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J. Biol. Chem., Vol. 279, Issue 46, 47912-47928, November 12, 2004

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Basic Fibroblast Growth Factor-induced Cell Death Is Effected through Sustained Activation of p38^MAPK and Up-regulation of the Death Receptor p75^NTR*

Andrew J. K. Williamson, Benjamin C. Dibling, James R. Boyne, Peter Selby, and Susan A. Burchill¶

From the Candlelighter's Children's Cancer Research Laboratory, Cancer Research UK Clinical Centre, St. James's University Hospital, Leeds LS9 7TF, United Kingdom

Received for publication, August 6, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Basic fibroblast growth factor (bFGF) induces cell death in cells of the Ewing's sarcoma family of tumors in vivo and in vitro. In this study we demonstrate that this is dependent on the rapid and sustained activation of p38^MAPK, in contrast to the transient activation of p38^MAPK associated with bFGF-induced cell proliferation. Stem cell factor-induced survival of TC-32 cells was also associated with transient activation of p38^MAPK. Inhibition of p38^MAPK by SB202190 and p38^MAPK small interfering RNA reduces bFGF-induced death in TC-32 cells, consistent with the hypothesis that activation of p38^MAPK is essential for induction of death by bFGF. This appears to be dependent on sustained activation of p38^MAPK, demonstrated by inhibition of bFGF-induced cell death following addition of SB202190 to TC-32 cells 5 min after exposure to bFGF (20 ng/ml) and activation of p38^MAPK. Prolonged activation of p38^MAPK is accompanied by a rapid and sustained phosphorylation of Ras and ERK; inhibition of ERK phosphorylation using the MEK-1 inhibitor PD98059 rescued 30% of cells from bFGF-induced death suggesting ERK plays a secondary role in the induction of death. This hypothesis is supported by observations in the A673 cell line; bFGF induced sustained activation of ERK and transient activation of p38^MAPK, which was not associated with cell death. These data demonstrate that sustained activation of p38^MAPK is essential for activation of the death cascade following exposure of Ewing's sarcoma family of tumors cells to bFGF and provide evidence that activation of p38^MAPK results in an up-regulation of the death receptor p75^NTR.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Ewing's sarcoma family of tumors (ESFT)¹ encompasses a group of malignancies, including Ewing's sarcoma, Askin's tumor of the chest wall, and peripheral primitive neuroectodermal tumor, which are thought to be of neural histogenesis (1–3). ESFT exhibit a common genetic rearrangement involving fusion of the 5' end of the EWS gene on chromosome 22 to the 3' portion of members of the Ets gene family of transcription factors. In over 90% of cases the Ets gene family member is fused to the Fli1 gene located on chromosome 11 (4). This results in the generation of a fusion gene, the protein product of which has been implicated in development of the transformed ESFT phenotype (5–7). ESFT typically arise in the bone or soft tissue of adolescents and young adults, 15–30% of patients presenting with metastatic disease. The outcome for this group of patients is particularly poor despite the use of aggressive therapeutic regimes, emphasizing the need for new therapeutic strategies.

A role for autocrine and/or paracrine growth factor survival loops in ESFT is well documented; the blockade of insulin-like growth factor/insulin-like growth factor receptor 1 (8–11) or stem cell factor (SCF)/c-Kit (12, 13) circuits results in a decrease in ESFT cell number in both in vitro and in vivo models. Previous studies (14) have also suggested that a basic fibroblast growth factor (bFGF)/fibroblast growth factor receptor autocrine/paracrine survival loop may be important for the survival and proliferation of ESFT. However, we have found no evidence of such a survival loop, and we demonstrated recently that treatment of ESFT with bFGF results in the up-regulation of the death receptor p75^NTR and induction of cell death (15, 16).

The intracellular signaling pathways leading to the induction of cell death following exposure of ESFT cells to bFGF are unknown. Although our preliminary results have shown that incubation of ESFT cells with bFGF causes phosphorylation of fibroblast growth factor receptor 1 and activation of the downstream signaling molecules Ras and ERK (16), whether these events are important effectors of bFGF-induced cell death is not clear. Following receptor activation, phosphorylated tyrosines function as binding sites for a number of downstream adapter and signaling proteins, including the docking protein FRS2 that recruits several signal transduction molecules leading to activation of the mitogen-activated protein kinase (MAPK) cascade and the phosphatidylinositol 3-kinase-AKT anti-apoptotic pathway (17, 18). Recruitment of guanine nucleotide exchange factors (e.g. hSOS) leads to the conversion of the small GTPase Ras from an inactive GDP-bound state to an active GTP-bound state and activation of the extracellular signal-regulated kinase (ERK) pathway (19). Activation of the Ras-ERK pathway has been shown to mediate such diverse cellular processes as proliferation (20), survival (21–23), apoptosis (24), senescence (25, 26), and differentiation (20).

Specificity of response is achieved by the influence of the MAPK superfamily of proteins on gene expression and regulation of downstream kinases or transcription factors. This family of proteins shares many structural similarities and includes the extracellular signal-regulated kinases ERK 1 and ERK 2, the p38^MAP kinases, and the c-Jun N-terminal kinases (JNK; also known as the stress-activated protein kinases). They are usually activated by distinct extracellular stimuli; ERK is typically stimulated by growth factors and mitogenic stimuli; p38^MAPK and JNK are primarily activated by cellular stress including heat, osmotic, and oxidative stress (19, 27, 28). Five p38^MAPK (p38, p38, p38, p38, and p38-2) and three JNK (JNK 1, JNK 2, and JNK 3) isoforms have been identified. Both control activation of transcription factors, p38^MAPK-activating transcription factor 1 (ATF 1), ATF 2, CHOP, growth arrest and DNA damage inducible gene 153 (GADD153), and p53 (29–35), whereas JNK most frequently activates c-Jun, Jun B, Jun D, ATF-2, and AP-1 (36–39). Although p38^MAPK and JNK may be co-activated by some agonists, the upstream signaling kinases are specific, i.e. p38^MAPK is activated via MKK 3 and 6 (and in some cases MKK 4), and activation of JNK is catalyzed by MKK 4 and 7 (40–42).

