Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints

The synovium from patients with rheumatoid arthritis (RA) and LEW/N rats with streptococcal cell wall (SCW) arthritis, an experimental model resembling RA, is characterized by massive proliferation of synovial connective tissues and invasive destruction of periarticular bone and cartilage. Since heparin binding growth factor (HBGF)-1, the precursor of acidic fibroblast growth factor (FGF), is a potent angiogenic polypeptide and mitogen for mesenchymal cells, we sought evidence that it was involved in the synovial pathology of RA and SCW arthritis. HBGF-1 mRNA was detected in RA synovium using the polymerase chain reaction technique, and its product was immunolocalized intracellularly in both RA and osteoarthritis (OA) synovium. HBGF-1 staining was more extensive and intense in synovium of RA patients than OA and correlated with the extent and intensity of synovial mononuclear cell infiltration. HBGF-1 staining also correlated with c-Fos protein staining. In SCW arthritis, HBGF-1 immunostaining was noted in bone marrow, bone, cartilage, synovium, ligamentous and tendinous structures, as well as various dermal structures and developed early in both T-cell competent and incompetent rats. Persistent high level immunostaining of HBGF-1 was only noted in T-cell competent rats like the disease process in general. These observations implicate HBGF-1 in a multitude of biological functions in inflammatory joint diseases.     

E5 oncoprotein transmembrane mutants dissociate fibroblast transforming activity from 16-kilodalton protein binding and platelet-derived growth factor receptor binding and phosphorylation.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44-amino-acid, hydrophobic polypeptide which localizes predominantly in Golgi membranes and appears to transform cells through the activation of tyrosine kinase growth factor receptors. In fibroblasts, E5 interacts with both the 16-kilodalton vacuolar ATPase subunit and the platelet-derived growth factor receptor (PDGF-R) via its hydrophobic transmembrane domain and induces autophosphorylation of the receptor. To further analyze the correlation between E5 biological activity and its ability to bind these cellular proteins, a series of nine E5 transmembrane mutants was evaluated. In 32D mouse hematopoietic cells, there was an incomplete correlation between the abilities of the E5 mutant proteins to associate the PDGF-R and to transform cells. However, all transforming E5 mutant proteins induced PDGF-R tyrosine phosphorylation. In NIH 3T3 and C127 mouse fibroblasts, both transforming and nontransforming E5 mutant proteins were defective for PDGF-R binding. In addition, while most of the transforming E5 proteins induced PDGF-R phosphorylation, one hypertransforming mutant (serine 17) neither bound nor induced receptor autophosphorylation. These findings support the hypothesis that the transformation of fibroblasts by E5 transmembrane mutants can involve alternative cellular targets or potentially independent activities of the E5 protein. In addition, these results underscore the critical role of the transmembrane domain in mediating E5 biological activities.     

Identification of receptor and heparin binding sites in fibroblast growth factor 4 by structure-based mutagenesis

Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-A resolution. FGF4 adopts a beta-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the betaC'-betaE loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O- and 6-O-sulfates in heparin to exert its optimal biological activity.     

Ligand dependent and independent internalization and nuclear translocation of fibroblast growth factor (FGF) receptor 1

Basic fibroblast growth factor (FGF-2) is one of the prototype members of a rapidly expanding family of polypeptides. FGF-2 acts on cells via a dual-receptor system consisting of high-affinity tyrosine kinase receptors (FGFR) and low-affinity receptors comprised of heparan sulfate proteoglycans. Following ligand binding and subsequent internalization, both FGF-2 and FGFR1 are translocated to the nucleus where they have activities distinct from those expressed at the cell surface. Despite the growing number of growth factors and receptors shown to translocate to the nucleus, little is known about the mechanisms of internalization and translocation and how these processes are regulated. In the studies reported in this paper, we examined the roles of clathrin-dependent and -independent endocytosis in the uptake of FGFR1 and one of its ligands, FGF-2. While the uptake of FGF-2 occurred at least partly by a caveolar-dependent mechanism, that of FGFR1 was independent of both caveolae and coated pits. Surprisingly, neither the uptake of FGF-2 nor FGFR1 required the activity of the receptor tyrosine kinase. In addition, we identified a cell cycle-dependent pathway of FGFR1 nuclear translocation that appears to be independent of ligand binding.     

