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MAPK signaling in human neuroblastoma cells
Experimental Cell Research 310 (2005) 218 – 228 www.elsevier.com/locate/yexcr
Research Article
Regulation of the Notch target gene Hes-1 by TGFa induced Ras/MAPK signaling in human neuroblastoma cells Marie-The´re´se Stockhausen, Jonas Sjo¨lund, Ha˚kan Axelson* Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, University Hospital MAS, Entrance 78, S-205 02 Malmo¨, Sweden Received 23 February 2005, revised version received 8 June 2005, accepted 24 July 2005 Available online 24 August 2005
Abstract Ras and Notch signaling have recently been shown to cooperate in the maintenance of neoplastic transformation. Here, we show that TGFa, a known activator of Ras signaling, can drive cell proliferation and at the same time induce the expression of the Notch target Hes-1 in the neuroblastoma cell line SK-N-BE(2)c. The up-regulation of Hes-1 occurred both at the transcriptional and protein levels and by use of EGFR and MEK inhibitors we could show that the Hes-1 response was dependent on activation of the MAP kinase ERK. Blocking Notch activation by g-secretase inhibition did not profoundly affect the Hes-1 levels, neither in untreated nor in TGFa treated cells. The upregulation of Hes-1 was associated with down-regulation of its pro-neuronal target gene Hash-1. Taken together, these results show that TGFa is a potent mitogen of neuroblastoma cells and suggest a connection between activation of ERK and Hes-1, thus providing a link between the Ras and Notch signaling pathways. D 2005 Elsevier Inc. All rights reserved. Keywords: Neuroblastoma; Notch; Hes-1; Hash-1; Ras/MAPK signaling; ERK; TGFa
Introduction Neuroblastoma is a childhood malignancy that originates from immature cells of the sympathetic nervous system [1]. Several lines of evidence suggest that the cells are halted at an early, embryonic state of development with maintained proliferative capacity. For example, the cells have a phenotype resembling sympathetic neuroblasts and they express several differentiation-related genes normally expressed during embryogenesis, such as the basic helix-loop-helix (bHLH) transcription factors human achaete-scute homo-
Abbreviations: bHLH, basic helix-loop-helix; Hash-1, human achaetescute homologue-1; Mash-1, mammalian achaete-scute homologue-1; TPA, phorbol ester; RA, retinoic acid; i.c. Notch, intracellular Notch; f.l. Notch, full-length Notch; T-ALL, T-cell acute lymphoblastic leukemia; MAPK, mitogen-activated protein kinase; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; TGFa, transforming growth factor a. * Corresponding author. Fax: +46 40337322. E-mail address: [email protected] (H. Axelson). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.07.011
logue-1 (Hash-1) and dHAND [2]. In addition, amplification of the N-myc gene, which serves as a key prognostic marker associated with an advert outcome of the disease [3 – 5], is normally involved in controlling the differentiation process of sympathetic neurons [6]. It has for a long time been known that neuroblastoma cells can be induced to differentiate in vitro, using phorbol ester (TPA), retinoic acid (RA), or a combination of growth factors [7,8]. The differentiated phenotype is characterized by induction of a number of neuronal differentiation marker genes and decreased proliferation along with induction of neurites. Thus, in order to develop novel differentiation-based treatment modalities, it is of vital importance to understand the mechanisms that control the process of neuroblastoma cell differentiation. Notch signaling is based on cell – cell contact since the Notch ligands (Delta like-1, -3, -4, and Jagged-1, -2) are primarily membrane bound [9]. They bind and activate Notch receptors (Notch-1 to -4) on neighboring cells, which ultimately leads to g-secretase-mediated cleavage of the intracellular part of the receptor (i.c. Notch) that translocates into the nucleus and associates with the transcription factor
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CSL (CBF-1, Suppressor of Hairless, LAG-1; also known as RBP-Jn), thereby converting it from a transcriptional repressor to an activator. In several tissues, genes belonging to the Hes and HRT/HERP/Hey families of transcriptional repressors are important targets of Notch signaling and these transcriptional repressors mediate many of the known effects of activated Notch [10]. In neuronal cells, Hes proteins negatively regulate transcriptional activators of the pro-neuronal bHLH family. During development of the sympathetic nervous system, mammalian achaete – scute homologue-1 (Mash-1) is one important bHLH target protein and gene ablation experiments in mice show that the gene is essential for proper formation of this part of the nervous system [11]. Based on these observations and in conjunction with the fact that Hes-1 and Hash-1 are regulated by Notch signaling, we have studied the role of this cascade in neuroblastoma cells. We noticed that during RA- or TPA-induced differentiation of neuroblastoma cells, there was an induction of Notch signaling activity, as indicated by transient elevation of the Hes-1 transcriptional repressor, with a concomitant decrease in Hash-1 expression levels [12]. Constitutive Notch-1 signaling also blocked RA- and TPA-induced differentiation of neuroblastoma cells [12], supporting the notion that Notch signaling might be involved in neuroblastoma cell differentiation and that active Notch signaling might maintain neuroblastoma cells in an undifferentiated state [13]. The most clear-cut example of how Notch signaling is linked to oncogenesis comes from the findings that the receptor can be activated by chromosomal translocation or point mutations in T-cell acute lymphoblastic leukemia (TALL) [14,15]. This leads to constitutive expression of the intracellular domain of the receptor and several experiments show that i.c. Notch-1 functions as an oncogene in this cell type [16,17]. Furthermore, in a mouse mammary tumor model system, retroviral insertion in the Notch-4 gene leads to constitutively active Notch signaling and tumorigenesis [18]. The cellular effects of Notch signaling are however highly cell-type dependent as illustrated by the situation in keratinocytes in which active Notch signaling is associated with induced differentiation and decreased proliferation [19]. Accordingly, Notch has been proven to be a tumor suppressor in this cell type [20]. Numerous studies have shown that Hes-1 is a primary Notch target gene, and as a consequence, Hes-1 expression has been considered a good surrogate marker for Notch signaling activity. However, it should be noted that in many cases, regulation of Hes-1 by Notch signaling has been studied in experiments in which the intracellular part of the receptor is overexpressed, resulting in supra-physiological levels of the protein. With regard to physiological regulation, it is important to note that gene targeting of Notch-1 in mice led to decreased levels of Hes-5 and HERP1/2 while the expression of Hes-1 remained unaffected [21 – 23]. Also, RBP-Jj gene ablation in mice led to reduced expression of Hes-5 but not Hes-1 [21].
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Thus, Hes-1 expression seems to be regulated both in a Notch-dependent and -independent manner. Regarding the Notch-independent regulation, it has been shown that Hes-1 can regulate its own expression through N boxes (CACNAG) in the Hes-1 promoter [24]. In addition, Hes-1 expression can also be induced upon serum stimulation in a variety of cell lines. In synchronized cells, this induction results in an oscillating expression pattern of Hes-1 mRNA and protein mediated through the autorepressive capacity of Hes-1 [25]. Other studies have shown that growth factor stimulation leads to an induction of Hes-1 [26], but it is currently not clear whether this occurs in a Notch-dependent or -independent fashion. In this report, we have analyzed the link between mitogen-activated protein kinase (MAPK) signaling and Hes-1 regulation by stimulating neuroblastoma cells with the transforming growth factor a (TGFa), which in previous studies have shown to be a mitogen of neuroblastoma cells [27]. We show that Hes-1 expression is directly correlated to the MAPK activity and that this induction is not mediated by increased signaling activity of the Notch receptors. In TGFa-stimulated cells, Hes-1 expression was inhibited both by MAPK and epidermal growth factor receptor (EGFR) inhibitors. In contrast, only the MAPK inhibitor could affect the expression of Hes-1 in non-stimulated cells. In addition, up-regulation of Hes-1 led to a rapid down-regulation of Hash-1 indicating a role in maintaining an undifferentiated and thus proliferative phenotype of neuroblastoma cells. Taken together, these findings suggest that Hes-1, an important mediator of Notch signaling, can be regulated both by Notch-independent, mitogen-driven and Notchdependent, mitogen-independent cues in neuronal cells.
Materials and methods Cell culture and reagents The human neuroblastoma cell line SK-N-BE(2)c was maintained in Eagle’s Minimum Essential Medium (MEM, Invitrogen Inc., Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 Ag/ml) at 37-C in an atmosphere of 5% CO2. The neuroblastoma cell line SH-EP was maintained in the same media as above, but with the addition of 15% FCS. For experiments, cells were seeded 1 day prior to initiation of treatment with L-685,458, DAPT, AG1478, SB203580 (all purchased from Calbiochem, La Jolla, CA, USA), U0126 (Cell Signaling Technology, Beverly, MA, USA), or LY294002 (Upstate, Lake Placid, NY, USA) for indicated times. All control experiments include the corresponding volume of the vehicle DMSO. The volume of vehicle never exceeded 1% of total volume. TGFa was purchased from R&D Systems (Minneapolis, MN, USA) and dissolved in 10 mM acetic acid containing 0.1% BSA, aliquoted and stored at 20-C until used.