In this study we have investigated the role of the Ras-ERK, p38^MAPK, and JNK cascades in mediating growth factor-induced survival or death in ESFT cells, and we examined the hypothesis that activation of p38^MAPK, JNK, and/or ERK may induce p75^NTR expression leading to cell death in these cells following exposure to bFGF. We have shown that exposure of ESFT cells to bFGF induces sustained activation of Ras-ERK and p38^MAPK and that the sustained activation of p38^MAPK is an important effector of bFGF-induced cell death. This sustained activation leads to up-regulation of the p75^NTR death receptor, which appears to be independent of JNK activation. In contrast, the survival effects of SCF in ESFT and the proliferative effects of bFGF in neuroblastoma cells are associated with transient activation of p38^MAPK.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Antibodies, Growth Factors, and Small Molecular Weight Inhibitors
Anti-ERK, anti-Ras, and anti--tubulin antibodies were purchased from Transduction Laboratories. The anti-Ras antibody (OP-40) used for immunoprecipitation was obtained from Merck. Anti-phosphorylation-specific ERK and p75^NTR antibodies were obtained from Promega. Pan-anti-p38^MAPK, anti-phosphorylation-specific p38^MAPK antibodies, and the nonradioactive p38^MAP kinase assay kit were obtained from Cell Signaling Technology. Total JNK and phosphorylated JNK 1 and 2 were detected by using the PhosphoPlus® SAPK/JNK (Thr-183/Tyr-185) antibody kit also from Cell Signaling Technology. p38^MAPK antibody was purchased from Upstate Biotechnology, Inc., and the p38^MAPK-specific antibody was obtained from Zymed Laboratories Inc. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Sigma. Alexa Fluor 680-labeled secondary antibodies were purchased from Molecular Probes. bFGF and SCF were obtained from Sigma and were reconstituted in PBS containing 0.1% (w/v) bovine serum albumin, aliquoted, and stored at –20 °C. The inhibitors of ERK (PD98059) and p38^MAPK and - (SB202190 and SB203580) were purchased from Calbiochem, reconstituted in dimethyl sulfoxide (Me₂SO), aliquoted, and stored at –20 °C.

Cell Culture
Cells were cultured on Primaria plastic (Falcon) for all experiments. RDES, TC-32, and TTC-466 ESFT cells were grown in RPMI 1640 media (Sigma) supplemented with 10% fetal calf serum (FCS) (Seralab, Sussex, UK); culture media for TTC-466 cells were supplemented with 10% conditioned media. A673 cells were grown in Dulbecco's modified Eagle's media (DMEM) (Sigma) and 10% FCS. SK-ES1 cells were grown in McCoy's media (Sigma) plus 15% FCS and SK-N-MC cells in DMEM/F-12 (Sigma) plus 10% FCS. SK-N-SH neuroblastoma cells were maintained in a 1:1 mixture of DMEM and modified Eagle's media supplemented with 10% FCS. TC-32 cells were a gift from Dr. J. Toretsky (Georgetown, University Medical School, Washington, D. C.), and the TTC-466 cell line was a gift from Dr. P. Sorenson (British Columbia Children's Hospital, Vancouver, Canada). All the other cell lines were purchased from the American Type Culture Collection.

Constructs
pGEX-RBD was a kind gift from Dr. J. Downward (Cancer Research UK, London, United Kingdom) and contains the Ras-binding domain of Raf (amino acids 1–149) fused to glutathione-Sepharose transferase.

Preparation of Cell Lysates for Western Blotting, Affinity Precipitation, or Immunoprecipitation
Cells were seeded in 60-mm plates and allowed to adhere for 24 h prior to treatment with growth factors or inhibitors at the concentrations and times indicated. To analyze the effect of growth factors in the absence of serum, media were aspirated, and the cells were washed briefly with serum-free media and cultured under serum-free conditions for 24 h prior to growth factor stimulation. Stimulation of the cells with growth factors and/or incubation with inhibitors was terminated by placing the cells on ice, aspiration of the culture media, and washing the cell monolayer with ice-cold phosphate-buffered saline (PBS). Cells were lysed in lysis buffer (50 mM Hepes (pH 7.5), 100 mM NaCl, 1 mM EGTA, 1 mM dithiothreitol, 10 mM MgCl₂, 1% (v/v) Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM sodium orthovanadate (Sigma), 25 mM sodium fluoride (Sigma)) for 30 min on ice. Cellular debris was removed by centrifugation at 11,600 x g for 10 min at 4 °C, and the supernatant was retained for analysis by immunoprecipitation, Western blotting, or affinity precipitation. Protein content of extracts was determined by using the DC protein assay (Bio-Rad).

Immunoprecipitation
Cell lysate was denatured by adding SDS to a final concentration of 0.1% and heating for 3 min at 100 °C before cooling on ice. Pan-Ras antibody (1 µg) and protein A-Sepharose^TM CL-4B (30 µl of 50% slurry; Amersham Biosciences) were added to cell lysate (500 µg) and incubated with mixing at 4 °C overnight. Beads were isolated by centrifugation and washed three times in ice-cold PBS and 0.1% (w/v) SDS. They were then resuspended in SDS-Laemmli buffer and analyzed by immunoblotting using anti-Ras antibodies.

Immunoblotting
Cell lysate (50 µg) was size-fractionated by SDS-PAGE, and proteins were transferred onto nitrocellulose membrane (Hybond-C, Amersham Biosciences) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) using a mini transblot system (Bio-Rad) overnight at 4 °C. The membranes were then hybridized with antibodies and analyzed using ECL or for quantitative analysis using the Odyssey infrared imaging system (Li-Cor). To aid resolution of phosphorylated ERK proteins, N,N'-methylenebisacrylamide was added to the separating gel at 200 parts acrylamide to 1 part N,N'-methylenebisacrylamide.

