Peroxynitrite modulates acidic fibroblast growth factor (FGF-1) activity

To establish peroxynitrite (ONOO(-)) as a mediator of acidic fibroblast growth factor (FGF-1) function, preparations of recombinant human FGF-1 were treated with the pro-oxidant in vitro and identified amino acid modifications were correlated with biologic activity. The sequence of FGF-1 amino acid modifications induced by increasing concentrations of ONOO(-) was from cysteine oxidation to dityrosine formation, and to tyrosine/tryptophan nitration. Low steady-state ONOO(-) concentrations (10-50 microM) induced formation of dityrosine, which involved less than 0.1% of the total tyrosines. Treatment of FGF-1 with ONOO(-) induced a dose-dependent (10-50 microM) loss of sulfhydryl groups that correlated with formation of reducible (dithiothreitol, arsenite) FGF-1 aggregates containing 50% latent biologic activity. Treatment with 0.1-0.5mM ONOO(-) induced increasing formation of non-reducible, inactivated FGF-1 structures. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that six residues (Y29, Y69, Y108, Y111, Y139, and W121) were nitrated by ONOO(-). ONOO(-) treatment (0.1mM) of an active FGF-1 mutant (cysteines converted to serines) induced dose-dependent, non-reversible inhibition of biologic activity that correlated with nitration of Y108 and Y111, both of which reside within a conserved domain encompassing the putative FGF-1 receptor binding site. Collectively, these observations predict a role for low levels of ONOO(-) during secretion of FGF-1 as an extracellular complex containing latent biologic activity. High steady-state levels of ONOO(-) may induce extensive cysteine oxidation, critical tyrosine nitration, and non-reversible inactivation of FGF-1, a potential inhibitory feedback mechanism restoring cellular homeostatis during the resolution of inflammation and repair.     

Acidic fibroblast growth factor (FGF-1) signaling inhibits peroxynitrite-induced cell death during pancreatic tumorigenesis

Previous immunohistochemical studies have demonstrated enhanced appearance of FGF-1 and nitrotyrosine, a footprint of reactive nitrogen species peroxynitrite (ONOO(-)), in human pancreatic adenocarcinoma. We have examined the consequences of constitutive exposure to FGF-1 in nontumorigenic rat ductal epithelial cells (ARIP). ARIP cells were transduced with either a secreted chimera of FGF-1, ARIP(FGF-1), or a control plasmid, 65 RIP(betag). These cells were evaluated for alteration in growth and morphology, responses to ONOO(-) (protein tyrosine nitration/phosphorylation), and in vivo tumor formation. ARIP(FGF-1) cells, in contrast to 65 RIP(betag), demonstrated a transformed morphology, a 2-fold increased growth rate, and enhanced protein tyrosine phosphorylation. Treatment with 150 microM ONOO(-) resulted in 86 and 7% (p <.01) death of ARIP(betag) and ARIP(FGF-1), respectively. Exposure of 65 RIP(betag) cells to ONOO(-) enhanced tyrosine phosphorylation and tyrosine nitration of several polypeptides. Cell signaling by FGF-1 enhanced both phosphorylation and nitration of tyrosine residues in target proteins modified by ONOO(-). ARIP(betag) cells failed to exhibit tumor formation in nude mice, but at d 7 in vivo cells were TUNEL and nitrotyrosine positive and FGF-1 negative. ARIP(FGF-1) cells readily formed tumor nodules, exhibiting features of pancreatic adenocarcinoma and demonstrating FGF-1-positive, nitrotyrosine-positive, and TUNEL-negative epithelium. These results suggest an interdependent role between FGF-1 and ONOO(-) during the development and progression of pancreatic adenocarcinoma.     