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Trypan blue exclusion assay Cell viability was analyzed using trypan blue exclusion of dead cells. In short, cell culture media was collected and adherent cells harvested by trypsination. The fractions were pooled, the volume was measured, and the cells were mixed with trypan blue solution (0.4%) (Sigma, St. Louis, MO, USA). Both dead and viable cells were then counted in a Bu¨ rker chamber. Each experiment was performed in triplicates and repeated at least three times. Western blot analysis For Western blot analyses, cells were harvested by centrifugation, snap-frozen in liquid nitrogen and stored at 70-C. Cell pellets were then lysed with NP40 lysis buffer (1% NP40, 10% glycerol, 20 mM Tris – HCl [pH 8.0], 137 mM NaCl) and 4% complete protease inhibitor cocktail mix (Roche, Mannheim, Germany) for total cell protein preparation. Proteins (50 Ag) were separated by SDS-PAGE followed by transfer to PVDF Immobilon-P filters (Millipore Corp., Billerica, MA, USA). The filters were probed with polyclonal anti-Hes-1 antiserum diluted 1:8000 (kindly provided by Dr. Tetsuo Sudo, Japan), monoclonal antiMash-1 antibody diluted 1:125 (Pharmingen, San Diego, CA, USA), polyclonal anti-phopho-ERK1/2 antiserum 1:500 (Cell Signaling), polyclonal anti-ERK antiserum 1:500 (Cell Signaling), or monoclonal anti-GAPDH antibody diluted 1:500 (Labora Chemicon International, Temecula, CA, USA). The blots were then incubated with secondary antibodies: donkey anti-rabbit 1:1000 (Amersham Life Science, Solna, Sweden), goat anti-mouse 1:7000 (Jackson, West Grove, PA, USA), or sheep anti-mouse 1:5000 (Amersham) all coupled to horseradish peroxidase. The immunoreactivity was detected using the enhanced chemiluminescence method (Pierce, Rockford, IL, USA). Real-time quantitative RT-PCR Total RNA was purified with RNeasy kit (Qiagen, Hilden, Germany) as described by the manufacturer. cDNA was synthesized as described earlier [28] and used as template in quantitative real-time PCR reactions with SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA). Relative quantification of expression levels was performed by using the comparative Ct method and normalized to the expression of three housekeeping genes (SDHA, YWHAZ, and UBC). The genes analyzed were: Hes-1 forward, 5V-AGC GGG CGC AGA TGA C-3V; Hes-1 reverse, 5V-CGT TCA TGC ACT CGC TGA A-3V; SDHA forward, 5V-TGG GAA CAA GAG GGC ATC TG-3V; SDHA reverse, 5V-CCA CCA CTG CAT CAA ATT CAT G3V; YWHAZ forward, 5V-ACT TTT GGT ACA TTG TGG CTT CAA-3V; YWHAZ reverse, 5V-CCG CCA GGA CAA ACC AGT AT-3V; UBC forward, 5V-ATT TGG GTC GCG GTT CTT G-3V; UBC reverse, 5V-TGC CTT GAC ATT CTC
GAT GGT-3V. Data shown are representative for one out of three experiments performed in triplicates. Luciferase reporter assay SK-N-BE(2)c cells were grown to 80 – 90% confluence in 24-well plates. Cells were then transiently transfected by using Lipofectamine 2000 reagent (Invitrogen) with 0.4 Ag Hes-1 promoter construct ( 467 to +46) (a kind gift from R. Kageyama, Kyoto University, Japan) [29], 0.4 Ag 8 CSL luciferase reporter construct (kindly provided by S.D. Hayward, Johns Hopkins School of Medicine, USA) [30], 0.8 Ag full-length Notch-1 (f.l. Notch-1) expression vector (kindly provided by J.C. Aster, Harvard Medical School, USA) [31], and 20 ng phRL-TK renilla expression vector (Promega, San Luis Obispo, CA, USA) as a control for transfection efficiency. Post-transfection, cells were treated with L-685,458, DAPT, U0126, AG1478, SB203580, or LY294002 with or without the addition of TGFa as indicated in the figure legends. DMSO and/or 10 mM acetic acid containing 0.1% BSA were added in control experiments in volumes corresponding to inhibitors or TGFa. Cells were lysed and assayed for luciferase and renilla activity on a TD-20/20 Luminometer (Turner Biosystems, Sunnyvale, CA, USA) according to the DualLuciferase Reporter Assay System manufacturer Promega’s instructions. Each condition was tested in replicates of two to four and repeated at least twice. Data are presented as representative experiments.
Results TGFa stimulation promotes growth of neuroblastoma cells and induces Hes-1 expression We have previously reported that RA- or TPA-induced differentiation of neuroblastoma cells led to a transient upregulation of Hes-1 followed by an almost complete downregulation [12]. During these conditions, the differentiated phenotype was associated with ceased proliferation, and we hypothesized that a maintained Hes-1 expression might be incompatible with terminal differentiation. In order to clarify the regulation of Hes-1 during mitogenic stimulation of neuroblastoma cells, we treated SK-N-BE(2)c cells with the EGFR ligand TGFa. In line with data published by other groups [27], we found a growth-promoting effect of TGFa, detected already at 24 h and maintained after 48 h in culture (Figs. 1a and b). Trypan blue exclusion experiments showed that this was not a consequence of differences in cell death between control and TGFa-stimulated cells (Fig. 1c). Having confirmed the mitogenic effect of TGFa on neuroblastoma cells, we next studied the expression of Hes-1 after 24 h of TGFa stimulation. We noticed a dose-dependent effect of TGFa on Hes-1 expression with a considerable induction already at 0.05 ng/ml (Fig. 2a). To verify that
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luciferase reporter assays using a Hes-1 reporter construct. The reporter assay recapitulated to a large extent the protein expression data, i.e., increased concentrations of TGFa led to increased activity of the Hes-1 promoter. Maximum activity was achieved at 10 ng/ml of TGFa, but a modest induction was detected already at the lowest concentration used (Fig. 2b). Similar results were obtained using a synthetic reporter containing eight repetitive CSL sites (8 CSL) (Fig. 2c). These data suggest that that the TGFa-induced Hes-1 expression was primarily mediated by CSL. In addition, real-time quantitative RT-PCR showed that Hes-1 mRNA levels increased in a dose-dependent manner upon TGFa stimulation (Fig. 2d). In a time-course experiment using 1 ng/ml of TGFa, elevated Hes-1 levels were detected already after 2 h and maintained until the end of the experiment at 24 h (Fig. 3). As expected, TGFa induced a rapid and persistent ERK1/2 phosphorylation, which was not due to up-regulation of total ERK. Interestingly, the fast induction of Hes-1 correlated with a rapid down-regulation of Hash-1, underlining the inverse expression pattern of Hash-1 and Hes-1 in neuroblastoma cells. This effect is less pronounced at later time points, but is still detectable at 24 h. These data show that the mitogenic effect of TGFa on human neuroblastoma cells is associated with increased phosphorylation of ERK and a concomitant up-regulation of Hes-1 expression together with a rapid down-regulation of Hash-1. Reporter gene assays and real-time quantitative RTPCR results indicate that the elevated Hes-1 levels are due to increased promoter activity and thus are a consequence of increased transcription.
Fig. 1. TGFa induced cell growth in the SK-N-BE(2)c neuroblastoma cell line. (a) Number of viable cells was determined after 0, 1, and 2 days of treatment with TGFa (1 ng/ml) using trypan blue exclusion and cell counting in a Bu¨rker chamber. (b) Total number of cells was counted after 24 h of TGFa treatment (1 ng/ml) and (c) dead cells determined using trypan blue staining. Each experiment was performed in triplicates and repeated at least three times. Results are shown as mean T SD of representative experiments.
