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Endocrinology, doi:10.1210/en.2003-1661
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Endocrinology Vol. 145, No. 9 4222-4231
Copyright © 2004 by The Endocrine Society

Expression of Pituitary Tumor Transforming Gene (PTTG) and Its Binding Protein in Human Astrocytes and Astrocytoma Cells: Function and Regulation of PTTG in U87 Astrocytoma Cells

Jacob Tfelt-Hansen, Shozo Yano, Sanghamitra Bandyopadhyay, Rona Carroll, Edward M. Brown and Naibedya Chattopadhyay

Division of Endocrinology, Diabetes, and Hypertension (J.T.-H., S.Y., E.M.B., N.C.), Department of Medicine and Membrane Biology Program and Department of Neurosurgery (R.C.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; Osteoporosis and Bone Metabolic Unit (J.T.-H.), Department of Clinical Biochemistry and Endocrinology, Copenhagen University Hospital Hvidovre, DK-2650 Copenhagen, Denmark; and Genetics and Aging Research Unit (S.B.), Department of Psychiatry-Neuroscience, Massachusetts General Hospital, Charlestown, Massachusetts 02129

Address all correspondence and requests for reprints to: Jacob Tfelt-Hansen, Laboratory of Molecular Cardiology, Department of Cardiology, University of Copenhagen, 20 Juliane Maries Vej, Section 9312, DK 2100 Copenhagen O, Denmark. E-mail: tfelt{at}dadlnet.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human securin, pituitary tumor transforming gene (PTTG), is a protooncogene. Here we report expressions of PTTG and its interacting protein, PTTG-binding factor in human astrocytic cells. PTTG expression was higher in malignant cells than in primary astrocytes, whereas PTTG-binding factor was not. Using a xenotransplantable, glioma cell line (U87), we observed that knocking down PTTG mRNA by RNA silencing inhibited serum-induced proliferation by approximately 50%. Furthermore, in U87 cells PTTG expression was up-regulated by promalignant ligands epithelial growth factor (EGF) and TGF{alpha}, both at the protein and mRNA levels. PTTG induction by EGF receptor (EGFR) ligands could be blocked by the specific EGFR inhibitor, AG1478. Hepatocyte growth factor (HGF) also induced PTTG but to a lesser extent than EGF. Although EGF stimulates HGF secretion in U87 cells, the effect of EGF on PTTG mRNA expression is independent of HGF as neutralizing antibody against HGF failed to abolish EGF-induced up-regulation of PTTG mRNA. PTTG mRNA was unchanged by incubating U87 cells with the promalignant growth factor TGFß, apoptosis inducing TNF{alpha} and ligands for nuclear receptors, such as retinoic acid and retinoid X receptors and peroxisome proliferator-activated receptor-{gamma}, known for their growth-inhibitory and apoptosis-inducing effects on gliomas. In addition, 17ß-estradiol and Ca2+, known to activate PTTG expression, did not change PTTG mRNA levels in U87 cells. In summary, we show higher PTTG expression in astrocytoma than normal astrocytes and secondly, PTTG is involved in glioma cell growth. Finally, regulation of its expression has glioma-specific features and is selectively regulated by promalignant cytokines including EGFR ligands and HGF.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY TUMOR TRANSFORMING gene (PTTG), the human homolog of securin, was discovered in rat pituitary adenoma cells by differential mRNA display PCR (1). Soon thereafter the human gene for this oncogene was cloned from human testis (2). Roles of PTTG in cell cycle progression, chromosomal stability, and cell division have been revealed from studies in mice in which PTTG was deleted (3). PTTG participates in the initiation of sister-chromatid separation during the anaphase (4), and its overexpression causes aneuploidy in live cells (5). It has been shown to induce proliferation and transformation in vitro and promote tumor formation in athymic nude mice harboring PTTG overexpressing NIH-3T3 fibroblasts (1, 6). Another promalignant characteristic of PTTG overexpression is the induction of angiogenesis through the production of basic fibroblast growth factor (bFGF) (7). Besides its tumorpromoting actions, PTTG also plays an important role in normal developmental processes such as the proliferation of developing ß-cells of pancreatic islets (8).

The current understanding of the regulation of PTTG expression is scanty. However, we do know that it is inhibited by cyclosporin A and hydrocortisone in T lymphocytes (9), up-regulated by estrogen and bFGF and down-regulated by a peroxisome proliferator-activated receptor (PPAR)-{gamma} agonist, rosiglitazone, in pituitary adenomas (10, 11); and up-regulated by extracellular Ca2+ acting via calcium-sensing receptor in H-500 testicular Leydig cancer cells (12).