Enhanced Chemiluminescence—Membranes were blocked with a 5% nonfat milk solution in TTBS (0.05% Tween 20, 20 mM Tris-HCl (pH 7.5), 500 mM NaCl) for 2 h. Immobilized antigen was detected following incubation with primary antibody diluted in 1% nonfat milk solution in TTBS for 2 h. Blots were then washed using TTBS and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody in 1% nonfat milk solution in TTBS for 45 min. After the blots were washed using TTBS, they were developed using ECL (Amersham Biosciences).

Odyssey Infrared Imaging System—Membranes were blocked using Li-Cor blocking buffer for 2 h and then incubated for a further 2 h with the primary antibody diluted in a 1:1 mixture of Li-Cor blocking buffer and PBS supplemented with 0.1% Tween 20. Blots were then washed using TPBS (0.1% Tween 20, 20 mM Na₂HPO₄ (pH 7.4), 120 mM NaCl)) and incubated with the appropriate Alexa Fluor 680-labeled secondary antibodies (Molecular Probes) for 2 h. After the blots were washed with TPBS, they were analyzed using the Odyssey infrared imaging system (Li-Cor) to analyze the relative density of protein bands.

Affinity Precipitation of Ras-GTP Using Immobilized GST-RBD
Affinity precipitation was carried out essentially as described previously (43). Briefly, BL21 (Invitrogen) Escherichia coli harboring pGEX-RBD was grown at 37 °C until they reached an absorbance of 0.4–0.6, as measured using a spectrophotometer. Expression of GST-RBD was induced with 1 mM isopropyl--D-thiogalactopyranoside (Sigma) for 3 h. E. coli cells were harvested by centrifugation (500 x g for 15 min at 4 °C), and the supernatant was discarded, and the pellet was resuspended in lysis buffer (1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100 (Sigma), 10 µg/ml aprotinin (Sigma), 10 µg/ml leupeptin (Sigma) in PBS) and sonicated. Debris was removed by centrifugation (21,500 x g for 20 min at 4 °C). The supernatant was incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) for 2 h at 4 °C, after which the beads were washed four times with wash buffer (20 mM Hepes (pH 7.5), 100 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Beads were stored at 4 °C in wash buffer containing 0.02% (w/v) sodium azide until required. Cell lysate (500 µg) was incubated with immobilized GST-RBD for 45 min on a rotating wheel at 4 °C. Beads were isolated by centrifugation at 11,600 x g, washed three times in ice-cold wash buffer (10 mM MgCl₂, 0.1% (v/v) Triton X-100 in PBS), and resuspended in SDS-Laemmli sample buffer. The levels of GTP-Ras were determined by immunoblotting using anti-Ras antibodies.

Transfection of p38^MAPK siRNAs
The presence of p38^MAPK and p38^MAPK in TC-32 cells was confirmed by RT-PCR (results not shown). siRNAs to p38^MAPK (p38 MAPK SMARTpool® siRNA reagent) and a nonspecific control SMARTpool® siRNA were obtained from Upstate Biotechnology, Inc. Two different p38^MAPK siRNAs, designated p381 and p382, were designed and purchased from Qiagen: p381 siRNA sequence 5'-AACTGGATGCATTACAACCAA-3' and p382 siRNA sequence 5'-AAGGAGCTCACTTACCAGGAA-3'. siRNA (100 nM) was delivered to 5 x 10⁶ TC-32 cells by electroporation. Cells were trypsinized, resuspended in 400 µl of growth media, mixed with either one of the p38^MAPK-specific siRNAs, a nonspecific scrambled siRNA control or a buffer negative control, and electroporated. Electroporation was carried out in 4-mm GenePulser cuvettes (Bio-Rad) at 500 millifarads and 400 V using the GenePulser system (Bio-Rad). The cells were seeded into fresh growth media and allowed to adhere for 24 h. The cells were then either harvested and protein extracted for Western blotting or treated with bFGF for 48 h and then analyzed by flow cytometry of PI and annexin V-positive cells. These transfectants were then either incubated for 48 h prior to annexin V and PI labeling or treated with bFGF for 48 h prior to labeling.

Viable Cell Counts
Cells (1 x 10⁵) were seeded in Primaria 6-well plates (Falcon) and incubated in normal media with serum for 24 h. For viable cell counts in the presence of serum, the media were removed, and the cell monolayer was washed once with normal culture media and 2.5 ml of culture media containing FCS alone, supplemented with bFGF, or SCF was added. Cells were incubated at 37 °C in a 95% air, 5% CO₂-humidified atmosphere for 24–72 h. After incubation the cells were harvested using EDTA (0.05%) and trypsin (0.1%); viable and nonviable cell number was counted using the trypan blue exclusion assay and a Neubauer hemocytometer. Results are shown as mean ± S.E. (n = 4). For viable cell counts in the absence of serum, cells were seeded and allowed to adhere to plates overnight in the presence of serum; media were then aspirated; cells washed in serum-free media, and experiments were carried out as above but in media with no serum.

Detection of Cell Death
Cell death was quantified by using the annexin V-fluorescein isothiocyanate apoptosis detection kit I (Pharmingen) according to the manufacturer's instructions. Briefly, cells were treated as indicated and harvested by trypsinization and centrifugation (500 x g for 5 min), and the cell pellet was washed in ice-cold PBS. Cells were resuspended in binding buffer and then labeled with annexin V (1 µg/ml) and propidium iodide (PI; 1 µg/ml) for 30 min in the dark. Cells were analyzed by flow cytometry (BD Biosciences FACSCaliber^TM) coupled with the Cellquest software (BD Biosciences). Dead cells were scored as necrotic (PI-positive/annexin V-negative) or apoptotic (annexin V-positive/PI-negative and annexin V/PI-positive). A minimum of three independent experiments was carried out. Cell death was visualized by electron microscopy. Cell pellets were fixed in glutaraldehyde and osmium tetraoxide, infiltrated with araldite, embedded in fresh resin, and polymerized. Sections (80 µm) were cut and mounted on gold grids before staining first with uranyl acetate in methanol followed by Reynold's lead citrate.