TGFa stimulation activated the Ras/MAPK cascade, we also monitored the phosphorylation status of the MAPK extracellular signal-regulated kinase (ERK). Increased TGFa concentrations led to a dose-dependent induction of ERK1/2 phosphorylation. This increase in ERK1/2 phosphorylation was not due to increased amounts of total ERK levels, as shown by probing with a pan-ERK antiserum. Furthermore, Hash-1 levels were decreased in a dosedependent manner upon TGFa stimulation, suggesting a retained capacity of Hes-1 to repress Hash-1 expression under these circumstances (Fig. 2a). To clarify whether the induced Hes-1 expression was a consequence of increased transcriptional activity of the Hes1 promoter or due to protein stabilization, we performed
TGFa-induced Hes-1 expression is not mediated by Notch activation Studies in other cell systems indicate that MAPK activity can lead to increased signaling activity of the Notch receptors [32]. In order to clarify whether this was the case in neuroblastoma cells, we employed two compounds (DAPT and L-685,458) that inhibit g-secretase activity and thereby prevent the activating cleavage of the Notch receptors. By this method, we would be able to clarify whether the Hes-1 induction upon TGFa treatment was a consequence of increased Notch receptor activity or a Notch-independent effect of MAPK activity. Treatment with DAPT did not affect the Hes-1 levels neither in the absence nor presence of TGFa (Fig. 4a). Similar results were obtained when treating the cells with L-685,458 (Fig. 4b). Reporter assays using the same Hes-1 reporter construct as above showed that DAPT only could inhibit the activity of the promoter to a small extent whereas an even less pronounced effect was observed with L-685,458 (Fig. 4c). When inducing promoter activity with TGFa (1 ng/ml), treatment with DAPT resulted in a small decrease of activation though not down to basal levels (Fig. 4d). Treatment with L-685,458 had no effect (Fig. 4d). The
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Fig. 2. Effect of TGFa stimulation on Hes-1 expression in neuroblastoma cells. (a) Western blotting of phosphorylated ERK1/2, total ERK, Hes-1, and Hash-1. GAPDH was used to confirm equal loading. Results shown are representative of three experiments. (b, c) Luciferase reporter assay using the (b) Hes-1 promoter or (c) 8 CSL coupled to a luciferase reporter. Results are shown as mean T SD and are representative for two experiments. (d) Real-time quantitative RT-PCR using Hes-1-specific primers. Data shown are representative for three experiments with similar results. Cells were treated with indicated concentrations of TGFa for 24 h whereafter Western blotting, reporter assays, and real-time quantitative RT-PCR were performed as described in Materials and methods.
results obtained with the Hes-1 reporter construct were recapitulated with the 8 CSL construct, though DAPT treatment resulted in a partial down-regulation of both basal and TGFa-induced promoter activity (Figs. 4e and f). The effective dose of g-secretase inhibitors is highly celltype specific and since we could only detect minute effect of the inhibitors on SK-N-BE(2)c cells, we wanted to confirm that the concentrations used actually inhibited g-secretase activity in human neuroblastoma cells. We therefore treated the neuroblastoma cell line SH-EP with L-685,458 and DAPT for 24 h. SH-EP expresses high levels of both Notch receptors and Hes-1 and thus we speculated that inhibition of g-secretase would be detected as a decrease of Hes-1 expression. Indeed, both L-685,458 and DAPT clearly
decreased the Hes-1 levels as shown by Western blotting (Fig. 5a). To further verify g-secretase inhibition in the SKN-BE(2)c cell line used in this study, we transiently transfected the cells with an expression vector coding for full-length Notch-1 (f.l. Notch-1), which is dependent of gsecretase activity for its activation, together with the 8 CSL luciferase reporter construct. Treating these cells with either L-685,458 or DAPT clearly decreased reporter gene activity, showing that the g-secretase activity indeed was inhibited, thereby verifying that the inhibitors were functional at the concentrations used (Fig. 5b). We therefore concluded that the elevated Hes-1 levels detected after treatment with TGFa were not primarily a consequence of increased Notch signaling activity. EGFR and ERK inhibition prevents TGFa-induced Hes-1 expression
Fig. 3. Time-course experiment using TGFa-treated SK-N-BE(2)c cells. Western blotting showing phosphorylation of ERK1/2, total ERK, Hes-1, and Hash-1 levels. GAPDH was used as a control of equal loading. Cells were stimulated with TGFa (1 ng/ml) for indicated times whereafter Western blotting was performed as described in Materials and methods. Experiment is a representative of three showing similar results.
Since Notch activation did not seem to be responsible for the TGFa-induced Hes-1 expression, we next wanted to analyze the effect of inhibitors that target pathways downstream the EGFR. The inhibitors tested include the EGFR inhibitor AG1478, the MEK inhibitor U0126, the PI3-K inhibitor LY294002, and the p38 MAPK inhibitor SB203580. Both the EGFR inhibitor and the MEK inhibitor led to a considerable down-regulation of Hes-1 along with decreased ERK1/2 phosphorylation in the presence of TGFa (Fig. 6a). In contrast, no obvious effect on Hes-1 expression
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Fig. 4. Effect of g-secretase inhibition in unstimulated neuroblastoma cells or cells treated with TGFa. Cells were grown with or without the addition of TGFa and treated with (a) DAPT (10 AM) or (b) L-685,458 (5 AM) for 24 h. Western blotting probing for Hes-1 and GAPDH was performed according to Materials and methods. Results shown are representative for at least three independently performed experiments. (c, d) Luciferase reporter assays using the Hes-1 promoter coupled to a luciferase reporter construct. Cells were grown in the absence (c) or presence (d) of TGFa and treated with L-685,458 (5 AM) or DAPT (10 AM) for 8 h. (e, f) Luciferase reporter assays using 8 CSL coupled to a luciferase reporter construct. Cells were grown in the absence (e) or presence (f) of TGFa (1 ng/ml) and treated with L-685,458 or DAPT for 24 h. DMSO was used at the same volumes as DAPT or L-685,458 to exclude effects of the solvent.
could be detected after treatment with the p38 inhibitor SB203580 (Fig. 6b). Treatment with LY294002 led to a decrease of Hes-1 to levels similar to those in untreated cells (Fig. 6c). It should be noted that inhibition of PI3-K but not p38 affected ERK1/2 phosphorylation (data not shown), supporting the notion that there is a correlation between ERK activity and Hes-1 expression, though effects through PI3-K target genes such as AKT cannot be excluded.
We also studied the effect of the various inhibitors on Hes-1 promoter activity in the presence of TGFa. As shown in Fig. 6d, treatment of SK-N-BE(2)c cells with AG1478 or U0126 led to down-regulation of the Hes-1 reporter gene construct, completely abolishing the effect of TGFa induction. In contrast to the results obtained with Western blotting, treatment with LY294002 did not affect the promoter activity whereas SB203580 led to increased
Fig. 5. g-Secretase inhibition is functional in human neuroblastoma cells. (a) Western blot analysis of SH-EP cells treated with L-685,458 or DAPT for 24 h showing Hes-1 expression. GAPDH was used to confirm equal loading. (b) Luciferase reporter assay using SK-N-BE(2)c cells transiently transfected with the 8 CSL reporter construct together with f.l. Notch-1 and treated with L-685,458 or DAPT for 24 h.
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Fig. 6. Effect of EGFR, MEK, p38, and PI3-K inhibition in TGFa-stimulated neuroblastoma cells. SK-N-BE(2)c cells were grown in the presence of TGFa (1 ng/ml) and treated with (a) AG1478 (40 AM), U0126 (20 AM), (b) SB203580 (10 AM), or (c) LY294002 (15 AM) for 24 h. Cells were then harvested and analyzed by Western blotting as described in Materials and methods. Filters were probed for the expression of phosphorylated ERK1/2, total ERK, and Hes-1. GAPDH was used for equal loading. (d) Reporter assay using a luciferase reporter under the control of the Hes-1 promoter. Cells were grown in the presence or absence of TGFa (1 ng/ml) and treated with AG1478 (40 AM), U0126 (20 AM), SB203580 (10 AM), or LY294002 (15 AM) for 8 h. The cells were then assayed for luciferase activity according to Materials and methods. Data are representative of two experiments and presented as mean T SD of quadruplicate samples. (e) Luciferase reporter assay using 8 CSL coupled to a luciferase reporter construct. Cells were stimulated with TGFa (1 ng/ml) in the absence or presence of 20 AM U0126. DMSO was used at the same volumes as AG1478, U0126, SB203580, or LY294002 to exclude effects of the solvent.
