Human type II alveolar epithelial cells (A549, American Type Culture Collection, CCL-195), were kindly provided by Dr. R. Danesi, University of Pisa, Pisa, Italy. The chemicals 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy) phenyl]pyridin-2-ylsulfanyl] acetamide (BAY 60-6583) and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamide (MRS1706) were purchased from Tocris Bioscience (Bristol, United Kingdom). The RNeasy minikit was obtained from Qiagen. The Script cDNA synthesis kit was furnished by Bio-Rad s.r.l. Fluocycle II SYBR was from Euroclone (Milan, Italy). TGF-β1 was purchased from Sigma–Aldrich. All other reagents were obtained from standard commercial sources and were of the highest commercially available grade

Human type II alveolar epithelial cells (A549) were maintained in DMEM-F12 containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified 5% CO² atmosphere. For the different experiments, cultures of cells were maintained in serum-free DMEM for 8 h prior to stimulation with cytokines or other reagents.

A549 cells were untreated or treated with DMSO (CTRL) or BAY 60-6583 (100 nM) in the absence or presence of TGF-β1 (10 ng/ml) and MRS 1706 (1 μM) for the indicated time. At the end of treatments, cells were collected, and total RNA was extracted using Rneasy^®Mini Kit (Qiagen, Hilden, Germany) as previously reported (Da Pozzo et al., 2014); residual DNA was removed using RNase free DNAse set (#79254, Qiagen, Hilden, Germany). The RNA purity was checked measuring the A260/280 ratio. cDNA synthesis was performed with 400 ng of RNA using i-Script cDNA synthesis kit (BioRad, Hercules, CA, United States) following manufacturer’s instructions. Real-time RT-PCR reactions consisted of 25 μL Fluocycle^®II SYBR^® (Euroclone, Milan, Italy), 1.0 μL of both 10 μM forward and reverse primers, 2.5 μL cDNA (100 ng), and 20.5 μL of H₂O. All reactions were performed for 40 cycles using the following temperature profiles: 98°C for 30 s (initial denaturation); 55°C for 30 s (annealing); and 72°C for 3 s (extension). The primer sequences were listed in Table 1. When possible, the primers used were designed to span intron/exon boundaries and the β-actin was used as the housekeeping gene. The mRNA levels for each sample were normalized against β-actin mRNA levels, and the relative expression was calculated by using a Ct value. PCR specificity was determined by both the melting curve analysis and gel electrophoresis.

A549 cells (3.5 × 10⁴ cell/cm²) were maintained untreated or treated with DMSO (CTRL) or BAY 60-6583 (100 nM) in the absence or presence of TGF-β1 (10 ng/ml) and MRS 1706 (1 μM) for 48 h. At the end 200 μl RIPA buffer were added for 60 min at 4°C to lyse the cells. Fifty microgram of total proteins was diluted in Laemmli solution, resolved by SDS-PAGE (7.5%), transferred to PVDF membranes and probed overnight at 4°C with primary anti-A₁AR (diluted 1:200, sc-28995; Santa Cruz Biotechnology), anti-A_2AAR (diluted 1:200, sc-13937; Santa Cruz Biotechnology), anti-A_2BAR (diluted 1:150, sc-28996; Santa Cruz Biotechnology), anti-A₃AR (diluted 1:200, sc-13938; Santa Cruz Biotechnology), anti-E-cadherin antibody (diluted 1:200; sc-7870; Santa Cruz Biotechnology), anti-N-cadherin antibody (diluted 1:200; sc-7939; Santa Cruz Biotechnology) anti-Vimentin (diluted 1:1000, #5741; Cell Signaling Technology) or β-actin antibody (diluted 1:1000; MAB1501, Merck KGaA, Darmstadt, Germany). The primary antibody was detected using appropriate secondary antibody. The peroxidase was detected using a chemiluminescent substrate (ECL, Perkin Elmer), and the images were acquired by photographic film or by LAS4010 (GE Health Care Europe, Uppsala, Sweden). Immunoreactive bands were quantified performing a densitometric analysis with Image J Software (version 1.41; Bethesda, MD, United States).