Phosphorylated p38^MAPK and Total p38^MAPK ELISA
The p38^MAPK(total) and p38^MAPK (Thr(P)-180/Tyr(P)-182) ELISAs (BIOSOURCE) were performed as directed by the manufacturer's instructions. Briefly, cells were treated as indicated and lysed in the recommended lysis buffer (10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma)). A 1:25 dilution of the lysate was applied to the p38^MAPK antibody-coated 96-well plate along with standards and controls. Phosphorylated or total p38^MAPK was detected by using the supplied detection antibody, horseradish peroxidase-conjugated secondary, and stabilized chromagen. Absorbance was measured at 450 nm using a Titertek Multiscan® MCC plate reader. The amount of phosphorylated and total p38^MAPK was determined by reading the measured absorbance from a standard curve. p38^MAPK(total) was measured by ELISA to control for the amount of protein assayed between samples, and the amount of phosphorylated p38^MAPK (Thr(P)-180/Tyr(P)-182) was adjusted accordingly.

p38^MAPK Kinase Assay
By using the nonradioactive p38^MAPK assay (Cell Signaling Technology), the level of active p38^MAPK was analyzed according to the manufacturer's instructions. Briefly, treated cells were lysed in the lysis buffer provided (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerol phosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin) and supplemented with 1 mM phenylmethylsulfonyl fluoride. p38^MAPK was immunoprecipitated from 200 µl of whole cell lysate using an immobilized phospho-p38^MAPK (Thr-180/Tyr-182) monoclonal antibody overnight at 4 °C with constant agitation. The immunoprecipitates were washed twice with lysis buffer and twice with the kinase buffer (25 mM Tris (pH 7.5), 5 mM -glycerol phosphate, 2 mM dithiothreitol, 0.1 mM Na₃VO₄, 10 mM MgCl₂) provided with the kit. The immunoprecipitated p38^MAPK was incubated for 30 min at 37 °C in kinase buffer containing 200 µM ATP and 2 µg of ATF-2 fusion protein. The reaction was terminated by adding 2x Laemmli buffer and heating the samples to 95 °C for 5 min. The samples were separated on a 12% SDS-PAGE and immunoblotted for phosphorylated ATF-2.

Statistical Analyses
Analyses were undertaken by using SPSS. Data were analyzed by one-way analysis of variance when comparing three or more conditions. When variation among the means was considered significant (p < 0.05), the treated samples were compared with controls (untreated) using a Dunnett's post-hoc multiple comparison test where appropriate. Differences were considered significant when p < 0.05. To investigate the effect of p38^MAPK and ERK inhibitors on bFGF-induced cell death, data were adjusted as relative to the percentage of cell death induced by bFGF. The three-factor interaction effects (estimated effects of SB202190, PD98059, and bFGF) were not statistically significant (p = 0.75); the estimated effects of the combination of factors was calculated from the relative death log score mean.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
bFGF Induces Cell Death in ESFT Cells through Apoptosis and Necrosis—Treatment of TC-32 and TTC-466 cells with bFGF (20 ng/ml) resulted in a significant reduction in viable cell number at 48 and 72 h (Fig. 1a, i and ii). This reduction in viable cell number reflected an induction of cell death demonstrated by electron microscopy (Fig. 1b) and an increase in annexin V- and PI-positive cells detected by flow cytometry (Fig. 1c). TC-32 and TTC-466 cells treated with bFGF exhibited classical features of apoptosis, i.e. chromatin margination, nuclear condensation, and the formation of apoptotic bodies, and characteristics of necrosis such as an increase in cytoplasmic volume, vacuolation, and disruption of the plasma membrane (Fig. 1b). These observations are consistent with our previous data (15, 16).

View larger version (55K): [in this window] [in a new window]   FIG. 1. bFGF induces apoptosis in the TC-32 and TTC-466 cell lines but not the SK-N-SH cell line. a, cells were treated with either bFGF or SCF (20 ng/ml), and at defined time points the cells were harvested, and a viable cell count was taken using the trypan blue exclusion assay. Results from three independent experiments are shown as mean ± S.E. (error bars). b, TC-32, TTC-466, and SK-N-SH cells were left untreated or treated with bFGF (20 ng/ml) (TC-32 and TTC-46 cells for 72 h and SK-N-SH cells for 48 h) and then visualized by using electron microscopy. c, TC-32 and TTC-466 cells either untreated or treated with 20 ng/ml bFGF for 48 h were stained with annexin V and PI and analyzed using flow cytometry. The number in each quadrant refers to the proportion of positive cells present and is expressed as a percentage of total cells. Data shown are representative of four independent experiments. Viable cells are in the lower left quadrant. Early phase apoptotic cells are found in the lower right quadrants, later phase apoptotic cells are found in the upper right quadrant, and dead cells are in the upper left quadrant. *, p < 0.05; **, p < 0.001.

bFGF Induces Sustained Activation of the Ras-ERK Pathway in ESFT Cells—Treatment of TC-32 and TTC-466 cells with bFGF (20 ng/ml) resulted in a rapid increase in Ras-GTP, detected after 2 min of exposure (Fig. 2a, i and ii). Levels of active Ras remained elevated above basal levels 2 h post-treatment (Fig. 2a, i and ii). Total levels of Ras protein were constant, demonstrating equal loading of protein for the GST-RBD assay (results not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Ras and ERK are activated in response to bFGF and SCF treatment; however, although the activation is sustained in TC-32 and TTC-466 cells, it is transient in SK-N-SH-treated cells and TC-32 cells exposed to SCF. a, activation of Ras protein was analyzed by using bFGF-treated TC-32 (i) and TTC-466 (ii) cell lysates and the GST-RBD affinity pull-down assay. b, total cell lysates were obtained from six different ESFT cell lines and one neuroblastoma cell line (SK-N-SH). Western blot analysis was performed using anti-Pan ERK and anti--tubulin (-Tub) antibodies, and the figures are representative of three independent experiments. c, TC-32 cells were left untreated or treated with bFGF (20 ng/ml) for the defined times. After incubation total cell lysates were obtained, and Western blot analysis was performed using (i) a twice phosphorylated ERK antibody and (ii) an anti-ERK 2 antibody; this experiment was repeated with TTC-466 cells (d). The phosphorylation status of ERK 2 (i) and Ras (ii) was analyzed in bFGF (20 ng/ml)-treated SK-N-SH cells (e) and SCF (20 ng/ml)-treated TC-32 cells (f).

