Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides

Signalling by fibroblast growth factors (FGFs) through FGF receptors (FGFRs) depends on the cell-surface polysaccharide heparan sulphate (HS) [1] [2]. HS has an ordered domain structure of highly diverse saccharide motifs that present unique displays of sulphate, carboxyl and hydroxyl groups [3]. These motifs interact with many proteins, particularly growth factors. HS binds both to FGFs [4] [5] [6] and FGFRs [7], and probably activates signalling by facilitating ligand-induced receptor dimerisation [8] [9]. Nevertheless, the extent to which specific HS saccharide sequences play a regulatory role has not been established. By screening a library of structurally diverse HS decasaccharides in bioassays of FGF signalling mediated by three different FGFR isoforms, we found that saccharides showed specificity for both ligands and receptors; some saccharides selectively activated FGF signalling through different FGFR isoforms, others acted as negative regulators. We conclude that HS saccharides play critical roles in dictating the specificity of ligand-receptor interactions in FGFR signalling. Controlled alterations in HS structures [10] would provide a mechanism for regulation of cellular responsiveness to growth factors that bind HS.     

Anti-apoptotic Bcl-2 enhancing requires FGF-2/FGF receptor 1 binding in mouse osteoblasts

In this study, we investigated the role of prostaglandin F2alpha (PGF2alpha) in mouse osteoblast survival and the function of fibroblast growth factor 2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) in this process. In particular, for the first time, we demonstrated that PGF2alpha increased osteoblast survival in a dose-dependent manner and we showed that the effect is correlated with an increase in Bcl-2/Bax ratio. Furthermore, we demonstrated that PGF2alpha caused a decrement of the active caspases 9 and 3. By blocking FGF-2 with the specific neutralizing antibody and by depletion of FGFR1 gene with a specific siRNA, we showed that FGFR1 and FGF-2 are critical for the increment of Bcl-2/Bax ratio and the decrement of the active caspases 9 and 3, induced by PGF2alpha. Moreover, transmission electron microscopy studies showed that PGF2alpha increased binding of FGF-2 and FGFR1 and co-localization of reactive sites at plasma membrane level. In conclusion, we report a novel mechanism in which PGF2alpha induces FGF-2 binding to its specific cell surface receptor 1 leading to a cascade pathway that culminates with increased mouse osteoblast survival.     

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.     

The cell surface proteoglycan syndecan-1 mediates fibroblast growth factor-2 binding and activity

Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases (FGFRs) and signaling is facilitated by binding of FGF to heparan sulfate proteoglycans (HSPGs). There are multiple families of HSPGs, including extracellular and cell surface forms. An important and potentially controversial question is whether cell surface forms of HSPGs act as positive or negative regulators of FGF signaling. This study examines the ability of the cell surface HSPG syndecan-1 to regulate FGF binding and signaling. HSPG-deficient Raji lymphoma cells, expressing a transfected syndecan-1 cDNA (Raji S1 cells), were used as HSPG “donor” cells. BaF3 cells, expressing an FGFR1 cDNA (FR1C-11 cells), were used as FGFR “reporter” cells. Using Raji S1 cells preincubated with FGF, it was found that they formed heterotypic aggregates with FR1C-11 cells in the presence of FGF-2, but not FGF-1. In addition, the FR1C-11 cells demonstrated FGF-2, but not FGF-1, dependent survival when cultured on fixed Raji S1 cells. Thus, Raji syndecan-1 (1) differentially regulates the binding and signaling of FGFs 1 and 2 and (2) acts as a positive regulator of FGF-2 signaling. J. Cell. Physiol. 174:310–321, 1998. © 1998 Wiley-Liss, Inc.     