Zinc binding to Alzheimer's Abeta(1-16) peptide results in stable soluble complex

Aggregation of the human amyloid beta-peptide (Abeta) into insoluble plaques is a key event in Alzheimer's disease. Zinc sharply accelerates the Abeta aggregation in vitro, and the Abeta region 6-28 was suggested to be the obligatory zinc binding site. However, time-dependent aggregation of the zinc-bound Abeta species investigated so far prevented their structural analysis. By using CD spectroscopy, we have shown here for the first time that (i) the protected synthetic peptide spanning the fragment 1-16 of Abeta binds specifically zinc with 1:1 and 1:2 stoichiometry under physiologically relevant conditions; (ii) the peptide-zinc complex is soluble and stable for several months; (iii) zinc binding causes a conformational change of the peptide towards a more structured state. These findings suggest the region 1-16 to be the minimal autonomous zinc binding domain of Abeta.     

Identification of novel inhibitors of fibroblast growth factor (FGF-2) binding to heparin and endothelial cell survival from a structurally diverse carbohybrid library

Inhibitors of FGF-2 binding to a heparin-albumin conjugate were identified by ELISA from a library of glucuronic acid derivatives. These compounds were also inhibitors of endothelial cell survival that is dependant on FGF-2 and heparin or heparan sulfate proteoglycans. The results indicate that these bioactive compounds may prove useful as lead structures for the further development of pharmaceutical agents capable of modulating biological activity of FGF-2.     

Towards a resolution of the stoichiometry of the fibroblast growth factor (FGF)-FGF receptor-heparin complex

The 22 members of the fibroblast growth factor (FGF) family have been implicated in cell proliferation, differentiation, survival, and migration. They are required for both development and maintenance of vertebrates, demonstrating an exquisite pattern of affinities for both protein and proteoglycan receptors. Recent crystal structures have suggested two models for the complex between FGFs, FGF receptors (FGFRs) and the proteoglycan heparan sulphate that mediates signalling, and have provided insight into how FGFs show differing affinities for the range of FGFRs. However, the physiological relevance of the two different models has not been made clear. Here, we demonstrate that the two complexes can be prepared from the same protein components, confirming that neither complex is the product of misfolded protein samples. Analyses of the complexes with mass spectrometry and analytical ultracentrifugation show that the species observed are consistent with the crystal structures formed using the two preparation protocols. This analysis supports the contention that both of the crystal structures reflect the state of the molecules in solution. Mass spectrometry of the complexes suggests that the stoichiometry of the complexes is 2 FGF1:2 FGFR2:1 heparin, regardless of the method used to prepare the complexes. These observations suggest that the two proposed complex architectures may both have relevance to the formation of an in vivo signalling complex, with a combination of the two interactions contributing to the formation of a larger focal complex.     

Differential effects of a heparin antagonist (hexadimethrine) or chlorate on amphiregulin,basic fibroblast growth factor,and heparin-binding EGF-like growth factor activity

Amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF) are two recently identified members of the EGF family. Both AR and HB-EGF share with EGF the ability to interact with the type-1 EGF receptor; however, AR and HB-EGF differ from EGF in that both of these mitogens bind to heparin while EGF does not. To determine whether interactions with heparin-like molecules on the cell surface influence binding of AR and HB-EGF with EGF receptors and the subsequent mitogenic activity exerted by these growth factors, murine AKR-2B and Balb/MK-2 cells were treated with either an inhibitor of proteoglycan sulfation (chlorate) or a heparin antagonist (hexadimethrine). As expected, neither treatment significantly altered the specific binding of ¹²⁵I-EGF on AKR-2B cells. Interestingly, treatment with either chlorate or hexadimethrine inhibited the ability of AR to compete with ¹²⁵I-EGF for cell surface binding and also attenuated AR-mediated DNA synthesis. Thus, as has been suggested for other heparin-binding growth factors such as basic fibroblast growth factor (bFGF), the interaction of AR with an EGF-binding receptor appears to be facilitated by interaction with cell-associated sulfated glycosami-noglycans or proteoglycans. Unexpectedly, however, neither chlorate nor hexadimethrine treatment caused an inhibition of HB-EGF-induced mitogenic activity. Chlorate treatment did not significantly alter the ability of HB-EGF to compete with ¹²⁵I-EGF for cell surface binding sites, however, heparin and hexadimethrine reduced the ability of HB-EGF to compete for ¹²⁵I-EGF binding. These results suggest that, in AKR-2B cells, HB-EGF may mediate its mitogenic response at least in part through a receptor which appears to be selective for HB-EGF and permits HB-EGF-mediated mitogenic responses in the presence of hexadimethrine or heparin. Finally, hexadimethrine inhibited the specific binding and mitogenic activity of bFGF, suggesting that this cationic polymer can function as an antagonist of heparin-binding mitogens other than AR. © 1995 Wiley-Liss, Inc.     

