Control of fibroblast growth factor (FGF) 7- and FGF1-induced mitogenesis and downstream signaling by distinct heparin octasaccharide motifs

Variation in length, disaccharide composition, and sulfation of heparan sulfate (HS) affects fibroblast growth factor (FGF) signaling. However, it is unclear whether the specific distribution of groups within oligosaccharides or random variations in charge density underlies the effects. Recently we showed that a mixture of undersulfated octasaccharides exhibiting 7 and 8 sulfates (7,8-S-OctaF7) generated from heparin had the highest affinity for FGF7 monitored by salt resistance (>0.60 M salt) of octasaccharide-FGF7 complexes. 7,8-S-OctaF7 also had the highest specific activity for formation of a complex with dimeric FGFR2IIIb competent to bind FGF7. Here we show that when endogenous HS was inhibited by chlorate treatment, 7,8-S-OctaF7 specifically supported FGF7-stimulated DNA synthesis and downstream signaling in FGFR2IIIb-expressing mouse keratinocytes. It failed to support FGF1 signaling in both HS-deficient mouse keratinocytes and 3T3 fibroblasts. In contrast, abundant, more highly sulfated and heterogenous mixtures of octasaccharides with lower affinity (0.30-0.60 M salt) for FGF7 supported FGF1-induced signaling in both cell types. In contrast to the two-component 7,8-S-OctaF7 mixture from FGF7, the high affinity octasaccharide fraction from FGF1 was a heterogeneous mixture with components ranging from 8 to 12 sulfates with 11-S-octasaccharides the most abundant. The high affinity fraction exhibited similar properties to the lower affinity fractions from both FGF1 and FGF7. Octasaccharide mixtures eluting from FGF1 between 0.30 and 0.60 M and above 0.60 M salt were nearly equal in support of FGF1 signaling in fibroblasts and keratinocytes. Both were deficient in support of FGF7-induced signaling in keratinocytes. The results show that both variations in overall charge density and specific distribution of charged groups within HS motifs exhibit FGF-specific control over formation of FGF-HS-FGFR complexes and downstream signaling.     

Evidence that the intracellular domain of FGF receptor 2IIIb affects contact of the ectodomain with two FGF7 ligands

Models of the oligomeric FGF signaling complex, including those derived from crystal structures, vary in stoichiometry and arrangement of the three subunits comprised of heparin/heparan sulfate chains, FGFR tyrosine kinase and activating FGF. Here, using covalent affinity crosslinking of radiolabeled FGF7 to binary complexes of FGFR2IIIb and heparin, we show that two molecules of FGF7 contact each FGFR2IIIb. This supports models that propose a dimeric complex of two units with stoichiometry 1 FGF:1 FGFR in which each FGF contacts both FGFR. The bivalent FGF7 contact was dependent on the full-length amino terminus of FGF7alpha and the intracellular domain of FGFR2IIIb extending through the juxtamembrane domain and the beta1 and beta2 strands of the kinase which is required for ATP binding. We propose that the differences in crosslinking report differences in relationships among subunits in the ectodomain of the complex that are affected by the amino terminus of FGF and the FGFR intracellular domain. From this, we suggest the corollary that conformational relationships among subunits in the ectodomain are transmitted to the intracellular and ATP binding domains during activation of the complex.     

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.     

Structure-based mutational analyses in FGF7 identify new residues involved in specific interaction with FGFR2IIIb

Receptor binding specificity is an essential element in regulating the diverse activities of fibroblast growth factors (FGFs). FGF7 is ideal to study how this specificity is conferred at the structural level, as it interacts exclusively with one isoform of the FGF-receptor (FGFR) family, known as FGFR2IIIb. Previous mutational analysis suggested the importance of the beta4/beta5 loop of FGF7 in specific receptor recognition. Here a theoretical model of FGFR2IIIb/FGF7 complex showed that this loop interacts with the FGFR2IIIb unique exon. In addition, the model revealed new residues that either directly interact with the FGFR2IIIb unique exon (Asp63, Leu142) or facilitate this interaction (Arg65). Mutations in these residues reduced both receptor binding affinity and biological activity of FGF7. Altogether, these results provide the basis for understanding how receptor-binding specificity of FGF7 is conferred at the structural level.     