PTTG is a multifunctional protein that regulates mitosis, gene regulation, cell transformation, and DNA repair. Several of these actions are mediated via its interaction with PTTG-binding factor (PBF) (13). In the central nervous system, PTTG expression has very recently been detected in developing neurons and has been implicated in human neurogenesis as an important cell cycle regulator. Astrocytes comprise the most abundant glial cells in the central nervous system and maintain the blood-brain-barrier. Also, astrocyte transformation leading to malignancy is the most common, and one of the most aggressive, forms of adult cancer. The purpose of this study was: 1) to determine whether PTTG and its interacting protein, PBF, are expressed in human astrocytic cells (both primary cells and cell lines), 2) investigate whether there is any correlation in PTTG mRNA expression between normal and transformed astrocytes and their grades of malignancy, 3) determine whether PTTG regulates cellular proliferation of glioma cells; and 4) profile the regulation of PTTG expression by promalignant, antimalignant, and apoptosis-inducing agents, particularly those relevant in the context of glioblastoma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
All routine culture media were obtained from Life Technologies, Inc.-BRL (Grand Island, NY). Human primary astrocytes were purchased from Clonetics-Biowhittaker (Walkersville, MD), and U87 cells from the American Type Culture Collection (Manassas, VA) and were maintained in monolayer culture in DMEM supplemented with 10% fetal bovine serum. TGF{alpha}, epithelial growth factor (EGF), 17ß-estradiol, bone morphogenetic protein (BMP)-2, TGFß, TNF{alpha}, TNF-related apoptosis-inducing ligand (TRAIL), a retinoic acid receptor ß/{gamma}-specific ligand (TTNP3), I3CRA, ciglitazone, and prostaglandin J2 (PGJ2) were obtained from Calbiochem (La Jolla, CA).

Specimen selection and tissue samples
Nine glioma samples were collected at the time of surgery in patients who underwent craniotomy for glioma resection with human study approval. All operations were performed by Dr. Peter Black at the Brigham and Women’s Hospital in Boston. A senior neuropathologist at the hospital evaluated all specimens and classified them in accordance with World Health Organization standard criteria. At the time of surgery, all tissue specimens were immediately snap frozen and stored in liquid nitrogen for RNA isolation. Primary astrocytes were obtained from Clonetics (San Diego, CA) that are derived from a fetal source. The primary astrocyte cultures are more than 90% pure as certified by the company obtained by assessing glial fibrillary acidic protein expression. U87, T98G, and U343 are all malignant glioma cell lines [derived originally from human high-grade astrocytoma (AS), American Type Culture Collection].

Northern blot analysis
To study whether TGF{alpha} affects the expression of PTTG mRNA, we performed Northern blot analysis as described elsewhere (14). In brief, cellular RNA was isolated (15) using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Poly (A+) RNA recovered was quantitated by spectrophotometry, and aliquots of 2.5 µg total RNA from U87 cells incubated with or without EGF or TGF{alpha} were loaded on a formaldehyde agarose gel after denaturation (14). The gel was stained with ethidium bromide to visualize RNA standards and rRNA to document equal loading of RNA from the various experimental samples. The RNA was then blotted onto nylon membranes (Duralon, Stratagene, La Jolla, CA). Blots were hybridized with a cDNA probe for PTTG and washed under high-stringency conditions as described previously (16). Equal loading was also confirmed by reprobing the membranes with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Specific radioactive signals were analyzed on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) with the ImageQuant program.

Quantitative real-time
PCR To amplify human PTTG1 (assignment no. NM_004219) and human GAPDH cDNA, sense and antisense oligonucleotide primers were designed based on the published cDNA sequences using the Primer Express (version 2.0.0, Applied Biosystems, Foster City, CA). Oligonucleotides were obtained from Genosys (Woodlands, TX). The sequences of the primers were as follows: 5'-CGG CTG TTA AGA CCT GCA ATA ATC-3' (PTTG sense, 18–41), 5'-TTC AGC CCA TCC TTA GCA ACC-3' (PTTG antisense, 119–99), 5'-TTC AAT GGC ACA GTC AAG GC-3' (GAPDH sense), and 5'-TCA CCC CAT TTG ATG TTA GCG-3' (GAPDH antisense). cDNA was synthesized with the Omniscript reverse transcription (RT) kit (Qiagen, Valencia, CA) using 2 µg total RNA in a 20-µl reaction volume. For real-time PCR, the cDNA was amplified using an ABI PRISM 7000 sequence detection system (PE Applied Biosystems). The double-stranded DNA-specific dye SYBR Green I incorporated into the PCR buffer QuantiTech SYBR PCR (Qiagen) to allow for quantitative detection of the PCR product in a 25-µl reaction volume. The temperature profile of the reaction was 95 C for 10 min, 40 cycles of denaturation at 95 C for 15 sec, annealing at 60 C for 30 sec, and extension at 72 C for 30 sec. An internal housekeeping gene control, GAPDH, was used to normalize differences in RNA isolation, RNA degradation, and the efficiencies of the RT. The size of the PCR product was first verified on a 1.5% agarose gel, followed by melting curve analysis.

