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Am J Physiol Regul Integr Comp Physiol 293: R775-R783, 2007. First published June 6, 2007; doi:10.1152/ajpregu.00737.2006
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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Effects on neurite outgrowth and cell survival of a secreted fibroblast growth factor binding protein upregulated during spinal cord injury

Elena Tassi,1 Sharon Walter,1 Achim Aigner,2 Rafael H. Cabal-Manzano,1 Ranjan Ray,1 Paul J. Reier,3 and Anton Wellstein1

1Lombardi Comprehensive Cancer Center, Georgetown University, Washington, District of Columbia; 2Department of Pharmacology, School of Medicine, Philipps University, Marburg, Germany; and 3Department of Neuroscience, University of Florida, Gainesville, Florida

Submitted 18 October 2006 ; accepted in final form 31 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The fibroblast growth factor binding protein (FGF-BP; GenBank accession no. NP_005121) is a secreted protein that mobilizes FGFs from the extracellular matrix, protects them from degradation, and enhances their biological activity. Several previous studies reported that FGF-BP is an early response gene upregulated during tissue repair processes including wound healing and atherogenesis. In this study we analyzed whether FGF-BP expression was impacted by spinal cord injury and could have an effect on neuronal cell viability. Immunohistochemical and in situ hybridization studies revealed a dramatic upregulation of FGF-BP protein and mRNA levels following unilateral hemisection and contusion injury of adult rat spinal cord. In spinal cord sections of laminectomized rats, increased FGF-BP expression was observed in the fibers and cell bodies ipsilateral to the lesion site but was absent in the uninjured spinal cord tissue contralateral to the lesion. Increased expression of FGF-BP was observed at all postinjury time points, examined with peak levels occurring at day 4, a time when injury-induced increased levels of FGF2 have also been reported to be maximal. Moreover, using PC12 cells as a neuronal model, we observed that exogenous FGF-BP increased the capacity of FGF2 to stimulate neurite outgrowth and to increase cell survival. At the molecular level, FGF-BP enhanced FGF2-induced protein tyrosine phosphorylation and AKT/PKB activation. Collectively, these results suggest that FGF-BP is an early response gene after spinal cord injury and that its upregulation in regenerating spinal cord tissue may provide a molecular mechanism for enhancing the initial FGF2-mediated neurotrophic effects occurring after such tissue damage.

PC12 cells; neurite outgrowth; apoptosis

THE FAMILY OF FIBROBLAST GROWTH FACTORS (FGFs) encompasses, to date, more than 20 distinct polypeptides that play pivotal roles in several biological processes, such as embryonic development (66), angiogenesis (56), and tumorigenesis (22). Several studies have found that multiple FGFs, but predominantly FGF1 and FGF2, are expressed in the central nervous system (CNS) and are key regulators of CNS development and functions (13, 27, 28, 75). Interestingly, although both FGF1 and FGF2 are highly expressed in developing brain and adult CNS, their cellular localization patterns are different. Whereas FGF2 accumulates in both neuronal and nonneuronal cells, FGF1 is found primarily in neurons (13, 59). Evidence of FGF1 and FGF2 mitogenic and neurotrophic activities includes their ability to enhance the survival and outgrowth of various neuronal cell types, such as neocortical, hippocampal, cerebellar, dopaminergic, spinal cord, and sensory neurons isolated from adult CNS (13, 30, 44, 54). They also stimulate neuronal cell fate determination (3), migration, and differentiation (59). Moreover, both FGF1 and FGF2 and their cognate receptors are upregulated after experimental central and peripheral nervous system injury (20, 24, 32, 43, 46). The ability of FGFs to act as potent neurotrophic factors has been extensively demonstrated. FGF2 prevents axotomy-induced death of glutamatergic neurons (53). Administration of FGF2 in vivo improves rat sensorimotor functions, reduces focal ischemia-induced infarct size (40), stimulates functional recovery of rats subjected to motor cortex lesion (48), and promotes rat hippocampal cell survival upon kainic acid-induced seizure (42). Proliferation and differentiation of hippocampal cells after seizure or cerebral ischemia failed to occur in FGF2 –/– mice (18, 76). Similarly, rats treated with anti-FGF2 neutralizing antibody displayed decreased cholinergic sprouting following the removal of hippocampal entorhinal cortical inputs (18). Moreover, various FGFs, such as FGF1, FGF2, FGF4, FGF5, and FGF9, have been shown to be highly expressed in and promote survival of spinal motoneurons (13, 15, 26, 31, 33, 70) upon axotomy or spinal cord injury (SCI) (9, 39, 45, 64, 65). In addition, FGF2 knockout mice display neuronal defects in the cervical spinal cord region (14). These studies together suggest that endogenous FGF is likely to help rescue injured neurons and to promote nerve regeneration.

FGF1 and FGF2 lack a signal peptide required for cellular secretion; nevertheless, they are found immobilized on extracellular matrix (ECM) heparan sulfate proteoglycans (47, 55). One mechanism for mobilizing FGFs from ECM requires heparinases or other glycosaminoglycan-degrading enzymes (7, 55, 61, 72). An independent mechanism involves a secreted FGF binding protein (FGF-BP) that functions as an extracellular chaperone for locally stored FGFs. In this study we analyzed the FGF-BP protein initially named HBp17 (FGFBP1; GenBank accession no. NP_005121). Studies from our and other laboratories have indicated that FGF-BP is a binding partner for FGF1, FGF2, FGF7, FGF10, and FGF22 (5, 49, 62), and our group (74) has delineated the FGF-BP COOH terminus as the interaction domain for FGF2. Moreover, FGF-BP is likely to complement the role of heparan sulfate in mediating FGF-dependent signaling and mitogenesis (57). It has been shown that FGF-BP binds to perlecan, and heparan sulfate or heparinoids can compete with FGF-BP for binding to FGF2 in vitro, thereby supporting the role for FGF-BP as a chaperone molecule for FGFs immobilized in the ECM (49, 62). Furthermore, FGF-BP protects FGF proteins from acid inactivation (73) and enhances their biochemical and biological activity (1, 57, 62). FGF-BP is overexpressed in a number of human cancers (1, 63). FGF-BP mRNA and protein are dramatically upregulated during early stages of colon and pancreatic cancer (i.e., dysplastic lesions), and this overexpression is sustained throughout cancer progression and metastasis (58, 63). In addition, FGF-BP appears to be upregulated during preneoplastic stages of carcinogen-treated human skin, concomitantly with an increase in angiogenesis (37). The biological significance of FGF-BP expression was revealed by ribozyme-mediated depletion of endogenous FGF-BP from human colon and squamous cell carcinoma cell lines that resulted in decreased tumor growth and angiogenesis in vivo (10).

