Glycobiology Advance Access originally published online on March 3, 2008
Glycobiology 2008 18(5):395-407; doi:10.1093/glycob/cwn016
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The AP-2
transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of a PTEN gene
2 Department of Biological Science, Molecular and Cellular Glycobiology Unit, SungKyunKwan University, 300 Chunchun-Dong, Jangan-Gu, Suwon City, Kyunggi-Do 440-746
3 Division of Molecular and Life Science, Hanyang University, 1271 Sa-1 dong, Sangnok-gu, Ansan, Kyunggi-Do 425-791
4 Department of Biotechnology, Dong-A University, Hadan-Dong, Saha-Gu, Busan 604-714
5 Proteome Research Center, Korea Research Institute of Bioscience and Biotechnology, Yusong-Gu, Daejeon 305-600, Korea
1 To whom correspondence should be addressed: Tel: +82-31-290-7002; Fax: +82-31-290-7015; e-mail: chkimbio{at}skku.edu
Received on October 16, 2007; revised on January 23, 2008; accepted on February 11, 2008
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Abstract |
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Ganglioside GM3 inhibits the growth of several cancer cells and induces cell cycle arrest by regulating cellular signal pathways. Our previous results have shown that GM3 suppresses tumor suppressor PTEN-mediated cancer cell proliferation. However, the precise molecular mechanism(s) for the transcriptional regulation of a PTEN gene induced by GM3 remains unclear. Here, we show, for the first time, that GM3 induces transcription factor AP-2
-mediated PTEN expression in colon cancer cells. The enhanced expression of PTEN by GM3 in both HCT116 and p53-null HCT116 cells has been shown to be not associated with p53 function. Thus, to further determine the mechanism underlying the regulation of PTEN gene expression by GM3, we characterized the promoter region of the PTEN gene. Promoter analysis of the 5'-flanking region of the PTEN gene showed that the region between –1175 and –1077 from the translational initiation site, which contains the AP-2 binding site, functions as the GM3-inducible promoter in colon cancer cells. Furthermore, gel shift assays, site-directed mutagenesis, and chromatin immunoprecipitation assay obviously indicated that the AP-2 is essential for the expression of PTEN in GM3-stimulated colon cancer cells. Moreover, siRNA against AP-2 diminished the enhancement of AP-2 and PTEN expressions in GM3-induced colon cancer cells. The transient expression of AP-2 also results in the induction of PTEN transcription in AP-2-negative colon cancer cells. Additionally, GM3 induced AP-2-mediated PTEN expression through the inhibition of autocrine-ligand-mediated EGFR activation. These results suggest that the AP-2 transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of the PTEN gene.
Key words:
AP-2
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colon cancer cells
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ganglioside GM3
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PTEN
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transcriptional regulation
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflict of interest statementReferences |
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Glycosphingolipids (GSLs) are ubiquitous components of the plasma membrane in all mammalian cells, and they are concentrated in specialized microdomains for cell signaling (Hakomori 2000
). Gangliosides, GSLs with sugar chains containing sialic acid, have been implicated in fundamental cell processes such as proliferation, differentiation, adhesion, and signal transduction (Svennerholm 1980; Hakomori 1990; Varki 1993; Birkle et al. 2003). Specifically, gangliosides are abundant in mammalian neurons and modulate the central nervous system (Svennerholm 1980). One of these, ganglioside GM3, is a precursor for simple and complex gangliosides. Previous studies have shown that GM3 especially plays an important role in the suppression of cancer progression. It has been found that GM3 inhibits an intrinsic tyrosine kinase activity of various receptors such as the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) (Hanai et al. 1988; Sachinidis et al. 1996). Furthermore, ganglioside-specific sialidase gene transfection enhances the activity of EGFR associated with the induction of cell survival, spreading and migration in squamous carcinoma, and ovarial epidermoid carcinoma cells, indicating the activation of EGFR as a result of decreased GM3 (Meuillet et al. 1999; Wang et al. 2003). Additionally, the overexpression of GM3 synthase has been shown to reduce invasion as well as tumorigenesis of bladder cancer cells and to increase cellular apoptosis (Watanabe et al. 2002).
The phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a tumor suppressor gene located at chromosome 10q23.3, are frequently mutated in human cancers including breast, lung, prostate, bladder, and glioblast cancer (Li and Sun 1997; Stambolic et al. 1998; Bonneau and Longy 2000; Dahia 2000). PTEN dephosphorylates phosphatidylinositol 3,4,5-triphosphate (PIP-3) produced by phosphatidylinositol 3-kinase (PI-3K) and negatively regulates cell survival mediated by PI-3K/AKT signaling (Myers et al. 1997; Stambolic et al. 1998). In addition, it displays weak tyrosine phosphatase activity, which may inhibit integrin signaling that involves focal adhesion kinase (FAK) and Shc (Tamura et al. 1998; Yamada and Araki 2001). The heterozygous disruption of the PTEN gene results in the spontaneous development of tumors in several tissues (Di Cristofano et al. 1998). Commonly, the event in cancer cells having the loss of PTEN function is the constitutive activation of AKT (Sun et al. 1999), which protects cells from the apoptotic death by phosphorylating proapoptotic substrates such as Bad, procaspase-9, p21WAF, p27kip1, and forkhead family transcription factors (Nakamura et al. 2000; Mayo and Donner 2002; Hara et al. 2005). PTEN also blocks cell survival induced by degrading a tumor suppressor p53 protein through PI-3K/AKT-mediated MDM2 phosphorylation (Mayo et al. 2002; Choi et al. 2006). Recent studies have shown that the frequent inactivation of PTEN is due to hypermethylation of the promoter in a variety of tumor cells, leading to the inhibition of PTEN gene expression (Goel et al. 2004; Schondorf et al. 2004). Although PTEN significantly functions in the survival or apoptosis of cancer, very little is known about the regulation of PTEN gene expression in response to numerous apoptotic stimuli (Stambolic et al. 2001; Virolle et al. 2001; Vasudevan et al. 2004).