Ligand binding properties of binary complexes of heparin and immunoglobulin-like modules of FGF receptor 2

Epithelial cells, which express FGFR2IIIb, bind and respond to FGF-1, FGF-7 and FGF-10, but not FGF-2. Stromal cells, which bind and respond to FGF-1 and FGF-2, but not FGF-7 and FGF-10, express FGFR2IIIc or FGFR1IIIc. Here we show that when both isolated FGFR2betaIIIb and FGFR2betaIIIc or their common Ig module II are allowed to affinity select heparin from a mixture, the resultant binary complexes bound FGF-1, FGF-2, and FGF-7 with nearly equal affinity. In addition, FGF-2 and FGF-7 bound to both heparin-Ig module IIIb and IIIc complexes, but FGF-1 bound to neither Ig module III. The results show that in isolation both Ig modules II and III of FGFR2 can interact with heparin and that each exhibits a binding site for FGF. We suggest that the specificity of FGFR2IIIb and FGFR2IIIc is dependent on the cell membrane environment and heparin/heparan sulfate. Ig modules II and III cooperate both within monomers and across dimers with cellular heparan sulfates to confer cell type-dependent specificity of the FGFR complex for FGF.     

FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development

FGF receptor 2 isoform IIIb (FGFR2b), originally discovered as a receptor for FGF7, is known to be an important receptor in vertebrate morphogenesis, because FGFR2b null mice exhibit agenesis or dysgenesis of various organs, which undergo budding and branching morphogenesis. Since FGF7 null mice do not exhibit marked defects in organogenesis, it has been considered that other FGF(s) than FGF7 might function as a major ligand for FGFR2b during organogenesis. One of the candidate ligands is FGF10, because FGF10 binds to FGFR2b with high affinity and the formation of the limb and lung is arrested in FGF10 null mice as found in FGFR2b-deficient mice. Previous analyses of FGF10 null mice revealed that FGF10 is required for limb and lung development. To elucidate the role of FGF10 in wide-range organogenesis, we further analyzed the phenotypes of the FGF10 knockout mice. We found diverse phenotypes closely related to those for FGFR2b-deficient mice, which includes the absence of thyroid, pituitary, and salivary glands, while minor defects were observed in the formation of teeth, kidneys, hair follicles, and digestive organs. These results suggest that FGF10 acts as a major ligand for FGFR2b in mouse multi-organ development.     

Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2.

Fibroblast growth factor-4 (FGF4), like other FGFs, shares a high affinity for the anionic glycosaminoglycans heparin and heparan sulfate (HS), which in turn enhance FGF-receptor (FGFR) binding and activation. Here we demonstrate using a cell free system that, at low concentrations of heparin, FGF4 binds only to FGFR-2, while much higher heparin levels are required for binding to FGFR-1. Chemical crosslinking of radiolabeled FGF4 to the soluble FGF receptors confirms the preferential formation of FGF4-FGFR-2 complexes under restricted heparin availability, with maximal ligand-receptor interactions at almost 20-fold lower heparin concentrations then those required for the affinity labeling of FGFR-1. In accordance, HS-deficient cells expressing FGFR-2 proliferate in response to FGF4 at extremely low exogenous heparin concentrations, while FGFR-1 expressing cells are completely unresponsive under the same conditions. We suggest that FGFR-2 is the preferred receptor for FGF4 under restricted HS conditions and that the bioavailability of structurally distinct HS motifs may differentially control receptor specificity of FGF4 in vivo.     

Highly sulfated nonreducing end-derived heparan sulfate domains bind fibroblast growth factor-2 with high affinity and are enriched in biologically active fractions

Human fibroblast growth factor-2 (FGF2) regulates cellular processes including proliferation, adhesion, motility, and angiogenesis. FGF2 exerts its biological function by binding and dimerizing its receptor (FGFR), which activates signal transduction cascades. Effective binding of FGF2 to its receptor requires the presence of heparan sulfate (HS), a linear polysaccharide with N-sulfated domains (NS) localized at the cell surface and extracellular matrix. HS acts as a platform facilitating the formation of a functional FGF-FGFR-HS ternary complex. Crystal structures of the signaling ternary complex revealed two conflicting architectures. In the asymmetrical model, two FGFs and two FGFRs bind a single HS chain. In contrast, the symmetrical model postulates that one FGF and one FGFR bind to the free end of the HS chain and dimerization require these ends to join, bringing the two half-complexes together. In this study, we screened a hexasaccharide HS library for compositions that are able to bind FGF2. The library was composed primarily of NS domains internal to the HS chain with minor presence of non-reducing end (NRE) NS. The binders were categorized into low versus high affinity binders. The low affinity fraction contained primarily hexasaccharides with low degree of sulfation that were internal to the HS chains. In contrast, the high affinity bound fraction was enriched in NRE oligosaccharides that were considerably more sulfated and had the ability to promote FGFR-mediated cell proliferation. The results suggest a role of the NRE of HS in FGF2 signaling and favor the formation of the symmetrical architecture on short NS domains.     