A549 cells were seeded at 3.5 × 10⁴ cell/cm² in chamber slide (BD Biosciences, San Jose, CA, United States). After treatment, cells were fixed in 2% paraformaldheyde in 0.1 M phosphate buffer, washed three times with PBS, rinsed, and blocked for 45 min with PBS containing 0.1% Triton-X 100 and 1% BSA. After washing, cells were incubated overnight at 4°C with mouse monoclonal anti-β-actin antibody (diluted 1:500; MAB1501, Merck KGaA, Darmstadt, Germany) or a rabbit polyclonal anti-A_2BAR (diluted 1:100, sc-28996; Santa Cruz Biotechnology) primary antibodies diluted in PBS containing 0.03% Triton-X 100 and 1% BSA overnight at 4°C. After washing, to visualize the staining, cells were incubated with Alexa Fluor 488- and Alexa Fluor 568-labeled goat anti-mouse (1:500) or anti-rabbit (1:500) antibodies for 2 h at room temperature. Then slides were covered with Vectashield conjugated with DAPI (Vector Laboratories, Burlingame, CA, United States). Images were obtained with a Nikon Ni-E microscope, using a 20× objective with 1.45 NA and a recommended pinhole size of less than 1.0 mm, and equipped with digital camera Nikon Mod.DS-Ri2. The images were processed with ImageJ software.

A549 cells were seeded at a density of 3 × 10³ cells/well in a black, clear bottom PerkinElmer 96-well CellCarrier^TM microplate (#6005550). After 24 h, the cells were starved for 8 h with non-complete medium; then, cells were treated with different concentrations of BAY 60-6583 (0.5 nM–1 μM) or TGF-β1 (5–20 ng/ml) alone or in combination for 48 or 72 h. Following the treatment period, cells were imaged with the EnSight^TM multimode plate reader equipped with well-imaging module, and Kaleido Data Acquisition and Analysis Software. Brightfield images were taken before and 48 or 72 h after treatments to allow for a Cell Count comparison between both time points. The cell count was determined using the pre-defined Brightfield Cell Count algorithm provided by the Kaleido software. Cell with a mean area ≥100 μm² were counted. Data were normalized to the cell count at t₀ and expressed as the number of cell per well.

A549 cells were seeded at a density of 3 × 10³ cells/well in a black, clear bottom PerkinElmer 96-well CellCarrier^TM microplate (#6005550). After 24 h, the cells were starved for 8 h with non-complete medium; then, cells were treated with different concentrations of BAY 60-6583 (0.1 nM–1 μM) in the absence or presence of TGF-β1 (10 ng/ml) in 100 μl of non-complete medium for 48 h. Cells were imaged with the EnSight^TM multimode plate reader. The desensitization experiments were performed incubating the cells with BAY 60-6583 (100 nM) in the presence or absence of TGF-β1 (10 ng/ml) for 48 h. At the end the medium was changed and cells were treated with different concentrations of BAY 60-6583 (0.1 nM–1 μM) for 15 min. Then, cells were lysed with 50 μl of 1X lysis buffer (PerkinElmer, #AL003C) supplemented with protease inhibitors and phosphatase inhibitors for 10 min with gentle shaking. Lysates were either tested immediately in AlphaLISA assays or frozen at –80°C for later testing. cAMP levels were quantified using cAMP AlphaLISA kit (PerkinElmer, #AL312) following manufacturer’s instructions. Standard curves for each AlphaLISA immunoassay were performed in the same diluent as the samples being tested (1X lysis buffer with supplements) using the recombinant standards provided in each kit. Curves were plotted with a sigmoidal concentration-response curve with variable slope. Quantitation of protein levels in cellular assays were interpolated off their respective standard curves.

