Cell Lines and Reagents
Human glioblastoma cell lines (A172, T98G, U87MG, U138MG, and U373MG) and human astrocytoma cell lines (SW1783 and SW1088) were purchased from American Type Culture Collection (Manassas, VA). Human glioblastoma cell lines were grown in Eagle’s minimal essential medium (EMEM) with 1mM MEM Sodium Pyruvate Solution (Gibco, Grand Island, NY), 2mM L-Glutamine (Gibco), 10 µg/ml gentamicin (Gibco), and 10% fetal bovine serum (FBS). Human astrocytoma cell lines were grown in Leibovit’s L-15 Media with 10 µg/ml gentamicin and 10% FBS. TGZ, Ciglitazone, Rosiglitazone, MCC-555, Pioglitazone, the PPARα agonist Wy14643, EP4 agonist PGE1-OH, and anti-EP2, anti-EP4, and anti-PPARγ antibodies were purchased from Cayman Chemical Co., Inc. (Ann Arbor, MI). The MEK-1/Erk Inhibitor PD98059 and the p38 inhibitor SB203580 were purchased from EMD Biosciences (San Diego, CA) and the PI3-kinase Inhibitor Wortmannin, anti-phospho-Erk MAPK (Thr202/Tyr204), and anti-Egr-1 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Sp-1 (sc-59), anti-Sp-3 (sc-644), anti-Erk 1 (sc-93), anti-Erk 2 (sc-154), anti-phosphothreonine (sc-5267), and anti-actin (sc-1615) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). EP 4 antagonist AH23848 was purchased from Sigma-Aldrich (St. Louis, MO). All chemicals were dissolved in 0.1% Me2SO and stocked at appropriate concentrations according to manufacturer’s information.
Colony formation in Soft agar assay
T98G cells were resuspended at 2 × 103 cells in 1 ml of 0.35% agar solution containing MEM, 10% FBS, 1mM MEM Sodium Pyruvate Solution, 2mM L-Glutamine, and the final concentration of TGZ (20µM), EP4 agonist (PGE1-OH) (10µM) or EP4 antagonist (AH23848) (30µM), then layered on top of a 0.7% agar layer in 6-well plates. For EP4 knock-down or overexpression, T98G cells were transfected with 100 nM of EP4 siRNA (M-005714-00, Dharmacon) or 3 µg of EP4 cDNA that was sub-cloned into pcDNA3.1 from UMR cDNA Resource Center (Rolla, MO). The effect of EP4 knock-down or overexpression was confirmed by Western blot analysis and real-time RT-PCR (data not shown). After 24h transfection, the cells were trypsinized and resuspended in 0.35% agar solution. Plates were incubated for 2 weeks at 37 °C in a 5% CO2 humidified atmosphere. Cell colonies were visualized following an overnight stain with 0.5 ml of p-iodonitrotetrazolium violet (Sigma-Aldrich) and examined microscopically. These were represented as mean colony number examined in 10 randomly chosen microscope fields.
Western Blot Analysis
Total cell lysates were isolated in RIPA buffer [1X PBS, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3 VO4, 1 mM NaF, 1 µM Okadaic acid, 10 mM β-glycerolphosphate, and Complete Mini protease inhibitor cocktail tablets from Roche (Indianapolis, IN). Quantitation of protein was performed by BCA assay (Pierce, Rockfold, IL) and 50 µg of total proteins were separated by SDS-PAGE 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA) and transferred onto a nitrocellulose membrane (Invitrogen). The blots were blocked for 1 h with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) (Sigma-Aldrich) and probed overnight at 4 °C with 5% skim milk in TBS-T with each primary antibody. After washing with TBS-T, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1h at room temperature with 5% skim milk in TBS-T and washed several times in TBS-T. Proteins were detected by the enhanced chemilluminescence system (Amersham Biosciences, Piscataway, NJ).
1 mg of nuclear extracts was prepared with Nuclear Extract kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. The nuclear extract was precleaned with 60 µl of Protein A/G PLUS-Agarose (sc2003, Santa Cruz) by incubating for 1h at 4 °C. After pelleting agarose beads, the supernatant was transferred to a new tube and immunoprecipitated with 5 µg of anti-Sp-1 antibody overnight at 4 °C. For a negative control, normal rabbit IgG (sc-2027, Santa Cruz) was added to the sample instead of anti-Sp-1 antibody. Immunocomplex was collected by adding 40 µl of agarose beads and incubating for 3h at 4 °C. After removal of the supernatant and washing the beads four times with RIPA buffer, the beads were resuspended in 30 µl of 1X LDS buffer and boiled for 5 min at 100 °C. The samples were separated by SDS-PAGE 4–12% Bis-Tris gel and transferred onto a nitrocellulose membrane. The immunoprecipitated proteins were analyzed by immunoblotting using anti-phosphothreonine (p-Thr) antibody diluted 1:50.