Activator protein 2 is a sequence-specific DNA-binding transcription factor that is known to regulate either activating or repressing effect on the target gene by directly or indirectly (Mitchell et al. 1987; Zutter et al. 1994; Bosher et al. 1995; Hennig et al. 1996; Gille et al. 1997). The protein consists of five homologous members including AP-2, AP-2β, AP-2
, AP-2
, and AP-2
(Williams et al. 1988; Moser et al. 1995; Bosher et al. 1996; Zhao et al. 2001; Tummala et al. 2003). Each of the proteins is encoded by a separate gene and their expression is cell-type specific. AP-2 homo- and heterodimers can activate transcription with a core recognition element sequence of 5'-GCCNNNGGC-3'. Among others, AP-2 plays an important role as the regulator of gene expression during vertebrate development, embryogenesis, and transformation (Schorle et al. 1996; Somasundaram et al. 1996; Zhang et al. 1996; Zeng et al. 1997). Recently, several literatures have elucidated that, in various cancers, loss or reduced expression of AP-2 has been associated with cancer progression, indicating a tumor suppressor gene (Bar-Eli 1999; Heimberger et al. 2005; Schwartz et al. 2007). In addition, a dominant negative mutant of AP-2 is found to expand tumor growth and metastasis in human melanoma cells (Bar-Eli 2001; Gershenwald et al. 2001). Inversely, the overexpression of AP-2 has been shown to suppress the invasion of ovarian cancer cells as well as mammary gland growth and morphogenesis (Zhang et al. 2003; Sumigama et al. 2004). Similarly, in vivo studies involving colon carcinoma have reported a decrease in AP-2 expression levels in Dukes’ stage adenocarcinomas (Ropponen et al. 2001).
Recently, we have been reported that GM3 had an effect on the expression of tumor suppressor PTEN resulting in cell cycle arrest through the up-regulation of p21WAF1 and p27kip1 in colon cancer cells (Choi et al. 2006). However, the precise molecular mechanism for the transcriptional regulation of the PTEN gene induced by GM3 in colon cancer cells remains unclear. In this study, we functionally characterized the ability of the PTEN promoter region to direct the up-regulation of reporter gene transcription in response to GM3. The present results, for the first time, suggest that the AP-2 binding site of the PTEN promoter plays an important role in the transcriptional regulation of the PTEN gene by GM3. In addition, the transcription factor AP-2 has an effect on the upexpression of the PTEN gene in GM3-stimulated colorectal cancer cells.
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Materials and methods |
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Cell line and cell culture
Human colon cancer cell lines HCT116, HCT116 p53(–/–) and RKO were cultured in Dulbecco's modified Eagle's medium (DMEM; JBI, Daegu, Korea) supplemented with 10% fetal bovine serum, 100 unit/mL penicillin, and 100 µg/mL streptomycin at 37°C under 5% CO2. Human colon cancer SW480 cells and HT29 cells were maintained in a RPMI 1640 medium (JBI) supplemented with 10% fetal bovine serum.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA from each cell was isolated using the Trizol reagent (JBI), and 2 µg of RNA was subjected to reverse transcription with the oligo dT primer using AccuPower® RT-PreMix (Bioneer Co., Daejon, Korea), according to the manufacturer's protocol. The cDNA was amplified by PCR with the following primers using EF-Taq polymerase (SolGent, Seoul, Korea): PTEN (460 bp), 5'-TGCAATCCTCAGTTTGTGGTCTGCCA-3' (sense) and 5'-GAAGTTGAACTGCTAGCCTCTGGATTTGA-3' (antisense); AP-2 (1314 bp), 5'-ATGAATTCATGCTTTGGAAATTGACGGATAATATCAAG TAC-3' (sense) and 5'-ATCTCGAGTCACTTTCTGTGCTTC TCCTCTTTGTCACTGC-3' (antisense); and β-actin (247 bp), 5'-CAAGAGATGGCCACGGCTGCT-3' (sense) and 5'-TCCTTCTGCATCCTGTCGGCA-3' (antisense). The use of equal amounts of mRNA in the RT-PCR assay was confirmed by analyzing the expression levels of β-actin. The PCR products were separated by gel electrophoresis on 1.5% agarose-containing ethidium bromide with a 0.5x Tris–acetate–EDTA (TAE) buffer. After electrophoresis, the intensity of the bands obtained from the RT-PCR result was estimated using TotalLab software of the Frog Gel Image Analysis System (CorebioSystem, Seoul, Korea).
