The Neurotrophic Activity of Fibroblast Growth Factor 1 (FGF1) Depends on Endogenous FGF1 Expression and Is Independent of the Mitogen-activated Protein Kinase Cascade Pathway
1996; Elsevier BV; Volume: 271; Issue: 5 Linguagem: Inglês
10.1074/jbc.271.5.2801
ISSN1083-351X
AutoresFlore Renaud, Sophie Desset, Lisa Oliver, Guillermo Giménez‐Gallego, Emmanuel Van Obberghen, Yves Courtois, M. Laurent,
Tópico(s)Nerve injury and regeneration
ResumoThe expression of fibroblast growth factor (FGF) 1, a potent neurotrophic factor, increases during differentiation and remains high in adult neuronal tissues. To examine the importance of this expression on the neuronal phenotype, we have used PC12 cells, a model to study FGF-induced neuronal differentiation. After demonstrating that FGF1 and FGF2 are synthesized by PC12 cells, we investigated if FGF1 expression could be a key element in differentiation. Using the cell signaling pathway to determine the effects of FGF1 alone, FGF1 plus heparin, or a mutated FGF1, we showed an activation to the same extent of mitogen-activated protein (MAP) kinase kinase and MAP kinase (extracellular regulated kinase 1). However, only FGF1 plus heparin could promote PC12 cell differentiation. Thus, the MAP kinase pathway is insufficient to promote differentiation. Analysis of the PC12 cells after the addition of FGF1 plus heparin or FGF2 demonstrated a significant increase in the level of FGF1 expression with the same time course as the appearance of the neuritic extensions. Transfection experiments were performed to enhance constitutivly or after dexamethasone induction the level of FGF1 expression. The degree of differentiation achieved by the cells correlated directly with the amount of FGF1 expressed. The MAP kinase pathway did not appear to be involved. Interestingly, a 5-fold increase in FGF1 in constitutive transfected cells extended dramatically their survival in serum-free medium, suggesting that the rise of FGF1 synthesis during neuronal differentiation is probably linked to their ability to survive in the adult. All of these data demonstrate that, in contrast to the MAP kinase cascade, FGF1 expression is sufficient to induce in PC12 cells both differentiation and survival. It also shows that auto- and trans-activation of FGF1 expression is involved in the differentiation process stimulated by exogenous FGFs through a new pathway which remains to be characterized. The expression of fibroblast growth factor (FGF) 1, a potent neurotrophic factor, increases during differentiation and remains high in adult neuronal tissues. To examine the importance of this expression on the neuronal phenotype, we have used PC12 cells, a model to study FGF-induced neuronal differentiation. After demonstrating that FGF1 and FGF2 are synthesized by PC12 cells, we investigated if FGF1 expression could be a key element in differentiation. Using the cell signaling pathway to determine the effects of FGF1 alone, FGF1 plus heparin, or a mutated FGF1, we showed an activation to the same extent of mitogen-activated protein (MAP) kinase kinase and MAP kinase (extracellular regulated kinase 1). However, only FGF1 plus heparin could promote PC12 cell differentiation. Thus, the MAP kinase pathway is insufficient to promote differentiation. Analysis of the PC12 cells after the addition of FGF1 plus heparin or FGF2 demonstrated a significant increase in the level of FGF1 expression with the same time course as the appearance of the neuritic extensions. Transfection experiments were performed to enhance constitutivly or after dexamethasone induction the level of FGF1 expression. The degree of differentiation achieved by the cells correlated directly with the amount of FGF1 expressed. The