Real-time RT-PCR assays were performed using an ABI Prism 7700 (Applied Biosystems, Foster City, CA). Real time RT-PCR fluorescence detection was performed in 96-well plates with Quantitect SYBR Green buffer (Qiagen). The sequences of PCR primers (Invitrogen) used by real-time RT-PCR were designed according to published data [10
] and are as follows: Human EP2 5’-CGAGACGCGACAGTGGCTTCC-3’ (sense), 5’-CGAGACGCGGCGCTGGTAGA-3’ (antisense), Human EP4 5’-TCGCGCAAGGAGCAGAAGGACAC-3’ (sense), 5’-GACGGTGGCGAGAATGAGGAAGGA -3’ (antisense), Human actin 5’-GCCGATCCACAGGGAGTA-3’ (sense), 5’-CCTGGCACCCAGCACAAT-3’ (antisense). The experiments were performed in duplicate three times with individual time-matched vehicle-treated controls for each gene tested. Amplified product size was routinely checked by gel electrophoresis on a 1% agarose gel in the presence of 0.1 µg/ml ethidium bromide then visualized under UV light to confirm that only one product was formed.
Constructions of Plasmid
Human EP4 promoter luciferase constructs were generated by PCR using human genomic DNA (Promega, Madison WI). At first, the EP4 promoter region of −1236 to −42 was generated using the primers 5’-GCAGATGGGAAGAGGTTTTT-3’ (sense) and 5’-TTCTCCTCCTCCAAGTTTCC-3’ (antisense). The primer designs were determined based on the previously published sequence of the 5’-flanking region upstream of the transcription initiation site for human EP4 [21
]. To construct the intact EP4 promoter region (pEP4-1, −1238 to +1), which contains Nhe
I (upstream) and Hind
III (downstream) restriction sites, PCR was subsequently carried out using the incomplete EP4 constructs (−1236 to −42) as a template and the primers were designed as follows: 5’-GGGCTAGCCTGCAGATGGGAAGAGGTTTTTCCAGGAATTTAAA-3’ (sense), 5’-GGAAGCTTTGGAGCTCGCGTGCTGCGGCCTTTCCACCCTCTGTACAAACTTTTCTCCTCCT-3’ (antisense). PCR products and the pGL3-basic vector (Promega) were digested with Nhe
I and Hind
III restriction enzymes (New England Biolabs, Beverly, MA) and then purified with QIAquick® PCR purification kit (Qiagen). Purified products were ligated using DNA Ligation kit Ver.2.1 (TaKaRa, Shiga, Japan) and sequenced-verified. Another EP4 promoter deletion constructs were generated using the primers of following sequences: pEP4-2 (−238 to +1): 5’-GGGGCTAGCCTCCGAGGGCGTGAAA-3’ (sense), pEP4-3 (−197 to +1): 5’-GGGGCTAGCGCCCAGCCCCGCCCCA-3’ (sense), pEP4-4 (−160 to +1): 5’-GGGGCTAGCAGTCTTCCCTGCGGC-3’ (sense). The sequence of antisense primer for all EP4 deletion constructs is as follows: 5’-GGAAGCTTTGGAGCTCGCGTGCTGCGGCCTTTC-3’. The pEP4-3 constructs incorporated point mutations in Sp-1 or AP-2α binding sites were created using QuikChange® II site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. Each Sp-1 or AP-2α binding site was point-mutated to the two TT base pairs (indicated by underline) in pEP4-3 constructs and primer designs were as follows: mut. Sp-1A pEP4-3: 5’-GCGCCCAGCCCTT
CCCCAGCCCAGAC-3’, mut. Sp-1B pEP4-3: 5’-CAGCCCAGACACTT
CCCCCCGCCAG-3’, mut. AP-2 pEP4-3: 5’-CAGCCCAGACACCGCCCCTT
GCCAG-3’. Each construct was sequenced-verified to confirm the incorporation of the appropriate mutation. The PPARγ wild type plasmid was a kind gift from Dr. Cary E. Clay (Department of Cancer Biology, Wake Forest University Baptist Medical Center, Medical Center Boulevard, Winston Salem, North Carolina, 27157 USA). The Sp-1-dependent reporter plasmid containing 6 Sp-1 binding sites (pGAGC6) and the control plasmid (pGAM) were kindly provided by Professor Jeffrey E. Kudlow (Division of Endocrinology, Diabetes and Metabolism, The University of Alabama at Birmingham, Birmingham, Alabama, 35294 USA). The Sp-1 expression plasmid was reported previously by our laboratory [22