Western blot analysis
Cells were homogenized in a buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.02% NaN3, 100 µg/mL phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL aprotinin and 1% Triton X-100. Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad, Richmond, CA). Thirty-microgram samples of total cell lysates were size fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electrophoretically transferred to nitrocellulose membranes using the Hoefer electrotransfer system (Amersham Biosciences, Buckinghamshire, UK). To detect the target protein, we incubated the membranes with the PTEN (NeoMarker, Fremont, CA), AP-2 (Santa Cruz Biotechnology, CA), glyceraldehydes-3-phosphode-hydrogenase (GAPDH) (Chemicon, Temecula, CA), VEGFR-2 (Santa Cruz Biotechnology), p-VEGFR-2 (Cell Signaling, MA), EGFR (Santa Cruz Biotechnology), and p-EGFR (Santa Cruz Biotechnology) antibodies, respectively. Detection was performed using a secondary horseradish peroxidase-linked anti-mouse antibody and an anti-rabbit antibody, and an ECL chemiluminescence system (Amersham Biosciences). After Western blotting, the intensity of the bands obtained from the film result was estimated using TotalLab software of the Frog Gel Image Analysis System (CorebioSystem).
Preparation of plasmids and mutagenesis
For the reporter analysis of the PTEN promoter, the plasmids pro1517, pro621, pro448, and pro65, were prepared and the plasmids constructed previously (Chung et al. 2003). The other constructs were amplified by PCR with the following sense primers containing a BglII site and antisense primer containing a HindIII site, individually: pro1253, 5'-GGAAGATCTTGGGTTTCTGGGCAGAGGCC-3' (sense); pro847, 5'-GGAAGATCTTCAGTTCTCTCCTCTCGGAA-3' (sense); pro454, 5'-GGAAGATCTTGAGTCGCCTGTCACC ATTT-3' (sense); PTEN proR, 5'- GCAAGCTTGTCTGGGA GCCTGTGGCTGAAGAAAAAGGA-3' (antisense) or pro279, 5'-GGAAGATCTGCAGGCTGGCGGCTGGG-3' (sense); pro181, 5'-GGAAGATCTAGAGCTACCGCTCTGCCCCC-3' (sense); and PTEN N-proR, 5'-CCCAAGCTTGAGTCCCGC CACATCACC-3' (antisense). In addition, pro357 was amplified by PCR with the pro1253 (sense) primer and PTEN N-proR (antisense) primer. Amplified DNA fragments were subcloned into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). To construct full-length form of AP-2, PCR amplification with LA-Taq polymerase (Takara Shuzo, Shiba, Japan) was performed with a sense primer AP-2 full 5' containing an EcoRI site (5'-ATGAA TTCATGCTTTGGAAATTGACGGATAATATCAAGTAC-3'), an antisense primer AP-2 full 3' containing an XhoI site (5'-ATCTCGAGTCACTTTCTGTGCTTCTCCTCTTTGTCACT GC-3'), and cDNA from human cervix epithelioid carcinoma cells (Hela) as a template. The PCR product was subcloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA). Each constructed plasmid was confirmed by sequence analysis. Mutation with base substitution at the AP-2 binding site of the PTEN promoter region was accomplished using a QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol with the following oligonucleotide primers: AP2-1-MuF, 5'-AGCAAAAGCTTAATAGCTACACTGGGCATGCTCAG-3'; AP2-1-MuR, 5'-TAGCTATTAAGCTTTTGCTCGGGCCGGT TCCCAG-3'; AP2-2-MuF, 5'-GTAGAAAGCTTAATTTGGGG ACTCTGCGCTCGCAC-3'; and AP2-2-MuR, 5'-CCAAA TTAAGCTTTCTACTGAGCATGCCCAGTG-3' (mutated nucleotides underscored). The presence of mutation was verified by sequence analysis.
Transfection and luciferase assay
Cells were plated onto 6-well plates at density of 105 cells/well and grown overnight. Cells were cotransfected with 0.5 pmol of PTEN promoter-luciferase reporter constructs and 0.5 µg of β-galactosidase reporter plasmid by the WelFect-EXTM PLUS method (JBI). Cells were cultured in a medium containing 10% FBS and incubated with GM3 (Alexsis, CA) for 24 h. Luciferase activity and β-galactosidase activity were assayed by using the luciferase and β-galactosidase enzyme assay system (Promega). Luciferase activity was normalized with the β-galactosidase activity in the cell lysate and calculated as an average of three independent experiments.