A potential role for FGF-BP as an early response gene for nonmalignant conditions is suggested by recent observations of a transient upregulation of FGF-BP in wounded skin (5, 37), as well as during the regeneration of renal tubular epithelial cells in pediatric patients affected by human immunodeficiency virus (HIV) infection associated with hemolytic uremic syndrome (41, 57). Hence, it is tempting to speculate that FGF-BP plays crucial early roles in the repair of tissue with aberrant FGF activity, by enhancing FGF-mediated angiogenesis and therefore accelerating wound healing or contributing to the healing of renal capillaries and tubules. These findings were extended to another nonmalignant condition when FGF-BP was found to be upregulated at both protein and mRNA levels in the aorta of a mouse model susceptible to early atherogenesis (51).

On the basis of these observations, we investigated the regulation of FGF-BP expression during nerve injuries and studied a potential contribution to the repair process reflected in cell survival and neurite outgrowth. In this report we show that FGF-BP is upregulated at both protein and mRNA levels following contusion injury or unilateral hemisection of the adult rat spinal cord, with elevated expression restricted to the fibers and cell bodies ipsilateral, but not contralateral, to the lesion sites. We also observed that FGF-BP synergistically enhances FGF2-induced neurite outgrowth and FGF2-dependent survival of rat pheochromocytoma cells (PC12) that served as a neuronal model system. Finally, we show that these cells, when costimulated with exogenous FGF-BP and suboptimally effective concentrations of FGF2, exhibit a differential protein tyrosine phosphorylation profile as well as an enhancement of AKT activation.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression and purification of recombinant FGF-BP. Expression and purification of recombinant human FGF-BP from Sf-9 insect cells was described elsewhere (62).

Cell culture and neurite outgrowth assays. Rat adrenal medullary pheochromocytoma PC12 cells, obtained from the American Type Culture (Manassas, VA), were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and 5% horse serum (Invitrogen) in an environment of 37°C and 5% CO2. PC12 cells (4 x 104 cells/well) were seeded on poly-D-lysine-coated (Sigma, St. Louis, MO) 24-well plates in complete medium. After 24 h, cells were cultured in DMEM containing 2.5% FBS and supplemented with nerve growth factor (NGF; 50 ng/ml; Invitrogen) or human recombinant FGF2 (Invitrogen) and/or recombinant FGF-BP at the concentration indicated. The culture medium was replaced every 2 days. The outgrowth of neurites was assessed after 8 days in 10 random fields of each well by using a phase-contrast microscope. A cellular projection was scored as a neurite if it was longer than the diameter of the cell body and possessed a growth cone. For each condition, the average number of neurites per field was based on quadruplicate experiments. The overall number of neurites per well was expressed relative to the number of viable cells, as determined using Cell Proliferation Reagent WST-1 (Roche Molecular Biochemicals, Indianapolis IN) according to the manufacturer's instructions. Neurites were counted by two independent observers who were blinded to the experimental design.

Survival assay. PC12 cells were seeded in six-well plates at a density of 106 cells per well. After overnight growth in complete medium, the cells were washed twice with phosphate-buffered saline (PBS) and maintained for 24 h in serum-free medium containing recombinant FGF-BP (6 ng/ml) and/or human recombinant FGF2 (5 ng/ml). Control cells were left untreated. Apoptosis was measured by flow cytometry with the aid of TACS Annexin V-FITC (Trevigen, Gaithersburg, MD), as indicated by the manufacturer.

Western blot and phosphorylation studies. Subconfluent PC12 cells were serum-starved for 16 h in DMEM and then stimulated with recombinant FGF-BP (3 ng/ml) and/or human recombinant FGF2 (1 or 10 ng/ml) for 15 min at 37°C. Cell lysates were prepared as previously described (62). Total cellular proteins (50 µg) were separated by 4–10% gradient SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Immunoblot studies for identifying tyrosine-phosphorylated proteins were performed with a monoclonal anti-phosphotyrosine antibody (clone 4G10; Upstate Biotechnology, Lake Placid, NY) as previously described (62). Immunoblot studies for anti phospho-AKT and total AKT as well as for phospho-MAPK were performed with the respective rabbit polyclonal antibodies (1:1,000 dilution; Cell Signaling, Danvers, MA). Actin was detected with mouse anti-actin monoclonal antibody (1:1,000 dilution; Chemicon International, Temecula, CA). Visualization was performed using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL) with horseradish peroxidase-linked donkey anti-mouse or anti-rabbit immunoglobulin G as a secondary antibody (Amersham Biosciences).

Experimental spinal cord injury. Seven adult female Sprague-Dawley rats (200–250 g; Zivic-Miller, Allison Park, PA) were deeply anesthetized with a mixture of ketamine (Ketaset; 40 mg/kg; Aveco, Fort Dodge, IA) and xylazine (Rompun; 6.7 mg/kg; Haver Lockhart, Shawnee, KS). Laminectomy was performed at spinal C4-C5, and a midline dural incision was made. Thereafter, a right unilateral hemisection lesion cavity of 3 mm in length was created by aspiration. After hemostasis was obtained, the lesion site was filled with Gelfoam (Amersham Pharmacia Biotech, Piscataway, NJ), and the dural incision was closed with 10-0 sutures. The animals were given a subcutaneous injection of penicillin (100,000 units) and an intraperitoneal injection of isotonic saline following a treatment with buprenorphine as an analgesic and penicillin procaine G (3,000 units daily) for the first week following surgery. Tissue specimens for immunocytochemical analysis were obtained at 2 (n = 3), 4 (n = 2), and 7 days postinjury (n = 2). To perform contusion injury, laminectomy was performed at spinal cord level T8. Tissue specimens were collected at 1, 4, and 7 days postinjury (n = 3 per time point). Intermediate-grade contusion injuries were made with the NYU electromechanical impact device (4).