activity for reasons currently unknown. Using the 8 CSL reporter construct, we could show that U0126 efficiently down-regulated the TGFa-induced reporter activity with approximately the same efficiency as when using the Hes-1 promoter construct (Fig. 6e). Expression of Hes-1 in unstimulated neuroblastoma cells is primarily mediated by ERK activation Even though TGFa strongly induces Hes-1 expression in neuroblastoma cells, data presented in this and our previous work show Hes-1 is expressed in exponentially growing cells in the absence of TGFa stimulation [12,28,33]. To investigate what factors are responsible for this basal expression, we treated SK-N-BE(2)c cells with the various inhibitors in the absence of TGFa. The MEK inhibitor U0126 effectively inhibited both ERK1/2 phosphorylation and Hes-1 expression, indicating that Hes-1 expression in neuroblastoma cells growing in full serum is dependent of a basal ERK activity (Fig. 7a). Interestingly, inhibition of EGFR neither affected ERK1/2 phosphorylation nor Hes-1 levels (Fig. 7a). These results underline the strong connection between ERK activity and Hes-1 expression levels and show that the basal ERK1/2 phosphorylation is not a consequence of EGFR activity. In addition, LY294002 was
able to decrease Hes-1 expression (Fig. 7c) while no effect could be detected upon treatment with SB203580 (Fig. 7b). In a Hes-1 reporter gene assay performed as above, treatment with U0126 led to a strong decrease in Hes-1 promoter activity while no effect of AG1478 treatment could be observed (Fig. 7d). Again, SB203580 for unknown reasons led to an induction of promoter activity. In addition, treatment with LY294002 also increased the activity of the luciferase reporter construct, showing a discrepancy between the Western blot results and promoter activity (Fig. 7d) possibly a consequence of unspecific effects on the control renilla reporter gene activity. Again, U0126 inhibited promoter activity of the 8 CSL reporter construct to the same extent as when the Hes-1 promoter construct was used (Fig. 7e ). Taken together, these results indicate that the basal expression of Hes-1 in the neuroblastoma cell line SK-NBE(2)c is dependent of ERK activity in an EGFRindependent manner while Notch signaling activity seems to play a minor role. When stimulated with TGFa, Hes-1 expression is induced in a dose-dependent fashion due to increased transcription mediated through CSL. This induction seems primarily to be dependent of ERK activity and to a lesser extent due to Notch activation. Concomitantly with induced Hes-1 expression, the pro-neuronal bHLH protein
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Fig. 7. Effect of EGFR, MEK, p38, and PI3-K inhibition in unstimulated neuroblastoma cells. Cells were treated with (a) AG1478 (40 AM), U0126 (20 AM), (b) SB203580 (10 AM), or (c) LY294002 (15 AM) for 24 h whereafter protein expression of phosphorylated ERK1/2, total ERK, Hes-1, and GAPDH was analyzed by Western blotting as described in Materials and methods. (d) Luciferase reporter assay using the Hes-1 promoter coupled to a luciferase reporter. Cells were treated with AG1478 (40 AM), U0126 (20 AM), SB203580 (10 AM), or LY294002 (15 AM) for 8 h and then assayed for luciferase activity according to Materials and methods. Data are representative of two experiments and presented as mean T SD of quadruplicate samples. (e) Reporter assay using a luciferase reporter gene under the control of 8 CSL sites. Cells were grown with or without the addition of 20 AM U0126.
Hash-1 was down-regulated showing that Hes-1 is functional as a repressor, compatible with a scenario in which Hes-1 can maintain an undifferentiated and proliferating phenotype in the presence of the mitogen TGFa.
Discussion In this study, we have shown that a key mediator of Notch signaling, Hes-1 also can be regulated by the Ras/ MAPK signaling pathway. Dose response experiments showed that Hes-1 expression was induced simultaneously with increased ERK1/2 phosphorylation in TGFa-stimulated neuroblastoma cells. Reporter gene assays indicate that the Hes-1 induction was a consequence of increased Hes-1 promoter activity, probably mediated through CSL. The mechanism behind the ERK1/2-induced CSL activity is currently unknown and requires further studies. Real-time quantitative RT-PCR confirmed that the Hes-1 induction was due to increased transcription as shown by an increase in Hes-1 mRNA. Furthermore, the Hes-1 induction could be blocked by treatment with inhibitors of EGFR, MEK, and PI3-K. Even though the Notch signaling cascade is functional in neuroblastoma cells as shown by Hes-1 upregulation in cells overexpressing i.c. Notch-1 [28], active Notch signaling does not seem to significantly contribute to neither basal nor the TGFa-induced expression of Hes-1 as indicated by the minute effect of g-secretase inhibition. Induction of Hes-1 was closely followed by Hash-1 down-
regulation showing that Hes-1 is capable of repressing Hash-1 expression. We therefore suggest that an elevated expression of functional Hes-1 is an integral part of the mitogenic effect of TGFa on neuroblastoma cells, incompatible with a differentiated phenotype. Hes-1 is believed to exert its repressive function through different mechanisms. The principle mechanism is by binding to N boxes (CACNAG) in target gene promoters and recruiting co-repressors such as Groucho/ TLE through the C-terminal WRPW motif [24,34 –37]. It has recently become clear that Hes proteins can form heterodimers with HERP that bind with high affinity to target sequences [38]. Alternatively, Hes-1 can form complexes with ubiquitously expressed bHLH proteins such as E47 and E2-2 [39], thereby inhibiting other functional E-protein complexes. Despite extensive studies regarding the repressive function of Hes-1, little is known about direct target genes. However, in neuronal cells, one important target gene is Hash-1/Mash-1 [40,41]. Our results show that Hash-1 expression was down-regulated at the protein level when neuroblastoma cells were stimulated with increasing amounts of TGFa. In addition, this repression was rapid, occurring already after 2 h of stimulation. These results indicate that the TGFa-induced Hes-1 is capable of repressing Hash-1. Interestingly, it was recently shown in Drosophila that EGFR stimulation attenuated the repressive function of the Hes co-repressor Groucho, thus antagonizing the Notch signaling cascade [42]. On the other hand, it has been reported that Hes-1
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itself can mediate phosphorylation of Groucho and thereby potentiating its repressive function in mammalian cells [43]. These results show that Hes and its cofactors cooperate in a species- and tissue-specific manner. In addition to its role in the bHLH network, Hes-1 can also associate with and modulate the activity of other types of transcription factors. Of particular interest is the ability of Hes-1 to potentiate JAK/STAT signaling through interaction with STAT3 [44]. Thus, it is tempting to speculate that one aspect of Ras/MAPK potentiation of STAT signaling is mediated trough a Hes-1-dependent mechanism. Dysregulated Notch signaling through translocations, point mutations, and retroviral insertion has been implicated in the genesis of T-ALL and murine mammary carcinoma [14,15,18]. In addition, it was recently shown that Notch signaling might play a role as a mediator of the well-established oncogenic Ras/MAPK signaling pathway [32,45]. In particular, in an experimental model system where primary human cells were transformed by introduction of hTERT, SV40 large T, and oncogenic Ras, it was shown that Notch-1 signaling activity was required for maintaining the transformed phenotype through a p38mediated pathway [32]. Whether these effects were mediated through Hes-1 or not was however not clarified. The link between the mitogenic Ras/MAPK cascade and Notch signaling has been indicated also in human tumors. Ras is frequently mutated in pancreatic cancer and it was recently shown that several components of the Notch signaling pathway including ligands, receptors, and downstream targets were expressed at elevated levels [45]. Interestingly, overexpression of TGFa in exocrine pancreas led to metaplasia along with ectopic Notch signaling activation. Furthermore, in explant cultures, it was shown that Notch activation was required for the TGFa-induced acinar to ductal metaplasia. In pancreatic cells, the TGFa effects could be inhibited by g-secretase inhibition suggesting that activation of Notch was required [45]. It is clear from our data that the rapid induction of Hes-1 upon TGFa stimulation in neuroblastoma cells was Notch receptor-independent and in addition was not affected by p38 inhibition (Figs. 4 and 6). Long-term effects of an active Ras signaling might however result in increased Notch receptor activity, and thereby elevated Hes-1 levels, by other means than described in the present paper. This indicates that, depending on cell type and stimuli, the link between activated Ras and the Notch signaling cascade is mediated at different levels (Fig. 8). In addition to Notch being a mediator of Ras signaling, there are also some evidences for Ras as an effector of Notch. Fitzgerald et al. showed that transformation by Notch-4 required active Ras signaling in particular the activity of ERK and PI3-kinase [46]. Cross coupling to other central signaling pathways has also been reported. For example, Notch has been shown to mediate the differentiation-inhibiting effects of BMP stimulation by interacting with SMAD1 in muscle stem cells [47].
Fig. 8. Schematic illustration of proposed model for Hes-1 regulation in neuroblastoma cells. During TGFa stimulation, ERK1/2 is phosphorylated resulting in activation of the Hes-1 promoter leading to increased Hes-1 expression. Without the addition of TGFa, Hes-1 expression is maintained by phosphorylated ERK1/2 independent of EGFR activation. Ras signaling has also been shown to activate the Notch cascade, though it is currently not clear which components of the Ras pathway are involved. When Notch is activated by binding to its ligand, intracellular Notch translocates into the nucleus and initiates Hes-1 transcription by binding to CSL present on the Hes-1 promoter.
In neuroblastoma cells, we have shown that Hes-1 mediates a differentiation-inhibiting effect by suppressing the expression of the downstream pro-neuronal bHLH protein Hash-1. Based on our and others findings, we would like to suggest that this can be achieved in two different ways; in the first scenario, active Notch signaling through interaction with ligand-expressing cells induces Hes-related protein expression through an i.c. Notch-dependent mechanism and in the second Notch-independent scenario, a direct mitogen-driven Hes expression exerts the same function, i.e., resulting in inhibition of neuronal differentiation.
Acknowledgments H.A. has a Children’s Cancer Foundation of Sweden research position. Supported by grants from the Swedish Cancer Society, the Children’s Cancer Foundation of Sweden, Ollie and Elof ˚ ke Wiberg’s Foundation, the Ericsson’s Foundation, A Crafoord Foundation and Malmo¨ University Hospital Research Funds.