A549 cells were seeded at a density of 3 × 10³ cells/well in 96 multi-well plate. After 24 h, the cells were starved for 8 h with non-complete medium; then, cells were treated with BAY 60-6583 (100 nM) in the absence or presence of TGF-β1 (10 ng/ml) and MRS1706 (1 μM) for 5 min, 30 min, 6, 24, and 48 h. In some experiments, before incubation with BAY 60-6583 or TGF-β1, cells were pre-treated for 30 min with PD98059 (1 μM) (MEK, inhibitor). At the end of treatments, cells were fixed with 4% formaldehyde to preserve activation of specific protein modification. Levels of total and phosphorylated extracellular signal-regulated kinases (ERK1/2) were determined by ELISA assays, as previously reported (Giacomelli et al., 2015). Briefly, the cells were washed three times with wash buffer (0.1% Triton X-100 in PBS) and 100 μl of quenching buffer (1% H₂O₂; 0.1% sodium azide in wash buffer) was added and incubation was protracted for other 20 min. The cells were washed with PBS twice, and then 100 μl of blocking solution (1% BSA; 0.1% Triton X-100 in PBS) was added for 60 min. After blocking, cells were washed three times with wash buffer and the specific primary antibodies (anti-phospho ERK1/2, 1:500, sc-7383 Santa Cruz Biotechnology; anti-ERK1/2, 1:500, #4695 Cell Signaling Technology) were added on at 4°C. Subsequent incubation with secondary HRP-conjugated antibodies and developing solution allowed a colorimetric quantification of total and phosphorylated levels. Blanks were obtained by treating cells in the absence of the primary antibody. The relative number of cells in each well was then determined using Crystal Violet solution. The results were calculated by subtracting the mean background from the values obtained from each test condition; values were normalized to the number of cells in each well, and were expressed as the percentage of untreated cells (basal).

A549 cells were seeded at a density of 3 × 10³ cells/well in a black, clear bottom PerkinElmer 96-well CellCarrier^TM microplate (#6005550). After 24 h, the cells were starved for 8 h with non-complete medium; then, cells were treated with different concentrations of BAY 60-6583 (0.1 nM–1 μM) in the absence or presence of TGF-β1 (10 ng/ml) in 100 μl of non-complete medium for 48 h. In some experiments, before incubation with BAY 60-6583 or TGF-β1, cells were pre-treated for 30 min with PD98059 (1 μM, MEK, inhibitor), H89 (100 nM, PKA inhibitor) or 8-Br-cAMP (100 nM–1 μM, cAMP analog & PKA activator). Then, cells were imaged with the EnSight^TM multimode plate reader. After imaging, cells were lysed with 50 μl of 1X lysis buffer (PerkinElmer, #AL003C) supplemented with protease inhibitors and phosphatase inhibitors for 10 min with gentle shaking. E-cad were quantified using E-cadherin AlphaLISA kit (PerkinElmer, #AL370), and N-cad were quantified using N-cadherin AlphaLISA kit (PerkinElmer, #AL379) following the manufacturer’s instructions. Standard curves for each AlphaLISA immunoassay were performed in the same diluents as the samples being tested (1X lysis buffer with supplements), using the recombinant standards provided in each kit. Curves were plotted with a sigmoidal concentration-response curve with variable slope. Quantitation of protein levels in cellular assays were interpolated off their respective standard curves.

The Graph-Pad Prism program (GraphPad Software Inc., San Diego, CA, United States) was used for data analysis and graphic presentation. All data are the mean ± SEM of at least three different experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Dunnett post hoc analysis to compare the data to the control, or two-way ANOVA with Bonferroni correction and two-sided tests for multiple comparisons. EC₅₀ values were reported as mean of the values obtained in at least three independent experiments performed in duplicate ± SEM. P ≤ 0.05 was considered statistically significant.

The A549 human alveolar epithelial cells have been widely used to study the fibrotic process in the lung and related EMT mechanism (Kim et al., 2007; Ji et al., 2016). Furthermore, these cells were maintained in serum-free medium to increase the epithelial phenotype (Dong et al., 2014).