Immunohistochemistry. By using the lesion site as the central point, a 14-mm segment of injured spinal cord was removed, fixed in formalin overnight at 4°C, and embedded in paraffin. Immunohistochemical analysis on paraffin-embedded spinal cord tissue sections (10 µm) and quantitation was performed as described previously (63). Detection of rat FGF-BP was obtained by incubating the tissues with a rabbit anti-human FGF-BP polyclonal antibody (2) for 12 h at 4°C, followed by incubation with a biotinylated goat anti-rabbit secondary antibody (1:500; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Colorimetric reaction was performed with the Vectastain ABC kit (Vector Laboratories) according to the manufacturer's instructions.

In situ hybridization. In situ hybridization analyses were carried out as previously described (29, 63). The digoxigenin-labeled riboprobes were generated from bases 1296 to 1622 of the rat FGF-BP full-length coding sequence (2) subcloned into pRc/CMV 5.5 vector (Invitrogen). The vector was linearized with either Hind III or Xba I to generate antisense and sense probes, respectively.

Statistical analysis. Prism 4 for Mac (GraphPad, San Diego, CA) was used for statistical analysis. Data are means ± SE. Neurite outgrowth in PC12 cells was analyzed using two-way ANOVA with Bonferroni's post hoc test. Analysis of PC12 apoptosis was performed using one-way ANOVA with Tukey's multiple comparison test. Statistical significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of FGF-BP in adult rat spinal cords after injury. In previous work (2), the expression of FGF-BP protein was detectable in some rat adult nervous system tissues by immunohistochemical analysis. Strong immunostaining was detected mainly in the eye, particularly in the retina, outer plexiform layer, ciliary body, rods, and cones. In the brain, FGF-BP was found in the choroids plexus and in the Purkinje cells of the cerebellar cortex. In contrast, low or undetectable FGF-BP protein levels were found in most other tissues.

FGF-BP is an early response gene involved in the repair of injuries to multiple types of tissues, such as skin and kidney (37, 57). We conjectured that FGF-BP might also be regulated during injury to the nervous system. Most interestingly, FGFs were reported to be upregulated after SCI (45, 46), and we thus used injury to adult rat spinal cord as a model system. In a first experimental series, spinal contusion was performed on adult rats and the effect on FGF-BP protein levels was examined using immunohistochemical analysis. Spinal cord tissue contralateral to the lesion sites was used as the negative control. Although no FGF-BP staining was detected in the control tissues, strong FGF-BP immunoreactivity was observed in discrete populations of damaged fibers at 1, 4, and 7 days following injury, with peak levels occurring at 4 days postinjury (Fig. 1). Interestingly, this postinjury time course of FGF-BP expression is similar to that reported for FGF2, which was highest at 4 days after SCI, but predominantly detected in a different cell type (astrocytes) (45).


Figure 1
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  		Fig. 1. Fibroblast growth factor binding protein (FGF-BP) accumulates in contused rat spinal cords. FGF-BP protein was detected with a polyclonal anti-FGF-BP antibody, as described in MATERIALS AND METHODS. FGF-BP protein was not detectable in sections from uninjured tissue taken from the contralateral (ctrl) side at any time. In samples taken after injury, increased staining was detected on the ipsilateral side at 1, 4, and 7 days postinjury (1d, 4d, 7d). FGF-BP expression was highest at 4 days postinjury, where it was primarily localized to retraction bulbs, and leveled off at 7 days postinjury. Scale bar, 50 µm.

 
A second type of SCI, unilateral spinal cord hemisection, was also used to investigate the posttraumatic regulation of FGF-BP protein accumulation. These lesions were also accompanied by dramatically upregulated FGF-BP protein levels. This upregulation occurred in the proximal end bulbs of severed white matter tracts located immediately rostral and caudal to the lesion site (Fig. 2B) and only in tissue ipsilateral to the lesion. These FGF-BP-positive retraction bulbs were observed at all postlesion times monitored and generally occupied the lateral-most regions of spinal cord white matter (data not shown). In sections obtained from more ventral regions of the injured spinal cords, FGF-BP staining also was observed in motoneurons, which exhibited the hallmark features of Wallerian degeneration, such as a shifting of the nucleus and Nissl substance to the periphery of the cell body (Fig. 2D). Motoneurons and sensory cells of the dorsal root ganglia (DRG) contralateral to the lesion site did not exhibit either FGF-BP upregulation or other abnormal features (Fig. 2, A and C; DRG not depicted).


Figure 2
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  		Fig. 2. FGF-BP protein accumulates in rat spinal cord neurons following unilateral spinal cord hemisection. A: with the use of a polyclonal anti-FGF-BP antibody, FGF-BP protein was not detected contralateral to the T1 lesion in spinal cords of adult rats. B: ipsilateral to the hemisection, FGF-BP-positive retraction fibers (arrowheads) were observed directly rostral and caudal to the lesion site. C: alpha motoneurons (arrows) in ventral gray matter contralateral to the spinal cord hemisection appear healthy and did not contain detectable amounts of FGF-BP protein. D: ipsilateral to the lesion site, FGF-BP staining was observed in alpha motoneurons exhibiting chromatolytic changes commonly associated with neuronal injury (arrows). Scale bars, 50 µm in A and B and 10 µm in C and D.

 
To detect FGF-BP mRNA expression in laminectomized rats, we performed in situ hybridization. Results similar to the protein expression pattern were found: an antisense probe detected upregulation of FGF-BP mRNA primarily in large motoneurons in the ventral gray matter and in the large sensory neurons within the DRG ipsilateral (Fig. 3A), but not contralateral (Fig. 3B), to the lesion. The specificity of these positive signals was demonstrated by the absence of detectable staining when a control sense-strand probe was used (Fig. 3C). Consistent with our observations, Koshinaga et al. (36) found increased amounts of FGF1 and FGF2 protein in motoneurons of injured rat spinal cord.


Figure 3
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  		Fig. 3. Expression analysis of FGF-BP mRNA by in situ hybridization. A: FGF-BP mRNA was highly expressed by alpha motoneurons (dark staining) located in the ventral gray matter of the hemisected side of the rat spinal cord. B: lack of expression of FGF-BP mRNA in the side contralateral to the injury. C: negative control with FGF-BP sense probe using sections from the injured side of the spinal cord. Scale bar, 10 µm.