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Research Article
Regulation of the Notch target gene Hes-1 by TGFa induced Ras/MAPK signaling in human neuroblastoma cells Marie-The´re´se Stockhausen, Jonas Sjo¨lund, Ha˚kan Axelson* Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, University Hospital MAS, Entrance 78, S-205 02 Malmo¨, Sweden Received 23 February 2005, revised version received 8 June 2005, accepted 24 July 2005 Available online 24 August 2005
Abstract Ras and Notch signaling have recently been shown to cooperate in the maintenance of neoplastic transformation. Here, we show that TGFa, a known activator of Ras signaling, can drive cell proliferation and at the same time induce the expression of the Notch target Hes-1 in the neuroblastoma cell line SK-N-BE(2)c. The up-regulation of Hes-1 occurred both at the transcriptional and protein levels and by use of EGFR and MEK inhibitors we could show that the Hes-1 response was dependent on activation of the MAP kinase ERK. Blocking Notch activation by g-secretase inhibition did not profoundly affect the Hes-1 levels, neither in untreated nor in TGFa treated cells. The upregulation of Hes-1 was associated with down-regulation of its pro-neuronal target gene Hash-1. Taken together, these results show that TGFa is a potent mitogen of neuroblastoma cells and suggest a connection between activation of ERK and Hes-1, thus providing a link between the Ras and Notch signaling pathways. D 2005 Elsevier Inc. All rights reserved. Keywords: Neuroblastoma; Notch; Hes-1; Hash-1; Ras/MAPK signaling; ERK; TGFa
Introduction Neuroblastoma is a childhood malignancy that originates from immature cells of the sympathetic nervous system [1]. Several lines of evidence suggest that the cells are halted at an early, embryonic state of development with maintained proliferative capacity. For example, the cells have a phenotype resembling sympathetic neuroblasts and they express several differentiation-related genes normally expressed during embryogenesis, such as the basic helix-loop-helix (bHLH) transcription factors human achaete-scute homo-
Abbreviations: bHLH, basic helix-loop-helix; Hash-1, human achaetescute homologue-1; Mash-1, mammalian achaete-scute homologue-1; TPA, phorbol ester; RA, retinoic acid; i.c. Notch, intracellular Notch; f.l. Notch, full-length Notch; T-ALL, T-cell acute lymphoblastic leukemia; MAPK, mitogen-activated protein kinase; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; TGFa, transforming growth factor a. * Corresponding author. Fax: +46 40337322. E-mail address: [email protected] (H. Axelson). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.07.011
logue-1 (Hash-1) and dHAND [2]. In addition, amplification of the N-myc gene, which serves as a key prognostic marker associated with an advert outcome of the disease [3 – 5], is normally involved in controlling the differentiation process of sympathetic neurons [6]. It has for a long time been known that neuroblastoma cells can be induced to differentiate in vitro, using phorbol ester (TPA), retinoic acid (RA), or a combination of growth factors [7,8]. The differentiated phenotype is characterized by induction of a number of neuronal differentiation marker genes and decreased proliferation along with induction of neurites. Thus, in order to develop novel differentiation-based treatment modalities, it is of vital importance to understand the mechanisms that control the process of neuroblastoma cell differentiation. Notch signaling is based on cell – cell contact since the Notch ligands (Delta like-1, -3, -4, and Jagged-1, -2) are primarily membrane bound [9]. They bind and activate Notch receptors (Notch-1 to -4) on neighboring cells, which ultimately leads to g-secretase-mediated cleavage of the intracellular part of the receptor (i.c. Notch) that translocates into the nucleus and associates with the transcription factor
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CSL (CBF-1, Suppressor of Hairless, LAG-1; also known as RBP-Jn), thereby converting it from a transcriptional repressor to an activator. In several tissues, genes belonging to the Hes and HRT/HERP/Hey families of transcriptional repressors are important targets of Notch signaling and these transcriptional repressors mediate many of the known effects of activated Notch [10]. In neuronal cells, Hes proteins negatively regulate transcriptional activators of the pro-neuronal bHLH family. During development of the sympathetic nervous system, mammalian achaete – scute homologue-1 (Mash-1) is one important bHLH target protein and gene ablation experiments in mice show that the gene is essential for proper formation of this part of the nervous system [11]. Based on these observations and in conjunction with the fact that Hes-1 and Hash-1 are regulated by Notch signaling, we have studied the role of this cascade in neuroblastoma cells. We noticed that during RA- or TPA-induced differentiation of neuroblastoma cells, there was an induction of Notch signaling activity, as indicated by transient elevation of the Hes-1 transcriptional repressor, with a concomitant decrease in Hash-1 expression levels [12]. Constitutive Notch-1 signaling also blocked RA- and TPA-induced differentiation of neuroblastoma cells [12], supporting the notion that Notch signaling might be involved in neuroblastoma cell differentiation and that active Notch signaling might maintain neuroblastoma cells in an undifferentiated state [13]. The most clear-cut example of how Notch signaling is linked to oncogenesis comes from the findings that the receptor can be activated by chromosomal translocation or point mutations in T-cell acute lymphoblastic leukemia (TALL) [14,15]. This leads to constitutive expression of the intracellular domain of the receptor and several experiments show that i.c. Notch-1 functions as an oncogene in this cell type [16,17]. Furthermore, in a mouse mammary tumor model system, retroviral insertion in the Notch-4 gene leads to constitutively active Notch signaling and tumorigenesis [18]. The cellular effects of Notch signaling are however highly cell-type dependent as illustrated by the situation in keratinocytes in which active Notch signaling is associated with induced differentiation and decreased proliferation [19]. Accordingly, Notch has been proven to be a tumor suppressor in this cell type [20]. Numerous studies have shown that Hes-1 is a primary Notch target gene, and as a consequence, Hes-1 expression has been considered a good surrogate marker for Notch signaling activity. However, it should be noted that in many cases, regulation of Hes-1 by Notch signaling has been studied in experiments in which the intracellular part of the receptor is overexpressed, resulting in supra-physiological levels of the protein. With regard to physiological regulation, it is important to note that gene targeting of Notch-1 in mice led to decreased levels of Hes-5 and HERP1/2 while the expression of Hes-1 remained unaffected [21 – 23]. Also, RBP-Jj gene ablation in mice led to reduced expression of Hes-5 but not Hes-1 [21].
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Thus, Hes-1 expression seems to be regulated both in a Notch-dependent and -independent manner. Regarding the Notch-independent regulation, it has been shown that Hes-1 can regulate its own expression through N boxes (CACNAG) in the Hes-1 promoter [24]. In addition, Hes-1 expression can also be induced upon serum stimulation in a variety of cell lines. In synchronized cells, this induction results in an oscillating expression pattern of Hes-1 mRNA and protein mediated through the autorepressive capacity of Hes-1 [25]. Other studies have shown that growth factor stimulation leads to an induction of Hes-1 [26], but it is currently not clear whether this occurs in a Notch-dependent or -independent fashion. In this report, we have analyzed the link between mitogen-activated protein kinase (MAPK) signaling and Hes-1 regulation by stimulating neuroblastoma cells with the transforming growth factor a (TGFa), which in previous studies have shown to be a mitogen of neuroblastoma cells [27]. We show that Hes-1 expression is directly correlated to the MAPK activity and that this induction is not mediated by increased signaling activity of the Notch receptors. In TGFa-stimulated cells, Hes-1 expression was inhibited both by MAPK and epidermal growth factor receptor (EGFR) inhibitors. In contrast, only the MAPK inhibitor could affect the expression of Hes-1 in non-stimulated cells. In addition, up-regulation of Hes-1 led to a rapid down-regulation of Hash-1 indicating a role in maintaining an undifferentiated and thus proliferative phenotype of neuroblastoma cells. Taken together, these findings suggest that Hes-1, an important mediator of Notch signaling, can be regulated both by Notch-independent, mitogen-driven and Notchdependent, mitogen-independent cues in neuronal cells.
Materials and methods Cell culture and reagents The human neuroblastoma cell line SK-N-BE(2)c was maintained in Eagle’s Minimum Essential Medium (MEM, Invitrogen Inc., Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 Ag/ml) at 37-C in an atmosphere of 5% CO2. The neuroblastoma cell line SH-EP was maintained in the same media as above, but with the addition of 15% FCS. For experiments, cells were seeded 1 day prior to initiation of treatment with L-685,458, DAPT, AG1478, SB203580 (all purchased from Calbiochem, La Jolla, CA, USA), U0126 (Cell Signaling Technology, Beverly, MA, USA), or LY294002 (Upstate, Lake Placid, NY, USA) for indicated times. All control experiments include the corresponding volume of the vehicle DMSO. The volume of vehicle never exceeded 1% of total volume. TGFa was purchased from R&D Systems (Minneapolis, MN, USA) and dissolved in 10 mM acetic acid containing 0.1% BSA, aliquoted and stored at 20-C until used.