Myo-inositol hexasulphate and low molecular weight heparin binding to human acidic fibroblast growth factor: a calorimetric and FTIR study

The interaction of an amino-terminal-truncated 139 amino-acids form of human acidic fibroblast growth factor with myo-inositol hexasulphate and low molecular weight (3500 g mol(-1)) heparin has been studied by isothermal titration calorimetry, differential scanning calorimetry and Fourier transform infrared spectroscopy. A slightly higher affinity for the monosaccharide has been measured. The binding of the ligands causes an increase of 13--15 degrees C in the melting temperature of the free protein (45 degrees C). From measured enthalpy and heat capacity changes, calculations of changes in accessible surface areas have been made. These calculations, together with infrared spectroscopy data, indicate that a small conformational change is induced by the binding of both ligands. This conformational change would affect the tertiary structure, not the secondary one.     

Inositolhexakisphosphate (InsP6): An antagonist of fibroblast growth factor receptor binding and activity

Summary Fibroblast growth factors (FGF), which have been implicated in tumor cell growth and angiogenesis, have biological activities that appear to be mediated by both heparinlike extracellular matrix sites and transmembrane tyrosine kinase receptor sites. In the present study, we demonstrated that inositolhexakisphosphate (InsP₆) inhibits basic FGF (bFGF) binding to heparin. Our spectrofluorometric analyses demonstrated that InsP₆ not only bound to bFGF, presumably within the bFGF heparin-binding domain, but also protected bFGF from degradation by trypsin. Also, InsP₆ inhibited the cellular binding of bFGF and other fibroblast growth factor family members such as acidic FGF (aFGF) and K-FGF in a saturable and dose-dependent manner. Furthermore, concentrations as low as 100μM InsP₆ inhibited bFGF-induced DNA synthesis in AKR-2B fibroblasts, as well as the growth of bFGF-and K-FGF-transfected NIH/3T3 cells. Together, these results indicate that InsP₆ may serve as a useful antagonist of FGF activity.     

Phenylmethylsulfonyl fluoride (PMSF) inhibits nerve growth factor binding to the high affinity (type I) nerve growth factor receptor

Dorsal root ganglia were extirpated from 9-day old embryonic chickens and solubilized in phosphate buffered saline containing 0.5% Noniodet P 40 detergent. When nerve growth factor binding studies are performed on these samples, the expected curvilinear Rosenthal (Scatchard) plot is obtained. However, when the solubilized cell sample is made 1-2 mM in phenylmethylsulfonyl fluoride and nerve growth factor binding is determined, a linear Rosenthal (Scatchard) plot is obtained. The equilibrium dissociation constant obtained from the slope of the line is 1.9 X 10(-9) M, identical to the equilibrium dissociation constant of the low affinity receptor. A similar phenomenon is observed when rat pheochromocytoma cells are solubilized in the non-ionic detergent and nerve growth factor binding is determined. No high affinity binding can be detected for either cell type when detergent solubilized cells are incubated with phenylmethylsulfonyl fluoride.     

Basic fibroblast growth factor (bFGF): mitogenic activity and binding sites in human breast cancer.