Western blotting
For the determination of PTTG protein levels, monolayers of U87 cells were grown on six-well plates. Cells were incubated for 48 h with or without 30 ng/ml EGF or 100 ng/ml TGF{alpha} in serum-free, Ca2+-free DMEM containing 4 mM L-glutamine, 0.2% BSA, and 0.5 mM CaCl2. At the end of the incubation period, the medium was removed, the cells were washed twice with ice-cold PBS containing 1 mM sodium vanadate and 25 mM NaF, and then 100 µl ice-cold lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM sodium vanadate, 50 mM glycerophosphate, and a cocktail of protease inhibitors] was added (17). The protease inhibitors were aprotinin, leupeptin, soybean trypsin inhibitor, pepstatin, and calpain inhibitor (10 µg/ml of each), all from frozen stocks, as well as 100 µg/ml of Pefabloc. The sodium vanadate, NaF, and Pefabloc were freshly prepared on the day of the experiment. The cells were scraped into the lysis buffer, sonicated for 5 sec, and then centrifuged at 6000 x g for 5 min at 4 C. The supernatants were frozen at –20 C. After thawing, equal amounts of supernatant protein (20 µg) were separated by SDS-PAGE (18). The separated proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Kreene, NH) and incubated with blocking solution [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and 0.25% BSA] containing 5% dry milk for at least 1 h at room temperature. PTTG protein levels were measured by immunoblotting using an 18-h incubation with a 1:1000 dilution of rabbit polyclonal antiserum specific for PTTG (M-16) (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were washed for three 15-min periods at room temperature (1% PBS, 1% Triton X-100, and 0.3% dry milk) and then incubated for 1 h with a secondary goat antirabbit, peroxidase-linked antiserum (1:2000) in blocking solution. Blots were then washed again (3 x 15 min). Bands were visualized by chemiluminescence according to the manufacturer’s protocol (Supersignal, Pierce Chemical, Rockford, IL). The same membrane was used after stripping (Restore Western blot stripping, Pierce) to measure ß-actin. Protein concentrations were measured with the Micro BCA protein kit (Pierce).

RT-PCR
One-step RT-PCR (Qiagen) was used for determining the presence of PTTG transcript(s) using a pair of primers that would yield a 352-bp product spanning nucleotides 165–517 of the human PTTG cDNA (NM_004219). Primer sequences are: sense, 5'-AGT TTC AAC ACC ACG TTT TGG C-3', and antisense, 5'-GCT TTT CAA GCT CTC TCT CCT CG-3'. For PBF (NM_004339), these two primers were used: 1) sense, 5'-TGT TGA CTC ACA CGG CTT TTG C-3', and 2) antisense, 5'-TTC TTT CTT CTT GGG GTG GAC C-3'. These primers would yield a 463-bp product spanning nucleotides 1249–1712. In brief, we used the following procedure for RT-PCR: 2 µg total RNA were mixed with a master cocktail containing RT-PCR buffer, sense and antisense PTTG primers, deoxynucleotide triphosphates, RNase inhibitor, and an enzyme mixture containing reverse transcriptase (Omniscript and Sensiscript) and HotStart Taq DNA polymerase at the concentrations recommended by the manufacturer (Qiagen) to a final volume of 50 µl. The temperature-cycle protocol was as follows: 30 min at 50 C for RT reaction, followed by denaturation and activation of HotStart DNA polymerase for 15 min at 95 C and PCR amplification (30 sec at 94 C, 30 sec at 58 C, and 1 min at 72 C for 40 cycles). A final extension for 10 min at 72 C was performed after the end of 40 cycles. To eliminate amplification from contaminating genomic DNA, we omitted RT as a negative control for the RT-PCR for each sample. RT-PCR products were fractionated on 1.5% agarose gels. The presence of a 352-bp amplified product was indicative of a positive PCR arising from the presence of a PTTG-related sequence within the cDNA.

PTTG mRNA silencing in U87 cells
Cells were plated in 96-well plate with 60–70% confluency. Twenty-four hours after plating, cells were transfected with either negative control or two different PTTG mRNA silencing oligonucleotides, purchased from Ambion (Austin, TX). For PTTG RNA silencing (siRNA), we first tested two different siRNA oligonucleotide sequences designated as PTTG1.1 and PTTG1.2. The sense and antisense sequences used were: PTTG1.1, 5'-GAU CUC AAG UUU CAA CAC Ctt-3' (sense) and 5'-GGU GUU GAA ACU UGA GAU Ctc-3' (antisense); PTTG1.2, 5'-GUC UGU AA A GAC CAA GG GAtt-3' (sense) and 5'-UCC CUU GGU CUU UAC AGA Ctt-3' (antisense). For negative control, we used oligonucleotide (Ambion), and the sequences were: 5'-AGU ACU GCU UAC GAU ACG Gtt-3' (sense) and 5'-CCG UAU CGU AAG CAG UAC Utt-3' (antisense). Chemically synthesized annealed oligonucleotide of the abovementioned PTTG siRNA sequences were used. The 100-nM final concentration of siRNA sequences were used for transfecting the U87 cells. Efficacy of silencing was determined by real-time PCR of PTTG gene 48 h post transfection, and PTTG1.2 siRNA sequence was found to have 70–80% efficiency in reducing PTTG mRNA, compared with negative control, whereas PTTG (1.1) was less than 50% efficient. Therefore, we used PTTG (1.2) to study the role of PTTG in the growth of U87 cells. Transfection was performed by following siPort lipid protocol and using siPort lipid reagent (Ambion). In brief, transfection cocktail consisted of OptiMEM (Invitrogen), siPort lipid, and 20 nmol oligonucleotide. Four hours after transfection, 20% serum containing DMEM was added to the cells and cultured for 48 h. Cells were then pulsed with 5-bromo-2'-deoxyuridine (BrdU) for 4 h, and its incorporation was measured by a kit obtained from Roche Diagnostic (Indianapolis, IN).