Cooperative dimerization of fibroblast growth factor 1 (FGF1) upon a single heparin saccharide may drive the formation of 2:2:1 FGF1.FGFR2c.heparin ternary complexes

The related glycosaminoglycans heparin and heparan sulfate are essential for the activity of the fibroblast growth factor (FGF) family as they form an integral part of the signaling complex at the cell surface. Using size-exclusion chromatography we have studied the capacities of a variety of heparin oligosaccharides to bind FGF1 and FGFR2c both separately and together in ternary complexes. In the absence of heparin, FGF1 had no detectable affinity for FGFR2c. However, 2:2:1 complexes formed spontaneously in solution between FGF1, FGFR2c, and heparin octasaccharide (dp8). The dp8 sample was the shortest chain length that bound FGFR2c, that dimerized FGF1, and that promoted a strong mitogenic response to FGF1 through FGFR2c. Heparin hexasaccharide and various selectively desulfated heparin dp12s failed to bind FGFR2c and could only interact with FGF1 monomerically. These saccharides formed 1:1:1 complexes with FGF1 and FGFR2c, which had no tendency to self-associate, suggesting that binding of two FGF1 molecules to the same saccharide chain is a prerequisite for subsequent FGFR2c dimerization. We found that FGF1 dimerization upon heparin was favored over monomeric interactions even when a large excess of saccharide was present. A cooperative mechanism of FGF1 dimerization could explain how 2:2:1 signaling complexes form at the cell surface, an environment rich in heparan sulfate.     

Structural basis for interaction of FGF-1, FGF-2, and FGF-7 with different heparan sulfate motifs.

Stromal cell-derived FGF-7 binds and activates only the resident FGFR2IIIb in epithelial cells while FGF-1 and FGF-2 exhibit a broader interaction with multiple isoforms of FGFR. Here we report the structure of FGF-7 that has been solved to 3.1 A resolution by molecular replacement with the structure of a dual function chimera of FGF-7 and FGF-1 (FGF-7/1) which was resolved to 2.3 A. Comparison of the FGF-7 structure to that of FGF-1 and FGF-2 revealed the strongly conserved Calpha backbone among the three FGF polypeptides and the surface hydrophobic patch that forms the primary receptor-binding domain. In contrast, a decrease and dispersion of the positive surface charge density characterized the heparin-binding domain of FGF-7 defined by homology to that of FGF-1 and FGF-2 in complexes with heparin. A simple heparin hexasaccharide that cocrystallized with FGF-1 and FGF-2 and protected both against protease in solution failed to exhibit the same properties with FGF-7. In contrast to FGF-1 and FGF-2, protection of FGF-7 was enhanced by heparin oligosaccharides of increased length with those exhibiting a 3-O-sulfate being the most effective. Protection of FGF-7 required interaction with specifically the fraction of crude heparin retained on antithrombin affinity columns. Conversely, heparin enriched by affinity for immobilized FGF-7 exhibited anti-factor Xa activity similar to that purified on an antithrombin affinity matrix. In contrast, an FGF-1 affinity matrix enriched the fraction of crude heparin with low anti-factor Xa activity. The results provide a structural basis to suggest that the unique FGF-7 heparin-binding (HB) domain underlies a specific restriction in respect to composition and length of the heparan sulfate motif that may impact specificity of localization, stability, and trafficking of FGF-7 in the microenvironment, and formation and activation of the FGFR2IIIb kinase signaling complex in epithelial cells.     