First, the expression of the AR subtypes in A549 cells was evaluated after incubation in serum-free medium for 48 h (Figure 1A). All the ARs were expressed under this condition, and the A_2BAR subtype was the most expressed with a fold change of approximately 200 (Figure 1A).

TGF-β1 has been reported to affect the expression of several proteins (Zhang, 2017); thus, the effects of cytokine treatment on AR expression were evaluated (Figures 1B–D). Challenging the A549 cells for 48 h with increasing concentrations of TGF-β1 (5–20 ng/ml) modified the expression of the different adenosine subtypes. Particularly, A₁AR expression was significantly increased when a high concentration of TGF-β1 was used; conversely, A_2A and A₃ receptor expression was slightly decreased when increased concentration of the cytokine was applied. Regarding A_2BAR expression, low concentrations of TGF-β1 (5 or 10 ng/ml) were not sufficient to modify the receptor expression; by contrast, a higher concentration of the cytokine significantly decreased its expression. In this light, further experiments were performed using a maximum concentration of the cytokine of 10 ng/ml for 48 h to minimize the change in the receptor expression.

A_2BAR stimulation or blockade has been correlated to different levels of cell proliferation depending on the cell type and culture condition (Phosri et al., 2017; Zhou et al., 2017). Thus, the effects of A_2BAR stimulation in the absence or presence of TGF-β1 were evaluated in the lung cells (Figure 2). The selective agonist BAY 60-6583 was used to stimulate selectively the A_2BAR subtype. The compound could slightly increase the cell proliferation when used at low concentration (50–100 nM) for 48 or 72 h treatment (Figure 2A), conversely, it produced a decrease in proliferation when used at a high concentration (1 μM), with a significant effect after 72 h of treatment (13970 ± 488 n° of cells CTRL, 11038 ± 816 n° of cells BAY; P ≤ 0.05). TGF-β1 alone presented a hormetic concentration-response course: the lower concentration possessed positive effects on cell proliferation that are lost at higher concentrations (Figure 2B). The combined treatment with BAY 60-6583 and TGF-β1 for 48 h did not significantly affect the cell proliferation (Figure 2C). These data confirmed the diverse role of A_2BAR in the cell proliferative mechanism. Based on such results, further experiments were then performed using BAY 60-6583 at a concentration (100 nM) not affecting significantly the A549 cell proliferation.

Next, the effect of cytokine treatment on A_2BAR functionality was evaluated by measuring the agonist-mediated cAMP production (Figure 2D). The potency of BAY 60-6583 was in the nanomolar range (EC₅₀ = 17.2 ± 2.5 nM), as derived by agonist concentration-response curves, it is a value comparable to that described for the same agonist in transfected cells (Figure 2D) (Trincavelli et al., 2014). The presence of TGF-β1 did not significantly affect the agonist potency (EC₅₀ = 10.0 ± 1.8 nM), even if the maximal effect of BAY in the production of cAMP was slightly increased (E_max = 4.3 pmol – TGF-β1; E_max = 5.8 pmol + TGF-β1, P = 0.0565) even if it was not statistically significant. These results are in accordance with the effect of other cytokines on A_2BAR functionality (Daniele et al., 2017); in fact, TNF-α has been demonstrated to increase A_2BAR coupling to the G_s protein (Daniele et al., 2017).

In parallel, the BAY-induced ERK phosphorylation was evaluated in order to further assess the A_2BAR functionality (Figure 2E). The agonist was able to produce a concentration-dependent increase of pERK with a potency in the sub-micromolar range of (EC₅₀ = 114.8 ± 24.5 nM).

Typically, the EMT process is triggered and maintained by the action of different cytokines and extracellular stimuli that, through the activation of specific intracellular pathways, modify the expression of several proteins (Nieto et al., 2016). A549 cells were treated for 48 h with the A_2BAR agonist alone or in the presence of TGF-β1 (10 ng/ml), which has been reported to induce EMT (Kawata et al., 2012). First, a morphological analysis of the cells was performed (Figures 3A,B). TGF-β1 treatment induced a change in cell morphology: most of the A549 cells, which normally display an oval shape, showed an elongated shape with a fibroblast-like appearance. Surprisingly, cells challenged with the A_2BAR agonist lead to a partial morphological change, with only some cells presenting an elongated shape, suggesting the induction of partial EMT.