Determination of hepatocyte growth factor (HGF) secretion
To study HGF secretion, U87 cells were grown to 70–75% confluence in complete growth medium in 24-well plates. They were then serum starved overnight in growth medium minus fetal bovine serum containing 0.2% BSA along with various concentrations of EGF. Medium samples were cleared by centrifugation, and HGF was measured in this conditioned medium with an ELISA. The ELISA employs a quantitative sandwich, enzyme-linked immunoassay technique, using a monoclonal antibody specific for HGF that is bound to microtiter wells. Assay sensitivity was 125 pg/ml. Data are expressed as picograms per microgram protein.

Statistics
The data are presented as the mean ± SE of the indicated number of experiments. Data were analyzed by one-way ANOVA followed by Fisher protected least significant difference test. P < 0.05 indicates a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PTTG and PBF mRNAs in human primary astrocytes, ASs, and AS-derived cell lines
We first studied expression of PTTG mRNA and its interacting protein, PBF, in primary human astrocytes; glioblastoma multiforme (GBM) (high-grade AS); and a human malignant AS cell line, U87. One-step RT-PCR with primers designed from the human PTTG yielded amplified cDNA products having the predicted molecular size (Fig. 1AGo). The same technique revealed expression of PBF mRNA in these cells (Fig. 1BGo). Furthermore, bidirectional sequencing of these PCR products revealed approximately 99% sequence homology with the corresponding portion of the cloned human PTTG and PBF mRNAs. Although existence of polymorphism cannot be completely ruled out because these are human tissues, less than 1% nonhomology is likely to result from sequencing error and/or errors made by the Taq polymerase during the course of PCR amplification. Therefore, our data demonstrate that human astrocytic cells express PTTG mRNA and PBF similar to that cloned from human pituitary adenoma.



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  		FIG. 1. PTTG and PBF are expressed in astrocytes and glioblastoma multiforme. A, Expression of PTTG transcripts as assessed by one-step RT-PCR using PTTG-specific primers in samples from human astrocytes (AS), human GBM, and U87cells. A 352-bp amplified cDNA fragment is indicative of a product arising from authentic PTTG-derived transcript(s). Lane 1 shows a DNA ladder for size comparison. No such product was apparent when the reverse transcriptase was omitted (rt–) from the one-step RT-PCR. B, Expression of PBF mRNA in samples from human primary astrocytes (1AS), human glioblastoma, and U87cells. A 463-bp amplified cDNA fragment is indicative of a product arising from authentic PBF-derived transcript.

 
To determine whether PTTG expression correlates with astrocyte malignancy, we performed real-time PCR with primers designed from human PTTG and GAPDH to measure the levels of PTTG mRNA in these samples. GAPDH was used to normalize differences in input RNA, RNA degradation, and the efficiency of RT reaction. Table 1Go shows that, although PTTG expression is readily detectable in astrocytes, it is consistently lower than in malignant cells (both primary malignant cells and malignancy-derived cell lines). These data suggest that expression of PTTG mRNA correlates with the malignant state of astrocytes, being higher in tumor-derived cells than in normal cells.


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  		TABLE 1. PTTG expression in patient samples and three astrocytoma cell lines

 
PTTG is mitogenic in glioma cells
The major characteristic of glioma cells is uncontrolled growth, and U87 glioma cells display this feature. Because PTTG is implicated in cellular proliferation, we speculated that PTTG might serve a similar function in these cells. Therefore, we employed PTTG siRNA to reduce PTTG expression. Transfection efficiency was optimized as described in Materials and Methods, and PTTG mRNA expression was compared by real-time PCR in cells transfected with negative control and two PTTG siRNA oligonucleotides (designated PTTG1.1 and PTTG1.2) in several experiments. Forty-eight hours post transfection, PTTG siRNA reduced PTTG mRNA expression by approximately 80%, compared with negative control-transfected cells, whereas PTTH1.1 was less efficient (data not shown). Therefore, we used PTTG1.2 in subsequent experiments to measure serum-dependent cell proliferation of U87 cells transfected with PTTG siRNA and negative control oligonucleotides as described in Materials and Methods. GAPDH was at the same level in control and the transfected cells, indicating that the oligo constructs did not alter the U87 cells. BrdU incorporation assay showed approximately 50% inhibition of proliferation of PTTG siRNA-transfected U87 cells, compared with negative control-transfected cells (Fig. 2Go). This result unequivocally demonstrated that PTTG regulates glioma cell growth.