Cellular localization of fibroblast growth factor 2 (FGF-2) in benign prostatic hyperplasia

Fibroblast growth factor 2 (FGF-2, basic fibroblast growth factor) has been reported to be elevated in tissues from benign prostatic hyperplasia (BPH), the most frequent neoplastic disease in aging men. This suggests that FGF-2 may play a significant role in the development of BPH. In this study the cellular distribution pattern of FGF-2 in tissues from BPH has been investigated by immunohistochemical and molecular biological methods. Radioimmunoassay revealed high concentrations of FGF-2, ranging between 450 and 950 ng per g tissue. Immunoblots confirmed the presence of a 18 kDa FGF-2 in tissue extracts. By immunohistochemistry done with a polyclonal antibody to recombinant FGF-2 on paraffin sections, FGF-2 was localized in fibroblasts, endothelial cells and smooth muscle cells of tissue samples of BPH. Nuclei of these cells were labelled distinctly. Moreover the cytoplasm of smooth muscle cells was labelled moderately. No immunostaining was seen in prostatic epithelium. Non-radioactive in situ hybridization with digoxygenin-labelled oligonucleotides revealed the presence of mRNA for FGF-2 in smooth muscle cells of the prostatic stroma. These results provide evidence that FGF-2 may be produced locally in the human prostate as a stroma-specific mitogen and may play a causal role in the development of BPH.     

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.     

Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2

Members of the FGF family of growth factors localize to the nuclei in a variety of different cell types. To determine whether FGF receptors are also present within nuclei and if this localization is regulated by FGFs, nuclei were prepared from quiescent and FGF-2-treated Swiss 3T3 fibroblasts and examined for the presence of FGF receptors by immunoblotting with an antibody produced against the extracellular domain of FGF receptor-1 (FGFR-1). Little or no FGFR-1 is detected in nuclei prepared from quiescent cells. When cells are treated with FGF- 2, however, there is a time- and dose-dependent increase in the association of FGFR-1 immunoreactivity with the nucleus. In contrast, treatment with either EGF or 10% serum does not increase the association of FGFR-1 with the nucleus. When cell surface proteins are labeled with biotin, a biotinylated FGFR-1 is detected in the nuclear fraction prepared from FGF-2-treated, but not untreated, cells indicating that the nuclear-associated FGFR-1 immunoreactivity derives from the cell surface. The presence of FGFR-1 in the nuclei of FGF-2- treated cells was confirmed by immunostaining with a panel of different FGFR-1 antibodies, including one directed against the COOH-terminal domain of the protein. Fractionation of nuclei from FGF-2-treated cells indicates that nuclear FGFR-1 is localized to the nuclear matrix, suggesting that the receptor may play a role in regulating gene activity.     

FGF-2/fibroblast growth factor receptor/heparin-like glycosaminoglycan interactions: a compensation model for FGF-2 signaling.

Heparin-like glycosaminoglycans (HLGAGs) play a central role in the biological activity and signaling behavior of basic fibroblast growth factor (FGF-2). Recent studies, however, indicate that FGF-2 may be able to signal in the absence of HLGAG, raising the question of the nature of the role of HLGAG in FGF-2 signaling. In this study, we present a conceptual framework for FGF-2 signaling and derive a simple model from it that describes signaling via both HLGAG-independent and HLGAG-dependent pathways. The model is validated with F32 cell proliferation data using wild-type FGF-2, heparin binding mutants (K26A, K119A/R120A, K125A), and receptor binding mutants (Y103A, Y111A/W114A). In addition, this model can predict the cellular response of FGF-2 and its mutants as a function of FGF-2 and HLGAG concentration based on experimentally determined thermodynamic parameters. We show that FGF-2-mediated cellular response is a function of both FGF-2 and HLGAG concentrations and that a reduction of one of the components can be compensated for by an increase in the other to achieve the same measure of cellular response. Analysis of the mutant FGF-2 molecules show that reduction in heparin binding interactions and primary receptor site binding interactions can also be compensated for in the same manner. These results suggest a molecular mechanism that could be used by cells in physiological systems to modulate the FGF-2-mediated cellular response by controlling HLGAG expression.     