PGF2alpha increases FGF-2 and FGFR2 trafficking in Py1a rat osteoblasts via clathrin independent and importin beta dependent pathway

Previous studies showed that prostaglandin F2alpha (PGF2alpha) stimulated fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 2 (FGFR2) cytosolic and nuclear accumulation, however, the endocytic pathway has not been elucidated. This study demonstrates that although PGF2alpha increased the formation of clathrin-coated structures in Py1a rat osteoblasts, they were not involved in FGF-2 and FGFR2 trafficking. PGF2alpha increased binding of FGF-2 and FGFR2 and co-localization of reactive sites in addition to nuclear translocation at the nuclear pore complex level. FGF-2 and FGFR2 were in close spatial correlation with importin beta, further supporting nuclear import of the FGF-2/FGFR2 complex. Immunogold and immunofluorescence techniques as well as Western blotting demonstrated increased importin beta protein labeling in response to PGF2alpha. Similar to PGF2alpha, phorbol 12-myristate 13-acetate (PMA) also increased importin beta protein. These data strongly suggest that prostaglandins may regulate osteoblast metabolism via FGF-2/FGFR2/importin beta nuclear trafficking.     

Intracellular trafficking in neurones and glia of fibroblast growth factor-2, fibroblast growth factor receptor 1 and heparan sulphate proteoglycans in the injured adult rat cerebral cortex

The potent gliogenic and neurotrophic fibroblast growth factor (FGF)-2 signals through a receptor complex comprising high-affinity FGF receptor (FGFR)1 with heparan sulphate proteoglycans (HSPGs) as co-receptors. We examined the intracellular dynamics of FGF-2, FGFR1 and the HSPGs syndecan-2 and -3, glypican-1 and -2, and perlecan in neurones and glia in and around adult rat cerebral wounds. In the intact cerebral cortex, FGF-2 and FGFR1 mRNA and protein were constitutively expressed in astrocytes and neurones respectively. FGF-2 protein was localized exclusively to astrocyte nuclei. After injury, expression of FGF-2 mRNA was up-regulated only in astrocytes, whereas FGFR1 mRNA expression was increased in both glia and neurones, a disparity indicating that FGF-2 may act as a paracrine and autocrine factor for neurones and glia respectively. FGF-2 protein localized to both cytoplasm and nuclei of injury-responsive neurones and glia. There was weak or no staining of HSPGs in the normal cerebral neuropil and glia nuclei, with a few immunopositive neurones. Specific HSPGs responded to injury by differentially co-localizing with trafficked intracellular FGF-2 and FGFR1. The spatiotemporal dynamics of FGF-2-FGFR1-HSPG complex formation implies a role for individual HSPGs in regulating FGF-2 storage, nuclear trafficking and cell-specific injury responses in CNS wounds.     

Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes.

Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate the interaction of FGFs with their receptors (FGFRs) and are thought to play a critical role in receptor dimerization. Biochemical and crystallographic studies, conducted mainly with FGF-2 or FGF-1 and FGF receptors 1 and 2, suggests that an octasaccharide is the minimal length required for FGF- and FGFR-induced dimerization and subsequent activation. In addition, 6-O-sulfate groups are thought to be essential for binding of HS to FGFR and for receptor dimerization. We show here that oligosaccharides shorter than 8 sugar units support activation of FGFR2 IIIb by FGF-1 and interaction of FGFR4 with FGF-1. In contrast, only relatively long oligosaccharides supported receptor binding and activation in the FGF-1.FGFR1 or FGF-7.FGFR2 IIIb setting. In addition, both 6-O- and 2-O-desulfated heparin activated FGF-1 signaling via FGFR2 IIIb, whereas neither one stimulated FGF-1 signaling via FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required for activating FGFs is dictated by the specific FGF and FGFR combination. These different requirements may reflect the differences in the mode by which a given FGFR interacts with the various FGFs.     