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Trypan blue exclusion assay Cell viability was analyzed using trypan blue exclusion of dead cells. In short, cell culture media was collected and adherent cells harvested by trypsination. The fractions were pooled, the volume was measured, and the cells were mixed with trypan blue solution (0.4%) (Sigma, St. Louis, MO, USA). Both dead and viable cells were then counted in a Bu¨ rker chamber. Each experiment was performed in triplicates and repeated at least three times. Western blot analysis For Western blot analyses, cells were harvested by centrifugation, snap-frozen in liquid nitrogen and stored at 70-C. Cell pellets were then lysed with NP40 lysis buffer (1% NP40, 10% glycerol, 20 mM Tris – HCl [pH 8.0], 137 mM NaCl) and 4% complete protease inhibitor cocktail mix (Roche, Mannheim, Germany) for total cell protein preparation. Proteins (50 Ag) were separated by SDS-PAGE followed by transfer to PVDF Immobilon-P filters (Millipore Corp., Billerica, MA, USA). The filters were probed with polyclonal anti-Hes-1 antiserum diluted 1:8000 (kindly provided by Dr. Tetsuo Sudo, Japan), monoclonal antiMash-1 antibody diluted 1:125 (Pharmingen, San Diego, CA, USA), polyclonal anti-phopho-ERK1/2 antiserum 1:500 (Cell Signaling), polyclonal anti-ERK antiserum 1:500 (Cell Signaling), or monoclonal anti-GAPDH antibody diluted 1:500 (Labora Chemicon International, Temecula, CA, USA). The blots were then incubated with secondary antibodies: donkey anti-rabbit 1:1000 (Amersham Life Science, Solna, Sweden), goat anti-mouse 1:7000 (Jackson, West Grove, PA, USA), or sheep anti-mouse 1:5000 (Amersham) all coupled to horseradish peroxidase. The immunoreactivity was detected using the enhanced chemiluminescence method (Pierce, Rockford, IL, USA). Real-time quantitative RT-PCR Total RNA was purified with RNeasy kit (Qiagen, Hilden, Germany) as described by the manufacturer. cDNA was synthesized as described earlier [28] and used as template in quantitative real-time PCR reactions with SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA). Relative quantification of expression levels was performed by using the comparative Ct method and normalized to the expression of three housekeeping genes (SDHA, YWHAZ, and UBC). The genes analyzed were: Hes-1 forward, 5V-AGC GGG CGC AGA TGA C-3V; Hes-1 reverse, 5V-CGT TCA TGC ACT CGC TGA A-3V; SDHA forward, 5V-TGG GAA CAA GAG GGC ATC TG-3V; SDHA reverse, 5V-CCA CCA CTG CAT CAA ATT CAT G3V; YWHAZ forward, 5V-ACT TTT GGT ACA TTG TGG CTT CAA-3V; YWHAZ reverse, 5V-CCG CCA GGA CAA ACC AGT AT-3V; UBC forward, 5V-ATT TGG GTC GCG GTT CTT G-3V; UBC reverse, 5V-TGC CTT GAC ATT CTC
GAT GGT-3V. Data shown are representative for one out of three experiments performed in triplicates. Luciferase reporter assay SK-N-BE(2)c cells were grown to 80 – 90% confluence in 24-well plates. Cells were then transiently transfected by using Lipofectamine 2000 reagent (Invitrogen) with 0.4 Ag Hes-1 promoter construct ( 467 to +46) (a kind gift from R. Kageyama, Kyoto University, Japan) [29], 0.4 Ag 8 CSL luciferase reporter construct (kindly provided by S.D. Hayward, Johns Hopkins School of Medicine, USA) [30], 0.8 Ag full-length Notch-1 (f.l. Notch-1) expression vector (kindly provided by J.C. Aster, Harvard Medical School, USA) [31], and 20 ng phRL-TK renilla expression vector (Promega, San Luis Obispo, CA, USA) as a control for transfection efficiency. Post-transfection, cells were treated with L-685,458, DAPT, U0126, AG1478, SB203580, or LY294002 with or without the addition of TGFa as indicated in the figure legends. DMSO and/or 10 mM acetic acid containing 0.1% BSA were added in control experiments in volumes corresponding to inhibitors or TGFa. Cells were lysed and assayed for luciferase and renilla activity on a TD-20/20 Luminometer (Turner Biosystems, Sunnyvale, CA, USA) according to the DualLuciferase Reporter Assay System manufacturer Promega’s instructions. Each condition was tested in replicates of two to four and repeated at least twice. Data are presented as representative experiments.
Results TGFa stimulation promotes growth of neuroblastoma cells and induces Hes-1 expression We have previously reported that RA- or TPA-induced differentiation of neuroblastoma cells led to a transient upregulation of Hes-1 followed by an almost complete downregulation [12]. During these conditions, the differentiated phenotype was associated with ceased proliferation, and we hypothesized that a maintained Hes-1 expression might be incompatible with terminal differentiation. In order to clarify the regulation of Hes-1 during mitogenic stimulation of neuroblastoma cells, we treated SK-N-BE(2)c cells with the EGFR ligand TGFa. In line with data published by other groups [27], we found a growth-promoting effect of TGFa, detected already at 24 h and maintained after 48 h in culture (Figs. 1a and b). Trypan blue exclusion experiments showed that this was not a consequence of differences in cell death between control and TGFa-stimulated cells (Fig. 1c). Having confirmed the mitogenic effect of TGFa on neuroblastoma cells, we next studied the expression of Hes-1 after 24 h of TGFa stimulation. We noticed a dose-dependent effect of TGFa on Hes-1 expression with a considerable induction already at 0.05 ng/ml (Fig. 2a). To verify that
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luciferase reporter assays using a Hes-1 reporter construct. The reporter assay recapitulated to a large extent the protein expression data, i.e., increased concentrations of TGFa led to increased activity of the Hes-1 promoter. Maximum activity was achieved at 10 ng/ml of TGFa, but a modest induction was detected already at the lowest concentration used (Fig. 2b). Similar results were obtained using a synthetic reporter containing eight repetitive CSL sites (8 CSL) (Fig. 2c). These data suggest that that the TGFa-induced Hes-1 expression was primarily mediated by CSL. In addition, real-time quantitative RT-PCR showed that Hes-1 mRNA levels increased in a dose-dependent manner upon TGFa stimulation (Fig. 2d). In a time-course experiment using 1 ng/ml of TGFa, elevated Hes-1 levels were detected already after 2 h and maintained until the end of the experiment at 24 h (Fig. 3). As expected, TGFa induced a rapid and persistent ERK1/2 phosphorylation, which was not due to up-regulation of total ERK. Interestingly, the fast induction of Hes-1 correlated with a rapid down-regulation of Hash-1, underlining the inverse expression pattern of Hash-1 and Hes-1 in neuroblastoma cells. This effect is less pronounced at later time points, but is still detectable at 24 h. These data show that the mitogenic effect of TGFa on human neuroblastoma cells is associated with increased phosphorylation of ERK and a concomitant up-regulation of Hes-1 expression together with a rapid down-regulation of Hash-1. Reporter gene assays and real-time quantitative RTPCR results indicate that the elevated Hes-1 levels are due to increased promoter activity and thus are a consequence of increased transcription.
Fig. 1. TGFa induced cell growth in the SK-N-BE(2)c neuroblastoma cell line. (a) Number of viable cells was determined after 0, 1, and 2 days of treatment with TGFa (1 ng/ml) using trypan blue exclusion and cell counting in a Bu¨rker chamber. (b) Total number of cells was counted after 24 h of TGFa treatment (1 ng/ml) and (c) dead cells determined using trypan blue staining. Each experiment was performed in triplicates and repeated at least three times. Results are shown as mean T SD of representative experiments.