MAP kinase pathway did not appear to be involved. Interestingly, a 5-fold increase in FGF1 in constitutive transfected cells extended dramatically their survival in serum-free medium, suggesting that the rise of FGF1 synthesis during neuronal differentiation is probably linked to their ability to survive in the adult. All of these data demonstrate that, in contrast to the MAP kinase cascade, FGF1 expression is sufficient to induce in PC12 cells both differentiation and survival. It also shows that auto- and trans-activation of FGF1 expression is involved in the differentiation process stimulated by exogenous FGFs through a new pathway which remains to be characterized. INTRODUCTIONFGF1 ( 1The abbreviations used are: FGFfibroblast growth factorFGFRFGF receptorrFGFrat FGFhFGFhuman FGFNGFnerve growth factorMAPmitogen-activated proteinMAPKKMAP kinase kinaseERK1extracellular regulated kinase 1RTreverse transcriptasePCRpolymerase chain reactionChATcholine acetyltransferaseAchEacetylcholine esteraseEIAenzyme immunoassayPBSphosphate-buffered salineDMEMDulbecco's modified Eagle's mediumbpbase pair(s).) and 2 are widely distributed in the peripheral and central nervous systems in the adult. In rat brain, FGF2 is present in most neurons within the cerebral cortex(1.Pettmann B. Labourdette G. Weibel M. Sensenbrenner M. Neurosci. Lett. 1986; 68: 175-180Crossref PubMed Scopus (236) Google Scholar), hippocampus(2.Janet T. Miehe M. Pettmann B. Labourdette G. Sensenbrenner M. Neurosci. Lett. 1987; 80: 153-157Crossref PubMed Scopus (64) Google Scholar), and cerebellum(3.Gonzalez A.M. Buscaglia M. Ong M. Baird A. J. Cell Biol. 1990; 110: 753-765Crossref PubMed Scopus (464) Google Scholar). High levels of FGF1 expression have been observed in motor neurons, primary sensory neurons, and retinal ganglion neurons(4.Jacquemin E. Halley C. Alterio J. Laurent M. Courtois Y. Jeanny J.C. Neurosci. Lett. 1990; 116: 23-28Crossref PubMed Scopus (36) Google Scholar, 5.Elde R. Cao Y.H. Cintra A. Brelje T.C. Pelto-Huikko M. Junttila T. Fuxe K. Pettersson R.F. Hokfelt T. Neuron. 1991; 7: 349-364Abstract Full Text PDF PubMed Scopus (178) Google Scholar). In chick brain, the expression of FGF1 is developmentally regulated(6.Schnurch H. Risau W. Development. 1991; 111: 1143-1154PubMed Google Scholar). In bovine and rat embryonic retina, all neuronal layers express FGF1 with an appearance corresponding to their sequential differentiation(7.Jacquemin E. Jonet L. Oliver L. Bugra K. Laurent M. Courtois Y. Jeanny J.C. Int. J. Dev. Biol. 1993; 37: 417-423PubMed Google Scholar, 8.Bugra K. Oliver L. Jacquemin E. Laurent M. Courtois Y. Hicks D. Eur. J. Neurosci. 1993; 5: 1586-1595Crossref PubMed Scopus (43) Google Scholar). In rat, the level of FGF1 expression remains uniformly low throughout the embryonic period until postnatal day 7. Thereafter, it increases rapidly, reaching a maximum in the adult retina. In the intermediate central nervous system, subclasses of FGF receptors appear to be down-regulated during development(9.Heuer J.G. Von-Batheld C.S. Kinoshita Y. Evers P.C. Bothwell M. Neuron. 1990; 5: 283-296Abstract Full Text PDF PubMed Scopus (174) Google Scholar), and during retinal embryonic development, the expression of FGFR1 and FGFR2 follows the retinal layering(10.Tcheng M. Fuhrman G. Hartman M.P. Courtois Y. Jeanny J.C. Exp. Eye Res. 1994; 58: 351-358Crossref PubMed Scopus (66) Google Scholar). These patterns of FGF expression suggest that these growth factors are involved in the integrity, development, and differentiation of the central nervous system. In fact, in vitro studies have shown that FGF1 promotes the survival of photoreceptors (11.Hicks D. Courtois Y. FEBS Lett. 1988; 234: 475-479Crossref PubMed Scopus (48) Google Scholar) and the neuritic outgrowth of dissociated retinal ganglion cells(12.Lipton S.A. Wagner J.A. Madison R.D.D. Amore P.