As there was no relationship between basal levels of ERK expression and bFGF-induced apoptosis in the ESFT cells, we wanted to determine whether the amount and pattern of phosphorylation of ERK after exposure to bFGF were different in cells in which bFGF induced cell death compared with those that did not die. Dual phosphorylated ERK 1 and -2 were detected in both the TTC-466 and TC-32 cell lines within 2 min of exposure to bFGF (Fig. 2, c, i, and d, i). Comparable levels of total unphosphorylated ERK demonstrated equal protein loading (results not shown). ERK 2 phosphorylation was also analyzed by gel shift; phosphorylated ERK 2 was again detected 2 min after exposure to bFGF (20 ng/ml) (Fig. 2, c, ii, and d, ii). The peak of ERK 2 phosphorylation in the TC-32 and TTC-466 cell lines was after 5 min of treatment (Fig. 2 c, ii and d, ii). Levels of phosphorylated ERK 2 were sustained 2 h post-treatment with bFGF.

The sustained activation of Ras and ERK in ESFT cells that die after treatment with bFGF is in contrast to the effect of bFGF on these intracellular signaling molecules in the neuroblastoma SK-N-SH cell line where bFGF induces cell proliferation and an increase in viable cell number (Fig. 1a, iii). In these cells, bFGF-induced a transient activation (2–10 min) of both Ras and ERK 2 proteins (Fig. 2e, i and ii).

SCF Induces Transient Activation of the Ras-ERK Pathway in ESFT Cells—To determine whether the sustained activation of the Ras-ERK pathway in response to exogenous growth factor stimulation is a common response of ESFT cells, we analyzed the effect of SCF on ESFT cells. First, we sought to confirm previous findings that SCF increases viable cell number in ESFT cells; treatment of TC-32 cells with SCF (20 ng/ml) for 24, 48, and 72 h resulted in a significant increase in viable cell number in defined media containing no serum (Fig. 1a, iv).

Treatment of TC-32 cells with SCF (20 ng/ml) also induced rapid activation of Ras, which was detected at peak levels after 2 min exposure to SCF (Fig. 2f, ii). In contrast to the effects of bFGF, activation of Ras was transient, with levels of GTP-Ras returning to basal levels after 10 min. ERK 2 was also phosphorylated in response to treatment with SCF (20 ng/ml). Phosphorylated ERK 2 was detected after 2 min, reaching a peak at 5 min and returning to basal levels after 10 min (Fig. 2f, i).

Inhibition of ERK Phosphorylation Rescues Cells from bFGF-induced Cell Death—To determine whether sustained activation of the Ras-ERK pathway by bFGF is necessary for the induction of cell death, a MEK 1 inhibitor (PD98059) was used to prevent activation of ERK 1 and ERK 2. The ability of PD98059 to rescue cells from bFGF-induced death was evaluated in TC-32 and TTC-466 cells. In these cells incubation with 10 µM PD98059 for 1 h prior to treatment with bFGF (20 ng/ml) resulted in a marked reduction in the level of twice phosphorylated ERK 1 and ERK 2 (Fig. 3a, i and ii). Pretreatment with 50 µM PD98059 completely abolished bFGF-induced ERK activation; however, at this concentration the PD98059 alone inhibited TC-32 and TTC-466 cell growth (data not shown). A concentration of 10 µM PD98059 was employed for subsequent experiments. Total ERK 2 was used as a control for protein loading.

View larger version (41K): [in this window] [in a new window]   FIG. 3. The MEK1 inhibitor PD98059 rescues a proportion of TC-32 and TTC-466 cells from bFGF-induced cell death. a, the MEK1 inhibitor PD98059 (10–50 µM) was added to cells (i, TC-32; ii, TTC466) for 1 h prior to the addition of bFGF (20 ng/ml). After 48 h of incubation, total lysates were prepared, and Western blot was used to analyze ERK status using the twice phosphorylated ERK antibody. To control for protein loading, total ERK 2 was analyzed using Western blot. b, TC-32 cells were treated with PD98059 (10 µM) or bFGF alone (20 ng/ml), pretreated with PD98059 for 1 h prior to bFGF addition, or left untreated. The cells were incubated for 48 h and then harvested, stained with annexin V and PI, and sorted using flow cytometry. The number in each quadrant refers to the proportion of positive cells present and is expressed as a percentage of total cells. Data shown are representative of seven independent experiments.

View larger version (30K): [in this window] [in a new window]   FIG. 4. Sustained activation of p38MAPK following exposure of TC-32 cells to bFGF but not in bFGF-treated SK-N-SH cells or SCF-treated TC-32 cells. a, total protein was extracted from six different ESFT cell lines and one neuroblastoma cell line (SK-N-SH); Western blot was performed using anti-total p38MAPK and anti--tubulin (-Tub) antibodies. b–d, i, TC-32 or SK-N-SH cells were treated with either 20 ng/ml bFGF or SCF and incubated for the indicated time, and total protein lysate was then prepared. Western blot was performed using anti-phospho-p38MAPK and anti-total p38MAPK. ii, total protein lysates from cells treated with bFGF for the indicated times were used for analysis on phospho-p38MAPK and total p38MAPK ELISA kits (BIOSOURCE). The graph represents phospho-p38MAPK results normalized to total p38MAPK and then compared with the untreated sample (0). The graph is also a representation of the means of four individual experiments (error bars ± S.E.).

