Inhibitors and antibodies

U0126, SB239063, SP600125, and LY24002 were purchased from EMD Biosciences (Darmstadt, Germany). [α-³²P]ATP and [γ-³²P]ATP were obtained from PerkinElmer Life Sciences (Waltham, MA). Fugene 6 was from Roche (Basel, Switzerland). Human recombinant FGF-2 was purchased from Invitrogen. Id-1 (C-20) and Egr-1 (588) antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Polyclonal anti-p42/p44 MAP kinases (137F5) and polyclonal anti-phospho-p42/p44 (Thr202/Tyr204) were from Cell Signaling Technology (Danvers, MA). Anti-Grb2 was from Transduction Laboratories (BD Biosciences, San Jose, CA).

Mutational analysis of Id-1 promoter

Seven 5′-deletion mutants of the Id-1 promoter were generated by PCR amplification from the full-length Id-1 sequence and subcloned into the pGL3 reporter vector.

Site-directed mutagenesis was performed using a plasmid-containing the Id-1 promoter sequence −986/+95 bp (pId-1 F5) as template for the PCR using the Quick-Change II site-directed mutagenesis kit (Strategene, La Jolla, CA) according to the supplier’s protocol. The primers were: Egr-1/Id-1 Fw: mut, 5′-GGCGACCGCCCATGCGGCGCCAGC-3′, and reverse. Egr-1-binding site is in bold and the altered nucleotides are underlined.

Transfection and luciferase assays

SK-N-MC cells were transfected using Fugene 6 (Roche). Cells were plated at a density of 5 × 10⁴ cells per well in a 24 wells plate 48 h prior to transfection. Co-transfection was performed according to the manufacturer’s protocol using 450 ng of reporter plasmid and 120 ng of Renilla luciferase pRL-TK control plasmid (Promega, Madison, WI) for 24 h. Following this period, the transfection mixture was removed and replaced with media with or without 40 ng/ml FGF-2 for 24 h. When required, cells were pretreated for 30 min with 15 μM of UO126, 10 μM of SB239063, 25 μM of SP600215, or 50 μM of LY24002 prior to FGF-2 induction. Cell extracts were subsequently prepared and assayed using the Dual Luciferase kit (Promega) as per the manufacturer’s instructions. Luciferase activities were normalized with Renilla values. Data are presented as the fold activation of the FGF-2 treated versus untreated samples. Results shown represent the mean of at least three independent experiments.

Western blots

Cell protein extracts were prepared by centrifugation and lysis of the cell pellet in the appropriate volume of RIPA buffer (50 mM Tris pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 mM sodium orthovanadate, phosphatase inhibitors, and protease inhibitor cocktails). Thirty to 50 μg of whole cell lysate were separated on a 4–15% SDS–PAGE (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane, followed by immunoreaction with the indicated antibodies.

Northern blots

Total RNA from SK-N-MC cells (6.5 × 10⁵ cells in 100 mm² diameter dishes) was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA (15 μg) was resolved on formaldehyde-containing 1.2% agarose gel and transferred to Hybond-N nylon membranes (GE Healthcare) and cross linked by ultraviolet irradiation. A 410 bp DNA fragment corresponding to the Id-1-coding region was radiolabeled with [α-³²P] dCTP and used as a probe. The filter was pre-hybridized for 1 h at 42°C in UltraHyb solution (Ambion, Austin, TX) and hybridized for 16 h at 42°C with the specific probe (10⁶ cpm/ml). The blot was then rinsed twice for 5 min with 2 × SSC 0.1% SDS at 42°C and washed twice with 0.2× SSC, 0.1% SDS at 42°C for 20 min. The filter was exposed at −80°C to a Fuji XAR-5 film with Kodak intensifying screen. To verify RNA loading, the filter was rehybridized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA probe.

Chromatin Immunoprecipitation assay

ChiP assay was performed with a kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s instructions. 1 × 10⁶ SK-N-MC cells were cross-linked in a 1% formaldehyde solution for 10 min at 37°C. Cells were then lysed in 200 μl of SDS buffer and sonicated to obtain DNA fragments of 500 bp average length. After centrifugation, the cleared supernatants were diluted 10-fold with ChIP buffer and incubated with 2 μg of α-Egr-1 or normal rabbit serum (NRS) at 4°C. Immune complexes were precipitated, washed, and eluted as recommended. DNA–protein cross-links were reversed by heating at 65°C for 4 h, and the DNA fragments were purified and dissolved in 20 μl of water. Two microliters of each sample were used as a template for PCR amplification. Id-1 oligonucleotide sequences for the PCR primers were: Id-1_ChIP Fw: 5′-CGCAAGAAACGCATTCCCAG-3′ and Id-1_ChIP Rev: 5′-AGCGGCTCAG ACCGTTAGAC-3′.