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2388-2392Crossref PubMed Scopus (148) Google Scholar). FGF1 also inhibits pigmentation of immature pigmented epithelium cells of embryonic chick retina and stimulates ganglion cell differentiation (13.Guillemot F. Cepko C.L. Development. 1992; 114: 743-754PubMed Google Scholar). FGF2 promotes the survival of neurons of the peripheral (14.Eckenstein F.P. Esch F. Holbert T. Blacher R.W. Nishi R. Neuron. 1990; 4: 623-631Abstract Full Text PDF PubMed Scopus (94) Google Scholar) and central nervous systems (15.Hatten M.E. Lynch M. Rydel R.E. Sanchez J. Joseph-Silverstein J. Moscatelli D. Rifkin D.B. Dev. Biol. 1988; 125: 280-289Crossref PubMed Scopus (321) Google Scholar, 16.Walicke P.A. J. Neurosci. 1988; 8: 2618-2627Crossref PubMed Google Scholar) and delays photoreceptor degeneration in a retinal degeneration model(17.Faktorovitch E.G. Steinberg R.H. Yasumura D. Matthes M.T. Lavail M.M. Nature. 1990; 347: 83-86Crossref PubMed Scopus (635) Google Scholar, 18.La Vail M.M. Unoki K. Yasumura D. Matthes M.T. Yancopoulos G.D. Steinberg R.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11249-11253Crossref PubMed Scopus (636) Google Scholar).These FGF activities in in vitro systems together with the temporal and spatial expression patterns of FGF in embryonic and adult neuronal tissues suggested that the FGF expressed by neuronal cells could be involved in the mediation of their neurotrophic activity. To investigate whether the expression of FGF1 by neuronal cells was implicated in the differentiation and neuronal survival, we have used PC12 cells as an in vitro model. This cell line was derived from a rat adrenal tumor (24.Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4822) Google Scholar) and responded to NGF and FGF by the extension of neurites and the acquisition of sympathetic neuronal phenotype. The transition of chromaffin phenotype to neuronal phenotype by NGF and FGF is accompanied by events mediated by the activation of high affinity tyrosine kinase receptors and the activation of the MAP kinase cascade. This is a main signaling pathway for cell proliferation, differentiation, and transformation and appears to mediate differentiation of PC12 cells induced by NGF and FGF2 (25.Tsao H. Aletta J.M. Greene L.A. J. Biol. Chem. 1990; 265: 15471-15480Abstract Full Text PDF PubMed Google Scholar, 26.Nguyen T.T. Scimeca J.C. Filloux C. Peraldi P. Carpentier J.L. Van Obberghen E. J. Biol. Chem. 1993; 268: 9803-9810Abstract Full Text PDF PubMed Google Scholar, 27.Peraldi P. Scimeca J.C. Filloux C. Van Obberghen E. Endocrinology. 1993; 132: 2578-2585Crossref PubMed Scopus (39) Google Scholar, 28.Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar).In this study, we show that PC12 cells expressed both FGF1 and FGF2. These cells were treated with different stimuli: FGF1-heparin, FGF2, and NGF (promotors of differentiation) or Lys-132-mutated FGF1 (FGF1K132E) and FGF1 alone (which do not promote differentiation). The expression of FGF1 at the transcript and protein level in parallel with the activation of the MAP kinase cascade was investigated in stimulated cells as a function of neuronal differentiation. The effect of FGF1 expression on neuronal differentiation was also examined in transfected cells in which FGF1 expression was under the control of a constitutive or dexamethasone-inducible promotor.We show that the expression of FGF1 was activated only by exogenous FGF stimuli (FGF2 and FGF1-heparin) which are neurotrophic for PC12 cells and was unchanged when cells remained undifferentiated