TGFa stimulation activated the Ras/MAPK cascade, we also monitored the phosphorylation status of the MAPK extracellular signal-regulated kinase (ERK). Increased TGFa concentrations led to a dose-dependent induction of ERK1/2 phosphorylation. This increase in ERK1/2 phosphorylation was not due to increased amounts of total ERK levels, as shown by probing with a pan-ERK antiserum. Furthermore, Hash-1 levels were decreased in a dosedependent manner upon TGFa stimulation, suggesting a retained capacity of Hes-1 to repress Hash-1 expression under these circumstances (Fig. 2a). To clarify whether the induced Hes-1 expression was a consequence of increased transcriptional activity of the Hes1 promoter or due to protein stabilization, we performed
TGFa-induced Hes-1 expression is not mediated by Notch activation Studies in other cell systems indicate that MAPK activity can lead to increased signaling activity of the Notch receptors [32]. In order to clarify whether this was the case in neuroblastoma cells, we employed two compounds (DAPT and L-685,458) that inhibit g-secretase activity and thereby prevent the activating cleavage of the Notch receptors. By this method, we would be able to clarify whether the Hes-1 induction upon TGFa treatment was a consequence of increased Notch receptor activity or a Notch-independent effect of MAPK activity. Treatment with DAPT did not affect the Hes-1 levels neither in the absence nor presence of TGFa (Fig. 4a). Similar results were obtained when treating the cells with L-685,458 (Fig. 4b). Reporter assays using the same Hes-1 reporter construct as above showed that DAPT only could inhibit the activity of the promoter to a small extent whereas an even less pronounced effect was observed with L-685,458 (Fig. 4c). When inducing promoter activity with TGFa (1 ng/ml), treatment with DAPT resulted in a small decrease of activation though not down to basal levels (Fig. 4d). Treatment with L-685,458 had no effect (Fig. 4d). The
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Fig. 2. Effect of TGFa stimulation on Hes-1 expression in neuroblastoma cells. (a) Western blotting of phosphorylated ERK1/2, total ERK, Hes-1, and Hash-1. GAPDH was used to confirm equal loading. Results shown are representative of three experiments. (b, c) Luciferase reporter assay using the (b) Hes-1 promoter or (c) 8 CSL coupled to a luciferase reporter. Results are shown as mean T SD and are representative for two experiments. (d) Real-time quantitative RT-PCR using Hes-1-specific primers. Data shown are representative for three experiments with similar results. Cells were treated with indicated concentrations of TGFa for 24 h whereafter Western blotting, reporter assays, and real-time quantitative RT-PCR were performed as described in Materials and methods.
results obtained with the Hes-1 reporter construct were recapitulated with the 8 CSL construct, though DAPT treatment resulted in a partial down-regulation of both basal and TGFa-induced promoter activity (Figs. 4e and f). The effective dose of g-secretase inhibitors is highly celltype specific and since we could only detect minute effect of the inhibitors on SK-N-BE(2)c cells, we wanted to confirm that the concentrations used actually inhibited g-secretase activity in human neuroblastoma cells. We therefore treated the neuroblastoma cell line SH-EP with L-685,458 and DAPT for 24 h. SH-EP expresses high levels of both Notch receptors and Hes-1 and thus we speculated that inhibition of g-secretase would be detected as a decrease of Hes-1 expression. Indeed, both L-685,458 and DAPT clearly
decreased the Hes-1 levels as shown by Western blotting (Fig. 5a). To further verify g-secretase inhibition in the SKN-BE(2)c cell line used in this study, we transiently transfected the cells with an expression vector coding for full-length Notch-1 (f.l. Notch-1), which is dependent of gsecretase activity for its activation, together with the 8 CSL luciferase reporter construct. Treating these cells with either L-685,458 or DAPT clearly decreased reporter gene activity, showing that the g-secretase activity indeed was inhibited, thereby verifying that the inhibitors were functional at the concentrations used (Fig. 5b). We therefore concluded that the elevated Hes-1 levels detected after treatment with TGFa were not primarily a consequence of increased Notch signaling activity. EGFR and ERK inhibition prevents TGFa-induced Hes-1 expression
Fig. 3. Time-course experiment using TGFa-treated SK-N-BE(2)c cells. Western blotting showing phosphorylation of ERK1/2, total ERK, Hes-1, and Hash-1 levels. GAPDH was used as a control of equal loading. Cells were stimulated with TGFa (1 ng/ml) for indicated times whereafter Western blotting was performed as described in Materials and methods. Experiment is a representative of three showing similar results.
Since Notch activation did not seem to be responsible for the TGFa-induced Hes-1 expression, we next wanted to analyze the effect of inhibitors that target pathways downstream the EGFR. The inhibitors tested include the EGFR inhibitor AG1478, the MEK inhibitor U0126, the PI3-K inhibitor LY294002, and the p38 MAPK inhibitor SB203580. Both the EGFR inhibitor and the MEK inhibitor led to a considerable down-regulation of Hes-1 along with decreased ERK1/2 phosphorylation in the presence of TGFa (Fig. 6a). In contrast, no obvious effect on Hes-1 expression
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Fig. 4. Effect of g-secretase inhibition in unstimulated neuroblastoma cells or cells treated with TGFa. Cells were grown with or without the addition of TGFa and treated with (a) DAPT (10 AM) or (b) L-685,458 (5 AM) for 24 h. Western blotting probing for Hes-1 and GAPDH was performed according to Materials and methods. Results shown are representative for at least three independently performed experiments. (c, d) Luciferase reporter assays using the Hes-1 promoter coupled to a luciferase reporter construct. Cells were grown in the absence (c) or presence (d) of TGFa and treated with L-685,458 (5 AM) or DAPT (10 AM) for 8 h. (e, f) Luciferase reporter assays using 8 CSL coupled to a luciferase reporter construct. Cells were grown in the absence (e) or presence (f) of TGFa (1 ng/ml) and treated with L-685,458 or DAPT for 24 h. DMSO was used at the same volumes as DAPT or L-685,458 to exclude effects of the solvent.
could be detected after treatment with the p38 inhibitor SB203580 (Fig. 6b). Treatment with LY294002 led to a decrease of Hes-1 to levels similar to those in untreated cells (Fig. 6c). It should be noted that inhibition of PI3-K but not p38 affected ERK1/2 phosphorylation (data not shown), supporting the notion that there is a correlation between ERK activity and Hes-1 expression, though effects through PI3-K target genes such as AKT cannot be excluded.
We also studied the effect of the various inhibitors on Hes-1 promoter activity in the presence of TGFa. As shown in Fig. 6d, treatment of SK-N-BE(2)c cells with AG1478 or U0126 led to down-regulation of the Hes-1 reporter gene construct, completely abolishing the effect of TGFa induction. In contrast to the results obtained with Western blotting, treatment with LY294002 did not affect the promoter activity whereas SB203580 led to increased
Fig. 5. g-Secretase inhibition is functional in human neuroblastoma cells. (a) Western blot analysis of SH-EP cells treated with L-685,458 or DAPT for 24 h showing Hes-1 expression. GAPDH was used to confirm equal loading. (b) Luciferase reporter assay using SK-N-BE(2)c cells transiently transfected with the 8 CSL reporter construct together with f.l. Notch-1 and treated with L-685,458 or DAPT for 24 h.
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Fig. 6. Effect of EGFR, MEK, p38, and PI3-K inhibition in TGFa-stimulated neuroblastoma cells. SK-N-BE(2)c cells were grown in the presence of TGFa (1 ng/ml) and treated with (a) AG1478 (40 AM), U0126 (20 AM), (b) SB203580 (10 AM), or (c) LY294002 (15 AM) for 24 h. Cells were then harvested and analyzed by Western blotting as described in Materials and methods. Filters were probed for the expression of phosphorylated ERK1/2, total ERK, and Hes-1. GAPDH was used for equal loading. (d) Reporter assay using a luciferase reporter under the control of the Hes-1 promoter. Cells were grown in the presence or absence of TGFa (1 ng/ml) and treated with AG1478 (40 AM), U0126 (20 AM), SB203580 (10 AM), or LY294002 (15 AM) for 8 h. The cells were then assayed for luciferase activity according to Materials and methods. Data are representative of two experiments and presented as mean T SD of quadruplicate samples. (e) Luciferase reporter assay using 8 CSL coupled to a luciferase reporter construct. Cells were stimulated with TGFa (1 ng/ml) in the absence or presence of 20 AM U0126. DMSO was used at the same volumes as AG1478, U0126, SB203580, or LY294002 to exclude effects of the solvent.