This primer set encompasses the Id-1 promoter segment from nucleotide −1,092 to −905, to generate a 187 bp amplification product which includes the Egr-1-binding site. PCR was performed at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s for 25 cycles. DNA samples were analyzed by electrophoresis on 1.2% agarose gels.

Electrophoretic mobility shift assay

Nuclear extracts were prepared as previously described (Andrews and Faller, 1991). Electrophoretic mobility shift assays (EMSAs) were performed by incubating 10 μg of nuclear extracts with [γ-³²P]-end-labeled double-stranded oligonucleotide probes (5.4 × 10⁴ cpm) in binding buffer containing 10 mM Tris (pH 7.5), 75 mM KCl, 10% glycerol, 0.1 mM EDTA, 2.5 mM MgCl₂, 0.25 mM DTT, and 1.0 μg poly(dI – dC) at 4°C for 20 min. Super shifts were performed by adding 2.0 μg antibody to the extract 30 min prior to incubation with probe. Protein/DNA complexes were resolved on 6% polyacrylamide gel run in 0.5× TBE, at 170 V at room temperature. The primers used for mutagenesis were also used for EMSA.

Generation of Id-1 shRNA lentivirus

The third generation-based ViraPower Lentivirus Expression System (Invitrogen) was used to generate lentiviruses encoding Id-1 or non-targeting (NT) shRNAs (Sigma Mission shRNA). Lentiviruses were produced by co-transfection of 293FT cells with four plasmids in the ratio of 1:4 (5 μg each) using calcium phosphate transfection kit (Promega). The medium was replaced with fresh medium after 8 h. After an additional 40 h the medium containing the viral particles was harvested and subjected to centrifugation, followed by filtration through a 0.45 μm membrane and frozen at −80°C.

Lentiviral transductions of SK-N-MC

SK-N-MC were plated at a density of 1 × 10⁵ cells per well in a six wells plate 48 h prior to infection with lentiviruses encoding Id-1 shRNA, or NT shRNA. In a pilot experiment, we determined the amount of virus that was non-toxic to the cells by diluting the initial stock as follows: undiluted, 1:2, 1:4, 1:8, 1:16, and 1:32. Cells were incubated with the virus for 16 h followed by a change of medium. Efficient reduction of Id-1 expression was evaluated after 24, 48, and 72 h by Western blot.

Cell survival assay

SK-N-MC were plated at a density of 5 × 10⁴ in 12-well plates 48 h prior to infection with lentiviruses encoding Id-1 shRNA, NT shRNA at a 1:4 dilution with complete medium. Twenty-four hours post-infection the medium was replaced with complete medium in the absence or presence of 40 ng/ml FGF-2 and incubated for an additional 1, 24, and 48 h. Cells were collected in 1 ml of DMEM medium and viable cells were counted with GuavaEasy Cyte system by using Guava CytoSoft ViaCount program according to the manufacturer’s recommendations (Guava Technologies, Hayward, CA).

Cell cycle analysis

SK-N-MC cells were infected as previous described. Aliquots of cells, 1 × 10⁵–10⁶/ml, were fixed in 70% ethanol for 30 min at 4°C. Cells were centrifuged at 1,600 rpm and the resulting pellets were resuspended in 200 μl of Guava Cell Cycle Reagent solution (Guava Technologies). Cell cycle distribution was analyzed with GuavaEasy Cyte system by using Guava CytoSoft cell cycle program according to the manufacturer’s recommendations.

FGF-2 activates Id-1 promoter through the ERK1/2 pathway

Previous reports have shown that FGF-2 treatment of SK-N-MC cells can result in a prolonged induction of ERK1/2 (Gentilella et al., 2008; Ma et al., 2008). FGF-2 has been reported to activate the PI3-K pathway in hippocampal neural progenitors (Peltier et al., 2007) and, to a lesser extent the p38 and JNK pathways (Gentilella et al., 2008; Ma et al., 2008). Therefore, in the next series of experiments we have investigated the contribution of the ERK1/2, PI3-K, p38, and JNK pathways in the up-regulation of Id-1. SK-N-MC cells were pre-treated with the MEK1 inhibitor UO126 (15 μM), the p38 MAPK inhibitor SB239063 (10 μM), the JNK inhibitor SP600215 (25 μM), and the PI3-K inhibitor LY24002 (50 μM), following by 1 h treatment with 40 ng/ml of FGF-2. Western blot in Figure 2A illustrates that among all the inhibitors used, only the ERK1/2 inhibitor UO126 (Fig. 2A, lane 3) significantly prevented the induction of Id-1 after 1 h treatment with FGF-2 (Fig. 2A, lane 2), while no major effect on Id-1 induction was observed for p38 MAPK, JNK, or PI3-K inhibitors (Fig. 2A, lanes 4–6).