upon treatment with FGF1 alone or mutated FGF1K132E. In contrast, the MAP kinase cascade was similarly activated in stimulated cells whether or not they differentiated in sympathetic neurons. Accordingly, in transfected cells the expression of FGF1 strictly correlated with the differentiated phenotype and increased the survival of PC12 cells. We thus propose that the activation of the MAP kinase cascade is insufficient to induce the differentiation and survival of PC12 cells, and that the expression of FGF1 stimulated by exogenous FGF stimuli is a key element in the neurotrophic activities of the FGF.EXPERIMENTAL PROCEDURESMaterialsHuman recombinant FGF1 was produced and purified in our laboratory (INSERM XR118). FGF1K132E was from one of our laboratories (G. G.-G.). In the mutant FGF1, the lysine 132 was mutated to glutamic acid, and this mutation decreased the affinity of FGF1 to heparin(29.Burgess W.H. Shaheen A.M. Ravera M. Jaye M. Donohue P.J. Winkles J.A. J. Cell Biol. 1990; 111: 2129-2138Crossref PubMed Scopus (91) Google Scholar). Human recombinant FGF2 was a kind gift from Farmitalia, Carlo Erba. The recombinant expression vector (pSVL-FGF1-134) was obtained from Dr. Jaye (Rhone-Poulenc-Rohrer)(30.Jaye M. Lyall R.M. Mudd R. Schlessinger J. Sarver N. EMBO J. 1988; 7: 963-969Crossref PubMed Scopus (98) Google Scholar). The FGF2-saporin, a cytotoxic fusion protein, was a gift from by Drs. Lappi and Baird (Whitter Institut, San Diego).Cell CulturesStock cultures of rat PC12 cells (originally obtained from P. Brachet, Angers) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 5% horse serum, and antibiotics at 37°C in a humidified atmosphere of 5% CO2, air. Culture media were renewed every 3 days. PC12 cells transfected with pSVL-FGF1-134 were maintained as described for the control PC12 cells. PC12 cells transfected with pLK-FGF1-134 were maintained in DMEM supplemented with 10% fetal calf serum and 5% horse serum which were both depleted in glucocorticoid by fixation onto 1% Norit-A and 0.1% dextran T70. FGF1 expression was induced by 5 × 10−7M dexamethasone.FGF1 Eukaryotic Expression Vectors and PC12 Cell TransfectionThe FGF1 expression vector (pSVL-FGF1-134) codes for 134 amino acids of the human FGF1 and is under the control of the early promotor from the simian virus 40(30.Jaye M. Lyall R.M. Mudd R. Schlessinger J. Sarver N. EMBO J. 1988; 7: 963-969Crossref PubMed Scopus (98) Google Scholar). The coding region of pSVL-FGF1-134 was subcloned into a glucocorticoid-responsive vector (pLK), which contained a variant murine mammary tumor virus-long terminal repeat promotor and the neomycin resistance gene(31.Wellinger R.J. Garcia M. Vessaz A. Diggelman H. J. Virol. 1986; 60: 1-11Crossref PubMed Google Scholar). The recombinant plasmid was named plK-FGF1-134. PC12 cells were cotransfected with 10 μg of FGF1 recombinant pSVL plasmid DNA and 1 μg of pSVL2-neomycin plasmid DNA (ratio 1 to 10) or with the FGF1-inducible expression vector pLK-FGF1-134 with Lipofectin (Life Technologies, Inc.) using the method previously described by Muller et al.(32.Muller S.R. Sullivan P.D. Clegg D.O. Feinstein S.C. DNA Cell Biol. 1990; 9: 221-229Crossref PubMed Scopus (62) Google Scholar). Briefly, the different plasmid DNA used for transfection were purified twice onto a cesium chloride gradient. The transfection medium containing 10 μg of plasmid DNA (9 μg of pSVL-FGF-134 and 1 μg of pSVL2-neomycin or 10 μg of pLK-FGF1-134), 60 μl of Lipofectin reagent (Life Technologies, Inc.) in 2 ml of serum-free DMEM was incubated 20 min at room temperature, then diluted with DMEM to a final volume of 6 ml and added to PC12 cells, plated the day before at the density of 105 cells per 100-mm diameter