FIGURE 3.

R(+)WIN55,212-2 regulates TLR3 signaling in a cannabinoid receptor-independent manner. A, total cellular RNA was prepared from HEK293 cells and subjected to first strand cDNA synthesis using SuperScript II reverse transcriptase and random oligonucleotide primers. PCR amplification was performed using Taq DNA polymerase and primers to selectively amplify regions of CB₁, CB₂, and GAPDH cDNA. B, D, and F, HEK293-TLR3 cells were cotransfected with pFA-IRF3 (30 ng) and pFR-regulated firefly luciferase (60 ng) and constitutively expressed TK Renilla luciferase (20 ng). Transfected cells were left overnight and then cells were pre-treated (1 h) with the inhibitors SR141716 (1 μm) (B), SR144528 (1 μm) (D), and PTX (50 ng/ml) (F) prior to exposure to R(+)WIN55,212-2 (20 μm; 1 h), and then stimulated with poly(I·C) (25 μg/ml) for 6 h. Cell lysates were assayed for firefly luciferase activity and normalized for transfection efficiency using Renilla luciferase activity. C, E, and G, HEK293-TLR3 cells were pre-treated (1 h) with the inhibitors SR141716 (1 μm) (C), SR144528 (1 μm) (E), and PTX (50 ng/ml) (G) prior to exposure to R(+)WIN55,212-2 (20 μm; 1 h), and then stimulated with poly(I·C) (25 μg/ml) for 4 h. cDNA was generated and assayed by quantitative real time PCR for levels of IFN-β mRNA. The expression level of IFN-β was normalized relative to expression of the housekeeping gene GAPDH. H, HEK293 cells were pre-treated with or without PTX (100 ng/ml; 24 h), SR141716 (SR1; 1 μm for 1 h), and SR144528 (SR2; 1 μm for 1 h) prior to treatment with ACEA (100 nm for 1 h) or JWH133 (100 nm for 1 h). Cells were then incubated with 3-isobutyl-1-methylxanthine (500 μm for 15 min) and stimulated with forskolin (30 μm for 30 min). Lysates were harvested and assessed for levels of intracellular cAMP using a cAMP parameter kit. Data represent the mean ± S.E. of triplicate determinations from three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared with vehicle-treated cells. +, p < 0.05; and ++, p < 0.01 compared with poly(I·C)-treated cells (B-G) and forskolin-treated cells (H). $$, p < 0.01 compared with cells treated with ACEA/JWH133 in the presence of forskolin.