Not all perlecans are created equal: interactions with fibroblast growth factor (FGF) 2 and FGF receptors

Human basement membrane heparan sulfate proteoglycan (HSPG) perlecan binds and activates fibroblast growth factor (FGF)-2 through its heparan sulfate (HS) chains. Here we show that perlecans immunopurified from three cellular sources possess different HS structures and subsequently different FGF-2 binding and activating capabilities. Perlecan isolated from human umbilical arterial endothelial cells (HUAEC) and a continuous endothelial cell line (C11 STH) bound similar amounts of FGF-2 either alone or complexed with FGFRalpha1-IIIc or FGFR3alpha-IIIc. Both perlecans stimulated the growth of BaF3 cell lines expressing FGFR1b/c; however, only HUAEC perlecan stimulated those cells expressing FGFR3c, suggesting that the source of perlecan confers FGF and FGFR binding specificity. Despite these differences in FGF-2 activation, the level of 2-O- and 6-O-sulfation was similar for both perlecans. Interestingly, perlecan isolated from a colon carcinoma cell line that was capable of binding FGF-2 was incapable of activating any BaF3 cell line unless the HS was removed from the protein core. The HS chains also exhibited greater bioactivity after digestion with heparinase III. Collectively, these data clearly demonstrate that the bioactivity of HS decorating a single PG is dependent on its cell source and that subtle changes in structure including secondary interactions have a profound effect on biological activity.     

Differential effects of Heparitinase I and Heparitinase III on endothelial tube formation in vitro

Heparan sulfate proteoglycans (HSPGs) play vital roles in many steps of angiogenesis under physiological and pathological conditions. HSPGs on endothelial cell surfaces act as co-receptors for a variety of pro-angiogenic growth factors such as FGF and VEGF and anti-angiogenic factors such as endostatin. However, the fine structural requirements of these binding interactions are dependent on the sulfation patterns of HSPGs. Previous studies have shown that Heparitinases, heparin lyases isolated from Flavobacterium heparinum, can cleave heparan sulfate chains. These enzymes have been shown to reduce tumor—derived neovascularization in vivo in mice. However, the results from these experiments could not conclusively pinpoint the origin of the HS fragments. Thus, in this study we utilized an in vitro assay to assess the differential effects of Heparitinase I (Hep I) and Heparitinase III (Hep III) on endothelial tube formation. Hep III was found to be a more potent inhibitor of tube formation than Hep I. In conclusion, differential cleavage of endothelial cell surface bound HS can affect the extent of inhibition of tube formation.     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

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.     

Effect of zinc on gene expression in osteoblastic MC3T3-E1 cells: enhancement of Runx2, OPG, and regucalcin mRNA expressions

Activation of fibroblast growth factor receptors (FGFRs) requires the formation of a ternary complex between fibroblast growth factors (FGFs), FGFRs, and heparan sulfate proteoglycans, which are all located on the cell surface and the basement membrane (BM)/extracellular matrix (ECM). Heparan sulfate proteoglycans appear to stabilize FGFs by inhibiting the rapid degradation of FGFs normally observed in solution. Because of the pivotal role of FGFs in proliferative and developmental pathways, a number of recent studies have attempted to engineer microenvironments to stabilize growth factors for use in applications in tissue culture and regenerative medicine. In this communication, we demonstrate that covalent linkage of FGF-2 to nanofibrillar surfaces (defined as covalently bound FGF-2) composed of a network of polyamide nanofibers resulted in the maintenance of the biological efficacy of FGF-2 when stored dry for at least 6 months at 25°C or 4°C. Moreover, covalently bound FGF-2 was more potent than FGF-2 in solution when measured in cellular assays of proliferation and viability using a variety of cell types. Covalently bound FGF-2 also strongly activated FGFR, extracellular signal-regulated kinase (ERK1/2), and c-fos. Hence cell-signaling molecules can be incorporated into a synthetic nanofibrillar surface, providing a novel means to enhance their stability and biological activity.     