Petri dishes. The transfection process occurred at 37°C for 4 h, then 6 ml of DMEM containing 20% FCS and 10% horse serum were added to the cells. Two days later, the cells were trypsinized and replated in four Petri dishes in selection medium (0.5 mg/ml geneticin in culture medium). The selection of the stably transfected clones was performed for 15 days, and the geneticin-resistant colonies were picked and allowed to grow.Neurite Outgrowth AssayPC12 cells were plated on poly-L-lysine-coated 12-well plates at a density of 3 × 104 cells/ml. After cell attachment (18 h), the cells were treated for 4 days with FGF2 (10 ng/ml), FGF1 (100 ng/ml), or FGF1K132E (100 ng/ml or 5 μg/ml). In some experiments heparin (10 μg/ml) was added when cells were treated with FGF1 or with FGF1K132E. The media and the growth factors were renewed every 2 days. The morphology of the PC12 cells stimulated by exogenous factors and of the transfected cells was examined after 3-4 days of culture.Cell Survival AssayPC12 cells and constitutive transfected cells were washed three times with PBS, dissociated in PBS, 10 mM EDTA, and plated in a 24-multiwell plate at a density of 5 × 104cells/ml in serum-free medium. At the indicated times, trypan blue exclusion tests were performed.RT-PCR AssayRNA preparations and RT-PCR assay were performed as described previously by Renaud et al.(33.Renaud F. Oliver L. Desset S. Tassin J. Romquin N. Courtois Y. Laurent M. J. Cell. Physiol. 1994; 158: 435-443Crossref PubMed Scopus (53) Google Scholar). Total RNA was isolated from cultured cells using the guanidium isothiocyanate method(34.Chirginn J.M. Przybyla A.E. MacDonald R.J. Rutter W. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16619) Google Scholar). One μg of RNA and 170 pg of tobacco leaf nitrate reductase transcripts were reverse transcribed in 30 μl of 50 mM Tris-HCl, pH 8.9, 3 mM MgCl2, 75 mM KCl, 2.5 μM random hexanucleotide primers, 300 units of Maloney murine leukemia virus (Life Technologies, Inc.) and 1/10 of the reverse transcripts were amplified in 100 μl of 50 mM Tris-HCl, pH 8.9, 7 mM MgCl2, 50 mM KCl, 15 mM ammonium sulfate, 0.17 mg/ml bovine serum albumin, 1 mM of dNTP mix, 15 pmol of each specific primer with 1 unit of Taq polymerase (Eurobio) as described previously(33.Renaud F. Oliver L. Desset S. Tassin J. Romquin N. Courtois Y. Laurent M. J. Cell. Physiol. 1994; 158: 435-443Crossref PubMed Scopus (53) Google Scholar). In a preliminary experiment, the level of FGF1 or FGF2 in PC12 cells was tested by RT-PCR after 20-40 cycles of amplification, and we determined that the linear exponential FGF1/FGF2 amplification phase is comprised between 26 and 33 cycles (data not shown). However, some small RT-PCR efficiency differences between each experiment occurred. Thus, to ensure that the amplification was in the exponential phase, for each experiment aliquots were withdrawn at three different cycles and analyzed. For FGF1 and FGF2 amplification, the oligomers were chosen in different coding exons to avoid amplification of genomic DNA. Specific oligomers were used for rat (rFGF1S, rFGF1AS) and human (hFGF1S, hFGF1AS) FGF1 amplification: rFGF1AS (5′-AAG CCC GTC GGT GTC CAT GG-3′) and rFGF1S (5′-GAT GGC ACA GTG GAT GGG AC-3′) generated a 135-bp fragment; hFGF1AS (5′-TCC GAG GAC CGC GTT TGC AG 3-′) and hFGF1S (5′-GAT CCT TCC GGA TGG CAC AG-3′) generated a 295-bp fragment. The two FGF2 oligomers (antisense primer 5′-CCC AGT TCG TTT CAG TGC CAC C-3′, sense primer 5′-CAT TCA AAG GAG TGT GTG CAA ACC-3′) generated a 174-bp fragment, the two NGF oligomers (antisense primer 5′-CTC CAA CCC ACA CAC TGA CA-3′, sense primer 5′-TCT GTC CCT GAA GCC CAC TG-3′) generated a 342-bp fragment, and the nitrate reductase-specific