Electrophoretic mobility shift assays (EMSAs)
Nuclear extracts from resting and GM3-induced HCT116 cell were prepared as described previously (Chung et al. 2003). EMSAs were performed using a gel shift assay system kit (Promega) according to the manufacturer's instructions. Briefly, double-stranded oligonucleotides containing the consensus sequence for AP2-1 (5'-CCGAGCAAGCCCCAGGCAGC TACAC-3') and AP2-2 (5'-CTCAGTAGAGCCTGCGGCTT GGGGACT-3') were end-labeled with [-32P]ATP using T4 polynucleotide kinase and used as probes for EMSA. Nuclear extract proteins (2 µg) were preincubated with the gel shift-binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM ethylenediamine tetra-acetic acid, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), and 0.05 mg/mL poly(deoxyinosine-deoxycytosine)] for 10 min and then incubated with the labeled probe for 20 min at room temperature. For the sample of a supershift assay, anti-AP-2, -SP1, -SP3, -Ets-1, and p53 antibodies were preincubated with the nuclear extract prior to the addition of labeled probes. Each sample was electrophoresed in a 4% nondenaturing polyacrylamide gel in a 0.5x TBE buffer at 250 V for 2 h. The resulting gel was dried and subjected to autoradiography.
Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed as outlined by the ChIP assay kit (Upstate Biotechnology, NY). DNA-binding proteins were crosslinked to DNA and lysed in a SDS lysis buffer containing 1x protease inhibitors. DNA was sheared to 200–500 bp fragments by 30 s sonications, each using a sonicator VC100 (Sonics & Materials Inc., Danbury). The chromatin solution was precleared with salmon DNA/protein A agarose 50% slurry (Upstate Biotechonolgy) for 30 min at 4°C. The precleared supernatant was incubated with anti-AP-2, SP1 polyclonal antibodies (Santa Cruz Biotechnology) overnight at 4°C. The region between –1268 and –970 of the PTEN promoter was amplified from the immunoprecipitated chromatin using the following primers: sense, 5'-TACCCTGCCTCCGGCTGGGTTT-3' and antisense, 5'-AAGACCGAGGGGAGGCGGGA-3'. The 299-bp PCR product was separated on a 2% agarose gel containing ethidium bromide and visualized under UV light.
Preparation and transfection of small interference RNAs (siRNAs)
An AccuTargetTM siRNA Set targeting TFAP2A (AP-2) was purchased from Bioneer Corporation. The coding sequence of plant chlorophyll a/b-binding protein mRNA of siRNA duplex was synthesized as a negative control by Bioneer Corporation. HCT116 cells were transfected with an AP-2 siRNA and a negative control siRNA, respectively, using WelFect-EXTM PLUS (JBI), according to the manufacturer's instructions. One day after transfection, transfection complexes were removed and replaced with a culture medium. After incubating with GM3 in the culture medium for 12 h, the transfected cells were used for experiments.
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Results |
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GM3 promotes the expression of the PTEN gene
To determine whether GM3 has the ability to regulate the expression of the PTEN gene, HCT116 colon cancer cells were exposed to various concentrations (0, 10, 30, 50 µM) of GM3 and were incubated for 0, 12, or 24 h after the treatment of 30 µM GM3. As shown in Figure 1A, the mRNA expression of the PTEN gene was increased gradually up to a concentration of 50 µM, and also increased dramatically for periods of 24 h at a concentration of 30 µM. In other human colon cancer cell lines of HT29, RKO, and SW480 cells, we further confirmed the enhancement of PTEN expression by GM3 at the transcription level. The RT-PCR analysis showed that GM3 up-regulated the PTEN expression in HT29 and RKO but not SW480 cells (Figure 1B). With regard to PTEN regulation, Stambolic et al. (2001
) demonstrated that the transcriptional activation of the PTEN gene was regulated by a transcription factor p53. Thus, we also investigated whether GM3 regulates the p53-mediated PTEN transcription using HCT116 cells lacking p53. However, as shown in Figure 1C, although the regulation of PTEN transcription by the p53 protein was known, GM3 in p53-negative HCT116 cells had an effect on the increase of PTEN expression as evidenced by RT-PCR and western blot analysis. These results indicate that enhanced PTEN expression by GM3 is not accompanied by the regulation of p53.
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Localization of the GM3-dependent positive element in the human PTEN promoter
To further confirm the region regulating the transcription activity of the PTEN gene in GM3-induced HCT116 cells, a genomic fragment containing 1517 bp of 5'-promoter region of the PTEN gene was subcloned into a pGL3-basic vector (pro1517). The luciferase constructs carrying 5'-deleted PTEN gene promoters (pro1253, pro847, and pro454 constructed from the translation initiation site and pro621, pro448, pro357, pro279, pro181, and pro65 constructed from the transcriptional start site) were also prepared (Figure 2A, left). After transfecting the luciferase constructs into HCT116 colon cancer cells and p53-negative HCT116 cells, we checked the activity of the PTEN gene promoter induced by GM3. As shown in Figure 2A, the region of the PTEN promoter localized between –1175 and –1077 from the translation initiation site (between pro 279 and pro 181) reflected the existence of several putative factors for the transcriptional regulation of the PTEN gene in GM3-induced both HCT116 colon cancer cells and p53-negative HCT116 cells. As shown in Figure 2B, analysis of a 621-bp genomic fragment corresponding to the region of the PTEN promoter between –1517 and –897 (pro621) using Matlnspector v6.2 program (core similarity 1.0, matrix similarity 0.75) of Genomatix revealed the existence of several putative transcription factor binding sites such as MZF-1, NF-
B, ATF-2, SP-1, AP-2, p53, EGR-1, and others. Furthermore, the 98-bp region between –1175 and –1077 from the translation initiation site, which was associated with the transcriptional activity of the PTEN promoter (between pro 279 and pro 181) induced by GM3, contained two AP-2 DNA binding sites and p53 binding site. Our previous data (Figures 1 and 2) clearly showed that GM3 induced p53-independent PTEN expression. Thus, we suggest that GM3 may regulate AP-2-mediated transcriptional activation of the PTEN gene.
