Animals and Cells
Female ICR mice were purchased from Japan SLC (Shizuoka, Japan) at 5–6 wk of age and maintained according to the Guidelines for the Regulation of Animals, provided by the Animal Experimentation Committee of Kyoto University. All animals were housed under controlled conditions of humidity (60 ± 5%), lighting (12-h light cycle), and temperature (24 ± 2°C). RAW264.7 mouse macrophages, U937 human monocytic lymphoma cells, THP-1 human acute monocytic leukemia cells, and MCF7 human mammary carcinoma cells were purchased from American Type Culture Collection (Manassas, VA). RAW264.7 macrophages and MCF7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% and 10% heat-inactivated fetal bovine serum (FBS), respectively, as well as penicillin (100 U/mL) and streptomycin (100 mg/mL). U937 and THP-1 cells were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 containing 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL). Each cell line was incubated at 37°C under a humidified atmosphere of 95% air and 5% CO2.
DMEM, RPMI 1640, and FBS were purchased from Invitrogen (Carlsbad, CA). LPS used in vitro was from Escherichia coli
0127:B8, while that used in vivo was from Salmonella typhimurium
ATCC 7823, and both were obtained from Sigma–Aldrich (St. Louis, MO). Antibodies were obtained from the following sources: rabbit anti-phospho-PI3K, anti-phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-phospho-p38, anti-JNK 1/2, anti-phospho-c-jun
, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG were from Cell Signaling Technology (Beverly, MA); goat anti-β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA); mouse anti-α-tubulin was from Oncogene (San Diego, CA); and HRP-conjugated anti-goat IgG and anti-mouse IgG were from Dako (Glostrup, Denmark). Anti-Pdcd4 antibody was described previously [13
]. All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan), unless otherwise specified.
The backs of 7-wk-old female ICR mouse were shaved with a surgical clipper, then TPA (8 nmol) dissolved in 200 μL of acetone was applied onto the shaved portion of each. Six hours later, the mice were killed by cervical dislocation, then the back skin was isolated and scraped with a razor to obtain epidermal protein samples [14
]. Each collected epidermal layer was lysed for Western blot analysis, which is described below.
Mouse Peritoneal Macrophages
Six-week-old ICR mice were killed, and ice-cold DMEM containing 10% FBS and 1% heparin was injected into the peritoneal cavity of each. Cells were collected twice by washing with the medium and cultured in 12-well plates. After 24 h of incubation at 37°C, nonadherent cells were removed by rinsing with phosphate-buffered saline (PBS), while adherent cells were treated with LPS (500 ng/mL) in DMEM without FBS. After 18 h, total RNA was isolated, as described below.
Western Blot Analysis
RAW264.7 macrophages were seeded 12 h before treatment. Prior to the experiments cells were rinsed with PBS and medium was exchanged to DMEM without FBS. After being treated with LPS (100 ng/mL) for various time periods, the cells were lysed with lysis buffer [protease inhibitor, phosphatase inhibitor (Sigma–Aldrich), 10 mM Tris (pH 7.4), 1% sodium dodecyl sulfate (SDS), 1 mM sodium vanadate]. The lysates from cells and mouse skin were then sonicated and centrifuged at 15 000g for 5 min. The protein concentration in the supernatant was quantified using a Bio Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) and standardized using γ-globulin as the reference. The lysates were denatured in a sample buffer containing SDS and 2-mercaptoethanol. An equal amount of protein (20–30 μg) was separated using SDS gel electrophoresis, subsequently proteins were electrotransferred to Immobilon-P membranes (Millipore, Billerica, MA). Proteins were detected with specific primary and secondary antibodies, and visualized using ECL reagents (GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed using Scion Image 0.4.0.3 (Scion Corporation, Frederick, MD).