The morphological changes, which are characteristic of cells undergoing EMT, are accompanied by a shift in the expression of epithelial genes to a mesenchymal gene repertoire (Nieto et al., 2016). Accordingly, challenging cells with TGF-β1 modified the expression of EMT markers, leading to a significant increase in the expression of the mesenchymal markers, Vimentin and N-cadherin (128.8 ± 6.5, P ≤ 0.01, and 162.7 ± 3.8% P ≤ 0.001 vs. CTRL, respectively; Figures 3C,D) and a concomitant decrease in the expression of the epithelial marker E-cadherin (6.7 ± 2.5% P ≤ 0.001 vs. CTRL; Figures 3C,D). The data were confirmed at the gene expression level by real-time RT-PCR analysis of E-cadherin (CDH1), Vimentin, and N-cadherin (CHD2) expression (Figure 3E).

A_2BAR stimulation was able to slightly decrease the expression of E-cad protein (Figures 3C,D) and increase the expression of a mesenchymal marker (N-cad, 119.8 ± 0.6% vs. CTRL; P ≤ 0.05). These effects are in accordance with the effects elicited by BAY on epithelial/mesenchymal gene transcription. Conversely, A_2BAR activation in the presence of TGF-β1 produced an opposite effect, significantly counteracting the induced EMT (Figures 3C–E). These effects were almost completely counteracted by concomitant treatment with the A_2BAR selective inverse agonist MRS 1706 (1 μM), demonstrating that they were mediated specifically by A_2BAR. The treatment with MRS 1706 alone was not able to significantly modify the expression of mesenchymal and epithelial markers in the absence or presence of TGF-β1 demonstrating that the adenosine in the medium was not sufficient to induce a basal A_2BAR activation.

The modulation of classical EMT centers on the transcriptional control of different transcription factors: Snail (SNAI1), Slug (SNAI2), ZEB1 and TWIST (Nieto et al., 2016). To gain insight into the mechanisms by which A_2BAR affects E-cadherin deficiency alone and in TGF-β1-induced A549 cells, the expression levels of the EMT-TFs were examined using real-time RT-qPCR (Figure 4).

A549 cells expressed Snail and Slug at low levels (Supplementary Figure 1), in accordance with the high expression of E-cad (Figure 2). Treatment with TGF-β1 for 48 h changed the expression pattern of these genes (Figure 4A), with a significant increase in ZEB1 (1.57 ± 0.13-fold; P ≤ 0.05), Slug (2.63 ± 0.25-fold; P ≤ 0.001), in accordance with the literature data (Ji et al., 2016). Next, the effects of A_2BAR stimulation (BAY 60-6583) or blockade (MRS 1706) were evaluated (Figure 4B). The MRS alone could not modify the expression of EMT-TFs, highlighting that the endogenous activation of the receptor was not sufficient to modify the related-EMT gene expression. Conversely, A_2BAR activation modified the gene repertoire, producing a significant increase in the Slug expression (1.65 ± 0.16-fold; P ≤ 0.05). These data are in accordance with the effects of A_2BAR activation on epithelial/mesenchymal markers (Figure 3).

Next, the role of A_2BAR stimulation on EMT-TF was evaluated in the presence of TGF-β1 (Figure 4C). As expected, the A_2BAR activation counteracted the effects of cytokine treatment, with a significant decrease in ZEB1 and Slug expression. When the A_2BAR inverse agonist was applied, the TGF-β1-induced decrease in gene expression was completely counteracted, demonstrating that these effects were mediated by A_2BAR.