 
Effect of FGF-BP on FGF2-induced neurite outgrowth. FGF-BP enhances FGF-induced growth of various cell lines, including human tumor cells and murine fibroblasts and endothelial cell lines (10, 11, 38, 57, 62). To examine the ability of FGF-BP to affect neuronal cell responses to FGF2, we used rat pheochromocytoma cells (PC12) as a model. PC12 cells are the immortalized counterpart of rat adrenal chromaffin cells, which are derived from neural crest progenitors (25) and can be differentiated toward neuronal cells by addition of exogenous growth factors. PC12 cells produce FGF2 protein and mRNA (23) and respond to exogenous FGF by growing neuritic processes (60, 68). Consistent with previous reports, we observed that PC12 cells were poorly adherent and grew only a few short filopodial extensions when cultured for 8 days in serum-free medium (Fig. 4A, ctrl). Nerve growth factor (NGF) caused these cells to grow long extensions (Fig. 4A, NGF), whereas FGF2 caused cells to grow shorter extensions that increased in number with increasing doses of FGF2 (Fig. 4A, FGF2 at 5 and 50 ng/ml, respectively). The effect of increasing FGF-BP doses on the FGF2-induced neurite outgrowth is shown in Fig. 4B. In the FGF-BP alone control, increasing doses of recombinant FGF-BP induced a modest, dose-dependent increase in neurite outgrowth. However, in the presence of a dose of FGF2 (5 ng/ml) insufficient to cause a significant increase in number of neurites, increasing concentrations of FGF-BP resulted in a significant, dose-dependent enhancement of FGF-mediated neurite outgrowth. No additional effect of FGF-BP was seen when used in combination with a maximally effective concentration of FGF2 (50 ng/ml; P < 0.0001 vs. the FGF-BP-only treatment). These observations support the conclusion that FGF-BP can positively modulate the neurite outgrowth activity of FGF2.


Figure 4
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  		Fig. 4. FGF-BP enhances FGF2-induced neurite outgrowth in PC12 cells. A: morphology of cells and their extensions under different treatment conditions: control (ctrl), nerve growth factor (NGF; 50 ng/ml), or FGF2 (5 and 50 ng/ml). Scale bar, 20 µm. B: quantification of the average number of neurites per field after treatment with FGF-BP only (ctrl) or combinations of FGF-BP (at 0, 10, or 30 ng/ml) and FGF2 at 5 or 50 ng/ml. Data are means ± SE (n = 4); ns, nonsignificant. *P < 0.05; ***P < 0.0001 vs. FGF-BP treatment only; 2-way ANOVA.

 
Effect of FGF-BP on FGF2-dependent survival of PC12 cells. PC12 cells undergo apoptosis after serum deprivation, and various molecules such as nitric oxide (35), NGF, and FGF2 can act as survival factors (71). To investigate whether FGF-BP can enhance the survival effect of FGF2 in serum-deprived PC12 cells, we evaluated apoptotic rates 24 h after serum withdrawal. As shown in Fig. 5, both FGF-BP alone and a suboptimal dose of FGF2 alone caused a modest, nonsignificant rescue from apoptosis. In contrast, the synergistic effect of FGF-BP and FGF2 resulted in significantly fewer apoptotic cells ( >50% reduction, P < 0.01). Thus FGF-BP can enhance FGF2-mediated antiapoptotic effects to levels comparable to those obtained by serum (a 70% reduction, P < 0.01 vs. no-serum control). These data show that FGF-BP can positively modulate FGF2 biological effects in neuroectodermal cells.


Figure 5
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  		Fig. 5. FGF-BP enhances the antiapoptotic effect of FGF2 on PC12 cells. Following serum withdrawal, PC12 cells were treated with FGF-BP (6 ng/ml) alone, 10% serum, or FGF2 (5 ng/ml) in the presence or absence of FGF-BP (6 ng/ml) for 24 h. Control cells were left untreated. Apoptotic cells were scored by cell sorting analysis after staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide. Data are means ± SE (n = 3) and are normalized to the percentage of apoptotic cells found under serum deprivation conditions (100%). **P < 0.01.

 
Effects of FGF-BP on FGF2-induced signaling. Several growth factors trigger downstream signaling cascades after they engage their cognate tyrosine-kinase receptors, such as ERK1/2 MAP kinase, p38, JNK, and phosphatidylinositol 3-kinase (PI3-K)/AKT and the associated pathways (16, 55). In addition to NGF receptors, PC12 cells express receptors for FGF, epidermal growth factor, and insulin that can trigger receptor type-dependent biological responses following their activation (12, 71). Since FGF-BP acts as a chaperone molecule able to enhance FGF2-mediated biochemical responses in vitro in various cell lines (57, 62), we sought to investigate whether FGF-BP could also affect FGF2-mediated signaling in PC12 cells. Serum-starved PC12 cells were treated with either recombinant FGF-BP or FGF2, or both, and phospho-tyrosine levels were determined by Western blot analysis using a phospho-tyrosine antibody. As shown in Fig. 6A, top, FGF-BP alone (3 ng/ml) or a low concentration of FGF2 (1 ng/ml) had undetectable effects on protein tyrosine phosphorylation, relative to control untreated cells. However, the simultaneous addition of FGF-BP and FGF2 (1 ng/ml) induced tyrosine phosphorylation of two proteins with an apparent molecular mass of 35 and 18 kDa, respectively. Comparable levels of tyrosine phosphorylation were observed with a 10-fold higher concentration of FGF2 alone (10 ng/ml) and could not be further increased with FGF-BP costimulation. The nature of these two bands has not been determined but is currently under investigation. Since PI3-K can be upregulated in FGF2-treated PC12 cells (34), and because the activation of AKT promotes FGF2-mediated survival of PC12 and embryonic rat hippocampal cells (17), we also tested the effect of FGF-BP on FGF2-induced AKT phosphorylation. Stimulation of PC12 cells with increasing concentrations of FGF2 resulted in a dose-dependent increase in phosphorylated AKT. Consistent with the protein tyrosine phosphorylation pattern described above, the ability of FGF-BP to enhance AKT phosphorylation was observed with low doses of FGF2 (1 ng/ml). Immunoblot analysis of p38 (not shown) and ERK1/2 MAP kinase phosphorylation (Fig. 6B) did not reveal a synergistic effect of FGF-BP on FGF2 stimulation under these experimental conditions. Collectively, these results indicate that FGF-BP acts as a positive modulator of FGF2-mediated signaling in PC12 cells, which leads to increased PC12 survival and differentiation.