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  		FIG. 2. Silencing PTTG mRNA by siRNA inhibits U87 proliferation. Cells were plated at 60–70% confluence in a 96-well plate (six wells each for negative control oligonucleotide and PTTG1.2 siRNA). Twentyfour hours after plating, cells were transfected with two oligonucleotides as described in Materials and Methods. Forty-eight hours after transfection, cells were pulsed with BrdU, and cell proliferation ELISA was performed. Data are pooled from three independent experiments (P < 0.05).

 
EGF receptor ligands induce PTTG expression in U87 cells
Having shown that PTTG mRNA is expressed in all astrocytic cells tested and is similar to that cloned from pituitary adenoma, we sought to study the regulation of its expression. A widely used model for high-grade glioma, the U87 cell line, was used for this purpose. Because EGF receptor (EGFR) stimulation is a well-documented promalignant pathway in AS and GBM, inducing robust proliferation (19), we first studied whether the ligands of the EGFR, such as EGF and TGF{alpha}, induce PTTG expression. We performed Western blot analysis on protein samples derived from U87 cells incubated with EGF (30 ng/ml) and TGF{alpha} (100 ng/ml) for 48 h using a primary antibody raised against PTTG and compared it with vehicle-treated cells. As shown in Fig. 3AGo, we observed more than a 2-fold increase in the intensity of the band representing PTTG protein (~22 kDa) in U87 cells treated with EGF and TGF{alpha}. These data clearly demonstrate that U87 cells, in addition to expressing PTTG mRNA, also express PTTG protein, which is increased by EGFR ligands.



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  		FIG. 3. TGF{alpha} and EGF induces PTTG protein and mRNA up-regulation in U87 cells. A, The cells were plated in a six-well plate, and after 3 d at 70–80% confluency, the medium was changed, and a serum-free medium without or with EGF 30 ng/ml or TGF{alpha} 100 ng/ml was added for 48 h. The blots were striped and reblotted with antibody against ß-actin to show equal loading. The Western blot is representative of three experiments. B, The cells were plated in a T-75 flask and, after 3 d at 70–80% confluency, were starved in serum-free medium for 4 h. The medium was changed, and a serum-free medium with EGF 50 ng/ml and TGF{alpha} 50 ng/ml was added for 18 h. mRNA was then prepared and made into poly(A+) mRNA as described in Materials and Methods. Northern blotting showing the effect of TGF{alpha} 50 ng/ml, compared with media with vehicle using a 1.9-kb PTTG-specific probe. Equal loading was also confirmed by reprobing the membranes with GAPDH cDNA. The Northern blot is representative of three independently performed blots.

 
We next studied whether the increase in PTTG protein due to EGFR activation involves up-regulation of its transcript. We performed Northern blot analysis on poly(A+) RNA samples from U87 cells treated with EGF or TGF{alpha} and probed with full-length rat PTTG cDNA (a generous gift of Dr. Shlomo Melmed, Cedars-Sinai Research Institute, University of California Los Angeles School of Medicine, Los Angeles, CA). We observed that treating the cells overnight (18 h) with 50 ng/ml TGF{alpha} resulted in up-regulation of the PTTG transcript (Fig. 3BGo). This result not only supports our Western blot data but also shows that EGFR activation induces PTTG mRNA in U87 cells. Furthermore, assessing PTTG up-regulation by EGFR ligands by gold standard methods such as Western and Northern blotting highlighted the reliability of the high-performance, real-time PCR method, which was used in subsequent studies.

Because PTTG is an oncogene, we next determined the time course of the effects of EGF and TGF{alpha} on its mRNA. Our data show that PTTG mRNA in U87 cells already starts to increase at 4 h [1.24 ± 0.09 (mean ± SE)-fold] after treatment with 30 ng/ml EGF, compared with vehicle-treated cells. PTTG mRNA then continues to increase to 2.18 ± 0.44-fold at 18 h and remained elevated at 40 h (1.74 ± 0.38-fold), compared with vehicle-treated cells (P < 0.05) (Fig. 4AGo). We further confirmed the involvement of the EGFR in ligand-induced up-regulation of PTTG by incubating the cells with EGF and TGF{alpha} in the presence of the specific EGFR inhibitor, AG1478 (0.7 µM), and observed that the inhibitor completely obliterated this effect, whereas AG1478 (at the same concentration) had no effect on the basal level of PTTG expression (Fig. 4BGo).