p38^MAPK Is Transiently Activated Following Addition of SCF to TC-32 Cells—SCF is a survival factor for ESFT cells (12, 13) and is substantiated by the increase in viable TC-32 cell number after exposure to SCF (20 ng/ml) in this study (Fig. 1a, iv). To ensure that sustained phosphorylation of p38^MAPK in ESFT cells is a specific response to bFGF and not the usual response of these cells following exposure to growth factors, TC-32 cells were treated with SCF, and the level of p38^MAPK phosphorylation was analyzed. SCF (20 ng/ml) induced phosphorylation of p38^MAPK within 5 min; however, the level of phosphorylated p38^MAPK had returned to basal levels within 30 min (Fig. 4d). This observation is consistent with the hypothesis that transient activation of p38^MAPK is associated with activation of cellular survival pathways.

p38^MAPK Is an Effector of bFGF-induced Apoptosis of TC-32 Cells—It has been shown that there is a sustained activation of p38^MAPK in TC-32 cells following exposure to bFGF. We have therefore investigated whether this is important for bFGF-induced cell death using the following: 1) pyridinyl imidazole inhibitors of p38^MAPK SB202190 and SB203580; 2) RNAi for p38^MAPK.

SB202190 and SB203580 function by competing for and binding to the ATP-binding site of p38^MAPK to inhibit activation of p38^MAPK and p38^MAPK (47). In most studies we have used SB202190 as this specifically inhibits p38^MAPK and - at the concentrations used, with no effect on JNK,² whereas SB203580 will also inhibit JNK. The inhibitors were added to the growth media to a final concentration of 20 µM for 1 h prior to addition of bFGF. The level of cell death (apoptosis and necrosis) was analyzed by labeling the cells with PI and annexin V and sorting by using flow cytometry. In Fig. 5a it was again shown that incubating TC-32 cells for 48 h with bFGF led to an increase in the number of cells undergoing apoptosis. Also, incubation with the inhibitors alone (SB202190 or SB203580) did not alter the apoptotic index of the cells when compared with the untreated cells (for SB202190, p = 0.87). However, following addition of the inhibitors 1 h prior to treatment with bFGF, the relative mean death was the same as the control untreated cultures (Fig. 5a and Table I). The vehicle for both inhibitors was Me₂SO; at the concentrations used this had no significant effect on viable cell number (p = 0.35). These results demonstrate that p38^MAPK is an important effector of bFGF-induced cell death.

View larger version (70K): [in this window] [in a new window]   FIG. 5. p38MAPK is involved in bFGF-induced death. a, TC-32 cells were either left untreated or treated with bFGF alone, inhibitor(s) alone, or preincubated with inhibitor(s) for 1 h prior to the addition of bFGF. The cells were then incubated for 48 h, harvested, stained with annexin V and PI, and sorted using flow cytometry. The number in each quadrant refers to the proportion of positive cells present and is expressed as a percentage of total cells. b, TC-32 cells treated with or without bFGF and SB202190 were stained using annexin V and PI and sorted using flow cytometry. # indicates that the cells were treated with SB202190 1 h prior to addition of bFGF, and * denotes cells that were treated with bFGF 5 min prior to the addition of SB202190. c, p38MAPK from TC-32 cell lysates was immunoprecipitated using an immobilized anti-p38MAPK antibody. The p38MAPK was then used to phosphorylate ATF 2, which was subsequently visualized by SDS-PAGE and immunoblotting for phosphorylated ATF 2. Protein loading was controlled by Western blot for -tubulin (Tub). i, activity of p38MAPK (measured by detection of phosphorylated ATF 2) 0–10 min. ii, activity of p38MAPK 48 and 72 h after initial addition of bFGF. DMSO, Me2SO.

Activity of the p38^MAPK inhibitor SB202190 was confirmed by using a p38^MAPK assay, measuring phosphorylation of ATF-2. When the cells were exposed to SB202190 alone, the inhibitor decreased phosphorylation of ATF-2 (Fig. 5c), although this decease in basal levels of p38^MAPK had no effect on viable cell number. Treatment of cells with bFGF increased the phosphorylation of ATF-2, consistent with the increase in phosphorylation of p38^MAPK demonstrated by Western blot (Fig. 4b). Preincubation of TC-32 cells with SB202190 for 1 h prior to the addition of bFGF successfully inhibited phosphorylation of ATF-2 5 and 10 min after exposure (Fig. 5c, i and ii). Cells were also treated with bFGF and harvested up to 72 h after initial exposure, to determine whether SB202190 inhibited activation of p38^MAPK at these extended times. However, because bFGF treatment of TC-32 cells for 48 and 72 h resulted in substantial cell death, the amount of protein extracted from treated cells was reduced. To control for differences in total protein extracted from different treatment groups, the relative densities of the phosphorylated ATF 2 and -tubulin bands were analyzed by using the Odyssey infrared imaging software (Li-Cor), and the amount of phosphorylated ATF 2 was adjusted relative to the -tubulin level to determine the percentage of p38^MAPK activity inhibited by SB202190. The increase in p38^MAPK activity (measured by ATF-2 phosphorylation) was still evident 48 h after initial exposure to bFGF; at this time the SB202190 inhibitor decreased p38^MAPK activity by 85%. This increased activity of p38^MAPK after 48 h of exposure to bFGF is consistent with the sustained activation of p38^MAPK demonstrated on Western blots (results not shown). However, at 72 h bFGF had no apparent effect on p38^MAPK, although at this time a high proportion of TC-32 cells was already dead. -Tubulin was analyzed to control for the amount of protein used in each sample.

