Identification of the hepatocyte mitogen in bovine spleen as heparin-binding growth factors.

Growth promoting activity for rat hepatocytes in bovine spleen was identified as three heparin-binding growth factors. All the features tested, such as heparin affinity, molecular mass, cross reactivity with antibody, and partial amino acid sequence, indicated that one of the three factors was identical to FGF-1 (fibroblast growth factor-1, acidic FGF), another one was related to FGF-2 (fibroblast growth factor-2, basic FGF), whereas it was more potent for hepatocytes than the FGF-2 purified from bovine brain. The third one was eluted from heparin-Sepharose column at 0.75M NaCl, of which activity was not abolished by anti-FGF-1 or FGF-2 antibodies. In addition, the mitogenic effect of this factor was synergistic with that of HGF (hepatocyte growth factor), a known potent hepatocyte mitogen, suggesting that it is a novel growth factor for hepatocytes.     

AMP-activated protein kinase positively regulates FGF-2-stimulated VEGF synthesis in osteoblasts

AMP-activated protein kinase (AMPK) is recognized as a regulator of energy homeostasis. We have previously reported that basic fibroblast growth factor (FGF-2) stimulates vascular endothelial growth factor (VEGF) release through the activation of p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of AMPK in FGF-2-stimulated VEGF release in these cells. FGF-2 time-dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an AMPK inhibitor, which suppressed the FGF-2-induced phosphorylation of AMPK, significantly inhibited the VEGF release stimulated by FGF-2. The AMPK inhibitor also reduced the mRNA expression of VEGF induced by FGF-2. The FGF-2-induced phosphorylation of both p44/p42 MAP kinase and SAPK/JNK was attenuated by compound C. These results strongly suggest that AMPK positively regulates the FGF-2-stimulated VEGF synthesis via p44/p42 MAP kinase and SAPK/JNK in osteoblasts.     

Modulation of cell growth and transformation by doxycycline-regulated FGF-2 expression in NIH-3T3 cells.

The single-copy fibroblast growth factor 2 (FGF-2) gene encodes four coexpressed isoforms of different molecular masses. The 18-kDa FGF-2 is primarily localized in the cytoplasm, whereas the higher molecular mass isoforms (HMW FGF-2) localize to the nucleus and nucleolus. The overexpression of either 18-kDa FGF-2 or HMW FGF-2 promotes cell transformation in a dose-dependent manner. In NIH 3T3 cells, the selective overexpression of HMW FGF-2 but not of 18-kDa FGF-2 confers upon the cells the unique phenotype of growth in low serum-containing medium. Thus, the distinct intracellular localization and the level of expression of FGF-2 are pivotal requirements for the differential effects of FGF-2 isoforms on the cellular phenotype. On this basis, we established a doxycycline-regulatable FGF-2 expression system that permitted us to regulate the expression of each isoform in a time- and dose-dependent manner. We analyzed the growth properties of cells in the presence and absence of doxycycline in both normal and low serum-containing medium and in soft agar. The doxycycline-activated expression of 18-kDa FGF-2 did not allow growth in low serum medium. The growth of cells expressing HMW FGF-2 was increased by doxycycline under all three conditions, and a relationship between the level of HMW FGF-2 expression and cell growth was observed for all three conditions. This doxycycline-regulatable FGF-2 expression system provides a mechanism to analyze changes in FGF-2 targeted pathways and genes and to characterize pathways specifically activated by either the 18-kDa FGF-2 or the HMW FGF-2 isoforms.     