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Fig. 2

MEK pathway contributes to regulation of Id-1 expression. A: Western blot analysis to detect Id-1 (upper part) in SK-N-MC cells treated with FGF-2 (40 ng/ml) in the absence (c, lane 2) or presence of UO126 (15 μM, lane 3), SB239063 (10 μM, lane 4), SP600215 (25 mM, lane 5), and LY24002 (50 μM, lane 6) after 1 h treatment with FGF-2. Anti-Grb2 antibody (lower part) was used as loading control.

B: Luciferase assay to monitor full-length (2,114 bp) Id-1 promoter activity in SK-N-MC cells after treatment with FGF-2 (40 ng/ml) in the presence or absence of UO126 (15 μM), SB239063 (10 μM), SP600215 (25 μM), and LY24002 (50 μM). Fold activation of luciferase values was determined from three independent experiments after normalization with renilla activity. The asterisk represents P < 0.05. N.S., not statistically significant. C: Western blot analysis to detect Id-1 in SK-N-MC cells treated with FGF-2 (40 ng/ml) in the absence (lanes 2–5) or presence of UO126 (15 μM; lanes 6–9) at the indicated time points. Immunoblot was performed using anti-Id-1 (first part), phosphorylated ERK1/2 (second part), total ERK1/2 (third part), and anti-Egr-1 (fourth part) antibodies. Anti-Grb2 was used as loading control.

Promoter functional assay further confirmed that the activation of transcription of the full-length human Id-1 promoter sequence induced by FGF-2 was reduced in SK-N-MC cells pre-treated with UO126 (Fig. 2B; P < 0.05). Pre-treatment of SK-N-MC cells with the p38 inhibitor SB230963 had no effect on the activation of Id-1 promoter. Treatment with the JNK inhibitor SP600215 resulted in a higher basal level of Id-1 promoter activation, which further increased with the addition of FGF-2. Those changes, however, were not statistically significant compared to the control without inhibitor. LY24002 had only a partial effect on the FGF-2-mediated activation of Id-1 promoter.

The efficacy of UO126 in inhibiting Id-1 transcription was further confirmed by Western blot analysis (Fig. 2C). Similarly to the results depicted in Figure 1A, up-regulation of Id-1 protein was observed between 30 min and 4 h after FGF-2 treatment. FGF-2 treatment results in a marked and sustained phosphorylation of ERK1/2, which also paralleled with increased expression of Egr-1. Pre-treatment of cells with UO126 prior to induction with FGF-2 prevented up-regulation of Id-1 and, as expected, efficiently inhibited ERK1/2 phosphorylation and Egr-1 expression.

To investigate which region in the Id-1 promoter was involved in the FGF-2-mediated activation of transcription, we have generated a series of reporter constructs containing the full-length human Id-1 promoter sequence and seven 5′ deletion mutants (Fig. 3A), and we have tested their responsiveness to FGF-2 treatment in SK-N-MC cells. Figure 3B shows that treatment with FGF-2 enhanced the activity of Id-1 promoter full-length (F1). Luciferase activity was not affected by removal of the Id-1 promoter sequences spanning nucleotides from −2,114 to −986 and the deletion mutants F2, F3, F4, and F5 behaved essentially as the full-length. Conversely, removal of sequences beyond the −986 nucleotide drastically reduced Id-1 promoter activity both at basal level and after FGF-2 induction (F6; P < 0.001). Examination of the Id-1 promoter sequence spanning the region between −986 and −904 using Transfac algorithms (www.biobase-international.com) revealed the presence of overlapping Egr-1/SP-1/YY1 element-binding sites (−959 to −976) and one putative CREB-binding site at position −922 from the transcription start site.

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Fig. 3

FGF-2 induces Id-1 promoter activity. A: Diagram showing the full-length Id-1 promoter sequence spanning from −2,114 to +95 bp, and the various 5′ deletion mutants, all cloned into the pGL3 reporter plasmid. B: FGF-2-induced transcriptional activation of deletion mutants of Id-1 promoter. Fold activation of mutated Id-1 promoters were calculated from FGF-2-induced transcriptional activities over transcriptional activities of the same mutant in the absence of FGF-2. Luciferase values were normalized by renilla values. Results are representative of three independent experiments with standard deviation. The double asterisk represents P < 0.001.