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  		FIG. 4. Time course for EGF-induced PTTG mRNA in U87 cells. Cells were plated in 60-mm dishes and, after 72 h at 80–90% confluency, were starved in serum-free medium for 4 h. Then cells were then incubated with or without EGF 50 ng/ml for 0.5, 4, 18, or 40 h. mRNA isolation, cDNA synthesis, and real-time PCR were performed as described in Materials and Methods. A, PTTG mRNA is up-regulated after 4 h, peaks at 18 h, and remains elevated at 40 h after stimulation by EGF, compared with media with no EGF. B, The effect of EGF and TGF{alpha} at 18 h on PTTG up-regulation was blocked by the EFGR inhibitor AG1478 (0.7 µM). The data are pooled from three independent experiments. GAPDH was used to normalize differences in RNA isolation, RNA degradation, and/or the efficiency of the RT reaction. (*, P < 0.05, n = 3)

 
HGF up-regulates PTTG mRNA in U87 cells
HGF is a promalignant cytokine that acts via its membrane bound receptor, c-Met, and induces proliferation and migration of AS cells as well as inducing angiogenesis in ASs (20). Previously, we reported expression of functional c-Met in U87 cells (14). This allowed us to study the effect of HGF on PTTG mRNA in these cells. Overnight (18 h) incubation of U87 cells with 5 ng/ml HGF resulted in a 2.27 ± 0.31-fold increase in PTTG mRNA, compared with vehicle-treated cells (P < 0.05) (Fig. 5AGo).



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  		FIG. 5. EGF and TGF{alpha} induce HGF release, but the effects of EGF on PTTG are independent of an autocrine action of HGF. A, Dose-dependent stimulation of HGF secretion from U87 cells by EGF. U87 cells were incubated with TGF{alpha} and EGF for 18 h, and conditioned media were collected and subjected to HGF determination by ELISA, as described in Materials and Methods. A, Significantly lower than vehicle (P < 0.05). B, Neutralizing antibody against HGF does not abolish EGF up-regulation of PTTG. U87 cells were plated, treated, and processed as described above. EGF and HGF up-regulate PTTG; the effects of EGF are not abolished by neutralizing antibody against HGF. Pooled data are from three independent experiments (P < 0.05, compared with cells treated with IgG).

 
Because EGFR and c-Met share many common features such as being receptor tyrosine kinases and promoting malignant features in GBMs, we were interested in their possible interrelationship. We observed that exogenous addition of EGF and TGF{alpha} up-regulated HGF secretion in U87 cells (Fig. 5AGo). This led us to hypothesize that the EGF-induced up-regulation of PTTG mRNA is indirect and could be mediated via HGF. To test this paracrine hypothesis, we incubated U87 cells with 30 ng/ml EGF in the presence of 1:50 dilution of 0.1 mg/ml antirabbit antibody HGF antibody or similar concentrations of rabbit IgG; the dose of neutralizing antibody was in a previous study found effective (21). As shown in Fig. 5BGo, cells treated with EGF in the presence of the neutralizing antibody against HGF induced PTTG mRNA to an extent comparable with treatment with EGF plus rabbit IgG, thus demonstrating that activation of EGFR exerts a direct effect on the up-regulation of PTTG mRNA.

Various agonists/ligands acting via respective cognate receptors do not alter PTTG mRNA in U87 cells
We have previously shown that high Ca2+, acting via the calcium-sensing receptor (CaR), induces PTTG mRNA in H-500 Leydig cancer cells, a model for hypercalcemia of malignancy (12). The CaR is expressed in U87 cells and activates a maxi-type Ca2+-activated K+ channel (22, 23). We were, therefore, interested in studying whether CaR activation by high Ca2+ yields a similar result in U87 cells. To our surprise, we observed that high Ca2+ (3.5 mM) had no effect on PTTG mRNA (Fig. 6Go).



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  		FIG. 6. EGF and TGF{alpha} through the EGFR up-regulate PTTG mRNA expression, whereas BMP-2, Ca2+, TGFß, TNF{alpha}, or TRAIL has no effect. Cells were plated in 60-mm dishes and, after 72 h at 80–90% confluency, were starved in serum-free medium for 4 h. Then cells were then incubated with or without one of the promalignant factors or known up-regulators of PTTG for 18 h (see text for detail). The data are pooled from three or more independent experiments. GAPDH was used to normalize differences in RNA isolation, RNA degradation, and/or the efficiency of the RT reaction (*, P < 0.05).

 
Figure 6Go also shows that TGFß (5 ng/ml) and BMP-2 (100 ng/ml) did not alter PTTG mRNA expression in U87 cells over a period of 18–20 h incubation with these cytokines. Similarly, apoptosis-inducing TNF superfamily ligands such as TNF{alpha} (5 ng/ml) and TRAIL (5 ng/ml) failed to alter PTTG expression in these cells (Fig. 6Go). Estrogen has been shown to be important in PTTG induction in pituitary tumors (11, 24). Interestingly, 17ß-estradiol (10–4 M), which binds with its nuclear receptors and induces PTTG mRNA in pituitary tumors, failed to do so in U87 cells.