Glycated fibroblast growth factor-2 is quickly produced in vitro upon low-millimolar glucose treatment and detected in vivo in diabetic mice

Angiogenesis impairment in hyperglycemic patients represents a leading cause of severe vascular complications of both type-1 and -2 diabetes mellitus (DM). Angiogenesis dysfunction in DM is related to glycemic control; however, molecular mechanisms involved are still unclear. Fibroblast growth factor-2 (FGF-2) is a potent angiogenic factor and, according to previous evidence, may represent a key target of molecular modifications triggered by high-sugar exposure. Therefore, the purpose of this study was to investigate whether short incubation with hyperglycemic levels of glucose affected FGF-2 and whether glucose-modified FGF-2 was detectable in vivo. Biochemical analyses carried out with SDS-PAGE, fluorescence emission, mass-spectrometry, immunoblot, and competitive ELISA experiments demonstrated that human FGF-2 undergoes a rapid and specific glycation upon 12.5-50 mm glucose exposure. In addition, FGF-2 exposed for 30 min to 12.5 mm glucose lost mitogenic and chemotactic activity in a time- and dose-dependent manner. Under similar conditions, binding affinity to FGF receptor 1 was dramatically reduced by 20-fold, as well as FGF receptor 1 and ERK-1/2 phosphorylation, and FGF-2 lost about 45% of angiogenic activity in two different in vivo angiogenic (Matrigel and chorioallantoic-membrane) assays. Such glucose-induced modification was specific, because other angiogenic growth factors, namely platelet-derived growth factor BB and placental-derived growth factor were not significantly or markedly less modified. Finally, for the first time, glycated-FGF-2 was detected in vivo, in tissues from hyperglycemic nonobese diabetic mice, in significantly higher amounts than in normoglycemic mice. In conclusion, hyperglycemic levels of glucose may strongly affect FGF-2 structure and impair its angiogenic features, and endogenous glycated-FGF-2 is present in diabetic mice, indicating a novel pathogenetic mechanism underlying angiogenesis defects in DM.     

A novel recombinant basic fibroblast growth factor and its secretion.

Basic fibroblast growth factor (FGF-2) is a pleiotropic mitogen which plays an important role in cell growth, differentiation, migration, and survival in different cells and organ systems. Recently, several clinical applications for FGF-2 gene transfer are being evaluated in wound healing and collateral artery development to relieve myocardial and peripheral ischemia due to the ability of FGF-2 to regulate the growth and function of vascular cells. However, FGF-2 lacks a classical hydrophobic secretion signal peptide, the FGF-2 chimeras containing various signal sequences have been explored. In this study, a novel recombinant 4sFGF-2 was constructed by replacing nine residues from the amino-terminus of native FGF-2 (Met1 to Leu9) with eight amino acid residues of signal peptide of FGF-4 (Met1 to Ala8) to better increase the secretion level of FGF-2. When the recombinant FGF-2 gene, cloned into the expression vector with CMV promoter, was expressed in COS-7 cells, the recombinant 4sFGF-2 was highly secreted into the media. The secreted 4sFGF-2 showed the same biological activity as the native FGF-2 in the dose-response effects on DNA synthesis and cell growth of rat aortic smooth muscle cells (RASMCs) and NIH3T3 cells. The 4sFGF-2 also was able to activate MAPK as wild FGF-2 in RASMCs. These results indicate that a novel recombinant 4sFGF-2 may be useful as clinical applicability of angiogenic growth factor gene transfer.     

The endogenous fibroblast growth factor-2 antisense gene product regulates pituitary cell growth and hormone production

Basic fibroblast growth factor (bFGF; FGF-2) is one of 19 related members of a growth factor family with mitogenic and hormone-regulatory functions. In Xenopus laevis oocytes, a 1.5-kb FGF-2 antisense (GFG) RNA complementary to the third exon and 3'-untranslated region (UTR) of FGF-2 mRNA has been implicated in FGF-2 mRNA editing and stability. The human homolog has been cloned, and we localized this gene by yeast artificial chromosome (YAC), somatic cell, and radiation hybrid panels to the same chromosomal site as FGF-2 (chromosome 4, JO4513 adjacent to D4S430), confirming this as a human endogenous antisense gene. The full-length GFG antisense RNA encodes a 35-kDa protein, which is highly homologous with the MutT family of antimutator nucleosidetriphosphatases (NTPases). We show that human pituitary tumors express FGF-2 and its endogenous antisense partner GFG. While normal pituitary expresses GFG but not FGF-2, pituitary adenomas express FGF-2 and have reduced levels of GFG; aggressive and recurrent adenomas expressed more FGF than GFG mRNA. To examine the effects of this antisense gene in the pituitary, we transfected the pituitary-derived GH4 mammosomatotroph cell line with constructs encoding the full-length human GFG cDNA. Transiently and stably transfected cells expressed the 35-kDa GFG protein that was localized to the cytoplasm. These cells exhibited enhanced PRL expression as documented by transiently transfected PRL-luciferase reporter assay and by endogenous PRL protein. GFG expression in these cells did not alter endogenous FGF-2 expression but increased the proportion of the higher molecular mass 22-kDa form of GH. Moreover, GFG expression inhibited cell proliferation as shown by [(3)H]thymidine incorporation, proliferating cell nuclear antigen (PCNA) nuclear staining, and cell cycle analysis. We conclude that the GFG-encoded protein has divergent hormone-regulatory and antiproliferative actions in the pituitary that are independent of FGF-2 expression. GFG represents a novel mechanism involved in restraining pituitary tumor cell growth while promoting hormonal activity.     

Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines

Basic fibroblast growth factor (FGF-2) and its respective tyrosine kinase receptors, form an autocrine loop that affects human melanoma growth and metastasis. The aim of the present study was to examine the possible participation of various glycosaminoglycans, i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell's proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous glycosaminoglycan/proteoglycan production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor, genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.     

Regulation of FGF-1 mitogenic activity by heparan sulfate oligosaccharides is dependent on specific structural features: differential requirements for the modulation of FGF-1 and FGF-2

The interaction of heparan sulfate (HS) (and the closely related molecule heparin) with FGF-1 is a requirement for enabling the growth factor to activate its cell surface tyrosine kinase receptor. However, little is known about the regulatory role of naturally occurring cell surface HS in FGF-1 activation. We have addressed this issue by utilizing a library of HS oligosaccharides, which are defined in both length and sulfate content. Mitogenic activation assays using these oligosaccharides showed that HS contained both FGF-1 activatory and inhibitory sugar sequences. Further analysis of these oligosaccharides showed a clear correlation between FGF-1 promoting activity and their 6-O-sulfate content. The results, in particular with the dodecasaccharide sequences, suggested that specific positioning of 6-O-sulfate groups may be required for the promotion of FGF-1 mitogenic activity. This may also be true for 2-O-sulfate groups though the evidence was not as conclusive. Differential activation of FGF-1 and FGF-2 was also observed and found to be mediated by both oligosaccharide length and sulfation pattern, with different specific O-sulfate positioning being implicated for the promotion of different growth factors. These results suggest that variation and tight control of the fine structure of HS may allow cells to not only control their positive/negative responses to individual FGFs but also to change specificity towards promotion of different members of the FGF family.     

Immunolocalization of fibroblast growth factor-2 (FGF-2) in the developing root and supporting structures of the murine tooth

Summary Epithelio–mesenchymal interactions are active during the development of the root of the tooth and are regulated by a variety of growth factors, such as fibroblast growth factors. FGF-2, 3, 4, and 8 have all been shown to play a role in the development of the crown of the tooth, but less is known about the factors that govern root formation, particularly FGF-2. The aim of this study was thus to elucidate the spatial and temporal expression of FGF-2 in the root of the developing tooth, as this growth factor is believed to be a mediator of epithelio–mesenchymal interactions. Parasagittal sections of the maxillary and mandibular arches of post-natal mice were utilized and the roots of the molar teeth were studied. Immunocytochemistry utilizing an antibody to FGF-2 was performed on sections of teeth at various stages of development. Intense immunostaining for FGF-2 was observed in differentiating odontoblasts at the apical end of the tooth and in the furcation zone of the developing root at all the stages examined. FGF-2 localization was also observed in cementoblasts on post-natal days 16, 20 and 24. The pattern of localization of FGF-2 in the developing root suggests that this growth factor may participate in the signaling network associated with root development.     

Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2)

Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1–155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.     