We investigated binding characteristics of basic fibroblast growth factor (bFGF) on membranes prepared from 4 human breast cancer cell lines and 38 primary BC biopsies. Competitive binding experiments were performed and analyzed using the "Ligand" program. Furthermore bFGF mitogenic activity was measured by [3H]thymidine incorporation into DNA from breast cancer cell lines. The presence of high-affinity binding sites was demonstrated in each cell type (MCF-7: Kd = 0.60 nM; T-47D: Kd = 0.55 nM; BT-20: Kd = 0.77 nM; MDA-MB-231: Kd = 0.34 nM). The presence of these high-affinity binding sites was confirmed with saturation experiments. A second class of low-affinity binding sites was detected in the 2 hormone-independent cells (BT-20: Kd = 2.9 nM; MDA-MB-231: Kd = 2.7 nM). bFGF stimulated the proliferation of MCF-7, T-47D, BT-20 but not MDA-MB-231 cell lines. With competition experiments, binding sites were detectable in 36/38 breast cancers; high-affinity binding sites (Kd less than 1 nM) were present in 19/36 cases and low-affinity binding sites (Kd greater than 2 nM) were present in 29/36 cases (the two classes of binding sites were present in 12 breast cancers). No relation between bFGF binding sites and node involvement, histologic type or grading of the tumor was evidenced. There were negative correlations (Spearman test) between total bFGF binding sites and estradiol receptor (P = 0.05) or progesterone receptor (P = 0.009). The demonstration of (1) bFGF specific binding sites in breast cancer membranes, and (2) bFGF growth stimulation of some breast cancer cell lines indicates that this factor may be involved directly in the growth of some breast cancers.     

Basic fibroblast growth factor (FGF) promotes cartilage repair in vivo

Although it has been clearly established that basic fibroblast growth factor (FGF) is a potent mitogen for chondrocytes in vitro, there is little evidence that it can stimulate this cell type in vivo. In an effort to address this problem, we examined the effect of an intraarticular administration of basic FGF. Alzet osmotic pumps delivering the mitogen to the site of injury promotes the healing of intra-chondrial lesions by stimulating chondrocyte proliferation and the formation of extracellular matrix. The observation that chronic infusions of basic FGF can elicit a repair response at the site of injury suggests that this growth factor may have therapeutic applications that extend beyond its capacity to induce neovascularization. The results also suggest that one of the ways that the perichondrium mediates cartilage repair may be by the local production of FGF-like mitogens.     

Polysialylation promotes neural cell adhesion molecule-mediated cell migration in a fibroblast growth factor receptor-dependent manner, but independent of adhesion capability

Polysialic acid (PSA), a carbohydrate polymer mainly present in the neural cell adhesion molecule (NCAM), promotes neural plasticity; however, its mode of action in tumor malignancy remains largely unknown. In this study, we investigated the influence of polysialylation on cell migration. PSA consistently promoted cell migration on different extracellular matrices (ECMs) but differentially affected cell adhesion. All of these actions were reversed by endo-N-acetylneuraminidase treatment, and PSA-driven migration was inhibited by the specific fibroblast growth factor receptor (FGFR) inhibitor Su5402. Consistent with this latter observation, PSA-stimulated migration on different ECMs was paralleled by activation of the FGFR and its downstream signaling components, PLC-γ, focal adhesion kinase and extracellular signal-regulated kinase 1/2. In contrast, the pattern of p59(fyn) activation correlated with differential adhesion to different ECMs. Collectively, these results indicate that PSA-conjugated NCAM potentiates signal transduction by the FGFR pathway and thereby enhances cell migration independent of adhesion capability, providing additional insights into the role of PSA in cancer development.     

Basic fibroblast growth factor binding is a marker for extracellular neurofibrillary tangles in Alzheimer disease

Neurofibrillary tangles (NFT) are abnormal filamentous inclusions that develop in neurons in Alzheimer disease and other disorders. When neurons die, the neurofibrillary tangles that persist in the extracellular space show ultrastructural and antigenic changes. Both intra- and extracellular NFT have recently been shown to contain heparan sulfate proteoglycans (HSPGs). HSPGs are also present in other amyloid deposits in the brain and in systemic amyloidoses. Basic fibroblast growth factor (bFGF) is a heparin binding growth factor which is involved in angiogenesis and also has neurite promoting activity. We now report that bFGF binds avidly to extracellular NFT. Alz-50, a monoclonal antibody (MAb) to an abnormal form of tau and bFGF binding label mutually exclusive subpopulations of neurofibrillary tangles. bFGF binding is abolished by heparinase or heparitinase treatment and therefore is most likely based on the presence of HSPG. Binding of bFGF is a specific and sensitive morphological method to distinguish intra- from extracellular NFT. As intracellular NFT, which also contain HSPGs, are not labeled by bFGF binding, this finding also suggests that HSPGs are modified when the NFT become extracellular.     