A_2BAR couples to G_αs proteins, leading to an increase in intracellular cAMP, and to the G_q-PLC pathway, which induces the activation of the MAPK/ERK pathway (Schulte and Fredholm, 2003; Sun and Huang, 2016). Thus, activation of these two intracellular signaling pathways were investigated following A_2BAR stimulation, in the absence or presence of TGF-β1 (Figure 5). MRS 1706 alone did not affect the activation of the two pathways, demonstrating that the adenosine levels in our cellular model were not sufficient to activate the receptor. BAY was able to increase the intracellular cAMP concentration (Figure 5A), and significantly enhance ERK1/2 phosphorylation (178.9 ± 10.5% vs. CTRL, P ≤ 0.05; Figure 5B), producing transient activation of these kinases (Figure 5C). The effects evoked by A_2BAR stimulation were almost completely reversed by the A_2BAR inverse agonist MRS1706, demonstrating the specific involvement of the A_2BAR subtype. As expected, TGF-β1 did not produce a significant increase in the cAMP levels (Figure 5A); conversely, it was able to significantly increase ERK1/2 phosphorylation (341.7 ± 16.2% vs. CTRL, P ≤ 0.001; Figure 5B). Both BAY and TGF-β1 induced transient activation of ERK1/2 but with different kinetics; whereas ERK phosphorylation induced by BAY peaked within 30 min and then returned to the basal value within 6 h, TGF-β1 caused a more sustained ERK activation up to 48 h of cell treatment (48 h, 155.2 ± 7.8%, TGF-β1; 85.3 ± 8.8%, BAY; P ≤ 0.05; Figure 5C). These data highlighted a difference in the final reorganization of the intracellular pathways activated by the cytokine and the receptor.

When A_2BAR was activated in the presence of TGF-β1, the receptor activation produced a significant increase in the intracellular cAMP concentration (56.4 ± 1.9 nM, CTRL; 96.6 ± 5.3 nM, TGF-β1 + BAY P ≤ 0.01; Figure 5A). This effect was in accordance with the slight increase in A_2BAR functionality that was noticed in the presence of the cytokine (Figure 2D). Regarding pERK1/2, the co-treatment led to a modest decrease in phosphorylation. The BAY activities on cAMP and pERK1/2 were most completely counteracted by cell pre-incubation with the A_2BAR inverse agonist MRS1706. Finally, TGF-β1 and BAY did not affect the amount of total ERK (Supplementary Figure 2).

To elucidate if the different activation of cAMP and ERK pathway in the presence of the cytokine, could be related to A_2BAR desensitization, the functionality of the receptor was evaluated after 48 h of cell treatment in the absence or presence of BAY and TGF-β1 alone or in combination (Figure 5D). In the basal condition (48 h without treatment), A_2BAR maintained its functional response. The cell treatment with BAY (100 nM) for 48 h induced the desensitization of the A_2B receptor (219.9 ± 7.7% in Basal condition; 113.3 ± 13.6% in BAY pre-treated cells; P ≤ 0.001). Cytokine alone was not able to significantly interfere on the receptor desensitization. Interestingly, when cells were treated with BAY in the presence of TGF-β1 for 48 h, the desensitization of the receptor was partially impaired (164.2 ± 8.7%; P ≤ 0.01).

The role of the cAMP pathway in EMT is controversial, depending on the cell type, the nature of the EMT inducers and maximum levels of the intracellular cAMP obtained (Weng et al., 2015). Thus, the effects of different levels of cAMP on EMT markers in our cellular model were investigated using the cAMP analog Br-cAMP (8-bromoadenosine 3′,5′-cyclic monophosphate) as the PKA activator (Figure 6). Br-cAMP (100 nm–1 μM) induced a concentration-dependent increase in the intracellular cAMP levels (Figure 6A). Surprisingly, different concentrations of the activator caused opposite effects on E-cad (Figure 6B) and N-cad (Figure 6C) expression. A high Br-cAMP concentration increased E-cad and decreased N-cad expression. A low Br-cAMP concentration (100 nM) was sufficient to determine a modest decrease of E-cad (1646 ± 56 ng/ml, CTRL; 1540 ± 93 ng/ml, Br-cAMP; Figure 6B) and an increase of N-cad (246 ± 35 ng/ml, CTRL; 270 ± 8 ng/ml, Br-cAMP; Figure 6C) even if these variations were not statistically significant. When TGF-β1 and BAY were used simultaneously, the activator produced higher cAMP levels and a concentration-dependent increase in E-cad and decrease in N-cad.