Mitogenic effects of fibroblast growth factors on chicken granulosa and theca cells in vitro

We have investigated the role that fibroblast growth factors (FGFs) may play in the rapid growth of preovulatory ovarian follicles in chickens. Granulosa and theca cells, dissected from the follicles of laying hens, were cultured in vitro and treated with FGF-1, FGF-2, FGF-5, and FGF-7. The synthesis of DNA by cultured cells was measured by incorporation of [(3)H]thymidine, which was added to the cultures. FGF-1 and -2 increased the synthesis of DNA in a dose-dependent manner in both cell types; however, FGF-5 and -7 had no effect in this respect. When genistein, a tyrosine kinase inhibitor, was added to these cultures, the synthesis of DNA due to FGF-2 was abolished. Treatment of cells with the glycosaminoglycans heparan sulphate and chondroitin sulphate had no effect on FGF-2-induced mitogenesis, while heparin inhibited it. Addition of a glycosaminoglycan antagonist, hexadimethrine bromide, to FGF-2-treated cultures inhibited DNA synthesis due to FGF-2, although not completely. Our data show that FGF-1 and FGF-2 are mitogenic for chicken granulosa and theca cells, and indicate that the actions of FGF-2 may be mediated via both tyrosine-kinase-type and glycosaminoglycan-type receptors on the surface of these cells.     

Molecular mechanisms of the antiproliferative effect of beraprost, a prostacyclin agonist, in murine vascular smooth muscle cells

The antithrombotic activity of heparin has largely been credited with the success found in some cancer treatment by heparin. There are, however, many potent growth factors involved in tumor and blood vessel growth that bind to heparin with high affinity and their regulation by heparin may play a role in heparin's efficacy. We therefore chose to study the activity of a heparin analog, sucrose octasulfate (SOS), which has been similarly shown to interact with heparin-binding growth factors. Using mouse melanoma and lung carcinoma models, we demonstrate in vivo inhibition of tumor growth by SOS. SOS, however, showed little effect in coagulation assays indicating that this activity was not a primary mechanism of action for this molecule. Studies were then performed to assess the effect of SOS on basic fibroblast growth factor (FGF-2) activity, a growth factor which promotes tumor and blood vessel growth and is produced by B16 melanoma cells. SOS potently inhibited FGF-2 binding to endothelial cells and stripped pre-bound FGF-2 from cells. SOS also regulated FGF-2 stimulated proliferation. Further, SOS facilitated FGF-2 diffusion through Descemet's membrane, a heparan sulfate-rich basement membrane from the cornea, suggesting a possible role in FGF-2 clearance. Our results suggest that molecules such as SOS have the potential to remove growth factors from tumor microenvironments and the approach offers an attractive area for further study.     

Potential mechanisms for the regulation of growth factor binding by heparin

Heparin and heparan sulfate proteoglycans (HSPG) bind many soluble growth factors and this binding is now recognized as an important mechanism for modulation of cell activity. Fibroblast growth factor-2 (FGF-2) is one of the best characterized of the heparin-binding growth factors and it has been shown experimentally that heparin regulation of FGF-2 activity is dependent on the level of cell HSPG and the concentration of heparin. In this paper, we explore, using mathematical modeling, proposed mechanisms for heparin regulation and determine how they impact FGF receptor binding. We demonstrate that the experimentally observed receptor binding phenomena can be reproduced if cells (1) express heparin-binding cell surface molecules and if either (2) these heparin binding sites are FGFR and bind heparin and FGF-2-heparin complexes or (3) are surface molecules able to bind FGF-2 and couple with FGF-2 receptors to form high-affinity FGF-2-bound surface complexes. The ability of heparin to directly interact with the FGFR and bind FGF-2 in the absence of this coupling function was not sufficient to explain heparin activity. These findings have implications with regard to regulation of heparin-binding growth factors and could help guide the development of highly specific growth regulatory molecules through specific regulation by heparin and HSPG.     