Investigating the mechanism of the assembly of FGF1-binding heparan sulfate motifs

Heparan sulfate (HS) chains play crucial biological roles by binding to various signaling molecules including fibroblast growth factors (FGFs). Distinct sulfation patterns of HS chains are required for their binding to FGFs/FGF receptors (FGFRs). These sulfation patterns are putatively regulated by biosynthetic enzyme complexes, called GAGOSOMES, in the Golgi. While the structural requirements of HS-FGF interactions have been described previously, it is still unclear how the FGF-binding motif is assembled in vivo. In this study, we generated HS structures using biosynthetic enzymes in a sequential or concurrent manner to elucidate the potential mechanism by which the FGF1-binding HS motif is assembled. Our results indicate that the HS chains form ternary complexes with FGF1/FGFR when enzymes carry out modifications in a specific manner.     

Synthetic Heparan Sulfate Oligosaccharides Inhibit Endothelial Cell Functions Essential for Angiogenesis

Background

Heparan sulfate (HS) is an important regulator of the assembly and activity of various angiogenic signalling complexes. However, the significance of precisely defined HS structures in regulating cytokine-dependent angiogenic cellular functions and signalling through receptors regulating angiogenic responses remains unclear. Understanding such structure-activity relationships is important for the rational design of HS fragments that inhibit HS-dependent angiogenic signalling complexes.

Methodology/Principal Findings

We synthesized a series of HS oligosaccharides ranging from 7 to 12 saccharide residues that contained a repeating disaccharide unit consisting of iduronate 2-O-sulfate linked to glucosamine with or without N-sulfate. The ability of oligosaccharides to compete with HS for FGF2 and VEGF₁₆₅ binding significantly increased with oligosaccharide length and sulfation. Correspondingly, the inhibitory potential of oligosaccharides against FGF2- and VEGF₁₆₅-induced endothelial cell responses was greater in longer oligosaccharide species that were comprised of disaccharides bearing both 2-O- and N-sulfation (2SNS). FGF2- and VEGF₁₆₅-induced endothelial cell migration were inhibited by longer 2SNS oligosaccharide species with 2SNS dodecasaccharide activity being comparable to that of receptor tyrosine kinase inhibitors targeting FGFR or VEGFR-2. Moreover, the 2SNS dodecasaccharide ablated FGF2- or VEGF₁₆₅-induced phosphorylation of FAK and assembly of F-actin in peripheral lamellipodia-like structures. In contrast, FGF2-induced endothelial cell proliferation was only moderately inhibited by longer 2SNS oligosaccharides. Inhibition of FGF2- and VEGF₁₆₅-dependent endothelial tube formation strongly correlated with oligosaccharide length and sulfation with 10-mer and 12-mer 2SNS oligosaccharides being the most potent species. FGF2- and VEGF₁₆₅-induced activation of MAPK pathway was inhibited by biologically active oligosaccharides correlating with the specific phosphorylation events in FRS2 and VEGFR-2, respectively.

Conclusion/Significance

These results demonstrate structure-function relationships for synthetic HS saccharides that suppress endothelial cell migration, tube formation and signalling induced by key angiogenic cytokines.     

Novel phosphotyrosine targets of FGFR2IIIb signaling

In partnership exclusively with the epithelial FGFR2IIIb isotype and a structurally-specific heparan sulfate motif, stromal-derived FGF7 delivers both growth-promoting and growth-limiting differentiation signals to epithelial cells that promote cellular homeostasis between stromal and epithelial compartments. Intercompartmental homeostasis supported by FGF7/FGFR2IIIb is subverted in many solid epithelial tumors. The normally mesenchymal-derived homologue FGFR1 drives proliferation and a progressive tumor-associated phenotype when it appears ectopically in epithelial cells. In order to understand the mechanism underlying the unique biological effects of FGFR2IIIb, we developed an inducible FGFR2IIIb expression system that is specifically dependent on FGF7 for activation in an initially unresponsive cell line to avoid selection for only the growth-promoting aspects of FGFR2IIIb signaling. We then determined FGF7/FGFR2IIIb signaling-specific tyrosine phosphorylated proteins within 5 min after FGF7 stimulation by phosphopeptide immunoaffinity purification and nano-LC-MS/MS. The FGF7/FGFR2 pair caused tyrosine phosphorylation of multiple proteins that have been implicated in the growth stimulating activities of FGFR1 that included multi-substrate organizers FRS2α and IRS4, ERK2 and phosphatases SHP2 and SHIP2. It uniquely phosphorylated CDK2 and phosphatase PTPN18 on sites involved in the attenuation of cell proliferation, and several factors that maintain nuclear-cytosolic relationships (emerin and LAP2), protein structure and other cellular fine structures as well as some proteins of unknown functions. Several of the FGF7/FGFR2IIIb-specific targets have been associated with maintenance of function and tumor suppression and disruption in tumors. In contrast, a number of pTyr substrates associated with FGF2/FGFR1 that are generally associated with intracellular Ca²⁺-phospholipid signaling, membrane and cytoskeletal plasticity, cell adhesion, migration and the tumorigenic phenotype were not observed with FGF7/FGFR2IIIb. Our findings provide specific downstream targets for dissection of causal relationships underlying the distinct role of FGF7/FGFR2IIIb signaling in epithelial cell homeostasis.     

Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.     

FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

Identification of residues important both for primary receptor binding and specificity in fibroblast growth factor-7

Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs). Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity. FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR. Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site. FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1. The sequences that confer on FGF-7 its specific binding to KGFR have not been identified. By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition. Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1. Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency. The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR. Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF.     

Specific heparan sulfate structures modulate FGF10-mediated submandibular gland epithelial morphogenesis and differentiation

FGF10, a heparan sulfate (HS)-binding growth factor, is required for branching morphogenesis of mouse submandibular glands (SMGs). HS increases the affinity of FGF10 for FGFR2b, which forms an FGF10.FGFR2b.HS ternary signaling complex, and results in diverse biological outcomes, including proliferation and epithelial morphogenesis. Defining the HS structures involved in specific FGF10-mediated events is critical to understand how HS modulates growth factor signaling in specific developmental contexts. We used HS-deficient BaF3/FGFR2b cells, which require exogenous HS to proliferate, to investigate the HS requirements for FGF10-mediated proliferation and primary SMG epithelia to investigate the structural requirements of HS for FGF10-mediated epithelial morphogenesis. In BaF3/FGFR2b cells, heparin with at least 10 saccharides and 6-O-, 2-O-, and N-sulfates were required for maximal proliferation. During FGF10-mediated SMG epithelial morphogenesis, HS increased proliferation and end bud expansion. Defined heparin decasaccharide libraries showed that 2-O-sulfation with either an N-or 6-O-sulfate induced end bud expansion, whereas decasaccharides with 6-O-sulfation alone induced duct elongation. End bud expansion resulted from increased FGFR1b signaling, with increased FGFR1b, Fgf1, and Spry1 as well as increased Aqp5 expression, a marker of end bud differentiation. Duct elongation was associated with expression of Cp2L1, a marker of developing ducts. Collectively, these findings show that the size and sulfate patterns of HS modulate specific FGF10-mediated events, such as proliferation, duct elongation, end bud expansion, and differentiation, and provide mechanistic insight as to how the developmental localization of specific HS structures in tissues influences FGF10-mediated morphogenesis and differentiation.     

Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.     

A novel form of FGF receptor-3 using an alternative exon in the immunoglobulin domain III

Four distinct FGF receptors were cloned and characterized and it was demonstrated that the ligand binding site of FGF receptors is confined to the extracellular immunoglobulin-like (Ig)-domain 2 and 3. The Ig-domain 3 is encoded by two separate exons: exon IIIa encodes the N-terminal half, and the C-terminal half is encoded by either exon IIIb or IIIc in FGFR1 and FGFR2, whereas FGFR4 is devoid of exon IIIb. Alternative usage of exons IIIb and IIIc determine the ligand binding specificity of the receptor. To analyze the arrangement of these exons in FGFR3 we cloned the genomic sequence between exon IIIa and IIIc of FGFR3 and identified an alternative exon, corresponding to exon IIIb of the FGFR1 and FGFR2. The sequence of this exon shows Ig-domain hallmarks, 44% identity with exon IIIb of other FGF receptors and 36% identity with exon IIIc of FGFR3. Using this exon as a probe for mouse RNA as well as PCR analysis, demonstrated that exon IIIb encodes an authentic form of FGFR3 that is expressed in mouse embryo, mouse skin and mouse epidermal keratinocytes. The results demonstrate that the presence of alternative exons for Ig-domain 3 is a general phenomena in FGFR1, 2 and 3, and represents a novel genetic mechanism for the generation of receptor diversity.