Figure 6
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  		Fig. 6. FGF-BP effect on FGF2-induced protein tyrosine, AKT, and MAPK phosphorylation in PC12 cells. Western blot (WB) analysis for tyrosine-phosphorylated proteins (pY), phosphorylated AKT (pAKT; A), and phosphorylated MAPK (pMAPK; B) of PC12 cells treated for 15 min with different concentrations of FGF2 with or without a constant amount of FGF-BP (3 ng/ml). Protein extract (50 µg) was used for the analyses. Arrows in A indicate tyrosine phosphorylation of 2 proteins with an apparent molecular mass of 35 and 18 kDa, respectively. Arrowheads in B indicate p42 and p44 MAPK. Equal amounts of cell lysate proteins were loaded onto the gels as indicated by the blot for actin. Data are representative of samples from 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
FGF-BP plays a significant role in tumor growth and malignant transformation (1, 10, 11, 63). In addition, tissue-specific regulation of FGF-BP gene expression during embryonic development (2, 38) and skin wound healing (37) suggests a role for FGF-BP in these biological processes. On the basis of these earlier studies, we examined FGF-BP expression during two different kinds of SCI. Since FGFs, acting as potent neurotrophic factors, appear to have a significant role in the repair and recovery processes following SCI (45, 46), we hypothesized that FGF-BP also might be involved. Our previous data indicated that FGF-BP is highly expressed during early embryonic spinal cord development (2) but becomes downregulated in that tissue after birth (2, 11, 38). Our present immunohistochemical analyses are consistent with these earlier reports, since FGF-BP protein is undetectable in the spinal cords of adult rats. In this study we show for the first time that FGF-BP is dramatically upregulated in the spinal cord of laminectomized rats. Remarkably, the time course and pattern of FGF-BP upregulation observed in the present study parallels the profile of FGF upregulation observed in the injured rat spinal cord (45). Indeed, Mocchetti et al. (45) examined the expression of FGF2 in the adult rat spinal cord at the same postinjury time points (1, 4, and 7 days) and reported that FGF2 expression was highest at 4 days postinjury. Interestingly, we also observed the highest levels of FGF-BP protein at 4 days following SCI. Although having similar temporal patterns of upregulation after similar kinds of SCI, FGF-BP and FGF2 have very different spatial profile. Whereas FGF2 was mainly found in astrocytes (46), our data indicate that FGF-BP protein is found primarily in damaged neurons. In situ hybridization analysis indicates that FGF-BP transcript is abundantly expressed in the large motoneurons in the ventral gray matter, suggesting that these neurons synthesize most or all of the damage-induced FGF-BP. Since FGF2 and FGF-BP have similar expression time courses, it is tempting to speculate that the localization and upregulation of FGF-BP expression by injured neurons may accelerate the release of FGF2 from its glial source. Increased mobilization of FGF2 by FGF-BP may result in stronger effects on neighboring neurons, thus facilitating spinal cord repair and recovery and therefore enhancing FGF2 neurotrophic activity.

Interestingly, previous reports indicated that upregulation of FGF2 following SCI not only precedes upregulation of the p75 subunit of the nerve growth factor receptor (p75) but may, in fact, also induce its upregulation (6, 19, 21, 46). Most importantly, p75 is a key molecule playing a role during later stages of injured spinal cord repair and regeneration (46). Therefore, it is tempting to speculate that FGF-BP-mediated enhancement of FGF2 neurotrophic functions following SCI is likely to be one of the initial events during the phase of tissue repair. Consistent with our current findings, FGF-BP was found to be an early response gene dramatically upregulated during other tissue repair events, such as wound healing (5, 37) and regeneration of renal tubular epithelial cells in pediatric patients affected by HIV infection associated with hemolytic uremic syndrome (41, 57).

To ascertain the physiological relevance of our in vivo observations, we sought to investigate the potential role of FGF-BP on cultured neuronal cell growth and survival, in vitro processes in which the neurotrophic activity of FGF is well established (see Introduction). We report the first evidence indicating that FGF-BP can enhance FGF2-induced neuronal cell differentiation and survival, using a rat pheochromocytoma PC12 cell model. PC12 cells are a useful model for studying neuritic outgrowth in response to neurotrophins (67), because the neural crest origin of these immortalized adrenal chromaffin cells allows them to respond to FGF by elaborating a sympathetic neuronlike phenotype (60, 68). Consistent with previous reports, we observed that FGF2 was capable of inducing both neurite outgrowth and survival of PC12 cells upon serum deprivation in a dose-dependent manner (71). Although FGF-BP alone was capable of inducing only modest neurite outgrowth and survival of PC12 cells, its greatest effects were observed when PC12 cells were treated simultaneously with FGF-BP and low doses of FGF2. A synergistic relationship between FGF2 and FGF-BP could also be demonstrated at the signal transduction level, as evidenced by the differential phosphoprotein profile and activation of the AKT pathway. Although our data appear inconsistent with findings by Wert and Palfrey (71), who reported that activation of the Ras/MAPK pathway, but not the PI3-K/AKT pathway, appeared to mediate the effect of FGF2 on the survival of serum-deprived PC12 cells, they corroborate reports that the AKT pathway promotes FGF2-mediated antiapoptotic effects in PC12 cells, in H19-7 embryonic rat hippocampal cells (17), and in breast cancer cell lines (69). Most importantly, AKT activation has been linked with neuronal cell survival as well as axonal regeneration in response to traumatic brain injury (8, 50, 52).

Nervous system development and regeneration are extraordinarily complex processes, the success of which depends on multiple factors, including growth factors and extracellular matrix molecules. Although we observed no evidence of regeneration or sprouting of retraction bulbs at the time points examined in this study, the in vitro activity of FGF-BP, combined with its localization and time course of expression following SCI, support the notion that FGF-BP may enhance the growth and survival of neurons during CNS development and following SCI. These observations contribute to a more complete identification of the factors affecting growth and survival of cells bordering injured spinal cord tissue. A better understanding of how these factors function and interact is necessary before we can hope to enhance recovery and regeneration.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by National Cancer Institute Grant CA-71508 (to A. Wellstein).