To ensure the rescue of ESFT cells from bFGF-induced cell death following incubation with the p38^MAPK inhibitors was effected through inhibition of p38^MAPK and not due to nonspecific activity, RNAi for p38^MAPK and p38^MAPK isoforms was utilized. p38^MAPK siRNA resulted in a substantial but not complete knockdown of p38^MAPK in TC-32 cells (Fig. 6a, i). Two siRNAs were designed to inhibit p38^MAPK, and one of the siRNAs (p38^MAPK2) successfully down-regulated p38^MAPK expression (Fig. 6a, ii). To confirm the specificity of the siRNA knockdown, Western blots of p38^MAPK siRNA electroporated cells were probed for p38^MAPK and vice versa. siRNA for p38^MAPK had no effect on the expression of p38^MAPK2, and siRNA for p38^MAPK had no effect on p38^MAPK (Fig. 6a, i and ii). The effect of these knockouts on TC-32 cells was examined by flow cytometry of annexin V- and PI-labeled cells.

View larger version (30K): [in this window] [in a new window]   FIG. 6. p38MAPK siRNA reduces the amount of bFGF-induced apoptosis and necrosis. a, TC-32 cells were transfected with buffer (Buff), nonspecific scrambled siRNA (Scram), p38MAPK siRNA (p38), p38MAPK1(p381), or p38MAPK2(p382) siRNAs. Once transfected the cells were incubated for 48 h, and total protein lysates were prepared and used for analysis by Western blot probing with anti-p38MAPK (p38), anti-p38MAPK (p38), or -tubulin (-Tub) antibodies. b, electroporation with p38MAPK decreased the amount of bFGF-induced cell death in TC-32 cells 48 h after treatment with bFGF (20 ng/ml) compared with that in cells electroporated with scrambled siRNA or incubated in RNAi buffer only. Cells were electroporated with the different siRNAs, incubated for 24 h, and then treated with or without bFGF. After 48 h of incubation the cells were harvested, labeled with annexin V and PI, and analyzed using flow cytometry.

One-way Negative Regulation of ERK 1 and -2 Activation by p38^MAPK—A number of studies have identified cross-talk between the p38^MAPK and ERK pathways (48–50). To establish whether activation of p38^MAPK following exposure to bFGF had any effect on the activity of ERK 1 and -2, and vice versa, we have used the specific inhibitors SB202190 and PD98059, Western blotting, and phospho-specific ELISA. Although incubation of the TC-32 cells with bFGF for 5, 10, and 30 min increased the level of p38^MAPK phosphorylation, this was unaffected by pretreatment with the MEK inhibitor PD98059 (Fig. 7a, i and ii). In complimentary experiments, the TC-32 cells were preincubated with SB202190, and ERK phosphorylation was analyzed. SB202190 alone did not alter the level of phosphorylated ERK 1 or -2 in the TC-32 cells. Exposure of the TC-32 cells to bFGF for 5–120 min caused an increase in the level of ERK 1 and -2 phosphorylation compared with untreated control cells. Most interestingly, there appears to be an increase in phosphorylated ERK 1 and -2 when cells were preincubated with SB202190 for 1 h prior to addition of bFGF (Fig. 7b). These studies suggest that p38^MAPK might negatively regulate growth factor-induced activation of ERK 1 and -2 but that ERK 1 and -2 do not regulate p38^MAPK under these conditions. These relationships require further investigation.

View larger version (29K): [in this window] [in a new window]   FIG. 7. There is no cross-talk between ERK and p38MAPK. a, TC-32 cells were left untreated or treated with PD98059 alone (10 µM), bFGF alone (20 ng/ml), or pretreated with PD98059 (10 µM) for 1 h prior to addition of bFGF (20 ng/ml). After the cells were incubated for the indicated time, total protein lysates were prepared and used for analysis by Western blot probing with anti-phospho-p38MAPK or anti--tubulin (-Tub) (i), or for phospho-p38MAPK and total p38MAPK ELISA assays (BIOSOURCE) (ii). The graph represents phospho-p38MAPK results normalized to total and then compared with the untreated sample (0). The graph is also a representation of the means of four individual experiments (error bars ± S.E.). b, TC-32 cells were left untreated or treated with SB202190 alone (20 µM), bFGF alone (20 ng/ml), or pretreated with SB202190 (20 µM) for 1 h prior to addition of bFGF (20 ng/ml). After the cells were incubated for the indicated time, total protein lysates were prepared and used for Western blot. Blots were probed using anti-double phosphorylated ERK antibody and anti--tubulin.

View larger version (31K): [in this window] [in a new window]   FIG. 8. Treatment of TC-32 cells with bFGF and SCF only manages to elicit a transient phosphorylation of JNK. a, total protein was extracted from six different ESFT cell lines; Western blot was performed using anti-total JNK and anti--tubulin (-Tub) antibodies. b, i, TC-32 cells were treated with bFGF (20 ng/ml) for the indicated times or with 0.5 M sorbitol for 5 min as a positive control. Total protein lysates were prepared and used for Western blot. Blots were probed with anti-phospho-JNK and anti--tubulin antibodies as well as the appropriate Alexa Fluor 680-labeled secondary antibodies (Molecular Probes), and they were then analyzed using the Odyssey infrared imaging system (Li-Cor). ii, using the Odyssey infrared imaging system (Li-Cor), the relative density of the JNK protein band was determined and normalized to the respective density of the -tubulin band. +Sorb, sorbitol-positive control.

Sustained Activation of p38^MAPK Induces Up-regulation of p75^NTR—p75^NTR is a glycoprotein, which belongs to the superfamily of tumor necrosis factor receptors (51). Our previous studies have demonstrated up-regulation of p75^NTR in ESFT cells that die following exposure to bFGF (16), and we have therefore examined the hypothesis that sustained activation of p38^MAPK and/or ERK leads to induction of this death receptor. Consistent with our previous observations, the addition of bFGF to TC-32 cells increases expression of p75^NTR by 2-fold (Fig. 9, a and b). Incubation of TC-32 cells with the ERK inhibitor PD98059 had no effect on expression of p75^NTR; however, incubation with the p38^MAPK inhibitor SB202190 prevented up-regulation of p75^NTR consistent with the hypothesis that p38^MAPK is an important effector of bFGF-induced cell death and that this is mediated at least in part through up-regulation of the death receptor p75^NTR. Most interestingly, incubation of TC-32 cells with SB202190 alone decreased basal levels of p75^NTR expression; we are currently investigating the role of p38^MAPK in the transcriptional regulation of death receptor expression.