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The AP-2
is essential for the transcriptional regulation of the PTEN gene in response to GM3Our previous study has determined that GM3 induces PTEN expression through a p53-independent mechanism. Virolle et al. (2001
) has reported that the EGR-1 transcription factor directly activates PTEN during irradiation. However, GM3 had no effect on the luciferase activity of pro65 which contains the region between –962 and –897 from the translation initiation site of the PTEN gene and two EGR-1 binding sites (Figure 2). Additionally, Vasudevan et al. (Vasudevan et al. 2004) has demonstrated that NF-B negatively regulates tumor suppressor PTEN expression. Thus, we examined whether GM3 modulates PTEN expression by NF-B down-regulation. However, the expression of p65 protein, a subunit of a heterodimeric NF-B transcription activator, was not down-regulated by GM3 in HCT116 colon cancer cells. Furthermore, EMSA data showed that the intensity levels of the p65-shift bands in the nuclear lysates from GM3-induced HCT116 cells were unchanged compared to GM3-untreated HCT116 cells (data not shown). Therefore, we assumed that the transcriptional activity of PTEN could be regulated by GM3 through the two AP-2 binding sites. To evidence that the PTEN expression by GM3 is related to the AP-2 transcription factor, we investigated the effect of GM3 on the expression of AP-2. Interestingly, as shown in Figure 3A, the transcription of AP-2 was significantly increased in GM3-treated HCT116 cells as evidenced by RT-PCR. Western blot analysis also showed the gradual increase of AP-2 expression for periods of up to 24 h in the nuclear extract of GM3-treated HCT116 cells (Figure 3B). To determine whether the AP-2 binding site is associated with the regulation of PTEN transcription in HCT116 cells induced by GM3, we performed EMSA using double-stranded 32P-labeled oligo fragments containing the consensus sequence for specific AP2-1 and AP2-2 of the PTEN promoter and nuclear extract from GM3-treated HCT116 cells. As shown in Figure 3C, a complex that binds to 32P-labeled AP2-1 and AP2-2 oligo nucleotides was detected and the intensity levels of the AP-2-shifted bands in the nuclear lysates from GM3-induced HCT116 cells were higher than the nuclear lysates from GM3-uninduced HCT116 cells. These bindings were competed with a 100-fold molar excess of unlabeled AP2-1 and AP2-2 oligo fragments itself. To further clarify whether this band of DNA-protein complex containing two AP-2 oligo nucleotides (AP2-1 and AP2-2) could be associated with AP-2 protein, we performed a supershift assay in the presence of an anti-AP-2 antibody. As shown in Figure 3C, the incubation of nuclear extracts with the anti-AP-2 antibody resulted in a supershift of the observed complexes. The addition of anti-Ets-1, p53, SP1 and SP3 antibodies did not produce a supershifted complex but the anti-SP1 antibody did reduce the intensity of the shifted complex in extracts from the GM3-treated HCT116 cells. To better understand the interplay between AP-2 and SP1 family members in the regulation of the PTEN promoter in vivo, we analyzed a 299-bp fragment that spans the –1268 to –970 regions using a chromatin immunoprecipitation assay (Figure 3D). Interestingly, in the GM3-stimulated HCT116 cells, AP-2 was bound to the distinguished activity region of the PTEN promoter but not SP1. Although our gel shift data showed that AP-2 weakly bound to the –279 to –182 region in vitro, it clearly certified to its binding in vivo. In p53-negative HCT116 cells induced by GM3, we obtained the same results using Western blot and EMSA analysis like GM3-stimulated HCT116 cells (Figure 3E and F). To additionally confirm that GM3 is directly involved in the AP-2-mediated transcriptional activation of PTEN, AP2-1 Mu, AP2-2 Mu, and AP-2 double Mu were constructed by mutating AP-2 binding sites from pro279 plasmid using a site-directed mutagenesis kit (Figure 4A). As shown in Figure 4B, in GM3-treated HCT116 cells, the promoter activity of pro279 including two AP-2 binding sites was remarkably increased compared to the control vector pGL3-Basic. However, the promoter activity of both AP2-1 Mu and AP2-2 Mu, which were mutated at –1109 to –1101 and –1143 to –1135 of the PTEN promoter regions, respectively, was significantly reduced compared to pro279 in response to GM3. Furthermore, the promoter activity of AP-2 double Mu which was mutated at both AP2-1 and AP2-2 binding sites markedly reduced compared to pro279 in GM3-treated HCT116 cells. These results clearly show that AP-2 binding sites are required for the GM3-induced expression of PTEN and that the AP-2 binding to these sites is involved in the induction of PTEN by GM3.