oligomers (antisense primer 5′-GCT GGA TCC ATT GCA AAT TCC-3′, sense primer 5′-AGG AGC TGA TGT GTT GCC CGG-3′) generated a 75-bp fragment. The FGF1 or FGF2 or NGF and nitrate reductase-amplified products were electrophoresed on 10% polyacrylamide gel, blotted onto Hybond N+, and hybridized with specific FGF1, FGF2, NGF, and NR probes. X-Omat AR5 x-ray film (Eastman Kodak Co.) was exposed for different periods, depending on the intensity of the signal.FGF1 Expression Analysis by Enzyme Immunoassay (EIA) and Western BlotNative PC12 cells, FGF1-treated PC12 cells, and FGF1-transfected PC12 cells were lysed in PBS, pH 7.5, containing 2 M NaCl, 0.1% Triton X-100. The protein concentration of the lysates was determined using a BCA kit (Pierce). The lysates were used to assay FGF1 by EIA and Western blot. Cell lysate proteins (1 mg) were incubated with 100 μl of heparin-Sepharose (Pharmacia Biotech Inc.) in PBS, 0.6 M NaCl. After one night of absorption at 4°C, the heparin-Sepharose was washed twice with the binding buffer, and the heparin-binding proteins were eluted in PBS, 2 M NaCl. The FGF1 was quantified using a second antibody solid phase EIA as described previously by Oliver et al.(35.Oliver L. Raulais D. Vigny M. Growth Factors. 1992; 7: 97-106Crossref PubMed Scopus (32) Google Scholar). For Western blotting analysis, 50-150 μg of lysate protein from inducible and constitutive transfected cells and from control PC12 cells were incubated with 30 μl of heparin-Sepharose in 0.6 M NaCl as for EIA. Heparin-binding proteins were eluted in 30 μl of Laemmli buffer and electrophoresed on 18% SDS-polyacrylamide gel electrophoresis. After transfer to nitrocellulose membranes (Optitran BA-S 83, Schleicher & Schuell), the proteins bound to heparin were incubated for 1 h at 37°C with rabbit polyclonal FGF1 antibody (diluted 1:500) in 150 mM NaCl, 100 mM sodium phosphate buffer, pH 7.6, containing O.1% milk protein. The FGF1 antibody, directed against human recombinant FGF1, has been produced in our laboratory, and its specificity has been previously described(35.Oliver L. Raulais D. Vigny M. Growth Factors. 1992; 7: 97-106Crossref PubMed Scopus (32) Google Scholar). The filter was washed three times in the aforementioned buffer, incubated with biotinylated anti-rabbit IgG from donkey (diluted 1:400; Amersham Corp.), then with streptavidin-horseradish peroxidase congugate (diluted 1:400; Amersham Corp.), and finally demonstrated with ECL Western blotting detection reagents (Amersham Corp.) according to the manufacturer's suggested protocol.ERK1 or MAPKK AssayNative PC12 cells or constitutive transfected PC12 cells were plated at 2 × 105 cells/ml. After 2-3 days of culture in complete medium, cells were incubated overnight in 0.25% fetal calf serum and 0.2% bovine serum albumin prior to stimulation and incubated for specified times with growth factors. The cells were lysed in 50 mM Hepes, pH 7.5, 150 mM NaCl, 100 mM NaF, 10 mM EDTA, 10 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, and a mixture of protease inhibitors. The solubilized cell extracts were incubated for 2 h with antibodies to ERK1 (diluted 1:50) or MAPKK (diluted 1:50) preabsorbed on protein A-Sepharose beads in HNT buffer (30 mM Hepes, pH 7.4, 30 mM NaCl, O.1% Triton X-100). Anti-ERK1 is a rabbit antiserum directed against a synthetic peptide from the carboxyl-terminal tail of the MAP kinase ERK1 (36.Scimeca J.C. Ballotti R. Nguyen T.T. Filoux C. Van Obberghen E. Biochemistry. 