Basic fibroblast growth factor (FGF-2) protects rat cochlear hair cells in organotypical culture from aminoglycoside injury

Given the evidence that basic fibroblast growth factor (FGF-2) can protect neural and retinal cells from degeneration, we evaluated the potential of this growth factor to protect sensory cells in the inner ear. When sensory cells of the organ of Corti are exposed to aminoglycoside antibiotics such as neomycin either in vivo or in vitro, significant ototoxicity is observed. The in vitro cytotoxic effects of neomycin are dose and time dependent. In neonatal rat organ of Corti cultures, complete inner and outer hair cell destruction is observed at high (mM) concentrations of neomycin while inner hair cell survival and severely damaged outer hair cells are noted at moderate (μM) concentrations, with a maximal effect observed after 2 days of culture. Approximately 50% of cochlear outer hair cells are lost at a dose of 35 μM neomycin, and most surviving cells show disorganized stereocilia. Inner hair cells show primarily disorganization of their stereocilia. A significant protective effect is observed when the organ of Corti is pre-treated with FGF-2 (500 ng/ml) for 48 hours, and then FGF-2 is included with neomycin in the culture medium. A greater extent of outer hair cell survival and a significant decrease in stereociliary damage are noted with FGF-2. However, disorganization of inner hair cell stereocilia is unaffected by FGF-2. The protective effect of FGF-2 is specific, since interleukin-1B, nerve growth factor, tumor necrosis factor, and epidermal growth factor are ineffective, while retinoic acid and transforming growth factor alpha show only a moderate protective effect. These results confirm the potential of molecules like FGF-2 for preventing cell death due to a variety of causes. © 1996 Wiley-Liss, Inc.     

Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.     

The role of fibroblast growth factor-2 (FGF-2) in hematopoiesis

Basic fibroblast growth factor (bFGF or FGF-2) is an angiogenic and pleiotropic growth factor involved in the proliferation and differentiation of numerous cell types. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and is thought to play an important role in the mesoderm induction. Although hematopoietic cells derive from the mesoderm, relatively few studies have, until recently, addressed the role of FGF-2 in hematopoiesis. FGF-2 is expressed in cells of the bone marrow including stromal cells, and possibly cells from several hematopoietic cell lineages. It is stored in the bone marrow extra-cellular matrix and released by enzymes such as heparanase, plasmin, or phospholipase C and D. FGF-receptors (FGF-Rs) are expressed in leukemic cell lines and in hematopoietic cells. FGF-2 positively regulates hematopoiesis, by acting on stromal cells, on early and committed hematopoietic progenitors, and possibly on some mature blood cells. The action of FGF-2 is most likely indirect since its action, on megakaryocytopoiesis for example, is abrogated by anti-IL6 antibodies. It synergizes with hematopoietic cytokines, or antagonizes the negative regulatory effects of TGF-β Taken together, these results demonstrate that FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.     

Overexpression of collagenase 1 (MMP-1) is mediated by the ERK pathway in invasive melanoma cells: role of BRAF mutation and fibroblast growth factor signaling

Melanoma progresses as a multistep process where the thickness of the lesion and depth of tumor invasion are the best prognostic indicators of clinical outcome. Degradation of the interstitial collagens in the extracellular matrix is an integral component of tumor invasion and metastasis, and much of this degradation is mediated by collagenase-1 (MMP-1), a member of the matrix metalloproteinase (MMP) family. MMP-1 levels increase during melanoma progression where they are associated with shorter disease-free survival. The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is a major regulator of melanoma cell proliferation. Recently, BRAF has been identified as a common site of activating mutations, and, although many reports focus on its growth-promoting effects, this pathway has also been implicated in progression toward metastatic disease. In this study, we describe four melanoma cell lines that produce high levels of MMP-1 constitutively. In each cell line the Ras/Raf/MEK/ERK pathway is constitutively active and is the dominant pathway driving the production of MMP-1. Activation of this pathway arises due to either an activating mutation in BRAF (three cell lines) or autocrine fibroblast growth factor signaling (one cell line). Furthermore, blocking MEK/ERK activity inhibits melanoma cell proliferation and abrogates collagen degradation, thus decreasing their metastatic potential. Importantly, this inhibition of invasive behavior can occur in the absence of any detectable changes in cell proliferation and survival. Thus, constitutive activation of this MAPK pathway not only promotes the increased proliferation of melanoma cells but is also important for the acquisition of an invasive phenotype.     