These data demonstrate that, in our cellular model, when the cAMP was increased up to high concentrations, it could negatively affect the EMT process. Conversely, when cAMP levels remained low, it seems to mediate the opposite effects, promoting a slight decrease in epithelial markers and an increase in the mesenchymal markers. This controversial nature of the cAMP analogue on the EMT process is in accordance with the recent data reported by Zuccarini et al. (2017).

Nevertheless, high levels of cAMP counteract EMT, and ERK phosphorylation mediated by MAPK activation has been demonstrated to promote EMT (Singh et al., 2017). Thus, the ability of A_2BAR to modify the amounts of both pERK and of cAMP could be a possible mechanism by which the receptor agonist affects epithelial/mesenchymal markers. In this respect, the involvement of A_2BAR in the regulation of EMT markers and transcription factors was investigated in the presence of PKA inhibitor (H89, 100 nM) and a mitogen-activated protein kinase inhibitor (PD98059, 1 μM) (Figure 7, Supplementary Figure 3). The BAY-mediated increase in the intracellular cAMP levels was not affected by H89 treatment (Figure 7A); conversely, PD98059 was able to block BAY-mediated ERK phosphorylation (Figure 7B). In this respect, the inhibitors were used to unveil the involvement of the two signaling pathways in A_2BAR mediated triggering of EMT.

BAY decreased the protein density of E-cad and increased that of the N-cad one; these effects were lost in the presence of the inverse agonist MRS 1706, in accordance with the abovementioned data (Figure 3). The ability of BAY to decrease the expression of E-cad was augmented by pretreatment with the PKA inhibitor (2718 ± 58 ng/ml, CTRL; 2045 ± 157 ng/ml, BAY; P ≤ 0.01; 1483 ± 122 ng/ml, BAY + H89; P ≤ 0.001 vs. CTRL, P ≤ 0.01 vs BAY, Figure 7C). Similarly, A_2BAR mediated increase in N-cad was enhanced when the cAMP/PKA axis was abrogated (239 ± 20 ng/ml, CTRL; 406 ± 29 ng/ml, BAY; P ≤ 0.05; 558 ± 72 ng/ml, BAY + H89; P ≤ 0.001 vs. CTRL, P ≤ 0.05 vs BAY; Figure 7D). These effects highlighted that cAMP, through PKA activation, could partially antagonize A_2BAR-mediated EMT induction. When the MEK inhibitor was used, the changes induced by A_2BAR stimulation were almost completely counteracted, restoring the basal conditions (E-cad, 2586 ± 90 ng/ml, BAY + PD98059; P ≤ 0.05 vs BAY; N-cad, 260 ± 49 ng/ml, BAY + PD89059; P ≤ 0.05 vs BAY, Figures 7C,D). These results are in accordance with the effects of the inhibitor treatments on A_2BAR-mediated alteration of the expression of CDH1, CDH2 and vimentin genes (Figure 7E).

Finally, the modulation of the EMT-TF gene expression by BAY was evaluated in the presence of H89 and PD98059. Similarly, regarding the EMT markers, the PKA inhibitor slightly exacerbated the A_2BAR-mediated effects; conversely, the MEK inhibitor counteracted the BAY-mediated increase in gene expression. The PD98059 activity was more pronounced on Slug expression, causing a significant decrease of gene expression (1.65 ± 0.16-fold, BAY; 1.61 ± 0.20-fold, BAY + H89; 0.93 ± 0.09-fold vs. CTRL, BAY + PD98059; P ≤ 0.05). These data are consistent with the reported role of ERK phosphorylation in the induction of Slug expression (Choi et al., 2007; Joannes et al., 2014).