1991; 30: 913-919Crossref Scopus (15) Google Scholar) and the anti-MAPKK is directed against the 17-amino acid amino terminus (37.Frodin M. Peraldi P. Van Obberghen E. J. Biol. Chem. 1994; 269: 6207-6214Abstract Full Text PDF PubMed Google Scholar). ERK1 activity was measured as the ability of immunopurified ERK1 to stimulate myelin basic protein phosphorylation, MAPKK activity was measured in a reconstitution assay as the ability of immunopurified MAPKK to activate added recombinant rat ERK1. Both assays were performed as described previously by Frodin et al.(37.Frodin M. Peraldi P. Van Obberghen E. J. Biol. Chem. 1994; 269: 6207-6214Abstract Full Text PDF PubMed Google Scholar).ERK1 and MAPKK Expression Analysis by Western BlotPC12 cells and constitutive transfected cells were lyzed in 50 mM Hepes, pH 7.5, 150 mM NaCl, 100 mM NaF, 10 mM EDTA, 10 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, and a mixture of protease inhibitors. 330 μg of cellular protein were electrophoresed in a 7.5% SDS-polyacrylamide gel electrophoresis, electrotransferred to nylon membranes, and immunoprobed for ERK1 and MAPKK (both antibodies were diluted 1:2000). The antigen-antibody complexes were incubated with a peroxidase-anti-rabbit-IgG (diluted 1:4000; Amersham Corp.) and revealed using an ECL kit for detection (Amersham Corp.).ImmunocytochemistryNative PC12 cells and FGF1 constitutive transfected PC12 cells were plated onto poly-L-lysine-coated glass coverslips in 12-well plates at a density of 5 × 104 cells/ml, treated for 3 days with FGF1 (100 ng/ml) in the presence or absence of heparin (10 μg/ml). The cells were fixed with 4% paraformaldehyde, incubated with anti-ERK1, 1/500, or nonimmune serum, 1/500, in 0.3% Triton X-100. The antigen-antibody complexes were detected with fluorescein isothiocyanate-anti-rabbit antibodies (1/100; Amersham Corp.).Choline Acetyltransferase (ChAT) Activity and Acetylcholine Esterase (AchE) AssaysNative PC12 cells, FGF1-treated PC12 cells, and differentiated FGF1 transfected cells (B18) were lysed in 50 mM sodium phosphate buffer, pH 7.4, containing 0.5% Triton X-100. ChAT activity was measured as described by Fonnum(38.Fonnum F. J. Neurochem. 1975; 21: 1109-1120Crossref Scopus (94) Google Scholar), and AchE activity was determined by the spectrophotometric method of Ellman et al.(39.Ellman G.L. Courtney K.D. Andress V. Featherstone K.D. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (20930) Google Scholar).RESULTSExpression of FGF1 and FGF2 Transcripts during the Differentiation of PC12 CellsPC12 cells express a basal level of FGF1 and FGF2 mRNAs (Fig. 1A). When treated with FGF1 and heparin, PC12 cells extend neurites and increase their steady state levels of FGF1 (Fig. 1B) and FGF2 (Fig. 1D) mRNAs. The level of FGF1 transcripts remained constant during the first hours of treatment when no neuritic extensions are observed and increased progressively from day 1 to 5 with the same time course as the neuritic extension process (Fig. 1E). Short extensions appeared after 1 day of treatment and increased greatly in size from day 3 to 5. In contrast, the amount of FGF1 transcripts was similar in proliferating and confluent control cells cultured for 1, 3, and 5 days without FGF (Fig. 1C).Neuritic Extensions in PC12 Cells Treated with FGF1, Mutated FGF1, FGF2, and NGFCells were treated in DMEM in the presence (Fig. 2) or absence of serum (data not shown), with FGF1 (100 ng/ml), FGF1K132E (5 μg/ml) in the presence or absence of heparin (10 μg/ml), FGF2 (10 ng/ml), and NGF (50 ng/ml). After 4 days of treatment, cells extended neurites only when exposed to FGF1 and heparin (Fig. 2, 3), to FGF2 (Fig. 2, 4), or to NGF (2, 5). PC12 cells remained undifferentiated when treated with FGF1 alone (Fig. 2, 2) or with FGF1K132E (Fig. 2, 7) even in the presence (Fig. 2, 6) of heparin. This mutated FGF1, known to have a lower affinity for heparin, binds to the FGFRs but has no mitogenic activity (29.Burgess W.H. Shaheen A.M. Ravera M. Jaye M. Donohue P.J. Winkles J.A. J. Cell Biol. 1990; 111: 2129-2138Crossref PubMed Scopus (91) Google Scholar, 40.Burgess W.H. Shaheen A.M. Hampton B. Donohue P.J. Winkless J.A. J. Cell. Biochem. 