Chemicals
The selective A2A AdoR agonist 2-hexynyladenosine-5′-N-ethylcarboxamide (HENECA) was purchased from Abcam (Cambridge, UK) and TNFa from R&D Systems (Minneapolis, MN). The selective A2A AdoR antagonist 4-(2-(7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5] triazin-5-ylamino)ethyl)phenol (ZM241385) and the membrane-permeable N6,2′-O-dibutyryl cAMP (dbcAMP) were purchased from Sigma Aldrich (St. Louis, MO). TNFα and dbcAMP were dissolved in de-ionized water while HENECA and ZM 241385 were dissolved in dimethyl sulfoxide (DMSO) for cellular administration.
Cell culture
MH7A cells were obtained from Riken Cell Bank (Saitama, Japan) and cultured in RPMI 1640 medium (Thermo Fisher Scientific, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific) and 4× Antibiotic-Antimycotic liquid (Thermo Fisher Scientific) at 37°C under an atmosphere of 5% CO2.
Cyclic AMP assay
MH7A cells were seeded at 1.0 × 106/well on flat-bottomed 24-well microplates. After 90 min, cells were treated with HENECA (0–250 nM) for 0–60 min. Intracellular cAMP was measured using a cAMP EIA System (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s protocol.
Measurement of adenosine receptor and MMP-3 mRNA expression
MH7A cells were seeded at 4 × 105 /well on flat-bottomed 6-well microplates and cultured for 24 h. Cells were then treated for an additional 24 h with TNFa (0–1000 pg/ml) or HENECA (0–100 nM). Total RNA was extracted from MH7A cells using an RNeasy Mini kit and QIAshredder (QIAGEN, Tokyo, Japan) according to the manufacturer’s instructions. Aliquots of RNA were reverse transcribed using a Transcriptor Universal cDNA Master (Roche Diagnostics K.K., Tokyo, Japan), and PCR reactions performed using TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer’s protocol on a LightCycler 480 instrument with LightCycler 480 Gene Scanning software version 1.5 (NIPPON Genetics, Tokyo, Japan). The primers used for real-time PCR were as follows: A1 AdoR (Hs00181231 _ m1), A2A AdoR (Hs00169123 _ m1), A2B AdoR (Hs00386497 _ m1), A3 AdoR (Hs01560269 _ m1), MMP-3 (Hs00968305 _ m1), and GAPDH (Hs02758991 _ g1) (Thermo Fisher Scientific). The amplification protocol consisted of 10 min at 95°C followed by 55 cycles of 15 s at 95°C, 1 min at 60°C, and 1 s at 72°C. Expression of MMP-3 mRNA was normalized to GAPDH mRNA expression.
Western blot analysis
Primary polyclonal antibodies against human adenosine receptor A2A were purchased from Abcam, while antibodies against human phospho-p38 mitogen-activated protein kinase (p-p38 MAPK), human phospho-activating transcription factor 2 (p-ATF-2), and ß-actin was purchased from Cell Signaling Technology (Tokyo, Japan). MH7A cells were seeded on 6-cm dishes at 1 × 106/dish for 24 h, then treated for an additional 24 h with TNFa (0–1000 pg/ml) (R&D Systems). Membrane- and cytoplasmic-protein fractions were extracted using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific), while total cellular protein was extracted using the M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific). The protein concentrations in each sample were measured using a BCA Protein Assay Reagent Kit (Thermo Fisher Scientific). Proteins were separated at 10–35 mg per gel lane by SDS-PAGE according to standard protocols and transferred onto PVDF-nylon membranes (Merck Millipore, Tokyo, Japan). Membranes were blocked with 5% non-fat milk in tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. After a brief wash, membranes were incubated overnight at 4°C with rabbit polyclonal antibodies against adenosine receptor A2A (1:1000), p-p38 MAPK (1:1000), p-ATF-2 (1:500), and ß-actin (1:1000). Blotted membranes were then washed 3 times with TBST and incubated in horseradish peroxidase (HRP)-conjugated donkey-α-rabbit IgG (1:2500, ECL Western Blotting Detection System, GE Healthcare) for 60 minutes at room temperature. Protein bands were captured, digitized, and quantified using ImageQuant LAS4000 mini (GE Healthcare).
Measurement of total MMP-3 secretion
MH7A cells were seeded on flat-bottomed 24-well microplates at 8 × 104 /well. After 24 h, cells were treated for an additional 24 h with TNFa (25 pg/ml) alone or in combination with dbcAMP (0–100 mM) or HENECA (0–100 nM) as indicated. Supernatant MMP-3 concentrations were measured using Panacurea MMP-3 (Sekisui Medical, Tokyo, Japan).
Statistical analysis
All experiments were repeated at least three times, and representative results are shown. Data are presented as mean ± standard deviation (SD). Treatment group means were compared using two-tailed unpaired t‑tests. A P < 0.05 (two-tailed) was considered significant for all tests.