]. The mThr453
Sp-1 expression plasmid, which has two mutations of residues Thr453
, was produced using QuikChange® XL site-directed mutagenesis kit (Stratagene) and the sequences of PCR primers were described previously [23
Luciferase Reporter Assay
T98G cells were seeded in 6-well plates at 2 × 105 cells/ well in EMEM and grown to 50–60% confluence. The plasmid mixtures, containing 2 µg of EP4 promoter luciferase construct and 0.05 µg of pRL-null (Promega), were transfected using FuGENE 6 Transfection Reagent (Roche) according to the manufacturer’s protocol. The co-transfection experiment was carried out using plasmid mixtures containing 1 µg of pEP4-3 luciferase construct, 1 µg of expression plasmid (Sp-1 or mutant Sp-1), and 0.05 µg of pRL-null. The pcDNA3.1 empty vector (Invitrogen) was used as a negative control for the expression plasmid. After 24h transfection, the cells were treated with indicated concentrations of PPARγ ligands (reported in the figure legends), 10 µM Wy14643, or Control (0.1% Me2SO) for an additional 24h. For PD98059 treatment study, the cells were pretreated with 20 µM PD98059 for 1h prior to the additional 24h treatment of 20 µM TGZ. Finally, the cells were harvested in 1 × luciferase lysis buffer (Promega) and luciferase activity was measured and normalized with the values of pRL-null luciferase activity using a dual luciferase assay kit (Promega).
Short Interfering RNA (siRNA) Transfection
The Sp-1 siRNA (M-026959-00), Sp-3 siRNA (M-023096-01), and control siRNA (D-001206-08-05) were purchased from Dharmacon (Lafayette, CO). T98G cells were grown to 70–80% confluence in antibiotic-free EMEM medium and transfected with each siRNA at 100nM using Lipofectamine™ 2000 reagent (Invitrogen) and Opti-MEM® I medium (Gibco) according to the manufacturer’s instructions. After incubating for 5h, the cells were washed and changed to the complete media and recovered overnight. After confirming the knock-down of target genes by Western blot analysis, the cells were subsequently treated for 48h and the effect of EP4 expression by Sp-1 or Sp-3 knock-down was investigated with Western blot analysis.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s protocol. Briefly, T98G cells (3 × 107) were treated with the indicated conditions for 24h and then fixed with 1% formaldehyde for 10 min at 37 °C. The fixed cells were scraped into conical tubes, pelleted, and lysed in SDS lysis buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml pepstatin A. DNA was sheared to fragments of 200–800 bp by sonication 10 times for 20 s at a 50% constant maximum power. The sonicated cell supernatant was diluted 10 fold in the ChIP dilution buffer and 1% of the diluted cell supernatant was kept as a positive control (Input). The chromatin was precleared with salmon sperm DNA/protein A-agarose slurry for 1 h at 4 °C. The precleared supernatant was incubated with antibodies against Sp-1 (sc-59X, Santa Cruz), Sp-3 (sc-644X), or normal rabbit IgG overnight at 4 °C. The immunocomplexes were eluted with elution buffer (1% SDS, 0.1 M NaHCO3, 10 mM DTT). 5M NaCl was added into eluted samples to reverse histone-DNA crosslinks and the samples were heated overnight at 65 °C. Purified DNA samples were used as a template for PCR amplification. The region between −238 and −103 of the human EP4 promoter was amplified using the following primers: 5’-CTCCGAGGGCGTGAAAAC-3’ (sense), 5’-CATTGGCCGGATTGGAAG-3’ (antisense). The 136 bp products were resolved on a 2% agarose gel and visualized under UV light.
All statistical differences between experimental groups were evaluated by the two-tailed unpaired Student’s t test.