Phosphorylation of fibroblast growth factor (FGF) receptor 1 at Ser777 by p38 mitogen-activated protein kinase regulates translocation of exogenous FGF1 to the cytosol and nucleus

Exogenous fibroblast growth factor 1 (FGF1) signals through activation of transmembrane FGF receptors (FGFRs) but may also regulate cellular processes after translocation to the cytosol and nucleus of target cells. Translocation of FGF1 occurs across the limiting membrane of intracellular vesicles and is a regulated process that depends on the C-terminal tail of the FGFR. Here, we report that translocation of FGF1 requires activity of the alpha isoform of p38 mitogen-activated protein kinase (MAPK). FGF1 translocation was inhibited after chemical inhibition of p38 MAPK or after small interfering RNA knockdown of p38alpha. Translocation was increased after stimulation of p38 MAPK with anisomycin, mannitol, or H2O2. The activity level of p38 MAPK was not found to affect endocytosis or intracellular sorting of FGF1/FGFR1. Instead, we found that p38 MAPK regulates FGF1 translocation by phosphorylation of FGFR1 at Ser777. The FGFR1 mutation S777A abolished FGF1 translocation, while phospho-mimetic mutations of Ser777 to Asp or Glu allowed translocation to take place and bypassed the requirement for active p38 MAPK. Ser777 in FGFR1 was directly phosphorylated by p38alpha in a cell-free system. These data demonstrate a crucial role for p38alpha MAPK in the regulated translocation of exogenous FGF1 into the cytosol/nucleus, and they reveal a specific role for p38alpha MAPK-mediated serine phosphorylation of FGFR1.     

Fibrolast growth factors in the nervous system

Fibroblast growth factors (FGFs) exhibit widespread mitogenic and neurotrophic activities. Nine members of the family are currently known, and FGF-1 and FGF-2 are present in relatively high levels in CNS. FGF-1 is expressed by a subset of neuronal populations, while FGF-2 is expressed by astrocytes. FGF-1 and FGF-2 lack signal peptides and appear to be present mainly in inracellular compartmens. This suggests that the factors may act as initiators of a repair response after injury. Support for this notion comes from observations that FGF-1 and FGF-2 levels are low during critical phases of development, but high in the adult CNS. A family of transmembrane tyrosine kinase receptors (FGFRs) mediates the effects of FGFs. Four different genes coding for FGF receptors are currently known, three of which are expressed in cell type-specific patterns in the CNS The main receptor variants present in this tissue, however, can by themselves not distinguish between FGF-1 and FGF-2. Additional selectivity may be established by interaction of the FGFs and their receptors with select heparan proteoglycans (HSPGs). Therefore, the precise physiological role of FGFs is determined by the combination of cell type-specific patterns of expression of FGFs, FGFRs and HSPGs together with the mechanisms that regulate the extracellular availability of FGFs. 1994 John Wiley & Sons, Inc.     

Fibroblast growth factor (FGF)-2 directly stimulates mature osteoclast function through activation of FGF receptor 1 and p42/p44 MAP kinase

We previously reported that fibroblast growth factor-2 (FGF-2) acts not only on osteoblasts to stimulate osteoclastic bone resorption indirectly but also on mature osteoclasts directly. In this study, we investigated the mechanism of this direct action of FGF-2 on mature osteoclasts using mouse and rabbit osteoclast culture systems. FGF-2 stimulated pit formation resorbed by isolated rabbit osteoclasts moderately from low concentrations (>/=10(-12) m), whereas at high concentrations (>/=10(-9) m) it showed stimulation on pit formation resorbed by unfractionated bone cells very potently. FGF-2 (>/=10(-12) m) also increased cathepsin K and MMP-9 mRNA levels in mouse and rabbit osteoclasts. Among FGF receptors (FGFR1 to 4) only FGFR1 was detected on isolated mouse osteoclasts, whereas all FGFRs were identified on mouse osteoblasts. FGF-2 (>/=10(-12) m) up-regulated the phosphorylation of cellular proteins, including p42/p44 mitogen-activated protein (MAP) kinase, and increased the kinase activity of immunoprecipitated FGFR1 in mouse osteoclasts. The stimulation of FGF-2 on mouse and rabbit osteoclast functions was abrogated by PD-98059, a specific inhibitor of p42/p44 MAP kinase. These results strongly suggest that FGF-2 acts directly on mature osteoclasts through activation of FGFR1 and p42/p44 MAP kinase, causing the stimulation of bone resorption at physiological or pathological concentrations.     