FIF [fibroblast growth factor-2 (FGF-2)-interacting-factor], a nuclear putatively antiapoptotic factor, interacts specifically with FGF-2

Numerous evidence indicates that some of the activities of fibroblast growth factor 2 (FGF-2) depend on an intracrine mode of action. Recently, we showed that three high molecular mass (HMM) nuclear forms of FGF-2 are part of a 320-kDa protein complex while the cytoplasmic AUG-initiated form is included in a 130-kDa complex. Consequently, the characterization of FGF endogenous targets has become crucial to allow the elucidation of their endogenous activities. Through the screening of GAL4-based yeast two-hybrid expression libraries, we have isolated a gene encoding a nuclear protein of 55 kDa, FIF (FGF-2-interacting-factor), which interacts specifically with FGF-2 but not with FGF-1, FGF-3, or FGF-6. In this system, FIF interacts equally well with the NH2-extended 24-kDa FGF form as with the 18-kDa form, indicating that the FIF-binding motif is located in the last 155 amino acids of FGF-2. Nevertheless, coimmunoprecipitation experiments showed an exclusive association with HMM FGF-2. The predicted protein contains a canonical leucine zipper domain and three overlapping hydrophobic heptad repeats. The region spanning these repeats is, together with a region located in the N-terminal part of the FIF protein, implicated in the binding to FGF-2. In contrast to the full-length FIF protein, several deletion constructs were able to transactivate a lac-Z reporter gene. Furthermore, the COOH-terminal part, but not the full-length FIF protein, has previously been shown to exhibit antiapoptotic properties. Thus we discuss the possibility that these activities could reflect a physiological function of FIF through its interaction with FGF-2.     

Role of AKT/PKB signaling in fibroblast growth factor-1 (FGF-1)-induced angiogenesis in the chicken chorioallantoic membrane (CAM)

Transfection of chicken chorioallantoic membranes (CAMs) with a chimeric secreted version of fibroblast growth factor-1 (sp-FGF-1) gene construct leads to a significant increase in vascularization. Though FGF-stimulated angiogenesis has been extensively studied, the molecular mechanisms regulating FGF-1-induced angiogenesis are poorly understood in vivo. This study was designed to investigate the role of the AKT (PKB) kinase signaling pathway in mediating sp-FGF-1-induced angiogenesis in the chicken CAM. The involvement of the AKT pathway was demonstrated by up-regulation of AKT1 mRNA expression in sp-FGF-1 compared to vector alone control transfected CAMs as demonstrated by real-time RT-PCR. Western analysis using an antibody specific to the activated AKT (phosphorylated AKT), demonstrated an increase in AKT activity in sp-FGF-1 compared to vector control transfected CAMs. More importantly, the AKT inhibitor ML-9 significantly reduced sp-FGF-1-induced angiogenesis in CAMs. These results indicate that AKT signaling plays a role in FGF-1-stimulated angiogenesis in vivo and the AKT pathway may serve as a therapeutic target for angiogenesis-associated diseases.     

