invasive MDA-MB-231 cells. Quantitative polymerase chain reaction for the levels of FGF receptor (FGFR) isoforms revealed that MCF-7 cells have greater levels of FGFR1 and that MDA-MB-231 cells have greater relative levels of FGFR2. Cross-linking demonstrated that FGF-2/HSGAGB primarily activated FGFR1, which in turn up-regulated the activity of mitogen-activated protein kinase; in contrast, FGF-1/HSGAGA led to the phosphorylation of equal proportions of both FGFR1 and FGFR2, which in turn led to the up-regulation of Src and p125(FAK). MDA-MB-231 cells were particularly responsive to vitronectin substrates in the presence of FGF-1/HSGAGA, and blocking antibodies established that they used the alpha(v)beta(3) integrin to bind to it. These results suggest that the clustering of particular FGFR configurations on breast cancer cells induced by different HS chains leads to distinct phenotypic behaviors.

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.

Synergistic effects of FGF and non-ridge ectoderm on gene expression involved in the formation of the anteroposterior axis of the chick limb bud in cell culture.

Skeletal patterning of the vertebrate limb is controlled by the zone of polarizing activity (ZPA), apical ectodermal ridge (AER) and dorsal ectoderm. In the present study, to understand the involvement of fibroblast growth factor (FGF) and non-ridge ectoderm in anteroposterior (AP) axis formation, gene expression in chick limb bud mesenchymal cells in culture was investigated by reverse transcription-polymerase chain reaction and in situ hybridization. It was found that Shh expression was locally maintained in the mesenchymal cells underneath and near non-ridge ectoderm in coculture with the posterior mesenchymal cells and non-ridge ectoderm in the presence of FGF-4 by in situ hybridization. In Shh-expressing anterior limb bud mesenchymal cells cultured with non-ridge ectoderm, it was also discovered that Bmp-2 was activated in the presence of FGF-2, -4 and -8, while Hoxd-13 was activated in the presence of FGF-4 and that FGF-2 had a similar effect but FGF-8 did not. This result indicates that Hoxd-13 activation by SHH depends on non-ridge ectoderm and FGF-2 or FGF-4, and that there may be a difference in the effect on AP axis formation of the limb bud between FGF-2, -4 and -8. Possible roles of these genes and signal molecules in AP pattern formation are discussed.