Cancer stem cells in solid tumors: elusive or illusive?

Background

The fibroblast growth factor receptor (FGFR) interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS) co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known.

Results

We have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44^MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2). In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (≥ 100 ng/mL) have little effect. At optimal concentrations (300 pg/mL) FGF-2 stimulates a sustained dual phosphorylation of p42/44^MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44^MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine.

Conclusions

These results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient) of phosphorylation of p42/44(MAPK) are varied, and so differing cellular responses are produced.     

Fibroblast growth factor-2 stimulation of p42/44MAPK phosphorylation and IkappaB degradation is regulated by heparan sulfate/heparin in rat mammary fibroblasts

Fibroblast growth factor-2 (FGF-2) interacts with a dual receptor system consisting of tyrosine kinase receptors and heparan sulfate proteoglycans (HSPGs). In rat mammary fibroblasts, FGF-2 stimulated DNA synthesis and induced a sustained phosphorylation of p42/44(MAPK) and of its downstream target, p90(RSK). Moreover, FGF-2 also stimulated the transient degradation of IkappaBalpha and IkappaBbeta. PD098059, a specific inhibitor of p42/44(MAPK) phosphorylation, inhibited FGF-2-stimulated DNA synthesis, phosphorylation of p42/44(MAPK) and p90(RSK), and degradation of IkappaBbeta. In contrast, in chlorate-treated and hence sulfated glycosaminoglycan-deficient cells, FGF-2 was unable to stimulate DNA synthesis. However, FGF-2 was able to trigger a transient phosphorylation of both p42/44(MAPK) and p90(RSK), which peaked at 15 min and returned to control levels at 30 min. In these sulfated glycosaminoglycan-deficient cells, no degradation of IkappaBalpha and IkappaBbeta was observed after FGF-2 addition. However, in chlorate-treated cells, the addition of heparin or purified HSPGs simultaneously with FGF-2 restored DNA synthesis, the sustained phosphorylation of p42/44(MAPK) and p90(RSK), and the degradation of IkappaBalpha and IkappaBbeta. These results suggest that the HSPG receptor for FGF-2 not only influences the outcome of FGF-2 signaling, e.g. cell proliferation, but importantly regulates the immediate-early signals generated by this growth factor.     

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.     

Requirement for anticoagulant heparan sulfate in the fibroblast growth factor receptor complex.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the fibroblast growth factor (FGF) receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation between self-associated FGFR and determines specificity for and mediates binding of activating FGF. Here we show that only the fraction of commercial heparin or rat liver heparan sulfate which binds to immobilized antithrombin formed an FGF-binding binary complex with the ectodomain of the FGFR kinase. Conversely, only the fraction of heparin that binds to immobilized FGFR inhibited Factor Xa in the presence of antithrombin. Only the antithrombin-bound fraction of heparin competed with (3)H-heparin bound to FGFR in absence of FGF, whereas both antithrombin-bound and unretained fractions competed with radiolabeled heparin bound independently to FGF-1 and FGF-2. The antithrombin-bound fraction of heparin was required to support the heparin-dependent stimulation of DNA synthesis of endothelial cells by FGF-1. The requirement for divalent cations and the antithrombin-binding motif distinguish the role of heparan sulfate as an integral subunit of the FGFR complex from the wider range of effects of heparan sulfates and homologues on FGF signaling through FGFR-independent interactions with FGF.