Reverse Transcription-(Quantitative) Polymerase Chain Reaction (RT-qPCR)
U937 cells were stimulated with TPA (10 nM) for various time periods. RAW264.7 macrophages were treated with LPS (100 ng/mL), conditioned medium from RAW264.7 macrophages (RAW-CM), or tumor necrosis factor-α (TNF-α; 2500 pg/mL) for various time periods. MCF7 cells were exposed to CM from U937 cells (U937-CM) or TNF-α (20 ng/mL). For pathway analysis, cells were pretreated with various inhibitors (PD98059, MEK1/2 inhibitor, 50 μM; SP600125, JNK1/2 inhibitor, 10 or 50 μM; SB203580, p38 MAPK inhibitor, 10 μM; LY294002, PI3K inhibitor, 30 μM; 0.5% DMSO as control) for 30 min before exposure to LPS (100 ng/mL), RAW-CM, or TNF-α (2500 pg/mL) for 24 h. Cells were preincubated with vehicle (0.5% DMSO) or each inhibitor for 30 min, then treated with or without LPS (100 ng/mL) or TNF-α (2500 pg/mL) for 24 h, followed by incubation with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). From the result of this assay, the indicated concentration of each inhibitor did not have any effect on cell viability.
Total RNA was extracted using TRIzol Reagent (Invitrogen), according to the manufacturer’s protocol. mRNA was reverse transcribed using the iScript cDNA synthesis kit (BioRad, München, Germany) according to the manufacturer’s instructions. Expression was analyzed using PCR (10× PCR buffer, dNTP mixture, MgCl2
, Taq DNA polymerase, 5 U/μL; Takara, Otsu, Japan). Amplified DNA was separated by agarose gel electrophoresis and stained with SYBR Gold (Invitrogen). Image analysis was performed using Scion Image 0.4.0.3. Lack of PCR saturation was confirmed by titrating each cDNA amount (data not shown). Alternatively, mRNA expression was analyzed by quantitative PCR using the Absolute Blue SYBR Green fluorescein kit assay (ThermoScientific, Hamburg, Germany) according to the manufacturer’s protocol. Primer sequences and PCR conditions were designed based on specificity and suitability for qPCR analysis (GC-content, length) or selected from previous reports [15
]. They were as follows: pdcd4
(mouse), 300 bp (5′-TAATCAgTg-CAAgCgAAATTAAggAA-3′ and 5′-CCTTTCCCA-gATCTggACCgCCTATC-3′), at 94°C for 15 s, 55°C for 30 s, and 72°C for 45 s; TNF
-α (mouse), 402 bp (5′-CCTgTAgCCCACgTCgTAgC-3′ and 5′-TTgACCT-CAgCgCTgAgTTg-3′), at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s; interferon (IFN
)-γ (mouse), 237 bp (5′-AACgCTACACACTgCATCTTgg-3′ and 5′-gACTT-CAAAgAgTCTgAgg-3′), at 90°C for 40 s, 62°C for 40 s, and 72°C for 60 s; CC chemokine ligand
)-1 (mouse), 125 bp (5′-CgTgTggATACAggATgTTgA-CAg-3′ and 5′-AggAggAgCCCATCTTTCTgTAAC-3′), at 95°C for 10 s, 56°C for 5 s, and 72°C for 30 s; Ccl-20
(mouse), 86 bp (5′-CAgAAgCAgCAAgCAACTACgA-3′ and 5′-CTgTCTTgTgAAACCCACAATAgC-3′), at 94°C for 20 s, 60°C for 20 s, and 72°C for 20 s; interleukin (IL
)-10 (mouse), 195 bp (5′-CgggAAgA-CAATAACTg-3′ and 5′-CATTTCCgATAAggCTTgg-3′), at 94°C for 30 s, 58°C for 30 s, and 72°C for 60 s; cyclophilin
(mouse), 240 bp (5′-gCCAggACCTgTAT-gCTTCA-3′ and 5′-TTgggTCgCgTCTCgTTCgA-3′), at 95°C for 30 s, 50°C for 30 s, and 72°C for 60 s; hypoxanthine guanine phosphoribosyl transferase (HPRT
) (mouse), 196 bp (5′-gTAATgATCAgTCAAC-ggggAC-3′ and 5′-CCAgCAAgCTTgCAACCTTAACC-A-3′), at 95°C for 30 s, 55°C for 25 s, and 72°C for 45 s; pdcd4
(human), 134 bp (5′-ACAgTTggTgggCCAgTT-TATTgC-3′ and 5′-TCAgAAgCACggTAgCCTTATC-CA-3′), at 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s; TNF
-α (human), 215 bp (5′-TCTCgAACCCC-gAgTgACA-3′ and 5′-gAggAgCACATgggTggAg-3′), at 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s; 18S
(human), 151 bp (5′-gTAACCCgTTgAACCCCATT-3′ and 5′-CCATCCAATCggTAgTAgCg-3′), at 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s.