   ACKNOWLEDGMENTS
 
We thank Dr. Wolfgang Streit (University of Florida) for important input during the initial phases of this project. Dr. Ali Al-Attar (Georgetown University) kindly supplied recombinant FGF-BP protein, and Barbara O'Steen provided excellent surgical assistance. Also, we thank Dr. Jean Wrathall (Georgetown University) for review, discussion, and input.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Wellstein, Lombardi Comprehensive Cancer Center, Research Bldg. E311, Georgetown Univ., 3970 Reservoir Road, N.W., Washington, DC 20057 (e-mail: wellstea{at}georgetown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abuharbeid S, Czubayko F, Aigner A. The fibroblast growth factor-binding protein FGF-BP. Int J Biochem Cell Biol 38: 1463–1468, 2006.[CrossRef][ISI][Medline]
  2. Aigner A, Malerczyk C, Houghtling R, Wellstein A. Tissue distribution and retinoid-mediated downregulation of an FGF-binding protein (FGF-BP) in the rat. Growth Factors 18: 51–62, 2000.[ISI][Medline]
  3. Anderson DJ. Cell fate determination in the peripheral nervous system: the sympathoadrenal progenitor. J Neurobiol 24: 185–198, 1993.[CrossRef][ISI][Medline]
  4. Basso DM, Beattie MS, Bresnahan JC. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol 139: 244–256, 1996.[CrossRef][ISI][Medline]
  5. Beer HD, Bittner M, Niklaus G, Munding C, Max N, Goppelt A, Werner S. The fibroblast growth factor binding protein is a novel interaction partner of FGF-7, FGF-10 and FGF-22 and regulates FGF activity: implications for epithelial repair. Oncogene 24: 5269–5277, 2005.[CrossRef][ISI][Medline]
  6. Brunello N, Reynolds M, Wrathall JR, Mocchetti I. Increased nerve growth factor receptor mRNA in contused rat spinal cord. Neurosci Lett 118: 238–240, 1990.[CrossRef][ISI][Medline]
  7. Buczek-Thomas JA, Nugent MA. Elastase-mediated release of heparan sulfate proteoglycans from pulmonary fibroblast cultures. A mechanism for basic fibroblast growth factor (bFGF) release and attenuation of bFGF binding following elastase-induced injury. J Biol Chem 274: 25167–25172, 1999.[Abstract/Free Full Text]
  8. Cheung ZH, Chan YM, Siu FK, Yip HK, Wu W, Leung MC, So KF. Regulation of caspase activation in axotomized retinal ganglion cells. Mol Cell Neurosci 25: 383–393, 2004.[CrossRef][ISI][Medline]
  9. Cuevas P, Carceller F, Gimenez-Gallego G. Acidic fibroblast growth factor prevents death of spinal cord motoneurons in newborn rats after nerve section. Neurol Res 17: 396–399, 1995.[ISI][Medline]
  10. Czubayko F, Liaudet-Coopman ED, Aigner A, Tuveson AT, Berchem GJ, Wellstein A. A secreted FGF-binding protein can serve as the angiogenic switch in human cancer. Nat Med 3: 1137–1140, 1997.[CrossRef][ISI][Medline]
  11. Czubayko F, Smith RV, Chung HC, Wellstein A. Tumor growth and angiogenesis induced by a secreted binding protein for fibroblast growth factors. J Biol Chem 269: 28243–28248, 1994.[Abstract/Free Full Text]
  12. Dailey L, Ambrosetti D, Mansukhani A, Basilico C. Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev 16: 233–247, 2005.[CrossRef][ISI][Medline]
  13. Dono R. Fibroblast growth factors as regulators of central nervous system development and function. Am J Physiol Regul Integr Comp Physiol 284: R867–R881, 2003.[Abstract/Free Full Text]
  14. Dono R, Texido G, Dussel R, Ehmke H, Zeller R. Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice. EMBO J 17: 4213–4225, 1998.[CrossRef][ISI][Medline]
  15. Elde R, Cao YH, Cintra A, Brelje TC, Pelto-Huikko M, Junttila T, Fuxe K, Pettersson RF, Hokfelt T. Prominent expression of acidic fibroblast growth factor in motor and sensory neurons. Neuron 7: 349–364, 1991.[CrossRef][ISI][Medline]
  16. Eswarakumar VP, Lax I, Schlessinger J. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 16: 139–149, 2005.[CrossRef][ISI][Medline]
  17. Eves EM, Xiong W, Bellacosa A, Kennedy SG, Tsichlis PN, Rosner MR, Hay N. Akt, a target of phosphatidylinositol 3-kinase, inhibits apoptosis in a differentiating neuronal cell line. Mol Cell Biol 18: 2143–2152, 1998.[Abstract/Free Full Text]
  18. Fagan AM, Suhr ST, Lucidi-Phillipi CA, Peterson DA, Holtzman DM, Gage FH. Endogenous FGF-2 is important for cholinergic sprouting in the denervated hippocampus. J Neurosci 17: 2499–2511, 1997.[Abstract/Free Full Text]
  19. Follesa P, Wrathall JR, Mocchetti I. Increased basic fibroblast growth factor mRNA following contusive spinal cord injury. Brain Res Mol Brain Res 22: 1–8, 1994.[Medline]
  20. Gomez-Pinilla F, Cotman CW. Transient lesion-induced increase of basic fibroblast growth factor and its receptor in layer VIb (subplate cells) of the adult rat cerebral cortex. Neuroscience 49: 771–780, 1992.[CrossRef][ISI][Medline]
  21. Gomez-Pinilla F, Lee JW, Cotman CW. Basic FGF in adult rat brain: cellular distribution and response to entorhinal lesion and fimbria-fornix transection. J Neurosci 12: 345–355, 1992.[Abstract]
  22. Grose R, Dickson C. Fibroblast growth factor signaling in tumorigenesis. Cytokine Growth Factor Rev 16: 179–186, 2005.[CrossRef][ISI][Medline]
  23. Grothe C, Meisinger C. The multifunctionality of FGF-2 in the adrenal medulla. Anat Embryol (Berl) 195: 103–111, 1997.[CrossRef][Medline]
  24. Grothe C, Meisinger C, Claus P. In vivo expression and localization of the fibroblast growth factor system in the intact and lesioned rat peripheral nerve and spinal ganglia. J Comp Neurol 434: 342–357, 2001.[CrossRef][ISI][Medline]
  25. Grothe C, Meisinger C, Holzschuh J, Wewetzer K, Cattini P. Over-expression of the 18 kD and 21/23 kD fibroblast growth factor-2 isoforms in PC12 cells and Schwann cells results in altered cell morphology and growth. Brain Res Mol Brain Res 57: 97–105, 1998.[Medline]