Studies of binding of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to fibroblast growth factor inducible 14 (Fn14)

To perform highly sensitive cellular binding studies with TNF-like weak inducer of apoptosis (TWEAK), we developed a bioluminescent variant of soluble TWEAK (GpL-FLAG-TNC-TWEAK) by fusing it genetically to the C terminus of the luciferase of Gaussia princeps (GpL). Equilibrium binding studies on human (HT1080 and HT29) and murine (Renca and B16) cell lines at 37 °C revealed high affinities of human TWEAK from 53 to 112 pm. The dissociation rate constant of the TWEAK-Fn14 interaction was between 0.48×10(-3) s(-1) (HT29) and 0.58×10(-3) s(-1) (HT1080) for the human molecules, and the association rate constant obtained was 3.3×10(6) m(-1) s(-1) for both cell lines. It has been shown previously that oligomerization of soluble TWEAK trimers results in enhanced Fn14-mediated activation of the classical NFκB pathway. Binding studies with GpL-FLAG-TNC-TWEAK trimers oligomerized by help of a FLAG tag-specific antibody gave no evidence for a major increase in Fn14 occupancy by oligomerized ligand despite strongly enhanced induction of the NFκB target IL8. Thus, aggregated complexes of soluble TWEAK and Fn14 have a higher intrinsic activity to stimulate the classical NFκB pathway and qualitatively differ from isolated trimeric TWEAK-Fn14 complexes. Furthermore, determination of IL8 induction as a function of occupied activated receptors revealed that the intrinsic capability of TNFR1 to stimulate the classical NFκB pathway and IL8 production was ～100-fold higher than Fn14. Thus, although ～25 activated TNFR1 trimers were sufficient to trigger half-maximal IL8 production, more than 2500 cell-bound oligomerized TWEAK trimers were required to elicit a similar response.     

Suramin interrupts androgen-inducible autocrine loop involving heparin binding growth factor in mouse mammary cancer (Shionogi carcinoma 115) cells

The androgen-dependent clonal cell line SC-3, derived from Shionogi carcinoma 115, secretes a fibroblast growth factor (FGF)-autocrine growth factor in response to androgen, which is able to bind to FGF receptors. In SC-3 cells, FGF receptor expression is upregulated by the SC-3-derived growth factor, providing a means of amplifying an autocrine loop of cell growth. In the present investigations, the effect of the polysulfonated naphthylurea suramin on this autocrine loop and its amplification in SC-3 cells were studied. Suramin inhibited androgen-dependent growth of SC-3 cells in a concentration-dependent fashion: ～50% inhibition was observed at 25 μM. [³H]Thymidine incorporation into the cells stimulated with partially purified SC-3-derived growth factor was inhibited by suramin in a similar way. Additionally, suramin inhibited acidic (a) or basic (b) FGF-induced cell proliferation, though relatively high concentrations were necessary to achieve the maximal inhibition. Pretreatment of SC-3 cells with suramin decreased cell surface ¹²⁵I-bFGF binding without altering dissociation constant (Kd) of the binding sites. When the cells were incubated with 250 μM suramin for 24 h, the maximum binding (Bmax) decreased to almost 50% of the control. Treatment with suramin also decreased the levels of FGF receptor-1 mRNA to a similar extent, whereas it appeared not to affect the levels of β-actin mRNA. Moreover, suramin completely blocked androgen- or bFGF-induced accumulation of FGF receptor-1 mRNA. The inhibitory effects of suramin on FGF receptor expression were reversed by simultaneous addition of high concentrations of bFGF. These results indicate that suramin exerts its potent antiproliferative action on SC-3 cells through inhibition of an androgen-inducible autocrine loop involving SC-3-derived growth factor and FGF receptor. © 1993 Wiley-Liss, Inc.     

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.