1991; 45: 131-138Crossref PubMed Scopus (36) Google Scholar). The dependence of FGF1 on heparin for its neurotrophic activity allowed us to discriminate between the neurotrophic activity of this growth factor and its other properties.Figure 2:Effects of FGF2, FGF1, and FGF1K132E on neuritic extension in PC12 cells. PC12 cells were plated onto poly-L-lysine-coated dishes in DMEM supplemented with 10% fetal calf serum and 5% horse serum. Twenty-four hours after plating, the cells were treated with 100 ng/ml FGF1 (2), 100 ng/ml FGF1 plus 10 μg/ml heparin (3), 10 ng/ml FGF2 (4), 50 ng/ml NGF (5), 5 μg/ml FGF1K132E plus 10 μg/ml heparin (6), 5 μg/ml FGF1K132E (7), or 10 μg/ml heparin (8). Untreated cells were used as a control (1). The morphology of the cells was examined after 4 days of treatment (magnification × 75).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression of FGF1 Transcripts during Differentiation of PC12 Cells Treated by FGF1, Mutated FGF1, FGF2, and NGFThe steady-state level of FGF1 transcripts in cells treated, as above, with FGF1 (100 ng/ml), FGF1K132E (5 μg/ml) in the presence or absence of heparin (10 μg/ml), and FGF2 (10 ng/ml) was examined by RT-PCR after 30 cycles, using rFGF1S and rFGF1AS oligonucleotides (Fig. 3). NGF (50 ng/ml)-treated cells were introduced as a control of differentiated cells. An increase in FGF1 transcripts was only observed in FGF differentiated cells. After 3 days of treatment with FGF1 and heparin (lane 3) or FGF2 (lane 4), the steady state level of FGF1 transcripts in differentiated cells increased 5.7 and 3.5 times, but remained unchanged when differentiation was induced by NGF (lane 5). Cells treated with FGF1 alone (lane 2), or FGF1K132E and heparin (lane 6) remained undifferentiated and expressed the same level of FGF1 transcripts as control cells (lane 1). These data strongly suggested that an up-regulation of FGF1 gene expression was implicated in the differentiation of PC12 cells. To test this hypothesis the effect of overexpression of FGF1 was analyzed by transfecting PC12 cells with plasmid constructs leading to constitutive overexpression of FGF1.Figure 3:Analysis by RT-PCR of FGF1 transcripts in differentiated and undifferentiated PC12 cells. Total RNA was extracted from PC12 cells after 3 days of treatment with 100 ng/ml FGF1, in the absence (lane 2) or presence (lane 3) of heparin (10 μg/ml), with 10 ng/ml of FGF2 (lane 4), with 100 ng/ml of NGF (lane 5) or with 100 ng/ml FGF1K132E plus heparin (10 μg/ml) (lane 6). Untreated cells cultured for 3 days were used as control (lane 1). One μg of each RNA preparation was assayed along with nitrate reductase transcripts to RT-PCR as in Fig. 1A. After 28 cycles, the amplification products were electrophoresed, analyzed by Southern blotting, and hybridized with FGF1 and nitrate reductase-specific probes. This experiment has been independently performed three times with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Isolation and Morphological Analysis of FGF1-transfected PC12 CellsAfter transfection of PC12 cells with pSVL-FGF1-134, 52 clones with different morphologies were isolated. Most of the clones (32/52) (Fig. 4, 1 and 3) were indistinguishable from the parental line. After 3 days in culture, the remaining clones (20/52) exhibited a differentiated phenotype with neuritic extensions of different lengths. The morphology of most of the differentiated clones (Fig. 4, 2 and 4) and of the PC1
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