Up-regulation of Id-1 expression involves activity of Egr-1

Based on the results obtained using various inhibitors (Fig. 2) and previous studies showing Egr-1-dependent transcription of Id-1 (Tournay and Benezra, 1996), we next investigated the possible interaction of Egr-1 with the Id-1 promoter sequence spanning the region comprised between −986 and −904 using chromatin immunoprecipation (Chipitsyna et al., 2004) assay (Fig. 4A). Cell lysates obtained from SK-NM-C cells untreated or treated with FGF-2 in the absence or presence of the MEK inhibitor UO126 were immunoprecipited using an Egr-1 antibody or NRS as a negative control. PCR amplification of Id-1 promoter containing the putative Egr-1-binding site was detected in SK-N-MC cells treated with FGF-2 for 1 h (Fig. 4A, lane 2), but not in the sample obtained from untreated cells (Fig. 4A, lane 1). As expected, significantly lower amounts of PCR product were observed in cells pre-treated with UO126 prior to stimulation with FGF-2 (Fig. 4A, lane 3) compared to cells untreated with the inhibitor (lane 2). The specificity of Egr-1 binding to the Id-1 promoter (Fig. 4A, lanes 4–6), equal loading and efficiency of DNA amplification (lanes 7–9) were evaluated by a Chromatin Immuno-Precipitation (Chipitsyna et al., 2004) assay performed with NRS and from the input DNA, respectively. Next, to confirm in vitro interaction between Egr-1 protein and its putative-binding sequences we performed EMSA and super shift analysis using nuclear extracts from SK-N-MC cells under various conditions. Sequence analysis of Id-1 promoter revealed that the predicted Egr-1-binding site differs one nucleotide from the canonic Egr-1-binding motif (Egr-1-binding motif 5′-GCGGGGGCG-3′, Egr-1/Id-1-binding site 5′-GCGCGG GCG-3′). Therefore, in this assay we have utilized the canonical Egr-1-binding motif as a positive control (Fig. 4B). A DNA–Egr-1 complex (asterisk) was detected in nuclear extracts obtained from cells treated for 1 h with FGF-2 when incubated with a DNA probe containing the canonical (Fig. 4B, lane 2) or the Egr-1-binding motif predicted in silico (Fig. 4B, lane 5). The difference in the intensity of the DNA/Egr-1 complex, Egr-1 consensus sequence versus Egr-1 putative sequence (lanes 2 and 5, respectively), might be due to the difference in the nucleotide sequence. Nonetheless, pretreatment of the cell cultures with UO126 followed by FGF-2 stimulation showed a strong reduction of DNA–protein complexes (Fig. 4B, lanes 3 and 6), further corroborating the FGF-2-mediated Egr-1/Id-1 pathway. In addition, mutation of two additional nucleotides in the putative Egr-1-binding site on the Id-1 promoter sequence further inhibited the complex formation (lane 14), confirming the specificity of the binding. Super shift assay in which the nuclear extracts were incubated with an antibody to Egr-1, prevented the formation of the DNA/Egr-1 complex (lanes 7–9), while no changes were observed when nuclear protein (treated under same condition) were incubated with NRS (lane 11).

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Fig. 4

Egr-1 interacts with Id-1 promoter after FGF-2 induction. A: Chromatin Immunoprecipitation assay. SK-N-MC cells were treated with FGF-2 in the presence or absence of UO126. Chromatin-bound Egr-1 was immunoprecipitated using a polyclonal antibody against Egr-1 (lanes 1–3) or NRS (lanes 4–6), followed by PCR amplification using Id-1-specific primers and analyzed by agarose gels. Inputs genomic DNA was used as positive control (lanes 7–9). B: Electrophoretic mobility shift assay (EMSA) was performed using nuclear extracts from SK-N-MC treated for 1 h with FGF-2 (40 ng/ml) in the absence (lanes 2, 5,7, 11, 14) or presence UO126 (15 μM; lanes 3, 6,9, 12,15). Samples were incubate with putative Egr-1-binding site sequences (lanes 1–3), Egr-1-binding site sequences on Id-1 promoter (lanes 4–6) or a mutant with two mutations in the Egr-1-binding site (lanes 13–15). Supershift assays were performed using a polyclonal rabbit antibody against Egr-1 (lanes 7–9) or NRS (lanes 10–12). Asterisk shows Egr-1/DNA complex. Experiments shown in (A)and (B) are representative of three independent experiments. C: Transcriptional activity of pId-1 F5 (−986/+95) deletion mutant and its variant obtained by a mutagenesis of Egr-1-binding site (−976/−968). Fold activation of luciferase values was determined from three independent experiments after normalization with renilla activity. The asterisk represents P < 0.05.

Finally, we have used the Egr-1-binding site mutant described in Figure 4B to confirm the role of Egr-1-binding domain in the activation of Id-1 promoter activity upon treatment with FGF-2. As shown in Figure 4C, a point mutation in the Egr-1-binding site of Id-1 promoter slightly decreased the level of activation of Id-1 transcript (P < 0.05), confirming a potential role of the Egr-1-binding site in the up-regulation of Id-1 upon treatment with FGF-2.

This work was supported by NIMH (MH079751) to F.P.

Contract grant number: MH079751.