  26. Grothe C, Wewetzer K, Lagrange A, Unsicker K. Effects of basic fibroblast growth factor on survival and choline acetyltransferase development of spinal cord neurons. Brain Res Dev Brain Res 62: 257–261, 1991.[Medline]
  27. Hattori Y, Miyake A, Mikami T, Ohta M, Itoh N. Transient expression of FGF-5 mRNA in the rat cerebellar cortex during post-natal development. Brain Res Mol Brain Res 47: 262–266, 1997.[Medline]
  28. Hattori Y, Yamasaki M, Konishi M, Itoh N. Spatially restricted expression of fibroblast growth factor-10 mRNA in the rat brain. Brain Res Mol Brain Res 47: 139–146, 1997.[Medline]
  29. Henke RT, Eun Kim S, Maitra A, Paik S, Wellstein A. Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization. Methods 38: 253–262, 2006.[CrossRef][ISI][Medline]
  30. Himmelseher S, Pfenninger E, Georgieff M. Effects of basic fibroblast growth factor on hippocampal neurons after axonal injury. J Trauma 42: 659–664, 1997.[ISI][Medline]
  31. Hughes RA, Sendtner M, Goldfarb M, Lindholm D, Thoenen H. Evidence that fibroblast growth factor 5 is a major muscle-derived survival factor for cultured spinal motoneurons. Neuron 10: 369–377, 1993.[CrossRef][ISI][Medline]
  32. Jungnickel J, Haase K, Konitzer J, Timmer M, Grothe C. Faster nerve regeneration after sciatic nerve injury in mice over-expressing basic fibroblast growth factor. J Neurobiol 66: 940–948, 2006.[CrossRef][ISI][Medline]
  33. Kanda T, Iwasaki T, Nakamura S, Ueki A, Kurokawa T, Ikeda K, Mizusawa H. FGF-9 is an autocrine/paracrine neurotrophic substance for spinal motoneurons. Int J Dev Neurosci 17: 191–200, 1999.[CrossRef][ISI][Medline]
  34. Kawamata T, Yamaguchi T, Shin-ya K, Hori T. Time courses of increased expression of signaling transduction molecules induced by basic fibroblast growth factor in PC12 cells. Neurol Res 23: 327–330, 2001.[CrossRef][ISI][Medline]
  35. Kim YM, Chung HT, Kim SS, Han JA, Yoo YM, Kim KM, Lee GH, Yun HY, Green A, Li J, Simmons RL, Billiar TR. Nitric oxide protects PC12 cells from serum deprivation-induced apoptosis by cGMP-dependent inhibition of caspase signaling. J Neurosci 19: 6740–6747, 1999.[Abstract/Free Full Text]
  36. Koshinaga M, Sanon HR, Whittemore SR. Altered acidic and basic fibroblast growth factor expression following spinal cord injury. Exp Neurol 120: 32–48, 1993.[CrossRef][ISI][Medline]
  37. Kurtz A, Aigner A, Cabal-Manzano RH, Butler RE, Hood DR, Sessions RB, Czubayko F, Wellstein A. Differential regulation of a fibroblast growth factor-binding protein during skin carcinogenesis and wound healing. Neoplasia 6: 595–602, 2004.[CrossRef][ISI][Medline]
  38. Kurtz A, Wang HL, Darwiche N, Harris V, Wellstein A. Expression of a binding protein for FGF is associated with epithelial development and skin carcinogenesis. Oncogene 14: 2671–2681, 1997.[CrossRef][ISI][Medline]
  39. Li L, Oppenheim RW, Lei M, Houenou LJ. Neurotrophic agents prevent motoneuron death following sciatic nerve section in the neonatal mouse. J Neurobiol 25: 759–766, 1994.[CrossRef][ISI][Medline]
  40. Li Q, Stephenson D. Postischemic administration of basic fibroblast growth factor improves sensorimotor function and reduces infarct size following permanent focal cerebral ischemia in the rat. Exp Neurol 177: 531–537, 2002.[CrossRef][ISI][Medline]
  41. Liu XH, Aigner A, Wellstein A, Ray PE. Up-regulation of a fibroblast growth factor binding protein in children with renal diseases. Kidney Int 59: 1717–1728, 2001.[CrossRef][ISI][Medline]
  42. Liu Z, D'Amore PA, Mikati M, Gatt A, Holmes GL. Neuroprotective effect of chronic infusion of basic fibroblast growth factor on seizure-associated hippocampal damage. Brain Res 626: 335–338, 1993.[CrossRef][ISI][Medline]
  43. Logan A, Frautschy SA, Gonzalez AM, Baird A. A time course for the focal elevation of synthesis of basic fibroblast growth factor and one of its high-affinity receptors (flg) following a localized cortical brain injury. J Neurosci 12: 3828–3837, 1992.[Abstract]
  44. Matsuda S, Saito H, Nishiyama N. Effect of basic fibroblast growth factor on neurons cultured from various regions of postnatal rat brain. Brain Res 520: 310–316, 1990.[CrossRef][ISI][Medline]
  45. Mocchetti I, Rabin SJ, Colangelo AM, Whittemore SR, Wrathall JR. Increased basic fibroblast growth factor expression following contusive spinal cord injury. Exp Neurol 141: 154–164, 1996.[CrossRef][ISI][Medline]
  46. Mocchetti I, Wrathall JR. Neurotrophic factors in central nervous system trauma. J Neurotrauma 12: 853–870, 1995.[ISI][Medline]
  47. Mohammadi M, Olsen SK, Ibrahimi OA. Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev 16: 107–137, 2005.[CrossRef][ISI][Medline]
  48. Monfils MH, Driscoll I, Vandenberg PM, Thomas NJ, Danka D, Kleim JA, Kolb B. Basic fibroblast growth factor stimulates functional recovery after neonatal lesions of motor cortex in rats. Neuroscience 134: 1–8, 2005.[CrossRef][ISI][Medline]
  49. Mongiat M, Otto J, Oldershaw R, Ferrer F, Sato JD, Iozzo RV. Fibroblast growth factor-binding protein is a novel partner for perlecan protein core. J Biol Chem 276: 10263–10271, 2001.[Abstract/Free Full Text]
  50. Namikawa K, Honma M, Abe K, Takeda M, Mansur K, Obata T, Miwa A, Okado H, Kiyama H. Akt/protein kinase B prevents injury-induced motoneuron death and accelerates axonal regeneration. J Neurosci 20: 2875–2886, 2000.[Abstract/Free Full Text]
