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
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
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 × 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.
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).
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.
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.
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).
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).
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.
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.
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.
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.
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.
These studies were supported by National Cancer Institute Grant CA-71508 (to A. Wellstein).
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.
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.
- Copyright © 2007 the American Physiological Society