Induction of stromelysin-1 (MMP-3) by fibroblast growth factor-2 (FGF-2) in FGF-2-/- microvascular endothelial cells requires prolonged activation of extracellular signal-regulated kinases-1 and -2 (ERK-1/2)

Basic fibroblast growth factor (FGF-2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF-2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP-3 (stromelysin-1), a broad-spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF-2-/- mice are highly responsive to exogenous FGF-2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF-2 results in strong induction of MMP-3 mRNA and protein expression. Upregulation of MMP-3 mRNA by FGF-2 requires de novo protein synthesis and activation of the ERK-1/2 pathway. FGF-2 concentrations (5-10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK-1/2 upregulate MMP-3 expression. In contrast, lower concentrations (1-2 ng/ml) that induce robust but transient (<8 h) ERK-1/2 activation are ineffective. Inhibition of ERK-1/2 activation at different times (-0.5 h to +8 h) of EC treatment with effective FGF-2 concentrations blocks MMP-3 upregulation. Thus, FGF-2 induces EC expression of MMP-3 with a threshold dose effect that requires sustained activation of the ERK-1/2 pathway. Because FGF-2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP-3 in vascular remodeling.     

Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.     

Differential tyrosine phosphorylation of fibroblast growth factor (FGF) receptor-1 and receptor proximal signal transduction in response to FGF-2 and heparin

The sulfated regions in heparan sulfate and heparin are known to affect fibroblast growth factor (FGF) function. We have studied the mechanism whereby heparin directs FGF-2-induced FGF receptor-1 (FGFR-1) signal transduction. FGF-2 alone stimulated maximal phosphorylation of Src homology domain 2 tyrosine phosphatase (SHP-2) and the adaptor molecule Crk, in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells expressing FGFR-1. In contrast, for phospholipase Cgamma(1) (PLCgamma(1)) and the adaptor molecule Shb to be maximally tyrosine-phosphorylated, cells had to be stimulated with both FGF-2 and heparin (100 ng/ml). Tyrosine residues 463 in the juxtamembrane domain and 766 in the C-terminal tail in FGFR-1 are known to bind Crk and PLCgamma(1), respectively. Analysis of tryptic phosphopeptide maps of FGFR-1 from cells stimulated with FGF-2 alone and FGF-2 together with heparin showed that FGF-2 alone stimulated a several-fold increase in tyrosine 463 in the juxtamembrane domain. In contrast, heparin had to be included in order for tyrosine 766 to be phosphorylated to the same fold level. Our data imply that tyrosine 463 is phosphorylated and able to transduce signals in response to FGF-2 treatment alone; furthermore, we suggest that FGFR-1 dimerization/kinase activation is stabilized by heparin.     

Dynamic regulation of fibroblast growth factor 2 (FGF-2) gene expression in the rat brain following single and repeated cocaine administration

Administration of drugs of abuse can produce long-lasting effects on brain function, which involve modifications at neurotransmitter level as well as changes in proteins important for structural alterations of selected brain regions. The contribution of trophic factors in these events has so far been underestimated. Here, we demonstrate that a single cocaine injection selectively up-regulated fibroblast growth factor 2 (FGF-2) mRNA levels in the striatum and prefrontal cortex within 2 h, an effect that vanished by 24 h. However, prolonged exposure (5 or 14 days) to cocaine treatment produced an enduring elevation of FGF-2 mRNA levels that was evident 72 h after the last injection in the prefrontal cortex and could even persist for 14 days in the striatum, raising the possibility that cocaine treatment primes the brain, resulting in longer-lasting FGF-2 up-regulation in regions that are highly innervated by dopaminergic projections. The expression of FGF-2 was also significantly increased in the midbrain following acute or 5-day injection, suggesting that modulation of FGF-2 biosynthesis in dopamine-producing cells occurs only during early stages of cocaine exposure. Our results point to important mechanistic conclusions as to how cocaine alters FGF-2 expression. Whereas cocaine-induced changes in FGF-2 gene expression following a single injection could be ascribed to increased release of transmitters (mainly dopamine), enhanced FGF-2 gene expression following repeated administration identifies the trophic factor as part of the adaptive changes set in motion by cocaine.