Activation of antimitogenic and proapoptotic nuclear receptor ligands does not alter PTTG mRNA in U87 cells
Ligands of various nuclear receptors, particularly those of retinoic acid receptor (RAR), retinoid X receptor (RxR), and PPAR{gamma}, inhibit cell growth and induce apoptosis in variety of tumor cells including ASs, which are expressed by U87 cells (14, 25). We studied, therefore, the effect of activating these receptors using their specific ligands. A synthetic agonist of RAR, TTNPB (a stilbene arotinoid), was used as a RAR agonist because it is 1000-fold more potent than natural agonists (14, 26). Using a sublethal concentration of 10 nM, which only inhibits cell growth (data not shown), we observed no alteration of PTTG mRNA in U87 cells, compared with the vehicle-treated cells (Fig. 7Go). Likewise, both the PPAR{gamma} agonists [the physiological ligand PGJ2 (100 nM) and the hypolipidemic, ciglitazone (100 nM)] failed to induce any change in PTTG mRNA, compared with their vehicle-treated counterparts.



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  		FIG. 7. Antimalignant and negative regulators of PTTG have no effect on PTTG mRNA expression in U87 cells. Cells were plated in 60-mm dishes and, after 72 h at 80–90% confluency, were starved in serum-free medium for 4 h. Then cells were incubated with or without TTNP3, I3CRA, ciglitazone, or PGJ2 (see text for detail) for 18 h. None of the antimalignant compounds had any effect on PTTG mRNA expression. The data are pooled from two independent experiments. GAPDH was used to normalize differences in RNA isolation, RNA degradation, and/or the efficiency of the RT reaction.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We first showed expression of PTTG and PBF mRNA by RT-PCR in human primary astrocytes, ASs, and several well-established AS cell lines derived from highly malignant tumors. Fibroblast growth factor-2, another protein that interacts with PTTG is known to express in normal and malignant astrocytes and plays an important role in determining malignant trait of human glioma. Our study demonstrates that in addition to already known high expression of fibroblast growth factor-2, human astrocytic cells express PBF, suggesting thereby that astrocyte-derived cells possess complete functional apparatus for PTTG.

Reports showing abundant PTTG expression in a variety of malignant cell lines but at comparatively lower levels in normal human tissues (27) suggest a role for PTTG in tumor progression. PTTG expression has been positively correlated with several malignancies, including breast, prostate, and thyroid (4). Therefore, we found it reasonable to ask whether such a phenomenon occurs in the case of astrocytic malignancies as well. Using quantitative real-time RT-PCR, we detected significantly higher levels of PTTG mRNA in primary ASs, glioblastomas, and all three high-grade ASderived immortalized cell lines, compared with primary human astrocytes. This finding strongly suggests that PTTG expression could positively correlate with the malignancy of astrocyte cells. In light of a recent study suggesting the possibility that PTTG and bFGF serve as prognostic indicators of thyroid cancer (28), a similar role for PTTG in ASs cannot be ruled out.

The mitogenic role of PTTG has been shown in National Institutes of Health 3T3 fibroblasts overexpressing PTTG (1), HeLa cells (29), and rat FRTL-5 thyroid carcinoma cells (30). On the other hand, PTTG overexpression in JEG-3 cells causes cell cycle arrest (31), whereas in neuronal NT-2 cells, it has a biphasic effect, being mitogenic at lower levels of expression and antiproliferative at higher levels (32). In our present report, we used PTTG siRNA technique to effectively down-regulate PTTG mRNA, which resulted in inhibition of serum-induced cell proliferation. This finding conclusively demonstrates that PTTG has a mitogenic role in glioma cells.

We next studied regulation of PTTG mRNA expression in U87 cells. Because of the heterogeneity of glioma cells, we decided to use U87 cells for the following reasons: they represent a well-established in vitro and in vivo model for studying high-grade AS owing to their ability to form tumors in athymic nude mice, and they express functional receptors for the ligands whose effects on PTTG expression we sought to study, such as the EGFR, TGFß receptors, BMP-2 receptors, Fas/CD95, CaR, estrogen receptor, RARs, RxRs, and PPAR{gamma}.

Studies with both glioma cell lines and primary tumors have shown that EGFR and its principal ligand, TGF{alpha}, induces proliferation of human glioma cells, which could be inhibited by tryphostin (AG1478), a protein tyrosine kinase inhibitor selective for the EGFR (33). Thus, we observed that PTTG mRNA and protein were up-regulated by EGFR agonists, and accordingly the effect could be abolished by AG1478. Our data in U87 cells are similar to a previous report showing transcriptional up-regulation of PTTG by EGF in NIH 3T3 cells using a PTTG reporter construct (34). Furthermore, the sustained nature of the up-regulation of PTTG by EGF and TGF{alpha} may suggest that PTTG mediates the mitogenic role of EGF in U87 cells.

HGF (like EGF) exerts various malignancy-promoting actions on gliomas including robust induction of proliferation, cell migration, and angiogenesis. In glioma cells, including U87, HGF acts in an autocrine mode via its receptor, c-Met (28). We observed that stimulation with HGF doubled the level of PTTG mRNA in U87 cells. Because both EGF and HGF are promalignant cytokines acting via two receptor tyrosine kinases and up-regulate PTTG mRNA, we hypothesized a possible relationship between the two. Incubating U87 cells with EGF increased HGF secretion robustly, which in turn raised the possibility that the effect of EGF on PTTG expression might be mediated by HGF. Therefore, we used a neutralizing antibody against HGF to determine whether the effect of EGF is changed. Our data showed that the neutralizing antibody against HGF has no effect on EGF-induced up-regulation of PTTG. Therefore, we conclude that EGFR ligands and HGF independently up-regulate PTTG expression.