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and AP-2 mutation elements are shown by 
. The transfected cells were incubated in the presence (solid bars) and absence (open bars) of 30 µM GM3 for 24 h. Relative luciferase activity was normalized with β-galactosidase activity derived from pCMVβ. The values represent the mean ± SD for three independent experiments with triplicate measurements.Transcription factor AP-2
enhances PTEN expressionPreferentially, our data showed that the expression of AP-2
in AP-2-positive Hela, AP-2-negative SW480, and HCT116 colon cancer cells was verified by Western blot analysis in the cytosol and nuclear extractions (Figure 5A). To further confirm AP-2 function, we investigated whether GM3 induces the enhanced PTEN transcription in AP-2-null SW480 colon cancer cells. Owing to SW480 cells lacking endogenous AP-2 expression, we found no change for the induction of PTEN transcription in response to GM3, as evidence by RT-PCR analysis (Figures 1C and 5B). To assess the contribution of the AP-2 transcription factor to tumor suppressor PTEN expression, we re-expressed transiently the AP-2 gene cloned into the pcDNA3 vector in the AP-2-null SW480 cells. RT-PCR as well as Western blot analysis showed that both AP-2 mRNA and protein in the AP-2-transfected SW480 cells were highly expressed, compared to the pcDNA3-transfected control cells (Figure 5B). Subsequently, we examined the extent of PTEN expression in the AP-2-transfected SW480 cells at transcription and translation levels. Interestingly, we confirmed for the first time that PTEN expression in the AP-2-transfected SW480 cells was induced, compared to pcDNA3 vector-transfected control cells (Figure 5B). To further investigate whether AP-2 could regulate the transcription of PTEN, pro279 construct of the PTEN promoter having the best activity and containing the AP-2 binding site was cotransfected with the AP-2 gene or the empty vector pcDNA3 into SW480 cells. Interestingly, the promoter activity of pro279 reporter in AP-2-transfected SW480 cells was increased 2-fold higher than that of the pcDNA3-transfected SW480 cells (Figure 5C). These results demonstrate for the first time that the transcription factor AP-2 is associated with PTEN expression.
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siRNA targeting an AP-2
gene reduces the expression of the PTEN gene induced by GM3To confirm whether the suppression of the AP-2
level by RNA interference in GM3-treated cells results in the reduction of PTEN expression, three siRNAs (siRNA 1, siRNA 2, and siRNA 3) against AP-2 and a siRNA as a negative control were utilized to the HCT116 colon cancer cells. As shown in Figure 6A, the expression of AP-2 induced by GM3 was apparently repressed in cells transfected with siRNAs 1 and 2 targeting AP-2, compared with GM3-exposed cells without AP-2 siRNA or GM3-treated cells transfected with a negative control siRNA, as determined by RT-PCR. Furthermore, the suppression of AP-2 expression by AP-2 siRNAs 1 and 2 in GM3-treated cells results in the decreased PTEN expression, compared with GM3-treated cells without AP-2 siRNA or GM3-treated cells transfected with a negative control siRNA, as evidenced by Western blot analysis (Figure 6B). These results clearly indicate that GM3 regulates AP-2-mediated transcription of PTEN in HCT116 colon cancer cells.
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Inhibition of EGFR activation by GM3 raises the level of an AP-2
proteinWe explore what is an upstream signaling pathway regulating AP-2
signaling. It is well known that GM3 inhibits EGFR tyrosine kinase (Hanai et al. 1988; Wang et al. 2003; Yoon et al. 2006). Thus, we checked whether GM3 suppresses EGFR activation in HCT116 colon cancer cells. As shown in Figure 7A, autoactivation of EGFR in HCT116 cells was inhibited by GM3. Furthermore, as described in Figures 1 and 3, our previous data have clearly shown that GM3 induces both expressions of AP-2 and PTEN. To further confirm whether EGFR activation leads to the regulation of AP-2 and PTEN expressions, we treated HCT116 cells with AG1478 known as a specific inhibitor of EGFR tyrosine kinase in HCT116 cells. As shown in Figure 7B, AP-2 and PTEN proteins were gradually increased in HCT116 cells from nuclear and cytosol fractions by AG1478 treatment in dose-dependent manners. Additionally, we investigated whether the inactivation of VEGFR by GM3 modulates AP-2 and PTEN expressions in HCT116 cells, which express vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR). However, although GM3 suppressed phosphorylation of VEGFR-2 in HCT116 cells (Figure 7C), VEGFR-2 inhibitor I (Calbiochem, CA) has no effect on AP-2 and PTEN expressions (Figure 7D). These results suggest that GM3 does not affect AP-2-mediated PTEN regulation through the VEGFR-2 signaling pathway. These results suggest that GM3 increases AP-2 expression through the suppression of autocrine ligand-mediated EGFR activation in HCT116 cells.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflict of interest statementReferences |
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The PTEN gene has been shown to be deleted or mutated in many human cancers including endometrial neoplasm, hematological malignancies, prostate, breast, and other cancer types (Ali et al. 1999