  51. Napoli C, de Nigris F, Welch JS, Calara FB, Stuart RO, Glass CK, Palinski W. Maternal hypercholesterolemia during pregnancy promotes early atherogenesis in LDL receptor-deficient mice and alters aortic gene expression determined by microarray. Circulation 105: 1360–1367, 2002.[Abstract/Free Full Text]
  52. Neary JT. Protein kinase signaling cascades in CNS trauma. IUBMB Life 57: 711–718, 2005.[ISI][Medline]
  53. Peterson DA, Lucidi-Phillipi CA, Murphy DP, Ray J, Gage FH. Fibroblast growth factor-2 protects entorhinal layer II glutamatergic neurons from axotomy-induced death. J Neurosci 16: 886–898, 1996.[Abstract/Free Full Text]
  54. Peulve P, Laquerriere A, Hemet J, Tadie M. Comparative effect of {alpha}-MSH and b-FGF on neurite extension of fetal rat spinal cord neurons in culture. Brain Res 654: 319–323, 1994.[CrossRef][ISI][Medline]
  55. Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 7: 165–197, 2000.[Abstract]
  56. Presta M, Dell'Era P, Mitola S, Moroni E, Ronca R, Rusnati M. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev 16: 159–178, 2005.[CrossRef][ISI][Medline]
  57. Ray PE, Tassi E, Liu XH, Wellstein A. Role of fibroblast growth factor-binding protein in the pathogenesis of HIV-associated hemolytic uremic syndrome. Am J Physiol Regul Integr Comp Physiol 290: R105–R113, 2006.[Abstract/Free Full Text]
  58. Ray R, Cabal-Manzano R, Moser AR, Waldman T, Zipper LM, Aigner A, Byers SW, Riegel AT, Wellstein A. Up-regulation of fibroblast growth factor-binding protein, by beta-catenin during colon carcinogenesis. Cancer Res 63: 8085–8089, 2003.[Abstract/Free Full Text]
  59. Reuss B, von Bohlen und Halbach O. Fibroblast growth factors and their receptors in the central nervous system. Cell Tissue Res 313: 139–157, 2003.[CrossRef][ISI][Medline]
  60. Rydel RE, Greene LA. Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal differentiation in cultures of PC12 cells. J Neurosci 7: 3639–3653, 1987.[Abstract]
  61. Saksela O, Rifkin DB. Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity. J Cell Biol 110: 767–775, 1990.[Abstract/Free Full Text]
  62. Tassi E, Al-Attar A, Aigner A, Swift MR, McDonnell K, Karavanov A, Wellstein A. Enhancement of fibroblast growth factor (FGF) activity by an FGF-binding protein. J Biol Chem 276: 40247–40253, 2001.[Abstract/Free Full Text]
  63. Tassi E, Henke RT, Bowden ET, Swift MR, Kodack DP, Kuo AH, Maitra A, Wellstein A. Expression of a fibroblast growth factor-binding protein during the development of adenocarcinoma of the pancreas and colon. Cancer Res 66: 1191–1198, 2006.[Abstract/Free Full Text]
  64. Teng YD, Mocchetti I, Taveira-DaSilva AM, Gillis RA, Wrathall JR. Basic fibroblast growth factor increases long-term survival of spinal motor neurons and improves respiratory function after experimental spinal cord injury. J Neurosci 19: 7037–7047, 1999.[Abstract/Free Full Text]
  65. Teng YD, Mocchetti I, Wrathall JR. Basic and acidic fibroblast growth factors protect spinal motor neurones in vivo after experimental spinal cord injury. Eur J Neurosci 10: 798–802, 1998.[CrossRef][ISI][Medline]
  66. Thisse B, Thisse C. Functions and regulations of fibroblast growth factor signaling during embryonic development. Dev Biol 287: 390–402, 2005.[ISI][Medline]
  67. Tischler AS, Greene LA, Kwan PW, Slayton VW. Ultrastructural effects of nerve growth factor on PC 12 pheochromocytoma cells in spinner culture. Cell Tissue Res 228: 641–648, 1983.[ISI][Medline]
  68. Togari A, Dickens G, Kuzuya H, Guroff G. The effect of fibroblast growth factor on PC12 cells. J Neurosci 5: 307–316, 1985.[Abstract]
  69. Vandermoere F, El Yazidi-Belkoura I, Adriaenssens E, Lemoine J, Hondermarck H. The antiapoptotic effect of fibroblast growth factor-2 is mediated through nuclear factor-{kappa}B activation induced via interaction between Akt and I{kappa}B kinase-beta in breast cancer cells. Oncogene 24: 5482–5491, 2005.[CrossRef][ISI][Medline]
  70. Vargas MR, Pehar M, Cassina P, Martinez-Palma L, Thompson JA, Beckman JS, Barbeito L. Fibroblast growth factor-1 induces heme oxygenase-1 via nuclear factor erythroid 2-related factor 2 (Nrf2) in spinal cord astrocytes: consequences for motor neuron survival. J Biol Chem 280: 25571–25579, 2005.[Abstract/Free Full Text]
  71. Wert MM, Palfrey HC. Divergence in the anti-apoptotic signalling pathways used by nerve growth factor and basic fibroblast growth factor (bFGF) in PC12 cells: rescue by bFGF involves protein kinase C delta. Biochem J 352: 175–182, 2000.[CrossRef][ISI][Medline]
  72. Whitelock JM, Murdoch AD, Iozzo RV, Underwood PA. The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases. J Biol Chem 271: 10079–10086, 1996.[Abstract/Free Full Text]
  73. Wu DQ, Kan MK, Sato GH, Okamoto T, Sato JD. Characterization and molecular cloning of a putative binding protein for heparin-binding growth factors. J Biol Chem 266: 16778–16785, 1991.[Abstract/Free Full Text]
  74. Xie B, Tassi E, Swift MR, McDonnell K, Bowden ET, Wang S, Ueda Y, Tomita Y, Riegel AT, Wellstein A. Identification of the fibroblast growth factor (FGF)-interacting domain in a secreted FGF-binding protein by phage display. J Biol Chem 281: 1137–1144, 2006.[Abstract/Free Full Text]
  75. Yamashita T, Yoshioka M, Itoh N. Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain. Biochem Biophys Res Commun 277: 494–498, 2000.[CrossRef][ISI][Medline]
  76. Yoshimura S, Takagi Y, Harada J, Teramoto T, Thomas SS, Waeber C, Bakowska JC, Breakefield XO, Moskowitz MA. FGF-2 regulation of neurogenesis in adult hippocampus after brain injury. Proc Natl Acad Sci USA 98: 5874–5879, 2001.[Abstract/Free Full Text]




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