TGFß has a tumorigenic role in gliomas. Our results show that PTTG gene expression remains unchanged in response to TGFß. In addition, because BMP-2 (a member of the TGFß superfamily) inhibits neurogenesis and concomitantly induces astrocytogenesis of mouse fetal neuroepithelial cells, we studied the effect of BMP-2 on PTTG expression. As in the case of TGFß, BMP-2 also failed to elicit any change in the PTTG mRNA expression in U87 cells, suggesting that ligands belonging to the TGFß superfamily have no effect on PTTG expression. Likewise, TNF{alpha}, Fas ligand, and TRAIL/Apo-2L, which serve as extracellular signals triggering apoptosis, did not alter PTTG mRNA expression in this cells.

We recently observed that high extracellular Ca2+, acting via the CaR, up-regulates PTTG mRNA in H-500 Leydig tumor cells (12). To our surprise, we failed to see any effect of elevated Ca2+ on PTTG mRNA in these cells, which points to the very interesting fact that the up-regulation of PTTG by a ligand seems to be tissue specific. From these results, we conclude that among the cell surface receptors, in AS cells PTTG is selectively regulated by receptor tyrosine kinases but not by G protein-coupled receptor (CaR in this study) or the receptors coupled to the phosphorylated mothers against decapentaplegic pathway (TGFß receptor family).

Retinoids strongly inhibit proliferation and migration of human ASs and are considered a potential tumor chemotherapy (35). A similar chemotherapeutic potential has been appreciated for PPAR{gamma} ligands (36). We and others have shown the presence of functional RARs, RxRs, and PPAR{gamma}s in a variety of primary gliomas as well as in cell lines including U87 (14, 25, 26). Also, down-regulation of PTTG mRNA has very recently been reported in pituitary adenoma cells on activation of PPAR{gamma} (10). In our experiments, we used sublethal concentrations of TTNPB, 13-cisRA, and PPAR{gamma} agonists (PGJ2 and ciglitazone). None of these ligands had any effect on the expression of PTTG mRNA. One interesting aspect of these data is the fact that whereas the cell growth-promoting factors, such as EGFR ligands and HGF, induce PTTG mRNA expression, growth-inhibitory agents, such as retinoids and PPAR{gamma} ligands, have no down-regulatory effect on its expression. The discrepancy between our result with the reported down-regulation in pituitary adenoma could be due to various reasons. The most plausible reason is cell-specific regulation of the PTTG gene. It is also possible that malignancy grade contributes to the ability of PPAR{gamma} agonists to down-regulate PTTG mRNA because U87 cells, like any other available AS cell line, are highly malignant. In the future, it would be interesting to study whether PPAR{gamma} agonists down-regulates PTTG mRNA in low-grade primary AS cells. Additional differences in the regulation of PTTG mRNA between pituitary adenoma and U87 cells are the product of its differential response to 17ß-estradiol. Whereas 17ß-estradiol up-regulates PTTG mRNA in pituitary adenoma (24), it has no such effect in U87 cells, despite the fact that estrogen has been shown to be biologically active in U87 cells (37).

In conclusion, we for the first time report that the protooncogene PTTG and its interacting protein, PBF, are expressed in astrocytes, ASs, and glioblastomas. Level of PTTG expression correlates positively with malignancy. PTTG has a mitogenic role in U87 glioma cells, and the ligands of EGFR and HGF up-regulate PTTG expression. Finally, the regulation of expression of PTTG mRNA in U87 glioma cell is different from other cells that have been studied.


   Acknowledgments
 
The authors are thankful to Paul Rooney and Deephti Kanuparthi for their technical assistance. The PTTG probe was a generous gift of Professor Shlomo Melmed.


   Footnotes
 
This work was supported by grants from Statens Sundheds Videnskablige Forskningsråd (to J.T.-H.) and the National Institutes of Health (DK41415, DK48330, and DK52005) (to E.M.B.) and AR02215 (to N.C.).

Abbreviations: AS, Astrocytoma; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; BrdU, 5-bromo-2'-deoxyuridine; CaR, calcium-sensing receptor; EGF, epidermal growth factor; EGFR, EGF receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBM, glioblastoma multiforme; HGF, hepatocyte growth factor; PBF, PTTG-binding factor; PGJ2, prostaglandin J2; PPAR, peroxisome proliferator-activated receptor; PTTG, pituitary tumor transforming gene; RAR, retinoic acid receptor; RT, reverse transcription; RxR, retinoid X receptor; siRNA, RNA silencing; TRAIL, TNF-related apoptosis-inducing ligand; TTNP3, a retinoic acid receptor ß/{gamma}-specific ligand.

Received December 8, 2003.

Accepted for publication May 25, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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