; Cantley and Neel 1999; Dahia 2000; Di Cristofano and Pandolfi 2000). Germ-line mutation in PTEN is found in autosomal dominant-inherited cancer syndromes, including Cowdens disease, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome (Liaw et al. 1997; Myers and Tonks 1997; Marsh et al. 1998; Cantley and Neel 1999). It is well known that PTEN, a tumor suppressor gene, negatively controls the PI-3K signaling pathway for the regulation of cell growth and survival (Stambolic et al. 1998; Sun et al. 1999). However, very little is known that the regulation of PTEN expression can be altered by various biological stimuli. It was reported that PTEN expression decreased in human keratinocytes induced by TGF-β (Li and Sun 1997) and dramatically increased during monocytic/granulocytic differentiation of myeloid leukemia cells (Hisatake et al. 2001), and also induced during neuronal differentiation (Ross et al. 2001). It has been reported that the p53, EGR-1, and NF-B (p65) transcriptional factors regulate PTEN transcription and functional p53, EGR-1, and NF-B binding sites were identified in the regions between –1190 and –1157 and between –947 and –939, and in nucleotide positions –1311, –1208, and –819 at upstream of translation start codon (Stambolic et al. 2001; Virolle et al. 2001; Vasudevan et al. 2004), respectively. This observation suggests that p53, EGR-1, and NF-B may be able to modulate the PI-3K/Akt/PKB pathway via regulating PTEN expression. Previously, we have shown that GM3 augmented the expression of PTEN and eventually it caused cell cycle arrest by accumulating p21WAF1 and p27kip1 in HCT116 colon cancer cells (Choi et al. 2006). Subsequently, in this study, we have demonstrated for the first time that GM3 regulates the PTEN transcription in colon cancer cells and it is due to AP-2 function on the transcriptional activity of the PTEN promoter containing two AP-2 binding sites. That is to say that GM3 modulates AP-2-mediated PTEN expression in HCT116 colon cancer. Here, our results show that the expression of PTEN mRNA strikingly increased up to 24 h after GM3 treatment in HCT116 cells. The PTEN gene also increased in a dose-dependent manner upon GM3 treatment. We further checked the induction of PTEN by GM3 in other colon cancer cells such as HT29 and RKO. GM3 also increased PTEN expression in HT29 and RKO cells. In accordance with Stambolic et al., it has been reported that the expression of PTEN was associated with the p53 protein. Furthermore, because our previous data have shown that GM3 induced PTEN expression in HCT116 cells, we guessed that GM3 does not induce in p53-lacking HCT116 cells. However, our data show that PTEN expression by GM3 was also increased in p53-null HCT116 cells. These data suggest that GM3 regulates p53-independent PTEN transcription in colon cancer cells.
Our results in this study show that the region between –1175 and –1077 of the PTEN promoter functions as the core promoter for the transcriptional activation of PTEN in GM3-induced HCT116 cells. This region as the GM3-inducible PTEN promoter in HCT116 cells contains several transcription factor binding sites such as CP2, AP2, p53, MUSCLE INI, and NF-B. Our present results obtained by site-directed mutagenesis analyses indicate that two AP-2 elements mediate GM3-dependent up-regulation of PTEN expression. Although the intensity levels of AP-2-shifted bands were faintly increased in EMSA and supershift results, we demonstrate that AP-2 binds to this site of the PTEN promoter in GM3-stimulated HCT116 cells, as evidenced by the ChIP assay. In addition, we show that GM3 raises the level of the AP-2 gene and protein in a time-dependent manner, being demonstrated by RT-PCR and Western blotting. Therefore, our data strongly suggest that AP-2 plays an important role in PTEN transcription induced by GM3.
The AP-2 family members regulate the expression of a variety of genes in differentiated tissues and tumor cells. There may also be differences in the functional activity of different family members. AP-2 can directly activate the promoter of several central growth- and differentiation-related genes such as p21WAF1/CIP1, Estrogen Receptor, SV40 enhancer region, and HER-2/neu (Mitchell et al. 1987; Bosher et al. 1996; Zeng et al. 1997; McPherson and Weigel 1999). Moreover, AP-2 proteins share similarities in their trans-activating properties for their structural conservation (McPherson and Weigel 1999) and the regulatory effect of AP-2 is more complex because of the AP-2 interacting molecules, which modify the transcriptional activity of AP-2. They include DNA binding factors such as p53, Sp1, Myc, retinoblastoma protein (RB), polyADP-ribose polymerase (PARP), adenomatosis polyposis coli (APC), and p300/cAMP response element-binding (CREB) protein (Batsche et al. 1998; Kannan et al. 1999; McPherson et al. 2002; Braganca et al. 2003; Li and Dashwood 2004; Mitchell et al. 2006). Among AP-2 family members, AP-2 acts as a tumor suppressor or suppressor-like activity evidenced by previous studies. In pancreatic ductal adenocarcinoma (DAC), AP-2 represses cell proliferation and MUC4 expression (Fauquette et al. 2007). In hepatoblastoma and colon carcinoma cell lines, AP-2 induces the expression of p21WAF1/CIP1, which has been thought to be the basis for the control of cell growth by AP-2 (Zeng et al. 1997; Wajapeyee and Somasundaram 2003). The loss of AP-2 expression is associated with an increase in tumorigenicity of colon and prostate cancers as well as the grade of human gliomas (Ruiz et al. 2004; Heimberger et al. 2005; Schwartz et al. 2007). The overexpression of AP-2 results in an inhibition of colony formation or induces apoptosis by repressing Bcl-2 (Wajapeyee et al. 2006). Based on these reports and our findings evidenced by GM3-induced PTEN promoter activity, we suggested that an AP-2 protein could be a candidate as a positive regulator of PTEN transcription in GM3-stimulated HCT116 cells. Our previous data have shown that GM3 induced PTEN transcription in colon cancer cells such as HCT 116, HT29, and RKO cells but not SW480 cells. Interestingly, we found that GM3 had no effect on PTEN transcription because of the lack of AP-2 expression in SW480 cells. Thus, to assess the role of AP-2 in PTEN expression, we established transient transfectants expressing AP-2 in SW480 cells. The re-expression of AP-2 resulted in the significant augmentation of PTEN expression without GM3 in AP-2-negative SW480 cells. We also observed enlargement in the activity of the PTEN promoter in the AP-2-transfected SW480, which can explain that AP-2 is an intermediate leading to PTEN transcription by GM3. On the other hand, the targeting expression of AP-2 in HCT116 cell with a siRNA resulted in down-regulation of PTEN expression although GM3 exists in HCT116 cells. These results obviously demonstrate for the first time that the AP-2 is required for the GM3-stimulated transcriptional regulation of the PTEN gene.