Preparation of Macrophage Conditioned Media
Following preincubation of RAW264.7 macrophages with regular growth medium, the cells were exposed to LPS (100 ng/mL) in DMEM without FBS. After 12 h of incubation, the medium was collected for the preparation of RAW-CM. U937 cells were exposed to TPA (10 nM) for 48 h. Adherent, that is, differentiated and activated, U937 cells were trypsinized, washed with PBS, and reseeded. After 24 h the medium was collected as U937-CM.
ELISA for TNF-α
RAW264.7 macrophages were seeded 12 h before experiments. After pretreatment with various inhibitors (PD98059, MEK1/2 inhibitor, 50 μM; SP600125, JNK1/2 inhibitor, 50 μM; SB203580, p38 MAPK inhibitor, 10 μM; LY294002, PI3K inhibitor, 30 μM; 0.5% DMSO as a control) for 30 min, cells were exposed to LPS (100 ng/mL) for 1 h. The concentration of TNF-α in the supernatants from culture media was determined using an ELISA kit (Endogen, Cambridge, MA), according to the manufacturer’s instructions.
Luciferase Assay for AP-1-Dependent Transactivation
Following 12 h of preincubation, RAW264.7 macrophages were transiently cotransfected with an AP-1-driven luciferase reporter (Clontech, Palo Alto, CA) and a pRL-TK plasmids (Promega, Madison, WI) using Lipofectamine (Invitrogen) in OPTI-MEM (Invitrogen). After 6 h, medium was changed to DMEM (10% FBS) for an additional 6 h. Then the cells were stimulated with LPS in DMEM without FBS. MCF7 cells were transiently transfected with the same constructs using Rotifect (Roth) according to the manufacturer’s protocol. After 16 h, medium was changed and cells were stimulated by U937-CM or direct co-culture with differentiated U937 cells for 24 h. Following stimulation, cells were lysed, and firefly and renilla luciferase activity assays were performed using a Dual Luciferase kit assay (Promega) and luminometer (PerkinElmer, Boston, MA), according to the manufacturers’ instructions.
Knockdown of TNF-α and pdcd4 With siRNA
RAW264.7 macrophages were seeded 24 h before siRNA transfection in regular growth medium. Control siRNA (Santa Cruz Biotechnology), TNF-α siRNA (Invitrogen), or Pdcd4 siRNA (Invitrogen) were mixed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol, and added to the cells at a final concentration of 75 nM. Six hours after transfection, the cells were recovered in DMEM containing 10% FBS for 24 h, then stimulated with LPS (100 ng/mL) for 6 or 12 h. The cell lysates and media were collected for RT-PCR, Western blotting, and ELISA, as described above.
PCR Array Analysis
RT2 Profiler™ PCR Array PAMM-011 (SA Biosciences, Frederick, MD) was used to assess the expression profiles of inflammatory cytokines and receptors in LPS-stimulated, Pdcd4-silenced RAW264.7 macrophages. Control or Pdcd4 siRNA-transfected RAW264.7 macrophages were exposed to LPS (100 ng/mL) for 6 h, which was followed by cDNA synthesis from 1 μg of total RNA. Thermal cycling was performed on 7300 Real Time PCR System using SYBR green PCR mix (Applied Biosystems, Foster, CA) according to the manufacturer’s protocol.
Data are shown as the mean ± standard deviation from more than three independent experiments and were evaluated using Student’s t-test, unless otherwise specified. Differences were considered statistically significant at the P <0.05 level.