View larger version (31K): [in this window] [in a new window]   FIG. 9. bFGF-induced up-regulation of p75NTR is effected through sustained activation of p38MAPK. a, total protein lysate was obtained from TC-32 cells treated with SB202190 (20 µM), PD98059 (10 µM), and/or bFGF (20 ng/ml). Western blot was performed using anti-p75NTR and anti--tubulin (-Tub) antibodies. b, using the Odyssey infrared imaging system (Li-Cor), the relative densities of the p75NTR protein band was determined and normalized to the respective density of the -tubulin band.

View larger version (49K): [in this window] [in a new window]   FIG. 10. A673 cells do not undergo apoptosis in response to bFGF; this is associated with sustained activation of ERK but transient phosphorylation of p38 MAPK, Ras, and JNK. a, A673 cells were treated with bFGF (20 ng/ml); at defined time points the cells were harvested, and a viable cell count was taken using the trypan blue exclusion assay. Results from three independent experiments are shown as means (error bars, ± S.E.). b, A673 cells either untreated or treated with 20 ng/ml bFGF for 48 h were stained with annexin V and PI and analyzed using flow cytometry. The number in each quadrant refers to the proportion of positive cells present and is expressed as a percentage of total cells. Data shown are representative of four independent experiments. c, A673 cells were left untreated or treated with bFGF (20 ng/ml) for 72 h and then visualized using electron microscopy. d, A673 cells were treated for the indicated time with bFGF (20 ng/ml); total protein lysates were then prepared. Western blot was performed using anti-ERK2 (i) and anti-total Ras and anti-Pan ERK antibodies (iii). Activation of Ras protein was analyzed using the GST-RBD affinity pull down assay (ii). e, with the use of Western blot (i), phospho-p38MAPK, and total-p38MAPK ELISA kits (ii) the phosphorylation status of p38MAPK in A673 cells treated with bFGF (20 ng/ml) was analyzed. f, i, Western blot analysis on total protein lysates of bFGF-treated (20 ng/ml) A673 cells using anti-phospho-JNK and anti--tubulin antibodies. ii, using the Odyssey infrared imaging system (Li-Cor) the relative densities of the JNK protein band were determined and normalized to the respective density of the -tubulin band.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mechanism by which p38^MAPK contributes to the apoptotic response following exposure to bFGF appears to be through an up-regulation of the death receptor p75^NTR, demonstrated by loss of p75^NTR expression and decrease in cell death following inhibition of p38^MAPK. This is the first report demonstrating that sustained activation of p38^MAPK effects an up-regulation of p75^NTR death receptor expression and suggests that p75^NTR may be a transcriptional target for p38^MAPK. Recent studies in p38^MAPK knockout mice have shown that p38^MAPK can sensitize cells to apoptosis through the positive regulation of FAS/CD95 (61). Together these data suggest that multiple death receptors may be transcriptional targets for p38^MAPK. We are currently investigating this possibility and the hypothesis that generation of high cell surface death receptor density might amplify the death response. Most interestingly, bFGF rapidly up-regulates expression of tumor necrosis factor receptor 1 in TC-32 cells. This expression is sustained at 24 and 48 h and is accompanied by an increase in the release of tumor necrosis factor-,³ which may enhance the level of bFGF-induced cell death. A pro-apoptotic role of p38^MAPK, following stimulation with tumor necrosis factor- (54) or in response to oxidative stress (62), has been linked previously to phosphorylation and/or translocation of members of the BCl-2 family leading to release of cytochrome c from the mitochondria (62–64). This is consistent with the hypothesis that up-regulation of p75^NTR is an important effector of p38^MAPK-induced death because p75^NTR stimulation has been associated with an increase in the mitochondrial pro-apoptotic effector proteins Bad, Bax, and Bik, a decrease in the mitochondrial pro-survival effector proteins phospho-Bad, Bcl-2, and Bcl-x_L, and induction of cytochrome c release from mitochondria during apoptosis (65–67). Our own studies have shown down-regulation of the survival protein BCl-2 and a loss of mitochondrial transmembrane potential following bFGF-induced expression of p75^NTR and cell death (16).

Because p38^MAPK plays such a critical role in bFGF-induced death, and sustained activation of the related stress-activated kinase JNK has been associated with the induction of apoptosis (44, 68, 69), we hypothesized that JNK might also be an effector of bFGF-induced cell death. However, JNK 1 and -2 were transiently activated in both ESFT cells that died and in those that did not die after treatment with bFGF. Although we cannot currently rule out some role for JNK activation in the initiation of bFGF-induced cell death (this may be investigated using JNK dominant negatives), it does not appear to be critical. This is supported by studies that have demonstrated apoptotic signaling in some cell types is mediated through a JNK-independent p38^MAPK stress-activated signaling pathway (70, 71), and other studies that have shown sustained but not transient activation of JNK are associated with induction of apoptosis (44, 45, 68, 69). These data are in contrast to results in PC-12 cells in which activation of both JNK and p38^MAPK is critical for induction of apoptosis (72).

Sustained activation of p38^MAPK following exposure of ESFT cells to bFGF was accompanied by prolonged activation of Ras-ERK. Activation of the Ras-ERK cascade has been implicated in the transmission of both cell death and survival signals (73–76), the cellular outcome most likely dependent on the geno- and phenotype of the cell studied and the time course of signaling protein activation (77). In this study we have shown sustained activation of Ras-ERK after exposure to bFGF, whereas the survival effect of SCF was associated with transient activation. These observations are consistent with the hypothesis that differential signaling kinetics provide a mechanism by which receptors can utilize a common signaling pathway to exert distinct biological responses. This hypothesis is based for the most part on evidence accumulated in PC12 cells, treatment of PC12 cells with nerve growth factor leading to the sustained activation of the Ras-ERK pathway, and induction of differentiation, in direct contrast to the induction of proliferation following exposure to epidermal growth factor and transient ac