The lipid moiety of the exogenous gangliosides mostly appears to be inserted into lipid bilayer of the plasma membrane (Callies et al. 1977; Radsak et al. 1982). Our previous results show that GM3 is localized on the surface of the membrane (Choi et al. 2006). For this reason, GM3 has been implicated in a variety of cell-surface events such as recognition phenomena and membrane-mediated signaling transduction (Laine and Hakomori 1973; Fishman and Brady 1976; Hakomori 1981; Hakomori 1990). Exogenously treated GM3 is rapidly incorporated into a plasma membrane and is responsible for numerous biological effects in vitro (Laine and Hakomori 1973; Choi et al. 2006). For example, the EGF-mediated proliferation of epidermal cells is inhibited by GM3 interaction with N-linked GlcNAc termini of a receptor (Yoon et al. 2006). GM3 also suppresses the PDGF- and basic fibroblast growth factor-mediated proliferations of fibroblast cells and neuroblastoma cells for blocking receptor dimerization (van Brocklyn et al. 1993; Rusnati et al. 1999). Based on these studies, we hypothesized that the suppression of receptor activity on the cell membrane associated with the proliferation of colon cancer cells by GM3 might relate to the regulation of AP-2 and PTEN expressions. The VEGF expressed in many solid tumors induces angiogenesis by interacting with a VEGFR expressed in the endothelial cell (Ferrara et al. 2003; Ferrara 2004). Furthermore, VEGF and VEGFR expressions in several human tumors stimulate tumor survival and proliferation through an autocrine pathway (Masood et al. 2001). Thus, we investigated whether the inactivation of VEGFR by GM3 modulates AP-2 and PTEN expressions in HCT116 cells having VEGF and VEGFR expressions. However, our results indicated that GM3 does not affect AP-2-mediated PTEN regulation through the VEGFR-2 signaling pathway. Whereas, it is well known that GM3 inhibits EGFR tyrosine kinase (Hanai et al. 1988; Wang et al. 2003; Yoon et al. 2006). The Brattain group has shown that autocrine TGF- as a member of the EGF family in the highly tumorigenic HCT116 cells induces the activation of EGFR associated with cell growth via interaction with EGFR (Howell et al. 1998). Thus, we examined whether GM3 suppresses the EGFR activation in HCT116 colon cancer cells. This study showed that the autoactivation of EGFR in HCT116 cells was inhibited by GM3. Furthermore, we further investigated whether EGFR activation has an effect on AP-2 and PTEN expressions. The treatment of HCT116 cells with AG1478 known as an inhibitor of EGFR tyrosine kinase activity resulted in the induction of AP-2 and PTEN expressions. Taken together, GM3 increases AP-2 expression through the suppression of autocrine ligand-mediated EGFR activation in HCT116 cells.
In conclusion, as illustrated in Figure 8, we demonstrate for the first time that the simple ganglioside GM3 modulates AP-2 expression through the EGFR pathway and the AP-2 is essential for the transcriptional regulation of the PTEN gene in response to GM3 for the suppression of cell proliferation and cell cycle arrest.
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Conflict of interest statement |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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This study was in part supported by 21C Frontier Research for Human Genome, KOSEF, funded by the Korean Government (no. FG-1-2 to C.-H. Kim) and the Korea Health 21 R&D Project, Ministry of Health and Welfare, Korea (A060052). H.J. Choi is a recipient of BK21 Biological Science of SKKU.
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Abbreviations |
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EMSA, electrophoretic mobility shift assays; FAK, focal adhesion kinase; GAPDH, glyceraldehydes-3-phosphode-hydrogenase; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; siRNAs, small interference RNAs; VEGFR, vascular endothelial growth factor; VEGFR, VEGF receptor
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