activity for reasons currently unknown. Using the 8 CSL reporter construct, we could show that U0126 efficiently down-regulated the TGFa-induced reporter activity with approximately the same efficiency as when using the Hes-1 promoter construct (Fig. 6e). Expression of Hes-1 in unstimulated neuroblastoma cells is primarily mediated by ERK activation Even though TGFa strongly induces Hes-1 expression in neuroblastoma cells, data presented in this and our previous work show Hes-1 is expressed in exponentially growing cells in the absence of TGFa stimulation [12,28,33]. To investigate what factors are responsible for this basal expression, we treated SK-N-BE(2)c cells with the various inhibitors in the absence of TGFa. The MEK inhibitor U0126 effectively inhibited both ERK1/2 phosphorylation and Hes-1 expression, indicating that Hes-1 expression in neuroblastoma cells growing in full serum is dependent of a basal ERK activity (Fig. 7a). Interestingly, inhibition of EGFR neither affected ERK1/2 phosphorylation nor Hes-1 levels (Fig. 7a). These results underline the strong connection between ERK activity and Hes-1 expression levels and show that the basal ERK1/2 phosphorylation is not a consequence of EGFR activity. In addition, LY294002 was
able to decrease Hes-1 expression (Fig. 7c) while no effect could be detected upon treatment with SB203580 (Fig. 7b). In a Hes-1 reporter gene assay performed as above, treatment with U0126 led to a strong decrease in Hes-1 promoter activity while no effect of AG1478 treatment could be observed (Fig. 7d). Again, SB203580 for unknown reasons led to an induction of promoter activity. In addition, treatment with LY294002 also increased the activity of the luciferase reporter construct, showing a discrepancy between the Western blot results and promoter activity (Fig. 7d) possibly a consequence of unspecific effects on the control renilla reporter gene activity. Again, U0126 inhibited promoter activity of the 8 CSL reporter construct to the same extent as when the Hes-1 promoter construct was used (Fig. 7e ). Taken together, these results indicate that the basal expression of Hes-1 in the neuroblastoma cell line SK-NBE(2)c is dependent of ERK activity in an EGFRindependent manner while Notch signaling activity seems to play a minor role. When stimulated with TGFa, Hes-1 expression is induced in a dose-dependent fashion due to increased transcription mediated through CSL. This induction seems primarily to be dependent of ERK activity and to a lesser extent due to Notch activation. Concomitantly with induced Hes-1 expression, the pro-neuronal bHLH protein
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Fig. 7. Effect of EGFR, MEK, p38, and PI3-K inhibition in unstimulated neuroblastoma cells. Cells were treated with (a) AG1478 (40 AM), U0126 (20 AM), (b) SB203580 (10 AM), or (c) LY294002 (15 AM) for 24 h whereafter protein expression of phosphorylated ERK1/2, total ERK, Hes-1, and GAPDH was analyzed by Western blotting as described in Materials and methods. (d) Luciferase reporter assay using the Hes-1 promoter coupled to a luciferase reporter. Cells were treated with AG1478 (40 AM), U0126 (20 AM), SB203580 (10 AM), or LY294002 (15 AM) for 8 h and then assayed for luciferase activity according to Materials and methods. Data are representative of two experiments and presented as mean T SD of quadruplicate samples. (e) Reporter assay using a luciferase reporter gene under the control of 8 CSL sites. Cells were grown with or without the addition of 20 AM U0126.
Hash-1 was down-regulated showing that Hes-1 is functional as a repressor, compatible with a scenario in which Hes-1 can maintain an undifferentiated and proliferating phenotype in the presence of the mitogen TGFa.
Discussion In this study, we have shown that a key mediator of Notch signaling, Hes-1 also can be regulated by the Ras/ MAPK signaling pathway. Dose response experiments showed that Hes-1 expression was induced simultaneously with increased ERK1/2 phosphorylation in TGFa-stimulated neuroblastoma cells. Reporter gene assays indicate that the Hes-1 induction was a consequence of increased Hes-1 promoter activity, probably mediated through CSL. The mechanism behind the ERK1/2-induced CSL activity is currently unknown and requires further studies. Real-time quantitative RT-PCR confirmed that the Hes-1 induction was due to increased transcription as shown by an increase in Hes-1 mRNA. Furthermore, the Hes-1 induction could be blocked by treatment with inhibitors of EGFR, MEK, and PI3-K. Even though the Notch signaling cascade is functional in neuroblastoma cells as shown by Hes-1 upregulation in cells overexpressing i.c. Notch-1 [28], active Notch signaling does not seem to significantly contribute to neither basal nor the TGFa-induced expression of Hes-1 as indicated by the minute effect of g-secretase inhibition. Induction of Hes-1 was closely followed by Hash-1 down-
regulation showing that Hes-1 is capable of repressing Hash-1 expression. We therefore suggest that an elevated expression of functional Hes-1 is an integral part of the mitogenic effect of TGFa on neuroblastoma cells, incompatible with a differentiated phenotype. Hes-1 is believed to exert its repressive function through different mechanisms. The principle mechanism is by binding to N boxes (CACNAG) in target gene promoters and recruiting co-repressors such as Groucho/ TLE through the C-terminal WRPW motif [24,34 –37]. It has recently become clear that Hes proteins can form heterodimers with HERP that bind with high affinity to target sequences [38]. Alternatively, Hes-1 can form complexes with ubiquitously expressed bHLH proteins such as E47 and E2-2 [39], thereby inhibiting other functional E-protein complexes. Despite extensive studies regarding the repressive function of Hes-1, little is known about direct target genes. However, in neuronal cells, one important target gene is Hash-1/Mash-1 [40,41]. Our results show that Hash-1 expression was down-regulated at the protein level when neuroblastoma cells were stimulated with increasing amounts of TGFa. In addition, this repression was rapid, occurring already after 2 h of stimulation. These results indicate that the TGFa-induced Hes-1 is capable of repressing Hash-1. Interestingly, it was recently shown in Drosophila that EGFR stimulation attenuated the repressive function of the Hes co-repressor Groucho, thus antagonizing the Notch signaling cascade [42]. On the other hand, it has been reported that Hes-1
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itself can mediate phosphorylation of Groucho and thereby potentiating its repressive function in mammalian cells [43]. These results show that Hes and its cofactors cooperate in a species- and tissue-specific manner. In addition to its role in the bHLH network, Hes-1 can also associate with and modulate the activity of other types of transcription factors. Of particular interest is the ability of Hes-1 to potentiate JAK/STAT signaling through interaction with STAT3 [44]. Thus, it is tempting to speculate that one aspect of Ras/MAPK potentiation of STAT signaling is mediated trough a Hes-1-dependent mechanism. Dysregulated Notch signaling through translocations, point mutations, and retroviral insertion has been implicated in the genesis of T-ALL and murine mammary carcinoma [14,15,18]. In addition, it was recently shown that Notch signaling might play a role as a mediator of the well-established oncogenic Ras/MAPK signaling pathway [32,45]. In particular, in an experimental model system where primary human cells were transformed by introduction of hTERT, SV40 large T, and oncogenic Ras, it was shown that Notch-1 signaling activity was required for maintaining the transformed phenotype through a p38mediated pathway [32]. Whether these effects were mediated through Hes-1 or not was however not clarified. The link between the mitogenic Ras/MAPK cascade and Notch signaling has been indicated also in human tumors. Ras is frequently mutated in pancreatic cancer and it was recently shown that several components of the Notch signaling pathway including ligands, receptors, and downstream targets were expressed at elevated levels [45]. Interestingly, overexpression of TGFa in exocrine pancreas led to metaplasia along with ectopic Notch signaling activation. Furthermore, in explant cultures, it was shown that Notch activation was required for the TGFa-induced acinar to ductal metaplasia. In pancreatic cells, the TGFa effects could be inhibited by g-secretase inhibition suggesting that activation of Notch was required [45]. It is clear from our data that the rapid induction of Hes-1 upon TGFa stimulation in neuroblastoma cells was Notch receptor-independent and in addition was not affected by p38 inhibition (Figs. 4 and 6). Long-term effects of an active Ras signaling might however result in increased Notch receptor activity, and thereby elevated Hes-1 levels, by other means than described in the present paper. This indicates that, depending on cell type and stimuli, the link between activated Ras and the Notch signaling cascade is mediated at different levels (Fig. 8). In addition to Notch being a mediator of Ras signaling, there are also some evidences for Ras as an effector of Notch. Fitzgerald et al. showed that transformation by Notch-4 required active Ras signaling in particular the activity of ERK and PI3-kinase [46]. Cross coupling to other central signaling pathways has also been reported. For example, Notch has been shown to mediate the differentiation-inhibiting effects of BMP stimulation by interacting with SMAD1 in muscle stem cells [47].
Fig. 8. Schematic illustration of proposed model for Hes-1 regulation in neuroblastoma cells. During TGFa stimulation, ERK1/2 is phosphorylated resulting in activation of the Hes-1 promoter leading to increased Hes-1 expression. Without the addition of TGFa, Hes-1 expression is maintained by phosphorylated ERK1/2 independent of EGFR activation. Ras signaling has also been shown to activate the Notch cascade, though it is currently not clear which components of the Ras pathway are involved. When Notch is activated by binding to its ligand, intracellular Notch translocates into the nucleus and initiates Hes-1 transcription by binding to CSL present on the Hes-1 promoter.
In neuroblastoma cells, we have shown that Hes-1 mediates a differentiation-inhibiting effect by suppressing the expression of the downstream pro-neuronal bHLH protein Hash-1. Based on our and others findings, we would like to suggest that this can be achieved in two different ways; in the first scenario, active Notch signaling through interaction with ligand-expressing cells induces Hes-related protein expression through an i.c. Notch-dependent mechanism and in the second Notch-independent scenario, a direct mitogen-driven Hes expression exerts the same function, i.e., resulting in inhibition of neuronal differentiation.
Acknowledgments H.A. has a Children’s Cancer Foundation of Sweden research position. Supported by grants from the Swedish Cancer Society, the Children’s Cancer Foundation of Sweden, Ollie and Elof ˚ ke Wiberg’s Foundation, the Ericsson’s Foundation, A Crafoord Foundation and Malmo¨ University Hospital Research Funds.
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