Antidepressant Acts on Astrocytes Leading to an Increase in the Expression of Neurotrophic/Growth Factors: Differential Regulation of FGF-2 by Noradrenaline

Recently, multiple neurotrophic/growth factors have been proposed to play an important role in the therapeutic action of antidepressants. In this study, we prepared astrocyte- and neuron-enriched cultures from the neonatal rat cortex, and examined the changes in neurotrophic/growth factor expression by antidepressant treatment using real-time PCR. Treatment with amitriptyline (a tricyclic antidepressant) significantly increased the expression of fibroblast growth factor-2 (FGF-2), brain-derived neurotrophic factor, vascular endothelial growth factor and glial cell line-derived neurotrophic factor mRNA with a different time course in astrocyte cultures, but not in neuron-enriched cultures. Only the expression of FGF-2 was higher in astrocyte cultures than in neuron-enriched cultures. We focused on the FGF-2 production in astrocytes. Several different classes of antidepressants, but not non-antidepressants, also induced FGF-2 mRNA expression. Noradrenaline (NA) is known to induce FGF-2 expression in astrocyte cultures, as with antidepressants. Therefore, we also assessed the mechanism of NA-induced FGF-2 expression, in comparison to amitriptyline. NA increased the FGF-2 mRNA expression via α1 and β-adrenergic receptors; however, the amitriptyline-induced FGF-2 mRNA expression was not mediated via these adrenergic receptors. Furthermore, the amitriptyline-induced FGF-2 mRNA expression was completely blocked by cycloheximide (an inhibitor of protein synthesis), while the NA-induced FGF-2 mRNA was not. These data suggest that the regulation of FGF-2 mRNA expression by amitriptyline was distinct from that by NA. Taken together, antidepressant-stimulated astrocytes may therefore be important mediators that produce several neurotrophic/growth factors, especially FGF-2, through a monoamine-independent and a de novo protein synthesis-dependent mechanism.     

Heparan Sulfate Proteoglycans Play a Dual Role in Regulating Fibroblast Growth Factor-2 Mitogenic Activity in Human Breast Cancer Cells

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of¹²⁵I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10–30 mM;higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.     

Decapeptide with fibroblast growth factor (FGF)-5 partial sequence inhibits hair growth suppressing activity of FGF-5

Earlier studies demonstrated that knock-out of fibroblast growth factor-5 gene (Fgf-5) prolonged anagen VI phase of hair cycle, resulting long hairs in the mice. We showed the activities on hair growth of the two Fgf-5 gene products, one of which, FGF-5 suppressed hair growth by inhibiting anagen proceeding and inducing the transition from anagen to catagen, and FGF-5S, a shorter polypeptide with FGF-5-antagonizing activity translated from alternatively spliced mRNA, suppressed this activity of FGF-5. As the results suggested that FGF-5 antagonist would increase hair growth, we synthesized various peptides having partial sequences of human FGF-5 and FGF-5S and determined their FGF-5 antagonist activity. Among them, a decapeptide designated P3 (95-VGIGFHLQIY-104) that aligns with receptor binding sites of FGF-1 and FGF-2 suppressed FGF-5-induced proliferation of BALB/3T3 A31 and NIH/3T3 murine fibroblasts, and FGF receptor-1c (FGFR-1c)-transfected Ba/F3 cell line (FR-Ba/F3 cells). IC50s of this peptide on these cell proliferations were 64, 28, 146 microM, respectively. On the other hand, IC50 of this peptide on binding of FGF-5 to the FGFR-1(IIIc)/Fc chimera was 483 microM. Examination in dorsal depilated mice revealed that the P3 peptide reduced the activity of FGF-5 to recover hair pigmentation and hair follicle lengths. The classification of histologically observed skin sections showed FGF-5-induced delations of anagen procedure had reduced by the P3 peptide. The anti-Ki67 antibody staining of hair follicles was inhibited by administration of FGF-5, and this inhibition by FGF-5 was recovered by administration of the P3 peptide. The P3 peptide alone did not affect hair follicle length and hair cell proliferation. These results indicate that the decapeptide antagonized FGF-5 activity in vivo, and reduced the inhibition of FGF-5 in hair growth, confirming that FGF-5 inhibitors are promising substances against hair loss and/or for promoting hair growth.